Document wKKV8wGMyGzY14VpmmBw8vKNJ

c\ b 4897 Texas Tech University Health Sciences Center SCHOOL OF MEDICINE / Department of Preventive Medicine and Community Health National Pesticide Telecommunication Network (NPTN) Lubbock, Texas 79430 (806) 743-3098 1-800-858-7378 Septem ber 7, 1988 Ben Nguyen C a r t w r i g h t , S l o b o d in , e t a l , C o u n s e lo r s a t Law 101 C a lif o r n ia S t r e e t , S u ite 2600 San F ra n c isc o , C a lifo rn ia 94111 D ear Mr. Nguyen: I am e n c l o s i n g c o p ie s o f l a b e l s f o r E s t e r o n 9 9 , F o rm u la 4 0 , a n d E s t e r o n 2 4 5 . I am a l s o i n c l u d i n g i n f o r m a t i o n on 2 ,4 - D , 2 ,4 ,5 - T a n d S iv e x w h ic h a re th e ch em icals in th e s e . The c o n te n ts o f each h e rb ic id e you l i s t e d is below : E stero n 245 - 2 ,4 ,5 -T Kuron - S i1vex Form ula 40 - 2,4-D 2,4-D ow Weed K i l l e r ( I c o u ld n o t f i n d t h i s p r o d u c t ) E ste ro n 99 - 2,4-D We do n o t h a v e some o f t h e i n f o r m a t i o n you r e q u e s t e d . I t h i n k Freedom o f In fo rm a tio n would be your b e s t so u rc e o r th e R e g is tr a tio n D iv isio n o f th e E nvironm ental P ro te c tio n Agency. The a d d re sse s a re below . Freedom o f In fo rm a tio n F0I O ffic e r (A-101) USEPA 401 M S t r e e t , SW W a sh in g to n , DC 20460 R ichard M ountfort R eg istratio n D ivision USEPA 401 M S t r e e t , SW W a s h in g to n , DC 20460 (TS-767C) T h an k you f o r w r i t i n g a n d l e t me know i f I c a n be o f a n y f u r t h e r h e l p . P esticid e S p ecialist V The LDso and How To Interpret It Pesticide toxicologists use rather simple' anim al toxicity tests to ran k pesticides according to their toxicity. Long before pesticides are registered with the Environm ental Protection Agency and eventually released for public use, the m anufacturer m ust delcare the toxicity o f their pesticide to the w hite ra t under laboratory conditions. T his toxicity is defined by the LD3o expressed as milligrams (mg) o f toxicant per kilogram (kg) o f body weight, the dose th at kills SO percent o f the test animals to which it is administered under experimental conditions. T he LDso is m easured in term s o f oral (fed to , o r placed directly in th e stom achs o f rats), dermal (applied to the skin o f rats or rabbits), and respiratory toxicity (inhaled). Combinad tabulation of pesticide toxicity classes. Routes of absorption Toxicity rating ldm Single oral dose for rats, mg/kg LDM Single dermal dose for rabbits mg/kg Probable lethal oral dose for man ' 6--Supertoxic 5--Extremely toxic A--Very toxic 3--Moderately toxic 2--Slightly toxic 1--Practically nontoxic <5 5 50-500 500-5,000 5,000-15,000 > 1500 <20 20-200 200-1,000 1,000-2,000 2^00-20,000 >20,000 A taste, a grain A pinch, 1 teaspoon 1 teaspoon to 2 tablespoons 1 ounce to 1 pint 1 pint to 1 quart >rt quart Source: Toxicity ratings modified from M. N. G leason, R. E . G leason, and H. C . Hodge. 1976. Sinica/ Toxicology o f Commercial Products. 4th ed. W illiam s and W ilkins Company, Baltimore, M d.p.6 * In LDW tests, 100% concentrates of the chem ical are used (unless otherwise stated), w hereas when you use the chem ical, it is diluted to a lower concentration which has a lower to x icity . Exam ple: Carbary! L D * m 500 mg/kg - Moderately to Very toxic 5% Savin Oust-*- LOM - 10,000 mg/kg - Slightly toxic (Carbary! is active Ingredient) The P esticide Book George W . W are. W. H. Freeman & Co. San Fran cisco - 1976 ( 4899 i FOR THE CONTROL OF TREES, BRI : .1 Low-Volatile Brush and W eed Herbicide for Industrial ; t ;i ACTIVE INGREDIENT: 2,4,5-Trichlorophenoxyacetic Acid, Propylene - Glycol Butyl Ether E ste rs........................................ ..............................69.2% INERT INGREDIENTS: .......................................... . . . .......... ....................30.8% 2,4,5-Trichlorophenoxyacetic Acid Equivalent --45-0% . >M 4 Pounds per Gallon . E.RA. Registration No. 464-205 l - ; E.P.A. E s t 464-MI-1 - JS t. - 3 "t-J # - i - t V . h t i *.< w 1^ ' i f 1 1 , ! r i . - llfl;i i PRECAUCION AL,-USUARIO: Si usted no lee ingls, no use este producto hasta que laetiquta le haya sido explicada ampliamente. TRANSLATION:'(TO THE USER: If you cannot read English, do not use this product untilthebel has been fully explained to you.) ; i r l - a i t i l l i i i i ' ; ; i : i -i SX . \ T;- r VV7* .* -* *ifJR** i-,-'4^' j>" " II 86-1064 PRINTED IN U . S . A . IfO EC EM B ER , 1980.. REPLACES SPECIMEN LABEL 86-1 Ob&^TTPPRRIINNTTED It l J0A/ NUARY, 1 9 8 0 . DISCARD PREVIOUS SPECIMEN LA B ELS. REVISION S INCLUDE: REPRINTED TO CORRECT ERROR IN PRECAUTIONS; CONTAINER GRAPHICS WERE CORRECT; SPECIMEN LABEL OF JANUARY 1930 4900 i i* ri- -. PSC'$ i* o LA B E L for Industrial Vegetation Control, Fencerows, and Rangeland ...69.2% ' K E EP OUT OF THE REACH OF CHILDREN MAY B E HARM FUL IF SW ALLOW ED MAY C A U SE IRRITATION Avoid Contact with E y e s, Skin and Clothing / Do Not Cut or Weld Container ,i \ In case of an emergency endangering life or I AGRICULTURAL CHEMICAL property involving this product, call collect | 517-636-4400 A * I Do Not Ship or Store with Food. Feeds. Drugs or Clothing , > , 5: iT r * ' * - -}:i - i i l - : l ; 1 | ' f ii '? x*1 ' :v r ; ? v 'w : `.1 -h f&. -a .*i }i ifM: ; ) 5 ali i -.ri-' i . *a - V ' . r V` ? k i : i -S >. i i. i } t;> f-; 4901 P -H llk FiON 245 HERBICIDE 1er Esters of 2.4,5-T Acid Equivalent: 4 Pounds per Gallon gallons of ESTERON 245 in 100 gallons of oil. Brush of average i feet high may take up to ISO gallons of spray mixture per acre, nt: Where growth is more than 6 to 8 feet tall, cut it close to the i the freshly cut stumps and stubs with 3 gallons of ESTERON (1 pint in 4 gallons) of oil, mixed thoroughly. For more resistant tons of ESTERON 24S in 100 gallons II pint in 3 gallons! of oil. IIexposed bark, as well as cut surfaces. This means spraying unn-off to the ground line is noticeable. Old or rough bark requires ne than young or smooth bark. Apply at any time, including ccept when ice. snow or water prevent spraying to the ground ire obtained on freshly cut stumps two inches across or larger, je normally requires from 10 to 100 gallons per acre depending nps and stubs. it: For large trees, make a singlehack girdle or "frill" of crvercompletely around the tree as close to the ground as feasible, roughly using a mixture of 2 gallons of ESTERON 245 in 100 n 3 gallons) of oil. latm ent: Use 1/4 pint of ESTERON 245 in 3 gallons of water all foliage, shoots, stems and bark without runoff. LOW VO LUM E SPR A YS tc sprays containing ESTERON 245 when foliage is well nts are actively growing. For best results on woody species, soil e sufficient to promote foliar growth. Spraying during proionger or after leaves have lost their normal green color and vigor sfactory control. Apply low volume sprays by air or ground hen spray drift will not be a problem --note use precautions. Usi w ered Knapsack Sprayer--Mix 1 1/2 to 2 gallons with ^ \ or kerosene to make 20 gallons of total spray soluportab. _ .tapsack mistblower to all sides of lower brush stems collar. Good coverage of the root collar is essential for best Slower at 1/4 to 1/3 throttle for best spray delivery and ximum drift control use a basal nozzle attachment and do not the horizontal position. I A P P LIC A T IO N FO R B R U SH CO N TR O L Itural Experiment Station, your local Extension Service Weed ts for best time to treat and need for re-treatment in your area, arty boot to milk stage where grass seed production is desired. >int ESTERON 245 plus 1/2 to 1 gallon of oil in enough water to I total spray per acre. Apply 40 to 90 days after first leaves ak: Use 1/2 to 1 quart of ESTERON 245 plus 1 gallon of oil in nake 4 gallons of total spray per acre, ck O aks: Use 2 quarts of ESTERON 245 plus 1 gallon of oil in nake 4 to 6 gallons of total spray per acre. U SE PRECAUTIONS e dairy animals on treated areas within 6 weeks after applicameat animals on treated areas within 2 weeks of slaughter, rr WITH 2.4.5-T SUSCEPTIBLE CROPS AND OTHER >AOLEAF PLANTS--ESTERON 245 Herbicide is injurious to mts. Therefore, do not apply directly to or otherwise permit nts to contact cotton, grapes, tobacco, fruit trees, vegetables. Is or other desirable plants susceptible to 2.4.5-T. Do not use <otise. IN THE VICINITY OF COTTON. GRAPES. TOBACCO. OTHER DESIRABLE 2.4.5-T SUSCEPTIBLE CROPS OR LANTS. DO NOT SPRAY WHEN WIND IS BLOWING TOWARDS SUSCEPTIBLE CROPS OR ORNAMENTAL PLANTS. AVOID SPRAY DRIFT--Applications should be made only when there is no hazard from spray drift since very small quantities of spray, which may not be visi ble. may severely injure susceptible crops during both growing and dormant periods. Use coarse sprays to minimize drift since, under adverse weather condi tions. fine spray droplets may drift a mile or more. The spray thickening agent, NALCO-TROL'. may be used with this product to aid in reducing spray drift. If used follow all use recommendations and precautions on the product label. 'NALCO-TROL-Trademark of NALCO Chemical Company GROUND EQUIPMENT--With ground equipment, spray drift can be lessened by keeping the spray boom as low as possible: by applying 20 gallons or more of spray per acre: by using no more than 20 pounds spraying pressure with large droplet producing nozzle tips: by spraying when wind velocity is 8 miles per hour or less. Do not apply with hollow cone-type insecticide or other nozzles that pro duce a fine-droplet spray. AERIAL APPLICATION--With aircraft, drift can be lessened by applying a coarse spray; by using no more than 20 pounds spray pressure at the nozzles; by using straight stream nozzles directed straight back; by using a spray boom no longer than 3/4 the wing span of the aircraft: and by spraying only when wind velocity is less than 6 mph. DO NOT APPLY BY AIRCRAFT WHEN AN AIR TEMPERATURE INVER SION EXISTS. Such a condition is characterized by little or no wind and with air temperature lower near the ground than at higher levels. The use of a continuous smoke column at or near site of application is suggested to indicate direction and velocity of air movement, and to indicate a temperature inversion by layering of the smoke. At high temperatures (above 95F) vapors from this product may injure suscepti ble plants growing nearby. Do not use in or near a greenhouse. Excessive amounts of this herbicide in the soil may temporarily inhibit seed germination or plant growth. Do not use around the home, recreation areas or similar sites. This product is toxic to fish. Keep out of lakes, streams, and ponds. Do not con taminate water by cleaning of equipment or disposal of wastes. Do not contaminate irrigation ditches or water used for irrigation or domestic pur poses. This product can be stored in an unheated building but if exposed to sub freezing temperatures, should be warmed to at least 40F and mixed thoroughly before using. Do not store near fertilizers, seeds, insecticides or fungicides. Do not reuse containers. To avoid injury to desirable plants, do not store, handle or apply other agricultural chemicals with the same containers or equipment used with ESTERON 245 except as specilied on this label. Rinse equipment and containers and dispose of waste by burying in non-crop lands away from water supplies. Containers should be disposed by punching holes in them and burying with waste or follow official local recommendations for container disposal. Local conditions may affect the use of herbicides. Consult your State Agricultural Experiment Station or Extension Service weed specialist for advice in selecting treatments from this label to best fit local conditions. Be sure that use of this pro duct conforms to all applicable regulations. Apply this product only as specified on this label. N O T IC E : Seller w arrants that the product conform s to its chem ical description an d is reasonably lit for the purpose stated on the label w hen used in accord an ce w ith d irections under norm al ccnd-tions of u se. but neither th is w arranty nor an y other w arranty of M ER C H A N T A B ILIT Y OR F IT N E S S FO R A P A R T IC U LA R P U R P O S E, exp ress o- kr^ lied . ex tends to the use ol th s nroriuct contrary to label in stru ctio n s, or under abnorm al co n d i tio n s. or under conditions not reasonably foreseeable to seder, and buyer assum es the risk o f an y su ch use 10586-00-9 m oan EMICcicMPANY ND SUBSIDIARIES lf*RA;- Hu nOBR/G* CEtNl ,- 4SWITZERLAND riJJONG KONG 1 3 4 . U S A 4 aRNIA.VONTABIC.ICAN AD A HE DOW C H E M IC A L *C O M P A Q ir^l f ? 4902 D O W ESTER! Contains Propylene Glycol Butyl Ether Esters DIRECTIONS FOR USE ESTERON 245 herbicide is recommended for industrial vegetation control, fencerows, and rangeland. Do not use in forest lands, rights-of-way. or pastures. This herbicide controls herbaceous and woody plants including such 2.4-0 resis tant species as--ash. black gum, brambles, groundcherry. hawthorn, horsenettle. maple, mesquite, oak, osageorange, palmetto, poison ivy. pricktypear cactus, redbay. salmonberry, sweetgum. wild blackberry, wild rose, and certain species of Ribes. Do not apply ESTERON 245 w here spray drift m ay con tact nearby 2.4.5-T susceptible crops or other desirable plants or may contam inate w ater intended for irrigation or dom estic purposes. Read and follow all Use Precautions given on this label. PREPARING THE SPRAY Use only diesel oil. No. 1 or No. 2 fuel oil or kerosene w here oil is recom m ended In the spray mixture. Oil sprays: Add ESTERON 245 to the required amount of oil in the spray tank or mixing tank and mix thoroughly. This,mixture can be made at any time before ac tual use and no separation will occur. Do not let any water, or oil-water mixture sprays get into the ESTERON 245 or into the finished mixture, as it may form a gel. W ater Sprays: Pill the spray tank about half full with clean water, add the re quired amount of ESTERON 245 and complete tilling the tank. Mix thoroughly and continue agitation while spraying. Caution: S e e NOTE fat paragraph on Oil-Water Mixture Sprays. Oil-Water Mixture Sprays: When vigorous agitation is used. 1 gallon of ESTERON 245 will emulsify up to 10 gallons of o i in 100 gallons of spray mature. First, premix the ESTERON 245 and oil in a separate container. Do not allow any water or mixtures containing water to get into the ESTERON 245 or the premix. Fill the spray tank about half full with water, then slowly add the premix with con tinuous agitation and complete tilling the tank with water. If the premix is put in the tank without any water, the first water added may form a thick "invert" (water in oill emulsion which win be hard to break. As an alternate procedure, the oE may be added after the ESTERON 245 is mixed in the water; but highly vigorous mechanical agitation is required and a poor emulsion may be formed. The premix method is preferred. NOTE: ESTERON 245 in water or oil-water sprays forms an emulsion, not a solu tion, and separation may take place unless sprays are agitated continousty. Mechanical agitation is recommended. H IG H V O LU M E S P R A Y S Basal Bark T reatm ent: Brush and small trees can be controlled by spraying the basal parts of brush stems and tree trunks to a height of 12 to 15 inches from the ground Bne. Use a solution of 3 gallons of ESTERON 245 in 100 gaUonsd pint in 4 gallons) of ok. With certain resistant species. 4 gallons of ESTERON 245 in 100 gallons (1 pint in 3 gallons) of o9. is effective. As only the basal portions of the brush are treated on a spot basis, the total amount sprayed per acre would not be _ expected to exceed 100 gallons. Knapsack or power equipment may be used, but complete wetting of the indicated area is necessary, particularly at the ground line. This means spraying untk run-down or run-off to the ground line is noticeable. Old or rough bark requires more spray than young or smooth bark. Low pressures are desirable. Apply a t any time, including the winter months, ex cept when snow, ice or water prevent spraying to the ground line. Often delayed response and killing can be expected . D orm ant Brush: Treat any time after brush is dormant and most of the foliage has dropped. Spray should be concentrated at the base of stems and in addition, the upper parts of the stems should be broadcast sprayed enough to wet them. Under rootsuckering species such as sumac, persimmon, sassafras and locust, also spray the ground area to control small root suckers tht may not be raadiy visible. Mix 1 1/2 gallons of ESTI density and 4 to 6 feet high may Stum p Treatm ent: Where grow, ground and spray the freshly cut 245 in 100 gallons (1 pint in 4 gall: species, use 4 gallons of ESTERC Wet thoroughly all exposed bark, til run-down or run-off to the groi more spray volume than young winter months, except when ice. line. Best results are obtained on Adequate coverage normally req on density of stumps and stubs. "Frill" Treatm ent: For large tre lapping axe cuts completely aroL Spray the friH thoroughly using . gallons (1/2 pint in 3 gallons) of S p o t Foliage T reatm ent: Use ' and spray to wet an foliage, shot LOW \ Apply low volume sprays con: developed and plants are actively moisture should be sufficient to p ed hot. dry weather or after leav may not give satisfactory contre equipment only when spray drift Basal Treatment Usinq^qwer of E S T E i r y i 5 with fT jor tkm. A p p v ^ /h a ________ (taps including the root collar. Good t results. Run mistbiower at 1/4 coverage. For maximum drift cor raise nozzle above the horizontal AIR APPLICATK Consult the Agricultural Experime or Range specialists for best time Do not use from early boot to mil- M esquite: Use 1 pint ESTERON i make 4 gallons of total spray pe appear. Sand Shinnery Oak: Use 1/2 to enough water to make 4 gallons ; Post and Blackjack Oaks: U se; enough water to make 4 to 6 gall USE F N ote: Do not graze dairy animals lion. Do not graze meat animals c AVOID CONTACT WITH 2.4.1 DESIRABLE BROADLEAF PLA most broadleaf plants. Therefore, even minute amounts to contact c flowers, ornamentals or other desi in or near a greenhouse. DO NOT APPLY IN THE VICI TOMATOES OR OTHER DESI; ORNAMENTAL PLANTS. iffl: * r * s-A Si l i i. '#I *s ;l i i : : * 4903 DOW 4 r V\% S ' , . -'J -`/S . . X -=.' . oc v ' W \ pr . V 3<'$"> N CONTAINS BUTOXYETHYI For the Control of Many Broadleaf Wee and Woody Plants Susceptible to 2,4-D in Grass Past ACTIVE INGREDIENT 2.4- Dlchlorophenoxyacetic add, Butoxyethyl E s t e r f ......................................................62.5% INERT INGREDIENTS ..........................................................37.5% 2.4- Dichlorophenoxyacetic Acid Equivalent: 43.2% -3.8 lb/gal tlsom er Specific by AOAC Method No. 6275-6.279 (13th Ed.) EPA Reg. No. 464-566 EPA Est.464-MI-1" *; 359-OR-15' Superscript used corresponds to letters in LOT number. PRECAUCION A L USUARIO: S i usted no lee ingls; no use este producto hasta que la etiqueta le haya sido explicada ampliamente. TRANSLATION: (TO TH E U S E R : If you cannot read English, do not use this product until the label has been fully explained to you.) ,, j IAvoid Contac In case of contact. irritation persists. H sticking finger down t unconscious person. This product istoxii likelv to occur. Do 1 8 .9 3 L / 5 g a l it ' * f '# : . 1 ? : $ 86-1674 PRINTED IN U .S .A . IN; SEPTEMBER, 1982". REPLACES SPECIMEN LABEL 86-1674~PRINTED IN MAY, 1982. 4904 DISCARD PREVIOUS SPECIMEN LABELS. REVISIONS INCLUDE: (1 ) REVISED WEED L IS T , "USE IN LIQUID NITROGEN H _cjeCt 4 F E R T IL IZ E " SECTION, "WEED C 0 T P 0 L IN SMALL G R A IN S ..." E r CTICN, 1 USE PREC-J~I0N.., AND STORAGE ANO DISPOSAL INSTRUCTIONS ' _ ) ADDED oid Contact w ith Skin, Eyes, or Clothing W ash Thoroughly After Handling j Statements of Practical Treatment ise of contact, iipmediately flush eyes or skin with plenty of water. Get medical attention if ition persists. If swallowed, induce vomiting immediately by giving two glasses of water and ing finger down throat. Call a physician. Do not induce vomiting or give anything by mouth to an insrious person, j Physical or Chemical Hazards , ' j Do Not Cut or Weld Container ; i ; - 1 Environmental Hazards product is toxic tp fish. Do not apply directly to any body of water. Do not apply where runoff is ito occur. Do nqt contaminate water by cleaning of equipment or disposal of wastes. Do not aminate irrigation ditches or water used for irrigation or domestic purposes. <' case of an emergency endangering life or opetty involving khis product call collect: 517*636-4400 > ; rvjr 5 : : : jg h or denting stag, apply 1 to 2 pints erenm ai w eeds, decrease weed seed d. cockfebur. dogbane, jimsonweed. with ` 'westing Do not torage or feed 'U S t JR A IN F IE L D S : Following the num. w ild g arlic and wild onion often 2 to 3 quarts per acre of ESTERO N 99 3rt of a control program . Do not torage -*r acre when sorghum is S to 15 inches ' to control som e w eeds but the chance it. Do not treat before the sorghum is S jg h stages. If sorghum is taller than 6 a s m uch a s possible. Temporary crop re and high a ir tem peratures Varieties -isitrwe Spray only varieties known to be .Mural Experim ent Station or Extension 9 am ount of w ater required for unrform iished stands in spnng from the tiller to seeding may be treated with the lower 1 w eed regrow th may be treated m the .M ASS P A STU R ES: N O TE: Do not graze ation. D o not use on bent grass, allalta. areas until g rass w e ll established. Do 3 production is desired. i-helder. MusktMstfte and Other B ra e * oer acre in the amount of water needed growing actively. 1 quart per acre win *1 w eeds may require repeated treat- m aking three applications (tau-spnngrmg. grass: Apply t to 2 quarts per acre use 1 quart m 5 gallons of oit or m 4 ircraft between May 15 and June is . On ->dapply by aircraft when foliage is fully acre m 2 to 3 gallons of oil or in 3 to 5 e 3 Quart rate is usually required. Brush d Retreatm ent may be needed, tebruah and C ertain Other Chaparral 3f w ta r. One gallon of fuel oil may be Jake applications by aircraft or ground ctive control, the brush m ust be fully nent may be needed, co p e o es susceptible to 2.4-0 m * \ anks. spray brush up to 5 to ft 1 4 Qw_. is of ESTER O N 99 Concentrate including foliage, stem s and berk. Ttua auate co verage of solid stand of brush. m e sp ray off the area being treated, ore frost as long as the so il moisture a esa effective m midsummerduring hot ire net atfrvety growing. O il or wetting sd effectivene ss. adleaf w eeds and brush on sites to be STERO N 99 Concentrate herbicide in ty 6 to 25 gallons. Applications can be apsack sprayer). Two to eight quarts of -iybe added to improve brush control, -iter o r spring to contro l susceptible rs. c u r a r e , cherry, service cherry and ;rak. u se ESTER O N 99 C oncentrate >r ground in sufficient spray volume tor in plantatio ns w here pete or tench era )hbte evergreen brush sp ecies, euch as m anzanita o r deciduous brush after its at rates up toftquarts per aere alone a o il or suggested rates of suitable STERO N 9ft Concentrate without oL -tart per acre but may cause injury or of sp ray tor good coverage of brush, enouaiy injured by treatment at these n. lack pm e. red pine, black spruce. * a harden oft and brush stiH actively *Concentrate peracre m enough water * ground to control certain competing 0nd w ihow Sm ce ttus treatment may >i cannot be tolerated. " ): Apply ESTER O N 99 Concentrate *<*pt<biby directing spray around the e unis of spray. Rates of ESTERO N 99 d-wpier. or water carrier at 10 to 100 v N S. G O LF CO U R SES. C EM ETER IES. AINAGE DITCH BA N KS: Apply 1 to 3 amount ot water needed for uniform *ed control under average conditions - on goft greens nor on dichondra or n r '-- or >g grasses such as bnt and ** nin g rass is waffesrat htned m l With spring application *>g v - ju m es ara usually damaged or riaeor *otd perenmai w eeds may u ~qt on- r a r e . E 4 T ER C N -9 Concentrate herbicide - e * ? l O ca' m s of water Spray to wet all foliage (400-800 gallons per acre). Addition of a wetting agent may be advisable Apply m the spring during flower head em ergence. Respray it needed when regrowth is 3 to 5 feel tall W EED CON TRO L ON FALLOW LAND: Use 1Wto 2% quarts per acre on annual broadleaf weeds and up to 3 quarts per acre on established perennial species, such as Canada thistle and lie u bindweed. Apply to actively growing weeds. Do not plant treated tallow land until three months alter treatm ent, or until chem ical has disappeared from soil. SPO T TREATM EN T: To control broadleaf weeds in small non-cropland araas with a hand sprayer, use Vi pint of ESTERON 99 Concentrate in 3 gallons of wstar and spray to thoroughly wet alt weed foilage. Keep spray m ixture agitated to prevent separation. CO N TRO L O F WOOOY W EEDS IN LO W -BU SH BLU EBERRY FIELD S IN M AIN E: How to u se: Mount a drum ft to 10 feet long or som e other suitable length, and 1Vrto 2 feet in diam eter on an axle su ch as an ok} hay raka frame. Cover the drum with watar absorbent yet tough cloth which win resist rapid wear and tear. Draw the cloth-covered drum across the blueberry field and at the sam e time spray evenly onto the full length of the top of the cloth-covered drum a spray m ixture made by diluting 1quart of ESTERO N 99 Concentrate in 50 gallons of water per acre. Have the drum mounted so that aa it revolves on its axis it is high enough to m iss most of the low bush blueberry stem s, yet low enough to forcibly brush the spray-saturated cloth-covered drum against the higher woody w eeds, principally sweet fern,wild cherry and poplar. Kaep the cloth wet enough to provide top coverage of the weeds, yet not so wet as to allow runoff of the liquid w hich could cause m/ury to the blueberry plants. W hentoU se: Applyduring June and Ju ly when weed tops have amarged sufficiently above the blueberry stem s to allow treatment ot the weeds and not the blueberry plants. Apply only during the year before the first bum. To use this method of weed control, two-year bum s should be extendad to three years. Caution: Do not allow tha spray baing applied to the cloth-covered drum to be directed onto the blueberries. Do not harvest-rake field during the herbicide treetment year or until a two-year interval thereafter. U SE PRECAUTIONS AVOID CON TACT WITH 2,4-0 S U SC EP TIB LE CRO PS AND OTHER D ESIR A B LE BRO AD LEAF PLA N TS: Do not apply directly to or otherwise permit even minute am ounts to contact cotton, grapes, tobacco, fruit trees, vegetables, ftowers, ornamentals or other desirable plants susceptible to 2.4-D. Do not use in or near a greenhouse. DO NOT APPLY IN THE VICINITY O F COTTON. GRAPES. TOBACCO. TOMATOES OR OTHER D ESIR A B LE 2.4-D SU SC EP TIB LE CRO PS OR PLANTS. DO NOT SPRAY WHEN WINO IS 8LOW ING TOWARDS SU SC EP TIB LE CRO PS OR ORNAMENTAL PLANTS. AVOID SPRAY D R IFT: Applications should be made only when there is no hazard from spray drift since very sm all quantities ot spray, which may not be visible, may severely injure susceptible crap s during both growing and dormant periods. Use coarse sp rays to minim ize drift sin ce, under adverse weether conditions, fine spray droplets may drift a m ile or more. A spray thickening agent sweh as NALCO TRO L1. maybe used with this product to aid m reducing spray drift. If used, follow all use recom m endations and precautions on the product lab el ' NALCO TR O L -- Trademark of NALCO Chem ical Company GROUND EQUIPM EN T: With ground equipm ent spray drift can be lessened by keeping the spray boom a s low as possible: by applying 20 gallons or more of spray per acre: by using no more than 20 pounds spraying pressure at large droplet producing nozzle tip s: by spraying when wind velocity is low: and by stopping all spraying whan wind exceeds 6 to 7 m iles per hour. Do not apply with ho&ow cone-type insecticide or other nozzles that produce a bnedroplet spray DETERM IN E AtR MOVEMENT AND D IRECTIO N S B EFO RE FO U A R APPLICA TIO N : U se a sm oke generator or other means at or near the application site for the detection of air movement a * stability or temperature inversions. Such a condition exists when there is Httte or no wind and air temperature n lower near the ground than at higher levels. U se appropriate drift control m easures or avoid application when sm oke a moving toward nearby desirable susceptible plants or sensitive areas. A ER IA L APPLICA TIO N : With aircraft, drift can be les sened by applying a coarse spray; by using no more than 20 pounds spray pressure at tha nozzles: by using straight stream nozzle directed stream back: by using e spray boom no longer that Vi tha wing or rotor span of tha aircraft: and by spraying only when wind velocity is lew than ft mph. Exce ssive am ounts of this herbicide in the so il may temporarily inhibit seed germ ination or plant growth. Violent wind storm s may move sod particles. If 2.4-0 is on so il particles and they ara blown onto the susceptible plants, visible symptoms may appear. Serious injury is unlikely The hazard of movement of 2.4-0 on dust is reduced rf treated fields era irrigated d r rt ram o ccurs shortly after application. AT HIGH TEM PERA TU RES. VAPORS FROM TH IS PROOUCT MAY IN JU RE SU S C EP T IB LE PLAN TS GROW ING NEARBY. To avoid injury to desirable plants, do not handle or apply other agricultural cham icala with the same equipment used lor ESTERO N 99 Concentrate unlew appropriately cleaned fu st Le e conditions may affect the use of herbicides. Consult your Stats Agricultural Experiment Station or Extension Service weed sp ecialists for cleaning methods which ara in com pliance with lo cal regulations and for advice in selecting treatments from this lab to best fit local conditions. Be sure that use of this product conform s to 1 applicable regulation. Apply this product only as specified on this tab. STORAGE AND DISPOSAL Do not contam inate water, food or feed by storage or disposal STO RA G E: Keep container tightly dosed when net in use. P ESTIC ID E D ISPO SA L: Pesticide, spray mixture or rinsafe that cannot be used according to label instructions must be disposed ot according to Federal. S t e . or lo cal procedures w ider the Resource Conservation and Recovery A ct CON TAIN ER D ISPO SA L:Triple rinee or equlvalant and offer for recycling or reconditioning, or dispose o< in a sanitary landtift. or by other approved State and local procedures. M ONEY BACK GUARANTEE fT T fftfT N ft rnnrew trats heittirkte Is QuerantaerthyTtu Oow Chem ical Com pany to tne tun extent of the purchase p rice: 1. To give satisfactory central of n ee d and brash sp ecies listed on conta in er when 2. To form a aultabto spray m ixture In any water At for spray usn. 3. To store satisfactorily at tem peratures a s few as -4 (rF. KOTiCC Shut orrenfeihet tie product conformslo m w w snd ttreaeonablyM ter Pie purpows m lM or' the laoei u m need ki eecorexrt* thow tw i .inoer normal condition* otuae. Out rvertT-vr -n mirmntf nor any Other w m n>| of MEACHANTA8H T> or FITNESS FOR A PARTICULAR PURPOSE, -xpree or xnohed. extend lo ttw m of mu product corverr to laoel instruction*. or undsr epiofim l conditions, or underconditionsnot rawoneOh t>sailer and buyer assume me nea o' any s>*ch wee 10596-025-2 20682-6992 THE DOW CHEMICAL COMPANY ANO SUBSIDIARIES M ID LAN D . M ICHIGAN 4BB4Q. U SA HO RGEN . S W U Z l R lA N O H O N G K O N G CO RAL G A B L E S . FLO RID A 3 3 1 3 4 . U SA SARN 'A O N TARIO . CAN ADA * tradem ark f THE OOW CMC M ica i OMPANr *0 *0 ?5<% ) 4906 ) V - 20OC>t * WEED LIST ESTERO N 99 Concentrate herbictoe <S recommended tor control oi num erous broadlsaf weeds end certain 2.4-D susceptible woody plants without injury to most established grasses. Species controlled include the following, plus many others' beggarlicke M terw eed bluew eed. T e sa s broom weed toucfcbrueh buckw heat, wNd burdock burhead carp etweed catnip cham ice chico ry cockle bur coffe tw eed cornflow er coyotebrush croton dandelion docks dogfennsl elderberry gaitneoge garde, erttd goatabeard halogeton hem p, wild fewetweed Jimaonereed ladyathumb tom bsquarter loco. Mgbend mattow, Venice m aruanlta marebalder mkkvetch mom Ingglory, annual nettlee onion, wild pennycrese (fanwee e d) pepperw eed, held pigweed" plantaina poorfoe rabbttbrueh radish, wfld ragw eed rap a, wild radatem a g a , co aataleaa g ab ru a h , big sa g e b ru sh , san d s a ls ify sa n d sh In n ary oak shephe rdspuree alcklepod sm ertw sad (annual) an ease w eed, bitter eowlM etle, ennuehepenlehneedles eum ec sunflow er sw sstclover teneyregw ort thistle, bull htsde. m usk # thistle, R u stan tum bleweed valve beef verveine vetch w ater plantain aHM m ustard willow wttchweed wormwood ye llow rocket yettow starlhiette ttT h e control of "hybrid** pigweeds appears to bo lass satisfactory from 2.4-0 products than formerly experienced on "non-hybrid" varieties. Since 2.4-0 herbicides are not as effective on the "hybrid" pigweeds, it is necessary to apply higher rates of 2.4-0 for control, especially later in the growing season. Higher rates injure som e crops, so le ss than satisfactory pigweed control may be experienced by the highest tolerated crop dosages. Therefore. The Dow Chem ical Company no longer includ es pigweed among the sp ecies covered by the perform ance guarantee statem ents on the lab els for ESTERO N 99 Concentrate herbicide. At this tim e, th is d o c tanner applies only to the High Ptams of Texas and western Oklahom a, including the Panhandles. All other guarantees on these product labels are unchanged by this disclaim er. DIRECTION S FOR U SE It is s violation of Federal law to usa this product in a m anner inconsistent with its labeling. Apply ESTERO N 99 Concentrate herbicide as water or o il spray during warm weather when w eedsor brush are actively growing. Application underdrought conditions often w illgive poor results. Use low spray pressure to minimize drift On cropland and along roadsides, do not excaed 20 psi pressure. Apply enough spray volume to provide uniform coverage of w eeds end brush, usually S to 20 gallons per acre by ground equipm ent end 3 le S gallons by aircraft. Higher galfonage may be used if desired to improve spray coverage. Generally, the lower dosages recommended on this label w ill be setafactory for young, succulent growth of sensitive weed sp ecies. For le ss sensitive sp ecies end under conditions w here control is more difficult, the higher dosages will be needed. For crop u ses, do not mix with o il or other adjuvants u nless specifically recommended on this label. Deep-rooted perennial w eeds such as Canada thistle end field bindweed end many woody plants usually require repeated applications for maximum control. Do not apply ESTER O N 99 Cancan b ats w here sp ray drift m ay contact nearby su scep tib le crepe or other desirable plants or m ay contam inate w ater 1er irrigation o r domestic u se. Reed end toMow sk U se P recautions given on th is lab el. N O TE: If there are uneetlam tiesconcem ing special local use situations or sp ecific crop variety tolerances to 2.4-0. consult your State Agricultural Experim ent Station or lo cal Extension S e rv io weed sp ecialists for advice. TO PREPA R E TH E SPR A Y: (1) Fin the spray tank about had full with water, then add the required amount of ESTER O N 99 Concentrate, with agitation, and finally the rest of the water. NOTE : ESTERO N 99 Concentrate in water form s an em ulsion w hich lends to separate u nless the mixture is kept agitated. (2) If oil is added, first mix the ESTERO N 99 Concentrate and the oil and then add this mixture to the water. However, with adequate agitation, the oil can be added after the ESTERO N 99 Concentrate n mixed in the water. (3) Hstraight oil is used, a solution formad and separation does not occur. Do not allow any water to get into the od-htrbicide mixtura to avoid formation of an invert em ulsion. U SE M LIQ UID N ITROGEN FER T ILIZ ER : ESTERO N 99 Concentrate may be com bined with liquid nitrogen fertilizer suitable lor foliar application to accom plish weeding end feeding of com . sm ell gram a or grass pastures m one operation. U se ESTERO N 99 Concentrate m accordance with recom m endations for these crops as given on this label. Use liquto fertilizer at ratas racommended by supplm r or Extension Service Specialist. Test for mixing com patibility using ds eired procedure and spray m a proportions m d e ar g lass jar b alers mixmg in spray tank. A com patibility aid su ch as UM TE"' OR CO M PEX*' may ba needed in som e situations Compatabitity bast with straight liquid nitrogen fertilizar solutions Mixmg with N-P-K solutions or suspensions may not ba satisfactory. even with addition of a com patabilrty aid. Premixing ESTERO N 99 Concentrate with 1 to 4 parts water m ay help m difficult situations. "'Tradem ark of Hopkins Chem ical Company * Trademark of Kalo Laboratories Ft the spray tank about half fun with the liquid fertilizer, than add the ESTERO N 99 Concentrate with agitation and com p u ts filling the tank with fertilizer. Apply im m ediately and continue agitation m the spray tank during application. Do not atora m o sp ray mfariure. Application during vary cold weather (near freezing) not advisable. W EED CON TRO L IN SM A LL GRAIN S NOT UN DERSECOCO WITH A LEG U M E: N O TE: Do not permit dairy anim als or m eat anim als being finished for slaughter to forags or graze treated gram fields within 2 w eeks after treatment. W heat S aHey and R ye: Appty to to 1 pint par sera. Spray when grain la in fuit tite r stag# (usually 4 to 6 inches taH) but before the boot stags and boot to dough stag*. For unproved control of difficult weeds including wild g arlic and wild onion or under dry or cool conditions, apply up to 2plnta per acre. Wild g an e ano wild onion may not ba kitted but dockagashould be reduced. Do not use higher rates unless pots ib is crop injury w ill be acceptth is . Consult State Agricultural Experim ent Station or Extension Service Weed Sp ecialists for recom m endations or suggestion s to fit lo cal conditions. S prlngS asdadO atatApptyW pint per sere at the full ittitf stage but before the oarfy boot stage O ats are Mss tolerant to 2.44) than wheat or ba1ley and are m ore likely to suffer sem e injury Fa9 Seeded O afs (Southern) Grown for G ram : Apply to t o i to pints par aera after futt tillering but before the early boot stage. Soma d if'-o ft weeds may require higher rates for maximum controlbut crop injury may result. Do not spr ycuring or nm edlatsty following cold weather Frsh a rv s>tTreatm ent: Apply 1to 2 oints iw< acrt when gram s a rt In the hard dough stag*' tc control large w eeds that may in tsrere w in n rv e sL Bast resude vrtH ba obtained when soi- m oisture is sufficient to cau sa suci uU*nt w-ed growth. N O TE: Do not feed treated t v s * to livestock. W EED CON TRO L W CO RN : Use onoMhfc Mowing in ree program s. P tsem srg eo ce: Apr iy t to 2 quarts per acre to so il anynm* *!> p *n:>ng but before com em erges Only em eraec twoedleafed w eeds are likely to be c o n to'ter Do not apply m ere than 1 quart per acre urnes* the increased risk ot crop injury can be to-e iteo Do not use on light sandy soil. Em ergence: Apply 1 pmt per sere just as corn plants are breaking ground. Fetto m eig an ca: Alter emergence of com use to omt per sere Aop eatmn of to to 1pmt per acre may be needed fo> m u inu m controlo! some weeds but such ra--s are mere kieyto injure th sco rn .it com -sever 6 >c,'estali.ueedropp-5zrtosto-e*ot*,e>or sy ch fie <i' n ftiageasm uchasooss< bi*.D ono: at pu from the tassslm g tc dougn vag- Co> oi j s - w :h *1 atiszm e or other adjuvant* C*oc m urv is more likely to occur il con- is gn w -c -a. >di. under high tem peraure and high so moisture conditions. To reduce breakage >f*' - s on tpm voisryonttleness caused by 2.--D d- ia\ cultivation for 8 to 10 days afi-r t*e.-.trr - - r 5 n:>t `orngeor feed com fodder tor 7 cay* fc io-vmg application, n o t e H>or*is . a w ' r m se t? r 4 -0 and som e are easily mju ec S : ra. only varieties known to be to e ra n to . - D ;i tar t seed company or you* Agricultura > 'im ent Station or Extension S**rv ce . sp-^ci ns-s *or this information V PREH A RV EST CORN TREATM EN T: After the hard dough or denting so per acre by a<r or ground equipment to suppress perennial w eeds r -production, and control tall-w eeds su ch as bindweed, cockiebur. dc ragweed, sunflow er, velvetfeal and vines that interfere with harvesting C corn Iodder for 7 days following application. CO N TRO L O F W ILD G A R LIC AND W ILD ONION IN STU B B LE GRAIN FI harvest of sm all gram , soybeans, com or grain sorghum, w ild g arlic a produce new tad growth. This should be sprsyed with 2 to 3 quarts p e r; Concentrate herbicide This is a useful practice as one part of a control pre for 7 days following application. W EED CO N TRO L M SORGHUM (M ILO):Apply to pmt peracre whan sore tall. A higher rate of to to 1 pint par acre may be needed to control some lor crop injury is likew ise increased. Do not use with oil. Do not treat bet m ches tall nor during the boot, flowering or early dough stages. If sore inches, use drop nozzles to keep the spray off the foliage as m uch a s poss injury may occur under conditions of high so il moisture and high sir le r vary in tolerance to 2.4-D end some hybrids are quits sensitive. Sprey onlyv tolerant to 2.4-0. Contact seed company or your Agricultural Experim ent Service weed sp ecialists for this information. G R A SS S EE D C R O P S : Use 1 to lto pints per sere in the amount of water application by air or ground equipment. Apply to established stands in sp early boot staoa Do nol spray m boot stage. New sprmg seedings may be t rata after the g rasses have at least fn * leaves Perennial weed regrowth r fall. W EED AND BRUSH CO N TRO L IN RANGELAND ANDG R A SS PASTU RES dairy anim als on tiaated areas withm 7days after application.Oo not usee clover , or other legum es. Do not use on newly seeded areas until g rass is not use from early boot to milk stags where grass seed production is de BW erw eed, Broom weed, Croton, D ocks. Kochia, M srshelde r. M uakthis: foef W eed s: Use 2 quarts o l ESTERO N 99 Concentrate per sere m the amc for uniform application if the w eeds are young and growing actively. ' provide control of som e sp ecies Oeeprooted perennial weed s may rec m ents m the sam e year or m subsequent years. W ild G arde and W ild O nion. Apply 2 to 3 quarts per acre, making three app fall or sprm g-fall-spnng) siartm g m late fall or early spring W eed Control In New ly Sprigged C oastal Berm udagrata: Apply 1 tc preem ergence and or postem ergence. Send Shbuiery Ook end Send Segebrush: On the oak. use 1 quart in 5 gallons p i water plus 1gallon of oil per sere. Apply by aircraft between Ma the sagebrush, use 1quart m 3 gallons of oil per sere and apply by atrcrati expanded and the brush a actively growing. S Iq Sag eb ru sh and RebM brw sh: Use 2 to 3 qusrts per acre m 2 to 3 gall gallons of oil-water em ulsion spray. For rabbitbrush, the 3 quart rat# is use should be leafed out and growing actively when treated. Retractm ent me Cham foe. M anzaM ta. Buckbrush. C o astal S a g s, Coyotebrush and Cart: tp se fo s: U se 2 to 3 quarts per acre m S to 10 gallons of water. One galic included in the spray m ixture lo r added effectiveness. Make applications equipm ent to obtam uniform spray coverage. For effective control, the . leafed out and growing actively when sprayed. fieteatment may be need W OODY FLAM T CON TRO L IN NON-CROP A R EA S: To control sp ecies s t nghts-ot-way. fonctrow s. roadsfoes. and along dramage ditch banks, spr foot tall after sprmg foliage is well developed, using3 to 4 quarts of EST Ef in 100 gallons of water and weftmg all parts ofthe brush ineluding foliag e.: may require up to 400 gallons of spray per acre for adequate coverage o f: Make application m su ch s way as to prevent drift of the spray off the Spraying can be effective at any time up to 3 w eeks before frost as tong a: sufficient for active growth of the brush. Control wiU be less effective in iruc dry we ather when so il m oisture a deficient and plants are not a ctively gtc agent may be added to the spray, if needed for increased effectiveness. Fore st BAe Preparation: For control of susceptible broadleal w eeds and planled m forests, use 1.5 to 8 quarts per acre of ESTERO N 99 C o n sufficient spray volum e lo r good plant coverage, usually 6 to 25 gallons meda by aw or ground (hand gun. boom, or powered knapsack sprayer). Tv d iesel o il per acre o re suitable surfactant or penetrant m aybe added to Inv Pereet C onifer R e le a se : For applications m fote winter or spring to deciduous brush sp ecies, such as alder, willow, poplars, cascara. chsrry vme m aple during early growth and before conifer budbreak, wee EST ER C rates up to 3 quarts par acre in diesel or stove oil by air orground msuftci< good plant coverage, usually 8 to 25 gallons. Do not u se In plantations wh i am ong 9 m d esired sp a d e s. For treatment before conifer budbrwskto controlsusceptible evergreen br tanoak. mendrone, chinquapin, ceanothus spp- and m anzanita or dec leafout o r broadleaf w eeds, uee ESTERO N 99 Concentrate at rates up to 3 c pr w ith O S to 2.0 gallons par sere of diesel or sim ilar pu or suggest surfactants or penetrants. After conifer budbreak. ESTERO N 99 Cone surfactant or penetrant can be used at rates up to 2 quarts par acre but suppression of the conifer growth. U se sufficient votum# of w xay for gooc usually to 25 gsttons. Som e sp ad es of pme may be seriously injured growth stages. After conifer species su ch as white pme ponder** pine, jack pm e. red wmtesp ru ce. red sp ru ce, and balsam fir cease growth and harden eft an d : g-ow ingm latt sum m er. L 5 lo 3 .0 qoartsofESTER C N -*9Concentrate pare tc obtain good plant coverage may be applied oy aw x oround to contre htrdw ood sp ecies su ch as aider, aspen b" Ch halt *>d wttiow. Sm ce cause occasio n al conifer mjury. do not use ><such n jn cannot be toter. D irected S p rays le Con Her F le a tH o m (Including pine): Apply EST ER herbicide at anytim e brush or broadieef wer-dsart susceptible by directs conifers to avoid epntact of nacdies w itr m ur.ou- a-nojnts o* spray. Ra C incentrata are net to exceed a quarts per acre ir o . c il-water. or wote? gallons per acre. - W EEO CO N TRO L M NON-CROP A R EA S SUCH AS LAWNS. G O LF CO O T' PARKS. A IR FIELD S , RO ADSIO ES. VACANT LOTS. DRAINAGE DITCH 6 quarts of ESTER O N 99 Concentrate par acre <n the- amount of water r application. UsuaNy 2 quarts par acre provides good weed control und er. Treat w een weeds are young and grow mg well. Do no* use on golf greens r o*ne- D' oa ile a l herbaceous ground cove*s. Do no* u*e cnc*ee ir g gras*' S* A .Qks i ne except for spot treating no* O'* newt, si ed-d *ur* ur til grass P -s--?d ng of treated areas sheuto be deia-.ed W ov. treatment. Ffith *-se-d n tne fall, with fall appticat.on reseed in the s:-- -g uscum e* are * * co not treat areas wrfere legume* s-e sss Oeeorcoted pe r -e; esied treatm ents m me sam e lease* o* w s. rs-quen* years TU LE(B U LR U SH )A N O O TH ER RU SH ES M *2Q iH ri c .*TERO N 4Co .*'d * g ton <i dw sel oil or kerosene, fn sr acd m t **-* e t- 1C 3g si* o* 4907 X ?- S o o z - 1 EU JG NiiiuN AND MANAGEMENT OF PESTICIDE POISONINGS DONALD P . MORGAN CHLOROPHENOXY COMPOUNDS GENERAL CHEMICAL STRUCTURE (or CH j) Cl ESTER GROUP Cl - 0 - SODIUM (Cl) A LK YL AMINE COMMON COMMERCIAL PESTICIDE PRODUCTS Several hundred commercial products contain chlorophenoxy herbicides in various concentrations and combinations. Following are names o f widely advertised formulations. In some cases, the same name is used for products with different ingredients. Exact composition must therefore be determined from product label. pZi`4-U, 2,dichlorophenoxyacetic acid (W eedonet, Agrotec, Amoxone, Aqua-Kleen, BH 2,4-D, Chipco Turf Herbicide " D " , Chloroxone, Crop R ider, DSO, Dacamine 4D, Ded-Weed, Desormone, Dinoxol, DMA4, Dor* m one, Emulsamine BK, Emulsamine E-3, Envcrt DT o r 17l, Eft?bH 9 9 C on centrate, Esteron Four, Esteron,Brush Killer, Estone, Fernoxone, Femimine, Ferxone, Fernesta, Formula 40, Hedonal, Herbidal, Lawn-Keep, Macondray, M iracle, Netagrone 600, Pennamine D, Planotox, Plantgard, Rhodia, Salvot, Spritz-Hormin/2,4-D, Spritz-Hormit/2,4-D, Superormone Concentre, Super D Weedone, Transamine, U46, Verton 2D, Visko-Rhap, Weed-B-Gon, W eedar, Weed-Rhap, Weed Tox, Weedtrol, De broussaillant 600, Lithate, Dicotox, Field Clean Weed Killer). 2,4-DB is the butyric acid homologue o f 2,4-D. Dichlorprop it the propionic acid homologue. r7 ,4 ,$ -T or 2,4,5-trichlorophenoxyacetic acid (Brush-Rhap, Dacamine 4T, Dbroussaillant Concentre, Ded-Weed Brush Killer, E&tt ron 24$1 Fence Rider, Forron, Inverton 245, Line Rider, Spontox, Super D Weedone, Tormona, Transamine, Trinoxol, Trioxone, U46, Veon 245, Verton 2T, Weedar, W eedone Envert T).; Common mixtures o f 2,4-D and 2,4,5-T are: Dacamine 2E72T, Esteron Brush Killer, Rhodia Low Volatile Brush Killer No. 2, U46 Special, Tributon, O Visko-Rhap LV2D-2T, and Transamine. > ------------------- A product of identical name containing pentachlorophenoi (Chapter 4) as } th e active ingredient has been discontinued by Amchem Products Co. '$ A product o f identical name marketed by the Crystal Chemical Com pany ^ contains cacodylic acid as the active ingredient (Chapter 10). r p-2j4;3-TPTSfl'Vtx)'is the propionic acid homologuc of 2,4,5-T.tKllrOh1S(.low volatile ester o f 2,4,5-TP. 2,4,5-TB is the butyric acid homologue of 2,4,5-T. Fenac or chlorfenac is 2,3,6-trichlorophenylacetic acid. Dicamba (feanvel) is dichloroanisic acid. MCPA, MCPB, MCPB-Ethyl, MCPCA and MCPP (Mecoprop) are 2-methyl, 4-chlorophenoxy aliphatic acids and eslets. W ^ TOXICOLOGY OF Some of the chlorophenoxy acids, salts, and esters are moderately irritating to skin, eyes, and respiratory and gastrointestinal linings. In a few individuals, local depigmentation has apparently resulted from prolonged ahd repeated dermal contact with chlorophenoxy materials. The chlorophenoxy compounds are absorbed across the gut wall, lung, and skin. They are not significantly fat storable. Excretion occurs within hours, or at most, days, primarily-in the urine. Given in large doses to experimental animals, 2,4-D causes vomiting, diar rhea, anorexia, weight loss, ulcers of the mouth and pharynx, and toxic injury to the liver, kidneys, and central nervous system. Myotonia (stiffness and in coordination o f hind extremities) develops in some species and is apparently due to CNS damage: demyelination has been observed in the dorsal columns of the cord, and EEG changes have indicated functional disturbances in the brains o f heavily dosed experimental animals. Ingestion o f large amounts o f chlorophenoxy acids has resulted in severe metabolic acidosis in hum ans. Such cases have been associated with electro cardiographic changes, myotonia, muscle weakness, myoglobinuria,' and ele vated serum creatine phosphokinase, all reflecting injury to striated muscle. Because chlorophenoxy acids are weak uncouplers of oxidative phosphoryla tion, extraordinary doses may produce hyperthermia from increased produc tion of body heat. Polychlorinated DibenzoDioxin (CDD) compounds are generated in the synthesis o f 2,4,5-T. The 2,3,7,8-Tetra CDD form is extraordinarily toxic to multiple mammalian tissues. Hexa- hepta-, and octa-compounds exhibit less systemic toxicity, but are the likely cause o f chloracne (a chronic, disfiguring skin condition) seen in workers engaged in the manufacture of 2,4,5-T, and certain other chlorinated organic compounds. Although toxic effects, notably chloracne, have been observed in manufacturing plant workers, they have not been observed in formulators or applicators regularly exposed to 2,4,5-T. The medical literature contains several reports o f peripheral heuropathy following what seemed to be m inor dermal exposures to 2,4-D. It is not certain that exposures to other neurotoxicants were entirely excluded in these cases. Single doses of 5 m g/kg body weight o f 2,4-D and 2,4,5-T have been administered to human subjects without any adverse effects. One subject consumed 500 mg of 2,4-D per day for 3 weeks without experiencing symptoms or signs o f illness. , p | ^ * (\j qq (\j h 0 0 ( < F R E Q ir - SYM PTO M S A N D SIG N S O F P O ISO N IN G Chlorophenoxy compounds are moderately IRRITATING to skin and mucous membranes. Inhalation of sprays may cause burning sensations in the nasopharynx and chest, and coughing may result. Prolonged inhalation some times causes dizziness. When INGESTED, high concentrations of chlorophenoxy compounds may irritate the ipouth, throat, and gastrointestinal tract. Prompt EMESIS, CHEST PAIN (from esophagitis), ABDOMINAL PA IN , and DIARRHEA commonly ensue. Injury to the GI tract does not usually progress to ulceration or perforation. Absorbed chlorophenoxy compounds have caused FIBRILLARY MUSCLE TWITCHING, skeletal muscle tenderness, and MYOTONIA (stiffness o f muscles of the extremities). Ingestion o f very large amounts has produced METABOLIC ACIDOSIS, fever, tachycardia, hyper ventilation, vasodilatation and sweating. Particular cases have been charac terized by coma and convulsions. CONFIRMATION OF DIAGNOSIS Gas-liquid chromatographic methods are available for detecting and mea suring the chlorophenoxy compounds in blood and urine. These analyses are useful in confirming and assessing the magnitude of chlorophenoxy absorp tion. Urine samples should be collected as soon as possible 'after exposure because the herbicides may be almost completely excreted in 24-72 hours, depending on the extent of toxicant absorption. Analyses can be performed at special laboratories operated by state health departments, chemical com panies, universities, and government facilities. If circumstances indicate strongly that excessive exposure to any of these compounds has occurred, ini tiate appropriate treatment measures immediately, not waiting for chemical confirmation of toxicant absorption. TREATMENT CO O to 1. BATHE aqd SHAM POO with soap and water to remove chemicals from skin and hair. Individuals with chronic skin disease or known sensitivity to chemicals should either avoid using these herbicides or take extraordi nary measqres to avoid contact. 2. FLUSH contaminating chemicals from eyes with copious amounts of clean water for 10-1S minutes. 3. If symptoms o f illness occur during or following inhalation o f spray, \ REMOVE victim FROM CONTACT with the material for at least two oo 4. c: days. Allpw subsequent contact with chlorophenoxy compounds only if effective respiratory protection is practiced. IF substantial amounts o f chlorophenoxy compounds have been IN GESTED, spontaneous emesis usually occurs. Ordinarily, this empties the stomach as effectively as intubation and lavage. If vigorous emesis has not occurred and IF VICTIM IS FULLY ALERT, inouce EMESIS with SYRUP O F IPEC A C (adults 12 years and older, 30 ml; children under 12 years, 13 ml), followed by 1-2 glasses of water. Following emesis, administer 30-30 gm o f ACTIVATED CHARCOAL in a slurry of 6-8 ounces tap water, to limit absorption o f herbicide remaining in the gut. 3. IF CONSCIOUSNESS LEVEL IS DEPRESSED or. other signs or NEUROTOXICITY appear, SUSPECT additional of alternative ingested toxicants. Evacuate the stomach by INTUBATION, ASPIRATION , and LAVAGE. Because petroleum distillates are commonly in cluded in chlorophenoxy formulations, gastric intubation incurs a risk of hydrocarbon pneumonitis from aspiration. For this reason: A. If victim is unconscious o r obtunded and facilities are at hand, in sert an ENDOTRACHEAL TUBE (cuffed, if available) prior to gastric intubation. B. Keep victim's H EAD BELOW LEVEL OF T H E STOMACH dur ing intubation and lavage (Trendelenburg, or left lateral decubitus, with head o f table lipped downward). Keep victim's head turned to left. C. ASPIRATE PHARYNX as regularly as possible to remove gagged or vomited stomach contents. After aspiration of gastric contents and washing of stomach, instill 30-30 gm o f ACTIVATED CH A RCO A L in 3-4 ounces o f water through the stomach tube to limit absorption o f remaining toxicant. Do NOT instill milk, cream, or other materials containing vegetable or animal fats, as these are likely to enhance absorption. 6. If bowel movement has not occurred in 4 hours and patient is fully con scious, give SODIUM SU LFA TE, 0.23 gm /kg, as a cathartic. Magne sium sulfate and citrate, in comparable dosages, are equally suitable if renal function is adequate. Retained magnesium may depress CNS func tion. 7. In SEVERE POISONINGS by very large amounts o f ingested chlorophenoxy acids, forced ALKALINE DIURESIS may save the victim's life. Assess serum electrolyte concentrations, and serum and urine pH . If a metabolic acidosis is present, infuse solutions of sodium bicarbonate at rates sufficient to keep the urine distinctly alkaline, continuing until plasma concentrations o f chlorophenoxy compounds are less than about 10 ftg/m l. (Prescott, L F. et al., Br. J . Clin. Pharmacol. 7:11 (1979)) 83 TOO () ^ qj ^ 0 (\] P I ^ * 01 PESTICIDE & TOXIC m uu is -i sStainf cie's r.afn2dr.tgi ennerha?l zi.asrsduoeussowf.a^srteegsu, lpateisotnicaidneds,lteogxisiclastuiobn- P age 10 O ctober 19, 1983 W <R> PESTICIDE & TOXIC CHEMICAL NEWS EPA NOTES INTENT TO END ALL USES OF 2 ,4 ,5 -T AND SILVEX Follow ing Dow Chem ical C om pany's w ithdraw al from th e 2 ,4 ,5 -T and silvex c a n c e lla tio n hearing and voluntary can cellatio n of reg istratio n of th e p ro d u cts, EPA has issued notices to o th er reg istran ts seeking to end all uses of the pesticides w ithout m ore hearings (See Aug. 10, Page 5; and Aug. 31, Page 2). To continue reg istra tio n s, hearings have to be req u ested w ith in 30 days of the a g e n c y 's O ct. 14 n o tice. T he O ct. 14 in ten t to cancel notice covers all rem aining non-suspended reg istratio n s of the products, those labeled for rice, range, sugarcane, orchard and m iscellaneous noncrop uses, m anufacturing use, and technical registrations. At the sam e tim e, EPA issued an en forcem ent policy s ta te m e n t " to prohibit the sale, d istrib u tio n , im p o rtatio n , or o th er tra n sfe r of u n reg istered 2 ,4 ,5 -T and silvex p ro d u cts, except as specifically allow ed by FIFRA Section 3(b)." The agency said it w as also "requiring each reg istra n t of a 2 ,4 ,5 -T or silvex p roduct to am end the confidential statem en t of form ula for th a t product to identify the source of each activ e ingredient in the product." EPA said failu re to request a hearing "w ill not prevent th e d istrib u tio n or sale of existing stocks of c e rta in products whose reg istratio n s are ca n ce lle d . T he agency will perm it the continued d istrib u tio n or sale of existing sto ck s of 2 ,4 ,5 -T and silvex p roducts which a re labeled for end uses subject to this notice, and whose reg istratio n s are cancelled pursuant to this n o tice, for no m ore than one year from the effectiv e d a te of such cancellation. H ow ever, th is provision will apply only to those existing sto c k s of 2 ,4 ,5 -T and silvex p ro d u cts which w ere packaged and labeled for such end uses and released for shipm ent prior to the d ate of receipt of this notice by the reg istran t or publication of this notice in the F ederal R eg ister, whichever occurs first. T h e agency w ill not p erm it th e continued d istrib u tio n , sale , or use of any can celled 2 ,4 ,5 -T or silvex product labeled for any end use subject to the 1979 em ergency suspension order or for m anufacturing use." The a g en cy 's le tte r to reg istran ts continued: "EPA n o tes th at many reg istra n ts receiving th is le tte r m ay hold reg istratio n s for products which w ere originally labeled for suspended uses of 2,4,5-T or silvex. R eg istran ts of p roducts w hich w ere labeled for one or m ore suspended uses and who elect not to p a rtic ip a te in ' any fu rth er hearing concerning such products m ay m eet the q u alifica tions for receiving indem nification paym ent under FIFR A S ection 15(a)." The agency also noted provision for EPA disposal of th e can celled p esticid es. T he le tte r to th e re g is tra n ts signed by Edw in L. Johnson, OPP D ire c to r, s ta te d : "T he tim e-consum ing and costly nature of continued litig atio n su p p o rts my b elief th a t it will be in th e public in te re s t if th e 2,4,5-T /silvex cancellation proceeding can be concluded w ith out the taking of additional testim ony. " If th e h earin g resu m es, it is e stim a ted th at it will tak e a t least one year to bring the adm inistrative proceedig to conclusion, allow ing for supplem ental p re -tria l activ ities including discovery, c presen tatio n of additional testim ony, briefing, and issuance of .recom m ended and fin al decisions, plus an o th er y ear o r m ore to resolve judicial appeals. "W hile m uch of th e evidence of risks asso ciated w ith use of 2 ,4 ,5 -T and silvex has been p resen ted , EPA a n tic ip a te s th a t ad d itio n al risk testim o n y would have to be p resen ted c o n c ern ing in fo rm atio n w hich has accum ulated during the tw o and o n e-h alf year period since the hearing was suspended. "In addition, since the benefits presentation had barely gotten underw ay w hen th e hearing was recessed, vitually th e en tire b en efits case w ill have to .be p resented." The ag en cy 's S ection 6(b)(1) cancellation notice said, "If a reg istran t whose 2,4,5-T or silvex product is lab eled for m ore than one of the u ses su b ject to this n otice or o th e r _ adversely a ffe c te d person elects to request a hearing concerning som e but not all a f fe c te d uses of th e p ro d u ct, the reg istran t also m ust subm it to the R eg istratio n Division, OPP, an application to am end the registration of such product to delete all other affected uses w ithin th e sam e 30-day period w ithin w hich a hearing may be requested. F ailure to subm it such an a p p licatio n for am ended re g istra tio n will result in can cellatio n of the e n tire reg istratio n of the product by operation of law ." It continued: " if a reg istran t or other adversely affected person requests a hearing pursuant to this notice for , y 2 ,4 ,5 -T or silvex p ro d u ct for which no hearing has previously been requested and th e lab el ing su b m itted for th a t p ro d u ct has not been previously am ended to d elete all uses w hich w ere subject to the em ergency suspension and Section 6(b)(1) cancellation notices for 2,4,5-T and silvex issued in 1979, th e re g istra n t also m ust subm it to th e R eg istratio n Division of th e O ffice of P esticide Program s an ap p licatio n to am end such labeling to d e le te such previously cancelled uses w ithin the sam e 30-day period w ithin which a hearing m ay be requested. F ailure to subm it such an ap p licatio n for am ended reg istratio n will resu lt in c a n ce lla tio n of th e e n tire re g istra tio n of the pesticide. "C oasequences of failure to file in a tim ely and effectiv e m anner. If no hearing has been re q u e ste d w ith in th e a p p licab le 30-day period for any 2 ,4 ,5 -T or silvex p ro d u ct w hich is labeled for one or m ore uses subject to this notice and the reg istran t has not subm itted to EPA an application to am end the reg istratio n of such product to d e le te all uses subject to this notice, th e e n tire reg istra tio n of th e p ro d u ct will be can celled . H ow ever, if a reg istra n t does not request a h earin g for a 2 ,4 ,5 -T or silvex p ro d u ct w hich is lab eled fo r one or m ore uses su b je c t to th is n o tic e , but did previously request a hearing concerning can cellatio n of one or m ore uses of th at pro d u ct which w ere su b ject to the em ergency suspension notices and notice of in ten t to cancel 2 ,4 ,5 -T and silvex issued in 1979, the reg istran t has the right to continue to p a rtic ip a te in a hearing w ith resp ect to th e la tte r uses. At the conclusion of the 2,4,5-T/silvex can cellatio n p ro ceed in g , re in sta te m e n t of such uses will be g ran ted to th e e x te n t p e rm itte d by the final adjudicatory order. Any registrant or form er registrant who requests reinstatem ent of a re g istra tio n for such uses pursuant to such a final ad ju d icato ry order will not be required to co m p ly w ith S e c tio n 3 (c)(1 )(D ) of F1FRA in order for such re in sta te m e n t to be g ra n te d ." Dow, in ending its p a rticip a tio n in the hearing and w ithdraw ing its hearing req u est, said, "2 ,4 ,5 -T and silvex a re s a fe , effectiv e herbicides th a t n e ith e r pose nor th re a te n u n reaso n V able adverse e ffe c ts on the environm ent and are th erefo re properly registered under the FIFRA . S cientific panels, regulatory authorities and judicial decision m akers in th is country and th ro u g h o u t the w orld, including E PA 's own S c ie n tific Advisory Panel in review ing p a rts of this proceeding, have reached sim ilar conclusions." ( 4911 'V-sooCo The com pany fu rth er sta te d , "in taking this actio n , Dow is hopeful th at resources h ereto fo re devoted to th is proceeding will be freed for m ore constructive use in addressing the broader issues of th e p resen ce of ch em icals, including dioxin, in the environm ent- F u rth er pursuit of this proceeding could d e tra c t from th at broader inquiry." Johnson, responding to D ow 's request for voluntary c a n cellatio n s, ac ce p ted the req u est, effectiv e im m ediately. He said, "existing stocks of such products which w ere fo rm u lated , p ack ag ed , labeled only for end uses which have not previously been suspended, and released for shipm ent by your firm on or before O ct. 14, 1983, may be distributed and sold until O ct. 14, 1984, but not thereafter." T he agency official concluded, "w hile we do not agree w ith all of the statem en ts co n cern -, ing 2 ,4 ,5 -T and silvex . . ., we do ag ree th at it is in th e public in te re st to bring th e 2,4,5-T /silvex proceeding to a close." In a n O ct. 14 le tte r to Alvin L. A im , EPA D eputy A d m in istra to r, D ow 's K .R . M cKennon, G roup V ice P resid en t, said, "fo rtu n ately , progress m ade by Dow and others in herbicide p ro d u ct developm ent since 1979 has also resulted in su b stitu te products being available for m ost uses of 2 ,4 ,5 -T and silvex. Dow is convinced th a t th ese su b stitu tes a re now su fficien tly available to satisfy its obligation to custom ers." He continued: "W ith th e issue of 2 ,4,5-T and silvex behind u s, Dow hopes th a t it can help to forge a lasting partnership w ith EPA and the many sc ie n tific groups, environm ental organizations, and industrial com panies having an in te re st and ex p ertise in dioxin m a tte rs." The c a n c e lla tio n n o tic e and en fo rcem en t policy w ere published in th e O ct. 18 F ederal R egister. 4912 'J J - S b o 7 t" * Occupational Health Guideline for 2,4,5-T * INTRODUCTION This guideline is intended as a source of information for employees, employers, physicians, industrial hygienists, and other occupational health professionals who may have a need for such information. It does not attempt to present all data; rather, it presents pertinent information and data in summary form. SUBSTAN CE IDENTIFICATION Formula: C lsCH iO CH aCO O H Synonyms: 2,4,5-Trichlorophenoxyacetic acid Appearance and odor: Colorless to tan odorless solid. PER M ISSIB LE EX P O SU R E LIMIT (PEL) T he current OSHA standard for 2,4,5-T is 10 milligrams of 2,4,5-T per cubic meter o f air (mg/m*) averaged over an eight-hour work shift. HEALTH HAZARD INFORMATION Routes of exposure 2,4,5-T can affect the body if it is inhaled or if it comes in contact with the eyes or skin. It can also affect the body if it is swallowed. Effects of overexposure Exposure to 2,4,5-T may cause abdominal pain, nausea, vomiting, diarrhea, and blood in the stool. It may also cause irritation o f the skin. Common contaminants of commercial preparations of 2,4,5-T may cause acne and liver damage. Animal experiments have shown that these contaminants may produce damage in unborn rats. Reporting signs and symptoms: A physician should be contacted if anyone develops any signs or symptoms and suspects that they are caused by exposure to 2,4,5-T. Recommended medical surveillance The following medical procedures should be made available to each employee who is exposed to 2,4,5-T at potentially hazardous levels: 1. Initial Medical Examination: --A complete history and physical examination: The purpose is to detect pre-existing conditions that might place the exposed employee at increased risk, and to establish a baseline for future health monitoring. Exami nation of the liver and attention to gastrointestinal complaints should be stressed. The skin should be examined for evidence of chronic disorders. 2. Periodic Medical Examination: The aforementioned medical examinations should be repeated on an annual basis. Summary of toxicology 2.4.5- T (2,4,5-trichlorophenoxyacetic acid) is o f low toxicity. T he oral LD50 for dogs is in the range of 100 m gA g or higher; effects are limited to a slight or moderate stiffness in the hind legs with development of ataxia. Contaminants of commercial preparations of 2.4.5- T have been 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent animal teratogen, and 2,3,6,7-tetrachlorodibenzo-p-dioxin (TCDD), a potent acnegenic agent which is hepatotoxic in animals; they are present as unwanted side products of synthesis of 2,4,5-T. In a study o f 73 workers in a 2,4,5-T manufacturing plant, 13 had moderate to severe acneform dermatitis (chloracne) and 22 had gastrointestinal complaints such as nausea, vomiting, diarrhea, abdominal pain, or blood in the stool; no significant liver dysfunction was found; al though no air sample results were reported, the chlor acne was thought to be a result of exposure to TCDD. 2.4.5- T dust is a slight irritant o f the skin. CHEM ICAL AND PHYSICAL PROPERTIES Physical data 1. M olecular weight: 255.5 2. Boiling point (760 mm Hg): Decomposes above melting point 3. Specific gravity (water = 1): G reater than 1 4. V apor density (air = 1 at boiling point o f 2,4,5-T): Not applicable 5. Melting point: 158 C (316 F) (decomposition) These recommendations reflect good industrial hygiene and medical surveillance practices and their implementation will assist in achieving an effective occupational health program. However, they may not be sufficient to achieve compliance with all requirements of OSHA regulations. UJS. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Centers for Disease Control National Institute for Occupational Safety and Health September 1978 U.S. DEPARTMENT O F LABOR Occupational Safety and Health Administration 491? 6. Vapor pressure at 20 C (68 F): Essentially zero 7. Solubility in water, g/100 g water at 20 C (68 F): 0.03 8. Evaporation rate (butyl acetate = 1): Not applica ble Reactivity 1. Conditions contributing to instability: Tem pera tures above 158 C (316 F) may cause sealed metal containers to burst. 2. Incompatibilities: None. 3. Hazardous decomposition products: Toxic gases and vapors (such as hydrogen chloride and carbon monoxide) may be released when 2,4,5-T decomposes. 4. Special precautions: None. Flammability 1. Not combustible Warning properties 2,4,5-T is not known to be an eye irritant. MONITORING AND M EASUREM ENT PROCEDURES General Measurements to determine employee exposure are best taken so that the average eight-hour exposure is based on a single eight-hour sample or on two four-hour samples. Several short-time interval samples (up to 30 minutes) may also be used to determine the average exposure level. A ir samples should be taken in the employee's breathing zone (air that would most nearly represent that inhaled by the employee). Method An analytical method for 2.4.5-T is in the N IO SH Manual o f Analytical Methods. 2nd Ed., Vol. 5, 1979, available from the Government Printing Office, Wash ington, D.C. 20402 (G PO No. 017-033-00349-1). R ESP IR A T O R S Good industrial hygiene practices recommend that engineering controls be used to reduce environmental concentrations to the permissible exposure level. H ow ever, there are some exceptions where respirators may be used to control exposure. Respirators may be used when engineering and work practice controls are not technically feasible, when such controls are in the process of being installed, o r when they fail and need to be supplemented. Respirators may also be used for operations which require entry into tanks or closed vessel's, and in emergency situations. If the use o f respirators is necessary, the only respirators permitted are those that have been' approved by the Mine Safety and Health Administration (formerly Mining Enforce ment and Safety Administration) or by the National Institute for Occupational Safety and Health. In addition to respirator selection, a complete respira tory protection program should be instituted which includes regular training, maintenance, inspection. 2 2.4.5-T cleaning, and evaluation. SANITATION Eating and smoking should not be permitted in areas where 2 ,4 ,5 ^ is handled, processed, or stored. Employees who handle 7,4,5-T should wash their hands thoroughly with soap o r mild detergent and water before eating, smoking, o r using toilet facilities. COMMON OPERATIONS AND CON TROLS T he following list includes some common operations in which exposure to 2,4,5-T may occur and control methods which may be effective in each case: Operation Formulation of herbicides and plant horm ones Controls P ro cess enclosure; local exhaust ventilation; personal protective equipment Application as herbicide, defoliant, and plant hormone Manufacture of 2,4,5-T Personal protective equipment P ro cess enclosure; local exhaust ventilation; personal protective equipment - EM ERGEN CY FIR ST AID PRO CEDURES In the event o f an emergency, institute first aid proce dures and send for first aid or medical assistance. * Eye Exposure If 2,4,5-T gets into the eyes, wash eyes immediately with large amounts of water, lifting the lower and upper lids occasionally. If irritation is present after washing, get medical attention. Contact lenses should not be worn when working with this chemical. Skin Exposure If 2,4,5-T or liquids containing 2,4,5-T get on the skin, wash the contaminated skin using soap or mild deter gent and water. If 2,4,5-T or liquids containing 2,4,5-T soak through the clothing, remove the clothing and wash the skin using soap or mild detergent and water. If irritation is present after washing, get medical attention. Breathing If a person breathes in large amounts o f 2,4,5-T, move the exposed person to fresh air at once. If breathing has stopped, perform artificial respiration. Keep the affect ed person warm and at rest. G et medical attention as soon as possible. * Swallowing When 2,4,5-T o r liquids containing 2,4;5-T have been swallowed and the person is conscious, give the person large quantities of water immediately. After the water has been swallowed, try to get the person to vomit by having him touch the back o f his throat with his finger. Do not make an unconscious person vomit. Get medical 4914 Sep tem b er 1978 attention immediately. Rescue Move the affected person from the hazardous exposure. If the exposed person has been overcome, notify some one else and put into effect the established emergency .escue procedures. D o *iot 'become a casualty. U nder stand the facility's emergency rescue procedures and know the locations of rescue equipment before the need arises. SPILL AND DISPOSAL PRO CEDURES Persons not wearing protective equipment and cloth ing should be restricted from areas o f spills until cleanup has been completed. If 2,4,5-T is spilled, the following steps should be taken: 1. Ventilate area of spill. 2. Collect spilled material in the most convenient and safe manner and deposit in sealed containers for recla mation, or for disposal in a secured sanitary landfill. Liquid containing 2,4,5-T should be absorbed in vermiculite, dry sand, earth, or a similar material. Waste disposal method: 2,4,5-T may be disposed of in sealed containers in a secured sanitary landfill. REFERENCES American Conference of Governmental Industrial lygienists: "2,4,5-T (2,4,3-Trichlorophenoxacetic Acid)," Documentation o f the Threshold Lim it Valuesfo r Substances in Workroom Air (3rd ed., 2nd printing), Cincinnati, 1974. Christensen, H. E., and Luginbyhl, T. L. (eds.): N IO SH Toxic Substances List, 1974 Edition, HEW Publication No. 74-134, 1974. Deichmann, W. B., and Gerarde, H. W.: Toxicology o f Drugs and Chemicals, Academic Press, New York, 1969. Drill, V. A., and Hiratzka, T.: "Toxicity of 2,4Dichlorophenoxyacetic Acid and 2,4,5Trichlorophenoxyacetic Acid," A.M.A. Archives o f In dustrial Hygiene and Occupational Medicine, 7:61-67, 1953. International Labour Office: Encyclopedia o f Occupa tional Health and Safety, McGraw-Hill, New York, 1971. Khera, K. S., and McKinley, W. P.: "Pre- and PostNatal Studies on 2,4,5-Trichlorophenoxyacetic Acid, 2,4-Dichlorophenoxyacetic Acid and Their Derivatives in Rats," Toxicology and Applied Pharmacology, 22:14-28, 1972. Patty, F. A. (ed.): Toxicology, Vol. II of Industrial Hygiene and Toxicology (2nd ed. rev.), Interscience, N ew York, 1963. Poland, A. P., et al.: "A Health Survey o f Workers in a 2,4-D and 2,4,5-T Plant," Archives o f Environmental Health, 22:316-327,1971. Spencer, E. Y.: Guide to the Chemicals Used in Crop Protection (6th ed.), Publication 1093, Research Branch Agriculture, Canada, 1973. SPECIAL NOTE The International Agency for Research on Cancer (IARC) has evaluated the data on this chemical and has concluded that it causes cancer. See I A R C Monographs on the Evaluation o f Carcinogenic Risk o f Chemicals to Man, Volume 15, 1977. Septem ber 1978 4915 So\o 2,4,5-T 3 g. 2 ,3 ,7 ,8-TETRACHLORODIBENZO-p-DIOXIN K Agency for Toxic Substances and Disease Registry U.S. Public Health Service ^^ t, i I > o <0 DRAFT TOXICOLOGICAL PROFILE FOR 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN Date Published -- December 1987 Prepared by: Syracuse Research Corporation under Contract No. 68-03-3228 for Agency for Toxic Substances and Disease Registry (ATSDR) U.S. Public Health Service in collaboration with U.S. Environmental Protection Agency (EPA) Published by: Oak Ridge National Laboratory under DOE Interagency Agreement No. 1425-1425-Al DISCLAIMER Mention of company name or product does not constitute endorsement by the Agency for Toxic Substances and Disease Registry. 0 FOREWORD The S u p erfu n d Amendments and R e a u th o r iz a tio n A ct o f 1986 (P u b lic Law 9 9 -4 9 9 ) e x te n d e d a n d am ended t h e C o m p re h e n siv e E n v iro n m e n ta l R e s p o n s e , C o m p e n s a tio n , a n d L i a b i l i t y A c t o f 1980 (CERCLA o r S u p e r f u n d ) . T h is p u b l i c law ( a l s o known a s SARA) d i r e c t e d t h e A gency f o r T o x ic S u b s ta n c e s a n d D is e a s e R e g i s t r y (ATSDR) t o p r e p a r e t o x i c o l o g i c a l p r o f i l e s f o r h a z a rd o u s s u b s ta n c e s w hich a r e m ost commonly fo u n d a t f a c i l i t i e s o n t h e CERCLA N a t i o n a l P r i o r i t i e s L i s t a n d w h ic h p o s e t h e m ost s ig n i f ic a n t p o te n tia l t h r e a t to human h e a lth , as d e te rm in e d by ATSDR a n d t h e E n v iro n m e n ta l P r o t e c t i o n A gency (E P A ). The l i s t o f t h e 100 m ost s ig n if ic a n t hazardous su b sta n c e s was p u b lish e d in th e F ederal R e g iste r on A p ril 17, 1987. S e c t i o n 110 (3 ) o f SARA d i r e c t s t h e A d m i n i s t r a t o r o f ATSDR to p rep a re a to x ic o lo g ic a l p r o f ile fo r each su b stan ce on th e l i s t . Each p ro file m ust inclu d e the fo llo w in g co n ten t: "(A) An e x a m in a tio n , summary, and i n t e r p r e t a t i o n o f a v a ila b le to x ic o lo g ic a l in fo rm atio n and epidem iologic e v a lu a tio n s on the hazardous substance in o rd er to a s c e rta in the le v e ls o f s ig n ific a n t human ex p o su re f o r th e su b sta n c e and th e a s s o c ia te d a c u te , subacute, and chronic h e a lth e ffe c ts , (B) A d e te r m in a tio n o f w h e th e r a d e q u a te in fo r m a tio n on th e h e a lt h e ffe c ts of each substance is a v a ila b le or in the p ro cess of developm ent to d eterm in e le v e ls o f exposure w hich p re s e n t a s i g n i f i c a n t r i s k t o hum an h e a l t h o f a c u t e , s u b a c u t e , a n d c h r o n i c ____ h e a lth e f f e c ts , and (C) Where a p p r o p r ia te , an i d e n t i f i c a t i o n o f t o x ic o lo g ic a l t e s t i n g n e e d e d t o i d e n t i f y t h e ty p e s o r l e v e l s o f e x p o s u re t h a t may p r e s e n t s ig n if ic a n t r is k o f ad v erse h e a lth e f f e c ts in hum ans." T his to x ic o lo g ic a l p r o file is p rep ared in accordance w ith g u i d e l i n e s d e v e lo p e d b y ATSDR a n d EPA. The g u i d e l i n e s w e re p u b l i s h e d i n the F ederal R e g iste r on A p ril 17, 1987. Each p r o file w ill be re v ise d and re p u b lish e d as n e c essa ry , b u t no le s s o fte n th an every th re e y e a rs, as r e q u i r e d by SARA. The ATSDR t o x i c o l o g i c a l p r o f i l e i s i n t e n d e d t o c h a r a c t e r i z e su c cin c tly the to x ic o lo g ic a l and h e a lth e ffe c ts in fo rm atio n fo r the hazardous sub stan ce b ein g d e scrib ed . Each p r o f ile id e n tif ie s and review s the key l ite r a tu r e th a t d e sc rib e s a hazardous s u b s ta n c e 's to x ic o lo g ic a l p ro p e rtie s . O ther lite r a tu r e is p rese n ted b u t d e scrib ed in le s s d e ta il th an th e key s tu d ie s . The p r o f ile is n o t in te n d e d to be an e x h a u stiv e docum ent; how ever, more com prehensive so u rc e s o f s p e c i a lt y in fo rm a tio n are referenced. iii Each to x ic o lo g ic a l p r o f ile b eg in s w ith a p u b lic h e a lth sta te m e n t, which d e sc rib e s in n o n te c h n ic a l language a s u b s ta n c e 's re le v a n t to x ic o lo g ic a l p ro p e rtie s . Follow ing th e statem en t is m a te ria l th a t p re s e n ts le v e ls o f s ig n i f ic a n t human ex p o su re and, w here known, s ig n if ic a n t h e a lth e f f e c ts . The adequacy o f in fo rm a tio n to d eterm in e a s u b s ta n c e 's h e a lth e f f e c ts is d e s c rib e d in a h e a lth e f f e c ts summary. R esearch gaps in to x ico lo g ic and h e a lth e ffe c ts inform ation are d escrib ed in the p ro file . R esearch gaps th a t are o f sig n ific a n c e to p r o t e c t i o n o f p u b l i c h e a l t h w i l l b e i d e n t i f i e d by ATSDR, t h e N a t i o n a l T o x ic o lo g y P ro g ra m o f t h e P u b l ic H e a l th S e r v i c e , a n d EPA. The f o c u s o f t h e p r o f i l e s i s on h e a l t h a n d t o x i c o l o g i c a l i n f o r m a t i o n ; t h e r e f o r e , we have in clu d ed th is in fo rm a tio n in th e f r o n t o f th e docum ent. The p r in c ip a l a u d ie n c e s f o r th e to x ic o lo g ic a l p r o f i le s a re h e a lth p ro fessio n als a t the fe d e ra l, s ta te , and lo c a l le v e ls, in te re ste d p r i v a t e s e c t o r o r g a n i z a t i o n s a n d g r o u p s , a n d m em bers o f t h e p u b l i c . We p la n to r e v is e th e s e docum ents in re s p o n s e to p u b lic comments and as a d d i t i o n a l d a ta becom e a v a i l a b l e ; t h e r e f o r e , we e n c o u ra g e comment t h a t w i l l make th e to x ic o lo g i c a l p r o f i l e s e r i e s o f th e g r e a t e s t u s e . T his p ro file r e f le c ts our assessm ent o f a l l re le v a n t to x ic o lo g ic a l te s tin g and inform ation th a t has been peer review ed. I t has been r e v ie w e d b y s c i e n t i s t s fro m ATSDR, EPA, t h e C e n t e r s f o r D is e a s e C o n t r o l , and th e N atio n al T oxicology Program . I t has a ls o been review ed by a p a n e l o f nongovernm ent p e e r re v ie w e rs and was made a v a ila b le f o r p u b lic review . F in a l r e s p o n s ib ility fo r th e c o n te n ts and view s ex p ressed in t h i s t o x i c o l o g i c a l p r o f i l e r e s i d e s w i t h ATSDR. Jam es 0 . M ason, M .D., D r. P.H . A s s ista n t Surgeon G eneral A d m i n i s t r a t o r , ATSDR iv CONTENTS FOREWORD ......................................................................................................................................... i i i LIST OF FIGURES ............................................................................. v ii LIST OF TABLES ........................................................................................................................... i x 1 . PUBLIC HEALTH STATEMENT ......................................................................................... 1 .1 WHAT IS DIOXIN? ............................................................................................... 1 .2 HOW MIGHT I BE EXPOSED TO 2 , 3 , 7 , 8-TCDD? ....................................... 1 .3 HOW DOES 2 , 3 , 7 , 8-TCDD GET INTO MY BODY? ....................................... 1 .4 HOW CAN 2 , 3 , 7 , 8-TCDD AFFECT MY HEALTH? ......................................... 1 .5 IS THERE A MEDICAL TEST'TO DETERMINE WHETHER I HAVE BEEN EXPOSED TO 2 , 3 , 7 , 8-TCDD? ............................................................... 1 .6 WHAT LEVELS OF EXPOSURE BY INGESTION AND BY SKIN CONTACT HAVE RESULTED IN HARMFUL HEALTH EFFECTS? .................................... 1 .7 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECTHUMAN HEALTH? ................................................................ 1 1 1 2 2 3 4 4 2 . HEALTH EFFECTS SUMMARY ............................................................ 2 .1 INTRODUCTION ................. 2 .2 LEVELS OF SIGNIFICANT EXPOSURE ............................................................. 2 . 2 . 1 Key S t u d i e s a n d G r a p h ic a l P r e s e n t a t i o n s ...................... 2 .2 .2 B io lo g ic a l M onitoring as a M easure o f Exposure a n d E f f e c t s .................................. ....................................................... 2 .2 .3 E nvironm ental L evels as In d ic a to rs o f Exposure a n d E f f e c t s .................................... .................................................... 2 .3 ADEQUACY OF DATABASE ....................................................... 2 . 3 . 1 I n t r o d u c t i o n ............................................................ 2 .3 .2 Adequacy o f the D atabase fo r H ealth E ffe c t End P o i n t s ............................................................................................ 2 .3 .3 Adequacy o f th e D atabase fo r O ther In fo rm atio n N eeded f o r R is k A s s e s s m e n t ..................................................... 7 7 8 8 18 19 21 21" 21 25 3 . CHEMICAL AND PHYSICAL INFORMATION ......................................... 3 .1 CHEMICAL IDENTITY .......................................................................................... 3 .2 PHYSICAL AND CHEMICAL PROPERTIES ...................................................... 29 29 29 4 . TOXICOLOGICAL DATA .................................................................................................... 4 .1 OVERVIEW ................................................................................................................. 4 . 2 TOXICOKINETICS .................................................................................................... 4 . 2 . 1 A b s o r p tio n ............................................................................................ 4 . 2 . 2 D i s t r i b u t i o n ....................................................................................... 4 . 2 . 3 M e ta b o lis m ............................................................................................ 4 . 2 . 4 E x c r e t i o n ............................................................................................... 4 .3 TOXICITY .................................................................. 4 . 3 . 1 L e t h a l i t y a n d D e c r e a s e d L o n g e v ity ..................................... 4 . 3 . 2 S y s t e m i c / T a r g e t O rgan T o x i c i t y ...................... ..................... 4 . 3 . 3 D e v e lo p m e n ta l T o x i c i t y ................................................................ 33 33 37 37 38 39 40 41 41 42 49 4920 v 4 . 3 . 4 R e p r o d u c t i v i t y T o x i c i t y ............................................................. 4 . 3 . 5 G e n o t o x i c i t y ....................................................................................... 4 . 3 . 6 C a r c i n o g e n i c i t y ................................................................................ 4 . 4 INTERACTIONS WITH OTHER CHEMICALS ........................ ............................. 5 . MANUFACTURE, IMPORT, USE, AND DISPOSAL ...................................................... 5 .1 OVERVIEW ................................................................................................................ 5 .2 PRODUCTION ............................................................................................................. 5 .3 IMPORT ....................................................................................................................... 5 .4 USE ....................................................................................... ...................................... 5 .5 DISPOSAL .................................................................................. 6 . ENVIRONMENTAL FATE .................................................................................................... 6 .1 OVERVIEW ....................................................................................... 6 .2 RELEASES TOTHE ENVIRONMENT ......................................................... 6 . 2 . 1 P ro d u c tio n and Use o f C e rta in H e rb ic id e s an d C h lo r o p h e n o ls ........................................................................... 6 . 2 . 2 P h o to c h e m ic a l R e a c t i o n s ............................................................. 6 . 2 . 3 T h e rm a l R e a c ti o n s ...................................... 6 . 2 . 4 Im proper D isp o sal o f C h lo rin a te d Chem ical W astes . 6 .3 ENVIRONMENTAL FATE .......................................................................................... 7 . POTENTIAL FOR HUMAN EXPOSURE ..................................................... 7 . 1`* OVERVIEW .................................................. _ 7 . 2 . LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT ................... 7 . 2 . 1 A i r ........................................................................ 7 . 2 . 2 W a te r ...................................... 7 . 2 . 3 S o i l ..................................... 7 . 2 . 4 O th e r ........................ 7 .3 OCCUPATIONAL EXPOSURES ........................ 7 .4 POPULATIONS AT HIGH RISK .......... ..................... .......................................... 8 . ANALYTICAL METHODS ........................ 8 .1 ENVIRONMENTAL MEDIA ................................................... .. . .............................. 8 . 1 . 1 A i r , W a te r, S o i l , a n d F o o d ....................................................... 8 .2 BIOMEDICAL SAMPLES ............ 8 . 2 . 1 F l u i d s / E x u d a t e s a n d T i s s u e s ...................................................... 9 . REGULATORY AND ADVISORY STATUS ........................................................................ 9 .1 INTERNATIONAL (WORLD HEALTHORGANIZATION) ..................................... 9 .2 NATIONAL............................. 9 . 2 . 1 R e g u l a t i o n s ............................................................ 9 . 2 . 2 A d v is o ry G u id a n c e ............................................................................ 9 . 2 . 3 D a ta A n a l y s is ...................................................................... 9 .3 STATE .......................................................................................................................... 1 0 . REFERENCES .......................................................................................................................... 1 1 . GLOSSARY ...................................................................... APPENDIXES A. PEER REVIEW .................................................................................. B. FEDERAL REGISTER ANNOUNCEMENT .................................................................. 50 53 56 60 61 61 61 61 61 61 63 63 63 63 64 64 64 65 67 67 68 68 68 69 69 73 73 75 76 76 76 76 83 83 83 83 83 83 84 85 109 115 117 LIST OF FIGURES 1 .1 H e a l th e f f e c t s from i n g e s t i n g 2 , 3 , 7 , 8 - T CD D ......................................... 5 1 . 2 H e a l th e f f e c t s from s k i n c o n t a c t w i t h 2 , 3 , 7 , 8 - T CDD ...................... 6 2 . 1 E f f e c t s o f 2 , 3 , 7 , 8- TCDD- or a l e x p o s u re ........................................................ 10 2 .2 E f f e c t s o f 2 ,3 ,7 ,8 -T C D D -d e rm a l e x p o s u re ................................................... 11 2 .3 L e v e ls o f s i g n i f i c a n t e x p o s u r e f o r 2 , 3 , 7 , 8- TCDD- or al ................. 12 2 . 4 L e v e ls o f s i g n i f i c a n t e x p o s u r e f o r 2 , 3 , 7 , 8 - T C D D - d e r m a l ............. 13 2.5 Adequacy of th e database on h e a lth e f f e c t s of 2,3,7,8-TCDD (hum an d a t a ) .................................................................................................................... 22 2.6 Adequacy o f th e datab ase on h e a lth e f f e c t s of 2,3,7,8-TCDD ( a n im a l d a t a ) ................................................................................................................. 23 4921 p - 5 09- Or . " LIST OF TABLES 3 . 1 C h e m i c a l i d e n t i t y o f 2 , 3 , 7 , 8 - T C D D .................................... 30 3 . 2 P h y s i c a l p r o p e r t i e s o f 2 , 3 , 7 , 8 - T C D D .......... 31 4 . 1 R e c o m m e n d e d T E F s f o r 2 , 3 , 7 , 8 - T C D D a n d I t s c o n g e n e r s ............ 36 4 . 2 G e n o t o x i c i t y o f 2 , 3 , 7 , 8 - T C D D i n v i t r o ............................... 54 4 . 3 G e n o t o x i c i t y o f 2 , 3 , 7 , 8 - T C D D i n v i v o ................................ 55 4.4 Summary of the oral carcinogenicity bioassay of Kociba e t al. ( 1 9 7 8 a , b ) ........................................................... 57 4.5 Other oral studies supporting the conclusion that 2 , 3 , 7 , 8 - T C D D is a n a n i m a l c a r c i n o g e n ................................ 58 7 . 1 L e v e l s o f 2 , 3 , 7 , 8 - T C D D i n s o i l f r o m d i f f e r e n t l o c a t i o n s ........ 70 8.1 -Analytical methods for environmental samples ................. 77 8 . 2 A n a l y t i c a l m e t h o d s f o r b i o m e d i c a l s a m p l e s ......................... 80 (5 ix y .b 0 1. P U B L I C H E A L T H S T A T E M E N T 1.1 WHAT IS DIOXIN? T he che m i c a l 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n ( 2 , 3 , 7 , 8-TCDD) is c o m m o n l y called dioxin. Dioxin, however, is a n i naccurate collo q u i a l name for 2,3,7,8 - T C D D . It is a colorless solid w i t h no distinguishable odor. 2 , 3 , 7 , 8-TCDD is n e i t h e r k n o w n to occur n a t u r a l l y n or is it intentionally manufactured b y any industry, except as a reference standard. It can be inadvertently produced in very small amounts as an impurity during the manufacture of certain herbicides and germicides, and during the incineration of municipal and industrial w a s t e s . At the pre s e n t time, 2,3,7,8-TCDD is not used for any purpose other than scientific research. 1.2 _MOW MIGHT I BE EXPOSED TO 2,3,7,8-TCDD? The m ain environmental sources of 2,3,7,8-TCDD are: Production and use of herbicides containing 2,4,5-trichlorophenoxy acids (2,4,5-T) Production and use of 2,4,5-trichlorophenol Production and use of hexachlorophene as a germicide Incineration of municipal and certain industrial wastes Small amounts formed during the burning of wood in the presence of chlorine Accidental transformer/capacitor fires involving chlorinated benzenes and biphenyls Exhaust from automobiles powered with leaded gasoline Improper disposal of certain chlorinated chemical wastes I n f o r m a t i o n on the level of 2 , 3 , 7 , 8 - TCDD in a m b i e n t a ir is n o t available. Detection of 2,3,7,8-TCDD in drinking water has not been reported. Th e c o n c e n t r a t i o n s o f 2 , 3 , 7 , 8 - T C D D in m o s t r u r a l soils is undetectable, but it can be present at trace levels in urban soils. In a waste oil-contaminated soil in Missouri, the 2,3,7,8-TCDD level was more than one million times higher than in soils from normal urban areas. 2,3,7,8-TCDD was detected in fish obtained from the contaminated sections of Lake Ontario, Saginaw Bay, and the Michigan rivers. In human milk, 2,3,7,8-TCDD has not been detected in the United States; however, using more sensitive methods of measurement, it has b e e n detected in several European countries. It has not b e e n detected in any other foods. 4923 i 2 Consumer sources: Skin contact with surfaces such as soil or vegetation contaminated by the chemical Consumption of root vegetables grown in contaminated soils Inhalation of air near improperly maintained dump sites or municipal incinerators Consumption of fish, livestock, and cow's milk from contaminated areas Breast-fed babies nursed by mothers who have 2,3,7,8-TCDD in their breast milk Since both 2,4,5-T-containing herbicides and hexachlorophene have been withdrawn from consumer use, they no longer will be future sources of exposure; however, exposures to these compounds may occur from hazardous waste sites. Occupational sources: Workers involved in the production or use of trichlorophenol and salts, hexachlorophene, and 2,4,5-T or other herbicides containing this chemical. The production of 2,4,5-T and 2,4,5-trichlorophenol, -- - h o w e v e r , h a s b e e n d i s c o n t i n u e d i n t h e U n i t e d S t a t e s . Workers at certain municipal and industrial incinerators Workers at certain hazardous waste sites Workers involved in the cleanup of certain accidental capacitor/transformer fires and in the salvaging of transformers Workers involved in spraying phenoxy herbicides such as Agent Orange . 1.3 HOW DOES 2,3,7,8-TCDD GET INTO MY BODY7 Absorption through skin from contaminated soils and other materials Ingestion of 2,3,7,8-TCDD through the consumption of contaminated fish, cow's milk, and foodstuffs Breathing contaminated ambient air may contribute very small amounts to total body intake. Inhalation of particulates such as fly ash, however, may constitute a major source of exposure. Intake of 2,3,7,8-TCDD from the consumption of drinking water should be negligible. 1.4 HOW CAN 2,3,7,8-TCDD AFFECT MY HEALTH? In humans, 2,3,7,8-TCDD causes chloracne, a severe skin lesion that usually occurs on the head and upper body. Unlike common acne, chloracne is more disfiguring and often lasts for years after the initial exposure. AQ 3 T h e r e is sugge s t i v e e v i d e n c e that 2 , 3 , 7 ,8-TCDD causes l iver damage in h u m a n s , as indicated by an increase in levels of certain enzymes in the blood. Animal studies have demonstrated severe liver damage in some species. T h e r e is sugge s t i v e e v i d e n c e that 2,3 , 7 , 8 - T C D D causes loss of appetite, w e ight loss, and digestive disorders in humans. A nim al exposure to 2,3,7,8-TCDD results in severe loss of body weight just prior to death. Although never demonstrated in humans, in animal studies, 2.3.7.8- TCDD produced toxicity to the immune system. This toxicity can result in greater susceptibility to infection. Although never demonstrated in humans, in some animal species, exposure to 2,3,7,8-TCDD r e s u l t e d ih spontaneous abortions. The m o n k e y is v e r y sensitive to this toxic pr o p e r t y of 2,3,7,8-TCDD. A l t h o u g h never d e m o n s t r a t e d in humans, in rodents, exposure to 2.3.7.8- TCDD during pregnancy resulted in malformations in the offspring. 2,3,7,8-TCDD has been demonstrated to be a carcinogen in animals. 1 .5 _^ IS T H E R E A M E D I C A L T E S T T O D E T E R M I N E W H E T H E R I H A V E BEEN EXPOSED TO 2,3,7,8-TCDD? There Is no common medical test available to demonstrate u n e q u i v o c a l l y that y o u ha v e b e e n e x p o s e d to 2,3,7,8-TCDD. It is b e l i e v e d that a blood test to detect certain enzymes indicating liver damage may be helpful in determining whether exposure has occurred. These tests do not indicate with certainty that you have been exposed to 2,3,7,8-TCDD, since other chemicals, as well as drinking alcohol, can cause similar results. W h e n tests for these enzymes have b e e n performed, changes in these enzymes were demonstrated only in some of the people suspected of 2.3.7.8- TCDD exposure. ________ There are other tests available, which' are not commonly conducted b y a physician but appear to more adequately indicate that you have been exposed to 2,3,7,8-TCDD. One test consists of removing a small piece of b o d y fat b y a simple surgical procedure; the fat is then analyzed for the presence of 2,3,7,8-TCDD. In another recently developed test, blood s e r u m is ob t a i n e d a n d a n a l y z e d for the p resence of 2,3,7,8-TCDD. The initial study appears to indicate that the me t h o d is sensitive enough to detect background levels of 2,3,7,8-TCDD. If the levels of 2,3,7,8-TCDD are higher than the determined average for people in the United States, the test indicates that you have probably been exposed to more 2.3.7.8- TCDD than the average population. In addition, detection of 2.3.7.8- TCDD in mother's milk would also indicate exposure; the level of 2.3.7.8- TCDD in the milk may provide some indication of whether exposure was to background levels or if additional exposure occurred. This method has not been used widely to evaluate human exposure. 4925 4 1.6 WHAT LEVELS OF EXPOSURE BY INGESTION AND BY SKIN CONTACT HAVE RESULTED IN HARMFUL HEALTH EFFECTS? The graphs on the following pages show the relationship between exposure to 2,3,7,8-TCDD and known health effects. In the first set of graphs labeled "Health effects from ingesting 2,3,7,8-TCDD" (Fig. 1.1), exposure is m e a s u r e d in millig r a m s o f 2 , 3 , 7 , 8 - T C D D pe r k i l o g r a m of bod y weight (mg/kg). In all graphs, effects in animals are shown on the left side and effects in humans on the right side. In the s e c o n d set o f graphs (Fig. 1.2), the same r e l a t i o n s h i p is represented for the known "Health effects from skin contact with p r o d u c t s c o n t a i n i n g 2 , 3 , 7 , 8 - T C D D ." E x p o s u r e s a g a i n a r e m e a s u r e d i n milligrams of 2,3,7,8-TCDD per kilogram of body weight (mg/kg). The levels mark e d on Fig. 1.1, as anticipated to be associated with minimal risk for effects other than cancer in humans, are based on information from animal studies; therefore, some uncertainty still exists. For cancer, the U.S. Environmental Protection Agency (EPA) has estimated that lifetime exposure to 1 nanogram of 2,3,7,8-TCDD per kilogram per day would result in 1560 or 1,560,000 additional cases of cancer in a population o f 10,000 or 10,000,000 people, respectively. It should be noted that these risk values are plausible upper-limit estimates!. Actual risk levels are u n l i k e l y to b e h i g h e r and m a y be lower. (One n a n o g r a m is one-bil l i o n t h of a gram.) ~ There was not enough information to prepare a graph for exposure by -- breathing. 1.7 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT HUMAN HEALTH? Both the EPA and the International Agency for Research on Cancer (IARC) have concluded that 2,3,7,8-TCDD causes cancer in animals, which suggests that 2,3,7,8-TCDD may cause cancer in humans. T h e E P A c a l c u l a t e d h e a l t h a d v i s o r i e s (HAs). f o r 2 , 3 , 7 , 8 - T C D D in d r i n k i n g water, t h a t is, e s t i m a t e s o f l e v e l s b e l o w w h i c h a d v e r s e h e a l t h effects are not expected to occur. The 1-day HA is 0.000001 milligrams per liter (mg/L) (10 parts per trillion, ppt) for a child; the 10-day HA is 0.000 0 0 0 0 1 m g / L (0.001 ppt) for a child. T he l o n g e r - t e r m H A is 0.0000001 mg/L for a child and 0.000000035 mg/L for an adult (0.1 and 0.035 ppt, respectively); the lifetime H A is 0.000000035 m g / L for adults (0.035 ppt). The EPA also calculated the amount of 2,3,7,8-TCDD in ambient water (lakes and rivers) which would be associated with increases in one additional incidence of cancer over background cancer incidence in a population of 1,000,000 to be 0.000000000013 mg/L. This calculated measurement takes into account that 2,3,7,8*-TCDD concentrates in fish; hence, exposure may occur through both the drinking of water and the eating of fish. The Federal Drug Administration has calculated that fish containing less than 25 ppt of 2,3,7,8-TCDD should pose no serious health concerns. 5 SHORT-TERM EXPOSURE (LESS THAN OR EQUAL TO 14 DAYS) EFFECTS IN ANIMALS DOSE (mg/kg/day) EFFECTS IN HUMANS LONG-TERM EXPOSURE (GREATER THAN 14 DAYS) EFFECTS IN ANIMALS DOSE (mg/kg/day) EFFECTS IN HUMANS 0.001 DEATH 0.00001 DEATH. 0.0005 0.0001 DEVELOPMENTAL < EFFECTS 0.00008 X T 0.00004 0.00002 1 T 0.000008 0.000006 0.000004 0.000002 1 T 0.000001. 0.000005 REPRODUCTIVE I TOXICITY AND CHLO RACN E________ 0.000001 0.0000008 0.0000006 LIVER DAMAGE < 0.0000004 i, T 0.0000002 MINIMAL RISK FOR EFFECTS OTHER THAN . CANCER 0.0000001 1 T 0.00000005 0.00000001 T 0.000000005 0.000000001 MINIMAL RISK FOR EFFECTS OTHER THAN CANCER Fig. 1.1. Health effects from ingesting 2,3,7,8-TCDD. f 4927 SHORT-TERM EXPOSURE (L E S S THAN OR EQ U A L TO 14 DAYS) EFFECTS IN ANIMALS DOSE (mg/kg/day) EFFECTS IN HUMANS LONG-TERM EXPO SURE (G R EA T ER THAN 14 DAYS) EFFECTS IN ANIMALS DOSE (mg/kg/day) EFFECTS IN HUMANS QUANTITATIVE DATA W ERE NOT AVAILABLE 0.01 QUANTITATIVE DATA W ERE NOT AVAILABLE 0.8 0.008 DEATH 0.6 0.4 0.2 0.006 0.004 C H L O R A C N E __________ 0.002 00 Fig. 1.2. Health effects from skin contact with 2,3,7,8-TCDD. 2. H E A L T H E F F E C T S S U M M A R Y 2.1 INTRODUCTION This section summarizes and graphs data on the health effects c o n c e r n i n g exposure to 2,3,7,8-TCDD. The purp o s e o f this s e c t i o n is to present levels of significant exposure for 2,3,7,8-TCDD based on key toxicological studies, epidemiological investigations, and environmental exposure data. The information presented in this section is critically e v a l u a t e d a n d d i s c u s s e d i n S e c t . 4, T o x i c o l o g i c a l D a t a , a n d S e c t . 7, Potential for Human Exposure. This Health Effects Summary section comprises two major parts. Levels of Significant Exposure (Sect. 2.2) presents b r i e f narratives and graphics for key studies in a manner that provides public health officials, physicians, and other interested individuals and groups with ( l ) ^ a n o v e r a l l p e r s p e c t i v e o f the t o x i c o l o g y o f 2 , 3 , 7 , 8 - T C D D a n d (2) a summarized depiction of significant exposure levels associated with various adverse health effects. This section also includes information on the levels of 2,3,7,8-TCDD that have been monitored in human fluids and tissues, and information about levels of 2,3,7,8-TCDD found in environmental media and their association with human exposures. The significance of the exposure levels shown on the graphs may differ depending on the user's perspective. For example, physicians concerned with the interpretation of overt clinical findings in exposed persons or with the identification of persons w i t h the potential to develop such disease may be interested in levels of exposure associated-- with frank effects (Frank Effect Level, FEL). Public health officials and project managers concerned with response actions at Superfund sites may want information on levels of exposure associated with more subtle effects in humans or animals (Lowest-Observed-Adverse-Effect Level, LOAEL) or exposure levels below which no adverse effects (No-ObservedAdverse -Effect Level, NOAEL) have been observed. Estimates of levels posing minimal risk to humans (Minimal Risk Levels) are of interest to health professionals and citizens alike. Adequacy of Database (Sect. 2.3) highlights the availability of key studies on exposure to 2,3,7,8-TCDD in the scientific literature and displays these data in three-dimensional graphs consistent with the fqrmat in Sect. 2.2. The purpose of this section is to suggest where t^iere m i g h t b e i n s u f f i c i e n t i n f o r m a t i o n to e s t a b l i s h l e v e l s o f significant human exposure. These areas will be considered b y the Agency for Toxic Substances and Disease Registry (ATSDR), EPA, and the National Toxicology Program (NTP) of the U.S. Public Hea l t h Service in order to develop a research agenda to provide this information. 7 8 2.2 LEVELS OF SIGNIFICANT EXPOSURE To help public health professionals address the needs of persons living or working near hazardous waste sites, the toxicology data summarized in this section are organized first by route of exposure--inhalation, ingestion, and dermal--and then by toxicological end points that are categorized into six general areas--lethality, systemic/target organ toxicity, developmental toxicity, reproductive toxicity, genetic toxicity, and carcinogenicity. The data are discussed in terms of three exposure periods--acute, intermediate, and chronic. T w o k i n d s o f gr a p h s are u s e d to d e p i c t the data. The first type is a "thermometer" graph. It provides a graphical summary of the human and animal toxicological end points (and levels of exposure) for each exposure route for which data are available. The ordering of effects does not reflect the exposure duration or species of animal tested. The second kind of graph shows Levels of Significant Exposure (LSE) for each route and exposure duration. The points on the graph showing NOAELs and LOAELs reflect the actual doses (levels of exposure) used In the key studies. No adjustments for exposure duration or intermittent exposure protocol were made. Adjustments reflecting the uncertainty of extrapolating animal data to man, intraspecies variations, and differences between experimental versus actual human exposure conditions were considered when estimates of_levels posing minimal risk to human health were made for noncancer end points. These minimal risk levels were derived for the most "sensitive noncancer end point for each exposure duration by applying u n c e r t a i n t y factors. These levels are shown on the graphs as a broken line starting from the actual dose (level of exposure) and ending with a concave-curved line at its terminus. Although methods have been established to derive these minimal risk levels (Barnes e t a l . 1987), shortcomings exist in the techniques that reduce the confidence, in the projected estimates. Also shown on the graphs under the cancer end point are low level risks (10*^ to 10'^) reported by EPA. In addition, the . -- actual dose (level of exposure) associ a t e d w i t h tumor incidence is ~ plotted. ;: = 2.2.1 Key Studies and Graphical Presentations It is d i f f i c u l t to assess the r i s k to h u m a n s from e xposure to 2.3.7.8- TCDD. There are many species differences in toxicity; the monkey and guinea pig are apparently the most sensitive, and the ham s t e r is the least sensitive. It is not known how sensitive humans are to 2,3,7,8TCDD. I n addition, 2,3,7,8-TCDD strongly adsorbs to materials such as soil, which may significantly affect the bioavailability and toxicity of this compound. Animal studies have generally been conducted with 2 . 3 . 7 . 8 - T C D D a d m i n i s t e r e d in oi l y v e h i c l e s fr o m w h i c h the c o m p o u n d is readily bioavailable. There are insufficient data available to consider the effect of bioavailability on the toxicity studies used to define the risk from exposure to 2,3,7,8-TCDD. Both bioavailability and species differences in sensitivity should be considered when evaluating the remainder of the data presented. .<0 9 Oral and dermal NOAELs and LOAELs are presented on "thermometer" graphs in Figs. 2.1 and 2.2, respectively. Although some qualitative d a t a are a v a i l a b l e for h u m a n s , quanti t a t i v e data were i n s u f f i c i e n t to graphically present inhalation effects. Levels of significant exposure are depicted in Figs. 2.3 and 2.4 for the oral and dermal routes. There were insufficient human and animal inhalation data for graphical representation. The intermediate minimal risk level for oral exposure was calculated by EPA (1985a) from a three-generation reproductive toxicity study in the rat, w ith effects obse r v e d in the fetuses. Since the effects were attributed to in utero fetal exposure, which chronologically represents an exposure of relatively short duration, as well as the chronic exposure of the dams, the same minimal risk level for chronic exposure was calculated from these data. 2.2.1.1 Inhalation No studies are available on the inhalation toxicity of 2.3.7.8- TCDD. Exposure through inhalation, however, may also have occurred to the po p u l a t i o n exposed to chemicals contaminated with TCDD in accidental releases or in the workplace (especially herbicide spraying). 2.2.1.2 Oral Lethality and decreased longevity. There have been no reports of death in humans as a result of oral exposure to 2,3,7,8 - T C D D . 2 , 3 , 7 , 8 -TCDD is h i g h l y toxic to all m a m m a l i a n species, even though t h e r e is a l a r g e d i f f e r e n c e i n s p e c i e s s e n s i t i v i t y . L D 50 v a l u e s r ange f r o m 0 . 6 /ig/kg i n m a l e g u i n e a p i g s ( S c h w e t z e t al. 1 973) to 5 5 0 0 /ig/kg in hamsters (Henck et al. 1981). These values are plo t t e d in Figs. 2.1 and 2.3 for the lethality of acute oral exposure. Death usually occurs 1 3 t o 1 8 d a y s a f t e r a s i n g l e e x p o s u r e . E x t e n d e d e x p o s u r e i n a 9 0 - d a y ______ feeding study in guinea pigs resulted in an estimated 50% mortality a f t e r c o n s u m p t i o n o f a t o t a l o f 0 . 8 /ig o f 2 , 3 , 7 , 8 - T C D D / k g ( 0 . 0 0 8 ftg/kg/day) (DeCaprio et al. 1986), whereas deaths did n o t occur at 0.0006 ^g/kg/day (NOAEL). Five of eight female monkeys that ingested a p p r o x i m a t e l y 0 . 0 1 /ig o f 2 , 3 , 7 , 8 - T C D D / k g / d a y f o r 9 m o n t h s d i e d ( A l l e n e t al. 1977). These FELs in guinea pigs and monkeys and the N O A E L in guinea pigs are shown in Figs. 2.1 and 2.3. Target organ/systemic toxicity. Four major toxic effects characteristic of 2,3,7,8-TCDD are chloracne, the wasting syndrome, hepatotoxicity, and immunotoxicity. Chloracne, immunotoxicity, hyperpigmentation, hyperkeratosis, hirsutism of the skin, possible hepatotoxicity, hypertriglycerid and hypercholesterolemia, aching muscles, loss of appetite, weight loss, digestive disorders, headaches, neuropathy, insomnia, sensory changes, and loss of libido have been observed in humans exposed to chemicals contaminated wi t h 2 , 3 ,7,8-TCDD. Because some herbicides and some industrial chemicals contain 2 . 3 . 7 . 8 - T C D D as a contaminant, the primary rout of e x p o s u r e is mos t likely to be dermal, although some oral and inhalation exposure probably also occurs. 4931 >.-!? 0 3 ? - ANIMALS (yg/fcg/day) 10.000 (-- HAM STER ID SINGLE DOSE 1,000 MOUSE. MONKEY. CHLORACNE. SINGLE DOSE 100 - K- HUNWNS QUANTITATIVE DATA WERE NOT AVAILABLE 1 -- MOUSE. DEVELOPMENTAL TOXICrTY. 10 DAYS. CONTINUOUS GUINEA PIG . LO *. SINGLE DOSE O MOUSE. DEVELOPMENTAL TOXICITY, 10 DAYS. CONTINUOUS RAT. DEVELOPMENTAL TO XICITY. 10 DAYS. CONTINUOUS 0.1 -- GUINEA PIG . LIV ER TO XICITY. SINGLE DOSE GUINEA PIG. IMMUNOTOXtCITY, B W EEKS. INTERMITTENT O RAT. DEVELOPMENTAL TO XICITY. 10 DAYS. CONTINUOUS 0.01 - MONKEY. DEATH. 9 MONTHS. CONTINUOUS: MONKEY, CHLORACNE. 7 MONTHS. CONTINUOUS f * GUINEA PIG. DEATH. 90 DAYS. CONTINUOUS [O GUINEA PIG . IMMUNOTOXICITY. 8 W EEKS. INTERMITTENT GUINEA PIG. WASTING SYNDROME. UVER TOXICITY. 90 DAYS. CONTINUOUS 0.001 - MONKEY. REPRODUCTIVE TOXICITY, 7 MONTHS. CONTINUOUS RAT. REPRODUCTIVE TO XICITY. 3 GENERATIONS: RAT. UVER TOXICITY. 2 YEARS, CONTINUOUS O GUINEA PIG . WASTING SYNDROME, UVER TO XICITY. 90 DAYS. CONTINUOUS O GUINEA PIG. DEATH. 90 DAYS. CONTINUOUS 0.0001 -- LOAEL O NOAEL Fig. 2.1. Effects of 2,3,7,8-TCDD--oral exposure. 11 A N IM A LS (ji^Vg/day) 1000 r- R A B B IT. L D SIN G LE DO SE 100 HUMANS Q UAN TITATIVE DATA W ER E NOT A V A ILA BLE 10 - M O USE. D ERM AL LES IO N S . 4 W E E K S . IN TERM ITTEN T 1 * LOAEL Fig. 2.2. Effects of 2,3,7,8-TCDD--dermal exposure. 4933 ACUTE (S14 D AYS) INTERM EDIATE (15-364 DAYS) C H R O N IC (2365 DAYS) D EV ELO PLETH A LITY M ENTAL (ugXg/day) TARGET D ECREASED REPRO- TARGET ORGAN LO N GEVITY DUCTION ORGAN TARGET ORGAN 10.000 s 1000 CA N CER 100 10 1 0.1 0.01 0.001- 0.0001 r g (LIV ER ) r k g k g (BODY W EIG HT, LIV ER ) r (LIV ER ) r 0.00001 0.000001 0.0000001 0.00000001 0.000000001 0.0000000001 0.00000000001 0.000000000001 0.0000000000001 * - j MINIMAL R IS K LE V E L , FOR E FFEC T S O THER s i/ THAN C A N CER g GUIN EA PIG k MONKEY m M OUSE r RAT S HAM STER ILO AEL AND NOAEL IN SAM E S P E C IE S LO A EL FO R ANIMALS O NOAEL FO R ANIMALS ' \ IO" * - 10"5 10"6 - ESTIM A TED HUMAN CA N CER R IS K LEV ELS 10-7 - MINIMAL R IS K LEV EL FO R CHRONIC EXTRA PO LA TED FROM IN TERM EDIATE EXPO SU RE Fig. 2.3. Levels of significant exposure for 2,3,7,8-TCDD--oral ACUTE ( 14 DAYS) LETHALITY (ng/kg/day) 10,000 r h INTERMEDIATE (15-364 DAYS) TARGET ORGAN CHRONIC (S 365 DAYS) QUANTITATIVE DATA WERE NOT AVAILABLE 1000 - 100 - 10 m (SKIN) 1 I- LOAEL m MOUSE h RABBIT Fig. 2.4. Levels of significant exposure for 2,3,7,8-TCDD--dermal. 935 14 Since chloroacne, the only lesion definitively identified in humans as resulting from 2,3,7,8-TCDD exposure, can only be detected in a few species, the investigation of this effect has been limited. In hairless mice, chloracne was produced after a single dose of 2,3,7,8-TCDD at 70 fig/kg (Greig 1984), whereas a single dose of 70 Mg/kg, or a dose of -0.01 /ig/kg/day in a 7 -month feeding study in monkeys, produced similar lesions on the face (McConnell et al. 1978, A l l e n et al. 1977). These data are indicated in Fig. 2.1. The available studies were not designed to define a dose-response relationship; only FELs are reported (see Fig. 2.1). The wasting syndrome is characterized b y extreme loss of b o d y weight. In acute studies, this syndrome is associated w i t h lethal doses. A dose-response relationship for the wasting syndrome has been defined in a 9 0 - d a y study in guinea pigs (DeCaprio et al. 1986). Fem a l e H a r t l e y guinea pigs were maintained on diets providing average 2,3,7,8-TCDD d o s e s o f 0, 0 . 1 2 , 0 . 6 8 , 4 . 8 6 , a n d 31 n g /kg/day. T h e h i g h dose r e p r e s e n t e d a F E L ; a 40% d e c r e a s e i n b o d y w e i g h t a n d d e a t h o c c u r r e d . T h e 4 . 8 6 - n g / k g ( 0 . 0 0 5 p g / k g ) d o s e r e p r e s e n t e d a L O A E L , w i t h a 13% d e c r e a s e in b o d y weight and no mortality. The 0.68 ng/kg (0.0007 pg/kg) is a NOAEL for this effect. The LOAEL and NOAEL for intermediate exposure are indicated in Figs. 2.1 and 2.3. 2,3-,"7,8 - T C D D is h e p a t o t o x i c i n a l l s p e c i e s t e s t e d ; h o w e v e r , t h e severity of the lesions depends on the species studied. Although liver damage is not as severe in the guin e a pig, the mo s t sensitive species -te s t e d with regard to lethality, liver changes such as focal necrosis and hypertrophy have been observed at very low doses by Turner and Collins (1983). In this study, a small group of male and female guinea pigs was given a single dose of 2,3,7,8-TCDD at 0.1, 0.5, 2.5, 12.5, or 20 /ig/kg. Effects on the liver occurred in all groups. The low dose was considered a LOAEL (see Figs. 2.1 and 2.3), but a NOAEL was not available. A minimal risk for effects of acute oral exposure (see Fig. 2 . 3 ) w a s c a l c u l a t e d f r o m t h i s L O A E L b y E P A ( 1 9 8 5 a ) , s i n c e i t w a s t h e __ most sensitive end point in acute studies. Similar liver damage was o b s e r v e d in guinea pigs in the 9 0 - d a y f e e d i n g study b y D e C a p r i o et al. (1986). The NOAEL was 0.68 ng/kg and the LOAEL was 4.86 ng/kg, the same as the NOAEL and LOAEL for the w a s ting syndrome (see Figs. 2.1 and 2.3). There are no chronic studies in guinea pigs; however, in chronic studies in rats, K ociba et al. (1978a,b) a n d NTP (1982a) reported "toxic hepatitis" and degenerative changes at the lowest dietary exposure that p r o v i d e d a d ose of 0 . 0 0 1 fig o f 2 , 3 , 7 , 8 - T C D D / k g / d a y in these 2 - year studi e s . Again, only a LOAEL, w h i c h is p l o t t e d in Figs. 2.1 a n d 2.3, is available for this end point after chronic exposure. The guinea pig also appears to be the most sensitive species to the i m m u notoxic effects of 2,3,7,8-TCDD. Vos et al. (1973) o b s e r v e d a decrease in thymus weight, total lymphocyte number, and total leukocyte number in groups of 10 guinea pigs given 2,3,7,8-TCDD weekly for 8 weeks a t d o s e s o f 0, 0 .008, 0.04, o r 0 . 2 ftg/kg. T h e h i g h d o s e w a s a F E L a n d also produced other toxic effects including loss of body weight. A LOAEL of 0.04 fig/kg and a NOAEL of 0.008 fig/kg were defined. At the LOAEL, b o d y weight was comparable to controls. The LOAEL and NOAEL for immunotoxicity are plotted on Fig. 2.1, b ut not on Fig. 2.3 because liver toxicity and wasting syndrome are more sensitive end points of 936 15 i n t e r m e d i a t e o ral e x p o s u r e in g u i n e a pigs. I m m u n o t o x i c i t y is n o t l i m i t e d to guinea pigs; T h i g p e n et al. (1975) r e p o r t e d that mice given 4 w e e k l y e x p o s u r e s to 2 , 3 , 7 , 8 - T C D D at doses as low as 1 /ig/kg w e r e more s e n s i t i v e to S a lm o n e lla -induced death. This dose caused no gross signs of toxicity in animals not exposed to S a lm o n ella . Developmental toxicity. There have been no reports of developmental toxicity in humans as a result of oral exposure 2,3,7,8-TCDD. to 2.3.7.8- TCDD produces anomalies in the fetus, including cleft palate and hydronephrotic kidneys in mice and internal organ hemorrhage in the rat. FELs in rats were reported to be 0.125 pg/kg/day after administration of the compound on days 6 through 15 of gestation (Sparschu et al. 1971a,b), whereas the n x t lower dose tested, 0.03 M g / k g , w a s a N O A E L (see Figs. 2 . 1 a n d 2.3). D o s e s o f 1 /ig/kg c a u s e d fetal death. Although developmental effects are also observed in mice, this species appears less sensitive, with FELs of -1 ^g/kg/day (when a d m i n i s t e r e d d u r i n g o r g a n o g e n e s i s ) a n d a N O A E L of - 0 . 3 ftg /kg ( Neubert and Dillman 1972) (see Figs. 2.1 and 2.3). Reproductive toxicity. There have been no reports of reproductive toxicity in humans as a result of oral exposure to 2,3,7,8-TCDD. "Tiurray et al. (1979) conducted a three- g e n e r a t i o n reproductive toxicity study in rats. 2,3,7,8-TCDD was administered in the diet at levels that p r o v i d e d doses of 0.001, 0.01, a n d 0.1 /ig/kg/day. The h i g h dose r e s u l t e d in d e c r e a s e d fetal survival. M u r r a y et al. (1979) concluded that the 0.01-dose represented a LOAEL (with effects observed on litter size and fetal and neonatal survival) and that the 0.001-dose was considered to be a NOAEL. Nisbet and Paxton (1982) reevaluated the above data, using different statistical methods, and concluded that the lowest dose tested produced dilated renal pelvises, decreased fetal weight, and changes in the gestational index, which indicated that 0 . 0 0 1 /ig/kg w a s i n f a c t a L O A E L ( s e e F i g s . 2 . 1 a n d 2 . 3 ) . E P A ( 1 9 8 5 a ) ______ calculated a minimal risk for intermediate and chronic oral exposure from the Nisbet and Paxton (1982) analysis (see Fig. 2.3). In a study with monkeys maintained on a diet for 7 months, which provided 2,3,7,8-TCDD at levels of 0.0015 and 0.01 pg/kg/day, there were spontaneous abortions in two-thirds of the monkeys at both dose levels (Allen et al. 1979). The 0.0015-/ig/kg/day level is indicated on Figs. 2.1 and 2.3. This study, which reported severe frank effects, indicates that monkeys may be the most sensitive species with regard to the reproductive toxicity of 2,3,7,8-TCDD. This study, however, only provided FELs, and additional data were not available for determining a NOAEL or the actual relationship between species sensitivity. Genotoxicity. There have been no reports of genotoxicity in humans as a result of oral exposure to 2,3,7,8-TCDD. 2.3.7.8- TCDD has produced mostly negative responses in tests for genotoxicity; however, there are a few positive responses, which may s u g g e s t t h a t 2 , 3 , 7 , 8 - T C D D is ge n o t o x i c (see Sect. 4 .3.5 o n genotoxicity). Some of the inconsistencies observed may be related to experimental difficulties in testing 2,3,7,8-TCDD, such as the very low solubility of this compound and the high toxicity in vivo (which limits 4937 16 the quantity that can be t e s t e d ) , rather than to inherent biological inactivity. Carcinogenicity. There have been no reports of increased cancer incidence in humans as a result of oral exposure to 2,3,7,8-TCDD. 2,3,7,8-TCDD has been demonstrated to be an animal carcinogen in both rats and mice in an NTP (1982a) bioassay, and in rats in a 2 -year b i o a s s a y b y Kociba et al. (1978a,b ) . EPA (1985a) used female rat data f r o m the K o c i b a et al. (1978a,b) s t u d y to de r i v e a q^*. In this derivation, the total incidences for tumors of the liver, lung, hard palate, or nasal turbinates, as reported b y K o ciba et al. (1978a,b), were combined. These data, along with a similar set of data derived for EPA b y Squire on the rvaluation of the histologic section from the K o c i b a et al. (1978a,b) study, were u s e d b y EPA (1985a) to derive a q^*. The tumor incidences reported b y K o c i b a et al. (1978a,b) for doses of 0, 0 . 0 0 1 , 0.01, a n d 0 . 1 /jg/kg/day w e r e 9/85, 3/48, 18/48, a n d 34/4 0 , respectively, whereas the respective values reported by Squire were 16/85, 8/48, 27/48, and 34/40. The lower dose of 0.01 pg/kg/day, associated with increased tumor incidence, is indicated on Fig. 2.3. The q^* thus calculated was 1.56 x 10^ ( m g / k g / d a y ) . For cancer, EPA has estimated that for a population of 10,000 people exposed to 0.6 pg/kg/day, the cancer ri s k is n ot like l y to e x c e e d 1/10,000, and similarLy-v that for a lesser exposure of 0.0006 pg/kg/day to 10,000,000 people, the expected cancer risk would not exceed 1/10,000,000 (Fig. 2.3) . Lethality and decreased longevity. There have been no reports of death in humans as a result of dermal exposure to 2,3,7,8-TCDD. S c h w e t z e t al. ( 1 9 7 3 ) r e p o r t e d a d e r m a l LD50 v a l u e o f 275 p g / k g i n rabbits. As in oral studies, there was a protracted length of time b e t w e e n a p p l i c a t i o n a n d d e a t h . N o o t h e r d a t a w e r e a v a i l a b l e . T h e LD50 is plotted on Figs. 2.2 and 2.4. T a r g e t o r g a n / s y s t e m i c toxicity. C h l o racne is the only substantiated effect in humans produced by certain compounds contaminated with 2,3,7,8-TCDD. As reviewed by Taylor (1979) and Suskind (1985), these persistent, deforming face and upper body lesions have been recognized for many years as resulting from exposure to certain h a l o g e n a t e d a r o m a t i c c o m p o u n d s , a n d it is b e l i e v e d t h a t 2 , 3 , 7 , 8 - T C D D is the m o s t e f f ective comp o u n d in p r o d u c i n g this lesion. T h e r e is, however, no information on the levels of exposure needed to produce chloracne in humans, and, thus, quantitative risk assessment cannot be performed. In addition, there are da t a that suggest that 2 , 3 , 7 , 8 - T C D D is hepatotoxic in humans. In populations exposed to herbicides and other industrial chemicals contaminated with 2,3,7,8-TCDD, there have been reports of increased serum levels of liver enzymes and the development of porphyria cutanea tarda (EPA 1985a). In all studies, however, exposure may have been to chemicals that also could cause liver damage, and as pointed out by Jones and Chelsky (1986) , the diagnosis of porphyria cutanea tarda in some of the studies may be questionable. It is thus difficult to assert that the presumed exposure to 2,3 , 7 , 8 - T C D D resulted in liver injury, and even if 2,3,7,8-TCDD induced liver damage, i 17 there are no human data available that could provide dose-response information. The only animal data that provide quantitative information on chloracne are provided by the study of Puhvel et a l . (1982), in which hairless mice given 0.1 pg of 2,3,7,8-TCDD per application three times per week for 4 weeks developed dermal lesions that resembled some features of chloracne in humans. A ssuming that a mouse weighs 0.03 kg, the dose is 3.3 pg/kg. This study, however, only used one dose which was a FEL; hence, it does not provide the necessary information for defining a d o s e - r e s p o n s e relationship. The dose is p l o t t e d on Figs. 2.2 a nd 2.4 for intermediate target organ toxicity of dermal exposure. Developmental toxicity. Studies of h u m a n populations exposed to herbicides and other industrial chemicals contaminated with 2,3,7,8-TCDD have suggested that 2,3,7,8-TCDD produces a variety of developmental effects (Hanify et al. 1981, N e l s o n e t al. 1979, M c Q u e e n et al. 1977, S m i t h et" a l . 1 9 8 2 ) .''"After r e v i e w i n g t h e s e s t u d i e s , E P A ( 1 9 8 5 a ) i n d i c a t e d that the data were not inconsistent with 2,3,7,8-TCDD adversely affecting development, but as a result of the limitations of the data, these studies could not prove an association with 2,3,7,8-TCDD exposure and the observed effect. The major limitations in these human studies were the concomitant exposure to other potentially toxic chemicals and t h e 1-ffck o f a n y s p e c i f i c q u a n t i t a t i v e d a t a o n t h e e x t e n t o f e x p o s u r e o f individuals within the study group. No animal studies were available on the developmental toxicity of 2.3.7.8- TCDD following dermal exposure. Reproductive toxicity. EPA (1985a) has reviewed human reproductive toxicity studies of groups exposed to herbicides and other industrial chemicals contaminated with 2,3,7,8-TCDD (EPA 1979, Field and Kerr 1979, N e l s o n et al. 1979, Thomas 1980, Dept, of H e a l t h New Zealand 1980, M c Q u e e n et al. 1977, A l d r e d 1978, S m i t h et al. 1982, B o n a c c o r s i et al. 1978, Reggiani 1980, Bisanti et al. 1980). These studies did not provide a scientifically valid indication that 2,3,7,8-TCDD adversely affects either male or female reproductive performance, or that exposure to 2.3.7.8- TCDD is without effect. The limitations of the studies are similar to those discussed in the section above. No animal studies were available on the reproductive toxicity of 2.3.7.8- TCDD following dermal exposure. Carcinogenicity. EPA (1985a) also reviewed a series of epidemiology studies of populations exposed to herbicides and other industrial chemicals contaminated with 2,3,7,8-TCDD. Some of these studies indicated an association of exposure with the development of soft tissue sarcomas at a variety of sites and an increase in nonH o d g k i n s lymphomas (Axelson et al. 1980; T h e i s s and F r e n t z e l - B e y m e 1977; H a r d e l l a n d S a n d s t r o m 1979; E r i k s s o n et al. 1979, 1981; H a r d e l l et al. 1980, 1981; Lynge 1985; Putoni et al. 1986). These studies also have the same limitations as those described above, as well as additional questions on whether tumors of various sites should have been grouped together for the analyses. There are also a number of epidemiology studies of populations with exposures to contaminated herbicides and industrial chemicals similar to those above where an elevated incidence 4939 Ji-S'cW o 18 of cancer was not detected (Ott et al. 1980, 1987; Zack and Suskind 1980; Cook et al. 1980, 1985, 1986; Cook 1981; Pazderova-Vejlupkova et al. 1981; J o h n s o n et al. 1981; Ri i h i m a k i et a l . 1982; Smith et al. 1983; W o l f e et al. 1984; S m i t h an d Pe a r c e 1985; F i n g e r h u t et al. 1984). Besides the confounding factors previously described regarding epidemiological studies of 2,3,7,8-TCDD, the studies that failed to demonstrate an association between cancer and exposure were additionally confounded by the lack of a sufficient time period to allow for the latency of tumor development, the apparent lack of a control population with no known exposure to 2,3,7,8-TCDD (as indicated b y the analysis of fat biopsies, the general population has some exposure), and inconclusive data on the actual levels of exposure in the exposed population. It was again concluded that the human data could neither p r o v e n o r disp r o v e tha t 2 , 3 , 7 , 8 - TCDD is a h u m a n carcinogen. There are some data from experimental animal studies regarding the dermal carcinogenicity of 2,3,7,8-TCDD. In Swiss mice, females, but not males, developed skin tumors following, dermal application of 2 , 3 , 7 , 8 - T C D D a t 0 . 0 1 ftg p e r a p p l i c a t i o n t h r e e t i m e s p e r w e e k (NTP 1982b). There has also b e e n m i x e d evidence that 2, 3 , 7 , 8-TCDD is a tumor promotor. P o l a n d et al. (1982) observed t u m o r - p r o m o t i n g activity in hairless mice, but not in mice heterozygous for the hairless trait. Similarly, Berry et al. (1978), Slaga and Nesnows (1985), and NTP (1982) have not b e e n able to demonstrate promo t i n g activ i t y in CD-I, Senear, or Swiss-Webster mice. 2.2.2. Biological Monitoring as a Measure of Exposure and Effects There are no adequate tests or analyses of biological samples that could indicate the extent of exposure to 2,3,7,8-TCDD; however, some qualitative indications can be obtained. Adipose tissue has been shown to be one of the primary storage sites for 2,3,7,8-TCDD, and tissue samples have been analyzed, although mixed results have been obtained. A s r e por t e d b y N y g r e n et al. (1986), Y o u n g et al. (1983) f ailed to _____ detect elevated levels of 2,3,7,8-TCDD in the adipose tissues of Vietnam veterans exposed to A g e n t Orange, whereas Gross et al. (1984) detected increased levels in veterans who had been exposed to high levels of A g e n t Orange. As reported by N y g r e n et al. (1986), hig h e r levels of 2.3.7.8- TCDD in adipose tissue have also been reported in individuals exposed during a transformer fire accident in Binghamton, New York, or the accident in Seveso, Italy. In another study, 39 exposed individuals in Missouri ha d med i a n adipose 2,3,7,8-TCDD levels of 17 ppt, w ith a range of 2.8 to 750 ppt, whereas the unexposed control group had a m e d i a n level o f 6.4 a n d a range o f 1.4 to 20 p p t ( P a t t e r s o n et al. 1986). Although six of the subjects in the exposed group had levels >5 times higher than the highest control, there was also extensive overlap between the groups. Although the monitoring of adipose tissue may provide some qualitative indication that exposure has occurred, there are no good correlations available between adipose tissue levels and the extent of exposure. In addition, the background level of 2 ,3,7,8-TCDD in the adipose tissue of individuals with no known h i s t o r y of exposure to 2 . 3.7.8- TCDD generally are in the range of 5 to 18 ppt. This would suggest an ubiquitous exposure to 2,3,7,8-TCDD, which makes it difficult 4940 / 19 to assess the contribution to body burden from any particular small additional exposure. A similar lack of correlation between estimated e xposure to 2,3,7,8-TCDD and sera levels of 2,3,7,8-TCDD was reported in a preliminary study in the MMWR (1987), in which Vietnam veterans with military histories indicating exposure to herbicides containing 2.3.7.8- TCDD were compared to non-Vietnam veterans with presumably no unusual exposure to 2,3,7,8-TCDD. In these preliminary results, at l e a s t , t h e r e w a s n o d i f f e r e n c e i n t h e r a n g e o f 2 , 3 , 7 , 8 - T C D D l e v e l s (1 to 9 ppt based on lipid weight) or the median 2,3,7,8-TCDD level (3.9 ppt for the presumably exposed group and 3.8 ppt for the nonexposed group). Biological monitoring, such as monitoring levels in breast milk, only provides possible qualitative indications of exposure. With commonly a v a i l a b l e a nalytical techniques, 2,3 , 7 , 8 - T C D D is no t d e t e c t e d in bo d y fluids, such as blood or urine, although a recent method with partsper-quadrillion sensitivity has detected 2,3,7,8-TCDD in human serum (Patterson et al. 1987b). There are also no clear tests for the effects of exposure to 2 . 3 . 7 . 8 - TCDD. Chloracne is the o n l y effe c t that is clea r l y assoc i a t e d wit h exposure to chemicals contaminated with 2,3,7,8-TCDD; however, ch l o r a c n e is also caused b y o t h e r h a l o g e n a t e d aromatic c o m p o u n d s . The d e v e l o p m e n t of chloracne in a n individual w ho m a y have b e e n expo s e d to 2 , 3 , 2-f8-TCDD w o u l d provide supportive evidence that exposure to this chemical had occurred. The development of chloracne, however, does not i n d i c a t e the e x t e n t o f expo s u r e . L i k e w i s e , the d a t a o f H o f f m a n et al. (1986) suggest that the development of immunotoxicity would provide supportive evidence for exposure to 2,3,7,8-TCDD; however, again, the e x t e n t of e x p o s u r e is n o t indicated. I n addition, there are i n s u f ficient d a t a to state w i t h ce r t ainty that i m m u notoxicity is a s s o c i a t e d wi t h 2.3.7.8- TCDD exposure. Other signs of toxicity observed in animal studies (i.e., liver damage, effects on lipid metabolism, and types of circulating lipids) have not been demonstrated in humans and are not useful in determining that exposure to 2,3,7,8-TCDD has occurred. 2.2.3 Environmental Levels as Indicators of Exposure and Effects 2.2.3.1 Levels found in the environment There are little data to associate environmental levels with significant human exposure. 2,3,7,8-TCDD has been monitored in the areas of extensive herbicide use and areas contaminated with 2,3,7,8-TCDD through industrial accidents; however, epidemiologic studies of inhabitants of these areas have lacked adequate exposure data that would permit the demonstration of a clear association between exposure and effects..The biological half-life of 2,3,7,8-TCDD in humans, calculated to be 5 years by Poiger and Schlatter (1986), would indicate that r e p e a t e d exposure to low levels of 2,3,7,8-TCDD could s u b s t antially elevate the body burden of this compound to a level equivalent to the level obtained after a single exposure to a high level. G i v e n the long half-life of 2,3,7,8-TCDD, the total exposure history of an individual has to be taken into account; hence, the environmental levels during a s i n g l e p a r t i c u l a r exposure scenario m ay be m i s l e a d i n g w i t h reg a r d s to either effects observed or levels of body burden. The difficulties of estimating safe environmental levels have been discussed by Kimbrough et 20 al. (1984), who concluded that >1 ppb of 2,3,7,8-TCDD are levels of concern in residential soil. 2.2.3.2 Human exposure potential It is n o t p o s s i b l e to s tate w i t h any p r e c i s i o n w h i c h r o u t e of exposure to 2,3 , 7 , 8 - T C D D is m o s t relevant to the h u m a n population. As w i t h many h u m a n expo s u r e scenarios, it is a n t i c i p a t e d that all routes of exposure occur, although there are no data available to quantify the relative contribution of each route. It is anticipated that a s i g n ificant route o f expo s u re is dermal. Dermal e xposure c an occur through direct dermal contact with the mist of liquid chemical formulations of a herbicide containing 2,3,7,8-TCDD as a contaminant, or as a result of the long environmental half-life of 2,3,7,8-TCDD, by contact with soil or foliage (soon after contamination) in areas sprayed with these herbicides or contaminated through industrial accidents or waste disposal. In contaminated areas, exposure may also occur through ingestion or inhalation of soil or dust containing adsorbed 2,3,7,8-TCDD. Inhalation may be of particular concern where contaminated soils are b e i n g excavated or dust is be i n g formed b y other activities. In a 5-month clean-up operation of a 2,3,7,8-TCDD-contaminated Superfund s i t e in M i s s o u r i , w h e r e - 6 in. o f soil w a s r e m o v e d w i t h a b a c k h o e f r o m a SOOO-ft^ area, average 2,3,7,8-TCDD levels in the air were between 1 and 1.5 pg/m^ (Fairless et al. 1987). 2,3,7,8-TCDD from industrial and municipal incinerators may also represent a source of long-term, lowlevel i n h a l a t i o n e x posure. E x p o s u r e fro m i n g e s t i n g c o n t a m i n a t e d food is 'also variable, w i t h c e r t a i n foods such as fish, w h i c h are k n o w n to concentrate 2,3,7,8-TCDD, providing potentially relatively high exposures. B a s e d o n d a t a o n d e r m a l a n d o r a l a b s o r p t i o n in a n i m a l s , it is a n t i c i p a t e d that a d s o r b e d 2 , 3 , 7 ,8-TCDD w i l l n o t b e as b i o a v a i l a b l e as the 2,3,7,8-TCDD used in experimental studies and administered in oily vehicles. In the studies available, oral absorption of 2,3,7,8-TCDD __ a d s o r b e d to s o i l was. s t i l l s u b s t a n t i a l b u t o n l y 5 0 % o f t h a t f r o m c o r n oil (a b s o r p t i o n o f 50 to 80%) ( M cConnell et al. 1984, L u c i e r et al. 1986, Umbreit et al. 1986a). B i o a v a i l a b i l i t y also v a r i e s w i t h the type of soil, as d e m o n s t r a t e d b y U m b r i e t et al. (1986b) for N e w J e r s e y and Missouri soils, where bioavailability from N e w Jersey soil was less than that from Missouri so i l s . The limited studies available may not be representative of the variation in bioavailability, since Poiger and Schlatter (1980) demonstrat ed that strong b i n d i n g vehicles, such as activated carbon, can apparently reduce bioavailability to zero, and P h i l i p p i et al. (1981) a n d H u e t t e r a n d P h i l i p p i (1982) d e m o n s t r a t e d that the strength of adsorption increases with contact time in soil. Hence, f a c t o r s such' as s o i l t y p e a n d c o n t a c t t i m e m a y a f f e c t t h e bioavailability of 2,3,7,8-TCDD. It is l i k e l y that m a n y of the same p h y s i c a l p r o p e r t i e s w h i c h result in strong binding to soil, such as extremely low water solubility and planar configuration, also result in very high bioconcentration factors. Because of the hig h lipophilicity and long half-life of 2,3,7,8-TCDD, exposure through ingestion of fatty tissues of fish that inhabit contaminated areas is anticipated to be significant. In addition, as a result of the lipophilic nature of milk, secretion of milk can provide a o f 21 relatively efficient mechanism for decreasing the body burden of 2,3,7,8-TCDD in females. As discussed by Graham et a l . (1986), this elimination of 2,3,7,8-TCDD through mother's milk can result in large exposures of the infant. Since both milk and the fatty tissues of fish are essentially providing an oily vehicle, it seems likely that these s ources w o u l d p r o v i d e 2,3 , 7 , 8 - T C D D i n a fo r m that is rea d i l y bioavailable. 2.3 ADEQUACY OF DATABASE 2.3.1 Introduct ion S e c t i o n 110 (3) o f S A R A d i r e c t s the A d m i n i s t r a t o r o f A T S D R to prepare a toxicological profile for each of the 100 most significant h a z a r d o u s s u b s t a n c e s f o u n d a t f a c i l i t i e s -on t h e C E R C L A N a t i o n a l Priorities List. Each profile must include the following content: "(A) An examination, summary, and interpretation of available toxicological information and epidemiologic evaluations on the hazardous substance in order to ascertain the levels of significant human exposure for the substance and the associated acute, subacute, and chronic health effects. B) "r* A determination of whether adequate information on the health effects of each substance is available or in the process of development to determine levels of exposure which present a significant risk to human health of acute, subacute, and chronic health effects. (C) W h e r e a p p r o p r i a t e , a n i d e n t i f i c a t i o n of t o x i c o l o g i c a l te s t i n g needed to identify the types or levels of exposure that may present significant risk of adverse health effects in humans." This s e c t i o n identifies da t a gaps in cur r e n t k n o w l e d g e rel e v a n t to developing levels of significant exposure for 2,3,7,8-TCDD. Such gaps a r e i d e n t i f i e d f o r c e r t a i n h e a l t h e f f e c t s e n d p o i n t s ( l e t h a l i t y , ------system/target organ toxicity, developmental toxicity, reproductive toxicity, and cancer) reviewed in Sect. 2.2 of this profile in developing levels of significant exposure for 2,3,7,8-TCDD, and for other areas such as human biological monitoring and mechanisms of toxicity. The present section briefly summarizes the adequacy of existing human and animal data, identifies data gaps, and summarizes research in progress that may fill such gaps. Specific research programs for obtaining data needed to develop levels of significant exposure for 2,3,7,8-TCDD will be developed by ATSDR, NTP, and EPA in the future. 2.3.2 Adequacy of the Database for Health Effect End Points 2.3.2.1 Introduction and graphic summary The adequacy of the 2,3,7,8-TCDD database for health effect end p o i n t s in h u m a n s a n d animals is d e p i c t e d on b a r graphs in Figs. 2.5 and 2.6, respectively. 4943 I D .. n O or HUMAN DATA a ADEQUATE V DATA J \ . SOME >~ DATA J NO DATA N> ro D CP LE T H A LIT Y ACUTE ZI_______ IN TERM ED IA TE CHRONIC _______________________________________/ S Y S T E M IC TO X IC ITY DEVELO PM EN TAL TO X IC IT Y R EPRO D U CTIVE TO X IC ITY CARCIN O O EN ICITV Fig. 2.5. Adequacy of the database on health effects of 2,3,7,8-TCDD (human data). A N IM A L D A T A A ADEQUATE V * DATA J SOME > DATA J NO DATA o CD S' LE T H A LIT Y ACUTE IN TERM ED IA TE CHRONIC Z _______________________________________________________________/ S Y S T E M IC TO X IC IT Y D EV ELO PM EN TA L REPR O D U C TIV E TO X IC ITY TO X IC IT Y CA RCIN O G EN ICITY Fig. 2.6. Adequacy of the database on health effects of 2,3,7,8-TCDD (animal data). 24 The bars of full height indicate that there are "adequate" data to meet at least one of the following conditions: 1. F o r n o n c a n c e r h e a l t h e n d p o i n t s , o n e o r m o r e s t u d i e s a r e a v a i l a b l e that meet current scientific standards and are sufficient to define a range of toxicity from no-effect levels (NOAELs) to levels that cause effects (LOAELs or FELs). 2. F o r h u m a n c a r c i n o g e n i c i t y , a s u b s t a n c e is c l a s s i f i e d as e i t h e r a "known human carcinogen" or "probable human carcinogen" by both EPA and International Agency for Research on Cancer (IARC) (qualitative), and the data are sufficient to derive a cancer potency factor (quantitative). 3. F o r a n i m a l c a r c i n o g e n i c i t y , a s u b s t a n c e c a u s e s a s t a t i s t i c a l l y signficant number of tumors in at least one species, and the data a r e s u f f i c i e n t t o d e r i v e a c a n c e r p o t e n c y f a c t o r . ~- 4. T h e r e are studieis w h i c h s h o w that the c h e m i c a l does n o t c a u s e this health effect via this exposure route. Bars of half height indicate that "some" data for the end point exist but_do not meet any of the criteria for "adequate" data. Although adequacy of data is indicated in Fig. 2.5 for dermal exposure only, the route of exposure in the available studies is not -clearly defined. Because of the nature of exposures to 2,3,7,8-TCDD, both inhalation and oral exposure are likely to occur along with dermal exposure; in some instances, exposure from these other routes will contribute substantially to the body burden. 2.3.2.2 Descriptions of highlights of graphs Human. Figure 2.5 indicates that there are very little human data o n t h e t o x i c o l o g i c a l e f f e c t s o f e x p o s u r e t o 2 , 3 , 7 , 8 - T C D D . T h e d a t a .. -- obtained from human studies are considered to be from dermal exposures, since most humans were exposed in contaminated areas considerably after the application or initial release of the chemicals cpntaining 2,3,7,8-TCDD. This long-term exposure would occur through contact of the skin with soil and other articles contaminated with 2,3,7,8-TCDD. Inhalation and oral exposures were also likely to occur through inhalation of contaminated dust and ingestion of contaminated food and dust. From reports of human exposure (acute accidental exposure as well as repeated e x p osure), there is a clear indication that chemicals c o n t a i n i n g 2,3,7,8-TCDD cause chloracne; there is also some i n d i c a t i o n that chemicals containing 2,3,7,8-TCDD are hepatotoxic; however, there are no data regarding the levels of exposure required for induction of these effects. The epidemiologic data on the effects of 2,3,7,8-TCDD on fetal development, human reproduction, and the development of cancer are too limited to either prove or disprove an association. As a result of mixed exposure and the lack of quantitative data on populations exposed to a c o m p o u n d in the environment, it is a n t i c i p a t e d that f i l l i n g data gaps on 2,3,7,8-TCDD will be difficult. 25 There are no additional data on the toxicity of 2,3,7,8-TCDD following inhalation exposure or oral exposure. Animal. Figure 2.6 indicates that there are considerably more data on the toxicological e f fects of 2,3,7,8*TCDD in animals compared to humans, although the data are primarily limited to oral studies. For animal data, it should be recognized that "adequate" may only apply to the specific species tested, since large species differences are observed with 2,3,7,8-TCDD. As a result of these large differences in s p e c i e s s e n s i t i v i t y (4 o r d e r s o f m a g n i t u d e f o r L D 50 v a l u e s ) , c o m p l e t e information on a specific end point will be difficult to obtain. For the complete understanding of a toxicological end point, the most sensitive species must be determined and adequately tested, or it must be clearly demonstrated which animal species is most representative of humans. The lack of adequate data on species sensitivity is a significant gap in the toxicological data on 2,3,7,8-TCDD. Although dermal exposure is a potentially significant route of human exposure, the only animal data available for this route are a single d e t e rmination of a lethal dose in rabbits (Schwetz et al. 1973) and a few studies of the effects of 2,3,7,8-TCDD in the two-stage tumorigenesis assay (EPA 1985a) of the mouse. Both of these types of studies provide only limited data on the dermal toxicity of 2,3, ?"78-TCDD. T h i s is p a r t i c u l a r l y the c ase w h e r e e x p o s u r e in h u m a n s is likely to occur from 2,3,7,8-TCDD which is adsorbed to soil or other material and may have only limited bioavailability compared with 2,3,7,8-TCDD in dermal studies where the chemical was applied in organic solvents. 2.3.2.3 Summary of relevant ongoing research A review of Federal Research in Progress will show that investigations of the toxicological properties of 2,3,7,8-TCDD are very active. This considerable research effort is too extensive to enumerate i n d i v i d u a l s t u d i e s . H o w e v e r , a v e r y i m p o r t a n t a r e a o f o n g o i n g r e s e a r c h ----is reviewed b y Y o u n g and K a n g (1985). This area consists of 15 ongoing epidemiology studies that are being conducted for the U.S. government and coordinated by the White House Agent Orange Working Group. Subject areas include mortality and morbidity, with particular concern for carcinogenic effects, anatomical birth defects, and the development of soft tissue sarcomas. A number of these studies are concerned with veterans exposed to the herbicide Agent Orange in Vietnam. 2.3.3 Adequacy of the Database for Other Information Needed for Risk Assessment 2.3.3.1 Pharmacokinetics and mechanisms of action M e c h a n i s m s o f action. T h e m e c h a n i s m o f 2 , 3 , 7 , 8 - T C D D t o x i c i t y is u n d e r extensive investigation. As reviewed b y Roberts et al. (1985), it has been proposed that 2,3,7,8-TCDD functions by a receptor-mediated mechanism. There is evidence that this m e c h a n i s m is as s o c i a t e d with many of the toxicological end points of 2,3,7,8-TCDD, as indicated by the segregation of the toxic properties of 2,3,7,8-TCDD with the Ah locus, w h i c h is n e a r the locus for the 2,3,7,8-TCDD receptor. Some toxicologic 4947 26 end points, however, do not segregate with the Ah locus. It has been s h o w n in tissue c u l tures w i t h h u m a n l y m p h o b l a s t o i d cells that there is a large genetic variation in aryl h y d r o c a r b o n hydroxylase inducibili ty (Nagaya et al. 1985, 1986), and the s u s c e p t i b i l i t y of these cells to the toxicity of 2,3,7,8-TCDD parallels the induction of this monooxygenase system. In addition, the levels of the receptors in a particular species do not necessarily correspond to species sensitivity. Thus, although a receptor-mediated mechanism is well established, the complete integration of this mechanism with the toxic action of 2,3,7,8-TCDD has not been fully characterized. Rozman et a l . (1985) have also described results of a study which indicates that the t h y r o i d h o r m o n e th y r o x i n e (T4 ) ha s the abil i t y to modulate the toxicity of 2,3,7,8-TCDD. Male Sprague-Dawley rats were divided into groups of normal rats, thyroidectomized rats, and thyroidectomized rats that received T4 replacement therapy. Following administration of 2,3,7,8-TCDD, mean time to death and percent mortality at 90 days were similar between the normal rats and the thyroidectomized rats that r e c e i v e d T 4 (35 days and 89%, a n d 37 days and 80%, respectively). Thyroidectomized rats that did not receive T4 survived longer (mean time to death of 63 days) and h ad lower mortality at 90 days (44%). It was also demonstrated that the 2,3,7,8-TCDD treatment resulted in a decrease in serum T4 levels in both normal and thyroidectomized T4 -supplemented rats. The modulation of the lethal effects of 2,3,7,8-TCDD was considered b y the authors to be related to thyroid hormone modulation of energy metabolism. The participation of this mechanism in the development of 2,3,7,8-TCDD toxicity needs further study. In addition, mechanisms for individual end points, such as the wasting syndrome and the development of cleft p a l a t e , have also been investigated. Although a number of mechanisms have been proposed for such end points, there has yet to be a definitive understanding of the underlining biochemical processes that result in the observed effects of 2,3,7,8-TCDD. Further investigation of these processes is n ecessary not only to understand how 2,3,7,8-TCDD induces certain toxic end points, b u t also h o w the different target organ responses relate to e a c h other. Target organ/pharmacokinetic profiles. There are few data to indicate that target organs (such as liver and thyroid) contain relatively higher levels of 2,3,7,8-TCDD than other, nontarget tissues. In general, 2,3,7,8-TCDD appears to distribute to organs in proportion to lipid content (Ryan et al. 1985b) r ather than in r e gard to the sensitivity of the organ to the toxic effect of 2,3,7,8-TCDD. Roberts et al. (1985), however, reported that the distribution of the 2,3,7,8-TCDD receptor may be a better indicator of target organ than the distribution o f 2,3,7,8-TCDD itself. Further work Is needed to clarify this issue. Further research is also n e e d e d to determine the as s o c i a t i o n between a species' capability to metabolize 2,3,7,8-TCDD and the sensitivity of that species to 2,3,7,8-TCDD-induced toxicity. Although t h e r e a r e d a t a s u g g e s t i n g t h a t the a b i l i t y to m e t a b o l i z e 2 , 3 , 7 , 8 - T C D D is Important in determining species sensitivity to this chemical, the data are not complete and a mechanism has hot been proven. 27 Ongoing research. There are a relatively large number of studies reported in Federal. Research in P rogress that are involved with the toxicokinetics and the mechanism of action of 2,3,7,8-TCDD. The list of p r o j e c t s is too e x t e n s i v e for i n c l u s i o n in this profile. 2.3.3.2 Monitoring human biological samples Adequate analytical methods are available to investigate 2,3,7,8TCDD levels in biological materials that are lipophilic and thus c o n c e n t r a t e 2 , 3 , 7 , 8 - T C D D . Y o u n g (1984) r e p o r t e d t h a t the m e t h o d o l o g y is not available for detecting 2,3,7,8-TCDD in blood or tissues with low lipid content, although a recent method reported the detection of 2,3,7,8-TCDD in parts-per-quadrillion levels in human serum (Patterson et al. 1987b). The d e v e l o p m e n t o f more sensitive a nalytical methods is necessary for environmental analysis, and-the resulting technology will be u s e d in monitoring biological samples. At present, the ability to m o n i t o r 2,3,7,8-TCDD in h u m a n tissues appears to exceed the ability to interpret the toxicological significance of the observed results. 2.3.3.3 Environmental considerations Analytical methods. Concentrations of 2,3,7,8-TCDD in ambient air and drinking water are so low that the existing analytical methodologies cannot"measure the levels in these two media. Bioavailability. Although some data are available from a few environmental media on the bioavailability of 2,3,7,8-TCDD, a data gap exists on this subject. Bioavailability data are needed, since existing data indicate that 2,3,7,8-TCDD can bind strongly to soils of high organic content, thus decreasing its bioavailability. At present, there are insufficient data on the mechanism of binding to permit adequate estimation of potential absorbed doses from dermal, oral, or inhalation e x p o s u r e to soils c o n t a i n i n g 2,3,7,8-TCDD. Environmental fate and transport. Some progress on the fate and transport of 2,3,7,8-TCDD in environmental media has been made in recent years. Substantial gaps still exist in quantitative data regarding its biodegr^dability, photolysis, and volatility from environmental media. Interactions w i t h o ther c o m m o n c o-contaminants There are no data to indicate that 2,3,7,8-TCDD will interact chemically with other contaminants under environmental conditions. The presence of 2,3,7,8TCDD in both biological systems and the environment, and the lack of any reactive groups, would support the conclusion that 2,3,7,8-TCDD will not easily react with other compounds. Ongoing research. Present research efforts are focused primarily on analytical methodology to develop more monitoring data. The objective o f m ost of this research is to develop ne w analytical methods that will unequivocally identify and quantify very small amounts of 2,3,7,8-TCDD in various environmental media with a faster turnover time. A n example o f such an ongoing research is the investigation n o w being perf o r m e d by Ro b e n s and Zabik (n.d.). Substantial research is in progress to determine the concentration of 2,3,7,8-TCDD in serum and human milk samples. Groups of investigators in the Centers for Disease Control, research organizations in Sweden, Rutgers University, the State j>5oifO 28 University of New Yor k at Binghamton, and the Canadian Food Research Division are involved in such efforts (Ryan 1987). 3. C H E M I C A L A N D P H Y S I C A L I N F O R M A T I O N 3.1 CHEMICAL IDENTITY The chemical identity of 2,3,7,8-tetrachlorodibenzo-p-dioxin, to be r e f e r r e d to as 2 , 3 , 7 , 8 - TCDD t hroughout this document, is g i v e n in Table 3.1. 3.2 PHYSICAL AND CHEMICAL PROPERTIES Selected physical and chemical properties of 2,3,7,8-TCDD are shown in T able 3.2. 2 , 3 , 7 , 8 - T CDD is stable t o w a r d heat, acids, a n d alkalies, b u t b e g i n s to d e c o m p o s e a t 5 0 0 C. T h e d e c o m p o s i t i o n is v i r t u a l l y c o m p l e t e w i t h i n 21 s at 800C. 2 , 3 , 7 , 8 - T C D D is susceptible to p h o t o degradation in the presence of ultraviolet light, particularly in the presence of a hydrogen-donating solvent. Gamma radiation degrades 2 , 3 , 7-7U - T C D D i n o r g a n i c s o l v e n t s ( E P A 1 9 8 4 a ) . 4951 29 > CP o\ Chemical name Trade name Chemical formula Wiswesser line notation I Table 3.1. Chemical identify of 2,3,7,8-TCDD 2.3.7.8- tetrachlorodibenzo[b,e]( 1,4)-dioxin; 2.3.7.8- tctrachlorodibcnzo-p-dioxin; dioxin; TCDBD; 2.3.7.8- TCDD; 2,3,7,8-tetrachlorodibenzodioxin; 2.3.7.8- tetrachlorodibenzo-l,4-dioxin (EPA 1985) N one (T h e com pound is not produced com m ercially.) (EPA 1985) c 12h 4c i 4o 2 TC666 BO IOJ EG FG LG MG or TC666 BO IOJ DG EG LG MG (H SD B 1987) O 0 o Chemical structure HH A Identification Nos. CAS Registry No. NIOSH RTECS No. EPA Hazardous W aste No. O H M -TA D S No. D O T /U N /N A /IM C O shipping No. STCC No. Hazardous Substances Data Bank No. National Cancer Institute No. 1746-01-6 (SA N SS 1987) HP3500000 (SA N SS 1987) Not assigned (H SD B 1987) 8300192 (SA N SS 1984) N ot assigned (H SD B 1987 Not assigned (H SD B 1987) 4151 (H SD B 1987) N C I-C 037I4 (SA N SS 1987) t 31 Table 3.2. Physical properties of 2,3,7,8-TCDD Property Value References M olecular weight Color Physical state Odor Melting point Boiling point Autoignition temperature Solubility W ater (ng/L) Organic solvents (m g /L ) Density (g /m L ) Partition coefficients Vapor pressure (mm Hg) H en ry 's Law constant Flash point Refractive index Flammability limits Conversion factors Vapor Liquid Solid 321.97 Colorless Solid at room tem perature Unknown 305'"C 412.2C (estim ated) NAa 7.91 (20-22C ), 19.3 (22C ), 317 (2 5 C ) o-Dichlorobenzenc, 1400; chlorobenzene, 720; benzene, 570; chloroform, 370; m ethanol, 10; acetone, 110 1.827 (estim ated) Log KoW: 6.15-7.28 Log K ^: 6.0-7.39 3.46 X lO'9 (30.1oC ), 3.51 X 10'9 (30.2C ), 1.4 X 10'9 (estim ated at 25C ) 2.1 X 10'6 atm -m 3/m o l (estim ated) Unknown Unknown Unknown 1 ppb = 13.384 /tg /m 3 at 20C 1 ppb (w /v) = 1 fig/L; 1 ppt (w /v) = 1 ng/L 1 ppb (w /w ) = 1 #ig/kg = 1 ng/g; 1 ppt (w /w ) = 1 n g /k g = 1 P g/g EPA 1985a EPA 1985a Schroy ct al. 1985 Schroy et al. 1985 Schroy et al. 1985. Adams and Blaine 1986, M arple et al. 1986a Schroy et al. 1985 Schroy et al. 1985 EPA 1985a, Schroy et al. 1985, Jackson et al. 1986, M arple et al. 1986b Rondorf 1986, Schroy et al. 1985, Palansky et al. 1986 Schroy et al. 1985 `'N ot available. 4953 //[/] fa -y 4. T O X I C O L O G I C A L D A T A 4.1 OVERVIEW Human and animal data indicate that 2,3,7,8-TCDD can be absorbed following ingestion, and although bioavailability is affected by binding to soils, the e x t e n t of a b s o r p t i o n is only de c r e a s e d b y -50%. In addition, anim a l data indicate that 2 , 3 , 7 , 8-TCDD is a b s o r b e d well t h r o u g h t h e s k i n . F o l l o w i n g a b s o r p t i o n , 2r, 3 , 7 , 8 - T C D D is d i s t r i b u t e d to tissues in proportion to the lipid content. 2,3,7,8-TCDD can cross the placenta with subsequent exposure of the fetus, and the newborn can be exposed following redistribution of 2,3,7,8-TCDD during lactation. Metabolism of 2,3,7,8-TCDD is currently thought to result primarily in detoxification and relatively rapid removal of the metabolites through e x c r e t i o n in the b i l e a n d urine. U n m e t a b o l i z e d 2 , 3 , 7 , 8 - T C D D is also e x c r e t e d t h r o u g h d i r e c t i n t e s t i n a l e l i m i n a t i o n in feces as w ell as through lactation. There are species and strain differences in both metabolism and elimination rates, with elimination half-lives varying fr o m 11 days in the hamster, w h i c h is r e l a t i v e l y re s i s t a n t to 2,3,7,8T C D D toxicity, to >1 y e a r in the monkey, w h i c h is s e n sitive to the toxicity of 2,3,7,8-TCDD. Although humans have been exposed to 2,3,7,8-TCDD as a contaminant of herbicides and industrial chemicals, there have been no reported deaths from acute exposure. Lethal oral doses of 2,3,7,8-TCDD vary from 0.6 to 5000 /ig/kg for g u i n e a p igs a n d h a m s t e r s , r e s p e c t i v e l y , w i t h other s p e c i e s t e s t e d h a v i n g L D 50 v a l u e s b e t w e e n t h e s e t w o e x t r e m e s . S u b c h r o n i c L D 50 v a l u e s f o r c u m u l a t i v e (total) e x p o s u r e d u r i n g a 9 0 - d a y o r a l s t u d y with guinea pigs were essentially the same as those observed after acute exposure. A 9-month feeding study of a small number of monkeys indicated t h a t a d o s e o f 2 t o 3 /ig/kg r e s u l t e d i n d e a t h . T h e a c u t e d e r m a l L D 50 v a l u e f o r 2 , 3 , 7 , 8 - T C D D i n r a b b i t s h a s b e e n r e p o r t e d to b e 2 7 5 /xg/kg; however, no other species have been tested, and rabbits are only intermediate in sensitivity to 2,3,7,8-TCDD in oral toxicity studies. Inhalation experiments have not been conducted. The only effect clearly demonstrated to be produced in humans f o l l o w i n g 2 , 3 , 7 , 8 -TCDD exposure is chloracne. This l e s i o n is a systemic toxic effect and not solely a dermal effect. Although animal models are available to study chloracne, there are limitations in investigating this toxicological end point. The lesions produced in animals are different from the lesions in humans, and the expression of this lesion may be limited to monkeys, specific strains of mice, and rabbits (observed only on the ears after dermal application). In animals, the m a j o r t oxic e f f e c t o f 2 , 3 , 7 , 8 - TCDD e x p o s u r e is the wasting syndrome, in which the animals progressively lose body weight prior to death, w i t h no clear signs of altered organ function. Although this syndrome is observed in all species tested, it occurs predominantly 4955 33 34 only at doses that are lethal or near lethal. The wasting syndrome has not been observed in h u m a n s . Immunotoxicity has also been observed in a variety of animal species and may be one of the most sensitive effects of 2,3,7,8-TCDD. Immunotoxicity, however, has not b e e n directly related to 2 , 3 , 7 , 8 -TCDD exposure in humans. Hepatotoxicity has been observed in a variety of animal species, and there is suggestive evidence that this effect also occurs in humans. The human data are not completely clear, however, since mixed exposure to other potentially hepatotoxic chemicals occurred. In addition, species variations in induction of hepatotoxic effects indicate that the two most sensitive species, guinea pigs and monkeys, develop relatively less severe hepatic lesions than many other species. O t h e r o r g a n systems are also a f f e c t e d b y 2 , 3 , 7 , 8 -TCDD. T h e r e is suggestive evidence in humans that the nervous system may be affected; in animals, there are data on effects on the digestive system and the kidney. These additional organ systems have not b e e n investigated as extensively as those described later. Also, they do not appear to be prime indicators of 2,3,7,8-TCDD toxicity or provide any special insight into extrapolating effects observed in animals to those in man. Many-'of the toxic end points d i s c u s s e d have b e e n observed to have extensive species and strain differences in sensitivity. These differences may be related to the receptor-mediated mechanism of -toxicity proposed for 2,3,7,8-TCDD. This mechanism follows the scheme of 2.3.7.8- TCDD binding to a soluble cytoplasmic receptor protein, which subsequently migrates into the nucleus of the cell as a receptor2, 3,7, 8 - T C D D c o m p l e x . O n c e i n t h e n u c l e u s , i t is p r o p o s e d t h a t b o t h transcription and translation of DNA may be affected. The receptor : p r o t e i n is k n o w n genet i c a l l y to se g r e g a t e w i t h the A h l o c u s , a n d m a n y of the strain differences in toxicity observed in mice are explained by this segregation. The receptor protein is known to be widely dispersed in different organ systems and in different species, although quantitative levels differ. These quantitative differences may help explain some of the species differences in sensitivity. The receptor mechanism cannot explain all the species and strain differences in 2.3.7.8- TCDD toxicity, because some toxic responses segregate w i t h the Ah receptor while others do not. The evidence from studies of human populations exposed to herbicides and other industrial chemicals known to be contaminated with 2.3.7.8- TCDD is inadequate to demonstrate that 2,3,7,8-TCDD is a h u m a n developmental toxicant. The data from studies in laboratory mice and rats, however, clearly demonstrate in these species that 2,3,7,8-TCDD induces a variety of developmental abnormalities. The data from human populations exposed to herbicides and other industrial chemicals contaminated with 2,3,7,8-TCDD are not adequate to determine if 2,3,7,8-TCDD adversely affects reproductive health in humans. A n i m a l studies cl e a rly d e m o n s t r a t e that 2 , 3 , 7 , 8 - T C D D is f e t o t o x i c in s e v e r a l s p ecies a n d c a u s e s s p o n t a n e o u s a b o r t i o n s . T h e r e is additional evidence that 2,3,7,8-TCDD' affects male hormone levels and the function of male reproductive organs. f ) \ :*- ''"-.v 35 Although 2,3,7,8-TCDD has not consistently produced positive results in genotoxicity assays, there are positive results in diverse b i o a s s a y s y s t e m s w h i c h m a y s u g g e s t t h a t 2 , 3 , 7 , 8 - T C D D is a g e n o t o x i c agent; however, the large numbers of negative results in assays usually p r e d i c t i v e of ge n o t o x i c agents s u g g e s t that 2,3,7,8-TCDD is no t a genotoxic agent. The h u m a n e v i d e n c e that 2 , 3 , 7 , 8 - T C D D is a c a r c i n o g e n is conflicting, with both positive and negative findings reported in cohorts exp o s e d to herbicides and other chlorinated chemicals known to be contaminated with 2,3,7,8-TCDD. As a result, the human data only p r o v i d e s u g g e s t i v e evidence that 2 , 3 , 7 , 8 - T C D D is a h u m a n carcinogen. The a n i m a l data, however, p r ovide c l e a r e v i d e n c e that 2,3,7,8-TCDD is carcinogenic in animals. Exposure to 2,3,7,8-TCDD in the environment is never to 2,3,7,8TCDD alone but to materials such as incinerator fly ash or industrial wastes, which contain 2,3,7,8-TCDD along with many other congeners of 2 , 3 , 7 , 8 - T C D D , as w e l l as other, p o t e n t i a l l y t o x i c m a t e r i a l s . T h e E P A h a s recognized the public and toxicological concerns resulting from exposure to these compounds, also the gaps in available information w i t h w h i c h to evaluate the human health potential from exposure (EPA 1987). In response to this problem, the Chlorinated dibenzo-p-dioxins/Chlorinated dibenzofurans Technical Panel of the Risk Assessment Forum has developed and recommended an interim method for assisting in estimating the risk from exposure to these chemical mixtures that can be used until the data gaps are filled. This procedure generates the "2,3,7,8-TCDD equivalence factors" (TEFs) of complex mixtures of chlorinated dibenzo-p-dioxins based on congener- and isomer-specific data, and the results are presented in Table 4.1. The TEFs are relative values and are a means of relating toxicity data for a chlorinated dibenzo-p-dioxin to an e q u i v a l e n t level of 2,3,7,8-TCDD. T h e T E F for 2 , 3 , 7,8-TCDD is defi n e d as unity, whereas all other TEFs are u n i t y or less, thus reflecting the lower toxic potency of most 2,3,7,8-TCDD congeners. These relative values were developed using a tiered approach to the evaluation of existing data. The toxicity d a t a were used to extrapolate between congeners and 2,3,7,8-TCDD and were segregated into data types that would provide differing degrees of.reliability for the extrapolation process. Using this system, definitive data on the human carcinogenicity of a congener of 2,3,7,8-TCDD would be the most appropriate data for establishing the TEF. If this human data were not available, however, animal carcinogenicity data would be used, followed by the data on reproductive toxicity (estimated exposure levels resulting in reproductive and carcinogenic effects are very similar for 2,3,7,8TCDD). Finally, if none of the above data are available, the TEFs are determined by the weight of evidence from in vitro tests (with particular weight placed on data from tests evaluating receptor binding interactions and induction of oxidative enzymes). The TEFs thus generated can be used, assuming additivity of the toxic response, for estimating the relative toxicity of a mixture containing a known distribution of congeners of 2,3,7,8-TCDD. 4957 36 Table 4.1. Recommended TEFs for 2,3,7,8-TCDD and its congeners Compound EPA current recommended values based on various effects MonoCDDs DiCDDs TriCDDs 2,3,7,8-TCDD Other TCDDs 2,3,7,8-PeCDDs4 Other PeCDDs 2,3,7,8-HxCDDs* Other HxCDDs 2,3,7,8-HpCDDs* Other HpCDDs OCDD 0 0 0 1 0.01 0.5 0.005 0.04 0.0004 0.001 0.00001 0 "Toxicity equivalence factors. 6Any isom er th a t contains chlorine in the 2,3,7,8 positions; CDDs = chlorinated dibenzo-p-dioxins. Source: E P A 1987. 37 4.2 TOXICOKINETICS 4.2.1 Absorption 4.2.1.1 Inhalation Pertinent data regarding the absorption of 2,3,7,8-TCDD by humans or animals following inhalation exposure were not found in the available literature. 4.2.1.2 Oral Human. The absorption data available are from an elimination study of 2,3,7,8-TCDD in which a male volunteer ingested ^H-2,3,7,8-TCDD in c o m oil at a dose of 1.14 ng/kg (Poiger and Schlatter 1986). Measurements of cumulative elimination in the feces and urine, as well as a determination of sequestering in fat via biopsy samples, indicated that >87% of the dose was absorbed. Following absorption, the half-life for elimination was calculated.to be 2120 days. Animal. Studies re v i e w e d in E PA (1985a) s h o w that 2,3,7,8-TCDD is generally well absorbed (50 to 80%) w h e n administered to rats, guinea pigs, or h a m sters in a lipophilic v e hicle b y gavage (Piper et al. 1973, Nola n _ e t al. 1979, Olson et al. 1980). There appeared to be no change in absorption rates with repeated dosing, but there was some decrease in absorption at higher dose levels. Absorption of 2,3,7,8-TCDD when administered in the diet was also between 50 and 60% (Fries and Marrow 1975). The vehi c l e in wh i c h 2,3,7,8-TCDD is administered has substantial effects on its gastrointestinal absorption. As described in EPA (1985a), Poiger and Schlatter (1980) observed a decrease in the absorption of 2.3.7.8- TCDD when administered in a soil suspension compared with a solution in 50% ethanol, whereas no absorption occurred whe n the compound was administered as a suspension of activated carbon. Since 2 . 3 . 7 . 8 - T C D D in the e nvir o n m e n t is likely to be a d s o r b e d to soil, M c C o n n e l l et al. (1984) and Lucier et al. (1986) compared the absorption of 2,3,7,8-TCDD from contaminated soil with that from 2,3,7,8-TCDD administered in c o m oil. As indicated b y biological effects and the amount of 2,3,7,8-TCDD in the liver, the absorption was -50% less from soil than from c o m oil. U m breit et al. (1986a) showed that 2,3,7,8TCDD-contaminated soil was less toxic than an equivalent amount of 2.3.7.8- TCDD, suggesting that binding to soil h a d an influence on bioavailability. These data indicate that substantial absorption occurs f rom contaminated soil; however, soil type and duration of contact, as suggested from the data that demonstrated decreased extraction efficiency with increasing contact time between soil and 2,3,7,8-TCDD (Phillipi et al. 1981, Huetter and Phillipi 1982), may substantially affect the absorption of 2,3,7,8-TCDD from soils obtained from different contaminated sites. 4.2.1.3 Dermal Human. Pertinent data regarding the absorption of 2,3,7,8-TCDD following dermal exposure in humans were not found in the available literature. 4959 ' 38 Animal. Data regarding dermal absorption in animals are limited. 2,3,7, 8 - T C D D is abso r b e d well through the skin, although as wi t h oral absorption the vehicle can substantially affect absorption. As discussed "in EPA (19 8 5 a ) , it was estimated that 40% of the dose was absorbed by rats when the compound was applied in methanol, whereas application in vaseline or polyethylene glycol resulted in 1.4 and 9.3% absorption, respectively (Poiger and Schlatter 1980). Applying 2,3,7,8-TCDD as a paste in soil or activated carbon resulted in poorer absorption than observed in oral studies, with absorption of <2% and nondetectable, respectively. 4.2.2 Distribution 4.2.2.1 Inhalation Pertinent data regarding the distribution of 2,3,7,8-TCDD following inhalation exposure of humans and animals were not found in the available literature. 4 . 2 . 2 . 2 O r a l ; - .. Human. Poiger and Schlatter (1986) estimated that -90% of the body burden of 2,3,7,8-TCDD was sequestered in the fat after a volunteer ingested~3H-2,3,7,8-TCDD in corn oil at a dose o f 1.14 ng/kg. During this study, which lasted 135 days, elevated radioactivity was detected in'the blood only during the initial 2 days following treatment. The -data would be consistent with the hi g h bioconcentration potential of 2 . 3 . 7 . 8 - TCDD in humans, as c a l c u l a t e d b y Geyer et al. (1986) from daily intake a s s u m p t i o n s l e v e l s in human adipose tissue, and pharmacokinetic models. Adipose tissue has been examined to determine if levels of 2.3.7.8- TCDD in this reservoir correlated with exposure. Adipose tissues of Vietnam veterans exposed to Agent Orange^and humans occupationally e x p o s e d to p o t e n t i a l sources of 2 , 3 , 7 , 8 - T C D D w e r e r e p o r t e d to h a v e u p to 10 times the level of 2,3,7,8-TCDD in unexposed control subjects (Schecter et al. 1985, Gross et al. 1984); however, another study b y - : Weerasinghe et al. (1986) failed to detect any difference b e t w e e n a group of veterans and unexposed subjects. Although a clear correlation with exposure was not demonstrated, it was apparent that adipose tissue i s a d e p o t f o r 2 , 3 , 7 , 8 - T C D D , :a n d d e t e c t a b l e l e v e l s w e r e o b s e r v e d i n subjects with no known exposure. R y a n et al. (1985b) further examined the distribution of 2,3,7,8-TCDD in two humans at autopsy. They determined on a weight basis that 2,3,7,8-TCDD distributed in descending order to fat (-6 ppt) and liver (-2 p p t ) , wit h levels in muscle and kidney below detection; however, 2,3,7,8-TCDD levels compared on a per lipid basis were similar between tissues. These data should be interpreted with caution, since only two subjects were examined and one of the subjects was suffering from fatty liver syndrome; therefore, the data cannot be generalized to the entire population. Animal. EPA (1985a) reviewed a number of early studies that described the distribution of 2,3,7,8-TCDD in rats, mice, and guinea pigs. In rats and mice, the liver contained the greatest amount of 2.3.7.8- TCDD, followed by the fat; however, in guinea pigs this was reversed. Similar patterns were observed after intraperitoneal administration. In studies of congenic mice which differ only at the Ah 39 locus, the distribution patterns w ere similar to those previously described, except that relatively higher levels of 2,3,7,8-TCDD were reported in the livers of the responsive mice compared with the n o n r e s p o n s i v e s t r a i n ( G a s i e w i c z e t al. 1983a,b; B i r n b a u m 1986). Thus, it appears that there is little potential for strain difference in the d i s t r ibution pattern of 2,3,7,8-TCDD. Lakshmanan et al. (1986) reported t h a t the tissue d i s t r i b u t i o n of 2,3,7,8-TCDD is m e d i a t e d through . transport in the lymph system in the rat, with initial high levels of 2,3,7,8-TCDD in the lymph decreasing rapidly with progressive a c c u m u l a t i o n in the fat d u r i n g the f irst 24 h. The h a l f - l i f e for redistribution of 2,3,7,8-TCDD out of adipose tissue and liver was 1.6 and 5.3 weeks, respectively. 2,3,7,8-TCDD crosses the placenta and accumulates in the mouse fetus after gavage administration (Weber and Birnbaum 1985). In this study, -0.5% of the dose was detected in the fetus. Similar results were reported by Krowke (1986) in mice treated with 2,3,7,8-TCDD by subcutaneous injection. Fetal distribution was not uniform, with 75% of the 2,3,7,8-TCDD located in the fetal liver. In addition to in utero exposure of the fetuses, Nau et al. (1986) reported that postnatal exposure of the young occurred via the milk after a single administration of 2,3,7,8-TCDD to pregnant mice between gestation days 14 and!17. Again, the fetal and pup liver was the predominant storage site. 4.2.2.3 Dermal H u m a n . Studies of humans exposed to herbicides or other industrial compounds known to be contaminated with 2,3,7,8-TCDD were discussed in Sect. 4.2.2.2 (oral distribution) for easier comparison with the only available human experimental data. Routes of exposure in these studies ar e no t clea r l y defined; the m o s t li k e l y route is p r o b a b l y dermal, although oral and inhalation exposure are also likely to occur. Animal. No studies are available. ------- 4.2.3 Metabolism Pertinent data regarding the metabolism of 2,3,7,8-TCDD following inhalation or dermal exposure of humans or an i m a l s , or following oral exposure of humans, were not found in the available literature. However, data regarding the metabolism of 2,3,7,8-TCDD following oral exposure of animals are available. 2,3,7,8-TCDD is relatively slow to metabolize, b u t once metabolites are formed they are rapidly excreted through the urine and b i l e as g l ucur o n i d e and sulfate conjugates (Olson et al. 1980, 1983, as reviewed in EPA 1985a). Recently, however, Olson (1986) repor t e d that up to 28% of the radioactivity in the tissues of guinea pigs treated with labeled 2,3,7,8-TCDD was associated w i t h metabolites, indicating that under the c o n d i t i o n s used, the m e t a b o l i t e s o f 2 , 3 , 7 , 8 - T C D D w e r e n o t e l i m i n a t e d as r a p i d l y as p r e v i o u s l y b e l i e v e d . S a w a h a t a et al. ( 1 9 8 2 ) i d e n t i f i e d 1hydroxy-2,3,7,8-tetrachlorodibenzo-p-dioxin and 8 -hydroxy-2,3,7trichlorodib e n z o - p - d i o x i n in vitro, and Poiger et al. (1982) identified 2-hydroxy-l,3,7,8-tetrachlorodibenzo-p-dioxin in dogs as major m e t a bolites of 2,3,7,8-TCDD. Other hydroxylated prod u c t s were ident||j|{l 40 as minor metabolites. Mason and Safe (1986a,b) synthezised some of these metabolites and demonstrated that they had considerably less biological activity than 2,3,7,8 - T C D D , which supports the observation by Weber et al. (1982) and Poi g e r et al. (1982) that m e t a b o l i t e s e x t r a c t e d f rom the bil e of dogs t r e a t e d w i t h 2 , 3 , 7 , 8-TCDD were less toxic to rats and guinea pigs, respectively, than equivalent amounts of 2,3,7,8-TCDD. Species and strain differences in the metabolism of 2,3,7,8-TCDD have been investigated to determine if differences in metabolism could a c c o u n t for the l a r g e o b s e r v e d d i f f e r e n c e in toxicity. G a s i e w i c z e t al. (1983b) reported that qualitative evaluations of the elution profiles of the urinary and biliary metabolites of 2,3,7,8-TCDD were similar for responsive and nonresponsive strains of mice. In dogs (in vivo) and rats (in vitro), pretreatment with 2,3,7,8-TCDD resulted in a 100 and 320% increase, respectively, in the rate of metabolism of a subsequent dose of 2,3,7,8-TCDD, whereas in guinea pigs (in vitro), pretreatment with 2,3,7,8-TCDD had no effect on the rate of metabolism of a subsequent dose of 2,3,7,8-TCDD (Poiger and Schlatter 1985, Wroblewski and Olson 1985, Olson and Wroblewski 1985). These data suggest that some of the differences in the toxicity of 2,3,7,8-TCDD may be related to the rate of metabolism as well as qualitative and quantitative differences in the metabolites formed. 4.2.4 Excretion - Pertinent data regarding the excretion of 2,3,7,8-TCDD following -inhalation or dermal exposure of humans or animals were not found in the available literature. However, data regarding the excretion of 2,3,7,8TCDD following oral exposure of humans and animals are available. 4.2.4.1 Human Poiger and Schlatter (1986) studied the elimination profile of 2,3,7,8-TCDD from a volunteer who ingested a single 1.14-ng/kg dose of the tritiated chemical. Urinary levels of radioactivity were never above background levels during the 135 days the individual was studied. Fecal elimination was initially rapid, with 11% of the dose eliminated in the first 3 days (speculated to be nonabsorbed m a t e r i a l ) , whereas during days 7 to 125, only 3.5% of the dose was eliminated. From these data, a half-life of 2120 days in the body was calculated assuming first-order ki n e t i c s . It is, however, n o t k n o w n w i t h a n y c e r t a i n t y that 2 , 3 , 7 , 8 -TCDD is a c t u a l l y elimin a t e d b y first-order kinetics, a n d other kinetic parameters would change the estimated half-life. 4.2.4.2 Animal 2,3,7,8-TCDD and its metabolites are eliminated predominantly in the feces and in the urine in an apparent first-order process. There appear to be no major differences whether the material was administered by the oral or intraperitoneal routes (EPA 1985a). Although metabolites were observed in the bile, unmetabolized 2,3,7,8-TCDD detected in the feces probably was derived from direct intestinal elimination, as d e m o n s t r a t e d b y O l s o n (1986) in g u i n e a pigs. N a u et al. (1986) r e p o r t e d that e l i m i n a t i o n of 2 , 3 , 7 , 8-TCDD thr o u g h l a c t a t i o n is s i g n i f i c a n t in mice. EPA (1985a) reported that between 91 and 99% of the excreted 41 2.3.7.8- TCDD-derived radioactivity was found in the feces of rats and guinea pigs; 54 and 72% was detected in the feces of mice, with greater fecal excretion in the responsive C57B1/6J strain; and 59% was observed in the feces of hamsters. Half-lives for elimination also varied with species and strain as follows (in decreasing order): guinea pigs (22 to 30 days); rats (17 to 31 days); mice (11 to 24 days, 11 days in responsive mice and 24 days in nonresponsive strains); and hamsters (10 to 15 days). O l s o n (1986) reported even longer half-lives of >90 days in guinea pigs. Although there are no adequate excretion data from monkeys, there are indications that 2,3,7,8-TCDD is v e r y persistent in this species, with a possible half-life of >1 year (EPA 1985a). 4.3 TOXICITY 4.3.1 Lethality and Decreased Longevity 4.3.1.1 Inhalation Pertinent data regarding lethality and decreased longevity f o l l o w i n g i n h a l a t i o n e x p o s u r e o f h u m a n s o r a n i m a l s to 2 , 3 , 7 , 8 - T C D D were' not found in the available literature. 4.3.1.2 Oral Human. No studies are available. Animal. Results of extensive investigations of the acute lethality of 2,3,7,8-TCDD indicate that this chemical is toxic at v e r y low levels in all species tested. Species differences in lethality cover 4 orders o f m a g n i t u d e i n d o s e l e v e l . L D 50 v a l u e s , a f t e r g a v a g e a d m i n i s t r a t i o n i n lipophilic solvents, were reported to range from 0.6 to 2.1 pg/kg in Hartley guinea pigs (Schwetz et al. 1973), 20 to 60 pg / k g in rats, 100 to 600 pg/kg in mice, and 1000 to 5000 p g / k g in hamsters (EPA 1985a, McConnell 1985). Rhesus monkeys have also been studied, but the lowest dose tested, 70 pg/kg, was lethal (McConnell et al. 1978). Reproductive-- toxicity studies indicate that female rhesus monkeys are very sensitive to the lethal effects of 2,3,7,8-TCDD. Eight of 16 pregnant monkeys died after treatment with a total dose of 1.0 pg/kg over a 9 -day period during gestation (McNulty 1985); toxic effects, as indicated by abortion, were observed at a total 2,3,7,8-TCDD dose of 0.2 pg/kg. F o l l o w i n g a c u t e a d ministration, d eath is g e n e r a l l y o b s e r v e d only after an extended period of time that ranges from 5 to 40 days; this lag time a p p e a r s to be, to s o m e e xtent, i n d e p e n d e n t o f d o s e a f t e r a t h r e s h o l d is reached. Other factors besides species differences affect the acute toxicity of 2,3,7,8-TCDD. These variables include the strain of animal tested, wit h responsive C57BL/6J mice demonstrated to be twice as sensitive to 2.3.7.8- TCDD-induced lethality as DBA/2J nonresponsive strains (Gasiewicz et al. 1983a,b), and differences in lethal dose (ranging from 165 to 320 pg/kg) reported for four strains of rats (Walden and Schiller (1985). Strain differences have not been investigated in other species b u t would be a n t i c ipated to occur. Also, the veh i c l e substantially affects the acute lethality of 2,3,7,8-TCDD, probably by altering the bioavailability of the compound. However, as discussed by Kaminsky et 42 al. (1985) and illustrated b y U m b r e i t el al. (1985, 1986a,b), the influence of m a t r i x on b i o a v a i l a b i l i t y is complex, with factors such as the properties of the matrix as well as duration of contact with the matrix substantially altering the bioavailability. The complexity of this issue is further illustrated b y the protective effect of orally administered activated charcoal on the lethality of subcutaneously or i n t r a p e r i t o n e a l l y a d m i n i s t e r e d 2 , 3 , 7 , 8 - T C D D (Manara et al. 1984), suggesting that some of the effects are the result of processes other than bioavailability. The lethal effects of subchronic exposure to 2,3,7,8-TCDD have been stu d i e d by DeCaprio et al. (1986) in Har t l e y guinea pigs. Gu i n e a pigs w e r e m a i n t a i n e d o n d i e t s c o n t a i n i n g 2, 10, 76, o r 4 3 0 p p t o f 2 , 3 , 7 , 8 T C D D for 90 days. At 76 and 430 ppt, mortalities of 10 and 70%, r e s p e c t i v e l y , w e r e observed, a n d a n L D 5 0 v a l u e o f 0.8 /ig/kg f o r the total consumption of 2,3,7,8-TCDD was calculated. This value is similar to that observed in the previously described study of the acute exposure o f m a l e g u i n e a p i g s t o 2 , 3 , 7 , 8 - T C D D . E x p o s u r e s o f 0..61 n g / k g / d a y r e s u l t e d in no deaths. In rhesus monkeys, however, Allen et al. (1977) observed deaths in 5 of 8 animals maintained for 9 months on a diet that contained 500 ppt of 2,3,7,8-TCDD. From food intake, the total dose i n g e s t e d w a s c a l c u l a t e d to b e 2 to 3 /xg/kg or 0 . 0093 to 0 . 0 1 4 ^ g / k g / d a y . 4.3.1.3 Dermal -- Human. No studies are available. it. Animal. There is on l y one s t u d y a v a i l a b l e r e g a rding the le t h a l i t y of 2 , 3 , 7 , 8 - T C D D b y the dermal route. Schwetz et al. (1973) d e t e r m i n e d t h a t the a c u t e LD50 o f 2 , 3 , 7 , 8 - T C D D in a c e t o n e w a s 275 /xg/kg (range, 142 to 5 3 1 /xg/kg) in N e w Z e a l a n d w h i t e r a b b i t s . A s w a s o b s e r v e d a f t e r o r a l administration, the time to death was protracted, with deaths observed b e t w e e n 12 and 22 days after treatment. 4.3.2 Systemic/Target Organ Toxicity 4.3.2.1 Chloracne Inhalation. Pertinent studies regarding the development of chloracne by humans or animals following inhalation exposure were not found in the available literature. Oral, human. No studies are available. ( *0 iP Animal. Greig (1984) observed typical skin lesions in male and female hairless A2G-hr/+ mice 4 weeks after a single gavage a d m i n i s t r a t i o n o f 2 , 3 , 7 , 8 - T C D D a t 75 /xg/kg. T h e f e m a l e s a p p e a r e d to b e m o r e s e v e r e l y a f f e c t e d than the males. M c C o n n e l l et al. (1978) o b s e r v e d acneform eruptions in rhesus monkeys given a single dose of 2,3,7,8-TCDD a t 7 0 /xg/kg. T h i s d o s e w a s s e v e r e l y toxic, r e s u l t i n g in m a n y g r o s s e ffects and ultimately death. Longer-term exposure of monkeys to 2,3,7,8-TCDD in the diet at 500 ppt (0.0094 to 0.014 /xgAg/day) for 9 m o n t h s also prod u c e d lesions that resembled chloracne (Allen et al. 1977). This dose was not a threshold for the formation of chloracne, since the lesions were severe and associated with other dermal effects such as subcutaneous edema and loss of facial hair. In addition, this AQ 43 level of 2,3,7,8-TCDD resulted in the deaths of 5 of 8 animals tested. Dermal, human. Since the turn of the century, chloracne has been observed in humans after a few days from the time of accidental exposure to a variety of chlorinated aromatic compounds. The prevalence of these lesions has bee n reviewed by Taylor (1979) and Suskind (1985). 2,3,7,8TCDD is known to be one of the most potent compounds in producing chloracne; however, sufficient data on exposure are not available to define the doses n e c e s s a r y to produce this lesion. It is k n o w n that chloracne appears prior to any other visible effects related to 2,3,7,8-TCDD exposure. The lesions usually appear on the face and upper trunk area. These lesions can be very disfiguring and can be persistent, lasting many years after exposure. After the Seveso accident, children appeared to develop chloracne more frequently than adults, but this may have b een related to greater exposure to -contaminated soils during play rather than to a greater sensitivity In the young. Additional signs of toxicity have been reported in case studies of small groups of people exposed to 2,3,7,8-TCDD. These signs include aching muscles, loss of appetite, weight loss, digestive disorders, easy fatigability, insomnia, loss of libido, headache, neuropathy, sleep disturbance, sensory changes, and uncharacteristic bouts of anger (Poland et al. 1971, Bauer et al. 1961, Kimmig and Schulz 1957, Schulz 1957 ^'Oliver 1975). Many of these symptoms are commonly observed with __ a c u t e e x p o s u r e t o c h e m i c a l s , a n d s i n c e t h e e x p o s u r e t o 2 , 3 , 7 , 8 - T C D D is always a mixed exposure, with 2,3,7,8-TCDD being only a minor component (on a percentage b a s i s ) , it is difficult to state w i t h certainty that these symptoms are produced from 2,3,7,8-TCDD. The other chemicals that provide concomitant exposure not only include the chlorinated compound o f w h i c h 2 , 3 , 7 , 8 - T C D D is a contaminant, but, in m a n y cases, such as w i t h h e r b i c i d e use, p e t r o l e u m solvents as well as ot h e r "inert ingredients" used in the formulation of the final product. Dermal, animal. Puhvel et al. (1982) app l i e d 0.1 p g of 2,3,7,8T C D D t o t h e s k i n o f h a i r l e s s S k h : H R - l m i c e 3 t i m e s / w e e k f o r 4 w e e k s , -- T h e ----m i c e d e v e l o p e d sk i n lesions w h i c h a p p e a r e d on h i s t o l o g i c e x a m i n a t i o n to resemble chloracne. Changes in the skin included hyperkeratinization, hyperplasia, absences of sebaceous glands, and buildup of keratin into dermal cysts. 2,3,7,8-TCDD was the most effective agent of the seven tested in inducing this dermal response, with the next most effective c o m p o u n d , 3 , 4 , 3 ' , 4 ' - t e t r a c h l o r o b i p h e n y l , i n d u c i n g a r e s p o n s e a t 2 0 0 ig per application. The use o f a single dose in this study precludes the determination of a threshold dose. T o t h et al. (1979) a d m i n i s t e r e d 2,3,7, 8 - T C D D to Swiss m i c e b y g a v a g e f o r 1 y e a r a t d o s e s o f 0, 0 . 0 0 7 , 0.7, o r 7 . 0 p g / k g / w e e k . Amyloidosis was observed in the kidney, spleen, and liver, along with dermatitis, in all treatment groups. The dermatitis had some s i m i l a r i t i e s to c hloracne, a l t h o u g h it is n o t c l e a r if the e t i o l o g y is the .same. If this dermat i t i s is similar to chloracne, t h e n the L O A E L in mice would be 0.007 pg/kg/week. G e n e r a l discussion. C h l o racne is a p e r s i s t e n t d e f o r m a t i v e ski n lesion that can be induced by single or multiple exposure to 2,3,7,8T C D D in humans. Chloracne is the first toxic ef f e c t u s u a l l y observed in humans exposed to chemicals contaminated with 2,3,7,8-TCDD and appears ^ 4965 . 44 to be a sensitive toxicological end point. It is believed that humans can develop chloracne following exposure to 2,3,7,8-TCDD by any route, but data are not available regarding the dose necessary to induce chloracne in h u m a n s . Hoses and Prioleau (1985) studied humans who had recovered from chloracne and determined that histologic examination of the skin was incapable of p r o v i d i n g any indication of past exposure to 2,3,7,8-TCDD. O n l y a few e x p e r i m e n t a l animals develop chloracne u p o n ex p o s u r e to compounds that produce this lesion in humans; the limitation of appropriate animal models has resulted in a poor understanding of this lesion and little information on dose-response relationships. In vitro s t u d i e s b y Gree n l e e et al. (1984) u s i n g h u m a n e p i d e r m a l c u l t u r e s suggested that 2,3,7,8-TCDD may affect the receptors for epidermal growth factors, which ultimately results in the typical dermal lesions associated with 2,3,7,8-TCDD exposure. 4.3.2.2 Vastlng syndrome Inhalation. Pertinent data regarding wasting syndrome in humans or animals following inhalation exposure to 2,3,7,8-TCDD were not found in the available literature. Oral, human. No studies are available. Animal. The wasting syndrome has been observed in several species after administration of acute lethal doses of 2,3,7,8-TCDD (EPA 1985a). As described in the above review, body weight will decrease after administration of 2,3,7,8-TCDD, possibly in a biphasic pattern, until death occurs 15 to 30 days after exposure. Studies with pair-fed rats suggest that the wasting syndrome results from 2,3,7,8-TCDD-induced appetite suppression rather than malabsorption or altered food energy u t i l i z a t i o n ( R ozman 1984; S e e f e l d a n d P e t e r s o n 1984; S e e f e l d et al. 1984a,b; K e l l i n et al. 1985; P o tter et al. 1986). Recently, however, Lu et al. (1986) demonstrated that total parenteral nutrition only protected Hartley guinea pigs partially from loss of weight. Treated guinea pigs maintained relatively stable body weight until a few days prior to death, demonstrating that decreased food consumption accounts for most of the observed loss in body weight but that other mechanisms must account for the final decrease in weight and ultimate death. Additional studies in cold-adapted Sprague-Dawley rats, which maintain high levels of food intake after treatment with 2,3,7,8-TCDD, support the observation that decreased food consumption only partially accounts for the wasting syndrome, since the cold-adapted rats lost weight twice as fast as rats treated at normal temperatures (Rozman and Greim 1986). Rhesus monkeys and Hartley guinea pigs also lose body weight after p r o l o n g e d exposure to diets conta i n i n g 2 , 3 , 7 , 8 - T C D D (Allen et al. 1977, D e C a p r i o et al. 1986). Weight loss was -20% in female monkeys Ingesting 2,3,7,8-TCDD at -0.01 pg/kg/day (the only dose level studied) for 9 months. Only eight monkeys were tested and five died at this dose. Guinea pigs (10 males and 10 females) maintained on diets for 90 days wh i c h provided doses of 2,3,7,8-TCDD of 0.0049 pg/kg/day had a 15% decrease in body weight, whereas guinea pigs given doses of 0.026 /ig/kg/day h a d a 40% decrease in b ody weight. All guinea pigs in the high-dose group died or were killed (when moribund) before the end of 45 the study. The 0 . 0 0 0 6 1 -/ig/kg/day level was a no-effect level. It appears that even in long-term studies, the severe loss of body weight, c h a r a c t e r i s t i c of exposure to 2 , 3 , 7 , 8 - T C D D , is associated w i t h dose levels that are lethal to the animal. Dermal. Pertinent data regarding the wasting syndrome in humans or animals exposed dermally to 2,3 , 7 , 8-TCDD were not found in the available literature. G e n e r a l discussion. The w a s t i n g syndrome is a charac t e r i s t i c sign of 2 , 3 , 7 , 8-TCDD toxicity in experimental animals. This syndrome is o b s e r v e d in b o t h acute and l o n g e r - t e r m studies and is most c o m m o n l y associated with lethal doses. The wasting syndrome has not been observed in humans. Since the m e c h a n i s m is n o t c l e a r l y understood, it remains to be determined whether the severe body weight loss associated with 2,3,7,8TCDD toxicity is the cause of the subsequent death of the animal or an effect that can be segregated from lethality. Mechanisms have been proposed by Aust (1984) which suggest that the effect of 2,3,7,8-TCDD on the thyroid results in activation of thyrotropin-releasing hormone, which has an anorectic action, and, in conjunction with 2,3,7,8-TCDDinduced vitamin A depletion, results in loss of body weight. Regardless of t h e - m e c h a n i s m or length o f exposure, the w a s t i n g syndrome appears to b e afT i n d i c a t o r o f i m p e n d i n g d e a t h r a t h e r t h a n a n e a r l y s i g n o f toxicity. No reports of abnormal weight change as a result of 2,3,7,8TCDD exposure in humans were found. 4.3.2.3 Hepatic effects Inhalation. Pertinent data regarding hepatotoxicity in humans or animals exposed by inhalation to 2,3,7,8-TCDD were not found in the available literature. Oral, human. No studies are available. Animal. The acute administration of 2,3,7,8-TCDD to rats and mid e results in toxic effects in the liver, whereas no severe changes in the liver are observed u p o n acute admin i s t r a t i o n of 2 , 3 , 7 , 8 -TCDD to guinea pigs and monkeys (EPA 1985a). Lesions in rodents consist of necrosis, proliferative changes, cellular membrane alterations, bile duct proliferation, altered lipid metabolism, and excess amounts of porphyrin. These liver effects have generally been observed following a s i n g l e e x p o s u r e at r e l a t i v e l y h i g h d o s e s o f 5 to 2 0 0 /jg/kg> Turner and Collins (1983), however, did observe morphologic changes in a small number of guinea pigs (groups of 1 male and 4 to 6 females, strain not reported) given a single gavage dose of 2 , 3 , 7 , 8 -TCDD at 0.1, 0.5, 2.5, 12.5, o r 20 /jg/kg- C h a n g e s i n c l u d i n g h y p e r t r o p h y , s t e a t o s i s , focal necrosis, and hyalin-like bodies were reported in all guinea pigs, although no indication of an association between dose and increased severity or incidence was reported. Deaths were reported for doses >0.5 a n d 1 2.5 fig / k g in m a l e s a nd females, r e s p e c t i v e l y . In a 9 0 - day f e e d i n g study with Hartley guinea pigs, similar mild liver changes were observed in animals maintained on diets that provided doses of 0.0049 ng/kg/day, liver changes were not observed at the next lower dose of 0.00061 M g / k g / d a y (DeCaprio et al. 1986). A l t h o u g h c h r o n i c s tudies are n o t 46 available in guinea pigs, studies reviewed by EPA (1985a) in rats (Kociba et a l . 1979, NTP 1982a) and mice (NTP 1982a) indicated that doses that represent a LOAEL of -0,001 ^g/kg/day for 2 years are also hepatotoxic. Dermal, human. Studies of the hepatotoxic effects of 2,3,7,8-TCDD on humans have been conducted on populations exposed to herbicides and other industrial chemicals contaminated with 2,3,7,8-TCDD. In these stu d i e s , it is c o n s i d e r e d t h at the p r e d o m i n a n t r o u t e o f e x p o s u r e is dermal, although some exposure by the oral and inhalation routes would also be expected. In addition, all individuals studied were exposed to multiple chemicals, which may have contributed to or been the cause of the effects observed. Reports reviewed by EPA (1985a), May (1973), Holden (1979), Bogen (1979) , and Holmstedt (1980) have reported "liver dysfunction" as one of the symptoms in workers and Vietnam veterans exposed to the herbicide 2,4,5-T contaminated with 2,3,7,8-TCDD. Since these were essentially case reports, no firm association'between exposure and effect could be m a d e . A more complete clinical study of the levels of serum liver enzyme was c onducted in children ages 6 to 10 who were potentially exposed to 2 , 3 , 7 , 8 - T C D D in the Seveso accident (Mocarelli et al. 1986). The children were examined yearly for 6 years following the accident. During the initial 2 years, elevated serum levels of gamma-glutamyltransferase (GGT) and alanine aminotransferase (ALT) were observed in both boys and girls who resided in the most highly contaminated area. The increase was slight, with a total incidence of 4.3% compared to 3.6% in the c o n t r o l group. V a l u e s r e t u r n e d to n o r m a l a f t e r 2 years. Ideo et al. (1982) reported elevated urinary D-glucaric acid in the urine of children in the Seveso area -2.5 years after the accident. They also noted that there was a strong correlation between glucaric acid levels and the activity of hepatic microsomal e n z y m e s . No liver e f f ects, as indicated by serum enzyme levels, however, were observed by Falk et a l . (1984) in a pilot epidemiologic study of 122 persons exposed in Missouri to chemicals contaminated with 2,3,7,8-TCDD. -- Liver involvement, as indicated by porphyria cutanea tarda, has also b e e n reported to be associated with 2,3,7,8-TCDD exposure. The major studies have been reviewed by Jones and Chelsky (1986), who reported that in all cases the porphyria cutanea tarda may not have been definitively diagnosed or that the other chemicals to which subjects were exposed may have been the causative agents. The pilot epidemiology study by Falk et al. (1984) of the Missouri accident failed to detect porphyria cutanea tarda in a population with known exposure. Dermal, animal. No studies are available. General discussion. Hepatotoxic effects have been investigated in h u m a n populations exposed to chemicals contaminated with 2,3,7,8-TCDD because of the known hepatotoxicity of 2,3,7,8-TCDD in rodents. Results of these studies have generally been inconsistent with regard to the detection of altered liver serum enzymes or porphyria cutanea tarda. These inconsistencies may be related to the populations studied, the level of contamination, or the bioavailability of the 2,3,7,8-TCDD under the specific conditions of the contaminated site. These confounding f a c t o r s , along with the ubiquitous nature of and causative agents for 47 slight changes in liver function, have made confirmation of 2,3,7,8TCDD-induced adverse effects on the liver in humans impossible. In rats and mice, 2,3,7,8-TCDD clearly produces adverse effects on the liver; however, there are no studies available that investigated the doser e s p o n s e relationship; thus, it is n ot k n o w n if initial changes in the liver are a first toxic manifestation of 2,3,7,8-TCDD exposure or an effect observed only after other toxic effects in other organ systems have been manifested. Although Turner and Collins (1983) reported mild liver effects in g u i n e a pigs (a species g e n e r a l l y resistant to 2 , 3 , 7 , 8 - T C D D - i n d u c e d liver d a m a g e ) a t d o s e s as l o w as 0 . 1 /ig/kg, the l a c k o f a s s o c i a t i o n b e t w e e n increasing dose and increasing severity of effect in this study makes it difficult to determine a threshold dose. Observing similar liver changes in a subchronic study in guinea pigs and in chronic studies in rats and mice, however, supports the observation of the acute study that hepatotoxicity is a sensitive indicator of 2,3,7,8-TCDD toxicity. Additionally, induction of hepatic microsomal enzymes has often been associated with exposure of laboratory animals to 2,3,7,8-TCDD. Early studies by Buu-Hoi et al. (1972) suggested that the toxicity of 2.3.7.8- TCDD was related to altered enzyme function. Later studies, however, have demonstrated that 2,3,7,8-TCDD toxicity appears in many cases-"to segregate w i t h induction of enzyme activity rather than be the cause of the toxic response. This has been demonstrated for DTdiaphorase (Beatty and Neal 1976) and for mixed function oxidase (Poland et al. 1979, Greig 1979, Ko c i b a and Schwetz 1982, P o l a n d a n d Knu t s o n 1982). In addition, the toxicity of structurally related chlorinated dibenzo-p-dioxins correlates well with the relative ability of these compounds to induce enzyme activity (Poland and Glover 1973). 4.3.2.4 Immunotoxicity Inhalation. Pertinent data regarding immunotoxic effects of 2.3.7.8- TCDD following inhalation exposure of humans or animals were_not_ found in the available literature. Oral, human. No studies are available. Oral, animal. 2,3,7,8-TCDD has been extensively investigated for immunotoxicity in mice, rats, and guinea pigs, and as indicated in the r e v i e w by EPA (1985a) and K n u t s e n (1984), this is a sensitive end point of toxicity. Most studies were conducted with weekly exposures for durations of between 4 and 8 weeks, and minimum effective doses ranged f r o m 1 (ig / k g / w e e k f o r m i c e t o 5 / x g / k g / w e e k f o r r a t s , a n d 0 . 0 4 x g/kg/week guinea pigs. As the study by Vos et al. (1973) indicates, the Hartley guinea pig may be the most sensitive species, with a LOAEL of 0.04 Mg/kg/day and a NOAEL of 0.008 ^g/kg/week.. In addition to species differences, strain differences in sensitivity to the immunotoxic effects of 2,3,7,8-TCDD have been observed in mice, with immunotoxic sensitivity segregating with the Ah locus. Segregation of immunotoxicity has also been demonstrated in fetal thymus organ cultures in vitro (Dencker et al. 1985). Thymus cultures from C57B1/6 mice, w h i c h are Ah r e s p o n s i v e , w e r e v e r y s e n s i t i v e t o t h e t o x i c i t y o f 2 , 3 , 7 , 8 - T C D D ( E C 50 of 1 0 * M) c o m p a r e d w i t h n o o b s e r v e d e f f e c t s o n t h y m u s c u l t u r e s f r o m t h e n o n r e s p o n s i v e D B A / 2 J m o u s e a t 3 x 1 0 " M, t h e h i g h e s t c o n c e n t r a t i o n TP 1 o -*0 48 tested. Effects of 2,3,7,8-TCDD on the immune system included decreases in thymus weight, sensitization to antigens (bacterial antigens, skin grafts, and tumor cell development), serum immunoglobins, and B but not T lymphocytes. T h i g p e n et al. (1975) d e m o n s t r a t e d that C 5 7 B L / 6 J F h m i c e that received as little as 1 ag/kg of 2 , 3 , 7 ,8-TCDD once a w e e k for 4 weeks were more susceptible to death from subsequent bacterial infection. This dose of 2,3,7,8-TCDD did not result in any gross signs of toxicity, suggesting that host susceptibility was an early effect of 2.3.7.8- TCDD exposure. Immunotoxic effects have also been reported in pups of Fischer rats and B6C3F1 mice following in utero exposure and postnatal exposure through lactation (Luster et al. 1982). The effective doses on a maternal weight basis were approximately the same as those that produced effects in adults. The study b y Greenlee et al. (1985), using thymic epithelium cell cultures, provides evidence that 2,3,7,8TCDD acts directly on the epithelium cells b y inhibiting the maturation of the thymocytes. Longer-term studies of the effect of 2,3,7,8-TCDD on the immune system are not available. Dermal, human. T h e r e is little i n f o r m a t i o n o n the immunotoxic effects of 2,3,7,8-TCDD in humans exposed to herbicides or other chemicals contaminated with 2,3,7,8-TCDD. In a pilot epidemiologic study of 82 high-risk and 40 low-risk subjects from areas in Missouri where 2.3.7.8- TCDD exposure occurred, Stehr et al. (1986) failed to detect any signs of-Immunotoxicity. In a study of 154 exposed and 155 unexposed persons in Gray Summit, Missouri, H offman et al. (1986) reported that the exposed group had an increased frequency of energy (11.8% vs 1.1%) and relative energy (35.3% vs 11.8%). The exposed group also h ad non- statistically significant increased frequencies of abnormal T-cell subset test results (10.4% vs 6.8%), a T4/T8 ratio of <1.0 (8.1% vs 6.4%), and an abnormality in the functional T-cell test results (12.6% vs 8.5%). Although the effects have not resulted in an excess of clinical illness in the exposed group, these data suggest that exposure to 2,3,7,8-TCDD may be associated with depressed cell-mediated Immunity; however, corroborative studies will be carried out. .... _ Dermal, animal. No studies are available. General discussion. There are abundant animal data that indicate that immunotoxicity may be one of the most sensitive toxicologic end points for 2,3,7,8-TCDD. There have been very limited investigations of this end point in humans; thus, the importance o f 2,3,7,8-TCDD-induced immunotoxicity in humans cannot be evaluated. The time of onset, the duration of the altered immune response, and the fact that evaluation of a l t e r e d immune r e sponse is a s p e c i a l i z e d an a l y s i s n o t r o u t i n e l y performed in clinical settings will make the assessment of this end point in humans difficult. T h e a n i m a l data, s h o w that 2 , 3 , 7 , 8 - T C D D p r o d u c e s i m m u n o l o g i c e f f e c t s in a number of species, and most of the studies have been concerned with the Investigation of alterations induced in the immune system. There are less data available regarding dose-response relationships and species and strain differences in sensitivity; these data would be of assistance in evaluating response with regard to human health. Also, no data are 4970 49 available on the immunotoxicity of 2,3,7,8-TCDD in monkeys, a species that appears to be very sensitive to other toxic effects of 2,3,7,8TCDD. 4.3.3 Developmental Toxicity 4.3.3.1 Inhalation Pertinent data regarding the developmental toxicity of 2,3,7,8-TCDD following inhalation exposure of humans or animals were not found in the available literature. 4.3.3.2 Oral Human. No studies are available. Animal. 2,3,7,8-TCDD has been extensively studied for developmental toxicity, and these studies, reviewed in EPA (1985a), indicate that 2,3,7,8-TCDD is teratogenic in mice and rats after gavage administration. In mice, the most commonly observed developmental . e f fects wer e h y d r o n e p h r o t i c k i d n e y a n d cleft p a l a t e (Moore et al. 1973, Neubert and D i l l m a n n 1972, Smith et al. 1976, Courtney 1976). Both of these anomalies have been observed at doses as low as 1 pg/kg (Courtney, 1976^-Smith et al. 1976), w i t h the kidney effects obse r v e d after only a single exposure on day 10 of gestation. In rats, gavage administration of 2,3,7,8-TCDD during organogenesis at doses of -0.125 to 0.25 pg/kg produced hemorrhage of internal organs and subcutaneous edema (Sparschu et al. 1971a,b; K h e r a and R u d d i c k 1973). No effects w e r e observed at 0.3 and 0.03 pg/kg, respectively, in NMRI mice and Sprague-Dawley rats. These teratogenic effects have been confirmed b y a number of studies in which 2,3,7,8-TCDD was administered subcutaneously (studies summarized in EPA 1985b). It has been demonstrated that both genetic susceptibility and concomitant exposure to other compounds affect the developmental toxicity of 2,3,7,8-TCDD. Poland and Glover (1980) and Dencker and Pratt (1981) demonstrated genetic differences in the susceptibility of mice; only responsive C57B1/6J mice developed the characteristic cleft palate and hydronephrotic kidneys after treatment. This indicates that developmental toxicity, in addition to many other toxicological end points of 2,3,7,8-TCDD, segregates with the Ah locus. Additionally, it was demonstrated that simultaneous exposure to 2,3,7,8-TCDD and specific p o l y c h l o r i n a t e d b i p h e n y l s ( B i m b a u m et al. 1985), or to the hormones hydrocortisone ( B i m b a u m et al. 1986) or thyroxine and triiodothyroxine (Lamb et al. 1986), increases the sensitivity of mice to the developmental effects of 2,3,7,8-TCDD. An additive effect was observed b y Weber et al. (1985) for simultaneous exposure to 2,3,7,8-TCDD and 2,3,7,8-tetrachlorodibenzofurans (TCDF). 4.3.3.3. Dermal Human. The EPA (1985a) evaluated several epidemiology studies of human populations exposed to herbicides contaminated with 2,3,7,8-TCDD. 50 It is assumed that the exposure in these studies w as predomi n a n t l y dermal; however, some oral and inhalation exposure was also likely. The studies reviewed were predominantly geographic correlation studies that reported elevated incidence of birth defects, including .cleft palate, cleft lip, neural-tube defects, he a r t abnormalities, hypospadias and epispadias, talipes, and cystic kidney disease, as well as increases in stillbirths (EPA 1979, Hanify et a l . 1981, Field and Kerr 1979). Other similar studies reviewed failed to demonstrate a correlation between birth defects and possible exposure to 2,3,7,8-TCDD (Nelson et al. 1979, T h o m a s 1980, Dept, o f H e a l t h N e w Z e a l a n d 1980, M c Q u e e n et al. 1977, A l d r e d 1978, Smith et al. 1982, B o n a c c o r s i et al. 1978, R e g g i a n i 1980, B i s a n t i et al. 1980). In addition, a case c o n t r o l s t u d y of the o f f s p r i n g of Vietnam veterans potentially exposed to 2,3,7,8-TCDD in Agent Orange detected increases in birth defects that included spina bifida, cleft palate, and certain congenital tumors (Erickson et al. 1984). W h e n all types of defects were combined, there was no elevated risk, and authors noted that the seemingly higher risk for individual birth defects may have resulted from chance or other unidentified risk factors. As a result of the inherent uncertainty in interpreting geographic correlation studies, the concomitant exposure to other potentially active compounds, and the review of similar studies that provided negative resu l t s , it was concluded that these investigations could neither prove nor disprove the hypothesis that 2,3,7,8-TCDD was a teratogen in humans. Animal. No studies are available. 4.3.3.4 General discussion The lack of a clearly defined exposed population, with adequate quantitative data on levels and duration of exposure, and the confounding presence of exposure to other chemicals that may in themselves be developmental toxicants, have made results of epidemiology data difficult to evaluate and inadequate for determining whether 2,3,7,8-TCDD induces developmental defects in humans. As with many developmental toxicants, the animal data indicated that the time of exposure was critical for the induction of effects, with treatment on days 8 or 11 producing maximal effects and treatment after 13 days being ineffective in mice (Neubert et al. 1973). A d d i t i o n a l u n c e r t a i n t i e s regarding quantitative data for the effects of 2,3,7,8-TCDD on development result from the lack of experimental data for the guinea pig, w h i c h is k n o w n to be extremely sensitive to the acute toxic effects of 2,3,7,8-TCDD, and for the monkey in w h i c h 2,3,7,8-TCDD is a p o t e n t fetotoxic agent. 4.3.4 Reproductive Toxicity 4.3.4.1 Inhalation Pertinent data regarding the reproductive toxicity of 2,3,7,8-TCDD in humans or animals following inhalation exposure were not found in the available literature. 51 4.3.A.2 Oral Human. No studies are available. Animal. The fetotoxicity of 2,3,7,8-TCDD has been demonstrated following short-term exposure in utero (rats and mice) as well as in a multigeneration study (rats). Fetal death and vaginal bleeding have been observed in developmental toxicity studies [reviewed by EPA (1985a)] at doses of between 2 and 9 ^g/kg/day. The three-generation study of Murray e t al. (1979) r e ported that d i e t a r y admini s t r a t i o n of 2 , 3 , 7 , 8 - T C D D at 0.01 and 0.1 /jg/kg/day resulted in adverse effects on litter size, fetal survival, and neonatal survival in Sprague-Dawley rats. The 0.1/xg/kg/day dose also resulted in a significant decrease in fertility. M u r r a y et al. (1979) consid e r e d the low dose of 0.001 p g / k g / d a y to be without substantial effects, since the only effect observed was an increase in dilated renal pelvises in the Fl generation. This effect was not observed at statistically significant levels in any other generation. The absence of effects at this lower dose level has been questioned by Nisbet and Paxton (1982), who reevaluated the data statistically and concluded that gestational index, decreased fetal weight, and the incidence of dilated renal pelvis were all increased at both the 0.01- and 0.001-/ig/kg/day doses. The analysis by Nisbet and Paxton (1982) indicates that reproductive performance and the fetus are very.__sensitive to the toxic properties of 2,3,7,8-TCDD. The monkey, however, appears more sensitive to the toxicity of 2,3,7,8-TCDD than either rats or mice. McNulty (1984, 1985) described the common occurrence of abortion in rhesus monkeys treated with a total o f 1 /xg/kg of 2 , 3 , 7 , 8 - T C D D o v e r d ays 20 to 4 0 of g e s t a t i o n ; a n d the lowest total dose tested, 0.2 pg/kg, produced abortion in 1 of 4 test monkeys. In fetuses examined, there was either only minimal indication (palatal abnormalities) or no indication of developmental toxicity. In an earlier feeding study, groups of eight monkeys were maintained on diets containing 50 or 500 ppt (the total dose ingested was 1.8 and 11.7 Hg, respectively) of 2 , 3 , 7 , 8 - T C D D for 7 months p r i o r to p r e g n a n c y and du r i n g pregnancy (Allen et al. 1979). In both groups, two-thirds of the pregnancies ended in abortion. As reported in EPA (1985a), chronic exposure of rats to 2,3,7,8TCDD in the diet results in a decrease in the weight of male r e p r o ductive organs. It was suggested b y Moore et al. (1985) that this effect on the reproductive organs might account for the reduced male Wistar rat reproductive performance following 2,3,7,8-TCDD exposure, which had been earlier observed by Khera and Ruddick (1973). Levels of circulating androgens in 2,3,7,8-TCDD-treated male rats were studied by M o o r e et al. (1985) and shown to b e d e p r essed as a r e s u l t o f treatment. It was speculated that these depressed hormone levels may participate in the reproductive dysfunction in males. Circulating estradiol levels in pregnant females, however, appear not to be affected by 2,3,7,8-TCDD exposure, although 2,3,7,8-TCDD does effect some estrogen-metabolizing pathways when studied in vitro (Shiverick and Muther 1983) . 4973 52 A.3.A.3 Dermal Human. Epidemiology studies have been performed on groups of individuals exposed to herbicides or industrial chemicals contaminated w i t h 2,3,7,8-TCDD. Alth o u g h the dermal route is considered to be the predominant route of exposure in these studies, some exposure by other routes would also occur. These studies, which include those reviewed by EPA (1985a) and Friedman (198A) and that of Forsberg and Nordstrom (1985), were conducted in groups of male herbicide application workers, chemical plant employees, and soldiers exposed to Agent Orange in Vietnam, and to both male and females exposed through major industrial accidents or inappropriate disposal, which permitted the escape of large amounts of chemical from production plants. Studies of these groups have not clearly demonstrated that 2,3,7,8-TCDD produced any adverse effects on reproductive performance, although as a result of the limitations of the study, particularly w i t h regard to the extent of exposure, it is not possible to interpret the results as indicative of the absence of r e p r o d u c t i v e t o x i c i t y f o r 2 , 3 , 7 , 8 ;T C D D i n humans. Animal. No studies are available. A.3.A.A General discussion The evidence that 2 , 3 , 7 , 8-TCDD is a reproductive toxicant in humans is l i m i t e d b y the l a c k o f e x p o s u r e d a t a a n d the c o n c o m i t a n t e x p o s u r e to other chemicals that may have been biologically active. The greatest exposure to 2,3,7,8-TCDD occurred in male herbicide sprayers, soldiers, and chemical plant w o r k e r s , whereas the studies conducted have been concerned mostly with fetotoxicity and spontaneous abortion. Although ^the possibility of pre-mating exposure in males resulting in f e t o t o x i c i t y a n d a b o r t i o n is of concern, this e n d p o i n t is d i f f i c u l t to a s s e s s , particularly when the exposure is temporally removed from the time of conception. In addition, there is a lack of data on the functioning of male reproductive organs during the time of potential exposure to 2,3,7,8-TCDD. There are no equivalent female populations _ studied that have had extended periods of high exposure to chemicals contaminated wit h 2,3,7,8-TCDD, and only limited populations [females in the v i c i n i t y o f Seveso (Bisanti et al. 1980, B o n a c c o r s i et al. 1978, Reggiani 1980) in the period immediately after the accident] from which to assess effects on reproduction in females exposed while pregnant. Given the above limitations, the present epidemiology studies are only consistent with observations in animals that 2,3,7,8-TCDD elicits adverse effects on reproduction; they do not provide sufficient evidence to p r o v e that this chemical is a reproductive toxicant. Animal studies i n d i c a t e tha t 2 , 3 , 7 , 8 - T C D D is a p o w e r f u l repr o d u c t i v e toxicant; however, differences in species sensitivity have been observed. The available data indicate that the monkey may be the most sensitive species, but tests in this species have been limited to rhesus monkeys, with only a few animals studied at any dose level, which provides insufficient data to fully evaluate the range and extent of potential toxic effects in this species. In addition, the sensitivity of humans compared to monkeys, rats, or mice can only be speculated. 53 4.3.5 Genotoxicity 4.3.5.1 Human Although there have b e e n no studies of hu m a n populations exposed to 2,3,7,8-TCDD alone, there are a number of studies on human populations exposed to chemicals contaminated w ith 2,3,7,8-TCDD, as reviewed in EPA (1985a). Czeizel and Kiraly (1976) reported that there was an increase in chromosomal aberrations of peripheral lymphocytes from workers in an East European 2,4,5-trichlorophenoxyethanol plant. Studies of soldiers (spouses and children of soldiers) exposed to Agent Orange (Mulcahy 1980) and of individuals involved in the Seveso accident (Reggiani 1980, M o t t u r a et al. 1981, D i L e m i a et al. 1982, Tenchini et al. 1983, Kaye et al. 1985) have, however, failed to detect chromosomal aberrations. Some of the individuals in these studies had skin eruptions consistent with exposure to 2,3,7,8-TCDD. The only chromosomal effect in the latter studies was a decrease in satellite association (SA), which is evidence of functional ribosomal genes, in 2,3,7,8-TCDD-exposed subjects ( D i L e m i a et al. 1982). The authors n o t e d that similar decreases observed after x - i r r adiation exposure may poss i b l y represent damage to the nucleolar organizing regions. All human data on chromosomal aberrations were confounded by mixed exposure to other potentially active^, com p o u n d s a n d the ina b i l i t y to d e t e r m i n e q u a n t i t a t i v e l y the extent of exposure to 2,3,7,8-TCDD. 4.3.5.2 Nonhuman A recent review by Giri (1986) of the mutagenic and genotoxic effect of 2,3,7,8-TCDD concluded that there is evidence for the genotoxic activity of 2,3,7,8-TCDD, but additional testing would be required to demonstrate this activity with certainty. The results of nonhuman genotoxicity studies are summarized in Tables 4.2 and 4.3. The e a rly positive results of H u s s a i n et al. (1972) and Seiler (1973) are likely artifacts resulting from extensive cell death and possibly from impurities in the test material. In vitro cytogenic tests in yeast in both the standard plate test and the intrasanguineous host-mediated assay (using CD-I mice) have produced positive results (Bronzetti et al. 1983). In in v i v o cytogenetic assays, Loprieno et al. (1982) observed that the positive responses depended on sampling time, with negative results obtained 24 h posttreatment and positive results 96 h posttreatment. Although the toxic properties of 2,3,7,8-TCDD usually s e gregate w i t h the A h locus, Meyne et al. (1985) o b s e r v e d negative responses in vivo after administration of 2,3,7,8-TCDD at hepatotoxic levels to both responsive, C57B1/6J, and nonresponsive, DBA/2J, mice w h e n s a m p l e d at 24 h. T h e r e are also l i m i t e d s u p p o r t i v e d a t a for the mutagenicity of 2,3,7,8-TCDD from the observation in vivo of the lowlevel binding of 2,3,7,8-TCDD to liver macromolecules (Poland and Glover 1979). 4.3.5.3 General discussion The nonhuman genotoxicity data on 2,3,7,8-TCDD are conflicting; negative results were reported in many of the assay systems, and when positive results were observed, the response was generally weak. These I S, % End point Table 4.2. Genotoxicity of 2,3,7,8-TCD D in vitro Species (test system) 1, Results with activation/w ithout activation References Gene mutation Salm onella typhim urium (reverse mutation) Cytogenetic Cell transform ation S. typhim urium (reverse mutation) 1 Escherichia coli (reverse m utation) Saccharom yces cerevisiae (reversion) L5I78Y mouse lymphoma cells (forward m utation) S. cerevisiae (gene conversion) S. cerevisiae (host mediated) Chinese ham ster cells (sister chromatid exchange) Baby hamster kidney cells BHK C 3 H /I 0 T I /2 cells , Not tested/+ N ot te s te d /4- + /~ t Not tested /+ , and not tested/ -- +/+ /N A " Not tested/ -- Not tested/+ Not tested/ -- M cCann 1978, G ilbert et al. 1980, Geiger and Neal 1981, Mortelmans et al. 1984 Hussain et al. 1972, Seiler 1973 Hussain et al. 1972 Bronzetti et al. 1983 Rogers et al. 1982 Bronzetti et al. 1983 Bronzetti et al. 1983 Toth et al. 1984 H ay 1982 Abernathy et al. 1985 "Not available. CD -3 I ) \ 1. Table 4.3. G enotoxicity o f 2,3,7,8-TC D D in vivo End point Species (test system) Results References Gene mutation Cytogenetic D rosophila (sex-linked recessive lethal) D rosophila (sister chromatid exchange) D rosoph ila (structural aberration) Rat (sister chromatid exchange) R ats - marrow cells (structural aberration) R ats - marrow cells (structural aberration) Mouse - marrow cells (structural aberration) Mouse - marrow cells (sister chromatid exchange) Mouse - marrow cells (structural aberration) Mouse - marrow cells (m icro n u cleu s) tO' a!JE . I Zim m ering et al. 1985 -- Zeiger 1983 -- Zeiger 1983 -- Lundgren et al. 1986 -- Green and M oreland 1975 + Green et al. 1977 + Loprieno et al. 1982 -- M cyne et al. 1985 -- Meync et al. 1985 Meync el al. 1985 " Ln Ui 56 conflicting data may result from technical difficulties in testing 2.3.7.8- TCDD rather than from a lack of biological activity. Testing difficulties arise from the extreme insolubility of this compound and the high toxicity observed in some test systems, which would be anticipated to result in a very narrow window for effective genotoxic doses. As a result of the largely negative data from nonhuman genotoxicity assays, some investigators, as discussed by Paustenbach et a l . (1986), have concluded that 2,3,7,8-TCDD is not a genotoxic agent. Sufficient data are not available to precisely define the toxicological mechanism of 2,3,7,8-TCDD and resolve these differences of opinion. Human studies are primarily limited b y the lack of data on the extent of exposure. The only indication of exposure in these studies was the development of gross skin lesions, which does not provide a good estimate of either the extent of exposure or the duration. In addition, the time after exposure that the cells were examined may not have been optimal for observing cytogenetic effects. Because of the limitations of the studies in humans, these data cannot be used to demonstrate that 2.3.7.8- TCDD does not pose a genotoxic hazard. A.3.6 Carcinogenicity A.3.6.1 Inhalation _ Pertinent data regarding the carcinogenicity of 2,3,7,8-TCDD following inhalation exposure of humans or animals were not found in the available literature. A.3.6.2 Oral, human. No studies are available. Animal. A number of bioassays have been conducted, and all have demonstrated that 2,3,7,8-TCDD is carcinogenic in animals via the oral route. The N T P (1982a) and Kociba et al. (1978a,b) studies are key bioassays of 2,3,7,8-TCDD. Although both studies used sufficient numbers of animals and exposure durations, the s t u d y b y K o c i b a et al. ( 1 9 7 8 a , b ) is relevant to h u m a n exposure scenarios b e c a u s e the compound was administered daily in the diet rather than biweekly by gavage as in the NTP (1982a) study. Furthermore, the study b y K o c i b a et al. (1978a,b) has proven to be the most sensitive indicator of the carcinogenic potency of 2.3. 7 . 8 - TCDD. The details of the Ko c i b a et al. (1978a,b) study are summarized in Table A.A. Other oral studies which support the conclusion t h a t 2 , 3 , 7 , 8 - T C D D is a n a n i m a l c a r c i n o g e n a r e p r e s e n t e d i n T a b l e A . 5. A.3.6.3 Dermal Human. Although there are no known cohorts that have been exposed solely to 2,3,7,8-TCDD, a number of cohorts that have been exposed to herbicides or industrial chemicals contaminated with 2,3,7,8-TCDD have been studied. The predominant exposure route for these cohorts was probably, dermal, although some inhalation and oral exposure was also likely. In addition, 2,3,7,8-TCDD was only a minor contaminant of these c o m pounds, and any effects observed may have been either caused or potentiated by the contaminated compound itself. 57 Table 4.4 Summary of the oral carcinogenicity bioassay of Kociba et al. (1978a,b) Animal Dose tested Sex (M g/kg/day) Tum or type Incidence Sprague- M Control Dawley rats 0.001 0.01 0.1 F Control 0.001 0.01 0.1 Squamous cell carcinoma of the tongue, adenoma of the adrenal cortex, and squamous cell carcinoma of the hard palate Squamous cell carcinoma of the tongue Squamous cell carcinoma of the tongue Adenoma of the adrenal cortex Squamous cell carcinoma of the tongue Adenoma of the adrenal cortex Squamous cell carcinoma of the hard palate Hepatocellular carcinoma Hepatocellular carcinoma Hepatocellular carcinoma Squamous cell carcinoma of the hard palate Hepatocellular carcinoma Squamous cell carcinoma of the hard palate Squamous cell carcinom a of the lung 0/85 1/50 1/50 2/50 3/50 5/50 4 /5 0 1/86 0/50 2/50 1/50 11/49 4/49 7/49 4979 50 fD O Table 4.5. Other oral studies supporting the conclusion that 2,3,7,8-TCDD is an animal carcinogen M ethod of exposure A nim al Sex/ number Doses tested Tum or type References Diet Gavage SpragueDawley rats OsborneMendel rats OsborncMendel rats M/IO M /50 F /5 0 B6C3F1 mice M /5 0 B6C3FI mice F /5 0 Swiss mice M /44 0.01,0.005, 0.05, 0.5, 1.0, or 5 ppb 0.01, 0.05, or 0.5 /ig/kg/w eek 0.01, 0.05, or 0.5 /ig /k g /w eek 0.01, 0.05, or 0.5 /ig/kg/w eek 0.01,0.05, or 0.5 /ig/kg/w cck 0.007, 0.7, or 7.0 /jg/kg/w cek Increase in total tum or incidence Van Miller et al. 1977a,b Follicular-cell adenomas and carcinomas of the thyroid Neoplastic nodules and hepato cellular carcinomas of the liver Hepatocellular carcinomas N TP 1982a N TP 1982a N TP 1982a Hepatocellular carcinoma and follicular-cell adenomas of the thyroid Hepatomas and hepatocellular carcinom as N TP 1982a Toth et al. 1979 4980 59 EPA (1985a) reviewed several epidemiology studies of humans exposed to herbicides contaminated with 2,3,7,8 - T C D D . The studies by Axelson et al. (1980) and Thiess and Frentzel-Beyme (1977) found an association between exposure and stomach cancer, and a series of other studies (Hardell a n d S a n d s t r o m 1979; E r i k s s o n et al. 1979, 1981; H a r d e l l et al. 1980, 1981; Lynge 1985; Palovi et al. 1986) reported an association between exposure and soft-tissue sarcomas (of various sites) and lymphomas. Other studies, however, reviewed by EPA (1985a) and Hiremath et al. (1986), failed to detect an a s s o c i a t i o n b e t w e e n herbicide e x p o s u r e a n d the i n d u c t i o n o f c a n c e r (Ott e t al. 1980; Z a c k and S u s k i n d 1980; Coo k et al. 1980, 1985; C o o k 1981; P a z d e r o v a - V e j l u p k o v a et al. 1981; J o h n s o n et al. 1981; R i i h i m a k i et al. 1982; S m i t h et al. 1983; Wolfe et al. 1984; Smith and Pearce 1985). Fingerhut et al. (1984), Cook e t al. (1986), a n d Ott et al. (1987) also d i d not d etect a n association b e t w e e n h e r b i c i d e exposure and cancer. The extent of exposure to 2.3.7.8- TCDD in some of the epidemiology studies is questionable, and precise quanti f i c a t i o n of exposure is not available for all of the studies. . An i m a l . T h e r e is o n l y l i m i t e d e v i d e n c e that 2 , 3 , 7 , 8 - T C D D p r o d u c e s tumors in laboratory animals following dermal exposure. NTP (1982b) administered 2,3,7,8-TCDD alone to Swiss mice. Female mice, but not male mice^-Fiad an increased incidence of fibrosarcomas in the integumentary system. As r e v i e w e d b y E P A (1985a), there is c o n f l i c t i n g e v idence that 2.3.7.8- TCDD acts as a tumor promotor w h e n applied to the skin. Berry et al. (1978) failed to detect any tumor-promoting activ i t y b y 2,3,7,8-TCDD in CD-I mice skin initiated with dimethylbenzanthracene (DMBA), and Slaga and Nesnow (1985) reported that unpublished data indicated that 2.3.7.8- TCDD either had no promoting activity or very weak promoting activity in Senear mice skin. NTP (1982b) also examined the ability of 2. 3 . 7 . 8- T C D D to act as a tumor prom o t e r in Swiss W e b s t e r mice, and animals t r e a t e d w i t h 2 , 3 , 7 , 8-TCDD alone h a d simi l a r t umor incidences to animals treated first with DMBA followed b y treatment w i t h 2,3,7,8-l'CDDT NTP (1982b) concluded, however, that "in the DMBA-TCDD experiment, failure to have included groups skin painted with only DMBA precludes i n t e r p r e t a t i o n o f these results." P o l a n d et al. (1982), however, not only demonstrated promotion, but also that genetic differences in CD-I mice affect the ability of 2,3,7,8-TCDD to act as a promotor. In HRS/J mice homozygous for the hairless trait, promotion with 2,3,7,8-TCDD after initiation with DMBA produced as many skin tumors (both incidence and multiplicity) as promotion with the kn o w n promotor TPA; whereas in mice heterozygous for the hairless trait (and wild type), skin tumors could only be promoted by TPA but not 2,3,7,8-TCDD. Even in the homozygous mice, 2,3,7,8-TCDD did not produce the commonly observed hyperplasia associated with promotors such as TPA, suggesting that 2.3.7.8- TCDD has a different mechanism of action. Other studies indicated that pretreatment with 2,3,7,8-TCDD could block the subsequent DNA binding of known carcinogens and also prevent tumor initiation (Berry et al. 1979, Cohen et al. 1979). A l t h o u g h not a dermal study, Pitot et al. (1980) also demonstrated prom o t i o n b y administering a single intragastric dose of the hepatocarcinogen diethylnitrosamine (DEN) followed b y repeated subcutaneous injections of 2 ,3,7,8-TCDD. No 60 hepatic tumors were observed in animals given DEN or 2,3,7,8-TCDD alone, but the combined treatment resulted in the development of hepatocellular carcinomas. 4.3.6.4 General discussion T h e e v i d e n c e f r o m h u m a n e p i d e m i o l o g y s t u d i e s that 2 , 3 , 7 , 8 - T C D D is c a r c i n o g e n i c is d i f f i c u l t to as s e s s b e c a u s e (1) e x p osure to 2 , 3 , 7 , 8 - T C D D is p o o r l y d o c u m e n t e d a n d (2) e x p o s u r e o c c u r r e d to o t h e r p o t e n t i a l l y active materials. The strongest evidence is from the induction of softtissue sarcomas at various sites, and it has been questioned whether combining tumor data from various sites is appropriate. With regard to the observed increase in stomach cancer, the two groups of workers studied were relatively small, and similar increases in stomach tumors have not been reported in other more extensive studies. T h e ani m a l data, however, c l e a r l y i ndicate that 2 , 3 , 7 ,8-TCDD is c a r c i n ogenic, a l t h o u g h there is some d i s a g r e e m e n t in the s c i e n t i f i c and international regulatory community as to whether 2,3,7,8-TCDD acts as a c o m p l e t e c a r c i n o g e n or as a c a r c i n o g e n p r o m o t o r (Shu et al. 1987). The rationale for describing 2,3,7,8-TCDD as a prom o t o r is b a s e d on the poor response of 2,3,7,8-TCDD in many short-term mutagenicity assays, the lack of strong evidence for binding to DNA, and positive results in in v i v o p r o m o t i o n assays using the c l assic skin p a i n t i n g technique (as described above) and the study of Pitot et al. (1980) using the twostage model in rat liver. The classic feeding study bioassays, however, w o u l d support the v i e w that 2 , 3 , 7,8-TCDD is a complete carcinogen. 4.4 INTERACTIONS WITH OTHER CHEMICALS There are few data on the interactions of 2,3,7,8-TCDD with other chemicals. As discussed in EPA (1985a), 2,3,7,8-TCDD is a strong inducer of microsomal enzymes; hence, prior exposure to 2,3,7,8-TCDD will alter the rate of metabolism and toxicity of many compounds that are either detoxified or activated by this enzyme system. The observed inhibition -- by 2,3,7,8-TCDD of the tumorigenic response of known tumorigens in the mouse skin b i o a s s a y (Berry et al. 1979, Cohen et al. 1979) m a y be an example of 2,3,7,8-TCDD-induced enzyme changes altering the metabolic fate and toxicity of another compound. The only other interactions that have been observed are the additive effect of 2,3,7,8-TCDD and similar polychlorinated dibenzofurans and polychlorinated biphenyls on the induction of cleft p a l a t e in mice (Weber et al. 1985, B i m b a u m et al. 1985), a n d the increased sensitivity of mice to 2,3,7,8-TCDD-induced cleft palate with the co-administration of the hormones thyroxine and triiodothyroxine (Lamb et al. 1986). W i t h regard to environmental exposure, the relevance of interactions of 2,3,7,8-TCDD and the high levels of hormones used in the latter study is unclear. A) & 5. M A N U F A C T U R E , I M P O R T , U S E , A N D D I S P O S A L 5.1 OVERVIEW 2.3.7.8- TCD D is n e i t h e r c o m m e rcially m a n u f a c t u r e d n or impo r t e d into the U n i t e d States. It is produced inadvertently in small amounts as an impurity during the manufacture of compounds for which 2,4,5- t r i c h lorophenol is a synthetic intermediate. A t the p r esent time, its on l y us e is in che m i c a l research. Several fie l d - t e s t e d a nd u n t e s t e d methods are available for the disposal of 2,3,7,8-TCDD-containing wastes. Some of the promising methods are incineration at high temperature, oxidative destruction with the aid of a catalyst, biodegradation by a fungus, photochemical destruction in the presence of a hydrogen-donating substrate, and stabilization (in case of soils) through the in situ addition of cementitious and asphaltic materials. 5.2 "''PRODUCTION 2.3.7.8- TCDD is synthesized on a laboratory scale primarily by two p r o c e s s e s : (1) c o n d e n s a t i o n o f d i c h l o r o c a t e c h o l w i t h s u b s t i t u t e d d i c h l o r o b e n z e n e s a n d (2) h a l o g e n a t i o n o f d i b e n z o - p - d i o x i n or its d i c h l o r o - s u b s t i t u t e d d e r i v a t i v e (EPA 1985b). 2 , 3 , 7 , 8 - T C D D is not commercially m a n u f a c t u r e d in the United States b u t is produced as an undesirable product during the manufacture of compounds for which 2,4,5-trichlorophenol is a synthetic intermediate (see Sect. 6.2 on Releases to the E n v i r o n m e n t ) . 5.3 IMPORT 2,3,7,8-TCDD is not imported into the U n i t e d States (EPA 1984b). 5.4 USE 2,3,7,8-TCDD has been tested for flame-proofing polyesters and as a control against insects and wood-destroying fungi in Germany; however, it has probably never been commercially produced or used for these p u r poses. A t p r e s e n t it is on l y u s e d as a r e s e a r c h c h emical (HSDB 1987). 5.5 DISPOSAL For the disposal of wastes and residues containing 2,3,7,8-TCDD, i n c i n e r a t i o n at a m i n i m u m t e m p e r a t u r e of 800 to 1 2 0 0 #C a n d a c o n t a c t time of >30 s was found to be satisfactory. Polychlorophenols containing 2,3,7,8-TCDD as an impurity have been disposed of b y dispersing or dissolving the material in water, or a nonnucleophilic organic solvent, and oxidizing with ruthenium tetraoxide catalyst at -70C (HSDB 1987). Satisfactory field-tested methods for the disposal of 2,3,7,8-TCDDcontaining soils were not available until recently. The following methods have either shown promise or are presently used for the disposal 61 62 o f 2 , 3 , 7 , 8-TCDD: (1) s t a b i l i z a t i o n t h r o u g h the in s i t u a d d i t i o n of c e m e n t i t i o u s a nd a s p h a l t i c m a t e r i a l s ; (2) d e g r a d a t i o n w i t h P h a n e r o c h a e te c h r y s o s p o r iu m , a w h i t e rot fungus; (3) a m o b i l e i n c i n e r a t i o n s y s t e m for the t h e r m a l d e s t r u c t i o n o f 2 , 3 , 7 , 8-TCDD; a n d (4) u l t r a v i o l e t p h o t o l y s i s in the presence of alkali polyethylene glycolate reagents (des Rosiers 1986). A m e t h o d (similar to m e t h o d 4) for p h o t o c h e m i c a l d e s t r u c t i o n of 2 , 3 , 7 , 8-TCDD in the p r e s e n c e of olive oil (a h y d r o g e n donor) w as us e d with soil from the area of the Seveso accident. A recent report discusses the disposal of 2,3,7,8-TCDD by a mobile poly(ethylene)glycolp o t a s s i u m hydroxide destruction u n i t (Rogers et al. 1987). 6. E N V I R O N M E N T A L F A T E 6.1 OVERVIEW The important sources of 2,3,7,8-TCDD in the environment are production and use of certain herbicides and chlorophenols, incineration of municipal and industrial wastes, and improper disposal of chemical wastes produced during the manufacture of 2,4,5-trichlorophenol, 2,4,5- T, a n d r e l a t e d herbicides, h e x a c h l o r o p h e n e , and chlorinated benzenes. The fate of 2,3,7,8-TCDD in the environment is n o t clearly understood. It appears that particulate-bound 2,3,7,8-TCDD in the air may undergo photolysis and may be removed by wet and dry deposition. The half-life of atmospheric 2,3,7,8-TCDD is such that 2,3,7,8-TCDD can b e transported l o n g d i s t a n c e s in the air. T h e u l t i m a t e s i n k o f a i r b o r n e 2 , 3 , 7 , 8 - T C D D is sediments of surface waters. The two processes that are likely to remove 2.3.7.8- TCDD from water and soils are vaporization and photolysis. The e s t i m a t e d h a l f - l i f e o f 2 , 3 , 7 , 8 - T C D D i n s u r f a c e w a t e r is > 1 year, a n d the u l t i m a t e sin k of a q uatic 2 , 3 , 7 , 8 - T C D D is sediments. The b i o c o n c e n t r a t i o n '' f a c t o r o f 2 , 3 , 7 , 8 - T C D D i n t h e f a t h e a d m i n n o w (P im e p h a le s p r o m e l a s ) is 7900 to 9300. 2,3,7,8-TCDD is immobile in most soils, b u t horizo n t a l movement of soil-bound 2,3,7,8-TCDD may occur in runoff water during flooding. As observed in Seveso, Italy, minimal vertical movement may occur in soils containing low organic matter. The estimated half-life of 2.3. 7 . 8 - TCDD is 1 to 3 years on soil surfaces and 10 to 12 years in the interior of soils. Although not accumulated, the level of 2,3,7,8-TCDD a b s o r b e d in parts of plants u n d e r g r o u n d is of the same order of magnitude as in soil, but the aerial parts of plants contain 50% lower concentrations. ------- 6.2 RELEASES TO THE ENVIRONMENT Although the following paragraphs discuss the sources of 2,3,7,8TCDD in the environment, the sources responsible for its background levels are not clear. 6.2.1 Production and Use of Certain Herbicides and Chlorophenols The phenoxy herbicide 2,4,5-T produced prior to 1960 contained up to 100 fig /g 2,3,7,8-TCDD. The level of 2,3,7,8-TCDD in commercial 2,4,5-T has been reduced in recent years to <0.1 p g /g , and most commercial 2,4,5-T available today may contain <0.02 p g /g 2,3,7,8-TCDD. Agent Orange, a 1:1 mixture of butyl esters of 2,4,5-T and 2,4-D produced before 1970, contained 0.02 to 54 p g /g 2,3,7,8-TCDD. Hexachlorophene, a germicide manufactured from trichlorophenol, contains 0.2 to 0.5 ng/g 2,3,7,8-TCDD. 2,4,6-Trichloro-, 2,3,4,6-tetrachloro-, and pentachlorophenol were found to contain <0.1 p g /g other tetra isomers but no 2,3,7,8-TCDD. 2,3,7-,8-TCDD w a s d e t e c t e d at a concentration <1 ng/g (2,3,7,8-TCDD detection limit of 0.03 ng/g) in all samples of sodium pentachlorophenate, 2,3,4,5-tetrachlorophenol, and 4385 63 64 hexachlorophene. 2,4,5-Trichlorophenol, on the other hand, contained up to 6 . 2 /ig/g 2 , 3 , 7 , 8 - T C D D . S i m i l a r l y , d i p h e n y l e t h e r h e r b i c i d e s w e r e found to contain other tetrachloro isomers but no 2,3,7,8-TCDD (EPA 1985b, HSDB 1987, Rappe 1984, Hagenmaier 1986, Weer e n and Asshauer 1985). From the analysis of sediments of a western Lake Ontario site, Czuczwa and Hites (1986) concluded that the likely source of tetrachlorodibenzo-p-dioxins was a pentachlorophenol production facility. The analytical method used, however, could not distinguish 2.3.7.8- TCDD from other tetra isomers. 6.2.2 Photochemical Reactions The photochemical reaction of phenoxy herbicides has be e n found to produce polychlorinated dibenzo-p-dioxins through photodechlorination a n d s u b s e q u e n t c o n d e n s a t i o n r e a c t i o n s ; h o w e v e r , t h i s p r o c e s s d o e s n o t produce 2,3,7,8-TCDD (Rappe 1984). Lower substituted dibenzo-p-dioxins are also formed during photodechlorination of higher chlorinesubstituted dibenzo-p-dioxins. Tr^ce amounts of 2,3,7,8-TCDD were observed from the photodechlorination of both 1,2,3,6,7,8-hexa- and 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (Buser 1979). 6.2.3 Thermal Reactions Small amounts of 2,3,7,8-TCDD have been detected in the flue gases from municipal incinerators. From the experimentally determined concentrations in flue gases of five municipal incinerators, the maximum average concentration of 2,3,7,8-TCDD in ambient air at ground level was e s t i m a t e d as 38 fg/g. Inc i n e r a t i o n of industrial wastes containing 2,4,5-T salts and esters, polychlorinated benzenes, and chlorophenoxy ethers also produced 2,3,7,8-TCDD (Rappe 1984, Barnes 1983). Upon analysis of sediments from Saginaw Bay, Saginaw River, and the Great Lakes, Czuczwa and Hites (1984, 1986) concluded that the source of tetrachlorodibenzo-p-dioxins was incineration, although the analytical method used was unable to separate 2,3,7,8-TCDD from other tetra isomers. Combustion of coal did not produce 2,3,7,8-TCDD at a detection limit of 1.2 ng/kg (HSDB 1987), but burning of woods did produce 0.65 /ig/kg 2 , 3 , 7 , 8-TCDD (EPA 1985b). E x h a u s t s f r o m a u t o m o b i l e s p o w e r e d w i t h leaded gasoline were reported to contain <0.05 to 0.3 ng 2,3,7,8TCDD/ 2 4 . 8 km, but no 2 , 3 , 7 , 8-TCDD was detected in exhausts of automobiles powered w i t h unleaded gasoline (Marklund et al. 1987). Accidental fires involving capacitors or transformers containing chlorobenzene will also release 2,3,7,8-TCDD to the environment. An example of such a contamination is the State Office Building in Binghamton, New York. 6.2.4 Improper Disposal of Chlorinated Chemical Vastes Improper disposal of certain chemical wastes produce^ during the manufacture of 2,4,5-trichlorophenol, 2,4,5-T, and related herbicides, hexachlorophene, chlorinated benzenes, etc., may be a source of 2.3.7.8- TCDD in the environment. Examples of such improper disposal leading to the contamination of the environment are the Love Canal, N iagara Falls, New York, sites where 2,3,7,8-TCDD up to a level of 672 /ig/kg w a s detected. S i m i l a r l y , s e v e r a l s ites in the s t a t e o f M i s s o u r i w e r e c o n t a m i n a t e d w i t h u p to 1 750 /ig/kg 2 , 3 , 7 , 8 - T C D D ( T i e r n a n e t al. 65 1985). 6.3 ENVIRONMENTAL FATE The fate of 2 , 3 , 7 , 8-TCDD in air, water, and soil is n o t u n d e r s t o o d w i t h certainty. Although some experimental efforts have been directed in recent years to elucidate its fate in different media, a substantial d a t a gap exists in this area. I n air, 2 , 3 , 7 ,8-TCDD is likely to be present predominantly in the gas phase. The two important processes that may remove 2,3,7,8-TCDD from the atmosphere are photochemical degradation and wet deposition. Even an estimate of the atmospheric h a l f - l i f e of 2 , 3 , 7 , 8 - T C DD is n o t available. O n the basis of photochemical experiments with 2,3,7,8-TCDD coated on silica gel, the half-life of atmospheric particulate 2,3,7,8-TCDD may be a few days. The half-life of atmospheric gas-phase 2,3,7,8-TCDD may be higher than p a r t i c u l a t e 2,3,7,8-TCDD. The lifetime of atmospheric 2,3, 7 , 8 - T C D D is such that it can be transported long distances in the air. The ultimate e n v i r o n m e n t a l sink of airborne p a r t i c u l a t e 2,3,7,8-TCDD is likely to be sediments of surface waters (Eitzer and Hites 1986, Czuczwa and Hites 1986, Choudhry and Hutzinger 1982). The b i o d e g r a d a t i o n of 2,3, 7 , 8 - T C D D in w a t e r is p r o b a b l y slow. The two processes that may be important for the removal of 2,3,7,8-TCDD are volatility and photodegradation. Although the photolysis of 2,3,7,8-TCDD in h y d r o g e n - d o n a t i n g solvents is a fast process, a s u s p e n s i o n of j 2,3,7,8-TCDD in distilled water showed no appreciable photodegradation. In natural waters, the presence of small amounts of hydrogen-donating substrate or the presence of photosensitizers may account for its observed photodegradation; however, the photochemical degradability of 2.3.7.8- TCDD in water, as provided by model ecosystem studies (Tsushimoto et al. 1982, M a t s u m u r a et al. 1983), has n o t p r o v i d e d definite evidence through mass balance that the observed loss of 2.3.7.8- TCDD attributed to photolysis was not due to its sorption on s e d i m e n t a n d b i o t a . T h e p h o t o d e g r a d a t i o n i s u s u a l l y a d e c h l o r i n a t i o n _____ process leading to the formation of tri- and dichlorinated dibenzo-pdioxins. In sediment-containing lake water, the estimated half-life of 2.3.7.8- TCDD is >1.5 years. In lake water alone, the e s t i mated half-life is >1 year. The ultimate sink of aquatic 2,3,7,8-TCDD is the sediment. Recent flow-through experiments with fathead minnows (P im ephales p ro a ela s) have shown that the bioconcentration factor for 2,3,7,8-TCDD in this species is 7900 to 9300 on a w e t w e i g h t basis (EPA 1985b, Adams et al. 1986). 2,3,7,8-TCDD is expected to be immobile in most soils b y irrigation and rainfalls. A downward movement of 10 cm in 12 years was observed with soil from Eglin Air Force Base. Although 2,3,7,8-TCDD usually does n o t l e a c h t h r o u g h soil, lea c h i n g is p o s s i b l e in rare instances fro m soils of very low organic carbon content as a result of 2<3,7,8-TCDD solvation with organic solvent or biotic mixing by earthworms or other soil invertebrates. A white rot fungus (P hanerochaete ch ryso sp o riu m ) has been shown to degrade 2,3,7,8-TCDD. This biodegradation does not occur significantly in natural soils, probably because of the lack of this or other degrading microorganisms. Both volatilization and photoreaction may remove some 2,3,7,8-TCDD from soil surfaces. The photoreaction on soil surfaces can be greatly enhanced by the presence of hydrogen- 4987 6? ^ 66 donating substrates (e.g., olive oil or arachis oil) in soil. The photoreaction will be insignificant beyond the surface soil layers. The estimated half-life of 2,3,7,8-TCDD on soil surfaces is 1 to 3 years, but the half-life in the interior of soil may be 10 to 12 years (EPA 1985b, Freeman and Schroy 1986, Bumpus et al. 1985, HSDB 1987). 2,3,7,8-TCDD present on leaves of plants as a result of spraying herbicides will photolyze with a half-life of a few hours. The chemical is a b s o r b e d b y h i g h e r p l a n t s and is p r o b a b l y translocated, b u t it is not accumulated. The absorption by underground parts may be at the same level as soil, but the aerial part contains -50% lower concentrations (Choudhry and H u t z i n g e r 1982, Sacchi et al. 1986). / CY* 7. P O T E N T I A L F O R H O M A N E X P O S U R E 7.1 OVERVIEW The concentration of total tetrachlorodibenzo-p-dioxin (not 2.3. 7 . 8 - TCDD alone) in amb i e n t air in Bloomington, Indiana, w a s 18 to 92 fg/m^ (femtograms per cubic meter). Assuming that the 2,3,7,8-TCDD isomer constitutes 3% of the total tetra.isomers, the concentration of 2.3.7.8- TCDD in Bloomington air would be 0.9 to 4.6 fg/m^. The concentration of 2,3,7,8-TCDD in the air around the stack of a municipal incinerator has b e e n estimated as 38 fg/m^. The accidental transformer fire in Binghamton, New York, produced a much higher level of 2,3,7,8T C D D -- at 0.23 to 0.47 pg/m^. The concentration of 2,3,7,8-TCDD in the a i r s u r r o u n d i n g a f i e l d a f t e r the a p p l i c a t i o n o f S i l v e x c o n t a i n i n g 15 ppm 2,3,7,8-TCDD was 0.62 pg/m^. Other than certain industrial effluents and leachates from chemical dump sites, no 2,3,7,8-TCDD has ever been reported in drinking water. The concentrations of 2,3,7,8-TCDD in most uncontaminated soils are below the detection limits of analytical methods. In u r b a n soils, the level of 2 , 3 , 7 , 8 - T C D D is in the range <0.0002 to 0.009 ng/g. Much higher levels have been detected in soils contaminated b y certain hazardous wastes, waste oils, and spillage of 2,4,5-trichlorophenol. A soil from Shenandoah Stables in Missouri contaminated b y waste oil containing 2,3,7,8-TCDD ha d up to 1750 ng/g of 2.3.7.8- TCDD. Fish samples collected from lakes and a selected Michigan river contained undetectable levels to 67 pg/g 2,3,7,8-TCDD. No 2.3.7.8- TCDD was detected in rice, soybeans, and crawfish in the United S t a t e s , o r i n C a n a d i a n c h i c k e n a n d p o r k s a m p l e s . S i n c e f a t i s t h e c h i e f ---contributor to the body burden of 2,3,7,8-TCDD in humans, it has been analyzed by many investigators. Levels of 2,3,7,8-TCDD in adipose tissue of exposed and control persons in Missouri have been determined. The level of 2,3,7,8-TCDD in adipose tissue in the general population in the U n i t e d States and Canada ranges from undetectable to 20 pg/g, with a m e a n value of 5 to 7 pg/g. Values as high as 99 pg/g were detected in an individual heavily exposed during the spraying operation in South Vietnam. 2,3,-7,8 - T C D D h as n o t b e e n det e c t e d in h u m a n m i l k f r o m exp o s e d and control areas in the United States; however, using more sensitive methods of measurement, 2,3,7,8-TCDD has been reported in human milk of the general population in Sweden at trace to 2.3 pg/g, with a mean value o f 0.6 pg/g, and in G e r many at 1.3 to 3.3 pg/g, w i t h a m e a n value of 1.9 pg/g. 2,3,7,8-TCDD was also found in human milk in other European countries. Although there are no data regarding exposure levels, workers at sites of improper chemical waste disposal and the general population residing near these sites m a y be susceptible to h i g h e r e x p o s u r e to 2.3.7.8- TCDD. Breast-fed babies nursed by mothers residing near improperly controlled municipal incinerators m a y also b e at hig h e r risk. The estimated daily h u m an exposure to 2,3,7,8-TCDD, expressed as picograms p e r k i l o g r a m of b o d y w e i g h t ( p g / k g ) , is 0.02 thr o u g h inhalation, 0.5 to 'P-SolO 67 68 5 through the consumption of milk, and 20 through the consumption of fish. The daily exposure of a 5 -kg b aby resulting from the consumption of 850 m L of b r e a s t mi l k is 20 to 200 pg/kg. F r o m the estimated bo d y burden and the half-life of 2,3,7,8-TCDD in the human body, the daily h u m a n intake of 2 , 3 , 7 , 8 - T C D D has b e e n e s t i m a t e d as 0.05 ng. 7.2 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT 7.2.1 Air The level of 2,3,7,8-TCDD in ambient air is so low that its d e t e c t i o n is b e y o n d the capabilities o f a n a l y t i c a l methods. The concentration of total tetrachlorodibenzo-p-dioxin isomers in both vapor and particulate phase in the ambient air in Bloomington, Indiana, was approximately 18 to 92 fg/m^. The analytical m e t h o d u s e d could not unequivocally identify 2,3,7,8-TCDD from other tetra isomers. Using the measured concentration in flue gases from five municipal incinerators and an air dispersion model, the maximum ambient concentration of 2.3.7.8- TCDD in the area around th stacks was estimated as 38 fg/m^. Since municipal incinerators are one of the prime sources of atmospheric 2.3.7.8- TCDD, there are a vast number of publications concerning the level of these compounds in the fly ashes of the incinerators; however, most of the publications failed to distinguish 2,3,7,8-TCDD from its isomersT~The levels of 2,3,7,8-TCDD in the flue gas of European incinerators are 0.05 to 1.3 ng/m^; in U.S. incinerators, the levels are a maximum of 3.5 ng/m^ (under normalized conditions of the emitted ~gases) (EPA 1985b, Barnes 1983, Nottrodt and Ballschmiter 1986, Marklund et al. 1986, Eitzer and Hites 1986). Accidents involving certain transformer/capacitor fires can release larger amounts of 2,3,7,8-TCDD in the air. The concentration of 2.3.7.8- TCDD in the air of the State Office Building in Binghamton, New York, following an accidental fire, was 0.23 to 0.47 pg/m^. Following an a c c i d e n t a l l o c o m o t i v e f i r e i n S w e d e n , t h e c o n c e n t r a t i o n o f 2 , 3 , 7 , 8 - T C D D __ was 50 pg/m^. The concentration of 2,3,7,8-TCDD in the air surrounding a field after the application of Silvex containing 15 ppm 2,3,7,8-TCDD was 0.62 pg/m^, b u t the concentration dropped to a level of 10 fg/m^ in 200 days (EPA 1985b, Smith et al. 1986, R a p p e et al. 1985). 7.2.2 Water No report is available on the detection of 2,3,7,8-TCDD in drinking water, using methods with detection limits in the nanogram-per-liter range; however, 2,3,7,8-TCDD has been detected in aqueous industrial effluents, sediments, and leachates from hazardous waste sites. The concentrations of tetrachlorinated dibenzo-p-dioxins, including 2.3.7.8- TCDD, in effluents from a trichlorophenol manufacturing facility ranged from none detected (detection limit, 10 to 30 pg/g) to 100 pg/g. The discharged wastewater effluent from Dow into the Tittabawassee River, in Michigan, has been reported to be approximately 15 pg/L. The leachate samples from a waste disposal site in Jacksonville, Arkansas, had a mean 2,3,7,8-TCDD level of 14 ng/L. The sump pump water from residences and leachates from the Love Canal area in New York contained 2.3.7.8- TCDD ranging from none detected to 1560 ng/L. The concentrations of 2,3,7,8-TCDD in sediments from storm sewers, residential sump water, 69 and surface water around the same site were none detected (detection limit, 10 to 100 pg/g) to 9570 n g / g (EPA 1985b, T i e m a n et al. 1985, L a mparski et al. 1986). 7.2.3 Soil Concentrations of 2,3,7,8-TCDD in most uncontaminated soils are below the detection limits of current analytical methods. In urban soils, the level of 2,3,7,8-TCDD is in the range of <0.0002 to 0.009 ng/g. 2,3,7,8-TCDD has been detected in samples that originated from certain industrial sites, waste disposal sites, and sites involved in accidental spillage of chemicals containing 2,3,7,8-TCDD. The levels of 2 . 3 .7.8- TCDD in soils from d i f f erent locations are g i v e n in Table 7.1. It is apparent from Table 7.1 that the accidental or improper disposal of still-bottom residue from the manufacture of 2,4,5-trichlorophenol (2,4,5-TCP) may be the largest source of 2,3,7,8-TCDD in soils. 7.2.4 Other There are limited data that 2,3,7,8-TCDD does not bioaccumulate in crop plants (Anonymous 1985). Crops grown in soil contaminated with 2.3.7.8- TCDD (up to 752 ppt) after the Seveso accident contained only a few parts per trillion of 2,3,7,8-TCDD in the aboveground portions. The roots_of these plants, however, contained higher levels of 2,3,7,8-TCDD than the surrounding soil, suggesting that a similar study using root crops would demonstrate the contamination of the edible p o r tion of the plant. Since aquatic organisms bioconcentrate 2,3,7,8-TCDD, a few investigators analyzed fish-eating birds as an indicator of possible pollution in the suspected water b o d i e s . A herring gull sample from Lake Huron contained 75 pg/g 2,3,7,8-TCDD. Similarly, herring gull eggs collected from the Great Lakes contained 12 to 101 pg/g 2,3,7,8-TCDD. Samples from Lake Ontario and Saginaw Bay had the maximum levels of 2.3.7.8- TCDD; those from Lake Michigan and Lake Superior had the minimum levels of c o n t a m i n a t i o n (Stalling et al. 1986, B u s e r a n d R a p p e 1984). Fish samples from the Great Lakes and selected Michigan rivers were shown to contain between undetectable amounts (detection limit, 2 pg/g) a n d 67 pg/g 2 , 3 ,7,8-TCDD (Fehringer et al. 1985, N i e m a n n 1986, R y a n et al. 1984). Y e l l o w pe r c h samples from Woods Pond, Massachusetts, were found to contain 26 pg/g 2,3,7,8-TCDD (Buser and Rappe 1984). No 2.3.7.8- TCDD was found in rice, soybean, and crawfish samples from Arkansas and Louisiana at a detection limit of 10 pg/g, or in Canadian chicken and p ork samples at a detection limit of 2 to 4 p g / g (Ryan et al. 1985a, Firestone et al. 1985). Since adipose tissue is the chief contributor to the b o d y burden of 2.3.7.8- TCDD, many investigators analyzed fat tissue from both exposed a n d control populations. That fat has the h i g h e s t b u r d e n is confirmed by the tissue analysis of a woman who died 7 months after the accident in Seveso, Italy. The following levels of 2,3,7,8-TCDD (pg/g) were found in d i f f e r e n t o r g a n s : fat, 1840; p a n c r e a s , 1 0 40; l i v e r , 150; t h y r o i d , 85; b r a i n , 60; l u n g , 60; k i d n e y , 40; a n d b l o o d , 6. T h i s a n a l y s i s s u g g e s t s that b l o o d is a po o r indicator of.the 2 , 3 , 7 , 8 - T C D D b o d y b u r d e n for Table 7.1. Lerels of 2,3,7,8-TCDD in soil from different locations TCD D concentration*4 Site Sample history (ng/g) References Love Canal, NY Jacksonville, AR M idland, MI St. Louis, M O Shenandoah Stables, MO Soils outside the dum p site W aste disposal site Inside DOW facility Urban sample of no obvious source of contamination Contam inated by waste oil N D (0.001-0.020) N D -2 .9 0.01-52 0.12 101-33,000 Timbcrline Stables, MO Bliss Farm , M O Bubbling Springs Ranch, M O M inker Resident, MO Times Beach, M O Urban areas, United States New Jersey New Jersey Lansing, MI Gaylord, MS Detroit, MI C ontam inated by w aste oil 30-42 C ontam inated by waste oil 382* ' C ontam inated by w aste oil 76-95 C ontam inated by w aste oil 50' C ontam inated by w aste oil 4.4-317 Urban samples of no obvious source of contamination <0.0002-0.009 Spillage of 2,4,5-TCP still bottom 26,000* Scrap yard where used reactor vessels were collected , 1,100* Urban sample N D (0.0007)-0.003 Urban sample N D (0 .0 0 0 2 ) Urban sample 0.0021-0.0036 EPA 1985b EPA 1985b Nestrick et al. 1986 EPA 1985b Tiernan et al. 1985, Kimbrough et al. 1977 Tiernan et al. 1985 Tiernan et al. 1985 Tiernan et al. 1985 Tiernan et al. 1985 Tiernan et al. 1985 Nestrick et al. 1986 Jackson et al. 1986 Jackson et al. 1986 Nestrick et al. 1986 N estrick et al. 1986 N estrick et al. 1986  V Table 7.1 (continued) Site Chicago, IL Akron, OH Nashville, TN Pittsburgh, PA Philadelphia, PA Brooklyn, N Y Arlington, VA Sample history Urban sample Urban sample Urban sample Urban sample Urban sample Urban sample Urban sample T<DD concentration4 ' ; (ng/g) References 0.0042-0.0094 0.0063 0.0008 0.0026 0.0009 0.0026 N D (0 .0 0 0 3 ) Nestrick et al. 1986 Nestrick et al. 1986 Nestrick et al. 1986 Nestrick et al. 1986 Nestrick et al. 1986 N estrick et al. 1986 N estrick et al. 1986 N D = not detected. ^Values within parentheses are detection limits. rOnly one sam ple was analyzed. CD CD 3 I 72 humans. The levels of 2,3,7,8-TCDD in adipose tissue in the general p o p u l a t i o n of the U n i t e d States a n d C a n a d a ra n g e d from u n d e t e c t a b l e to 20 pg/g, with a mean value of 5 to 7 pg/g. In a U.S. National Human Adipose Tissue Survey, 2,3,7,8-TCDD was detected with a frequency of 76%. In Europe, the range of 2,3,7,8-TCDD concentration in the adipose tissue of the general population is not detectable to 9 pg/g, w i t h a mean value of 3 pg/g. Instances of higher levels in adipose tissue have been reported in individuals exposed to this chemical either during spraying herbicides containing 2,4,5-T or during accidental capacitor or transformer fires. For example, the adipose tissue of a few exposed individuals in the State Office Building fire in Binghamton, New York, had 2,3,7,8-TCDD concentrations in the range 11.6 to 28.3 pg/g, with a mean value of 17.4 pg/g. The adipose tissue of a few heavily exposed individuals involved in spraying operations in Vietnam had 2,3,7,8-TCDD levels ranging from undetectable (detection limit, 3 pg/g) to 99 pg/g, w i t h a m e a n value of 37 pg/g; however, no difference in the level of 2,3,7,8-TCDD was found in lightly exposed, possibly exposed, and other Vietnam veterans who sought medical help compared with the control p o p u l a t i o n group (EPA 1986a; G r o s s et al. 1984; W e e r a s i n g h e et al. 1986; P a t t e r s o n et al. 1987c; N y g r e n et al. 1986; Schecter et al. 1985, 1986; G r a h a m et al. 1986; S t a n l e y et al. 1986; Y o u n g 1984). The m e d i a n concentrations of 2,3,7,8-TCDD (concentration range in parentheses) in control and exposed populations in Missouri have b e e n reported to be 6.4 pg/g (1.4 to 20.2 pg/g) and 17 p g/g (2.8 to 750 pg/g), respectively ( P a t t e r s o n et al. 1986). Human breast milk has also been analyzed for 2,3,7,8-TCDD. It has b e e n r e p o r t e d that h u m a n b r e a s t m i l k is the largest c o n t r i b u t o r towards the b o d y intake of 2 , 3 ,7,8-TCDD in b r e a s t - f e d b abies (Rappe et al. 1986). The following levels (the detection limits are given in parentheses) of 2,3,7,8-TCDD have been determined in human breast milk from different countries: United States, none detected (0.1 to 6.0 pg/g) in mothers from 2,4,5-T-exposed areas and in control areas; Seveso, Italy, 2.3 to 28.0 pg/g from mothers near accident area; South V i e t n a m , ~ none detected (0.5 pg/g) to 40 to 50 pg/g from mothers in sprayed areas; Sweden, trace to 2.3 pg/g, w i t h a m e a n of 0.6 pg/g; and Germany, 1.3 to 3-3 pg/g, with a mean of 1.9 pg/g. 2,3,7,8-TCDD was also detected in human milk obtained from Denmark, the Netherlands, and Yugoslavia (Heath et al. 1986, Patterson et al. 1986, Y o u n g 1984, Rappe et a l . 1986, J e n s e n 1987, N y g r e n et al. 1986, E P A 1985b). T h e r e is a l a r g e u n e x p l a i n a b l e difference in the v a l u e s of 2 , 3 , 7 , 8 - T C D D c o n c e n t r a t i o n s in milk from South Vietnamese mothers analyzed by two groups of investigators. In addition to 2,3,7,8-TCDD, mother's milk has been found to contain 1,2,3,7,8-pentachlorodibenzo-p-dioxin at levels >10 ng/g. This congener has rarely b e e n reported as a contaminant in any commercial product other than in commercial pentachlorophenol (Hagenmaier 1986); however, it has always been found in samples from municipal and industrial waste incinerators. Given this finding, a 1-year moratorium for the construction of new municipal incinerators in S w e d e n was p u t into effect (Nygren et al. 1986). Other human tissues, obtained from the autopsy of two subjects in Canada, were analyzed for 2,3,7,8-TCDD with the following results: liver, none detected to 2.5 pg/g; muscle, none detected; and kidney, / *0 73 none detected. The detection limit in these determinations were in the range of 1 to 4 p g / g (Ryan et al. 1985b). No 2 , 3 , 7 , 8 - T C D D was detected in the blood of exposed workers following the accident in Binghamton, N e w York, at a d e t e c t i o n limit of 1 to 2 p g / g (Schecter et al. 1985). Serum 2,3,7,8-TCDD levels in veterans who w e r e heavily exposed to Agent Orange in Vietnam during 1967-1968 have been reported by the Center for D i s e a s e Cont r o l to range b e t w e e n none d e t e c t e d (0.0013 p g/g) a n d 25 pg/g, with a median value of 3.8 pg/g. The same study reported serum 2.3.7.8- TCDD levels in a group of non-Vietnam veterans to range between none detected (0.0013 pg/g) and 12 pg/g, w i t h a median value of 3.9 pg/g. Serum levels in a group occupationally exposed to 2,3,7,8-TCDD prior to 1970, however, have been reported to be 30-fold higher in the same study (MMWR, 1987). 7.3 OCCUPATIONAL EXPOSURES Occupational exposures to 2,3,7,8-TCDD may occur during the production and use of hexachlorophene, trichlorophenol, and herbicides containing 2,4,5-T. The heaviest exposure may occur during the step that is u s e d to purify 2,4,5-T from its contaminants, since these products contain much higher levels of 2,3,7,8-TCDD than the purified products. No data on the occupational exposure to 2,3,7,8-TCDD during the manufacture of these chemicals are available (Rappe 1984). The indirect e v i d e n c e of o c c u p a t i o n a l e x p o s u r e to 2 , 3 , 7 , 8 - T C D D is the s i g n i f i c a n t l y higher adipose tissue levels of the compound in heavily exposed Vietnam veterans and in certain workers at the State Office Building in Binghamton, N e w York, following the transformer fire. 7.4 POPULATIONS AT HIGH RISK F r o m t h e m o n i t o r i n g d a t a d i s c u s s e d i n S e c t s . 7 .2 a n d 7.3, it is possible to predict the segments of the general population and of occupational groups that may be exposed to higher levels of 2,3,7,8TCDD. Among the occupational groups, workers involved in the production or use of trichlorophenol or its salts, hexachlorophene, and 2,4,5-T or other herbicides conta i n i n g 2,4,5-T are susceptible to exposure to higher levels of 2,3,7,8-TCDD than the general population. 2,4,5-T and 2,4,5-trichlorophenol and its salts, however, are no longer manufactured in the United States (SRI 1987). Since both flue gases and ashes from municipal and industrial incinerators contain 2,3,7,8-TCDD, workers in this profession are expected to be at higher risk. Populations residing nea r municipal incinerators may also be subjected to exposure. Workers at sites of improper chemical waste disposal (from trichlorophenol, hexachlorophene, 2,4,5-T, and associated industries) and the general p o p u l a t i o n residing near those sites are p o t e n t i a l l y exp o s e d to 2.3.7.8- TCDD. Breast-fed babies nursed by mothers residing near improperly controlled municipal incinerators or other sources of exposure are expected to receive 2,3,7,8-TCDD through th milk. Studies in humans have not demonstrated that there is a sensitive subpopulation. Animal studies, however, suggest that the fetus and newborn infants may represent such a sensitive population. As discussed in Sects. 4.3.3 and 4.3.4 on developmental and reproductive toxicity, 2.3.7.8- TCDD is a demonstrated teratogen in rats and mice, and also results in spontaneous abortions and fetal death in monkeys. Since these 4995 74 effects occur at low doses, and in the case of teratologic effects at doses that do not a p p e a r to adversely affect the mother, it is likely that, at certain stages of fetal development, the fetus represents a sensitive subgroup. Animal data also demonstrate that toxic levels of 2,3,7, 8 - T C D D c a n be i n g e s t e d d u r i n g nur s i n g a nd that l a c t a t i o n is a major route for elimination of 2,3,7,8-TCDD. This indicates that the newborn might receive greater exposure than the adult, since no data were found to indicate that newborns are more sensitive than adults. 8. A N A L Y T I C A L M E T H O D S Several methods are available for the analysis of 2,3,7,8-TCDD in different media. Some of the more recent methods are g i v e n in Tables 8.1 and 8.2. The methods listed in these tables are not exhaustive but are illustrative of a few recent methods. Methodologies for collecting samples before their analysis are important, since the concentrations of 2.3.7.8- TCDD in most samples are low. This is particularly important for stack samples that exist both in the vapor and particulate phase. The details of stack-sampling methods are available in Velzy (1986), Ozvacic (1986), and Brenner (1986). The accuracy of analysis has increased in recent years with the availability of stable isotope-labeled ( ^ C l and l^C) 2,3,7,8-TCDD for use as an internal standard in mass spectral analysis. With a combination of one of several methods available for sample cleanup, high-performance gas chromatography (GC) (HRGC), and h i g h -resolution m a s s ^ s p e c t r o m e t r y (MS) ( H R M S ) , u n e q u i v o c a l i d e n t i f i c a t i o n and quantification of 2,3,7,8-TCDD can be performed at very low levels. Although negative chemical ionization MS (NCI/MS) shows a higher sensitivity to all other polychlorinated dibenzo-p-dioxins than electron impact MS (EI/MS), it has a lower sensitivity for 2,3,7,8-TCDD (Buser et al. 1985). The analysis of fly ash samples poses a s p e c i a l challenge b e c a u s e of poor solvent e x t r a c t i o n recovery and the d i f f i c u l t y in the resolution of 2,3,7,8-TCDD from a large number of congeners present in these samples. Best results were obtained by using digestion with excess dilute HC1, followed by freeze-drying of the residue and hot extraction w i t h toluene. The HC1 t r e a t m e n t opens the pore st r u c t u r e of fly a sh to pe r m i t access to the solvent, and freeze-drying removes w a t e r to improve material transfer from the hydrophilic surface of the fly ash to the solvent (Stieglitz et al. 1986). Emphasis has been p l a c e d on the analysis of adipose tissue and mother's milk, because these two tissues may be indicators of the human body burden for 2,3,7,8-TCDD. The ana l y s i s of fish is also i m p o rtant since some b o t t o m fee d e r s (e.g., channel catfish and carp) and those fish that feed upon bottom feeders m a y be indicators of 2 , 3 , 7 , 8-TCDD-polluted water (Ryan et al. 1984a, Jensen 1987). Besides the commonly used analytical methods, other newly developed b u t y e t generally u n t e s t e d methods are available for the analysis of 2.3.7.8- TCDD. Some of these methods are GC with a polymeric liquid crystal capillary column (Naikwadi and Karasek 1986), GC with matrix isolation Fourier transform infrared spectrometry (Wurrey et al. 1986), a nd H R G C w i t h m i c r o w a v e - i n d u c e d p l a s m a d e t e ction (Mohamad et al. 1986). More detailed descriptions of the analytical methods for 2,3,7,8-TCDD c a n be found in Buser et al. (1985), T i e m a n et al. (1985), Rappe (1984), and EPA (1985b). 4997 75 76 8.1 ENVIRONMENTAL MEDIA 8.1.1 Air, Water, Soil, and Food See Table 8.1. 8.2 BIOMEDICAL SAMPLES 8.2.1 Fluids/Exudates and Tissues See Table 8.2. 0O lS'd CD CO CO ) V Sample matrix Tabic 8.1. Analytical methods for environmental samples Sam ple preparation Analytical method0 1 1 : Detection limit Accuracy0 References Ambient room air following accidental transformer Tire Ambient outdoor air S lack emission F ly ash from municipal incinerator Soil Vapor phase sample collected by silica gel; particulate sample collected on glass fiber filter, solvent extracted, and precleaned by alumina and carbon Vapor and particulate collected on polyurethane foam and glass fiber, precleaned by Florisil and modified silica V apor and particle collected on polyurethane and glass fiber, precleaned by silica and alumina Vapor and particle collected on X A D -2 and glass fiber by modified E P A method 5; solvent extracted and precleaned by silica, alumina, and Biobead Solvent extraction, H P L C separa tion on normal-phase and reversephase column Solvent extraction, precleaning by two adsorbent columns, and further fractionation on reverseand normal-phase H P L C Solvent extraction, precleaned on silica and alumina Solvent extraction, K O H wash, pre cleaned in alum ina, reversedphase H P L C , and carbon Solvent extraction, K O H wash, pre cleaned in chem ically treated silica, basic alumina H RG C/H RM S ' 0.003 pg/m' H R G C /N IC I/M S 0.1-0.2 pg/m5 (method detection limit too high for T C D D determination) H R G C /N IEC /M S-SIM N R ( T C D D not separated from other tetra isomers) H RO C/M S NR G C /M S or H R G C / F ID N R ( T C D D not separated from other tetra isomers) G C/LRM S 40 pg H RG C/LRM S H RG C/LRM S H RG C/LRM S NR 3 ng/g NR 131 27% at 5-10 pg/m 1 Sm ith et al. 1986 J N A Oehm e et al. 1986 N A Eitzer and H ites 1986 N R Hagenmaier el al. 1986 N R Tong et al. 1984, Tong and Karasek 1986 N R Lam parski and N cstrick 1980 N R Buser and Rappc 1980 N R Donnelly et al. 1986 N R Freeman et al. 1986 -vj Table 8.1 (continue^) Sample matrix Sam ple preparation Analytical method" W ater C hicken and pork Fish Fish and herring gull Sam ple passed through glass fiber filter and adsorbent cartridge; solvent extracted and precleaned by acid alumina, graphitized carbon and alumina Fat and liver solvent extracted, partitioned with concentrated l l , S 0 4, cleaned by Florisil and reverse-phase H P L C Homogenized fillet digested with ethanolic K O H and solvent extracted; extract cleaned by silica gel-supported H 2S 0 4 column and H P L C Homogenized fillet solvent extracted, partitioned with con centrated H jS 0 4, cleaned by Florisil Homogenate digested with concen trated H C I and solvent extracted; extract cleaned by silica gelsupported H jS 0 4, chem ically treated silica and alumina, and reverse-phase H P L C Digested with lalkali, solvent extracted, washed with concen trated H ] S 0 4, and cleaned up by site exclusion chromatography, normal- and reverse-phase H P L C Solvent extracted, cleaned up by potassium silicate-silica gel. cesium silicate-silica gel. carbon, and H , S 0 4-silica gel cesium silicate and alumina GC/M S H RGC/M S/M S H RGC/M S H RG C/H RM S HRGC/M S/M S H RG C/EC H RG C/LRM S Detection limit !!| p g / L Accuracy' 88% at 6.5 p g /L References O'Keefe et al. 1986 2-4 pg/g 5-10 pg/g N R Ryan ct al. 1985a N R Fehringer el al. 1985 2-10 pg/g <1 pg N R Ryan et al. 1984 N R Clement et al. 1986 12 pg/g 1-8 pg/g 105% at 18-45 pg/g Niemann 1986 N R Stalling et al. 1983, Rappe 1984 vj 00 rr \ Table 8.1 (continued) Sample matrix Sam ple preparation Fish, egg, or sediment Added M CI and solvent extracted; extract cleaned by gel permeation, trisodium phosphate, H ] S 0 4, alumina and carbon columns Analytical method" H RG C/EC/LRM S \ Detection limit '. NR Accuracy* 72% References Lawrence et al. 1986 * H R G C " High-resolution gas chromatography; N I C I / M S -- negative ion chem ical ionization mass spectrometer; N I E C / M S -- negative ion electron impact mass spectrometer; S I M " selective ion monitoring; F I D -- flame ionization detector; L R M S - low-resolution mass spectrometer; E C * electron capture detector; N R * not reported; N A -- not applicable. vcj cn o o *3 / o V o_ Sample matrix Hum an milk Adipose tissue Table 8.2. A nalytical methods1for biomedical samples Sam ple preparation Saponification with hot alkali; sol vent extraction, cleaned with concen trated H ] S 0 4i alumina Extraction with potassium oxalate and mixture of solvents, cleaned by gel permeation, Florisil A cid ic digestion, solvent extraction multiple cleanups with adsorbents and chem ically modified adsorbents, normal- and reverse-phase H P L C Tissue solvent extracted and subjected to eight different preparations: Cleaned through potassium silicate/ silica gel, carbon, H jS O ,/silica and alumina W ashed with concentrated H jS 0 4 and passed through silica, chemically treated silica, alumina, reverse- and normal-phase H P L C Digested in concentrated H C I and cleaned up by s ilic a / H 2S 0 4, alum ina, carbon/celite Saponified with alkali, solvent extracted, and cleaned up by alumina, charcoal/silica Saponified with alkali, solvent extracted, washed with concentrated H tS 0 4, and chromatographed on silica acid and alumina W ashed with H 2S 0 4 and chromatographed on Florisil W ashed with H 2S 0 4, chromatographed on alumina, charcoat/celite, alumina Passed through silica/potassium silicate, silica/carbon, potassium silicate/ H 2S 0 4/silica and alumina Analytical method" H R G C / H R M S and LRG C/H RM S Detection limit <Pg/g) T i u 0.6; 0.5-6 H RG C/LRM S 5 (fat basis) Accuracy" NR NR References H eath et al. 1986 Fuerst et al. 1986 LRG C/LRM S 0.5 (78 13)% at 1-12 pg/g Langhorst and Shadorr 1980 H RG C/LRM S H RG C/LRM S <5 At 50 pg/g: 70% 78% A lb ro et al. 1985 H RG C/LRM S H RG C/LRM S H RG C/H RM S 92% 88% 90% H R G C /C IM S H RG C/H RM S H R G C /N IC I/M S 72% 84% 90% co O \' v Tabic 8.2 (continued) Sample matrix Sam ple preparation Adipose (issue i Blood, liver, kidney and muscle Serum Automated extraction and enrichment apparatus consisting of solvent ex traction. cleanup by carbon and silicate/silica gel Homogenized sample solvent extracted, cleaned up with H jS O ,, chromatography, or Florisil Solvent extraction, concentrated sulfuric acid wash and cleanup by carbon and silicate/silica gel, followed by s ilic a ( e / H ,S 0 4/silica gel and alumina columns Analytical method0 ^election limit ' '(pg/g) IIR O C / M S or H R G C /IIR M S <2 IIR G C /M S/M S 14 Isotopic dilution with H RG C/H RM S 1.25 X I 0 ' 5 Accuracy0 >85% at 24 pg/g NR 89% at 5.0 jig / m L References Lap cza et al. 1986, Patterson ct al. 1987a R yan et al. I985b,c Patterson et al. 1987b H R G C -- High-resolution gas chromatography; N I C I / M S " negative ion chem ical ionization mass spectrometer; H R M S -- high-resolution mass spec trometry; M S -- mass spectrometry; L R M S -- low-resolution mass spectrometer; C I M S -- chem ical ionization mass spectrometry; N R " not reported. * cn o o \ o O i-'o- \< A 9. R E G U L A T O R Y A N D A D V I S O R Y S T A T U S 9.1 INTERNATIONAL (WORLD HEALTH ORGANIZATION) No World Health Organization standards were found. The World Health O r g a n i z a t i o n a d v i s o r y d e v e l o p e d b y IA R C is p r e s e n t e d below. 9.2 NATIONAL 9.2.1 Regulations The reportable quantity (RQ) for 2,3,7,8-TCDD is 1 lb, w h i c h places 2 , 3 , 7 , 8 - T C D D in c a t e g o r y X. The a u t h o r i t y for lis t i n g 2 , 3 , 7 , 8 - TCDD as a C E R C L A h a z a r d o u s s u b s t a n c e is p r o v i d e d b y S e c t i o n 307(a) o f the C l e a n W a t e r A c t (FR 50, No. 65, p. 13456). 9.2.2^ Advisory Guidance *^Air. N o h e a l t h a d v i s o r i e s (HAs) f o r levels of 2 , 3 , 7 , 8 - T C D D in air were encountered. Water. AGENCY ADVISORY EPA Drinking water advisories: 1-day HA--1.0 x 10'6 mg/L (child) 10-day HA--1.0 X 10'^ mg/L (child) Long-term HA--1.0 x 10*" mg/L (child) Long-term H A - -3.5 X 10* mg/L (adult) Drinking water equivalent level--3.5 x 10* mg/L 10"^ to 10'^ excess cancer risk--2.2 x 10* to 2.2 x 1 0 `H mg/L (EPA 1986b) EPA Ambient water quality criteria 10*^ to 1 0 "^ excess cancer risk--1.3 x 10*^ to 1.3 x 10*^2 m g/L (EPA 1984) Food. AGENCY ADVISORY FDA 9.2.3 Levels in fish: No serious health concerns--<25 ppt (EPA 1985b) / Data Analysis Reference doses (RfDs). EPA (1985a) calculated a chronic oral RfD for 2,3,7,8-TCDD based on the data from a three-generation study in rats b y M u r r a y et al. (1979), as r e a n a l y z e d b y N i s b e t a n d P a x t o n (1982). In this study, rats were exposed to diets containing 2,3,7,8-TCDD at levels 83 5 0 0 5 p - 5 |0fc 84 that provided doses of 0.001, 0.01, and 0.1 jjg/kg/day. The highest dose resulted in decreased fetal survival; the middle dose resulted in effects on litter size and fetal and neonatal survival. The lowest dose resulted in dilated renal pelvises, decreased fetal weight, and changes in the g e s t a t i o n a l index. Therefore, the dose of 0.001 p g / k g/day is a LOAEL, and the RfD was calculated as follows: R f D - (0.001 /ig/kg/day)/(100)(10) - 0.000001 pg/kg/day , where: 0 . 0 0 1 fig / k g / d a y - LOAEL, 100 - uncertainty factor for inter- and intraspecies extrapolation, 10 - uncertainty factor for use of a LOAEL. Carcinogenic potency, q^*. EPA (1985b) has classified 2,3,7,8-TCDD in CAG Group B2 w h e n 2,3,7,8-TCDD is c o n s i d e r e d alone, and in Group B1 w h e n 2,3,7,8-TCDD is considered in ass o c i a t i o n w i t h phenoxyherbicides and/or chlorophenols. Group B2 indicates that although evidence in humans is inadequate, there are sufficient animal carcinogenicity data to consider 2,3,7,8-TCDD a probable human carcinogen. Group Bl indicates that there are not only sufficient animal data but also limited human data to support the consideration that 2,3,7,8-TCDD, in conjunction with phenoxyherbicides and/or chlorophenols, is a h u m a n carcinogen. IARC (1982) h a s c l a s s i f i e d 2 , 3 , 7 , 8 - T C D D in G r o u p 2B, w h i c h is a n a l o g o u s to the_CAG classification of Group B 2 . NIOSH (1984) recommended that 2,3,7,8-TCDD be considered a potential occupational carcinogen and that exposure be limited to the fullest extent feasible. EPA (1985a) developed a quantitative unit cancer risk estimate b a s e d on the study b y Kociba et al. (1978a,b) a n d a reexam i n a t i o n o f the histologic evidence from that study conducted by Squire for the EPA. The calculations were based on the increased incidence of tumors of the lungs, liver, hard palate, and nasal turbinates in rats maintained on diets containing 2,3,7,8-TCDD for 2 years. The two different pathologic examinations p r o d u c e d differences in tumor incidence and, hence, different q^* values. The final value derived, 1.56 x 105 (mg/kg/day) was an average of the values for each pathologic evaluation. Carcinogenic potency, methods used by other agencies. Both the Center for Disease Control (CDC) and the Food and Drug Administration (FDA) have calculated a virtual safe dose for 2,3,7,8-TCDD, which corresponds to a n excess cancer risk of 10"^ (Hiremath et al. 1986, K i m b r o u g h et al. 1984). The FDA calculations w e r e b a s e d on the Ko c i b a et al. (1978a,b) study, whereas the CDC calculations w ere based on the S q uire e v a l u a t i o n of the Kociba et al. (1978a,b) study. Thus, h u m a n intake values for a virtual safe dose derived b y EPA, CDC, and FDA, respectively, are 6.4, 27.6, and 57.2 fg/kg/day. 9.3 STATE Minnesota, Michigan, and New York have set additional regulations on the allowable levels of 2,3,7,8-TCDD. In addition, other states may either have regulations or are in the process of promulgating regulations. (Regulations and advisory guidance from the states were still be i n g compiled at the time of printing.) 10. R E F E R E N C E S A b e r n a t h y DJ, Greenlee WF, H b a r d JC, Boreiko CJ. 1985. 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3H/10T1/2 cells. Carcinogenesis; 6:651-653. Adams WJ, Blaine K.M. 1986. A water solubility determination of 2 , 3 , 7 , 8 -TCDD. Chemosphere; 15: 1397-1400. A d a m s WJ , D e G r a e v e GM , S a b o u r i n TD", C o n n e y J D , M o s h e r G M . 1 9 8 6 . T o x i c i t y and bioconcentration of 2,3,7,8-TCDD to fathead minnows (P im ephales p ro m e la s). Chemosphere; 15: 1503-1511. A l b r o PW, C r u m m e t t WB, D u p u y AE, Jr., G r o s s ML, Hanson, M. 1985. Meth o d s for the quantitative determination of multiple, specific polychlorinated . dibenzo-p-dioxin and dibenzofuran isomers in human adipose tissue in the parts-per-trillion range. A n interlaboratory study. Anal Chem Nov; 57(13): 2717-2725. A l d r e d JE. 1978. Report of the Consultative Council on Congenital Abnormalities in the Yarrom District. Minister of Health, Melbourne, Victoria, Aust. (Cited in EPA 1985a) *A l l e n JR, Barsotti DA, V a n Miller JP, A b r a h a m s o n LJ, L a l i c h JJ. 1977. Morphological changes in monkeys consuming a diet containing low levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fd Cosmt Toxicol; 15:401-410^ A l l e n JR, Barsotti DA, Lambrecht LK, Mil l e r JP. 1979. Reproductive effects of halogenated aromatic hydrocarbons on nonhuman primates. Ann N Y Acad Sei; 320: 419-425. (Cited in EPA 1 9 8 5 a ) . Anonymous. 1985. Dioxins in the environment: No consensus on human h a z a r d . C h e m i c a l a n d E n g i n e e r i n g News; M a y 27, p. 41-44. A u s t SD. 1984. O n the m e c h a n i s m of a n o r e x i a a n d t o x i c i t y o f T C D D and r e l a t e d compounds. B a n b u r y Rep. V o l 18, Iss B i o l M e c h D i o x i n Action; 309-319. *Key study 5007 O - t> 0 $ 86 A x e l s o n 0, S u n d e i l L, A n d e r s o n K, E d l i n g C, H o g s t e d t C, K l i n g H. 1980. Herbicide exposure and tumor mortality: An updated epidemiologic investigation on Swedish railroad workers. Scand J Work Environ Health; 6: 7 3 - 7 9 . ( C i t e d i n E P A 1 9 8 5 a ) Barnes DG. 1983. Chlorinated Dioxins W o r k Group, EPA, Washington, DC; 12(4-5): 645-655. B a r n e s D, B e l l i n i J, D e R o s a C, e t al. 1 9 8 7 . R e f e r e n c e d o s e ( R f D ) : Description and use in health risk assessments. Appendix A in Integrated R i s k I n f o r m a t i o n S y s t e m S u p p o r t i v e D o c u m e n t a t i o n , V o l u m e I. W a s h i n g t o n , DC: EPA, Office of Health and Environmental Assessment. EPA 600/8-86032a. B a u e r H, Schulz KH, S p i e g e l b e r g A. 1961. 'Berufliche V e r g i f t u n g e n b e i der herstellung von chlorphenol-verbindunger. Arch Gewerbeath Gewerbehyg; 18: 538-555. (Ger.) (Cited in E P A 1984). Beatty PW, Neal RA. 1976. Evidence for a role for DT-Diaphorase induction in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Pharmacol; 18(2):211. (Cited in EPA 1985a) J B e r r y D L ^ - D i G i o v a n n i J, J u c h a u MR, B r a c k e n WM, G l e a s o n GL, Slaga TJ. 1978. Lack of tumor-promoting ability of certain environmental chemicals in-a two-stage mouse skin tumorigenesis assay. Res Commun Chem Pathol Pharmacol; 20(1): 101-108. (Cited in EPA 1985a) B e r r y DL, S l a g a TJ, D i G i o v anni J, J u c h a u MR. 1979. Studies w i t h chlorinated dibenzo-p-dioxins, polybrominated biphenyls and polychlorinated biphenyls in a two-stage system of mouse skin tumorigenesis: Potent anticarcinogenic effects. Ann NY Acad Sei; 405-414. (Cited in EPA 1985a) 320: Birnbaum LS. 1986. Distribution and excretion of 2,3,7,8tetrachlorodibenzo-p-dioxin in congenic strains of mice which the Ah locus. Drug Metab Dispos; 14(1):34-40. differ -- at B i r n b a u m LS, W e b e r H, Harris MW, Lamb IV, JC, M c K i n n e y JD. 1985. T o x i c interaction of specific polychlorinated biphenyls and 2,3,7,8tetrachlorodibenzo-p-dioxin: Increased incidence of cleft palate in mice. Toxicol Appl Pharmacol; 77:292-302. B i r n b a u m LS, Harris MW, M i l l e r CP, Pratt RM, Lamb IV, JC. 1986. Synergistic interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone in the induction of cleft palate in mice. Teratology; 33:29-35. i B i s a n t i L, B o n e t t i F, C a r a m a s c h i F et al. 1980. E x p e r i e n c e s f r o m th e accident of Seveso. Acta Morphol Acad Sei Hung; 28(1-2): 139-157. (Cited in EPA 1985a) Bogen G . 1979. Symptoms of Vietnam veterans exposed to Agent Orange. JAMA; 242(22): 2391. (Cited in EPA 1985a) 87 B o n a c c o r s i A, F a n e l l i R, T o g n o n i G. 1 9 78. I n the w a k e of S e v e s o . Arabio; 7(5-6):234-239. (Cited in EPA 1985a) Brenner KS. 1986. Pu-foam-plug technique and extractive co-distillation (Bleidner apparatus), versatile tools for stack emission sampling and sample p r eparation. Chemosphere; 15: 1917-1922. B r o n z e t t i G, Z e i g e r E, L e e I, S u z u k i K, M a i l i n g HV. 1983. M u t a g e n i c i t y study of TCDD and ashes from urban incinerator "in vitro" and "in vivo" u s i n g yeast D7 strain. Chemosphere; 12: 549-553. (Cited in EPA 1985a) B u m p u s JA, T i e n M, W r i g h t D, A u s t SD. 1985. O x i d a t i o n of p e r s i s t e n t environmental pollutants by a white rot fungus. Science; 228(4706): 1434-1436. Buser HR. 1979. Formation and identification of tetra- and pentachlorodibenzo-p-dioxins from photolysis of two isomeric hexachlorodibenzo-p-dioxins. Chemosphere; 4:251-257. B u s e r HR, R a p p e C. 1980. H i g h - r e s o l u t i o n gas c h r o m a t o g r a p h y o f the 22 t e t r a c h l o r o d i b e n z o - p - d i o x i n isomers. A n a l Chem; 52: 2257-2262. Buser-"HR, R a p p e C. 1984. I s o m e r - s p e c i f i c s e p a r a t i o n o f 2 , 3 , 7 , 8 substituted polychlorinated dibenzo-para-dioxins by high-resolution gas-chromatography mass-spectrometry. Anal Chem; 56(3): 442-448. B u s e r HR, R a p p e C, G e r g q v i s t PA. 1985. A n a l y s i s o f p o l y c h l o r i n a t e d dibenzofurans, dioxins and related compounds in environmental samples. Anal. Chem. 60:293-301. B u u - H o i NP, P h a m - H u u - C h a n h , S e s q u e G, A z u m - G e l a d e MC, S a i n t - R u f , G. 1972. Organs as targets of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) intoxication. Naturwissenschaften; 5 9 ( 4 ) :174-175. (Cited in EPA 1985a) C h o u d h r y GG, H u t z i n g e r 0. 1982. P h o t o c h e m i c a l f o r m a t i o n a n d d e g r a d a t i o n o f p o l y c h l o r i n a t e d d i b e n z o f u r a n s a n d d i b e n z o - p - d i o x i n s . R e s Rev; 84: 113-161. C l e m e n t RE, B o l e l i e B, T a g u c h i , V. 1986. C o m p a r i s o n o f i n s t r u m e n t a l methods for chlorinated dibenzo-p-dioxin (CDD) determination interim results of a round-robin study involving GC-MS, MS-MS and high r e s o l u t i o n MS. Chemosphere; 15: 1147-1156. C o c k e r h a m LG, Y o u n g AL, T h a l k e n CE. 1980. His t o p a t h o l o g i c a l and ultrastruetural studies of liver tissue from TCDD-exposed beach mice (P erow yscus p o l-io n o tu s ). J A D -A083 323/6 PC A 0 4 / M F A01. 61 p. (Cited in EPA 1985a) Co h e n GM, Bracken WM, Iyer RP, Berry DL, Selkirk JK, Slaga T J . 1979. Anticarcinogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene tumor initiation and its relationship to DNA binding. Cancer Res; 39(10): 4027-4033. (Cited in EPA 1985a) 5 0 0 9 D ' = ' 110 88 C o o k RR. 1981. Dioxin, c h l o r a c n e a n d s o f t - t i s s u e sarcoma. L a n c e t J; 618-619. (Cited in EPA 1985a) C o o k RR, T o w nsend JC, Ott MG, S ilverstein LG. 1980. M o r t a l i t y experience of employees exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). J O c c u p Med; 22; 530-532. (Cited in E P A 1985a) C o o k RR, Bond GG, Olsen RA, Ott MG. 1985. 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Airborne dioxins and d i b e n z o f u r a n s : Sources and fates. Environ Sci Technol; 20(2): 195-200. * D e C a p r i o AP, M c M a r t i n DM, O ' K e e f e PW, Rej R, S i l k w o r t h JB, K a m i n s k y LS. 1986. Subchronic oral toxicity of 2,3,7,8-tetrachlor o d i b e n z o - p - d i o x i n in the guinea pig: Comparisons with a PCB-containing transformer fluid pyrolysate. Fund Appl Toxicol; 6:454-463. D e n c k e r L, Pratt RM. 1981. A s s o c i a t i o n b e t w e e n the p r e s e n c e of the A h r e c e p t o r in em b r y o n i c m u r i n e tissues a n d s e n s i t i v i t y to TCDD-i n d u c e d cleft palate. Teratog Carcinog Mutag; 1:399-406. D e n c k e r L, H a s s o u n E, D ' A r g y R, A i m G. 1985. F e t a l t h y m u s o r g a n c u l t u r e as an in vitro model for the toxicity of 2,3,7,8-TCDD and its congeners. M o l Pharmacol; 27: 133-140. Dept, of Health, New Zealand. 1980. Report to the Minister of Health of an Investigation into allegations of an association between human congenital defects and 2,4,5-T spraying in and around teKuiti. New Z e a l a n d M e d J; 314-315. (Cited in EP A 1985a) ' des R o s i e r s PE. 1986. Metho d o l o g i e s for materials c o n t a m i n a t e d w i t h PCDDs and related compounds. Chemosphere; 15: 1513-1528. C, O' o io 89 D i L e m i a R, C r i m a u d o C, P a c c h e t t i G. 1982. T h e s t u d y o f x - r a y s a n d T C D D effects on satellite associations may suggest a simple model for application in environmental mutagenesis. Hum Genet; 61(1): 42-47. (Cited in EPA 1985a) Donnelly JR, V o n n a h m e TL, H e d i n CM, Niedenhut W J . 1986. Evaluation of R C R A m e t h o d 8 2 8 0 f o r a n a l y s i s o f d i o x i n s a n d d i b e n z o f u r a n s . R a p p e C, C h o u d h a r y G, K e i t h LH, eds. C h l o r i n a t e d D i o x i n s a n d D i b e n z o f u r a n s in Perspective. 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C a s e - c o n t r o l study on malignant mesenchymal tumors of the soft-tissue and exposure to c h e m i c a l substances. Lakartidningen; 76: 3872-3875. (Cited in E P A 1985a) E r i k s s o n M, H a r d e l l L, O ' B e r g N, M o l l e r T, A x e l s o n 0. 1981. S o f t - t i s s u e sarcomas and exposure to chemical substances: A case-referent study. Br J Ind Med; 38: 27-33. (Cited in EPA 1985a) 5011 90 F a i r l e s s B J , Bates D I , H u d s o n J, K l e o p f e r RD, et al. 1987. P r o c e d u r e s used to measure the amount of 2 , 3 , 7 , 8 -Tetrachlorodibenzo-p-dioxin in the ambient air near a Superfund site cleanup operation. Environ Sci Techno1 ; 21:550-555. Fa l k H, Stehr PA, S t e i n GF, et al. 1984. A p i l o t e p i d e m i o l o g y s t u d y of health effects due to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) c o n t a m i n a t i o n in Missouri. B a n b u r y Rep. V o l 18, Iss Biol M e c h D i o x i n Action; pp. 447-460. 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Freeman RA, Schroy JM. 1986. Modeling the transport of 2,3,7,8-TCDD and other low volatility chemicals in soils. Environ Progress; 5(1): 28-33. F r e e m a n RA, Schroy JM, H i l e m a n FD, Noble RW. 1986. Environmental mobility of 2,3,7,8-TCDD and comparison chemicals in a roadway soil m a t r i x . R a p p e C, C h o u d h a r y G, K e i t h LH, eds. C h l o r i n a t e d D i o x i n s a n d Dibenzofurans in Perspective. Chelsea, MI: Lewis Publishers, Inc.; 171183. Friedman JM. 1984. Does Agent Orange Cause Birth Defects? Teratology; 29:193-221. Fries GF, Marrow GS. 1975. Retention and excretion of 2,3,7,8tetrachlorodibenzo-p-dioxin by rats. J Agric Food Chem; 23(2): (Cited in EPA 1985a) 265-269. F u e r s t P, M e e m k e n HA , G r o e b e l W. 1 9 8 6 . D e t e r m i n a t i o n o f p o l y c h l o r i n a t e d dibenzodioxins and dibenzofurans in h u m a n milk. Chemosphere; 15: 19771980. f 5012 91 G asiewicz TA, O l s o n JR, G eiger LE, Neal RA. 1983a. 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Chemosphere; 15:1495-1502. in G i l b e r t P, S a i n t - R u f G, P o n c e l e t F, M e r c i e r , M. 1980. G e n e t i c e f f e c t s of chlorinated anilines and azobenzenes on S alm onella typhim urium . Arch Environ Contam Toxicol; 9(5): 533-541. (Cited in EPA 1985a) Giri AK. 1986. Mutagenic and genotoxic effect of 2,3,7,8tetrachlorodibenzo-p-dioxin, a review. Mutat Res; 168:241-242. G r a h a m M, H i l e m a n FD, O r t h RG, W e n d l i n g JM, W i l s o n JW. 1986. Chlorocarbons in adipose tissue from a Missouri population. Chemosphere; 15: 1595-1600. G r e e n S, M o r e l a n d FS. 1 9 7 5 . C y t o g e n e t i c e v a l u a t i o n o f s e v e r a l d i o x i n s i n th e rat. T o x i c o l A p p l P h a r m a c o l ; 33: 161. ( C i t e d i n E P A 1985a) G r e e n S, M o r e l a n d , S h e u C. 1 9 7 7 . C y t o g e n i c e f f e c t o f 2 , 3 , 7 , 8 tetrachlorodibenzo-p-dioxin on rat bone marrow cells. FDA, Washington, DC. F D A B y - L i n e s ; 6: 292. ( C i t e d in E P A 1 9 8 5 a ) G r e e n l e e WF, O s b o r n e R, H u d s o n LG, T o x c a n o W A Jr. 1984. S t u d i e s o n the m e c h a n i s m s o f t o x i c i t y of T C D D to h u m a n epidermis. B a n b u r y Rep; 18 (Biol. Lech. Dioxin A ction): 365-372. G r e e n l e e WF, D o l d KM, Irons RD, Osborne R. 1985. E v i d e n c e for dire c t a c t i o n of 2 , 3 , 7 , 8 - T C D D o n thy m i c epithelium. T o x i c o l A p p l Pharmacol; 79: 112- 120. Greig JB. 1979.-The toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin and its structural analogues. Ann Occup; 22:411-420. (Cited in EPA 1985a)* * G r e i g J. 1984. D i f f e r e n c e s b e t w e e n sk i n and l iver t o x i c i t y of 2,3,7,8tetrachlorodibenzo-p-dioxin in mice. Banbury Rep; 18 (Biol Mech Dioxin Action): 391-397. 92 *Gross ML, Lay Jr, JO, Lyon PA, et al. 1984. 2,3,7,8Tetrachlorodibenzo-p-dioxin levels in adipose tissue of Vietnam Veterans. Environ Res; 33:261-268. H a g e n m a i e r H. 1986. D e t e r m i n a t i o n o f 2 , 3 , 7 , 8 - T C D D i n c o m m e r c i a l chlorophenols and related products. Fresenius Z Anal Chem; 325: 603-606. H a g e n m a i e r H, K r a f t M, J a g e r W, M a y e r U, L u t z k e K, S i e g e l D. 1986. Comparison of various sampling methods for PCDDs and PCDFs in stack g a s . Chemosphere; 15: 1187-1192. H a n i f y JA, M e t c a l f P, N o b b s CL, W o r s l e y RJ. 1981. A e r i a l s p r a y i n g o f 2.4.5- T and human birth malformations: An epidemiological investigation. Science; 212:349-351. (Cited in EPA 1985a) H a r d e l l L, S t a n d s t r o m A. 1979. C a s e - c o n t r o l study: S o f t - t i s s u e s a r c o m a s a n d e x p o s u r e to p h e n o x y a c e t i c a cids or c h l o r o p h e n o l s . B r J Cancer; 39: 711-717. (Cited in EPA 1985a) H a r d e l l L, E r i k s s o n M, L e n n e r P. 1980. M a l i g n a n t l y m p h o m a a n d e x p o s u r e to chemical substances, especially organic solvents, chlorophenols and p h e n o x y - a c i d s . Lakartidningen; 77: 208-210. (Cited in E P A 1985a) H a r d e l l L, E r i k s o n M, L e n n e r P, L u n d g r e n E. 1981. M a l i g n a n t l y m p h o m a a n d exposure to c hemicals, especially organic s o l v e n t s , chlorophenols and phenoxy acids: A case-control study. Br J Cancer; 43: 169-176. (Cited in EPA 1985a) H a y A. 1982. T o x i c o l o g y of dioxins. In: T h e C h e m i c a l Scythe: L e s s o n s of 2.4.5- T and Dioxin. NY, London: Plenum Press; 41-47. (Cited in EPA 1985a) H e a t h RG, Harless R L Gross ML, et al. 1986. D e t e r m i n a t i o n of 2,3,7,8tetrachlorodibenzo-p-dioxin in human milk at the 0.1-10 partspertrillion level: Method va l i d a t i o n and survey results. A nal Chem (USA) 58(2): 463-468. Henck JW, New MA, Kociba RJ, Rao K S . 1981. 2 , 3 ,7,8-Tetrachlorodibenzop-dioxin: Acute oral toxicity in hamsters. Toxicol Appl Pharmacol; 59:405-407. (Cited in EPA 1985a) H i r e m a t h C, B a y l i s s D, B a y a r d S. 1986. Q u a l i t a t i v e a n d q u a n t i t a t i v e cancer risk assessment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8TCDD). Chemosphere; 15:1815-1823. H o f f m a n RE, Stehr - G r e e n PA, W e b b KB, et al. 1986. H e a l t h effects long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxini JAMA; 2031-2038. of 255: H o l d e n C. 1979. A g e n t O r a n g e f u r o r c o n t i n u e s to b u i l d . S c i e n c e ; 205: 77-772. (Cited in EPA 1985a) / Vi* 93 H o l m s t e d t , B. 1980. P r o l e g o m e n a to S e v e s o E c c l e s i a s t e s I 18. A r c h Toxicol; 44(4): 211-230. (Cited in EPA 1985a) HSDB (Hazardous Substances Data Bank). 1987. National Library of M e d i c i n e . O n - l i n e p r i n t - o u t . A p r i l 6, 1987. H u e t t e r R, P h i l l i p p i M. 1982. S t u d i e s o n m i c r o b i a l m e t a b o l i s m of T C D D u n d e r l a b o r a t o r y c o n d i t i o n s . P e r g a m o n S e r E n v i r o n S c i ; 5: 8 7 - 9 3 . ( C i t e d in EPA 1985a) H u s s a i n S, E h r e n b e r g L, L o f r o t h G, G e j v a i l T. 1 9 7 2 . M u t a g e n i c e f f e c t s o f T C D D o n b a c t e r i a l s y s t e m s . A m b i o ; 1: 32-33. ( C i t e d i n E P A 1 9 8 5 a ) H u t z i n g e r 0, B l u m i c h MJ, V a n d e r b e r g M, O l i e K. 1985. S o u r c e s a n d fa t e o f PCDDs and PCDFs: A n overview. Chemosphere; 14: 581-600. IARC ( I n t e m a t i o n Agency for Research on Cancer). 1982. World Health Organization-1ARC monographs on the Evaluation of the Carcinogenic Risk o f C h e m i c a l s to H u m a n s . L y o n s , F r a n c e : W HO; I A R C V o l . 1 - 2 9 , Supp l . 4; 238-243. 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A n a l y s i s o f p o l y c h l o r i n a t e d d i o x i n s a n d fu r a n s . A l l 75 PCDDs and 135 PCDFs can be identified by isomer-specific techniques. Environ Sci Technol; 18(3): 78A-90A. R a p p e C, N y g r e n M, S. M a r k l u n d , e t al. 1 9 85. A s s e s s m e n t o f h u m a n exposure to polychlorinated dibenzofurans and dioxins. Environ-Health Perspect; 60: 303-304. 5023 102 R a p p e C, M a r k l u n d S, K j e l l e r LO, T y s k l i n d M. 1 9 86. P C D D s a n d P C D F s in e m i s s i o n s from v a r i o u s inciner a t o r s . Chemos p h e r e ; 15: 1213-1218. R e g g i a n i G. 1980. A c u t e h u m a n e x p o s u r e to T C D D in Seveso, Italy. J Toxicol Environ Health; 6(1): 27-43. (Cited in EPA 1985a) R i i h i m a k i V, S i s k o A, H e m b e r g S. 1982. M o r t a l i t y o f 2,4dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid h e r b i c i d e a p p l i c a t o r s i n F i n l a n d . S c a n d J W o r k E n v i r o n H e a l t h ; 8: 37-42. (Cited in EPA 1985a) R o b e r t s EA, Shear NH, O k e y AB, M a n c h e s t e r DK. 1985. The A h r e c e p t o r and dioxin toxicity: From rodent to hum a n tissues. Chemosphere; 14:661-674. Rogers AM, Anderson ME, Back KC. 1982. Mutagenicity of 2,3,7,8tetrachlorodibenzo-p-dioxin and perfluoro-n-decanoic acid in L5178y mouse lymphoma cells. Mutat Res; 105:445-449. (Cited in EPA 1985a) R o gers CJ, Kornel A, P e t e r s o n RL. 1987. M o b i l e KP E G d e s t r u c t i o n u n i t for P C B s , dioxins, and furans in contaminated wastes. Presented at the 13th Annual Research Symposium: Land Disposal, Remedial Action. Incineration and Treatment of Hazardous Wastes, Cincinnati, OH. M a y 6-8, 1987. R o bens JF, Z abik M. n.d. Triple q u a d r a p o l e mass s p e c t r o m e t r y a n d R M temp phosphoresence-dioxin residue. Michigan State University. R o n d o r f B. 1986. Ther m a l p r o p e r t i e s o f dioxins, furans a nd r e l a t e d c o m p o u n d s . C h e m o s p h e r e ; 15: 1 3 25-1332. R o z m a n K. 1984. S e p a r a t i o n of w a s t i n g syndrome and l e t h a l i t y c a u s e d b y 2.3.7.8- tetrachlorodibenzo-p-dioxin. Toxicol Lett; 22:279-285. R o z m a n K, G r e i m H. 1986. T o x i c i t y o f 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n in cold-adapted rats. Arch Toxicol; 59:211-215. R o z m a n K, R o z m a n T, S c h e u f l e r E, P a z d e m i k T, H G r e i m . 1985. T h y r o i d hormones modulate the toxicity of 2,3,7,8-TCDD. J Toxicol Environ H e alth; 16: 481-491. R y a n JJ. 1987. Food Research Division, Health Protection Branch, Ottawa, Canada. Personal telephone communication to DK Basu, Syracuse Research C o r p o r a t i o n , NY. S e p t e m b e r 2, 1 9 8 7 . R y a n JJ, L a u PY, Pilon JC, et a l . 1984. Incidence a n d levels o f 2.3.7.8- tetrachlorodibenzo-p-dioxin in Lake Ontario commercial fish. Environ Sci Technol; 18(9): 719-721. / K e y s t u d y R y a n JJ, L i z o t t e R, S a k u m a T, M o r i B. 1985ba. C h l o r i n a t e d d i b e n z o - p a r a - d i o x i n s , chlorinated dibenzofurans and pentac h l o r o p h e n o l in Canadian chicken and pork samples. J Agric Food Chem; 33(6): 1021-1026. 103 R y a n JJ, S c h e c t e r A, L i z z o t e R, S u n WF, M i l l e r L. 1985bb. T i s s u e distribution of dioxins and furans in humans from the general p opulation. C h e m o s p h e r e ; 14: 929-932. R y a n JJ, L i z o t t a R, L a u BPY. 1985bc. C h l o r i n a t e d d i b e n z o - p - d i o x i n s a n d chlorinated dibenzofurans in Canadian human adipose tissue. Chemosphere; 14: 697-706. S a c c h i GA, V i g a n o P, F o r t u n a t i G, C o c u c c i SM. 1986. 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E f f e c t o f h e r b i c i d e 2,4,5-trichlorophenoxyethanol (TCPE) containing dioxin on mutation and induction of sister chromatid exchanges. Carcinogenesis; 5:1725-1728. (Cited in Giri 1986) Turner JN, Collins DN. 1983. Liver morphology in guinea pigs administered either pyrolysis products of a polychlorinated biphenyl transformer fluid or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl Pharmacol; 67: 417-429. U m b r e i t TH, P a t e l D, G a l l o MA. 1985. A c u t e t o x i c i t y o f T C D D c o n t a m i n a t e d soil from an i n d u s t r i a l site. Chemosphere; 14: 945-947. Umbreit TH, Hesse EJ, Gallo MA. 1986a. Bioava i l a b i l i t y of dioxin in soil from a 2,4,5-T manufacturing site. Science; 232: 497-499. ymbreit TH, Hesse E J , Gallo MA. 1986b. Comparative toxicity of TCDD contaminated soil from Times Beach, Missouri, and Newark, New Jersey. Chemosphere; 15: 2121-2124. V a n Miller JP, L a l i c h JJ, A l l e n Jr. 1977a. 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GLOSSARY Acute Exposure--Exposure to a chemical for a duration of 14 days or less, as specified in the Toxicological Profiles. Bioconcentration Factor (BCF)--The quotient of the concentration of a chemical in aquatic organisms at a specific time or during a discrete time period of exposure divided by the concentration in the surrounding water at the same time or during the same time period. Carcinogen--A chemical capable of .inducing cancer. Ceiling value (CL)--A concentration of a substance that should not be exceeded, even instantaneously. C hronix E x p o s u r e - - E x p o s u r e to a chemical for 365 days or more, as specified in the Toxicological Profiles. Developmental Toxicity--The occurrence of adverse effects on the developing organism that may result from exposure to a chemical prior to conception (either parent), during prenatal development, or postnatally t o t h e t i m e o f s e x u a l .m a t u r a t i o n . A d v e r s e d e v e l o p m e n t a l e f f e c t s m a y b e detected at any point in the life span of the organism. E m b r y o t o x i c i t y a nd F e t o t o x i c i t y - - A n y toxic effe c t on the conceptus as a result of prenatal exposure to a chemical; the distinguishing feature b e t w e e n t h e t w o t e r m s is t h e s t a g e o f d e v e l o p m e n t d u r i n g w h i c h t h e -----insult occurred. The terms, as used here, include m a l f o rmations and variations, altered growth, and in tero death. Frank Effect Level (FEL)--That level of exposure which produces a statistically or biologically significant increase in frequency or severity of unmistakable adverse effects, such as irreversible f u n c t i o n a l i m p a i r m e n t or. m o r t a l i t y , i n a n e x p o s e d p o p u l a t i o n w h e n compared with its appropriate control. EPA Health Advisory--An estimate of acceptable drinking water levels for a chemical substance based on health effects information. A health a d v i s o r y is n o t a l e g a l l y e n f o r c e a b l e f e d e r a l s t a ndard, b u t serves as technical guidance to assist federal, state, and local officials. Immediately Dangerous to Life or Health (IDLH)--The maximum environmental concentration of a contaminant from which one could escape within 30 min without any escape-impairing symptoms or irreversible health effects. 5031 110 Intermediate Exposure--Exposure to a chemical for a duration of 15-364 days, as specified in the Toxicological Profiles. Immunologic Toxicity--The occurrence of adverse effects on the immune sys t e m that m a y res u l t from exposure to environ m e n t a l agents such as chemicals. In vitro--Isolated from the living organism and artificially maintained, as in a test tube. In vivo--Occurring within the living organism. Key Study--An animal or human toxicological study that best illustrates the nature of the adverse effects produced and the doses associated with those effects. Lethal Concentration(LO) (LCLo)--The lowest concentration of a chemical in air which has b e e n reported to have caused death in humans or animals. Lethal Concentration(50) (LCso)--A calculated concentration of a c h e m i c a l in a i r to w h i c h e x p o s u r e fo r a s p e c i f i c l e n g t h of t i m e is expected to cause death in 50% of a defined experimental animal population. . Lethal Dose(LO) (LDLO)--The lowest dose of a chemical introduced by a route other than inhalation that is expected to have caused death in humans or animals. Lethal Dose(50) (LD50)--The dose of a chemical which has been calculated to cause death in 50% of a defined experimental animal population. Lowest-Observed-Adverse-Effect Level (LOAEL)--The lowest dose of chemical in a study or group of studies which produces statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control. Lowest-Observed-Effect Level (LOEL)--The lowest dose of chemical in a study or group of studies which produces statistically or biologically significant increases in frequency or severity of effects between the exposed population and its appropriate control. Malformations--Permanent structural changes that may adversely affect survival, development, or function. Min i m a l Risk L e v e l - - A n estimate of daily h u m a n exposure to a chemical that is likely to b e ' w i t h o u t an appreciable risk of deleterious effects (noncancerous) over a specified duration of exposure. ' M u t a g e n - - A substance that causes mutations. A m u t a t i o n is a change the genetic material in a body cell. Mutations can lead to birth defects, miscarriages, or cancer. in Ill Neurotoxicity--The occurrence of adverse effects on the nervous system following exposure to a chemical. No-Observed-Adverse-Effect Level (NOAEL)--That dose of chemical at which there are no statistically or biologically significant increases in frequency or severity of adverse effects seen between the exposed population and its appropriate control. Effects may be produced at this dose, but they are not considered to be adverse. No-Observed-Effect Level (NOEL)--That dose of chemical at which there are no statistically or biologically significant increases in frequency or severity of effects seen between the exposed population and its appropriate control. Permissible Exposure Limit (PEL)--An allowable exposure level in workplace air averaged over an 8-h shift. qj*--The upper-bound estimate of the low-dose slope of the dose-response curve as determined b y the multistage procedure. The q^* can be used to calculate an estimate of carcinogenic potency, the incremental excess cancer risk per unit of exposure (usually pg/L for water, mg/kg/day for food, and pg/m^ for a i r ) . Reference Dose (RfD)--An estimate (with uncertainty spanning perhaps an order of magnitude) of the daily exposure of the h u m a n population to a potential h a z a r d that is likely to be wit h o u t risk of deleterious effects during a lifetime. The RfD is operationally d e r ived from the NOAEL (from animal and human studies) b y a consistent application of uncertainty factors that reflect various types of data used to estimate RfDs and an additional m o d i f y i n g factor, w h i c h is b a s e d on a professional judgment of the entire database on the chemical. The RfDs are not applicable to nonthreshold effects such as cancer. R e p o r t a b l e Q u a n t i t y ( R Q ) - - T h e q u a n t i t y o f a h a z a r d o u s s u b s t a n c e t h a t -- i s --c o n s i d e r e d r e p o r t a b l e u n d e r C E R C L A . R e p o r t a b l e q u a n t i t i e s are: (1) 1 lb o r g r e a t e r o r (2) f o r s e l e c t e d s u b s t a n c e s , a n a m o u n t e s t a b l i s h e d b y regulation either under CERCLA or under Sect. 311 of the Clean Water Act. Quantities are measured over a 24-h period. Reproductive Toxicity--The occurrence of adverse effects on the reproductive system that may result from exposure to a chemical. The toxicity may be directed to the reproductive organs and/or the related endocrine system. The mani f e s t a t i o n o f such toxicity m a y be n o t e d as alterations in sexual behavior, fertility, pregnancy outcomes, or modifications in other functions that are dependent on the integrity of this system. t Short-Term Exposure Limit (STEL)--The maximum concentration to which wor k e r s can b e . e x p o s e d for up to 15 m i n continually. No more than four excursions are allowed per day, and there must be at least 60 min between exposure periods. The daily TLV-TWA may not be exceeded. 5033 112 Target Organ Toxicity--This term covers a broad range of adverse effects on target organs or physiological systems (e.g., renal, cardiovascular) e x t e n d i n g from those ar i s i n g thro u g h a single limited exposure to those assumed over a lifetime of exposure to a chemical. Teratogen--A chemical that causes structural defects that affect the development of an organism. Threshold Limit Value (TLV)--A concentration of a substance to which most workers can be exposed without adverse effect. The TLV may be e x p r e s s e d as a TWA, as a STEL, o r as a CL. Time-weighted Average (TWA)--An allowable exposure concentration averaged over a normal 8-h workday or 40-h workweek. Uncertainty Factor (UF)--A factor used in operationally deriving the RfD f r o m e x p e r i m e n t a l data. UFs a r e i n t e n d e d to a c c o u n t f o r (1) the variation in sensitivity among the members of the human population, (2) the u n c e r t a i n t y in e x t r a p o l a t i n g a n i m a l d a t a to the c a s e o f h u m a n s , (3) the u n c e r t a i n t y in e x t r a p o l a t i n g f r o m d a t a o b t a i n e d in a s t u d y that is o f less t h a n l i f e t i m e e x p o s u r e , a n d (4) the u n c e r t a i n t y in u s i n g L O A E L d a t a rat h e r than N O A E L data. U s u a l l y each o f these factors is set e q u a l to 10. APPENDIXES 5035 2$ 9 '5' APPENDIX A: PEER REVIEW A peer review panel was assembled for 2,3,7,8 tetrachlorodibenzop - d i o x i n ( T C D D ) . The p anel c o n s i s t e d of the f o l lowing members: Dr. Herbert Cornish, University of Michigan; Dr. Shane Que Hee, University of Cincinnati M e d ical Center; and Dr. James Olson, State Univer s i t y of New York at Buffalo, School of Medicine. These experts collectively have knowledge of 2,3,7,8-TCDD's physical and chemical properties, toxicokinetics, key health end points, mechanisms of action, human and animal exposure, and quantification of risk to humans. All reviewers were selected in conformity with the conditions for peer review specified in the Superfund Amendments and Reauthorization Act of 1986, Section 110. A joint panel of scientists from A T S D R and EPA has reviewed the peer reviewers' comments and determined which comments will be included in the-profile. A listing of the peer reviewers' comments not incorporated into the profile, with a brief explanation of the rationale __ f o r t h e i r e x c l u s i o n , e x i s t s a s p a r t o f t h e a d m i n i s t r a t i v e r e c o r d f o r this compound. A list of databases reviewed and a list of unpublished documents cited are also included in this record. 115 >-531 APPENDIX B: FEDERAL REGISTER ANNOUNCEMENT DEPARTMENT OF HEALTH AND HUMAN SERVICES AGENCY FOR TOXIC SUBSTANCES AND DISEASE REGISTRY ENVIRONMENTAL PROTECTION AGENCY ( A T S D R - 2; F R L - 3 2 6 9 - 7 ) NOTICE OF AVAILABILITY OF TOXICOLOGICAL PROFILES AGENCIES: Department of Health and Human Services (DHHS): Agency for Toxic Substances and Disease Registry (ATSDR); and Environmental Protection Agency (EPA). ACTION: Notice. SUMMARY: The Superfund Amendments and Reauthorization Act (SARA) (Public Law 99-499) amends the Comprehensive Environmental Response, - Compensation, and Liability Act (CERCLA or Superfund) (42 U.S.C. 9601 et seq.) by establishing certain requirements for the Agency for Toxic Substances and Disease Registry (ATSDR) of DHHS and EPA wit h regard to hazardous substances which are most commonly found at facilities on the CERCLA National Priorities List (NPL). Among these statutory r e q u i r e m e n t s is a mand a t e for the A d m i n i s t r a t o r of A T S D R to prep a r e toxicological profiles for each substance previously included on the first priority list of 100 chemicals. The list identified the first 100 chemicals which both Agencies determined posed the most significant potential threat to human health. This list was published in the Federal Register on April 17th, 1987 (52 FR 12866) as required b y SARA section 110. This notice announces the expected availability dates of the first 25 draft toxicological profiles for review and comment. 5039 117 118 AVAILABILITY: The following draft toxicological profiles are expected to be publicly available by the date indicated: Date/Proflie CAS # O c t o b e r 17, 1987: Benzo(a)anthracene Benzo(a)pyrene Beryllium Chloroform Chromium Chrysene D i b e n z o ( a ,h ) a n t h r a c e n e Heptachlor/Heptachlor epoxide Nickel N-Nitrosodiphenylamine 56-55-3 50-32-8 7440-41-7 67-66-3 7440-47-3 218-01-9 53-70-3 76-44-8 / 1024-57-3 7440-02-0 86-30-6 O c t o b e r 29, 1987: Aldrin/dieldrin Arsenic B e n z o ( b )fluoranthene PCRs - Aroclor 1260, 1254, 1248, . 1242, 1232, 1221, 1016 2,3,7,8- Tetrachlorodibenzo-p-dioxin 309-00-2 / 60-57-1 7440-38-2 205-99-2 11096-82-5, 11097-69-1, 53469-21-9, 11141-16-5, 12674-11-2 1746-01-6 12672-29-6 11104-28-2 N o v e m b e r 5, 1987 Benzene Bis(2-ethylhexyl)phthalate Cadmium 1,4-Dichlorobenzene Methylene chloride 71-43-2 117-81-7 7440-43-9 106-46-7 75-09-2 N o v e m b e r 30, 1987 Cyanide Lead Tetrachloroethylene Trichloroethylene Vinyl chloride 57-12-5 7439-92-1 127-18-4 79-01-6 75-01-4 119 A full 90-day public comment period will be provided for each profile, starting from the actual release date. The close of the comment period for each draft profile will be indicated on the front of each profile. R e q u e s t s for d r a f t t o x i c o l o g i c a l p r o f i l e s s h o u l d b e s e n t to: Ms. Georgi Jones Director, Office of External Affairs Agency for Toxic Substances and Disease Registry Chamblee 28 South > : 1600 Clifton Rd. - Atlanta, GA 30333 Specify the profiles you wish to review. One copy of each profile requested will be forwarded, free of charge, as they become available. In the case of undue delays, requestors will be notified. Five copies of all comments should be sent to Ms. Jones at the above address by the end of the comment period. All written comments and the draft profiles will be available for public inspection at the Agency for Toxic Substances and Disease Registry (ATSDR), Building 28 South, R o o m \103, 4770 Buford Highway, NE, Chamblee, GA, from 8am to 4:30pm, Monday through Friday, except legal holidays. Written comments and other ,,-data submitted in response to this notice and the draft toxicological ; profiles should bear the docket control number ATSDR-2.^ 5041 120 SUPPLEMENTARY INFORMATION: I . BACKGROUND O n O c t o b e r 17, 1986, the P r e s i d e n t s i g n e d the Sup e r f u n d A m e n d m e n t s and Reauthorization Act of 1986 (Public Law 99-499), which extends and amends the Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA or Superfund, 42 U.S.C. 9601 et seq.). Section 110 of SARA amends section 104(i) of CERCLA by establishing r e q u i r e m e n t s for the p r e p a r a t i o n of: (1) lists of hazardous subst a n c e s in o r d e r o f p r i o r i t y , (2) t o x i c o l o g i c a l p r o f i l e s o f those s u b s t a n c e s , a n d (3) a r e s e a r c h p r o g r a m to fill d a t a gaps a s s o c i a t e d w i t h the substances. In compliance with section 104(i)(2)(A) of CERCLA, ATSDR and EPA p u b l i s h e d o n A p r i l 17, 1987 (52 F R 12866) the first priority list of 100 hazardous substances. This priority list of 100 was further broken down into four groups of 25 chemicals. The first group of 25 was to be the subject of the second phase of the requirements, i.e., the development of the first set of toxicological profiles. Section 104(i)(3) of CERCLA spells out the content of these profiles and the timetable by which they must be developed. Profiles on at least 25 substances on the first priority list were to be completed within one year of the enactment of S A R A (by^October 17, 1987). The r e m a i n i n g seventy-five are to be completed at a rate of at least twenty-five per year with the total 100 completed within four years after the enactment of the SARA amendments. R e v i s i o n a n d r e p u b l i c a t i o n is m a n d a t e d as n e c e s s a r y but no less often than once every three years. Each profile is required to include an examination, summary and interpretation of available toxicological information and epidemiologic e v a l u a t i o n s . This information and data are to be used to ascertain the levels of significant human exposure for the substance and the associated health effects. The profiles must also include a determination of whether adequate information on the health effects of -- each substance is available or in the process of development. The Agencies' intention is that this information be u s e d to identify the key toxicological testing needs that w h e n filled will improve our ability to define significant human exposure levels. The toxicological profiles are to be provided to the States and made available to the public. The profiles are to be prepared in accordance with the guidelines developed b y A T S D R and EPA. These guidelines were published along with the priority list of 100 in the A p r i l 17, 1987 Federal R e g i s t e r N o t i c e (52 FR 12870). This current notice announces the projected availability dates of the first 25 draft toxicological profiles. The documents have undergone extensive internal review and have been subject to scientific and technical peer review by outside experts. We are now announcing their availability and encouraging public participation and comment on the further development of these profiles. Although the profiles will not be c o m p l e t e d b y the O c t o b e r 17, 1987 dea d l i n e , w e b e l i e v e that the e x t r a time g i v e n to p e e r r e v i e w and p u b l i c r e v i e w and comment is im p o r t a n t to the development of quality profiles of scientific merit. 5042 121 Although we are reasonably confident that the key studies for each of the 25 substances were considered during the profile development process, this Federal R e g iste r notice solicits any significant studies, including unpublished data, which may aid the revision of these draft profiles. II. LEVELS O F SIGNIFICANT H U M A N EXPOSURE The setting of specific levels of significant human exposure has presented a unique set of problems. The significance of a specific level o f a h a z a r d o u s s u b s t a n c e d e p e n d s o n the c o n t e x t i n w h i c h that level is evaluated. For example, a low level that may be insignificant with respect to causing acute, immediately debilitating symptoms may be highly significant with respect to causing gradual, chronic effects over a longer term. Since these profiles are intended for use b y a diverse group of people who have different situations in wh i c h to interpret the significance of specific l e v e l s , it was considered appropriate at this time to describe the range of exposures over which effects m ay occur ( w h e r e d a t a a r e a v a i l a b l e ) , a n d to' a l l o w t h e u s e r t o m a k e d e t e r m i n a t i o n s as to which type of effect is significant in any particular instance. A format for graphically displaying the levels of significant human exposure has b e e n developed and is used in the profiles to present the ranges-over which effects may be observed. We encourage public comment and recommendations on this specific ''issue. III. SOLICITATION OF PUBLIC COMMENT We are soliciting public comment on all phases of the development of the toxicological profiles. A previous Federal Register notice, p u b l i s h e d o n A p r i l 17, 1987 (52 F R 12866) s o l i c i t e d c o m m e n t on the first priority list of hazardous substances. We are currently reviewing those comments and are evaluating the impact that those comments may have on the priority list and the methods used in its development. As the first 25 toxicological profiles become available in draft form, we are eager to provide them to the States, industry, public health professionals, scientists and the general public. We welcome comment and feedback on the content of the profiles; the format and scope of the documents; the process used in the development of the levels of significant human exposure and the overall process used in the development of the profiles. There are specific items that we would like to draw to the attention of the reader and would strongly encourage as candidates for close attention during the comment period. A. PUBLIC HEALTH STATE M E N T / The d r a f t p r o f i l e s include a p u b l i c h e a l t h s t a t e m e n t w h i c h is intended to provide the lay public with a concise statement of the general health risks associated with the chemical of concern. The summary as originally planned should be able to stand alone. If removed -from the rest of the document, it should still be capable of conveying - 5043 122 to the public the substantive health concerns associated with the substance. We are also considering the development of more abbreviated versions of the public health statements and are evaluating a number of different f o r m a t s . This notice specifically invites comments on the existing public health effects statements in the draft profiles and solicits recommendations for alternative approaches. B. D A T A / S T U D I E S U S E D I N T H E D E V E L O P M E N T O F T H E P R O F I L E S In general, and for each chemical-specific profile, have the appropriate studies been used in the development of these documents? Our c o n c e r n here is that we capture the c r i t i c a l , or " k e y " , studies but not miss other data that may be important in the valid evaluation of the toxicological profile chemicals. C. F O R M A T A N D C O N T E N T O F T H E P R O F I L E S The draft profiles represent our best effort to provide the information required by Section 104 (i)(3) of CERCLA in the most useful format for the various identified users of the p r o f i l e s , given the constraints of the tight timeframe. Every effort has be e n made to define sections clearly and to format the documents in such a way that they can be use d as resource documents by many different audiences. We specifically request comment on the format and content of the initial set of profiles, including how the format might be modified for subsequent sets of profiles. - - D. L E V E L S O F S I G N I F I C A N T H U M A N E X P O S U R E What is the m o s t useful w a y of p r e s e n t i n g this type of information? For this first generation of profiles we have selected a graphic presentation that reflects a "range" of values that covers both upper and lower bounds of effect levels. Is this more useful than a single number? Are there other ways of presenting this type of information that would be more useful to the eventual user? E. I D E N T I F I C A T I O N O F S I G N I F I C A N T D A T A G A P S The process used to develop the draft profiles has resulted in the identification of the full range of health effects data gaps associated with each chemical. However, depending on individual circumstances some subset of the identified data gaps may be essential in determining levels of significant exposure, while other data gaps may be less immediate. ATSDR, EPA, and the National Toxicology Program (NTP) have been exploring ways to identify the critical data elements that are needed to establish significant human exposure levels. This notice specifically requests comment and suggestions for approaching this phase of the toxicological profile process. / DEPARTMENT OF HEALTH 4 HUMAN SERVICES Public Health Service Agoney lor Toxic Substances and Disease Registry Atlanta, GA 30333 Official Business Penalty for Private Use $300 ABRAHAM W. HSIE DEPT.PREVENTIVE MEDICINE k COMMUNITY HEALTH DIV OF ENVIRON TOXICOLOGY UNIVERSITY OF TEXAS MEDICAL BRANCH GALVESTON TX 77550 ' S P EC IA L FOURTH-CLASS RATE POSTAGE 4 F E E S PAID PHS/CDC Permit No. G 284 5045 / ISP's/ \5 Y. 1 ^ 0 ^ V i s i t s i -177 5047  ; ': ' '^ & ' k r S V :' ^ - 7 '# * ? $ & -$T.. ; :j' Biochemical R a m b k n t e jr THE DOW CHEMICAL COMPANY *** V ^ t ' 4 1 P - ,-.. . ..v;.>.0. - i ; ML. V PRELE-UNAEY INFORMATION RELATIVE T O ___, ; THE TOXICITX-OF 2, 4-DICHL0R0PHEN0XY- ' ,ACETIC ACID (E2372-3) - ..... . * : \r-*.- ;*> a*,*vC`w * t ~^V.. . w-.r . ;r- *. T* Technical Service & Deve?BJhent Div. "" - rrVFfc T23. 14- 11-1 -'.&. / : Or. I 219A v . ;*; v.v w a 9-27-44 >- V raw 10-19-44 ^ W o A B j H. A. Wyse * w *!* ... ' ...__ 1 .. .. :v . * . ~ V - Rappti. Br ' V . / < . Z j . (W. W. Allen) :f W. R. Veazey - -."Organic Research Laboratory .< ', v- 'l' f ' '*' : ^ -3 -yy ".V-IV-**.J #..'*'-** 1. -- v- V*` ; - .:v.. -.v- * ,v- `v : ;'f'/ r '-*-*- r^ - ; Preliminary tests have indicated that 2^4-dichloro- - .. ; \ v- phenoxyacetlc acid has a moderate "acute oral toxicity and that it does not produce marked skin irritation. iff:xj?'g.1. s . " ix * A.' .X.". " We would J not .expect serious difficulties to result . V * * " w from *\* * ';''.*v-.Y'* * *'"' 'T-'i handling this material in a reasonable manner., 'v "*. * Ji / * .' j - Similar tests vili be made on 2,4,5"trichloropheno^y- "'v acetic acid when a sample '- * ia-4 copies * * - ; r -, - . ' - 1219 25 25C 244 .. Ti-f . '-r ' Y . f . . V .- 'H - ->*:* THIS REPORT IS TH PROPERTY ' -OF THE DOW CHEMICAL COMPANY)0 0 8 9 5 0 4 8 C7\_ /V ^ rt n r> o a O L * 1-.. I : ' .1 I " * J-* ' > , * * :v *". 11I 1 ! -ir.:; j ;v - T o . . I- - ' F < . . . . i a ^. , , ! .. -'ut ; C i ` in* .* I w . * . . v. * . V. 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T r a n c i ' t* ci f r -a: ir.,.* Jjv J o s e p h f . itcA in h cv fi y 7 , 1 D 4 G CCOCeli fr Z.7 X~ C '-.-.C iT 0OVOM J wi. J j uoauai-j 3 3 V/ ^ #*f ) MN 07 3280 V. :/0 1 * , -J '--- !r^ 3 iyw3 Kja Ja 3, V.tU -w*--J --'IwWW.--';.] IwC t^aj ->a "*)J . . j ' 3 .: .a -j p -.ji 3. wie L:;*iculU^.al Lab "* * i * J - *> <w * i f l . <*ii-pi 9 />** _ _ . y <-- w V 3 ` ^V .' >/ A o J w*o J .;_ _____ l3 L'W^'ciX O w -- U' '. , r i l a r 3 . e t Ohio ta to r . i l v o i 'Gi y p rp o o n fco d : . . r e n e ? a t h o rcceir c o a t n p o t t h e . ..L . . v J 'C eh 'Z i'o l C o o t T e z e j C h o 31 2 2 .3 r.:oo . j - . e j ' o t iloi-oloV oJ m -3 will be polished !X C `l".pe U a.;u '-ul -ewewU--! c i b v - Very -ru! en ex ce ce t;cD/swP?- /. . . j r t a n C C 04G 13 V 5051 Za 5052 Za !00 H2156896 J* J Harrisson, Jos. V/. E., and Rees, Edward W. 2,4-D Toxicity-I. Toxicity Toward Certain Species of Pish. American Journal of Pharmacy 118, 422-425 (1946). The L D 50 concentration of 2,4-D for minnows is approximately 2,000 p.p.m.; for sunfish, 1,000 p.p.m.; and for catfish, 2,000 p.p.m. The upper safe limit for minnows is 1,50 0 p.p.m.; sunfish, 500 p.p.m.; and esti mated to be about 500 p.p.m. for catfish. These figures refer to a seven-day exposure period during which the concentration of 2,4-D was kept at a fixed level. (R.H. Summary) 0012205 5053 3 5054 3 DOW &00264 * T l [ i f 1 3. Toxicity of 2 ,4-Dichlorophenoxyacetic Acid For Experimental Animals. Edwin V. Hill and Harold Carlisle. Journal of Industrial Hygiene and Toxicology. Vol. 29 pp. 85-95. ^ March 1947. } V. b L L L 0002579 5055 DOW 500265 TOXICITY OF 2,4 DICHLOROPHENOXYACETIC ACID FOR EXPERIMENTAL ANIMALS* V.E d w in H il l 1 a n d H arold C arlisle* W IT H IN the past few months several articles ( 1) have appeared in the scientific press describing the action and use of 2 ,4* dichlorophenozyacetic (2,4-D ) ad d and its am* monium or sodium salts as herbicides and plant hormones. This compound has also been used in orchards to prevent the premature dropping of apples. 2,4-Dicblorophenoxyacetic add is prepared commercially by reacting chloracetic acid, 2,4* dichlorophenol, and aqueous alkali or by the direct chlorination of phenoxyacetic add. It can be applied to plants either as an aqueous solution or dissolved in tributylphosphate diluted with diesel oil. .Potential health hazards may exist through inhalation when disseminated in aqueous or tributyl phosphate-diesel oil mists or by eating leafy vegetables or fruits contaminated by traces of the compound. Practically no data have been presented in the literature on the toxidty of this and related com pounds. Experiments were performed to deter mine the toxidty of the free add and its salts for experimental animals when administered orally, parenterally, and by inhalation. M e t h o d s -a n d M a t e r ia l s Commerdal 2,4-D contains small amounts of phenolic impurities and related isomeric phe* noxyacctic adds. The following procedure was used to remove such impurities as well as inorganic contaminants and traces of colored material, a. Two hundred and twenty-one grams of 2,4-D were dissolved in 2 liters of 98-100 per cent ethanol with the aid of mild heating to increase rate of solution, b. T he resulting solution was suction filtered to remove small amounts of suspended matter which occasionally were present, c The filtered alcoholic solution was saturated with gaseous ammonia until a test portion Received for publication August 1 1 ,'lWd. This work was conducted at Camp Detrick, Frederick, Maryland, from December, 1944 to June, 1945. ` LL Col., MC, AUS. L l (Jg). USNR. of the clear supernatant liquid g a v e ' no further predpitate of the ammonium 2 , 4* dichlorophenoxyacctate. The solution was stirred continuously during the addition of the ammonia. d. The predpitated ammonium salt was suction' filtered, dissolved in 2 liters of hot distill* water (about 70* C.), and 10 grams of de colorizing char added to the ammoniacal solution, which was then allowed to A at 70* C. for one-half hour. e. The decolorizing char was removed by gravity filtration and the filtrate cooled at 10* C. The ammonium 2 ,4-dichlorophenoxyacctatc gradually crystallized in fine needles, the crystallization being complete after twelve hours. f. The ammonium salt was suction filtered, dissolved in 2 liters of hot water (70* C.) and the 2,4-dichlorophcnoxyacctic add pre dpitated by the addition of the alkaline solution, with stirring and cooling, to ISO ml. of concentrated hydrochloric add (38 per cent S.G. 1.19). g. The 2,4-dichlorophcnoxyacetic add was suction filtered, washed with distilled water until a test portion of the filtrate gave no predpitate or cloudiness with aqueous silver nitrate, and re-crystallized three times from hot water. h. The filtered product, vacuum dried over concentrated sulfuric add for seven (lays, consists of fine transparent necdldike crystals, m.p. 139* C. (A Fisher-Johns melting point apparatus was used for the determination), reported m.p. (literature) 138* C , 139-140* C The neutralization equivalent was determined for 3 samples, 1) 221.1, 2) 221.1, 3) 221.0. Toxidty determinations were made on crude and purified 2,4-D . Sodium and ammonium salts of the purified acid were prepared and also assayed for toxidty at concentrations of 1 or 4 per cent. Solutions of the acid or salt were pre pared in jihysiological saline and adjusted to a final pH of 7.0 to 7.2. In the parenteral toxidty tests on these four materials, physiological saline o m o. 5^ "V IZ 9 o m a z IM I eI i i I l i i 5056 0002680 Dov^ 500266 86 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY was ordinarily used as a volume control but in later experiments a 1.81 per cent sodium chloride solution was used as an osmotic pressure and sodium ion control. The preparation of oil solutions of crude-acid required the use of an intermediate solvent. The acid was fust dissolved in either tributyl phosphate or n-butyl alcohol and these solutions were then mixed with the proper amount of diesel oil. Var ious combinations of 2,4-D , solvent and 0 2 diesel oil were tested. Each combination tested will be described in detail as the experimental work on the material is presented. All injections were made by accepted technics. Forced feedings were carried out in the smaller animals by means of a 04 or $S woven doth ureteral catheter and in the larger animals by means of a 08 rubber ureteral catheter. The catheters were attadiud to syringes of appropriate size and were lubricated slightly with petrolatum before use. It was found advantageous to an esthetize white rats and guinea pigs before feeding was carried out. Since nothing was known about the length of time necessary for effects to occur, all animals were held for more than an adequate number of days during the fust part of the work. It was found that deaths seldom occurred after the fifth day so that in the later experiments animals were sacrificed on the sixth or seventh day. R esu lts r Tie Acute Tosieiiy of tUt Purified Sodium Sail of 2,4-D A total of 17 experiments was carried out to determine the toxicity of the purified sodium salt of 2,4-D for the various experimental animals when given by injection or feeding with a stomach tube. In these experiments approximately 450 mice, 150 white rats, 125 guinea pigs, 70 rabbits and 3 monkeys were used. Since the titrations proved to be more precise and repeatable than titrations involving living agents such os bacteria, it was found possible to arrive at a fairly accurate LDm by using only 10 mice, 6 rats, 6 guinea pigs, and 4 rabbits for each 4 or 5 dilutions, covering a comparatively small range o f concentrations. For example, the mouse titrations consisted of tho injection or feeding of 2.5 mg., 5.0 mg., 7.5 mg., and 10- mg. of 2,4-D . The amounts usually administered to_guinea pigs were 100 mg., 150 mg., 2C0 mg., 250 mg., and 300 mg. Most of the titrations were earned out at lc.'-'t twice and in every replicates agreed satit.fr.cicr;!/. The toxic and tolerated doses determined b y these 17 titrations are given in .Table 1. Tiro LDi of 2,4-D injected intravenously in rabbits is approximately 400 mg./kg. About 50 per cent of the animals die suddenly of acute ventricular fibrillation and those that survive this acute episode die within several hours to three days. The outstanding symptoms were referable to the skeletal muscle where a condition not unlike myotonia congcntia was present. The animals received symptomatic relief by the administration of quinine hydrochloride, but a fatal outcome could not be avoided. The symptoms observed in the smaller animals were, in order of appearance, stiffness of the extremities with some muscular incoordination, lethargy, paralysis of the hindquarters, stupor, coma, and finally, death. These symptoms were observed consistently regardless of the method of administration of the chemical. The data indicated that the toxic and tolerated doses are slightly higher when the material is given by stomach tube. As would be expected, animals fed with 2,4-D did not develop symptoms as early as did injected animals. In addition, symptoms were more variable in degree in animals fed by stomach tube and the 'final results of such titrations were less precise and repeatable. Table 2 summarizes the results of one of several ex periments carried out in which comparative data on toxicity by feeding and injection were obtained. Three monkeys were fed varying amounts of the material. One monkey receiving 0.75 gm. exhibited only slight atypical symptoms and re covered completely. Two additional monkeys were fed 1.0 gm. and 1.5 gm. each, but both p-mW.! became ill about four hours after lyin g fed and regurgitated a large portion of the material so that no information on lethal doses for monkeys by feeding is available. N o typical sym ptom s' appeared in any of the monkeys, and all were apparently normal seven days after being fed. One animal, which was fed 1.0 gnuof 2,4-D ,w as kept under observation for an addlr^n-t e i^ y days without the appearance of any delayed efforts. Two of these same monkeys were then used in injection experiments. The monkey which had been fed 0.75'gm . was injected mtrapcritoncally with 1.0 gm. of the purified sodium salt of 2 ,4 -D . U rn aaimal developed nausea and vom iting 5057 0 0 0 2 G51 orci JW7\ TOXICITY OF 2.4 DICHLOROriIEXOXYACETIC ACID 87 9200QM o a stillness o(.ihc less with some muscular incoordina tion, lethargy, hanging of the head and ptosis of the eyelids. The symptoms appeared about two which had been fed 1.5 gm. seven days previously, was injected intraperitoncally with 1.5 gm. of the same material, l i t i s animal showed symptoms TABLE l Toxic and " olesated Doses o r tbz Sodiuu Salt o r 2.4-D ro a ExrESUUEVTAL An iu a u av Inaction and F eeding IROU n n o o or AMiMt* tAtlOM AffafflUUATS VSIC3T 07 AMttlAl TOXICDOCS* Per Asiaci Bx-Af. TOtXSATXDDOSS** Per Aaiaul ms-Ae. White mouse..................... Intraperitoneal White mouse..................... Stomach tube 20 gm. 20 gm. 7.5 mg. 7.5 mg. 375 375 2.5 mg. 2.5 mg. 125 125 Guinea pig......................... Intra|>eritoneal Guinea pig........................ Stomach tube 300 gm. 209 mg. 666 100 mg. 300 gm. 300 mg. 1000 100 mg. 333 333 Rabbit................................ Intraperitoneal Rabbit................................ Stomach tube Rabbit................................ Intravenous 2.5 kg. 2.5 kg. 2.5 kg. 1.0 gm. 2.0 gm. 1.0 gm. 400 800 400 500 mg. 500 mg. 500 mg. 200 200 200 White rat.......................... Intraperitoneal 150 gm. ICO mg. 666 25 mg. 166 White ra t.......................... Stomach tube 150 gm. 100 mg. 666 50 mg. 333 Monkey.............................. Intraperitoneal Monkey.............................. Stomach tulie 3.5 kg. 3.5 kg. 1.5 gm. 750 mg. 428 214 * Toxic dose--50 per cent mortality. ** Tolerated dose--largest amount causing no deaths. TABLE 2 Cou?a2ison o r tbs Toxioty o r tux Pusitied odxdu Salt o r 2,4-DiarLoaornENOXYACETic Acid roa White Rats bv I njection and F eeding HO1,4-0 OlVXM soon AfPCASANa or SVUROHS MOBTA& in* 50 ICO 150 200 50 ICO 150 200 Saline control Saline control i.p. i.p. i.p. LP. Feed Feed Feed Feed LP. Feed 1 hour 30 min. 30 min. 30 min. 4-6 hours 2-3 hours 1 -2 hours 1 -2 hours None None 2/6 6/6 6/6 6/6 0/6 4/6 5/6 6/6 0/6 0/6 * Mortality data recorded as ratio of deaths to total animals used. Rats used in this experiment averaged 150 gm. in weight. hours after inoculation, persisted for about fortyeight hours, and then began to lessen in intensity. A t the end of five days, the animal had recovered and was apparently normal. The second monkey, similar to those seen in the fust injected monkey and by five days had recovered completely. Toxicity of Crude Venus Purified2,4-D Five titrations were carried out to com|>arc this toxicity of purified sodium salt of 2,4-1) with that of the crude material as it comes from the manu facturer. In these cx]>crimcnt<, the crude add was first converted to the sodium salt and was used in this form with all impurities present. The re sults, as presented in Table 3, indicate that there was no significant difference in the toxidty of the two materials cither by injection or feeding. The Toxicity ofPure 2,4-D Two experiments were carried out to test the toxidty of pure 2,4-D . Because of its insolubility, the material was suspended in S per cent gelatin solution and fed to white rats by means of a stomach tube. The addition of gelatin to the suspension prevented settling ami agglomeration of the partidcs. N o injection experiments were carried out, but the results of the two feeding titrations summarized in Table 4 indicated that 2,4-D possesses about the same toxidty as does its sodium salt. 5058 0002682 -j JOUHMAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY TASLE 3 f n --.. - rrn-f n- t~ Tnvrnrv n? PuiLTOD ANDCiUDE SODIO!! SAIT 07 2,4-D 3Y IirjTCROtt Atm 2Y*--- - 3 irscca KO./ZO. sons Parid uoatAUX*** Crai* CoEtrsla White mouse SCO 375 250 125 Saline control I.P.* I.P. I.P. I.?. . I.P. 10/10 3/10 0/10 0/10 10/10 4/10 0/10 0/10 0/10 White rat 1CG0 I.P. 5/6 6/6 666 LP. 5/6 4/6 333 I.P. 0/6 0/6 167 I.P. 0/6 0/6 Saline control I.P. 0/6 Guinea pig * 833 I.P. 6/6 4/6 667 I.P. 5/6 4/6 5G0 I.P. 1/6 2/6 33 I.P. 0/6 0/6 Saline control I.P. 0/6 Rabbit 4C0 I.P. 3/3 1/3 2C0 I.P. 0/3 0/3 Soline control I.P. 0/3 White rat 1C00 Feed** 5/6 5/6 666 Feed 3/6 2/6 333 Feed 1/6 1/6 Saline control Feed 0/6 * LP.--Intr&peritoneal injection of 2,4-D salts in physiological saline solution. ** Feed--Feeding by stomach tube of 2,4-D salts in physiological saline solution. *** Mortality data are recorded as ratio of deaths to total animals used. TA2LE4 A Coumusoh or m Toxicity o r P u u 2,4-D and tsx Ptnunxn Sonimi Salt o r 2,4-D by Fekoimo to White Rats and Guinea Pics in au White rat moJ eo . 1CC0 666 333 Gelatin control Soline control UOITAUTV* S oditi 1 .4 -0 S a ilo ( 2 .4 * 0 C o tr U 4/4 5/6 3/4 3/6 0/4 0/6 0/4 0/6 Guinea pig 33 0/8 0/8 Celatin control Soline control 0/4 0/4 * Mortality data recorded as.ratio of deaths to total animals used. The Toxicity oftheAmmoniumSait of2,4-D Experiments were carried out to compare the toxicity of the ammonium salt of 2 ,4 -D with that of the sodium salt. Amounts of the ammonium salt equivalent to the tolerated dose of the sodium salt for each species were fed to groups of 6 guinea pigs, 6 white rats, and 4 rabbits. l a this experi ment all animals survived, indicating that the ammonium salt is a t least no more toxic than the sodium salt by feeding. Two injection titrations were carried out on white mice and the results indicated that the ammonium and sodium salts possess about the some degree of toxicity by in jection also. The precoding two types of experi ments were carried out with both the purified salt and the crude salt and no difference in to x id ty was detected. One monkey was fed 1.0 gm. of the pure ammonium salt of 2t4-D. T h e monkey regurgitated a large`amount of the material about 500268 oo 000268 DOVV500269 forth IH 7) TOXICITY OF 2,4 DICXLOROPHENOXYACETIC ACID 89 three hours later and was apparently normal when observed the next day. N o symptoms other than the nausea were noticed. 'The results of the experiments on the ammonium salt of 2 ,4 -D arc outlined in Table 5. The Toxicity of Crude Acid 2,4-D Dissolved in either Tributylphosphate or n-Buiyl Alcohol and Oil The toxidty of crude add 2,4-D dissolved in tributylphosphate and oil was investigated to tributylphosphate in oil, and a feeding titration in guinea pigs using 4 per cent 2,4-D and 8 per cent tributylphosphate in oiL It can be seen from Table 6, which summarizes the results of the first two experiments, that when a 1 per cent solution was used deaths occurred at one-fifth the tolerated dose of 2 ,4-D and all control animals died, whereas, when a 4 per cent solution was used, only occasional deaths occurred in the control group and in the scries of animals fed the tolerated dose. In view of the results of the first two experiments it was TABLE 5 A Comparison o r ta x Toxzcmr or Poax aks Cam Annotami Salt with tmx Pu is Sooiun Salt or 2,4-D sn a zs White mouse K S -/30. 500 37S 250 125 Saline control BOOTS I.P. I.P. i.p. i .p . I.P. u o t i AllTT*** A m m onium S alt Pvt C rudo Sodium S a lt P u ra 9/10 7/10 10/10 4/10 3/10 3/10 0/10 1/10 0/10 0/10 0/10 0/10 C oatrola 0/10 Vhite rat 333 Feed* 0/6 0/6 0/6 Saline control Feed 0/6 Guinea pig Rabbit 33 - Saline control Feed Feed 0/6 0/6 200 Saline control Feed 0/4 Feed 0/4 0/6 0/4 Monkey 285 Feed 0/1 * I.P.--Intraperitoneal injection of 2,4-D salts in physiological saline solution. ** Feed--Feeding by stomach tube of 2,4-D salts in physiological saline solution. *** Mortality data are recorded as ratio of deaths to total animals used. determine whether or not the combination of solute and solvents was significantly more toxic than the solute alone. It was not the purpose of this investigation to determine the toxic levels of the solvent. One gram of 2,4-D is soluble in 2 ml. of tri butylphosphate and the resulting solution is miscible in all proportions in oiL Various con centrations of the agent in tributylphosphate and oil were prepared by altering only the amount of oil used and leaving the agent-solvent ratio con stant. The first two experiments on the 2,4-D tributylphosphate oil complex were preliminary in taturc and consisted of a feeding titration in white rats using 1 per cent 2,4-D and 2 per cent decided to test a constant amount of 2,4-D (the tolerated dose) in various amounts of tributyl phosphate and oiL For this work, concentrations of 1 per cent, 2 per cent and 4 per cent 2,4-D in oil containing 2 per cent, 4 per cent, and 8 per cent tributylphosphate respectively, were used for feeding experiments with guinea pigs, white rats and rabbits. When a 4 per cent solution of tributylphosphate in oil containing a tolerated dose of 2,4-D was fed to experimental animals in a comparatively small volume of oil, all or most of the animals survived. Only occasional deaths were observed in the groups of guinea pigs and white rats, and all of the rabbits survived. When the 2 per cent solution was used, b 0002684 DOW 500270 so JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY (m/. 29, 84.2 A-<thc were still sporadic but occurred with greater frequency than with the 4 per cent solution. However, when the l per cent solution was used, the tolerated dose in the large amount of oil killed all of the animals fed with the mixture. All of these expermenta pointed to the fact that the volume of oil and trilnitylphosphate fed governed the results of the toxicity tests, and that the 2 ,4 -D was secondary in importance. There was no evidence of any additive effect Itctween the 2,4-D ' and the tributylphosphatc-oil portion of the complex since deaths occurred in the trihutylphosphatc-oil control groups to almut the same extent an amount of 4 per cent 2,4-D in oil containing 0.5 grains 2 ,4-D . This volume of material, 12.5 CC-, caused very violent regurgitation soon after the feeding was carried out, but the animal was apparently normal the next day. N o symptoms other than the nausea were noticed. Three experiments were carried out to test the toxicity of n-butyl alcohol solutions of 2 ,4-D . These experiments were similar in nature to those carried out with the tributylphosphate-oil solu tions. Two groups of rabbits were fed the toler ated dose of 2,4-D (0.5 gram) in 2 per cent and 4 per cent solution and all animals survived. In TABLE 6 2,4-DT i t b a t o w o r O n e P e x C e n t a n d F o c a P e e C e n t in T u s o m r a o s p a A r z a n d O i l in W in n R a t s a n d G u in e a P ic s d v F e e d in o IIC Q U a c x x T l.t- A u n t m ? J.4 -B t u VOUTWt 0 * la V K N T MOATAXm** White rat Oil-T.U.1*. (2%) control Oil cwitrol 1 1 1 1 None None 10 mg. 25 mg. *50 mg. ICO mg. 5.0 mL 5.0 mL 1.0 mL 2.5 mL 5.0 mL 10.0 mL 4/4 4/4 . 2/4 . 3/4 ' 4/4 4/4 Guinea pig Oil--T.B.P. (8%) control Oil control 4. 4 4 4 4 None None 25 mg. 50 mg. *100 mg. 200 mg. 300 mg. 2.5 mL 2.5 ml. 0.625 mL 1.25 mL 2.5 mL 5.0 mL 7.5 mL 1/4 0/4 0/4 0/4 1/4 4/4 4/4 *Tolerated amount of 2,4-1) when in saline solution. ** Mortality data are recorded as ratio of deaths to total animals ui.il. as in the groups of animals fed the same volume of 2,4rD tributyipliasphatc mixture. No attempt was made to determine whether the toxicity of the trihutylphosphatc oil mixture was due to the tributylphosphatc or the oil. In view of the comlurativcly slight differences in toxicity Itctwccn the saline and nil solutions of 2,4-D in concentra tions greater than 2 per cent, it was decided to test a 3 |x .T cent solution of 2 ,4 -D in oil and to feed various species j of the tolerated dose. Such an experiment was carried out in white rats, guinea pigs, and rabbits, and all animals survived. The solution used was 3 per cent 2,4-D and 8.4 |>cr cent tributylphosphatc in 2 diesel oil. The amount of this material tolerated by the experi mental animals was equivalent to aliout 200 cc. for the average sized man. One monkey was fed comparable experiments in guinea pigs and white rats, only an occasional death occurred. Occa sional deaths were also observed in the control groups which received comparable volumes of n-butyl alcohol-oil mixture. Although not con clusive, these experiments indicated that n-butyl alcohol oil solutions of 2,4-D are at least of the same order of toxicity os the tributylphosphatc oil solutions. Sub-acute Intoxication in Dots A group of 8 dogs received 2 ,4 -D intravenously, varying from two injections of 200 m g./kg. each to six injections of 25 mg./kg., the injections being given at daily intervals. Two dogs (nos. 1 and 2) receiving two injections , each of JOQ m g./kg. died on the third and second 5061 0002655 DOVV500271 -Arch 1M7] TOXICITY OF 2,4 DICHLOROPHENOXYACETIC ACID 91 day respectively after the last injection. Tne first symptom was an unsteady gait, with the extremities somewhat stiff and extended. On the day following injection both animals were lethargic and stood up only when forced. The gait was still unsteady and extremities stiff. The deep reflexes of both animals were equal and active, pupils in mid dilation and equal. Corneal reflex in dog no. 1 was absent in contrast with dog no. 2. Nictitating membrane was sluggish in dog no. 1 covering one-half of the eye Iall; it was active and in normal position in dog no. 2. Two dogs (nos. 3 and 4) received two injections of 200 m g.A g. each on successive days. Both animals died on the second day. They were semi-stuporous for the first day after injection and responded slowly to stimulation. Pupils did not react to light and corneal reflex was either absent or extremely sluggish. Deep reflexes were equal and active in dog no. 3. The polymorpho-nudcar leucocyte count was 13,030 before injection and 1,400 the day following injection. Similarly, the lymphocyte count decreased from 3800 to 8C0. D og no. 4 showed no significant change in the number of polys but did show a reduction of ymphocytcs from 5,CC0 to 2,200. One dog (no. 6) was given six injections at daily intervals of 25 m g.A g. of 2,4-D . On the second day this animal showed blood oozing from the gums at the tooth margins which did hot increase in severity. On the fifth day after injections were terminated, decubitus ulcers developed over the outer aspect of the forelegs and right thigh which gradually became more extensive, deeper and infected. Neutropenia or lymphopenia were not apparent at any tme during the illness. The animal was sacrificed on the ninth day. Two animals (not. 7 and 8) received six injections, a t daily intervals, of 50 m g./kg. each. D og no. 3 died on the third day. This animal became lethargic and semi-stuporous after the third injec tion. On the second day after the series of injec tions were terminated conjunctivitis developed. Blood oozed from the gums and tooth margins, the gait became unsteady and coarse muscle tremors appeared in the extremities after moderate exercise. The deep reflexes were equal and active. The rectal sphincter was lax and allowed the escape of light, watery brown stools. The animal became more lethargic on the third day and died in the mid-afternoon. Polymorpho-nudcar leucocyte count decreased from 6,CG0 to 2,500 and the lymphocyte count from 3,500 to 200 as a result of the intoxication. The platelet count, originally 323,000, dropixai to 138,000 in the post-injection .period. Dog. no. 7 dcvcl<i|>cri necrosis of the gums on the right and left side of the lower jaw along the posterior as|)cct where the teeth of the upper jaw overlap those of the lower jaw. Blood oozed from the gum margins throughout the remainder of the mouth. The necrotic process gradually extended to involve the floor of the mouth, the buccal mucous membrane and exposed much of the mandibular bone. Decubitus ulcers developed on the outer aspect of the extremities on the Kth day. The animal was sacrificed on the sixteenth day when rectal temperature was elevated for the first time. This animal did not show any significant reduction in the polymorpho-nudcar count. There was, however, a reduction in the lymphocyte count from 2,600 to 1,100. It was interesting to note that smaller doses of 2 ,4-D appeared to have a cumulative action. The development of the decubitus ulcers and severe necrotizing lesions of the mouth occurred in all animals receiving 2,4-D over a protracted period. In general, the animals showed a slight reduction in the red count, hemoglobin level, and an increase in the plasma non-protein and urea nitrogen. There was no significant change in the total protein content of the plasma. At no time did any of the animals show any signs of icterus. Sub-acult Oral Toxicity in Rats Young male rats, weighing around ICO gnu. each, were put in individual metabolism cages and fed a stock diet of sucrose, Labco casein, Crisco, and salts. In addition they received a daily vitamin supplement containing pyridoxine, ribo flavin, nicotinamide, calcium pantothenate, choline chloride and thiamine. Following an observation period of one week, the animals were divided into 4 groups of 7 mts each. Croup 1 received 100 mg. of 2,4-D /kg. of diet; Group 2, 200 mg.; Group 3, 400 mg.; Group 4 served as controls. N o difference could be observed In the food consumption and growth rate of animals receiving 100 and 200 mg. of 2,4-D /k g. of diet. One animal in the group receiving 500 mg. of 2,4-D of diet died on the tenth experimental day, but In as much as no other member of the group was affected, the death was considered incidental. In view of the negative nature of the experiment, the diet of the rats of group 1 was changed on t h e 00025S WSOOS Moa 92 JOURNAL OF INDUSTRIAL HYGIZNE A N D TOXICOLOGY --i twenty-first experimental day to contain 1CC0 mg. of 2,4-D /k g. of diet. Despite this large dose there was no evidence of reduced food intake and co effect on the growth rate during the subsequent two weeks. The experiment was discontinued one month from the time 2,4-D was first intro duced into the diet. It was concluded that the rats* diet containing as much as 0.1 per cent 2 ,4-D was not intoxicating. Sub-acute Oral Toxicity in Guinea Pits Two groups of 6 guinea pigs each were fed daily 50 mg. and ICO mg. respectively of the purified sodium salt of 2,4-D . On two occasions it was impossible to carry out the feedings on schedule so that the animals actually received only ten feedings in twelve days. Five out of 6 of the guinea pigs receiving 50 mg. at each feeding sur vived. These 5 animals were fed a total of 0.5 gm. in twelve days. Three out of 6 of the guinea pigs receiving 100 mg. at each feeding survived. The total dosage of tins group was ID gm. in twelve days. These results are nullified to a certain extent by the fact that three deaths occurred in the saline controls, making it appear that all deaths might have been non-specific and caused solely by the rather rough treatment associated with daily feeding by stomach tube. Supporting this con tention is the fact that none of the animals showed the typical complete paralysis before death. Aside from these discrepancies, the fact remains that 3 guinea pigs survived a total of 1.0 gm. of 2 ,4-D , .which is about 3 or 4 times the amount necessary to kill when given at one feeding. The Toxicity oftheSodiumSaUof2,4-D by Inhalation Attempts were made to produce symptoms in guinea pigs by exposing them to a wet cloud of sodium salt of 2,4-D in a spray chamber produced by nebulizing an aqueous solution of the salt and to a dry cloud of the crude add produced by directing a jet of air against a finely pulverized dry powder in the bottom of a small chamber. Tn no case did any of the animals exhibit typical symptoms and there was no gross evidence of lung irritation, even though concentrations of the material in the air were high and exposure times were tong. In the first experiment carried out in the spray chamber the animals were exposed to a CT (mg. min. per M 1) of approximately 6000-8000. The second experiment was not quantitative in that it was impossible to calculate the CT given. I t hon been estimated that the animals were expored to & C T even greater than used in the spray chamber. N o data was obtained on the average ciac of the particles in the aerosols or th fraction of the material impinged out of the inspiratory air and absorbed by the animai. Pathelopeol Chanfes Rats and guinea pigs dying of massive doses of 2,4-D were sacrificed sixty to ninety-six hours after administration of the chemical were autopsied (the central nervous system was not examined). Constant findings on gross examination were con gestion of the viscera and enlarged swollen kidneys. On section of the kidneys, the parenchyma bulged from the cut surface and the capsule stripped almost spontaneously presenting a dull yellowishred surface. The cortex was swollen and poorly differentiated from the medulla. Microscopic examination of the kidney revealed the swollen cortex to be due almost entirely to a massive clouding swelling of the epithelium of the proximal convoluted tubules, which in many cases com pletely occluded the lumina. Some of the cell membrane had ruptured, with the escape of the cytoplasm and nuclei to form an acidophilic debris in the tubules which was latex converted to hyaline and cellular casts found in the collecting tubules. The glomeruli and blood vessels were unaffected. Occasional animals showed slight patchy pulmonary edema and alveolar hemor rhages. The livers did not show any significant pathological changes. A group of 6 rabbits receiving from four to thirteen daily (except Sunday) injections of 50 mg./kg. were sacrificed on the day of or the day following the last injection. N o striking patho logical changes were observed in this group of rabbits. The kidneys of all animal but one showed slight to moderate degenerative changes consisting of granular or vacuolar swelling of the cells of the proximal convoluted tubules. T he glomeruli were unaffected. Stimulation of hema topoietic and rcticulo-endothelial tissues was suggested by the occurrence of hyperplasia of the lymph nodes and spleen in several animals. Mod erate hyperplasia of the bone marrow associated with the extra-medullary hematopoiesis in the liver, spleen, sa d adrenals. This effect was not consistent throughout the group. Significant blood destruction, evidenced by considerable 0002SG? DOW 500273 i f arch 19J7\ ( TOXICITY OF 2,4 DICHLOROPHENOXYACETIC ACID 93 hemosiderosis of the 'spicvn, was present in only one animal. A review of the biood hemoglobin levels in this animal showed a decrease in the hemoglobin from a pre-injection level of 9.0- 9.7 gm. to 7.8 gm. after five injections. Of three other rabbits in this group receiving as many or more injections, two showed no significant change in hemoglobin level and one showed a slight decrease. The liver showed no significant changes. The lungs showed scattered patcchial hemorrhages in a few eases and moderate patchy edema in two. Another group of 6 rabbits receiving from three to seven daily injections of 100 mg./kg. were sacrificed on the day of or the day following the last injection. There was a significant decrease in hemoglobin and red ceils in one animal of this group, and a moderate decrease in hemoglobin in three others. No changes in the white cell count or differential count were observed. The kidneys of this group of animals also showed parenchymatous degeneration as the most constant change. Marked hyperplasia of the bone marrow, associa ted with extra medullary hematopoiesis, occurred ' i one animal. Lymphoid and splenic hyperplasia ere observed in several animals but was not a constant finding. Hemosiderosis of the spleen was observed only once, in the animal showing the considerable decrease in hemoglobin anrl red cells. Evidence of slight lymphoid destruction was ob served in the spleens of two animals of this group. The liver showed no significant changes. Dogs succumbing to massive doses of 2,4-D or sacrificed were autopsied. In contrast with the other species studied, dogs showed a considerable susceptibility to the development of liver damage. The hepatic lesions consisted of ccntro-lobular degeneration, atrophy and lysis of the parenchy mal cells about central veins of the lobules, with congestion and dilatation of the pericentral sinu soids. In one dog, dying at thirty houn after two injections of a relatively high dose (200 mg./kg-)i there was an extensive though early necrobiosis throughout the liver. The kidneys showed cloudy swelling of the tubular epithelium; the reversible nature of this lesion is indicated by its absence in animals sacri ficed or dying after some delay after the last injection. On the other hand, one dog receiving a large dose (4C0 mg./kg-) showed actual tubular epithelial necrosis, similar in type to that observed in mercury poisoning. Striking lymphoid necrosis the lymph nodes, thymus and spleen was evident, especially at the higher doses. Unequivo cal bone marrow changes were not observed, although it seems quite possible that with the proper dosages of the chemical such lesions might occur. An occasional animal showed evidence of blood pigment deposition in the spleen and lymph nodes. Minor focal parenchymal lesions were observed in the adrenal in several instances, accomitanicd by infiltration of the polymorpho nuclear leucocytes. D iscussion The investigations disclosed that 2,4-D is a relatively non-toxic compound for experimental animals, having an LDt*, expressed in mg./kg., of 375 for mice, 1000 for guinea pigs, 666 for rats and 800 for rabbits when administered orally in aque ous solution. All of these species reacted similarly to the chemical. There was no apparent differ ence in the toxicity between crude acid and a highly purified preparation, or between the sodium and ammonium salts of the acid. Monkeys were shown to be able to tolerate 428 mg./kg. of 2,4-D when administered intraperitoncally. However, when the material was given in large doses by mouth, 1 or 1 ) grams, the animal became nauseated and vomited a large portion of the material so that accurate information as to the toxicity of the compound administered by the oral route in this species is lacking. Three-fourths of a gram of the material was given to one monkey without the development of vomiting or serious illness. Based on the best data available, which arc recognized to be inadequate, monkeys can tolerate single dosages equivalent to 214 mg./kg. In any assessment of the acute toxicity of a chemical based on data obtained from laboratory animals it should be borne in mind that considera ble variation in species susceptibility may occur and that the data obtained cannot always be translated into the toxic doses for humans. In this case, however, all of the laboratory animals tested reacted in a similar fashion to the material so far as could be determined from signs and symptoms which developed and from the patho logical lesions which were present at autopsy. Assuming that man is no more resistant or suscep tible than the rabbit or monkey, then the largest tolerated dose for a 75 kg. man would be 15 gras. With the exception of the monkey, all of the laboratory animals used lacked the vomiting" reflex to that they were unable to relieve them- 0002S38 I. f `I 1 I f J1 I 1 b ! j- L L l i 500274 JOUIIXAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY d v c3 o irritating material by vomiting. The experiments conducted ia monkeys indicate that the material is a gastric irritant in large doses, so that the possibility of the occurrence of acute poisoning in humans would seem relatively remote because of the large dose which man could pre sumably tolerate. Assuming that man is no more susceptible than the most susceptible animal tested, the mouse, then the calculated oral LDi* for man would amount to approximately 28 gnu. Sub-acute intoxication was produced in dogs by giving daily injections of the material over a period of six da)?. With the administration of smaller doses, 25 mg./kg., there was a suggestion that the material had a cumulative anion. In contrast with the other species studied, dogs showed a considerable susceptibility to the development of liver damage. Occasional lymphoid necrosis in the lymph nodes, thymus, and spleen occurred especially with high doses. A few animals demon strated a significant reduction in the number of lymphocytes in the circulating blood. The de velopment of decubitus ulcers and their occasional association with a significant reduction in tho leucocyte count was a disturbing observation. Reduction in the leucocyte count did not occur in every ease, but its occasional development should direct attention to blood studies in cases of sus pected intoxication in man. Investigations on the sub-acute toxicity of 2 ,4-D by the oral route were limited to two species, rats and guinea pigs. Rats were fed varying amounts of the material up to ^ per cent by weight of their diet for period of one month without any significant effect on their food intake, rate of growth, or the development of any charac teristic signs of intoxication. Guinea pigs, which were fed 100 mg. per day of the material by stom ach tube over a period of twelve days until a total of 1 gram of the material had been administered, did not develop characteristic evidence of intoxica tion. In this particular experiment, non-specific deaths occurred in approximately the same per centage in both the test and control animals. All of these deaths were believed to result from trauma, associated with the passage of a stomach tube a t frequent intervals, in as much as none of the test animals developed the typical paralyses or skeletal muscular signs observed in poisoned animals. It was therefore concluded that guinea pigs could tolerare 1 gram of 2,4-D in divided doses over a period of twelve day3 without any harmful effects. The experiments to determine the toxicity of the sodium salt of 2,4-D by inhalation tend'd to indicate that the material was relatively non-toxic when wet or dry clouds were inhaled. It should be pointed out that in none of these experiments was the average particle size of the aerosol determined nor the amount of material retained and absorbed by the respiratory tract determined. Further studies on the toxicity of this compound by the respiratory portal of entry will be needed before any final conclusions can be made. In view of the fact that the material did not produce any evidence of lung irritation and that the toxic oral dose of the material for guinea pigs is relatively high, it would tend to indicate that the material would be relatively non-toxic by this route. 2 ,4 -D dissolved in a solvent complex of tri butyl phosphate and diesel oil has been recom mended for use as a herbicide. This particular form , of material has the advantage over the aqueous solution in that the tributylphosphate acts as a co-agent or synergist to the 2,4-D producing greater plant damage than could be accounted for by the acid alone. T he present study did not attempt to determine the toxic or tolerated dose of tributylphosphate or diesel oil. There was no evidence of a synergistic or addi tive effect when a tolerated amount of 2 ,4 -D was dissolved in the tolerated dose of tributylphosphate oil complex and administered to the experimental animal*. Production of chronic poisoning by this material was not attempted. S l'MUARY The LDm expressed in m g./kg. of 2,4-dichlorophenoxyacetic acid by mouth is 375 for mice, 666 for rats, 800 for rabbits, and 1000 for guinea pigs. The tolerated dose for these same species was 125, 166, 200 and 333 m g./kg. respectively. The largest dose administered to monkeys without ierious after effects was 214 mg./kg. Subacute intoxication waa produced in dogs by the daily administration of 25 m g./kg. over a six day period with the development of liver damage. Lcucopcnia was observed in a few nimnU. Rats were fed up to per cent by weight of their diet over a period of one month without any harmful effects. Guinea pigs can apparently tolerate 1 gram of the material administered in divided doses of 100 mg. over a period of twelve days. oo 5065 0002529 500275 l6rtklW \ TOXICITY OF 2,4 DICHLOROPUENOXYACETIC ACID A limited amount of work on the toxiaty of wet and dry clouds of the sodium salt indicated that it was relatively non-toxic. The toxicity of 2,4-D dissolved in tributylphos* phate and diesel oil was not enhanced by 'these solvents. AacMowuDOZunrr The authon wish to thank Major Arthur Glazier and Captain Morton Goldston of the Medical Division, Edgewood Arsenal for permission to use their data on subacute toxicity of 2,4-D for dogs and chronic toxicity in rats. BIBLIOGRAPHY (1) HanzBOAMs, E. M.: War on weeds. Science, 103: 465, 1946. ' S p e c ia l P o o le r s DivistOM, Csmncai W a x p a u Szavicz: Plant growth regulators. Science, 103: 469, 1946. VAM OVTSSIEX, J . AMD VXLXZ, ISUAXLt Use of 2,4-dlchlorophenoxyacetic acid as a selective herbicide in the tropics. Science, 103:- 472, 1946. Sio t h , F. G., H am m , C. L. am d C a o u o m , R. F.: Control of ragweed pollen production with 2,4- dichlorophenoxyacetic add. Sdencc, 103: 473, 1946. Hiuzsbamd, E. M.i Herbiddol scU'oa of 2,4dichlorophenoxyacetic sdd on the water hya cinth, eirhomia aassipca. Science, 103: 477, 1946. Emus, W. B., Tuoupsom, H. E. and S u m , H. H.t Tributyl phosphate at a solvent for preparing concentrated and oil-miscible solution* of 2,4= oichlorophenoxyacetlc add and similar sub stances. Science, 103:476,1946. 5066 0002630 5067 i- BIOCHEMICAL RESEARCH LABORATOR? THE DOW CHEMICAL COMPANY Subject TOXICOLOGY AND HYGIENE: --------- 2,4-DICHLOROPHENOXY ACETIC" ACID \ t Pile T23.14-11-1? Chg. 1219 Rec'd Pin'd 6-9-48 Work By E.M.Adams To: Britton's Div. Att: B. N. Schrauf R. C. Dosser J. B. Arnold Safety Dept. Att: John Knight Dr. H. H. Gay T2.2-10-1 chef k -Zc '1 ? Rept. By UNIT INDEX A summary of available information. INDEX HEADINGS cn cn Acetic acid, 2,4-dichlorophenoxy - SUMMARY OF HAZARD The toxicity is such as to present but minor hazards of systemic effects. Skin irritation is possible from heavy prolonged contact with strong solutions or the solid acid. of absorption through the skin. There is no hazard _1_ SUMMARY OP TOXICITY The systemic toxicity was moderately high as Indicated by single and repeated oral feedings to rats. For single doses: Largest dose survived by all rats 0.3 g./kg. Smallest dose to which all rats succumbed 1.0 g./kg. Por repeated (20) doses; 0.3 g./kg. rapidly produced serious injury and death, 0 .1 g./kg. had but very slight deleterious effect and 0.03 g./kg. had none THIS REPORT IS THE PROPERTY T111fC. w I-J , 000898C 5068 Biochemical Research Laboratory * T23.14-11-1 Page 2 Examination of the affected animals revealed minor effects In liver and kidneys, the major action being local Irritation In the stomach and gut. When lncorporated^lnto the diet of rats, there was no adverse effect at. concentrations which the rats would eat, 0 .03# and less. At concentrations of 0.1# and more, the rats refused to eat. The dichlorophenoxyacetic acid had but very slight effect the skin of when tested In solution upon/animals. There was no evidence of absorption through the skin. KO In making and processing 2,4-dlchlorophenoxyacetic acid c/i cn there appears to be no serious hazard of systemic effects. Atmos- q q cn pherlc contamination by dust offers the only concern and this would have to be quite severe to be excessive. Any such excessive exposure would probably be first Indicated by symptoms referable to irritation in the xipper respiratory passages or by gastrointestinal disturbances. By comparison with other common substances, it offers very little hazard of serious organic injury, even from repeated exposures. Animal results indicate little irritating action upon the skin. There is the possibility that prolonged heavy exposures to solid or to strong solutions could cause a dermatitis. This applies to both the free acid and the sodium salt. cc-c in the use of 2,4-dichlorophenoxyacetic acid, the solution concentrations are sufficiently low and the total amounts Involved sufficiently sma^l.-that there appears to be no hazard to the appli cator or to farm animals. 5069 ec*8 T + 0008987 :iit Mi i.t.l CU ILM , 8. Till MF-, On i ( < cn o o l i u\ I Y-\ of isonic trie recordn.0 , d ie m ovem ent o f the pin under a full load o f 1500 g . Is lim ited 0 0.04 m m. T h e bridge signal w as fed through a direct-coupled am plifier to a atlrodc ray oscillosco|K for photographic recording. T h e bridge ou tp ut w as linear hrough full-scale deflection. R esting tension of the m uscle w as m aintained a t a nnslant value throughout any one experiment, but varied from one experim ent to nothcr betw een 75 and 200 g. For indirect stim ulation the sciatic nerve was crushed and tied proxim ally. T he istal segm ent was placed in a p lastic insulating bed containing tw o silver stim u latin g cctrodcs. D irect stim ulation w as cITccled through steel needles placed a t op posite ids of the muscle. Stimuli, which were supramaximal in intensity, were presented s square waves generated by a trigger circuit, the duration of which was controlled y a variable resistance-capacitance circuit. Nerve action potentials were recorded through a Sherrington electrode from the Dial nerve isolated peripherally. Stim ulation w as effected a t the p elvic en d o f the :i'alic nerve which w as isolated centrally. Fleetromyograms were recorded through steel needles insulated to w ithin 1 to 2 nn. of their tips and placed in th e b elly of the m uscle ap proxim ately 5 m m . a p art, lie potentials were fed through a condenser-coupled amplifier to the cathode ray icilloscopc a ml recorded photographically. W hen the needles were inserted shortly ter section of the sciatic nerve, spontaneous random spike potentials were observed, hese spikes lessened in frequency and then disappeared in the course of 10 to 15 inutes. N o studies were carried out until spontaneous activity had subsided. Intra-arterial injection was made through a sm all cannula tied into the contratcral iliac artery and directed centrally. T he crural artery of the leg under study as ligated. The aorta was occluded during injection b y drawing up on a loose jalurc placed above the bifurcation. The injection volum e was 0.15 m l. T his eparalion preserved normal blood supply to the muscle under study but is under mtinuing developm ent to attain further restriction of the injection to th e triceps rac. T h e sm all size o f the rat's peripheral vessels enabled th e use o f a sim p le highcqucncy d esiccatin g apparatus (T lyfrccator', Bircher) to coagu late v essels and, iu s , to reduce bleeding. When required, denervation of the triceps surae was performed 10 d ays before cording by aseptic section of tbe sciatic nerve. Concentrations of potassium in scrum and m uscle were determ ined in control its of the same weight in whom equivalent am ounts of KC1 had been injected intracritoncally (i-p). Analyses were m ade in .a modified Bcrry-Chappcll-Barnes in:rnal standard flame photom eter (9, 10) . Prior to administration of curare the trachea was cannulatcd and artificial resiration provided by a pump. The concentrations of the agents used and their dosages follow: 1) Sodium pentobarbital, i f n ig /m l., tvas adm inistered i-p in doses of 40 to 50 m g /k g . b o d y eight. a) Sodium a , 4-diclilqrphi-noiyacctate m onohydrate (R a k e r) w as dissolved in w ater in a con* miration of j o m g/m l., and the f n w as adjusted to 7.4 w ith dilute I I C I for i-p in jectio n in doses of to ajo m g /lg . 5071 L '* .. j) d-Tubocurarinechloride* wasInjected -pindose*of 1 .j mg/kg. Thislsequ' Happroxt* matcly to one unit of cuiare per animal and produced total paralysis of all skeletal muvtie. 4) KCI was injected i-p as ao mg/ml. (170 mEq/l.) aqueous solution. Doses were 400 mg/kg. (5-4 mEq.). j) Quinine dihydrochloride was injected intramuscularly in aqueous solution of 45 mg/ml. and in doses of 150 mg/kg. 6) Disodium d-l-a-locophcryl phosphate* was injected i-p in an aqucouS solution of too mg/ml. in doses of 1 gAg. 7) Magnesium sulfate was injected i-p in doses of 150 mg/kg. 8) Calcium gluconate was injected i-p in doses of too mg/kg- RESULTS O ur observations are in full agreem ent with those of Bucher on the behavior of the ra t which has received 2 , 4-D (4). In both conscious and ancsthctiscd rats full development of myotonia appeared some 30 to 45 m inutes after i-p injection and lasted for hours. The injection of small am ounts (2 mg.) of 2 , 4-D into the vascular tree was followed by extreme generalised m yotonia in two m inutes. Even under deep anesthesia and full curarisation, the resistance to passive m otion of the ex tremities was appreciably enhanced. P rim ary cfTccts of 2 , 4 -D on muscle function arc illu strated in figures 1 an d 2. Increase in tension developed by an isometric tw itch, in response to a single supra maximal stim ulus to the nerve, was of the order of 25 to 30 per cent; duration of tw itch until half relaxation was greatly prolonged (figs. 1 A and 2A). T he electrom yogram of normal muscle stim ulated by a single shock to the nerve is a simple diphasic deflection (fig. iB ). W hen m yotonia had developed a fter in jection of 2 , 4-D the response became repetitive. The rapidly recurring, brief po tential changes were less than the initial spike, and occasionally they did not appear until after a short period of electrical silence, ro to 50 msec., following the initial spike. On one occasion the silence persisted for 600 msec. (fig. 3 C ). In some records the regular rhythm icity of the repetitive response suggested th at a single m otor unit was firing directly under the recording electrodes. T he duration of repetition was variable, ranging from 100 msec, to 6 sec. Prolongation of tw itch and repetitive firing decreased rapidly w ith repealed stim ulation; a t a stim ulus rate of 12 per min. myotonic features diminished rapidly during the first five or six consecutive single volleys (fig. 1 A, B ) .. R est for 10 m inutes resulted in complete return of the myotonic response. T he same changes were noted following stim ulation by a pair of nerve volleys delivered a t short intervals. M yotonia in m an and goat is characterised by exquisite sensitivity of muscle to mechanical stim uli. 2 , 4-D produced in the rat this same explosive electrical re sponse to tapping of the muscle or tendon and also to the insertion or movem ent of the recording electrodes (fig. 2). The site of development of the myotonic response to 2 , 4-D was delimited par tially by injection of sufficient d-lubocurarinc chloride to block completely any muscle *Generously provided by E. R. Squibb and Sons, Mew York, N\ V'., and by Abbott Laboratories, North Chicago, 111. 4Generously provided by IIofinian-LaRochc, Xutley, X. J. 0 0 m j 5 l M 0007163 F ig . ( . I ndirect stimulation o r the triceps surae bv the sciatic nerve. A . r . N orm a gram . s - j . Consecutive myograms, 110 m in. after 40 mg. of J , 4- D , in response to stim ul eicd a t a rate of la / m in ., illustratin g decreased duration and tbe phenom enon of `w arm -u p ' . Im e : Jo m sec. 7. T e n sio n : aoog. B. 1. N orm al electrom yogram , a - 5. Consecutive records 1 A . 6. V oltag e: a o o /<V . 7. T im e : 50 m sec. f F ig . *s. I ndirect stimulano.v and mechanical tapnko. A . 1. N o rm a l m yo gram . . min. after 30 m g. a , 4- D . 3 . 39 m in . after Co m g. K C I (sam e p reparatio n a s a). 4 . T im e : 50 Low er lin e: aero tension; middle lin e: resting tension of 143 g .; upper lin e: aoo g. B. a ktibmyogram in icsp o n sc to a brisk tap on tendon. 1 . N o rm a l, 3 . Co rresp o nd s to A s an d 3 . "'oltage: 1 jiV . 3. T im e : 30 msec. familiar sim ple diphasic electrogram o f ncr\*e stim ulated b y a brief single b recorded in figure 4 ( 11). One hour after adm inistration of 2, 4-D a similar iglfr. stim ulus evoked a volley of p oten tia ls w hich arose in th e n erve proper, ice,it had been isolated from both central and peripheral structures. Certain agents which modify spontaneous m yotonia were studied for their effects fth e myotonia produced by 2, 4-D . Potassium , 5.4 m E q /k g . i-p, elevated the rum concentration from a normal level of 5 m E q /l. to 10 to 14 m E q /1. in 20 to 30 Z:]'.-LW. ' 5072 007164 F ig . 3 - Completely curarized preparation; direct stimulation. A . r. N o r m a l m yo gram a. 4S m in . a fte r 40 mg. a , 4- D . 3 . T e n s io n : aoo g. 4. R e stin g te n sio n : 115 g. 5. T im e : 30 m sec. B. 1. N o rm a l m yogram , t. 43 m in. a fte r 40 mg. a , 4- D . 3 . t l m in . a fte r >0 m g. q u in in e . 4. $ m in. afte r ad d itio n al 10 mg. q u in ine. $. 10 m in. afte r 4. 6. T e n sio n : 300 g. 7. R e stin g te n sio n : 100 g. 8. T im e : 50 m sec. C. 1. N o rm a l clcctro m yog ram . a & 3 . C o n se cu tiv e sw eeps 40 m in . after 30 mg. a , 4- D , illu stratin g a n u n u su a lly prolonged period of silen ce (600 m se c.). 4. 5 m in . afte r 40 mg. q u in ine (irre g u larity of trace is ow ing to 1 ao cycle in terferen ce). 5 . V o lta g e : 1 >tV. 6 . T im e : 50 msec, (referring to 1- 3). 7. T im e : so m sec, (referring to 4). F ig . 4. D iphasic action potential from tibial nerve, i . N o rm a l, a. i j m in . afte r 30 mg. a , 4-D . 3. 60 m in. after a , 4-D . 4. V oltage: aoo jrV . 5. T im e : 30 m sec. m inutes. T his increase in serum concentration presum ably was reflected rapidly in the interstitial fluid surrounding the muscle cells, but analyses o f the total m uscle itself revealed no significant increase in total K content. W e have confirmed HZTTS l Mflfl '.`A . m u iile in response to a single stimu # ___________ --____ II course o f response to a , 4-D pro* . ; ;nttatiori o f th e m yoton ic response an d in degree o f rep etitiv e firing ( f i g n s B ) ^ Jrttr&sed se n sitiv ity to acetylcholine w as d em onstrated b y in jectin g to o ig. intftu Hally ( u ) . In the preparation described here, this injection elicited a few small tered^spike potentials in the electrom yogram . 4 A fter treatm en t w ith a , 4*D the; e am ount of acetylcholine evoked an explosive and prolonged burst of electrical vty-4. :iV. ;* I .. . Quinine has been shown to reduce or obliterate the m yotonic response occurring U ancously in m an ( 13, 14) and goat (6, 8) . T h e sam e effect w as produced on itonia induced in the rat by a , 4-D . T h e m yogram revealed a lowering of tension wing adm inistration o f quinine (fig. 3B ). T here are certain characteristics of response which warrant noting. First, tension developed after the second dose uinine fell to 54 per cent of the control tw itch; and second, the duration of the ch was not reduced. The effect of quinine on the responses induced b y 2, 4-D town in figure 3C where the characteristic repetitive firing of the muscle was Lerated. Alpha-tocophcryl phosphate, when injected i-p in relatively large doses into normal , produces profound generalized effects: somnolence, ataxia, m uscular flaccidity, a and, occasionally, death follow ing con vu lsion s ( 15) . W hen adm inistered per* ly to tw o p atients with m yotonic dystrophy a suggestive decrease in spontaneous Ionia was observed ( 16). Injection of a-tocophcryl phosphate in the rat, which received 2,-q D , obliterated the evidences of m yotonia as effectively as quinine. Similar suppression of repetitive response followed injection of magnesium and urn. D ISCU SSIO N ^Thc neurom uscular apparatus o f th e rat, treated w ith 9 ,4 -1 ), m im ics faithfully characteristics of myotonia occurring spontaneously in m an and goat. In both uhfcUnccs, m uscle exhibits exquisite sensitivity and repetitive response to several iSiitlr .... fcpcUtive phenomenon. Alpha-tocopheryl phosphate now m ay be added to the of agents inhibiting repetitive responses in both spontaneous and induced inia. These effects of 2 , 4-D , producing a m yotonic reaction so rem iniscent of the ltapcously occurring forms, are indistinguishable, likewise, from the consequences ctmcnt with a variety of substances; e.g., th e `vcratrin c' alkaloids derived from Urum and Schotnocaulon, aldehydes, tetraeth yl am m onium ions, phcnanlhrcne-9* oxylic acid and substituents, and dihydronaphthacridine carbonic acid v ' 1 M' 1' 0007165 FF( jcn co is group xVe h av e added recen tly p cntam eth*: Mahy, attd pcrHapi'all, of these diverse veratrinic substances, how ever, produce u m -iiq i titiv 'm p i e In n erve th a t th e y produce In m uscle. I t h a s n o t b een 1 'established in Spontaneous m yotonia whether or not this repetitive phenom enon ^occurs in nerve a s well as in m uscle; nevertheless, som e observations h ave suggested &*.that m yoton ia in m an is accom panied b y functional changes n ot lim ited to m uscle fiber proper ( 19) . In deed, it is reasonable to view repetition a s a stereo ty p ed re* '>S'f'gponse of excitab le tissu e (nerve, neurom uscular ju n ction and m uscle) to m a n y un* . ' related a ltera tiv es w hich m ay p la y rles o f differing in ten sity on a ll ex c ita b le stru c turcs. A ten tative exploration of this possibility m ight Begin b y exam ining the circumstances in which repetition has been observed. In order to lim it the analysis, only those instances of repetitive response to single stimuli have been considered; obviously, this lim itation excluded other im portant changes in excitability manifested b y spontaneous firing or b y m easurable changes in pre-discharge conditions, such as threshold, resting potential, recovery cycle etc. ( 20, 21) . T h e appearance in excitable tissue of rep etitive, rh ythm ic responses to a single stim ulus occurs under the m ost diverse of circum stances, as for exam ple: a) increased external h yd rostatic pressure ( 22); b) stim ulation b y co n sta n t rectangular currents ( 23); c) increased external concentration o f 11+ ( 20, 24) , K + ( 12) , a ceta te, lactate, citrate and oxalate ( 24) , B a ++ and guanidine ( 25) , ad en osine triphosphate ( 26), D D T and quinoline ( 27) ; d) decreased external con centration o f Ca++ ( 28); t) inhibition of cholinesterase activity at the neuromuscular junction b y cserine ( 11), neostigm ine ( 29) and D F P (30) ; / ) tetanus toxin affecting the neurom uscular ju n ctio n (31); g) treatm ent with that heterogeneous group of substances which produce veratrinic effects ( 17); and h) spontaneous myotonia in man and goat. In the face of our fragmentary knowledge of procsscs subserving excitability, the incongruity of those conditions and agents producing repetitiveness m akes it ex tremely difficult to apply any unifying concept which can explain all of the obser vations noted above. An exam ple of the difficulties encountered in a sim ple approach is contained in an attem pt to analyse the phenomenon of repetitiveness in term s of distortions o f cation ic vtilicit. Preservation of totally normal function dep en ds on maintenance of concentrations of N a+, K+ and Ca++ w ithin certain lim its. Could these m any agents act b y disturbing the required cationic patterns? Sim ple in spection of the structure of the veratrinic agents reveals their variation through al most full scale with respect to chemical activity, steric and physical characteristics; some are potential metal squestrants or prcipitants, others totally inactive. Lack of any com m on characteristic is emphasized not to im ply that a unitary' pattern of action is non-existent but to stress the necessity for fitting m any divergent obser vations into any proposed scheme. . It seem s likely that these m any different forces and agents m ay act at different loci in the com plicated train o f events responsible for the sm oothly integrated flow of energy which m aintains normal excitability. Only vague d ues exist to suggest certain possible sites of action. SJTfSTTdl IlfliflF orexam ple Lorentc de N 6, from an elaborate analysis in frog nerve, suggests Sfebin erlfii m i tudyjbf plants and micro-organisms com es suggestive,evidence' |-D *diitorts m echanism s involved in the transfer o f oxygen (32, 33) ^ C om patibly th,J>ut n o t proving, th is suggestion is th e suppressive action o f M g * + o n veratrinic cnomena ( 24) and the sim ilar a ctio n o f o-tocopheryl p hosp hate w hich is so p o te n tly :/! lioxidative as well as antiproteolytic ( 15). , , . .M odels of ex cita b ility or en ergy transfer expressed In term s o f ch em ical o r p hys1 structure, enzym atic activity, surface activity or membrane potential, m ay exlin Some or m any of the observations; b u t none, as y et, has been offered in satii:tory interpretation of all the d ata. , Jv.The phenom enon o f the silen t period intervening b etw een th e norm al single ke potential and th e outburst o f repetitive spikes follow ing treatm en t w ith 2, 4-D the same as that described b y Eichlcr in the frog treated with sm all am ounts of :ratrine* (34) . A sp cculative'in tcrp rctation o f this phenom enon m ig h t assum e ponential decay of facilitating and depressing processes initiated sim ultaneously the stim ulus (35). If under the observed circumstances the depressing process caycd so rapidly that the facilitating process remained unopposed, then a burst of tivity m ight be released. The data available do not permit a more specific analysis. S U in iA K Y ' 2, 4-dichlorphcnoxyacctatc produces in the neurom uscular apparatus o f th e rat xratnnic response marked by repetitive response to single stim uli in m uscle and rve. T h is results in increased `twitch* tension and prolonged `twitch* duration. iCrepetitive responses and their sequelae are accentuated b y K + and acetylch olin e d arc obliterated b y activity, quinine, Mg++, Ca++ and a-tocopheryl phosphate, esc phenomena arc indistinguishable from those occurring in the spontaneous m yoiia of man and goat, and also in response to several apparently unrelated chem ical, ysical and electrical agents. Si We ate deeply indebted to Dr. S. A. Talbot for designing and supervising construction of the cIronic instruments used in these studies. REFERENCES >N,W. A., R. E. Slade and W. G. T empleton. Brit. Patents 573, 919, 1941. . nhitauAN, P. W. and A. E. Hitchcock. "Cantrib. Boyce Thompson Inst, tt: j i t , 1941. ^Thompson, If. E., C. P. Swanson and A. G. Norman. Bet. Cot. 107: 476,' 1946. j Buche*, N. L. R. Proe. Soe. Exp. Biol. Jfc MeJ. 63: T04, 1946. l-T oomsen, J. A rth.f. Psythial. 6: 706, 1876. . Broun, G. L. and A. M. Harvey. Brain 61: 341, 1939. .'C lark, S. L., F. If. Luton and J. T. C utler. J . Ken. iftn l. Dit. 90: 297, 1939. | K ou, L. C. Bull. Johns ilapUns IJosp. 63: rrt, 193S. . Folk, B. P., K. L. Z ikrler and J. L. L ilientiial, Jr. Am. J . Physiol. 433: 381, 4948. i' Luientiial, J. L., Jr., K. L. Zierler, B. P. Folk and M. J. R ilev. In preparation. . Broun, G. L., II. II. Dale and \Y. F eldoerg. / . Physiol. 87: 394, 1936. . Walker, S. M. Am. J. Physiol. 149: 7, 1947. . Kolb, L. C., A. M. Harvey and M. R. Wiiiieiiill. Bull. Johns Hopkins llosp. 6r: *88, 4938. . Wolf, A .. Arch, i f enrol. Psythial. 36: 381, 4936. 5074 7t8 . ' Z i e u e r ,' K . J^-'a n d j . U L r u E N T iiA L , J r.' U n p u b lish ed o b s e r v a t i o n s ^ 1 V i^ ' v i / ^ R r a v m , O . a n d O . H . A ciieso m . Physiol. Re*. ttf: 3 8 3 ,1 9 4 8 . - .., :V.'- ' *.** : i 8 .;E y z a o u ir r e , C . a n d J. L . LntCN TH A t, J r. U n publish ed o b se rv a tio n s ..'* .^ ] ' ' i 19 / D e n n y - B r o w n , D . a n d S. N e v e r . B rain 6 4 : 1 , >941. ; to . E r l a n c e r , J . a n d H . S . G a s s e r . Electrical Signs o f N trvoui Activity. P h ila d e lp h ia : U n lv . 7V ; o f P e n n a . P ress, 1937. $ < s i. K atz, B . - Electric Excitation of Nerve. London: Oxford Unlv. Press, 1939. t i. G rondfest, II. Cold S frin t ITdrkor S ym f. Quant. Biot. 4:179, 1936. . s j. H o d g k in , A . L . . Phytiol. 1 0 7 :1 6 3 ,1 9 4 8 . -- 14. L o r e n t e d e N6, R . . A S tu d y o j Nerve Physiology. Studies Rockefeller In st. 1 3 1 - 1 3 * . *5. D on, F . T . and T . P . F eno. Chin. J . Physiol. 1 5 :4 3 3 ,1 9 4 0 . *6. B uckthal, F ., A . D eutsch and G . G . K nappeis. Acta Physiol. Stand. 8 : 1 7 1 , 1 9 4 4 . *7. Gordon, II. T . and J. H . Welsh. / . Cell. Comp. Physiol. 3 1 : 393, >938. *8. K opplf.r, S. W . J . Neurophysiot. 7 : 1 7 , 1944. *9. Harvey, A . M ., J. L. Loientual,J e. and S. A . T albot. Bull. Johns H opkins Hasp. 8 9 :3 * 9 , 1941. 3 a Harvey, A . M ., J. L. Lilienthal, J r ., D . G rob and S. A . T albot. B ull. Johns tlo p k in t Ilosp. 81: *6 7 ,19 4 7. 31. IIarvev, A. M. J . Physiol. 98: 348, 1939. 3 1. Smith, F . G . Plant Physiol. 23: 7 0 ,19 4 8 . 33. Worth, W . A ., Jr. and A . M . McC abe. Science 10S: 16 , 1948. 34. E i c iil f . r , W . Ztschr.f. Biot. 99: 243, 1938. 33. Harvey, A . M ., J . L. L ilientical, J r ., and S. A . T aldot. Bull. Johns H opkins Ilosp. 8 9 : 547i 94 . 9 7T T T r : l u n n 0G 07166 lo 507; Reference 4 Id ahoek, nepectlTelp. AS of the 10 eoatrola hewed atea, ap p a ra tip a trae antlanaphplaetle drag in th at it ahoek, S died, 1 m aeverely shocked, u d the r a t bowed Interfere with tha antigen-antibody reaction to pre- m oderate o r s l i d hock. rent or deereaae tha ontoward reanlta of tho challenging Three week later w m of tbo arTirois of both tho doae of antigen. 4 pcrimeatel and tha control granp w en again chaDcaged without-the drag with 0.6 cc of egg white, Lr. AU bowed TABU 1 X rrscta o r A oK in im u rion or 16 G atina ( J U G u) or A c a rru a u eru e Acta, n Divina Door. a Anarmtlac ric B eocs Ma. Doaose 831 333 13 fioiaa mia 3 i i m 337 30 041 03 43 M 43 a 347 36 34 3 33 3 38 03 b ra . 2 1 21 dar* later 3 4 1 #9 ao O0 0 ,, 1. CanrosLL, Bsaar. BaaonorasT, i n s D., aad Good, Bosaar A. ^rec. tu*, car- M . Jfad* 1047. 04. 261. 2. Coaran, A. F,, aad Karr. S . IL J. *. Jfad, 1041, T7. 173. 1. D naics, C. L . IliT rnrocn, C. A , aad Bwtvr. H, r . / . rUe. /aeeaf.. 1026. 6. 427. 4. Bomrasa, T. B ra . Beat, aeo. B id . Mad* 1040, 01 10L 6. Jaota. B. T. Brae. Amir. Pcd. He. Baa* 1047, 0 1 . R im . L A , Boiaraar, P. J., and trin a , W. W. Betaaee, 1040, 100, 332. 7. Bolliva*. C L P ia a n . T. V , aad Riaaaar, A W. P ra . Be. **. B iel Med., 1343, 07. 303. A Bwirr. H. P. J. ero. Bad.. 1322, 33. 738. Effects of 2,4-Dichloropbenoxyocedc Add on Chicks1 IfELVoi K. Bjoon and E x n r T. N o o n s 33 34 Mo0#m 34 aa 3 40 aa 43 44 a 40 os 4 M 04 a t 34 24 Department / Botany, Dnfearrify / Wyoming 1 1 With the inereadng n o of 2,4-D aa a harbieide it ia 1 4 2 important to inquire iato tho poaribh toxio offoets of tho # chemical oa animal. Several larcatigatora bara atndiad 2 tha effect of 2,4'D oa mammal. Tha lethal doea for 1 2 4 0410 mice, when injected aubentnneooolp or intraraocaly, haa been determined bp Bnehiar (1) to ba t86 mg/kg of body weight, MitebeU and K arth (B) reported that thay fad 200 mg of 2,4-D daily, to n a i l experimental aaimala with To teat tho ffet of aeatylaalleplla ad d oa histaariaa ao ill effects. aek, 11 rabbits weighing 1 -1 1 kg war* g in n 6 graiaa Of neetylaalieylle add, orally, a t StSO PAL oa ICareb 11, TABU 1 1047. Thla doaago waa rapante* a t 0:00 AAL oa March 12 and again a t 2:30 PAL the aaam dap. One boor later, C m n or AUAnoLAMina or 3,4-D on W arn Bocx C aicso each rabbit waa Injected intravenously with 1.73 mg of hkUmiae phosphate. S e ra of tha 11 died (# 4 abode), S bowed arer (# 3 ) shock, aad 3 bowed aMdorato (# 2 ) ahoek. DoMce (m s of acld/knof body welcht) lacera* la weight at end of fonr weoka (%> Tbo animala whieh wore preaedieatad with aeetplaaliepUe ad d bowed atrikiag proteetioa agalaat anaphy- lactle ahoek. I t aaap bo praw ned, oa tho baaU of tbo M en to n rerlewed sbovo, th at thla waa dao to a do* errai la the aatlbodlea a t tha time o f the ekeUengiag doae. Thero la aethiag to indicate anp protaetlro effect againat hlataalne ahoek, fo r though anaeeompanied bp aimoltanaoaa control, the aaorbidltp la tho boro aoriao waa d o lla r to th at reported la a predoni coauanaieatJoa (1). I t la iatam tln g to noto that the offoeta of tho drag were temporarp and th a t tho treated animala ahowed eoadderabla bppeneniitiritp 3 weoka later. Tho eontra d between tho reanlta with thla drag aad thoae reported earlier (p) fo r Beaadrpl ia completo. Benadryl gare good protection, In dm tlar sperimenta, ngalnat hlataalne ahoek bot waa withont affect oa an aphylactic ahoek. Tho p m e a t drag, aeatplaalieplie add, however, effect!reIp protect againat anaphylactic ahoek Lat not ngalnat biatamine. I t la, with tha other aalleyl- 0.00 (control) J 3.80 28.00 280.00 433 444 4M 427 373 la tha p ra e a t aspefimant Whlta Bock ehleka won naod. Tha dkanolamiaa of 2,4-D waa admialaterad orally through a pipette. In tho drat apartment, data for whieh ore giro ia Table 1, one part of tbo alkanolomlwo waa dilated with 10 parte of wnter. The dosages recorded are in terms of tho add equivalent. Five ehleka (each weighing approximately 60 gm a t tha beginning) w en used la each group. Tho chirk w an weighed and then won g ir a tho appropriate doaa (Table I) three time a week oa alternate dap* for n period of fonr weeks, making a total of 12 d o ra Aa the ehleka gained in weight, the >Coattibottoa Ma. 210 from the D eport of B ettor and tha Aock? Manatala Heibarlas^.Ualvanltr of Wromlns. CCIEN CE, O ctober 29, 194, VoL 10 XI N ( oc n 00 05 ro < p z 9 * 1\ >J < 2N mN3 Zo z z 0 5076 0002798 f mount administered u adjusted is ardor U m h U h the original dosage. A t tho o d e f four w b they am g ala weighed. T te table hows the perecatago lie m m is weight fer tho looM N tk period. The differences between the eeatrol group a id theee girea docagee of 0.28, S M , a id 28.00 m g/kg were le t d p ld e u t a t the 5% Im L The difference betweem the eeatrol groep a id the greap fire s doeage ef 280 m g/kg waa barely significant a t the t% lereL Next, experiments were started to determine the lethal does of the elhaaolamiae e f 2,4-D whea dilated 1:0 with water. Each ehiek of a group of 8 (each areragiag 160 gm) wae girea one dees of 280 mg/kg of body w eight These ehleha w eired. Each ehiek e f aaother greap e f S ehieha (arerage weight, 111 gm) waa girea a dees e f 768 m g/kg of body w eight All of these ehieha died. Postmortem examinetioa rereeled a fatty degeaetatioa with a pale se ttlin g ef the llrer, spleen, kidneys, aad h eart Hetnorrhagie gaatreeateritia waa abo erideat* Hence, for email ehieha the lethal does of dilated 2,4-D is eomewbere bo* tweca 280 and 768 mg/kg. Tho fact that a single does of 763 mg killed, bat not a total dees ef 2,260 mg administered orer a four-week period (280 mg/kg ehieha of Table 1), ladiestee that the alkanolamiae of 2,4-D U not a eamolatire poison. The question might be raiaed as tc tho poeeibility of ehieha being hilled by feeding ea plaata which had been sprayed with 2,4-D. A t a spraying rate of 1 lb of 2,4-D/ aero, a ehiekea weighing 1 kg would hare to consume oil of the 2,4-D applied ea 72 sq f t within a day or two to obtain a lethal dees. 1. B ecaaa . R . L . 1 Pree. gee. am . S M . UH.. 1040. S3, '20*. - 2. U ircaau , 1. W , aad Means, P. C. get. S u , 4 4 ,100. too. A New Histological Procedure for Whole . Tissue Cultures Grown in Plasma A m u i i u Conan aad Coautt Watkouth Chester B m ttg Jtcsrereh /astitate, Jt syol Cancer Jgaspital, f0tam flood, Aendoa, S .JFJ Standard taxtbook proeadaraa ( f , J ) reeeauaeaded for fixing and etaiaiag whole tissue outturn la plasma eoagula hare prorea uaaatlefmetory for the following reasonsi (1) The dense fixed eoagulum presents a nearly impermeable barrier to the stale, thus aeeemitatiag pro longed staining periods) (2) the aoagulum itself stains diffusely and haarily, presenting adequate contrast be* twees the cells end the medium aad aeeeaeitating careful aad tlme roneualng deeolorising aad deratoplag pro* eedores) ({} the reeuttlag^preparation, when mounted, is thick, hoe o tendency to form sir bubbles, does not T b s anthem i n sratefel to Hebert W. U adsestru tb, who msde tbs, eism lsstto es. 480 dry rsadily, end le generally Uadoquato fo r mietooeopU etndy. Tompkias, Cnnningbam, aad K lrk (d) mesutly attem pted te Imprese ternato by woohiag cattareo in a . salt salatina fo r 4 hra a t 7* C te remore eeluble p iatela before fixatioa, bat stlU had te reeert te prolongad etnialag (1 k r ia Delafleld's kem ateaylia) followed by aer* ria l baure of waahlng. Earle (2) haa dariaed empii* ratrd fixing, sU iaiag, nad meuaUag piocodwcs, too elab orate ta w arrant eraloatloa beta. P ia. 1. ntass appesisse* af staiard tteeae eeltam e: A, plsaam aadrltd : B. plasma drisd (a 1 2 ). VTe here found that most of the dlaadraatages e f the plasma eoagulum con bo eliminated by drying tho whole tissue culture preparation after fixation. The prosedore is as follows: The culture in its eoagulum on a eqror slip ia fixed orernight in 3-4% formol containing 02% acetic aeld or la Carney's fluid for 1 h r. la general, aeetioaleohol fixatioaa giro a m art granular aad more opeqae dried specimen than aeetie-fonaol fixatioaa. The sorer slip is then washed thoroughly ia raaalag w ater (after aeetie-fonaol) or la descending strengths of alcohol, aad water (after Carney's). A fter a final rinse ia dlitHlod water the carer slips are laid flat on a glass eurfaoa protected from dost (P etri dishes or etaiaiag dishes eaa be used) and allowed to dry slowly aad thoroughly in air. I f a preparation of a tube or a flask culture ia plasma is desired, the portion should be fixed aad washed in mtm, pried loess sad placed, like a paraffin sectton, ia a dish ef water, where it eaa ba manipulated with a dissecting needle sate a elide or eorer slip prerleueiy coated with a thin film ef M ayer's albumin, aad dried as abore. Cultures grown ia a fluid medium should aot be dried. The dried cultures may ba stained w ith say one of the hematoxylins for from 8 tie IS a la , depending ea the strength ef tee stain. W elgert*s iron-hematoxylin has been found eery satisfactory. The staining ia simple to eoatrol after a prelim inary-trial, mad prolonged wash*, lag to derelep the color is aot necessary. Ones stained, the cultures should net be allowed to dry again. Poliowlag 'this, tee eqror allpa ere passed through the aleohola in tee usual manner, cleared iu-xyleae, end mounted. I f a I r ishman or Olomea stain ia used, acetone and acetone* xyleae dehydrations should be employed Instead ef the alcohols. The dehydration end electing procedures take considerably leas time with dried cultures, sines each SC IE N C E , O ctober 2. 1948, VoL 108* DOW 508325 t ^ q ^ rj 00027S9 5078 Biochemical Research Laboratory TH E DOW CHEMICAL COMPANY R ES U LT 3 OP SK Z R IR R IT A T IQ R T E S T S OR VA RIO U S SA M PLES OP 2 ,* - D TA X ER A T V A RIO U S STA G ES IR T H E M ARUFACTUR2RO PRO CESS ^ y3 * fb. T23.1U11-V CH#. 1 2 1 9 - 0 2 ^ 5/53/*9 Fin'd 1 0 / 1 2 / ^ 0 W ork By V . X ROVO B r it t o n 's D iv is io n A t t i J . V . B r it t o n R C . D o sso r C . A . K lg h h lU Ckock H . R . H o y le / E s e c u tiv o R o s e s re h C o a a itte e O rg a n ic R e s o s re h la b o r a t o r y A t t i E. c. B r it t o n /d -/ -4TS T H i i ; i-v.'hT THE Pr<r Or T!!> DOW CMEr:CAL COMP -- Y .yk Ia p u r e 2 ,A - D i s e o n s id t r s b ly s e r e i r r i t a t i n g th a n th e p u r ifie d p ro d u c t. 2 vt- D ie h lo ro p h e n o x y a e e tla s e id A e e tlo a e id t 2 >t - d lc h lo ro p h cn o x y - J <0 >0 m ir\ o cn ** CD CD o cn A fe w e a s e s o f d a m a t lt ls h aw s b een o b se rv e d aro u n d p a r t ic u la r o p e r a t io n s in th e a a n o fa c tu r e o f 2 #A D . S a a p le s fr o n th e v a r io u s s ta g e s o f a s n u fa e tu re w e re s u b s it te d f o r s k in ir r it a t io n t e s t s to d e t e rs in e w h e th e r o r n o t th e re w as a s ig n if ic a n t d iffe r e n c e b e tw e e n th e v a r io u s p r e p a r a t io n s . M A TER IA L Rasw s 2 , A - D le h lo ro p h e n o x y a c e tle a c id < P o m u la t S tru e t u r a li E a p ir ic a lt C l fi H* C l -O-C-C-0-H H CgHgO ^Cl^ 0007478 S a a p le s o b ta in e d b y i H . R . H o y le f 5079 00jy . 549306 Biochemical Research Department T23.14-11-3 Page 2 Sample description: 3.R.D. K no. 2372-12 13 drier feed from plant no. 2. 267-Building containing 0.24 of 2,4-dichlorophenol. K2372-13 is drier feed fro m plant no.l, 267-Building containing 1.2ljfc of 2.4- dichlorophenol. K2372-14 is unwashed cake from the filter box, 267-Building containing 0.96# of 2.4- dichlorophenol. K2372-15 is cake from the BIrd f ilt e r , 489Bullding containing 0.10 of 2,4-dichlorophenol. X2372-16 is an especially purified sample of 2.4- D obtained from 267-Bulldlng, low in 2.4- dichlorophenol. EXPERIMENTAL RESULTS When the purified sample of 2,4-D, K2732-16, was repeatedly bandaged onto the shaven abdomen of a rabbit in a dry state, it produced no detectable irritation; when wet with water it produced only a very slight, simple irritation. <* When the 2,4-D cake from the Bird filter, X2372-15, was repeatedly bandaged to the shaven abdomen of a rabbit, it failed to produce any appreciable evidence of irritation except a rather bluish coloration of the dermal tissue; when wet it produced a slight amount of superficial denaturatlon, again with a rather marked purplish coloration. This lesion healed promptly after exposures were terminated. When the dry 2,4-D unwashed cake from the filter box in the 267-Buildlng,,2372-14, was repeatedly bandaged to the shaven abdomen of a rabbit, it promptly produced an appreciable amount of irritation, characterized by hyperemia and exfoliation; when dampened with water this sample of material produced a moderate to severe 0007479 5080 iiiachemica! <csearch Tapertnent T23.14-11-3 Page 3 response. This latter response was characterized by moderate exfoliation and necrosis and a great deal of soreness. When the 2,4-D drier feed from plant no. 1, 267-Building, <???T?-1 7 , was tested in a similar manner, it produced a slight, 3imple irritation, which was not enhanced by wetting the material. W h e n the drier feed from plant no. 2, 267-Building, ..T2372-12, wa3 tasted it produced no appreciable irritation. CONCLUSIONS It would seem fro m these results that impure 2,4-D is more likely to produce a significant amount of skin irritation than is pure material; and also, it seems that a damp product is more likely to produce irritation than is a dry one. 549307 bc-6 t 000'V480 v i. 5 0 8 1 8 5082 T 2 3 ' Y - i ' - y TQCOO# ca c e eo TM cow chsm cg m am n M in o m c a M t W T O * -CITY SAN rSAN C ISC O ph ila o cla h ia LOS AN O tLC S WASHINGTON S A IN T LO U IS C LIV C LA N O HOUSTON CH IC AG O (A T T U I D C TW O IT The Dow Chemical Company "MIDLAND MICHIGAN December lb, 1949 Dr. V. A. Drill Department of P h a rm a c o lo g y and Therapeutics Wayne University College of Medicine 1512 St. Antoine Street Detroit 26, Michigan xj ro 00 o Dear Dr. Drill: We are sending to you under separate cover, samples of 2 , 4 - D (2,4-dichlorophenoxyacet lc acid) and 2,4,1>-T (2,4,b-trlohlotophenoxyacetlc acid) for use in the toxicological studies on aogs. The sample of 2 , 4 - D being sent la regular production 1natei loi a s s a y i n g 9 8 .5^ a n d h a v i n g a f r e e z i n g point of 132.ti*C. The sample of 2 , 4 , 5 - T being sent is a lso regular procuctl:; m a t e r i a l a s s a y i n g 100)1 a n d h a v i n g a f r e e z i n g point of 148.8Vj. B o t h o f these m a t e r i a l s a r e being s u p plied to you in the aci d form. B o t h a r e f a irly s oluble In olive oil and both cui oni Lj be put In a q u e o u s s o l u t i o n w i t h caustic. I bolieve you will fin-! that a a l i g h t e x c e s s of ca u s t i c la r e q u i r e d to put theae material;-, solution and that once In solution, a large part of the excess al\nll c a n be neu t r a l i s e d . Hence, f o r a 5 .0^ a q u e o u s .o'.utlon of the b o -.u .: salt of 2,4-D, the pH after solution is effected, can be adjusted down to 7.2 with HC1; with 2,4,5-T the final pH of a 1.0 aqueous solution can probably be in the neighoorhood of 10 . W h e n 2,4-D or 2,4,5-1' is used ae an herbicide, the c o n c e n t r a t i o n usually ranges f r o m 0 .1^ to 0 .2,*. The a ctive luiteri-.l r.iay be a p p l i e d In a n y one of a numb e r of forms d e p e n d i n g upon tne particular r e q u i r e a e n t s of the job. fhe most common forms a. c- 1 . 09 sodium salt, alaanol amine salts, and esters. _ *1 ......... Almost all the toxicological work so far reportcu ^ w i t h 2 , 4 - D a d m i n i s t e r e d e i t h e r as the free acid In oI.L or r.' s o d i u m salt In a q u eous solution at a pil of ( . 2 . *e navt to... r. . a fair a m o u n t of toxicol o g i c a l work on the other f:rr.iO of L - - a l s o on 2 ,-*.S>-f in its various fornn. V.e nave outervc=; ii.di. . 5 0 8 3 of the ma t e r Lai :u*hei> little d i f f erence to u.u- .* ..),<. ue.. ;.ni .; - that, acutely,, u.ere is wot u.ucn d i f f erence j .1 0 , . .... ^onnn Dr. V. A. Drill Pig 2 December 13, 1949 The toxicity picture as jre Jiaee At Am *isn An Atom following tabulations! Toxicity of 2.4*0 when administered in Single Doses. Animal Route Source Aoorox. LD~0 (a.Ax.) Rat Oral Rat. Oral Mouse Oral Mouse Guinea Pig Sub-Cut. Oral Chloks Oral Lit. Na Salt Dow-Incomplete Na Salt Acid Isopropyl esters Lit. Na Salt Dow - Incomplete Acid Lit. Na Salt Lit. Na Salt Dov - Incomplete Na Salt ACld Lit. Na Salt Dow - Just started 0.666 0.70 0.5 - 0.6 0.5 - 0.7 0.375 0.3 - 0.5 0.280 1.000 0.5 - 0.7 0.3 - 0.7 0.38 - 0.76 Toxicity of 2.4-D when administered in Reseated Doses. Animal Route Source Notes Dogs Sub Cut or I.V. Lit. Guinea Pigs Orally (/) Lit. * Rats Mies Orally in diet Lit. Dow I.P. Lit. C G O 'S 2 3 Na Salt 0.200 g./kg/d*y-death in 2 da, 0.100 g./kg./day dsath in 2-4 days 0.030 g./kg./day dsath in 3 days 0.023 S A 6*/d*y marked affsets Na Salt 0 .1 0 0 g ./ k g '/ d * y 10 times in 12 uaj was tolerated O.lji tolerated 0 .1 -caused very slight effects 0. 03^ clear No marked histop&tftolSgical findings ho neoplaotic rowtna 5084 Reproduction G.K. o Dr. V. A. Drill Page 3 December 15, 19*9 Toxicity of 2*4-D jibn ad*inlstered in -Repeated Dotas. - Cont *d Anlaal Chicks Route Orally Orally In Diet Source Lit. Dow Motes 0.028 .A. 3 tlnaa/vaak for 4 weeks - 0.1 . 0.280 g./kg. 3 tinaaAaak for 4 waakt - growth daprattlon 0.1* for 1 waak - O.K. 0.3* for 1 waak - O.A. azcapt for slightly dapraated growth rata. Toxicity of 2,4,5-T Whan Adainlttarad in Singla Dotat Orally (This Information It from our data and It not eoaplata - We art finishing It.) Anlnal Rat Mous Oulnaa pig Chick Wotat LD30 for the acid H a t between 0.5-1.0 g./k LD50 for the isopropyl attar lias between 0.3 -1*0 g.Aff* LD30 for the aeld lias between 0.3-0.7 g . A s LD50 for the aeld lias between 0.3-1.0 g. A s Por both the aeld and Wa salt Work Just begun Neither 2*4-D nor 24,5-T are aore than vary wild tkin Irritants van in eonoantratad fora. In dllutad fora ready for uaa, they present no health hasard. Studies eonduetad upon guinea pigs by Hill and Carlisle on tha Inhalation of 2,4-dlchlorophenoxy aoatlo acid dust, either wat or dry, but of unknown particle site. Indicate that tha dust It not likely to causa aystaale Intoxication. This hat bean born out by our own ex perience with nan handling tha dust. Tha dust, however, it capable of causing Irritation of the note if the concentration in tha breathing zone becoast sufficiently high. ..... Tha following references nay be helpful if you wish to consult the literature for a aore detailed description of procedures, etc.: CCO-iT-iO inono Dr. V. A. Drill Page A December 13 1949 1. Toxicity of 2#4-Dlchlorophenoxy Acetic Acid for Experimental. Animals. Edwin AT. Hill and Harold Carlisle (Camp Detrick, Frederick, MD.) J. Ind. Hyg. Toxicol. 29 85-95 (1947) C.A. All 3217a (**y 20, 1947) 2. Tolaranca of Farm Animals to Faad Containing 2,4-Dlchloro phenoxy aeatlc aold. J. V. Mltchall, R. E. Hodgson and C. F. OastJens (U.S. Dapt. of Agr., Washington, D. C.) Animal Scl. 3 228-32 (1946) C.A. 40 (17); 5198. (Sapt. 10, 1946) 3. 2,4-D Toxicity. I. Toxicity Towards Certain Species of Fish. Jos. W. E. Harrlsson and Edward W. Reas (La wall and Harrlsson, Research Consultants, Philadelphia, Pa.) Am. J. Pharm. 118, 422-5 (1946) C.A. 41 (2529) (1947) 4. Effects of 2,4-Dlchlorophenoxy Acetic Acid on Experimental Animals. Nancy L. R. Bucher (Harvard University) Proc. Soc. Exptl. Biol. Med. 63 204-5(1946) C.A. 41* 808 (1947) Data on the acute oral toxicity of both 2,4-D and 2,4,5-T for dogs Is lacking. Therefore, It is suggested that the acute oral studies be started at your earlleat opportunity so that Information will be available for choosing appropriate dosage levels for the 90 day feeding tests. In the single dose experiments we suggest that a notation be made regarding any symptoms exhibited at the various dosage levels, body weights for each day during 3 or 4 days Immediately following administration and on alternate days during the rest of the 2-week observation period. We do not feel that It Is neoessary to do any elaborate tissue studies on acutely dosed animals. We would, however, like to see animals that die and those that survive autopsled and the gross condition of the Q.l. tract, liver, and kidneys noted, A few sections at critical dosage levels might also be desirable - at your discretion. CGG4c4i 5086 \. AArSn Dr. V. A. Drill Pag t> December la, 194y In the 90 day faading tests, we suggest that the material be administered with a part of the diet. Dosage levels cannot be decIdea upon until acute data la available. We would suggest that general ob servations, growth records, and periodic hematological examinations (initially, after 30 days, and after 90 days on the experiment) be con ducted during the experiment. At autopsy, gross and hlstopathologlcal studies on the liver, kidney, stomach, intestine, spleen, lung, heart, adrenal, testis or ovary, and possibly brain and vascular system are of Importance. We believe that negdttW^rfWIngs at upper dosage levels make similar studies at lower dosage levels unnecessary. The requisition for this work has been approved and I will see that an appropriate check is deposited as before. If there aze any details which are not clear or if any problems arise, please contact us at once. I regret that I did not have more time last week to spend with you. Nevertheless, I was very glad to get home early and avoid the snowstorm that settled on this area about 5t00 p.m. that evening. Please give ay regards to Dr. Hays. Sincerely yours, THE DOW CHSHICAL COMPANY VKR/uig / c <> V. K. Rowe Biochemical Research Department '42 5087 J-E8QSM09 Reference 6 ACUTE ORAL TOXICITY OP 2,4-DICHLOROPHENOXYACETIC ACID TO RATS, MICE, GUINEA PIGS AND CHICKS V. K. Rowe D. D. McCollleter and H. C. Spencer Biochemical Research Laboratory The Dow Chemical Company Midland, Michigan 5089 0002800 0o V / 508335 -1- The 2,4-dichlorophenoxyacetic acid was administered by oral intubation to rats, mice, guinea pigs and chicks as a solution in olive oil emulsified with 5-lOjS gum arable solution. For comparison, the sodium salt of 2,4-dlchlorophenoxyacetic acid was fed in aqueous solution to rats and guinea pigs. The. guinea pigs and the white rats were mature, young adult animals selected from the stock colonies of this laboratory. Young adult white mice were obtained from the Carworth Farms, ROckland, New York. The chicks (New Hampshire Red) were purchased from a commercial hatchery and used when about three weeks of age. Mixed sex groups of each species were used in these tests. Table I summarizes the concentrations and volumes of olive oil solutions of 2,4-dichlorophenoxyacetic acid administered to each species. TABLE I SINGLE ORAL ADMINISTRATION OF 2,4-DICHLOROPHENOXYACETIC ACID Species Per cent Material in Olive Oil Volume Given by Stomach Tube Minimum (ce.) Maximum (cc.) Rats Guinea Figs Mice Chicks 5, 10 10 5, 10 3 0.4 0.9 0.1 0.3 4.0 2.9 0.22 5.2 Mortalities resulting from the various single oral dosages of 2,4-dichlorophenoxyacetic acid and its sodium salt are given in 5090 002801 eoW 508336 Tables II and III. All surviving*animals were observed until it was certain that they had fully recovered (usually about two weeks). Symptoms of poisoning, particularly with the higher doses, were ataxia and myotonia. TABLE II MORTALITIES RESULTING FROM ADMINISTRATION OF SINGLE ORAL DOSES OF 2,4-DICHLOROPHENOXYACETIC ACID Dosage (gm./ k g m .) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1.0 Rats No. No. Fed Died 10 0 10 0 10 3 10 6 10 7 10 10 33 Guinea Pigs No. No. Fed Died 60 64 66 Mice. No. No. Fed Died 60 51 64 66 66 Chicks No. No. Fed Died 60 61 66 64 66 TABLE III MORTALITIES RESULTING FROM ADMINISTRATION OF SINGLE ORAL DOSES OF 2,4-DICHLOROPHENOXYACETIC ACID, SODIUM SALT Dosage (gm. A s m . ) Rats No. Fed No. Died Guinea Pigs No. Fed No. Died .0.3 10 0 6 0 0.5 10 2 6 4 0.7 10 . 4 6 6 1.0 14 14 The acute oral LD^q values with their 19/20 confidence limits v:ere determined for each according to the method described by Litchfield and Wilcoxon1. The results of the statistical treatment of the dosage- response data are presented in Table IV. 5091 1. Litchfield, J. T., Jr. and Wilcoxon, F.: J. Pharm. and s 1 -3- TABLE IV THE ACUTE ORAL LD50 VALUES FOR 2,4-DICHLOROPHENOXYACETIC ACID WHEN ADMINISTERED TO RATS, MICE, GUINEA PIGS AND CHICKS Species Form LD50 (19/20 Confidence Limits) Slope gm. A g m . Functi Rats Mice Guinea Pigs Chicks Oll-acld Oil-acid Oil-acid Oll-acld 0.375 (0.302-0.465] O .368 :0 .3 1 2 -0.434 0.469 0 .39 7-0.553 0.541 [0 .358-0 .8 17 ] 1.6 2 1.23 1.17 I.8I Rats Guinea pigs Aq.-Na salt Aq.-Na salt 0.805 [0 .610 -1 .063] O.55I [0 .4 1 7 -0 .727] 1.77 1.55 *The fold change In dosage required to produce one unit standard deviation In response along the line. DOW508337 The Litchfield and Wllcoxon test for parallelism of two lines revealed that the dosage-response Mine for the rats (oil-acid) deviated significantly (19/20 probability) from those for the mice and the guinea pigs receiving the 2,4-dichlorophenoxyacetic acid In olive oil. The line for the chicks also deviated significantly in parallelism from those for the guinea pigs and mice. The tests for parallelism of two lines and estimate of relative potency Indicated no statistically significant differences in comparison of rats versus chicks and mice versus guinea pigs. The dosage-response curves for guinea pigs (oll-acld) and guinea pigs (aqueous-sodium salt) did not deviate significantly In parallelism or relative potency. A comparison of the rats (oll-acld) versus rats (aq.-Na salt) revealed no significant difference In parallelism of the two lines,but the test for relative potency Indi cated that the free acid was significantly more toxic to rats than was the sodium salt. 5092 0028G3 to VO ' G ( 5 0 9 3 BOW 508338 SEMICHRONIC ORAL TOXICITY OP 2,4-DICHLOROPHENOXYACETIC ACID TO RATS I. Oral Administration - Repeated Doses by Stomach Tube. II. Oral Administration in the Diet V. K. Rowe D. D. McCollister and H. C. Spencer Biochemical Research Laboratory The Dow Chemical Company .. Midland, Michigan 5094 0002804 DOW 508339 i h/ - 1- I. ORAL ADMINISTRATION - REPEATED DOSES BY STOMACH TUBE Experimental Procedure Thirty-one female rats, 60 to 80 days old, were divided according to body weights into groups of five each (six in the control group) and \ maintained on the stock rat diet. Before the repeated oral administration of 2,4-dichlorophenoxyacetic acid was started, weight records were kept on the animals for a period of 21 days to make certain that the groups were well matched in respect to growth. The rats in each of the groups were given by means of a stomach tube repeated oral doses of 2,4-dichlorophenoxyacetic acid dissolved in olive oil and emulsified in 2 to 3 cc. of a 5-10 per cent gum arable solu tion. The dosages fed and the olive oil solutions used are given in Table I. TABLE I DOSAGES ADMINISTERED AND OLIVE OIL SOLUTIONS OP 2,4-DICHLOROPHENOXYACETIC ACID USED Dose g.Ag. Per cent 2,4Dlchlorophenoxy- acetic acid in Olive Oil Approximate Vol. of Olive Oil Solutlon Administered per Rat per Dose 0.0 O.OOJ 0.01 0.0'3 0.1 0.3 - 0.00 0.06 0.20 0.60 2.00 6.00 1.5 1.0 1.0 1.0 1.0 1.0 5095 0002805 DOW 508340, The animals were weighed at intervals of two and three days during the experiment, and a record was kept of the average growth of each group. Hematological examinations were made on the control rats and on those that had received 20 oral doses of 0.0? and 0.01 g./kg. 2,4-dichlorophenoxyacetic acid. At the end of the experiment, all of the surviving rats were killed by decapitation, and oxalated blood wa collected for urea-N determinations.^1^ Each rat was examined for gross pathological changes, organ (heart, liver, kidneys, spleen) weights were obtained, and tissues were saved for microscopic examination. Hematoxlln and eosin-stained sections of the following organs were prepared: lung, heart, liver, kidney, spleen, adrenal, pancreas, stomach and intestines. EXPERIMENTAL RESULTS The rats that received 20 oral doses In 28 days of 0.00?, 0.01, and 0.0? g./kg. 2,4-dichlorophenoxyacetic acid showed no 111 effects so far as could be judged from gross appearance and behavior, survival, growth, hematology (Table II), blood urea-N values (Table III), organ weights (Table III), gross appearance of organs, and hlstopathology. (1) Barker, S. B. The Direct Colorimetric Determination of Urea in Blood and Urine. J. Biol. Chem. 152: 45?-46?, 1944. ni .TABLE II AVERAGE HEMATOLOGICAL VALUES OBTAINED ON FEMALE RATS THAT HAD RECEIVED 20 ORAL DOSES IN 27 DAYS OF 2,4-DICHLOROPHENOXYACETIC ACID Dose g.Ag. Controls 0.0? 0.01 Number of rats in group Erythrocytes (million per cu.mm.) Hemoglobin (g./100 cc.) Leucocytes (thousand per cu.mm.) Differential Count: r.Neutrophils (per cent) Lymphocytes (per cent) 6 11.4 13-9 18.8 10 90 5 10.5 13.3 1 8 .> 15 85 5 10.0 12.9 17.4 9 91 TABLE III AVERAGE BODY WEIGHTS* ORGAN WEIGHTS AND BLOOD UREA-N VALUES OF FEMALE RATS THAT RECEIVED REPEATED ORAL DOSES OF 2,4-DICHLOROPHENOXYACETIC ACID Dose g-Ag- 0.00 0.003 0.01 0.03 0.1 0.1 0.3 No. of Doses 0 20 20 20 15 15 2 No. of Days 28 28 28 28 21 21 2 No. of Rats Surviving 6 5 5 5 3 2 2 B o d y Wt. (&) Initial Final* 195 203 191 197 191 195 187 192 190 126 19* 202 195 186 g.)Organ Weights (g./100 Heart Liver Kidney Spleen 0.42 0.40 0.42 0.41 0,51 0.43 0.48 3.40 3-37 3.38 3.25 4.68 4.08 3.08 0.75 0.75 0.79 0 .8 1 0.94 O .87 O .82 0.23 0.22 0.19 0.23 0 .16 0.23 0.17 Blood Urea-N mg. % 24.9 24.8 32.8 25.9 26.6 17.2 -- Fasted overnight. 002S07 mSOSMOQ cn o CO DOW 508342 -4- Of the five rata that received repeated oral doaea of 0.1 g./kg. 2.4- dichlorophenoxyacetic acid, three loat weight rapidly after the flrat few doaea and 8howed a marked diarrhea. On the other hand, the other two rata receiving 0.1 g./kg. maintained their initial body welghta and 8howed no evident 111 effecta so far as general appearance and behavior were con concerned. All of these rata were killed and autopsled after they had received fifteen doses In a period of twenty-one days. The outstanding finding In these animals was varying degrees of gastrointestinal irritation; there was also slight cloudy swelling of the liver with an increase in weight of this organ (Table III). Blood urea-N values obtained on these animals compared favorably with those for the controls (Table III). g*As*Three of the five rats fed 0.3 2,4-dichlorophenoxyacetic acid died after the second dose. Consequently, the two surviving animals were killed and examined; severe gastrointestinal Irritation was observed in both of these rats. SUMMARY AND CONCLUSIONS Female rats that received 20 oral doses In 28 days of 0.003, 0.01, and 0.03 g./ks* 2,4-dichlorophenoxyacetic acid showed no adverse effects as Judged by gross appearance and behavior, mortality, growth, hematological values, blood urea-N determinations, organ weights, and gross and micro scopic examination of the tissues. On the other hand, rats receiving repeated oral doses of 0.1 g . A s * 2.4- dichlorophenoxyacetic acid showed varying degrees of gastrointestinal irritation with marked weight loss in three out of five animals. Higher (0.3 g.Ag*)doses wre not tolerated upon repeated administration. 5098 0002808 -5- II. ORAL ADMINISTRATION IN THE DIET : Ol O Experimental Procedure ^ 00 The modified Sherman diet, which has been used successfully^ for several years In this laboratory as the stock ration for rats, served as the control and basic diet In this experiment. The follotving Ingredients were used In the preparation of this stock diet: Whole wheat, freshly ground Dried whole milk Dried extracted liver Dried yeast Iodized table salt Calcium carbonate Percent by weight 55 25 12 5 2 1 The experimental diets were prepared by thoroughly mixing finely ground 2,4-dichlorophenoxyacetic acid with the stock diet on a "percent by weight" basis. No diet preparations over a month old were used. Female white rats from the stock colony of this laboratory were used In this Investigation. These animals were the descendants of rats obtained In 1938 from the Wlstar Institute. Five rats were caged together In wire bottom cages. The animals were fed from alurai- V. num hoppers which were weighed and refilled three times a week. Each rat had access to food and water at all times. 5099 nftAnoi'a -6- DOW 508344 The rats were maintained on the stock diet fro m the time of v/eaning until they were 2 to 5 months of age, when they were divided according to body weights into well matched groups and started on the experimental diets. Groups of 5 female rats each were placed on diets containing 0.00 (control), 0.01, 0.03, 0.10, 0.30 and 1.00 percent 2,4-dichlorophenoxyacetic acid. All rats were weighed twice a week throughout the course of the experiment. Records were kept of body weight, general appearance, and estimated average daily food consumption of each animal. During the course of the experiment, rats that were obviously quite sick were killed and examined to determine the cause of illness. At the end of the experimental periods, all surviving rats were fasted overnight, weighed, killed by decapitation and examined. Certain organs (liver, kidneys, heart and spleen) of each rat were weighed and.the tissues of all animals of each group were saved for histopathological studies. Hematoxylin and eosin stained sections of the following organs were prepared: lung, heart, liver, kidney, spleen, adrenal and pancreas. In addition, stained sections were prepared from the stomachs of the rats that received the diets containing 1.00 and 0.30 percent of the test material. The concentration of urea-nitrogen of the blood was determined by the diacetyl monoxime method^ on the control rats and on those that had been maintained for 12 days on diets containing 1.00 and 0.30 per cent 2,4-dichlorophenoxyacetic acid and 113 days on diets containing 0. 10, 0.03 and 0.01 percent of the test material. 1. Barker, S. B., J. Biol. Chem. 1^2: ^53-463, 19^4. 5100 0 0 0 2 S 10 -7- Pood Consumption Exuerimental Results OOV/ 5063^5 The dally food Intake of the rats fed the 0.10, 0.03 and 0.01 percent diets was found to be from 10 to 15 grams per rat. It may be calculated that these rats, weighing about 200 grams, Ingested quantities of 2,4-dichlorophenoxyacetic acid of the order of 0.06, 0.02 and 0.006 gm./kgm./day, respectively. On the other hand, food consumption of the rats fed the 1.00 and 0.30 per cent diets was quite low, the exact amount eaten being uncertain because the animals wasted a great deal by pawing and scratching at the hoppers. Growth and Survival The rats on diets containing 1.00 and 0.30 percent 2,4-dichloro- phenoxyacetlc acid lost weight rapidly after the start of the experiment (see chart) so that they were killed and examined after 12 days. One rat on the 0.30 percent diet died (Table IV). The rats receiving the diet containing 0.10 percent 2,4-dichloro- phenoxyacetlc acid did not grow well. Only one rat survived the full experimental period (113 d a y s ). Two of the rats In this group were killed because of spontaneously occurring diseases (pneumonia and ear Infection), one was missing from the cage, and the fourth was found dead In the cage In such a condition that the cause of death could not be ascertained. The rats in the groups receiving 0.03 and 0.01 percent 2,4-di- chlorophenoxyacetlc acid In the diet grew as well as the animals re ceiving the control ration. Deaths In these groups were attributable to either pneumonia or ear infection. Hematological values obtained on the blood of these rats after 106 days on the diets as well as those of the control group are summarized in Table V. All results were within normal limits. 5101 9W S0SM O Q -8- TABLE IV SURVIVAL OP FEMALE RATS ON DIETS CONTAINING 2,4-DICHLOROPHENOXYACETIC ACID Days on Exnerlment 0 9 12 20 50 41 51 62 69 79. 90 100 113 Number of Rats Surviving on Each Dietary Level 0.00 0.01 0.05 0.10 0.50 1.00 555555 5 5 5 5 *a 5 5 5 5 5 4b 5b 5555 5 5 4C 5 5 5 4 4a 4d 5 4 4e 4543 4 5 3d 3 4 5 3 2d 4 *d 3 2 4432 4 4 2C l c a- dead In cage b- animals losing weight, sacrificed for hlstopathologlcal examination c- pneumonia d- ear Infection e- missing from cage 5102 H S?8 : -9- Gcneral Appearance The rats killed after 12 days on the 1.00 and 0.30 percent ^ diets appeared thin and unkempt. Pat depots were depleted. On the othercA O hand, the rat3 maintained for 113 days on the 0.10, 0.03 and 0.01 percentOO CO diets appeared to "be in good condition. ^ Body and Organ Weights A summary of the average final body weights, organ weights and blood urea-nitrogen is given in Table VI. Increased organ weights on the "grams per hundred grams" basis for the groups of rats receiving the 1 .00, 0.30 and 0 .10 percent diets as compared with the controls was due entirely to decreased body weights. The body weights and organ (heart, liver, kidneys, spleen) weights of the rats on the 0.03 and 0.01 percent diets were normal. TABLE V HEMATOLOGICAL VALUES FROM FEMALE RATS MAINTAINED FOR 106 DAYS ON DIETS CONTAINING 2,4-DICHLOROPHENOXYACETIC ACID Rat Number Percent 2.4-D Erythrocytes (million/ cu.mm.) Hemoglobin (gm./lOOcc.) Leucoeytes Diff.Count(percent) (thousand/ freutr. Lymnh. Eosin. cu.mm.) 8092 8076 7950 8099 0.0 0.0 0.0 0.0 9.0 9-3 8 .1 8.4 14.4 14.2 14.0 14.1 14.0 21.7 13.5 13.4 29 68 14 78 -- 20 71 3 8 9 7947 8079 0.1 0.1 9.2 9-.9 14.0 14.7 22.6 20.5 25 72 -- 3. - 8101 8088 0.03 0.03 9.0 9.0 15.1 13 .6 18.7 24.0 12 83 12 82 5 6 5103 0CC2S3 TABLE VI AVERAGE BODY WEIGHTS, ORGAN WEIGHTS A ND BLOOD UREA-NITROGEN VALUES PROM GROUPS OP FEMALE RATS MAINTAINED ON DIETS CONTAINING 2,4-DICHLOROPHENOXYACETIC ACID Percent In Diet 0.00 0.01 0.03 0 .10 0.30 1.00 Number of Rats f 4' 4 2 1 4 5 Days on Diet 113 113 113 113 12 12 Body Wt . (gnu) Initial Pinal* 174 206 175 207 182 203 176 175 177 145 175 127 Organ Weights igm./lOO gm. body wt .) Heart ^* liver Kidneys Spleen 0.40 0.39 0.36 0.38 0.41 0.39 2.44 2.33 2.39 3.14 3.57 4.72 0.73 0.72 0.75 0.78 O .89 O .85 0.33 0.30 0.25 0.34 0.39 0.47 Blood Urea Nitrogen 29.2 18.9 17.1 12.5 37.7 2 0 .1 1 0 1 A Fasted overnight o CJ o ro r- - o t 8^880SMO<1 DOW508349 * -11- Hlstopathology Microscopic examination of the tissues revealed a slight parenchymatous degeneration of the liver ceils In the rats on the 1.00 percent diet. Two of the four rats on the 0.30 percent diet showed slight cloudy swelling In the liver and In the proximal con voluted tubules of the kidneys. There was no evident Irritation of the stomach mucosa of the rats on the 1.0 0 and 0.30 percent dietary levels. The one rat surviving one the 0.10 percent diet showed only a very slight cloudy-swelling In the liver. None of the animals on the dietary levels of 0.03 and 0 .0 1 percent 2,4-dlchlorophenoxyacetic acid showed any changes upon hlstopathologlcal examination. SUMMARY AND CONCLUSIONS Groups of 5 female rats each maintained for 113 days on diets containing 0 .00, 0 .0 1 and 0.03 percent (0, 100, and 300 p.p.m.) 2,4-dichlorophenoxyacetic acid showed no adverse effects as Judged by growth, food consumption, general appearance, mortality, blood urea-nitrogen concentrations, hematological examination, organ weights, and gross and microscopic examination of the tissues. On the other hand, slight adverse effects were observed at concentrations of 0 .10 percent (1000 p.p.m.) fed In the diet for 133 days. Extreme weight loss of the rats on the 0.30 and 1 .0 percent (3000 and 10,000 p.p.m.) diets was associated with a low food Intake and necessitated sacri ficing the animals''on these dietary levels 12 days after feeding was begun. 5105 0 0 0 2 8 io AVERAGE BODY WEIGHT IN G R A M S >4, 230 220 210 200 190 180 170 160 150 140 0SC80S MOQ 90 5107 2 A . 5-T ACID Trichlorophenoxy Acetic Ac1.d Trichlcrphenol Sodium Chloride Freeze Point Color 9393 * 0.76% 0,6% 1 5 0 . 6C. Whits po cn CO co cn 2,h-D Dichlorcphenol Sodium Chloride Freeze Point Color 2,^-L ACID / ' Batch 7-2 99.35% 0.1Z % 0.035% 1 3 6 , 5C. White 3.&.S C . 2. 7 7 j.. /3<f. i* LCW/rJ Vll/50 Lm C. White Britten'8 Division 2-267 Building 0008887 5108 Ma 5109 ', r 1* Research Department THE DOW CHEMICAL COMPANY onin t e r im report the acute and subacute ORAL -TOXICITY O? 2 ,4,5 -TRICHLOnOPHENOXY- ACETIC ACID FOR DOGS. FUm T23.14-23-4 c h a. 5 8 6 2 Rac'd 12-16-49 F ia'd - 8 * 3 0 - 5 0 W ork By Dr.V.A.Drill Wayne Univ. 7* J. W. Britton ouch Executive Research Committee ij'*-- * Rapt. Bjr 2 - 3 / - S'* o u n it p t d ex vw ro A copy of A report of the acute oral studies and a progress . report of the 3 month studies being curried out by Dr. V. A. Drill of Wayne University on 2,4,5-trlohlorophenozyacetlc acid is given. ^ INDEX HEADINGS 2,4,5-Trichlorophenoxyacetic acid Aeetic Acids 2,4,5*trichlorophenoxy-, Phenoxyacetlc acids 2,4,5-trichloro-, MATERIAL The material usod in this study was a sample designated as K4568-5. Zt was supplied by L. C. White and bore the reference number 20W-103, Batch 138, Semple 6. The freezing point was 130.6C and an assay showed the material to be 98.93$ pure. PROBLEM 7 '- order to have some toxicological information relative to the effects of 2,4,5-T when fed repeatedly in single doses to a larger laboratory animal, the dog, a project was set up with Dr. Drill at Wayne University. A progress report of thiB work which was filed as an exhibit In evidence at the F.D.A. Hearing for the establishment of residue tolerances on fresh fruits and vegetables comprises a part of this report. OGCKkCjkiLl 5110 M O W 758853 Dloohcalcal Research Dapartnent T23.14-23-4 PQG 2 cohcluskhs The acute toxicity of 2,4,5-T for dogs la somewhat greater than for rats. Zn general, however, 2,4,5-T resembles 2,4-D In toxicity. 1 (5) C00483Q 5111 =' * L ":SV. -V'.- ^ \ O. \ 5113 b lo e w a lM l Research D tp ir ta n t T2~ . l * - l l - k Page 9 le a st 2, 1-^ lchlo ro p h eno vyacetlc s o ld P o ra u la t S tru ctu ral: C l -0-C-T-9-H Cl E a p lrlca l: " 8a 6: i 2': 3 B .R .D . K !o.t 2372-1'. M a t e r ia l r e c e iv e d i t -j * a . E . C o lb y , 2( 7 B u ild in g , 5-31-^9 s f . Mo.s l o r i r . - .. -y io CO CO to ..CD *:*V t t appears fro a the data th t hava obtained and a lso fro th a t which appears In the lit e r a t u r e th a t ?,b -D 1 M oderately to x ic and altho u g h I t would be n o a slb l f o r a person to eonaua* a s u f f ic i e n t aaount o f a a t e r i a l to be dangerous to l i f e , t h i s does not seen l i k e l y e x ce p t p o s s ib ly where co ncentrated solution. a re being handled and a re a v a ila b le to ch ild re n or liv e s to c k . It does not seen lik e ly that d ilu te so lu tio n s such a s a re used In ord inary rory teehnlours in the f ie ld eould present ary serio u s Ingestion hazard. u (IS) 00017-3 5115 5 K*No..Z & J 2J L :- S C. R. I . . F it s Name: , r, T - Structural formula: BIOCHEM. F REQUEST FOR BIOLOGICAL TEST TO BIOCHEMICAL RESEARCH LABORATORY 3 tU - Molecular formula. * D Q W 758854 Constituents in copolymers or mixtures: _ _ ------------------2 r * L = r _ 912:. v i/ r - " r I f) / r . .. .. .... L / Tr. \2/lA.C^lra^n. Suggesteo solvents: i.?* < o. 4 0 0ISCUSSED WITH--. Date Requested by--- .155^5^----Made by L . . C . r> -- 45 T2 1 .___ BP. Reference. . . . Sent.________ r^dJC^L 13 $ to ..C at_____m m . Hg Dens.---- at-- ---- #C/_________ C Handling Precautions Product state: Research.-.-- -- -- Development.-- -- -- __ Spec. Chem.--------- Plant------- Use CONTEMPLATED---------------------------------------------------------------------------- --------------------------------- --------------------------------------------------------- Tests requested: L ^* Y- / /*? , & uA A y Charge No . L it. Re f . -- , - * 1 ^ .1 1 . D iscl. No .________ Patent------------- (Please do not write selow this line) Tests By Date vt/ JA a a a ta a a a a a a a a a a a a a a a a a a a a a M a a M a * ia a a General screen ing................--...........--Su!....--............ Noted By__i. 7^:U7* .1.datd.z!>zln.. .By ,, 5112 S a m ple t o ___________________________ .........................D a t e ........... . CT C P a r M. IB. ASSOULY (Paris). , C c 'C c Le dsir d'obtenir de belles rcoltes a, de tous temps, t l souci perm anent des agriculteurs. P our y parvenir, ils ont essay divers moyens : enrichissement du sol p ar des engrais; accroissement du rendement par l'emploi de substances de croissance; cnlln, destruction des mauvaises herbes qui, en absorbant les engrais, l'eau et les matires minrales, entravent le dveloppement des plantes utiles. On est parvenu aujourd'h u i,'d an s le domaine des produits de croissance et des herbicides, A des rsultats tout--fait remarquables. Mais leur fabrication suscite des inquitudes dont le Conseil Suprieur "Hygine de France s'est fait l'cho dans sa sance du 13 fvrier dernier. On a d 'abord pens que ces produits pouvaient prsenter une action cancrigne, et cette apprhension s'explique fort bien quand on voit l manire dont ces substances agissent sur les plantes, o elles provoquent des tum eurs, des hyperplasies. Mais flnalcmcnt, cette action cancrigne a t carte p ar le pro fesseur Simonnet. P a r contre, le Conseil Suprieur d"Hygine de France a soulign le danger d'intoxication, que pouvaient prsenter ces produits pour les ouvriers qui les fabriquent. A vant de nous attacher l'tude de la toxicit des substances en cause, nous croyons utile de rappeler quelques notions techniques leur sujet. ... com position e t action d e s horm ones v g ta les Nous avons employ les mots d'hormones vgtales, parce que c'est ainsi que l'on dsigne couramment ces produits destins accrotre le rendement. Il conviendrait peut-tre d'employer plutt (comme le remarque Pierre Chouard) le nom de substances de croissance qui dsigne, sans prjuger de leur nature intim e, les agents chimiques de tous ordres, capables d'exercer, A de trs petites doses, une stim ulation puissante sur l'longation des cellules vgtales jeunes. Primitivement, ces substances ont t appeles auxines ou hormones de croissance vgtales ou phyto-hormones. E n ralit, les auxines sont seulement les substances fabriques par la plante elle- mme et qui exercent ce phnomne de stimulation, d'longation cellulaire p ar un . mcanisme horm onal.. - On s'est aperu, ces 10 dernires annes, qu'une m ultitude d'autres substances avaient le mme pouvoir que les auxines; on les a dnommes hormones vgtales. . . . . Ces hormones ont une action qui varie suivant le moment d'utilisation et le b u t : recherch : elles exaltent la croissance des plantes ou, au contraire, la ralentissent; elles acclrent la germination des graines, excitent la formation de racines; de germes, .d--e-- boutons, d---e---f--leur--s; ou, au contraire--7, -ralentissent ces fo--nctions;r e--l--le--s sp--e--r--m---e--t-t-e--n--t---la formation artificielle de*fruits sans graines, sans fcondation p ar le pollen; elles aid en t (1) Communication ia Sodili de Mdecine du Travail, le 20 novembre 1950. 0 (3 1 2 LO 0 / 5116 I d 9 9 8 9 6 |i H O.0 D S H E R B A N T S E T S U B S T A N C E S ' E R O IS S A N C E ' 27 , &la rparation des dgdtscauss p ar la gele, facilitent la transplantation, le bouturage ' C'est dire que leu r champ d'application est vaste. . Que 30nt hormones T -- Ce sont .des drivs oxy-actiqucs du noyau benzine . e t du noyau n ap h talin e; des acides plinyl, naphtyl et indol-actiquc, proplonlque et = . butyrique, ainsi que certains de leurs drivs, comme p ar exemple des nitrilcs, des-. esters alkyliques, des sels d 'alcali e t d'ainmonium, e tc .- L 'un des plus connus est.. ' l'acide 2-4 dichlorophnoxyactlque, qu'on appelle, par abrviation, le 2-4 D. * Chacun de ces produits a une-action particulire et leur emploi varie donc suivant le rsultat &obtenir ; p ar exemple, pour le bouturage, on utilise l'acide indol-actlque, - ' ' l'acide.indol-butyrique ou l'acide naphtaline-actique plutt que le 2-4 D. qui a une 1 . " - action trop dformante dans ce cas; contre la chute des fruits, on se sert de l'acide - naphtaline-actique ou de scs sels, ou bien encore du 2-4 O . mql grande dilution. Signalons aussi que le 2-4 D. appliqu en pulvrisations ou arosols, stimule puissam ment la m aturation des fruits. - Un nombre considrable de corps ont t ainsi tudis et mis au point et certains . " (comme le 2-4 D . dont nous parlons plus h au t) se sont rvls tre de l'ordre d'une centaine de fois plus actifs que l'axine proprem ent dite; si bien qu'aux doses ordinaires ce n 'est plus le pouvoir normal de croissance qui s'exerce, mois l'excs de ce pouvoir, - aboutissant & la toxicit. De cette toxicit a driv un emploi nouveau : l'utilisation - comme herbicide.. . . . . . . . . Cette utilisation des hormones comme herbicides slectifs est venue aussi de la con naissance de leur action, stim ulante sur les diverses fonctions de la plante, y compris la formation de tum eurs. L'usage des hormones, en ta n t que substances de croissance, provoque souvent, aprs l'application, des hyperplasies, des cals cicatriciels qui se form ent su r la surface de section d 'u n organe, tige ou ptiole en gnraL Les hormones, -- . doses herbicides, ont une action intensifie; elles provoquent une exaltation folle - des manifestations de croissance par hypertrophie et hyperplasie, une activit dsor- . donne qui entrane l'apparition de monstruosits les plus diverses, la consommation . Intense les rserves et finalement la m ort, au stade herbac. n s'agit donc d'une destruction radicale, puisqu'il n 'y a pas formation de graines. Cette action est, en outr, slective; les hormones s'attaq u en t aux mauvaises h erb es" mais respectent les plantes utiles. E t ceci s'explique facilement : la rsistance des ' plantes varie suivant les espces; celle des crales, par exemple, est beaucoup plus . forte pie celle des ravenelles, coijucllcots ou bleuets. C'est pourquoi lo traitem ent est - sans effet sur les crales, d condition cependant qu'elles aient dpass le stade 3 feuilles. - Il devient ais de composer un produit de telle manire pie seules les mauvaises herbes - soient touches, tan dis pus les autres plantes resteront insensibles d son action. Il convient, en outre, d 'observer, lors de l'emploi, certaines prcautions pour obtenir . un bon rsu ltat. E n gnral, on opre &la fiu-du printem ps, lorsque les tiges des plantes . ont attein t leur plein dveloppement et ont puis les rserves de leurs racines. On - vaporise le produit, en solution, sur le feuillage; il adhre fortement, pntre rapide- . : m ent dans les feuilles; la sve le vhicule jusqu'aux racines; il provoque alors, en mme tem ps qu'un arrt de croissance au stade herbac, une prolifration dsordonne des . cellules pii a b o u tit & la m ort, ainsi que nous l'avons vu. L a destruction est obtenue - en 15 jours ou 1 mois. " -Ces produits prsentent un avantage considrable sur les dsherbants habituellem ent - employs : chlorate de soude, qui dtruit toute vgtation sans distinction; acide sulfuriipie, qui acidifie le terrain, ou sulfate de cuivre, susceptible d'une lgre slec-.- Uvit, mai dont la manipulation prsente do graves dangers ou des inconvnients e t ; > ' d o n t l'action n 'e st pas - aussi totale. ; ,, -v ;: ;v" . . ; V FABRICATION DE L'ESTER DU 2-4 D " - w v L a fabrication de ces hormones vgtales s'effectue seulement penlant quelques mois, en gnral de fvrier m ai, c'cst--Uirc pendant la priode oii l'agriculteur a ' besoin de ces produits.; . n existe su r le m arch deux sortes de produits dsherbants; l'u n est d base d 'e ster de l'acide 2-4 D. que l'o n mulsionne dans de l'huile minrale avec un m ouillant. L 'utili' sateur procde d une seconde mulsion du produit dans l'eau et le projette ensuite . ] s 6889 S I ZH00 1 28 M. ASSQULY . sur les surfaces dsherber au moyen d'un pulvrisateur. En cas de pluie, le produit, llx par le mouillant, adhre nanmoins. Le second qui semble avoir la faveur des agriculteurs, ne contient ni mulsifiant, ni mouillant.. A base de sel de soude du 2*4 D. il est directement soluble dans l'eau. Mais il risque d'tre lav par la pluie, puisqu'il * ne contient pas de mouillant. De nouveaux produits base d'ester sont actuellement en fabrication, comprenant une plus grande quantit d'mulsiflants. On pourra alors verser le produit directe m ent dans l'eau. Le mlange se fera seul, sans qu'on a it mme besoin de l'agiter. Dans^'usine que nous avons visite, on procdait seulement &la raction d'estri fication de l'a d d e 2-4 D. qui arrive to u t prt d'une usine de province. ; Ouvrons une parenthse pour signaler que dans cette usine qui fabrique l'acide lui-mme, les ouvriers sont imprgns d 'une trs forte odeur de phnol, caractristique de ce produit. Cette odeur est extrmement tenace; clic persiste, mme lorsqu'ils q u itten t leurs vtements de travail. Ce qui leur a occasionn quelques dsagrments : on considre, par exemple, leur prsence indsirable dans les lieux publics, cafs, tram w ays, etc.... Fermons ici notre parenthse et revenons notre usine parisienne. La raction d'estrification a lieu partir du sel de soude de l'acide 2-4 D. sur lequel on fait agir de l'alcool thylique 95*. port une tem prature de 70*. A ce mlange, on ajoute ensuite de l'acide sulfurique 98*. ce qui entrane une lvation considrable de tem prature. La cuve est alors refroidie et maintenue 70*. Cette opration ayant lieu en vase clos, l'alcool distille et retombe dans la cuve. . Le produit est ensuite lav l'eau, toujours en vase clos et on procde h une dcan tation. 11 reste alors dans la cuve un liquide sirupeux qui contient environ 1 p.- 100 d'alcool et qui dgage encore une forte odeur de phnol. PATHOLOGIE _. \ V .;;v L'Interrogatoire des ouvriers affects & cette opration rvle qu'lis prsentent les . troubles suivants : somnolence avec sensation de jambes lourdes, irritation des voies ariennes suprieures, gastralgie avec perte d'apptit, got sucr dans la bouche avec ... . hypersallvation, sensation d'brit et d'hypersensibilit de l'oue, le moindre bruit faisant sursauter les ouvriers. Les ouvriers employs au conditionnement se plaignent d'asthnie avec jam bes - . lourdes, sensation d'brit et de tte vide,. A quoi pourraient tre dus les symptmes signals ci-dessus ? On peut imaginer qu'ils sont causs par le produit Uni lui-mme ou par les manations des diffrents consti- * tuants, au cours de la fabrication. C'est ainsi que l'irritation des voles ariennes suprieures est bien connue dans - l'Intoxication par les phnols, qui produit des catarrhes (laryngites, trachites). D 'autre p art, il est signal par les auteurs que l'intoxication chronique p ar le phnol -- d 'ailleurs discute -- peut se m anifester entre autres p ar des troubles digestifs, - J anorexie, ptyalisme, cphales, vertiges. Or, les ouvriers nous ont signal tous ces ' . signes, parmi les troubles qu'ils rattachent & l'exercice de leur profession. * : ' - On pourrait rattacher galement quelques-uns des symptmes relevs aux manations probables des vapeurs d'alcool en cours de fabrication. Cette opinion n'est toutefois . pas partage par l'Ingnieur que nous avons vu pendant notre visite, car, d'aprs lui, . d'autres fabrications m ettant en jeu des quantits importantes d'alcool ne prsen-. ` tcralcnt pas les inconvnients signals par lo personnel. E n rsum, on peut donc dire que la plupart des signes pathologiques peuvent tre dus au dgagement de vapeurs de phnol. Toutefois, des signes plus particuliers, tels que le got sucr, la sensation d 'hypersensibilit de l'oue paraissent originaux e t dus au produit fini lui-mme. Les troubles rapports ci-dessus pourraient constituer un syndrome caractristique du travail au contact d erp ro d u its en question, si d'autres auteurs les avaient signals. Nous avons procd des recherches bibliographiques, mois nous n'avons pu trouver ' - de rfrence, du moins dans les sources que nous avons utilises, concernant, leur - . : action sur le personnel employ la fabrication. . . . . -, 0012192 5118 D SH ERBAN TS E T SU B ST A N C E S DE CROISSANCE 29 . P a r contre, diverses publications signlcnL tes travaux effectus sur leur toxicologie. U nous parat intressant de rsumer ci-dessous les recherches effectues dans les diffrents pays du monde. 'toxicologie : Le professeur R. Fabre, dans un rapport prsent aux Journes Mdicales Interna tionales de Paris en 1937; signale les tum eurs qui se forment chez la plante autour .d'une piqre d'htro-auxine, acide indol 3-nctlque. U termine son rapport sur quelques questions pleines d 'Intrt : < Les auxincs vgtales ont-elles une action sur le m ta bolisme ou la croissance d'un organisme animal ? Aucune exprience dfinitive n'a t tente dans ce sens jusqu'ici. Mais il serait fort intressant d'tablir un parallle . entre l'action encore bien discute des hormones sexuelles sur le dveloppement des plantes e t celle des auxines sur le dveloppement des animaux. E t s'il y a action, sur quelle cellule de l'organisme porte-t-elle 7 A utant de question sans rponses, qui doivent tenter physiologistes et mdecins. De mme, cette question do la toxicit du 2-4 D. pour les humains fu t pose lors de la deuxime runion du N orth Central States Wced Contrl Confrence. Quelques membres relatrent alors des cas d'ingestions accidentelles de quantits variables, mais toujours sans altration apparente de la sant des accidents. K raus fit savoir . l'assemble qu'il avait lui-mme absorb quotidiennement 1/2 g de 2-4 D. p u r pendant 3 semaines, sans ressentir de dsagrment. . Des expriences furent tentes Bcltsvllle, afin d'prouver la toxicit du 2-4 D. sur les anim aux. L a premire consista foire patre, pendant 15 jours, des vaches et des moutons dans un pturage pralablem ent tra it avec l'herbicide. On avait mme doubl, la dose habituellement ncessaire la destruction des mauvaises herbes. Les' animaux furent ensuite examins par des vtrinaires, et trouvs en excellente sant. Dans la seconde exprience, on fit absorber une vache 5,5 g de 2-4 D. par jour, e t ce pendant 3 mois. L 'animal consomma facilement le grain d'alimentation avec lequel la substance chimique tait mlange. Sa production laitire ne subit aucune baisse et un veau, nourri entirement de son lait pendant 1 mois, ne m ontra aucun symptme de maladie. Une autopsio n'a pas rvl d 'effet apparent du 2-4 D. su r les diffrents organcs.de cette vache. M. K . B jom e t H . T . N orthen donnrent pendant 4 semaines une dose quotidienne de 200 mg/kg des poulets. Cela fu t sans effet, tais la dose de 765 mg/kg fu t mortelle. E . V. H1U, e t H . Carllsle, entreprirent des expriences sur 450 souris, 150 ra ts blancs, 125 cobayes, 70 lapins et 3 singes. Le 2-4 D. fu t administr avec les alim ents, en injec tions intra-veineuses, des doses croissantes pour atteindre e t dterminer la dose mortelle. A ce stade, les anim aux m eurent soudainement de fibHlatlon ventriculaire aigu. S'ils chappent la m ort instantane, on voit apparattre la raideur des extrm its, avec do l'Incoordination musculaire, la lthargie, la stupeur et finalement, le coma e t la mort. ; Ces symptmes sont constants, quelle que soit la faon d'adm inistrer lo 2-4 D. e t quel que soit l'anim al utilis pour l'exprience. . . .. - : L'Investigation m ontre que le 2-4 D . e st-u n compos relativem ent peu toxique dont la dose mortelle e st de : . 375 mg/lcg d* souris, '-10 0 0 -- de cobaye, ' \ -.* . 660 -- de rat, ' 800 ` ~ ' de lapin, '*; / .. . , : .,,; quand ces doses sont administres en solution aqueuse par la bouche. E n supposant que la tolrance pour l'homme soit similaire celle des anim aux, la plus forte dose que pourrait supporter un homme de 75 kg serait de 15 g, ' Cs expriences o n t t conduites avec du 2-4 D. utilis comme herbicide, softs que sa toxicit soit augmente p a r celle de ses solvants. Aucune exprience n 'a t entreprise pour m ettre en vidence l'Intoxication chro nique avec les effets de l'absorption p ar la voie respiratoire. De leur ct, K lng et Prnfound firent des essais en aquarium sur la brme et le bar.- Ils trouvrent que dans ce milieu clos, le 2-4 D. 1 p. 100 n 'est pas toxique pour ces poissons, mais qu'il le devient lgrement 100 p. 100, concentration qui ne peut tre quo temporaire et. locale en eau courante. ' - `- E n vrit, on se trouve 1&devant un produit nouveau dont le mode d'action lui-mme sur les vgtaux n'est pas encore trs dflni en agronomie et reste encore un peu mys- trieux. --r -- . . v. . E n ce qui concerne l'organisme humain, le problme est entier, autant du moins que .'n o s recherches.nous perm ettent de l'affirmer. nsuat ' " ,r -.V ' 'f On a donn le nom d 'hormones vgtales &certaines substances chimiques capables d'exercer, h de trs petites doses, une stimulation puissante sur l'longation des cel- Iules vgtales jeunes, action exerce habituellement par une substance labore p a r la plante elle-mme. Ces hormones sont des drivs oxy-nctiques du noyau benzne et du noyau naphtalne, des acides phnyl, naphtyl et indol-actique, propiontque et butyrique, ainsi que certains de leurs drivs : nitriles, esters alkyllques, sels d'alcali, .. d'am m onium, etc_ L 'u n des plus connus est l'acide 2*4 dicblorophnoxyactique, . appel couramment 2?4 D. . L'action intensive de ces hormones les a fait utiliser comme herbicides slectifs. Elles agissent en provoquant une exaltation folle des manifestations de croissance, une activit dsordonne qui entrane l'apparition des monstruosits les plus diverses, . la consommation intense des rserves, et flnnlement la m ort au stade herbac. - Les ouvriers employs &la fabrication de l'ester du 2*4 D. se plaignent de somno lence avec sensation de jambes lourdes, d'irritation des voies ariennes suprieures, de gastralgie avec perte d'apptit, de got sucr dons la bouche-avec hypersalivation, de sensation d'brit et d'hypersensibilit de l'oufe, le moindre bruit les faisant, sursauter. .. -, Des expriences faites sur les animaux ont montr que le 2-4 D. absorb avec les aliments on inocul en injections intra-veineuses, n'tait toxique qu' doses assez leves. - -< x . . *" ' . : amuopasmix . . Bjorn, M. K. et N oirm sif, H. T. -- Elfects of 2-4 Dlchlorophenoxy -ectie acid on chieics ' Science, 29. 1948, p. 479-480. - -- Bmassb-Orossabo, L. -- Les mauvaises herbes. Cuovaro, P. -- Les progrs rcents dans la connaissance et l'emploi des substances de crois sance. Revue Internationale de Botanique applique et d'Agrieullure tropicale, n" 307,308; 309, 310. 311. 312,1948; n - 313. 314, 317, 318, 319. 320, 1949. - Cayouxttx, Richard. -- Ce m erveilleux 2-4 D. Ministre de l'Agriculture, Qubec. Faurb, R . :-- Les hormones vgtales. Rapport prsent aux Journes /dittes Internationales l&37f A iitt MtTTr n Ki.r.. J . W ., Hoooso.v, R. E. et Oaetjkxb, C F . -- Tolrance of farm nimnt to feed contnining 2-4 O. acid. J , Animal Science, S, 1946, p. 228. Fam m fA, L. T. -- 2-4 D. Indus. Hgq. Neunletter, 8,1918, p. 9. . l l u , B. Y. et Carlislx, H. -- Toxicity of 2-4 O. for experimental animais. J . Indus. Ilgg, et Toxicol. 29. n* 2 , 1947, p. 85-05. Janot. -- Phytohonnoncs. Confrence faite h la Aioiso.a de la Chimie, le 15 dcembre 19-11. 0012194 5120 5121 IH 5122 T-CN PHENOXYACETIC ACID. 2,4- n. DICHLORO Selective Herbicides and Growth Substances. Pathologic Effects on Man During the Manufacture of the Ester of 2,4-D. M. Assouly. Arch. Mai. Profess. 12^, 26-30, (1951). (French). Workers employed In the manufacture of an ester of 2,4-dlchlor phenoxyacetic acid complain of somnolescence with heaviness of the legs, Irritation of the upper respiratory passages, gastralgia with loss of appetite, of a sweet taste in the mouth with Increased salivation, a sensation of drunkenness, and hypersen sitivity of hearing, the least sound causing them to jump. In animal experimentation it was shown that when 2,4-D was Ingested or injected intravenously, high doses were required to produce intoxication. -- Biol. Absts. Industrial Hygiene Digest Vol. 16, No. 4 - April 1952 Zift9 I 7*nn ! 0012195 5123 Tndlreot Effects of Herbicides C. J. W i l l a r d Under `'Indirect Effects of Herbicides" wa have Included all effects other than direct damage to the weeds or crnz plants to which tney are.spoiled* The discussion will concern the newer organic herbicides only* This la an extensive U t r e r a t u r e on the effects of sodium chlorate and arsanicals, which does not need reviewing here* DOW 755434 The indirect effects.we have considered are: 1* Eff e c t s out s i d e the treated field due to drift.or vapors of tne herbicides. 2. Unforaeen effecta on'the "balance of nature". 3* D e s t r u c t i o n of wild flowers, ram cover, etc., in roadside spraying. 4, Poiaonoua offocte of herbicides, & Direct effecta b* Pciocnlng from poiaonoua plants, not usually eaten, but eaten after spraying, c* P o i s oning from ordinarily harmless plants made poisonous by tne effeot of sprays on tnem. 5* E f f e c t s o n the c o m p o s i t i o n of crops. The first problem, drift of dust, droplets, or voore to adjacent or not-so-adjacent susceptible plants, has been so widely discussed that I will talcs time to m ake just one point about vapors. Any effect.of vapors must be more or lees a mass action effect. Dr. Alban and T have U9ed experimental amounts of esters around susoertible plants for years, with no effects on them. But those were small plots. If you spray 10 acres with esters and wind moves slowly across this area (Have you noticed how almost every story of vapor damage begins "There was hardly any wind"?) the air may easily pick up a toxic load of vapor. As weed control men, we.must constantly emphasise the hazards Involved In using these extremely powerful chemicals, and p e r s o n a l l y and by precept, see to It. that every pr e c a u t ion Is taken against damage where no damage is intended. This oannot be said too often, or made too emphatic. Carelessness in application has already resulted in some severely restrictive laws, and otners will follow if they seem to be needed. Several articles have appeared expressing tne fear that we "weed men" will run wild and exterminate local flora of all kinds In all places. Killing plants even with modern chemicals, still takes time a n d material, w h i c h cost money, a are riot'likely to uso e i t h e r when we do not have c.n economic reason for doing so. In so d o i n g vp may kill some plants toat we do not `ntenl to M i l , - simply b e c a u s e tnay are in bad comnanv, CGG5C13 5124 UUW 755435 a Gtiould not be, nor > ; /., n f- .i-J o r oblivious to changes In the balance of should study .those c h a n t s r^nd, so far on poosiblo, avoid r e c o m m e n d a t i o n s thot produce un- fr.vorr.blo changes, o r suggest ways of overcom.1 n?r them. For example, repeated roadside spraying may take out legumes and, on many s^lls, reaulre nitrogen fertilisation to-maintain a healthy sod* The complaint about killing wild flowers elong v * asides 1 g one to w h i c h >*a must, at least partially, plead guilty, ~ut without apologies* Home of these complaints remind me of the nntlvivlsect l c r 'roups thr o u g h o u t the country, to whom, if one wore to Judea b y either actions, the 11ft of a st^oy dnn> \p more aacrec than the lives of children, tfe wil l always hove some unreasonable c o m p l a i n t s , .So far ns In us ll9s, we should he "wise as serpents and h a r m l e s s as d o v e s ". We should listen to these complaints, *t-udy them, and then, If the weight of evidence 13 then on the side of weed control, a n s w e r the m c o o peratively and court e o u s l y and (to ahead. Dr, E, P, S y l w e e t a r has done this b e a u t i f u l l y -In h1a roadside spraying campalffn in Iowa, He started tnls In cooperation with the Highway Dore-traent In 19U, The unsightly unremoved, deed brush along the road started c r l t 'cs talking so vigorously that the campaign was "ooft-podalod" In l4d, resulting In poorer results and gre; ter e x p o n s g f o r the H i g h w a y Department, So, this spring, Dr. Sylvester arranged for a prin t e d debate between himself and one of his active opponents in the Sunday Dao Moines Register and Tribune, wnlch has & Sunday circulation of nearly half a million. He aloo defended the roadside spraying program at some 5 m e e tings t h r o u g h o u t the state, A.s a result, the Iowa roadalde spraying program is going ahead full steam, with relatively little criticism. His handling of the program was a model of public relations for all of us, 9o far as anything I might say here defending roadside spraying is concerned, I could oniy say what Dr, Sylwester said, 'or say it less veil. That article has been reprinted several times, most r e c e n t l y In "Down to E a r t h " (Fall, 1950), I sue-gest that y o u get It. It is a t r e n c h a n t defense of the beauty, safety, and economy of grasacovercd roadsides, produced by spraying, a9 contrasted with the miles o f p o i s o n ivy, w i l d carrot, brush, hemp, reftweed, etc,, etc,, which still corstitute the average roadside. Folson! nt by herbicides la n more serious matter, F o r c u n ? `rely our problems are small compared tc those of the entomologists, but we defini t e l y do have problems. In the first place, any n e w thinsr gets blamed for a n y t h i n g that happens 'n its vicinity# 'vhen D", of o u r Department mad e a veeV*o tnu^ of northwestern.Ohio..in p u t t i n g on a few s p r a y i n g and d u s t i n g d e m o n e t ^ a c t l ^ ns wi fn ?, 4-D, two r e p o r t s of p o i s o n i n g followed - potn l a t e r sorwn to he rrrneous, but i l l u s t r a t i n g tne tendenev to blame t r o u b l e s onto n n v f M n r new. Last s umm e r I I n v e s t i g a t e d s case of allege:! sheep nol soninr: by a CGQ'IRLS 5125 "b r u s h k i l l e r " u s e d b y .a n e l e c t r i c coop. Investi g a t i o n showed that there w a s t o r e a s o n a b l e chance that the arraying war. responsible, but the spray tr u c k p a s s e d alone; a l l t t l 9 before the sheep took sick, no the two things were linked, both by the farmer and by the veterinarian. Of the n o w herbicides, T C A la caustic to the skin and the dlnltros a r e d e f i n i t e l y poleonoun. The Dii*s are a serious hazard to the p e r s o n apply i n g them -- de a t h has resulted from long e m o 3 u r e to air c o n t a m i n a t e d by droplets of DN npray - but so for no co-clatnts of livestock poisoning from DN*s have come to our attention, and grazing experime n t a l l y sprayed areas with them at four tires the r e c o m m e n d e d doses has g i v e n no serious results (3). The same is C true of TCA () 5" m Thero have been a few reports of persons who are allergic _ to or affected by 2,4-D. There is no reason to suppose that at least coma of t h e s e r e p o r t s e r e not true. These are i m p o rtant to ''3 the few individuals concerned, and worthy of serious study, but we now have experience enough to say that they are highly exceptional. ^ To halo put the matter In proper perspective, in my personal ^ experience I know more peonie who are poisoned by e^vs then I have definitely h e a r d of b e i n g u n f a v o r a b l y affec t e d by 2,k-o, M a n y teste (, 11, 1?) h a v e shown that stock w i l l consume grass sprayed with 2,4-D and readily and without injury under ordinary conditions. These materials have been directly fed to pilkcows without Injurious effects on the cattle. Host reports also state that thero was no effect on the milk - Dr, Grigsby in a letter reporting unpublished work-this summer ( l ^ O ) says that cows fed large q u a n t i t i e s o f 2,4-- 3 gave m i l k w i t h a charactari sti c p h e n o l flavor during tho feeding and for 12 hours after the l^st cose of 2,4-1) h a d b e e n given. These experiments and the experiences of thousands of formers w i t h s p r a y e d p a s t u r e s p r e t t y w e l l dispose of direct 2,-'-0 toxicity to stock. However, almost all of ua have seen sprayed plants eaten that h a d not b e e n eater, b e f o r e - all the way from 9. M. B a l e i g h 's case of r odents c h o o s i n g to eat rows of corn treated p r e - e n e r g e n c e w i t h 2,k-D to stock eating Canada thistles (0, Loe}, velvet leaf (J, L. Hutchison), Jimaon weeds (F. W. 9 1 1 f c ) t wild p a r s n i p (Geo. 3riggs, C, J. Willard), sunflowers (!, F. Yost), docks (Crafts a n d Harvey), r o u n d leaved m a l l o w (N. Shafer) and un p a l a t a b l e w e e d s in gen e r a l (Chas* J. Gilbert, II. S. Wood). Clearly, if any of these weeds were injurous or poisonous, their sudden consumption after spraying could cause sickness or death of the stock. Actual instances are few but suggestive, C. J. G i l b e r t m e n t i o n s oasee of p o isonous range weeds c a u sing death whe n eaten after spraying.' Bot h W a r r e n Shaw and O l i v e r Lee re>rort poisoning of cattle from ?,^-D-crrnved wild cherry and if this O G O ie lS 5126 - a- 0004816 If r*t definitely p o i s o n o u s plant, la p r e s e n t in an area to be opraysd, it would c e r t a i n l y bo well to kee p stock out of it. J. K. F l eetwood reports h e a r i n g of a case of awl no eating* arrayed mature cockle-burs with severe sicknoss following* Crafts and Harvey (U) make a e l d l a r report concerning arrayed thiotles eaten by lambs* Virgil Freed mentions oeveral inatances of ragwort colsonlng livestock - a f t e r being sprayed with ?,U-D, He writes! "normally, livestock ^ become poisoned on this plant only when they are forced to graze it by lack of other vegetation, particularly In the late winter and ^ early soring monthsi However, In the instances mentioned above, ^ it was a case of the a n imal a c t u a l l y seeking out this olant two or- c. three days after spraying and consuming sufficient quantities to.be ^ toxic. Plants that had been treated for a oorloi longer than one to three day s d i d n o t a p p e a r to be pala t a b l e to .the animal and the initial palatabillty was attributed to th9 marked Increase in sugar that accompanies application of ?,U-B to these plants* formally t he h i g h e r s u g a r con t e n t extends f rom F^.to ?2 hour a f t e r spraying, thereafter declining steadily," A wise precaution, therefore, would be to k eep stock out of s p r a y e d .'paaturas containing any poisonous weeds for 3 t o - d a y s to a week. Much the most serious possible 2,4-D poisoning effect that has been reported was due to the accumulation of nitrates in sugar beet leaves that have been sprayed with sub-l9thal amounts of 2,^-0, For those data I a m almost entirely Indebted to an unpublished paper submitted to "Science" by L* M, S t a b l e r and 2 iih!teheed, which I am abstracting In part. Nitrate polooning of stock has been known for some time 5# 7)(1, 2, especially in connection with oat hay. Beet leaves from a Manitoba field damaged by airplane spraying of an adjacent whest field in lbbg caused the dorth of several steers, A local veterinarian.recognized the symptoms ae those of nitrate poisoning. The standard treatment, injection with methylene blue, revived living but recumbent steers within an hour. The leaves were analyzed '"for nitrate and showed nitrate levels for above those known to be toxic. In August, lQ^Q, acres of beets on seven f BT'rr.s In North D a k o t a w ere sprayed wit h insecticide c o n m i n ot'ei with it 19'ves fr'1m each fiel-3 and. flelds,' The lewvea frorr untreated fhfields a v e r a g e d 0 , ? ? p e r c e n t K?vO?; p. r. or the trod e>3 V*nge of 1,f ! l to matter of forgro ?.77 1a c n T)0rceut. n1.ie*,ed toxic. Is tr, nr.t :i"ac', but nltr1r. v;-.1cii \n n: 1i-A. V "i .`-p r* a JT , fi: t:1 Lun>., t.;V3 "ii i<i t. re i,:1v-!'<" h t r r n y -1r r. vft e-,r ce 0ri r.outh. i-'nlos9 *r =.0te'l KID*! UUW Vi>i>436 promptly, death cay coco In a few hours or loss. Pir.;ocd i^zor anthna r o t r o f l a y u s ) and l&mb3auarter*9 (Chennondt ^ I b r v a ) a r e c o r - o n ouj cioeely rbl'nicu ;o bm? 1 3 v.'1eh Olson '-.na w n i t c h a a d ( 1 3 3 a t S o u t h Dako t a h a v a sue wo to a c c u m u l a t e nitrates in h a z a r d o u s amounta, oven without c,too treatments* J. '*. Zahnley wrltoo no of unpublished work by Harold Jones of Kansas state College, o h o u l n g that lambocuartora, clcwoed, and smortwoed (iolygonur: so.) treated ;;ith 2 3to-D core -ostreacly hlefc In nitrate, wr.lle"trooe not treated contained very little. This oeored to be duo to the 2, c h e c k i n g the a s s i m i l a t i o n of n i t rates Into oroteln, ao that nitrate ac c u m u l a t e d In the t r e a t e d elnr.tn, but wee uned ur In the untreated ones. It w o u l d surely be rlGky to eoray a pasture contiilnln^ t.l - weeds Presumably, If they are killed outrlcht, they will not a c c u m u l a t e c ore ni t r a t e b e fore d e a t h , but. with t h e ot vjc M c & I lrregu- laritlos in mjrsylr.f? wo cannot be sure that t.Mo will occur* Corn, aorghum, orooo, a u n f l o w e r (H o l i n n t h u s ar">) gur..;ood Cdrlnclell* fton, ) a nd opldortjort (T r a d o o c n n t l n a n r . ) hove also been indVroFel Tr^) na c o n t a i n i n g lstfcol auantiicio'a of nltrata under some conditions. Hr. C* D* Floyd, State Apiarist for Klnnocnta, reports one rather cloer ccae o? poloonlnr of bees pasturing on mustard s p r ayed a f t o r it car.a I n bloom* Ho was not ablo to ducllc'-te the roault tfca nc^tt yoar, oven by f e e d i n g the boos oyruo contaminated by ouprooodly tho oomo 2,b-D formulation used the year before. However, aor.^thlrr? k i l l e d the beaa the p r e c e d i n g y e a r (lntog}# Hr, Floyrl V o c o r t o of InJury wore recolved In 1^50. Apparently tho trouble is at loaat unusual. Tfcsre will bo many of these problems, reel and 1mug! r.ary, ae w e e d control c o n t l n u o n to develop. C u r ettl.tu.1n, aa good weed control men, r.uat ba to c o n s i d e r every complaint, even those /ri cl. look absurd, cautiously ond carefully, neither anylng "Impoedible" without investigation, or fourfully accenting unproven rearonelblltty,, There h ave beer many reports of 2,too I n c r easing the protein content of grain (c, t Ik), In gonb^al, there offoeta soon physiologically to be like the Increased protein obtained In a dry se&eoni that to, the protein elements In the grain arc laid down first, e n d If the grain does not "fll*." veil, it. will b? higher In protein than one that does fill* Usually, at leant, the p r o t M r p e r acre hr.a not b3n I n c r e a s e d by spraying. However, m^ny o f t h o e f f e c t s on the v e g e t a t i v e p a r t s o f p l c n t e ( 1 0 , 3) a r e not. so s im p le and r v l 3o f i e l d i s open f o r the s t u d y o f th e e f f e c t s r f '2 , k --n t r . 1 rel*-t.pd compounds%on f o r n r e . The v e g e t a b l e r.en >now w e l l t h e -Trustor development o f f i b e r lr: rsr^rsp-uo, e t c , , f o l l o w i n g 2 , to--1> t r r r i t n e n t s * r n f i e l d c^^rp wo have no 1i>+s s 9 y e t , but tho problem l r hi c h iy 1 rro"t..nt. 0004517 5128 < - 6- litoraturn Cited 1. nr-adloy, \l9 B ai Crpcan, H. F. 5? Ponth, O. A* Ltveetnck p o l o o n ir.% b y .es 127 a n d b t b n r p l n n t n containing nitrate, '/yominar As?r. Srp. S t a t i o n Sul. *! lko. .2 C o o k , B . L * affect of aoll tyro and fartliltora on nitrate c o n s e n t o f Zita ospron*?cl ono and the total nitrogen content of t h o . t i c s a c s of t h o c r n l l grcrtnn. J. Aa. soc f.raron 2?: ?r,7 lio? (1OT0) m #v i oof,on t 3 Corna, W. G. B f f o e t of 2 , U D a n d jotl n o i nture on the catalase aatlvitv, roapivatlon, and protein content of bean nlunta. Con. Jour. Poa. 2^:393~u05(lqW k. G r a f t o # '-a . 8* avA Barvoy, it* A* tteed Control* Advancon In Agren. li20o*32C(19^) 5* ravidaon# tf. I?.# at nl* Nit r a t e polaontnrr of livestock. Can. Jour, of C o s p .t!od. Vol. 5, l^Ui* 6 . Srlclrcon# I. C.,, Sooly# C. I.# end Vlncoa# K. H. Cffoct of 2 , U - D uron o r o t o i n c o n t e n t of vheflt3. Jour. Aner* Boo. Arron. ^ 0 : (j 5 ^ 5 < 3 0 U . ^ 3 > 7. Gilbert# C. ^^roon, B. F,# Srndley, V, 9, A Reoth, 0, A, 'Jltrstc? accu^nlr.tion in c u l t i v a t e d p l n n t a anfl weoda. 'Wyo. Ap t . B*r?, station#; B a ll, 2 7 7 # 1 9 U6 . g. Grlpoby# 9, R# ft Fnpwcll# B, 1), Bora e ffect of h e r b i cide on m a t u r e and o n (rraRins? ltvastoek. Rich. A?r, Uxo, station ^uavt, sail. Vol* 32# Ko. 3# Fob. l ^ O * Q. UQlu'OGoiij j. i*. T b o offcct o f 2,&-D on vrhoat. Proc* -th Ann. xV M o o t i n g Fo r t h C o n t r o l Wood Control Conference# Bee* l^^f (Topol?n# ICnnaao) op. 7 7 * 3 ? 10. H u l l 1near# C. H# Eff e c t o f 2,* -I? o n the n l t r o j a n ond c a rbohydrate octabollaa of tho corn Rea* Boo. N.C.W.C.C# Sprlnarfleld, 111# 1PU, Bee. VIII# A t t r a c t 5, 11 Kaphart, L. M o d e r n t o r of discussion at f>t* iul meotlnc* of N . C . 'a .C.C# Proc. 2nd Ann. M e e t i n g J.C.W.C.O.# at. Paul, Finn. 1<*5. PP. bg-75. 12. Mitchell, J, v.# et al* Tolerance of farm animals--to feed cen tal nl nff c# ^ - D acid. Jour. Anii.% r,c \ . 13* Olaon, C, E, * V h l t a h e a d . Dakota plants. ` i ^ r a t e c o n t e n t o r 3 0 m e r.ourh Ac a . of 'Jci. Pf'J0,r 1C-.1 ( i oitr-) 5129 7" C.: U lla r , 0 , J# Cfoofc o f 2#M> on o ro tein csr/J-.iS o f noocarcH iioport II.C.U.C.C. Reooaroh Cocualtte e 77? 15 ia n n j? , :Ja 4- u f c o a o f 2 , U-D on ancrai boato. C ryatal-lzed fdOwG p-D'a oO';o.r b eota. V ol. XV, Ho. 1, Jon. 1959* Ctbc~3 'O 'j nono only aro from unoubltchod co-munlcntIona to t*I:o vvUSo** . . . n r r. r* V * ,B CG04E13 m 755911 . R-& D REPORT & jCL .> ', . DOW C H E M IC A L U.S:A. RESTRICTED: lor ul hin Th Dow Chmicol Company only. " t wfcN' '<".*" Environmental Sciences Research _ " t o x i c i t y of e l e v e n h e r b i c i d e s t o daphnids ES-274 M a r c h 16, 1979 1, 9 1 9, 0 ; 0 0. 0 0 3 20 ac ui as * - ; h i 3 Z W. M. M c C a r t y ac aw*mi p $ s c '* r fe s U /*; " fc^ t n e s s i j r i p T j A t .^ /J.O DESCRIPTIVE SUMMARY WITH CONCLUSIONS: , ? AZE ;;.3:3PQj9l ': //O / ' / This 4/c/-?<> report 1 s: 1 1 interim 1 Xj =!NAL and mctnly: [_X] n e * n Hnclua m ?hn space references *o 3o?a books, and ro earner rej*C'j '<" is, potems and publications.; i i 1 Eleven herbicides were evaluated for acute toxicity to the a q u a t i c i n v e r t e b r a t e , D a o h n i a m a g n a S t r a u s . T h i s s t u d y wa-s conducted to procure environmental data on these products. The 48-hour LC5O'values and their confidence intervals for the sample's e v a l u a t e d a r e as follows: S atipie i .i 2,4-Dichlorophenoxy acetic acid B u t y l e s t e r 2,.4-D.'\ . .. D O W A I I O L * PIB;E3*te'r" of. S i l v e x ' " 4(11 i D O W A N O L P I 3 E s t e r 8 <4 ^ O W A I I O L P I 3 E s t e r T .o c S 'l' I s o - o c t y l 2.,'4-D -* h leC 4 F o r m u l a 4 0 ' of2,4-D o f - '2 , 4 , 5 - T * ' . ' . ; n R -c rb i.tfn K - 114 5 7 DMA 6 Unseq. MCP amine DR.#lcf-4/1 r - n 2 .2 -' 3utoxy ethanol ester of dichlorprop DOWAIIOL E 3 ester 2,4-D 48-Hour LC50 1262 ( 2 4 8 - 2 6 ) - t m q / L ''iO. 6 '.(3-1*4) m g / L 1 . 2 ( 0 . 7 - 1 ' . 6 ) '. m g / L 13*. 2 (.9.9-16,-5)' m g / L 4'6. ( 22-r.) m g / L . : 13".7 ( 0 . . 0 0 - 3 3 . 5 ) m g / L .456 (394-528)' m g / L 4 6 7 C4 2 5 - 5 0 6 ) ' m g / L 146 (36-223) mg/L 14.7 (11.7-19.2) mg/L 32.6 '25.6-43.9) m g / L 951 Confidence Interval `T r a d e m a r k o f T h e D o w C h e m i c a l Li DISTRIBUTION? p = 1 2 a -1L i j n * a 7:C.* Xn * 3 a l REPORT NO < s i r 1** ; ; r - - -4 ^ , * ,r i C604S2Q 5131 5132 t HN073280 D O W 'j9 5 G 2 m - . .. lie d } in United taU of America Bnrist! fra AssoaraoM cwFooa it Dnoa Otkcims erras Ucnco Crttct Vol. XV, N'.. t, Ottnbtr ItSI CHEMICALS IN FOC3: A IIEPOIIT TO THE ASSOCIATION OF FOOD AND DRUG OFFICIALS ON CURRENT DEVELOPMENTS. PART II. PESTICIDES ARNOLD J. LEHMAN, M.D. . Chief, Dleition of Pharmacology, V. B. Pood and Drug Adminitiraiion INTRODUCTION Tho pesticides bs discussed under the sectioned headings of acute toxicity, dermal toxicity, subacute and chronic toxicity, biochemistry, end pathology. In the tabutar eununarics to follow it will bo necessary to mention many of tho individual pesticides a number of times. Since their chemical names arc too cumbersome for this purpose ths common or short* term designation will bs given preference. A list of tho pesticides to be considered is given below. In this listing the short term is given firet with the raoro customary chemical designation, wherever applicable, presented in tho second column. Because of the diversified character of tho pesticides from both the chemical and utility standpoint, a classification based upon either one or the other of these aspects did not appear to be practical; hence, in an attempt to cegrcgato tho compounds into some useful order an arrangement was devised which is based partially upon chemical similarity and partially upon use. Chemical names will not bo repeated in tabulations and the groupings as presented below will be maintained. 1 . Dorris 2. Rotations 3. Pyrethrins 4. Adothrin 3. Nicotino 0. Sobadiila 7. Jlyonlo 8. Dihydrorotonono 9. Th&nito 10. Lethauo-C0 11. Lotbano-334 12. Lelhano flpecisi 13. Copper chloride 14. Copper carbonate 16. Coppor sulfnto 10 . Calomel 17. Corrooivo sublimate P lant Product! and Derivatioae Rotonono and rotenoids Oleoresins Allyi cinorin Vcratrino-Iiiio aiknioido Alkaloids: ryanodine, rynoino, and others Thtocyanatea Isobornyl-thiocynno acctato Uota-ihiocyano-cthyl esters ot aliphatic acids with 10-18 carbon atoms Ecta-butoxy-heta-thiocyano-dicthyl ether Lcthanc-00,3 parts and Lcthano-331,1 part Copper hfer-ury Merci. us chlorido Mercuric 'hiorida 122 0GG4S4C 5133 . I S v~t< y _ir 5S5G3 ASSOCIATION OV FOOD AND DRNQ 0 F71CTAL0 123 18. E thyl msrcurio pbozphato 19. Thonyl mercurio triothanol ammonium lactate Arsenic 29. Arcenio trioxide 2 1. Potassium oroanito 22. Paris croon Coppor acato-arsenite 23. Calcium nreonnte 24. Load areauato 28. Dariuia fluoailleato Fluoride / 23. Cryolito Sodium aluminum fluorido 27. Sodium fluorido 23. Sodium fluosilicato Afiacellaneout Mclala 29. Sodium eelonito or cclonato 29. Cadmium chlorida 31. T a rta r ematio Potassium antimony! tartrnto Chlorinated Hydrocarbons 32. D D T Dlchloro-diphcnyl-lrichloroothano 33. T D E Dlchloro-diphonyl-dichloroethana 34. D FD T Difluoro-diphonyl-trichloroothnno 35. Mothoxychlor Dlmothoxy-diplionyl-trichloronthana 30. TI11I Technical bonzone hesaehlorids 37. A im Alpha corner of bcnznno hoxnchlorido 33. n m i Bota lsomor of bensenn hexochioride 39. n u n Delta isomer of benzeno hasachlorida 40. Lindauo Gamma iaomer of bensono hoxacblorida 41. Toxaphsno Chlorinated campbcna 42. Chlordano l,2,4,0,0,7,fl,8-Octochloro-2,3,3a,4,7,7a-liexo- hydro-4,7-math,-moindece 43. Aldrin 1,2,3,4,10,10 cxachloro 1 ,4,40,8 ,8 ,8 1 hoiahydro-l,4,8,8-d:m thanonapbtiialone 44. Dioldrin 1,2,3,4,10,10 Hoxschioro 0,7 epoxy 1,4,40,5,5,7,8,8a - ootahydro 1,4,5,8 dlmothnnonaphthalona 45. Iloptachlor 1,4,5,0,7,8 ,8 llcptachloro - 3a,4,7,7a tcira- hydro-4,7-metiianoindono 48. Prolan 2-H itro-l, 1 .bia(p-chlorophcnyi) propane 47. Bulan 2-Nitro-l,l-biu(')-chloropl)snyl) Lutano Organic Pknpkaiia 48. Parathiou 0,0-Diotbyl-p.nitrnphepyl-thiophwiphatrj 49. D im ethyl paratiiion 0 ,0 'D ito c th y l'p .iiitro p h r.n y l-th io p h o ep h ale CO. Paraozone 0 ,O.Diothyl-p-r<itrophcnyl-phnsphr.ta 51. OMPA Octamctbyl pyrophosphorumide 82. EPN E thyl p.nitrophcnyl'thionGlionzanfiphoRpho. note C3. T E P P Tc trnethy1pyroph ospha to 54. III5TP Ilozaetliyltetrapkr.xplislo 55. AB1M7 Totruelhyiditkionopyrcphonphato o O * -3 QG04E44 ?S a g . ^ SVSaM. DOW75&564 124 ASSOCIATION 0 7 FOOD AND MtUO OFFICIAL za. E-GC3 Diethoxy thiophonphoric ccid cuter of 7-hy- droxy-4-wethyl-cou.'narin Dinitro Compounds 67. DNOC 3,5-Dinitro-o-crojol 53. DN-111 2,4-dinitro-o-cycloho):yl phenol dicyclohcxyl- amino Ilerbicidu 59. 2,4-D 2,4-DiehIorophcnuxy acetic acid 60. 2 ,4 ,5-T 2,4,5<Trichlorophcnoxy ncew acid 61. Naphthalene acetic acid 62. M ethyl-l-saphthalena aeotlo acid 63. Ammate Ammonium culfanutc 64. Pontschlorophonol 65. Endothal 3,0-EndoxohoxahydropLthalic acid C6. Mnleio hydroxide Acticafore 67. n-Propyl-isome di - n - Propyl - 0,7 mcthylencdioxy 3 - methyl - 1 ,2 ,3,4 tctrahyuronnphthalcne - 1 ,2-dicarbcxylato 68. Piperonyl butoxido (Butylcarbityl) (0-propyl piperonyl) ether 69. Van Dyke 23-1 n-Octyl-bicycIohcpt&ne dienrhoximido 70. Sulfoxide n-Octyl-eulfoxido 71. Piperonyl cyclohexanone Alkyl-6-(3,4-mcthy]cnedioxy)-phenyl-3-3-cyclo- hoxen-S-ono Rodcnticidzs 72. 1030 Sodium monofluornacetato 73. Castrix 2 -Chloro-4-dimcthylainino-0-iaothylpyrimidin3 74. ANTU Alpha-nnphthylthiourca 75. W arfarin 2 Alpha phenyl > beta acetyl ethyl 4 hydroxy-coumarin 76. Strychnine Strychnine nitrato 77. Thallium oulfato 78. Usd cquill 79. Zinc phosphide Miteellantoun 80. Phygon 2,3-Dichloronaphthaquionno 81. Spergon Tctrachlorobcnznquinone 82. Phsnothiazine Thiodiplionylamins 83. Aramito Beta' - chloristhyl - beta - (p - tert. - butyl phenoxy)-alpha -methy 1-cl hy 1 r.ulfit c 84. Arasan Tctrainctliyl-thuirum-disulfide 85. Fungicide 311 C Glyoxalidino acetate SO. Dithar.c Zinc and r.odium iedtn of ethvlcnc-bia-dithio- carhr.mic acid SEC TIO N I. A C U T E O IIA L T O X IC IT Y The acute toxic dose valuer for the pesticides arc listed in Table I. Practically all of the data were obtained in the laboratories of the Division CG04C4S 5135 ASSOCIATION OF FOOD AND DUUG OFFICIALS 125 of Pharmacology, and, with a very few exceptions, the rat was employed as the test animal. The values for a few of the older compounds ouch as derrta, sodium fluoride, and arsenic trioxido cro so well documented in the literature that they were not necessarily checked in thb laboratory. In some instances technical difficulties would not permit the calculation of ,, an LDhi hence, in the interest of accuracy tho term "approximate LD" ia given as the column heading. Wherever possible ths pure compound dissolved in an innocuous solvont was employed, and dosages were ad* ministered by stomach tube to fasted animals. In an attempt to present as much useful information as possible in tabular form, the chief characteristics of the poisoning symptoms, tho time elements involved in their appearance and duration, end the clinical cource of the poisoning as observed in the onimal3 are briefly summarized. A few additional comments on tho various groups of pesticides follow. Thiocyanaiea: Except for thanite, tho acute toxicity was determined on the commercially available concentrates which aro 50% solutions in dec* domed kerosene. The LDeo's as tabulated were calculated on the basis of the pure compound. Copper: Although the compounds listed are not used as pesticides, their toxicity doss ccrvo to give eome estimation of tbs toxicity of copper oxy* chloride, oxide, phosphate, silicate, zeolite, oxychloridcsulfatc, and Bor* deaux Mixture. Mercury: The general trend of phenyl mercuric compounds is an J,Do of 50 mg.A s- or less. Arsenic: The ratio of toxicity of arsenites to arsenates may be considered cs 1:1.6; hence, other arsenical pesticides not listed, such os sodium and zinc arsenites, would have an LD*a of about 13-14 mg./kg., and copper, magnesium, and manganese arsenates would be in tho 22-23 mg./kg. range. Miscellaneous Metals: The value for sodium selenite or sclcnate is calcu lated on the basis of selenium. The toxic dose of cadmium chloride is listed because this metal is a constituent of a t least one fungicide, a com* bination of cadmium copper zinc chromate and calcium sulfate. CONTINUED IN THE N E X T ISSUE o a to ci C 5136 1. Dorris 3. Rotonona 3. Pyrothrins 4. Allothrin 5. Nicotine 0. Echadilla t A m t s t.-J II: Lily A rra '" * u v.3 i U c c d - J G ^ is n iili (hr:t f C^ rjU ra Natural produca cad drrivctiras -1CS1 Frequent c'cu7ubivo Within minutes 1 to 3 days crisuraa followed by to fito 0 hours drprczsica cf central errvous cyctsa DOW75S5G! C .a h d C w :::a r i . i Death by respiratory failurs in 21 hours. Fatalities ram after Crd day although death c a y fcs delayed 10 cr 12 day' 153 Caca sa for arria 33 Central norvous eyatom Usually within 10 hours irritation; hypsrascit- hour ability, trasera, and convulsions Death may bo delayed for as long as 8 day?, respiratory paralysis is a major cuuts of death C3> Tremors and canvulaiona COminutes 0 bosra Fatalities nppesr to b# rare 21 hours after poisoning. Death due to respiratory paralysis CO-CO Clonlo convulsions Within minutes 8 to 20 minutes Curaro-liko psralyds of respiratory mus cles and death teso Hatching, nuasubr Within S min- 2-1 hours eparms, otada, coma tC3 Gymptomorimllar to aeenito.Death from respiratory or cardiao paralysis may occur within 3 to 10 minutes after tha ingestion m a fatal dess 7. R yania 780 Depression ef central ner 6 to 8 minutes 2 to 10 hours Continuous depression up to term inal vous oyctem convulsions. D eath usually within 24 hours although largo dccce may ba fatal within the hour after ingestion 8. Dihydrorotonons 0. Thanlto 10. L othsns-00 1 1 . Lcthano-ESl 12. Lethono Special 13. Copper chlorids _ 14. Copper carbonate 18. Coppar sulfate 1C. Calomel 17. Corrosive cublim ita CO 1000 6G0 CO 400 140 180 SCO (210) 37 es for dsrna Thiocyanates Deep depreoeion, cyano- Within a few Rapid callapcs Occasionally oymptoms m ay be delayed cis, dyopnes and tonio minutes within min- for a few hours. D eath isuus to respira- convulsiona UtC3 tory paralyis Copper Violent retching, rcuscu- Within a few lar epaems end collapse minutes If dors is ratai nod could bo fatal in 1 hour Symptomo of gastrointestinal irritation may cubsido, b u t dcath may foiluw csvcral daya inter bcccuss of damage to im portant organe ouch as livcr and kidnsys Mercury Gyrapt oms of morcurialism ut tliis dore but no desths Sheet, dyeautsry, and Immdiats anuria Symptoms Shoak clTcct o lits irritan t may be fatal nernet till within 21 honre. Acuta kidnay ila rangs death m.By ho fatal by iho tlird dey 13. E thyl mercuric phew- pkata 19. Phenyl mercuric tri- cthunoi ammonium lactate 3 Similar to corrosivo cublimate l 30 i C0'g4 ? 5137 20. Aresuio trixido 21. Potom iun amenito 32. Pttria_grc3n 23. Caloium nrconato 21. Lead arssnsta 2fi. Barium flaosilicato 23. Cryolite 27. Sodium fluorido 23. Sodium fluosilicate 20. Sodium eolenito or eelensto 30. Cadmium chloride 31. T a rta r emetto ARSENIC 13 Violent c ^ trc o n to ri'ia, Almost A fow hours to Death from exhaustion and dehydration. M dinrrkca, rico water immediately several days Convulsions and general paralysis may 3 stools oesur before onset of B.-u.trccnterilie. 0 D eath -within -a-fow hours to several ICO days Fluorides * 175 Abdominal diotrers, diar- Immediately Stupor and In fatal poisonings death can result in 2C9 rhea, cyanosis, dye?* and ur. to 0 woakness a fow minutes 200 non, fibrillation of skcl- houra . may persist 125 ota! rouaclea for 23 hours Miccolliu'couj Metala 2.5 Garlio breath, nervous- V/ithiu 15 min 12 to 18 hours D eath duo to respiratory failure ness, central nervous system depression utes i 83 Salivation, vomiting, 15 to 0'm in diarrhea utes Gastroenteritis may be a contributing eauso of death. 115 Percllolo thoss given for aresnio poisoning D eath within a few hours to several daye 32. D D T 33. TDE 3-1. D FD T 35. Mcthcxychlor 30. TB II 37. ABH 53. BBH 30. L'J-il j ^~ C C c t Chlorinated hydrocarbons 2S0 Extreme excitability, W ithin com m 2 1 hours tremors, twitching, utes convulsions, eoma, death 3100 Lethargy W ithin 21 hours 2 to 1 days 1120 Similar to DDT C000 eco eco coco Largely depression of Within 21 centra! nervous system houro 2 to i days Convulsions which may givo W3y to a central nervous sjviem depres sion Within comm utes 2 weeks H ypercxeitability end About 1 hour 2 weeks convulsions Principally tremors 2 to b hours 2 dayj KvO Depressant to the cent ml 1 to 2 hours nervous system 1 day Time interval for a fatal outcome is var iable, but death usually occurs within 21 houra Convulsione do not appear to ko a major symptom. If death has not occurred by the 1 th day prognosis appears to be favorable Apparently the f.uurico part, of tho mole cule does not contributo to tho symp toms of ecuto poiconing Tremors havo b/.en noted but tr s not a prominent symptom Symptoms may persist for 2 weeks and deaths have bean observed lata in tho 2 wsoko observation Deaths delayed up to 2 weeks with con vulsions pcf.aistin* to the cr.d I'jflth occurs between tho i':id and day Death may Le delayed to between liu: 2nd And Olh il.-i.y ^ 513 8_ - ; <V 49. Ltidans 125 l7yp:tt:n.UivjSy and 13 isusj convukiuns j! lililaO lLj'J--ii/ 1`ith.ft L. tr/O-ilvL? |.C: idle? l a ^ i k a . Couvalncsa pin. tnthniud 41. ToxcpLsns es IlyFSK-aaUvlty, trem 1J bou erai end convulsiona 21 bou bloat of tho fatalities occur within bourn. Dels e i dratha have bean no' uptotLoCihda7 43. Chiodano, technical i* u 457 Hypsieresltivity, trcm< 16 minutas era, end convulsiona 2 ayo Treeless brgin in lOminutesjcouvnloio' appear efts? about 21 hours end tn persist for 2 days; deaths may bo c layed up ta tho Cih day 43. Aldrin 07 Trcmc-m ned convulsiona Within 1 hour 2 aya Tims of death variable; may bo delay up to tha 6th day 44. Jloldrin 87 Tremo, muscular Usually within 2 days epanres, ccnvukions tho hour Ccatha may ba delayed up to tha Cth d 07 longer 45. Hcptachlcr C9 Tremo and convulsiona SO minutes to 2 days on hour Clmilar to aldrin, dieldrin end calorda 45. Proion 4000 Tremo end eonvuldoco Within about 1 Prolonged for Persistant convulsions up' to time hour days dcatn which may not occur within houm 47. Bulan 3S9 Convulsiona About 2 hours 4 bou Fatalities uaually occur within 4 h ^ 43. Parethion 49. Dimethyl parethion 49. Parsozono 61. OMPA S3. EPI7 63. T15?.? 64. TOTIP 63. AOP-47 63. E-003 67. DNOC 63. DN-111 9. 2,4-D 5139 GGOS343 cr cc v Organio Phosphates 3 16.2 Generaliced fibrillary lGccinutea to 1 12 hours 7.6 tremoi ealivetion, hour 13.6 lecrimation, diarrhea 14.5 end convulsions 1.3 7 6 19 Tha symptomatology cf tha individua organic phosphates iaeimilar. Dcatfc usually occur in 1 to 21 hours Dicto Compounds 23 Increased respirator}' ZZO rato Within tho 21 hou hour Time of death variable hut usuali within 21 hours with byperpyrcai contributing causo Herbicides C0 A taxia, m yctcria, gas 29 minutes trointestinal irritation Several hou D eath occurs Govern! hours after exes tion CO. 2 ,4 ,6-T 220 Similar to 2,4-D 61. Naphthaicno acctio ncid 62. Mctbyl-1-naphthalone acetic acid 1053 2140 G astroenteritis, depres CO minutes sion arid paraiyeia Lesa then 21 Most fatalities occur within tho first hours hours 63. Amicato &3. acra Lintlcccucrs TX Tr* .* in.-l W ithin m inute: 21 Lourd r<n .. Tremors have ce :n noted with very hi.jh dosed and death within 1C m iaaioj. Sar rival i-iicr 21 L r.jrj ij iorow.iji^ cu.v.pletu recovery : ^- * w . Eudaihnl C3. M oldo hydracids C-.3 oJ 07. n-Propyl b e a C3. Flporonyl butosido 00. Von D y to 23 u 70. Sulfoado 1223 11C50 exa 2220 71. Flperonyl cycloha:nono 02 ) 72. 1020 73. Cestri* 74. ANTU 1.7 1.7 0 M uidor Tierno, inusada; eject Depression (.V 21 L-cm It) to CO min* 3 hours utca . A ttirato L\.v.Lj e: L*.V..-,:ra 1 ami 2i hour D sa'.ks within 0 hours Depression 23 minutes Coverei dsyo Delayed deaths cp to ons week Gone bypercncitability Z m inutes followed by depression 2 clays M eet fatalities occur within tbo 2-day period Tremors, covcro dspres- 10 to 20 a io * 2-1 hours or Continuous covcro depression till dontb d on, coma ates moro Survivals may bavo cover symptom attending beyond tbs 21-hour period Death usually within three days T rem e, convulsions Rcdonticidss 15 minutes 1 weak Convulsions IS minutes 12 hours Dyspnea; occasionally 15 m inutes convulsions 21 houra Intermittent convulsions for as long e 10 dayu before death Survival fur m o than 12 hours appear to be a favorable prognostic sign Death usually occurs w ithin 24 hours 75. V/nrfnrin 76. Stryehuino 77. Thallium sulfate 78. lied squill *V 79. Zinc phosphide E0. I'hygou 81. SpCTEon 82. Fhenothiezino 83. Arami to 84. A rsisr S5. Fungicide 341 C 85. D ithane ICO Severe depression; gener alised hemorrhages 10.2 Tonic convulsions 10 minutes 12 hours 25 Depression 48 hours 72 hsura 200 Central nervous oyotem 12 houra stimulation 21 hours 450 Depression 24 hours Miscellaneous 1500 D iarrhea and cover de pression 4200 Depression 72 hours 5000 Secondary anemia &20 Denrersiou 3 hours S5 Hyperevcitability, Ba vero diarrhea 4720 Deprejoion, hsmorrh.'-go fr:>a the noia 5GG3 |: D iarrhea 3 dayo 1 0G04630 _____ 5140 Deaths era usually delayed but in sever', poisoning death could oscur within half hour after ingestion Deaths from medullary paralysis and ex haustion and usually occur within ; 12-hour period Most of the fatalities occur within th. 72-hour period. Renal damage b th principal causa Animals exhibit circular m ovem ents; re pealed rotation on long axis of body Majority of deaths fall within tb s 21 hour period Meat deaths occur within 72 hours D eaths occur within 21 houra Depression may bo prolonged for day. before death Recovery from temporary seconder; anemia if poisoning is not serious; residual toxic ciTccto Delayed dentbs over 1 wce'x Most deaths occur within 5 days Delayed deaths up to 2 wcakj 1 5141 223961 Parto b O topaala 00 ! U 4 *0 3 # ^ a plooo --vllfel *' V-.-;VS? f o n o os fo o . n a atibada eaoaaBM tfela r o M lly , t t w a r o U lt s Ufe M tin ta tram a tba atemafe tafea, fiarlas tfea ata? tfea h M I j n r t fei| ta ita a aaa*. o str a l bloc eouata aara tafeaa b a fw * fea felU atrtt 1 aa 1 toa ru a U a J-Hfe ay tac aa tba Ota ay t taa toadla t i t a cf tfea I t ataa^JiCblfe aaa abarato* atut a p b etoalactrta col Timbar (6 ) . Qpafe tfea laatb a* t tba ecap laltaa of tba atudy, taa aalaala 1 a ata a M , tu a a a a ara takaa froa taa la, srt, llva.-, kldaay, adraaai, aplata, lataatifefe tfeyroU^M r. I. wrary * taataa acd flxa ta l foraalla. "*31* m a. Jyajteaa jruduatd. Tba a ffa a t of m rlou* oaaa o 2,4*0 a eurvival for 1 aya ta aojan ta rabia 1. Iba oral IB^q 1 appraxlaatal? 1>J aa/kicx. 1, la do-a taat 41 iba ayaptona produce by 2,4 -0 varia fraa a a a ilJ ataxia ano atlfTaaaa lo tba b la l '..a to a a fta ita ayoteala. IfeltlaX c ffc c ta aa.-a o ftaa nota b Ouura a fta r tba oral a b a la la tra tlo * of 2.4*6. .'ta aataala aar* cura Tulat taaa a o r v l aa a altgfet atabla aaa oaaaalnfeal ly preaos: at th la t la a . Iba blad l ^ a ara alaoya affaotad f l r o t , tba faralaes la ta r aot at a l l . rhara aaa uaually a p r o n ta s Iva lfearaaaa la apaaa la tba oui la .a aaa je lata attb laoraaalm atabla aaa ama asa apealaosanal? aitaooa tba hind lim a la a ip aatla aoaamat la a tla a faa aoaefes. Tfea nao j-rk aaa atib ar n o r l hyparactiva. la toa arly ata.aa * aaata plaosla tfea afeifefelfe otra aamll? t u ia t . If tba a a im l la llf t a aa M a to aalk tba ffesfe aa a t a la ara b o tica , at feas aalk* t<w a afeUa tfea apsafe aa abfebU t M f tb aaraasfe* lo tfea la ta r ata* of U to x lo a tiefe tba aaifefel, afeo* pi fe afe I I I f n t , ! 514 0 0 0 1 7 0 3 J* ire* . a i . l i fc* c o i a i <roaa eaaac a notad ara ii. q q m c a n r a i u m l. JfcKtiLllZ- da*a rM tT iac 2, 3 cr 10 a s or 2.4-0 yar k U i M * l n 4 tta 90 day u i t f i r M . Beth daa r M * t< U | u * 20 jar k ilo dlad durine tha atudy. Tha aaaa to t a l d o lly dooo dlvtdad aad ndaihln- tarad tola* a day to too dogs eauaad aaath. o fto r a loacar tla o in te r v a l. la only ooa an U al (Tabla 2) . 2. Ooooral ayaptoaa. Doa that aurrlTod tha oral adm inistration rf 2 ,4 -0 f * ?0 daya ooro froa of any ay sfto a a . Tha thraa dago t in t dlad oh ila r seal Ice tha hl<0aot dooo of 2 A -D ' teooad ayhftoM aad alas d Iffarin g aoaaonat tram thuoo obaarrad la tha aeate atad Isa . Tha doth oara .iu la t, aaakar and laaa r*apo.-.ala taaa aoraal. aoaala toaaa aoo hlhar la tha bind laaa. p a r tic u la r ly on paaalva axteaaloa Tha hlad laa aara held aora s t i f f l y than uaual ahan *a 11 1n.- aad a a l l t t ataxia aoa praaant. Durla tha la a t ; to j daya of thalr a w v lv a l 2 a t tea data hossd d if f ic u lt y la chaaiac cr aaal lo sin g aod aaantiailly aaaa rafuaad a . -9 a a a ll bolua of eaaaad doe food (u a g a ily ra a d lly caoauaad) Thara " a alao note oaolac of blood froa tha guns and buccal aaionaa. 3# Body a a l A t . Chaasaa la body aolRht ara lla te d U Tabla 2s Tha oaly alRO lflaont ebaota a*a oosarvad lo tha doga th at fa lla d to sw v lv a tha atudy. Tha an igh t looa la thsaa a a la a la hagan 7 to 12 daya boforo tha daath Of tha doc. k . Blood aount. Tha a d a la la tr stio n a t 7,4-D did not boos aay a ffa o t oa tha haaoBlohla or rad t a l l count a t a o iM la that aw load dlad * U | tha atudy (Tabla 3 ) . or tha aalaala that dlad. aha da (Nn. 104) ahaaad a aadara t a tear aaaa la ta ta i a a lta t a i l aouat. NaMvar, a a a la llt v haw a n o m a d 1 a a s t r a l 1 (Na. 42) i t aha atea l uted th a t th U a hat a tl B lf laah t a ffa o t af tha dr. Tha d lf fa m a t lh l m a t M hat & jv-t,3* - * - * ..i; -> - ;v ' m : yCs ;V - V A " Vf ; . . "ilciiftittti^ t f f H M hjr 2.4-0 tti ik* poMiki* m t r U a T -** 0.7- ' r tai H * ' t u * - - - ' l* , & og w i t t u t a t r a K h * par at of t rfa i toaA a M t praaaatt U dog Ma. 104 ja t h f a t 4 a i ' ' ; ".*</-J.v OrM l iM M U t t t UAMW l M A 1 :td r4 arooa (1>2 a > ) a n | n i a > ta Ma lie b t dlfffeaa rodaoaa o f ta* daadaaol a n o --. ft**- b . C rr a -- U n t o . n * r o *oa ao a ls a lfla a a t Utk* r ta * t a y r o i d . a d r e n a l. a * a r t , a r a r o r k i d a t r t a aalMla that a r r l r t t i o la y w a t p e r io d . t iaor**ao l a aoa* a t t a * oc** a le t t a aa bo w w 4 in t-v 'U, H and n a tn*t r * e i r * d ta* h lA o o t doro o f 2 ,1 -fi aad A o dlad tu.-lrv u>* ip -r ta o t. asi g p4 ^ V *0 CU C J 1 VJ Ua U -i !OU u*-pT*a C iio m it.a rh* a w t o a o produced by ta* k l^ i a la c i# oralo doaoa :" 0 i i*rrr.-. -a itw y aa not o o * * rr* d ta ta * p ro a a a l atudy. J o m of A a dosa jy ia . . i' tu acut iiM a *n o **d * a l l d to d lffu aa r*da*aa <f tb* duodeaaai . - u ; -.< * imi * d c a * i .t n i i l l y r * * * la t b * lu a^ t h a t *pp**rod t o b* boaorrbacla* r--* S i . t i - r du**. f tu ..raaucad n a r a t t a aad oaaoqaoat N l ^ t io * * . a * tn fr^oi l.ir g * a ia ^ l* a * * l aoa*a o f 2, 4*0 la doloyad rad orrrra la y a * f t * r *.n . a u t : * d . c i a t * t r * i t o a . r h r i r r i U t i c o oad p ala a * arraod ta i J w i *to tr. a r l a ' f '.h a n r c r o f t n r a a ra la e ro apod m y la d lo a to a m * ^ in n a r i i r r i t a t i o n . end u.* t t a n a l oc and e r o o o la f of ta blad ta fa ta araa ^ ir. a , *r.o l i f t * .y t a * Miuuidara. m>y point to (p in o l w oaafer*! l aal A t a M* tarant* atady ta* ara aure lead doaad f ! 5 **d 19 A f . ( - per t i l o o f ( i d ; o a t^ tt. Ocaoa o f cO par fella . k M W t 4M ar " t ; 5147 produca daatb aban a d a lb is ta ra d o**r a r la d a t 11 to } a o j o , rb ia la la a o a fa -m lty l u aub-aauta atu d laa la Ic a a a r f-r a a d by M ill aad l a r l l a l a ( i l s rfeay a d a la la ta ra d 2 , -0 lntraaan ou aly la dooaa o f 25 -T j l a<. par d i o f b la j a e t l o n * . oueb n ia a la d a d <r oora a a c r ifle a o bataaaa t t * ira - a i la ta day o f t t * atuo y . I t `.a a id * a t toan tr.at tar* la sos* .- la u la t i* * i k i * a t 2*4-0 la Uta atu d laa a t K i l l ad a r i la la an i la i l a p r***at i i --iy a lia tba 20 g . par a llo doao. Ifea doga aur l a i a* ia * ab raaie aooaa a i . . - I act ma n j *i - a l f l a a a t sbaaga la b aa o glo b la blood oouat- 7h* ioaaa - f - ',- - 1 j ;4 n..t a l g a l f l o a a t l y a ttu a * tha afeito blood o ju b i. I d ooa a a la a l th at d a d tsa ra ' a a a a aarfead f o i l l a tba par co at of lyapborytaa la tba l l f f a r a a t t a l - aunt bfefar* daatfe. B i l l and a r lla l# ( i ) , a d a tn ia ta r la - -S * ;.J ^ . a t 4-I . r f e ll * lfe"a***o ualy fo r a l i da y a , bava obaaraad a f a l l la tao poi yjor^o aouei--r _ la lafeaaayta cotait and la tba por ta n t o f lyapbocytaa. altaougb B o tta li d<%*. Tfea lyaptaaa produood la an t e l a dying fro * taa 20 ag. par a lio V - feaaa rf 2 4 -fld iffn m d an--abat fro tto a a a a a a la tfea aauta atudlaa- i t t tfea arasi* d a lf e la t r a t lf tfea aala a la aafelbltad e b ia fly a atlffn a a a - f t l i felad la ca a i ataxia* anraaaa* aad b isadla fro taa guaa. dosa of taa ,Ut*r y- y . n a t e l a a*laa. L.'a'dao*aad *d lf f la u l t y la ha*lag or aaalloalac' JBBC1 1* ni* * oral LB50 T 2*44--0 la a p p r o a te ta ly 130 w fella* Pitia la tfel rana*' felgfear pradaa* a daflalta yataalu afelofe :p ? m u feaaoapfefeladfey a oraa la gfe laoa o f a l# t * < ,,a**. * ta taradla a l f aadlag tfea natela anted daaaa o , j C T ' ,.-Tc atf 1 * | fella for afe da m ithaat affati a body ---- < abtgkt, blaf ai fM 1^ 1* W m i-- l l f I M Mfef radati ST3 J f 9 of tfea H M H W W I M a 1fepd f fedfegfe ffefetlvffel 20 da fV tu r 1 4 4 dUd I r i n parla* Uataal"vpfeaaa ^ . / '^PoW 223968 Spf  r ~ -7*. 1 aa femlo*la. tatal llM l u i 4iff*rr*a*attli*al iMr iiUta i&iM '.i .* Dtffwttiui -jai ur j a . & mt utt im a 2 e M M d I> 2 0 > 7 2 17,SoO yt~ W _____ o 1_____ B L 3# 13.82 5.33 1 5 .* 83 23 5 .7 30 13.70 4.08. H.3V> 71 27 3 0 4.01 10.800 <4i 11 3 0 0 36 14.72 " 4.44 13U5 73 12 / o a ...Hatt,., 4.14 - Z * l > 9 - - 2 3 -- 21 5.12 >47 13.550 J J2 0 3 0 --1 to 8.74 > 8 8 I8 0 OO 7 41/ ,. 2 . V - 1>31 4 . >84 14.450 _2L 54 ; ,v : 36 14.81 > 3 8 17.850 30 14.74 4.97 15.530 54 70 12 -U . 33 23 3 3 30 0 o 3 05 9 3. 5 ' 0 ' * JN S S T a r r y TM < r 10.43 4.55 20,450 71 20 ^ -. `S.\ ' " 30 12.47 4.47 14.200 47 ?3 ___ 22.-19*28-. 3.42 -1 3 .1 3 2 ---SL---- 12-- 0 14.06 5*92 14.350 43 22 2 3 2 0 .0 0 7 *. 7 5 * <a** 36 14.53 5.74 * 15.55 42 11.57 h * tf 4 9 9 30 10 o 14.40 4 0 5 17,350 73 5 18 15 19 0 3 13.35 4.24 17.300 60 28 "nr M M-M ----- 14*34---- 7.03 11.4S0 71 0 1 4 0 4 *.33 15.200 34 -L.1 3 30 14.78 4.21 17.500 45 4 TT" 30 U.; 0 uTT .7*31_U a i S ^ 4 l 15.000 __ 14_______Jir____ L. 3 30 11 U1.13#* 117.8000 2222 545* 2 3 9 - 1233___2 II422___p ______ U ____ L 20 0 10.43 4.02 13.3 . 8 0 0 4 * 2 7 5 36 12.44 4.14 15.400 *4 28 99 11*32 V. ft 12*199___ 12____ 22. *20 0 1 3 m > > U . 8 5 0 54 36, 14.44 5*86 LL. 47 44 4 73 la 7 X. . 3 1 ii *77 14.34 ^ 4 7 m13.15 .. a - i Ak*tuT litt 3*191 - U - -44. 11 JL. -ii. _L M . K U | I m t l T l M H I H a u iiU r H t la * .4 . u 4 la c .a , ft TLt.ff`i'V'7 5155 DOW 882090 MNO02 527 Made in United State of America Reprinted from A w o c u n o x o r F ood Ac Dptra O m c u u o r t n U m ttsb St a t u V oL X V I , No. 1, JnnuAry H U CHEMICALS IN FOODS: A REPORT TO TH E ASSOCIATION OF FOOD AND DRUG OFFICIALS ON CURRENT DEVELOPMENTS. PART II. PESTICIDES SECTION II. DERMAL TOXICITY ARNOLD J. LEHMAN Chief, Divirion of Pharmacology, U. S . Food and Drug AdminUtration Pesticides may have uses other than those related to agriculture. A num ber of them may nd clinical application in the control of ectoparasites on m an and animals, some may find use as insect repellents, and still others may be useful in treating fungus diseases in human and veterinary medicine. The development of these various uses involves application to the skin; consequently, dermal toxicity studies are of considerable impor tance. M any of the pesticides are not amenable to clinical use, hence, the list of compounds investigated for dermal toxicity is not as extensive as th at presented in Section I dealing with acute toxicity. The general method of approach in evaluating dermal toxicity was to determine the effect of a single acute dermal exposure, the pesticide being employed in the dry form or in solution in an innocuous solvent, dimethyl phthalate. If these initial tests indicated a favorable toxicity, subacute studies were undertaken. These were either daily applications of a series of dosage levels for a period of 21 or 90 days, or occasionally wear-test experiments in which a definite quantity of the compound under test was incorporated in each square foot of cloth and the animal permitted to wear the treated cloth as a jacket for 21 days. Only limited wear-test data are included j n the tabular summaries. Frequently animals failed to survive the scheduled 21- or 90-day test period. This is shown in Table I I I by indicating the number of doses which were fatal to the animals. Dosage levels were determined to a great extent by the proposed use o f the compound. If the results of single or multiple exposure experiments indicated th at the compound was too toxic to warrantfurther consideration, additional experiments were not done. This accounts for the numerous instances of listing dermal toxicity values as less than ( < ) the value pre sented in the tables. Identity of the compounds listed in Tables II and I I I will be found in the preceding article of this series.1 In summarising the results it may be stated that insecticidal materials which are solids or formulated as wettable powders and dusts are poorly absorbed through the skin and a single massive exposure may not be serious. At least, if prompt remedial procedures are invoked immediately after large quantities are spilled on the skin the chances of injury appear 1 Quarterly Bull., Assn, of F. and D . Officials of U. S ., XV, N o. 4, p. 122. t 3 GGG5G10 4 ASSOCIATION OF FOOD AND DRUG OFFICIALS vxsn cD i TABLE II Dermal toxicity: Single acute (24 how) exposure in rabbit* T O U n ilA T lO X am oxncan LDUMO./EO. sm uLxm cn H B i a t u r im Botenone Pyrethrin A llathrin Nicotin R y ania Plant products and derivatives 10% technical end in dim ethyl phthalato >0 20% pyrethrina in aoya >1880 b*a oil 8 w u U in Undiluted alkaloid la powdered to m 80 >00 Severe skin irritation Slight body anight to e, generalised weaIrate, partial paralysis and iaocftrdination of aetrem itiea No d p iillim t local No systotaie affacte Ida effecta; severe irritation of abraded kia No local kin effects No effect Convulsions, respiratory failure and daath No yatemic effects Thiocyanates T h an ite Lethana-60 Lethaoe-3St L eth ase Special Undiluted technin! (rada 60% coaeanttate in petroleum distillate (.0 ml. lO Jm L 50% eoaeentrate in pe 0AS-0J mL troleum diatillate Lethane-60, 3 parte Lthaaa-38t, 1 part L0 ml. No local Ida affacte L ittle evidence of pononiag until lethal dcae 1reached Ifodarate Ida lrrita- Little evidence of p-- 'm tioa due to petro until lethal data ia ap leum diatillate proached No significant local ir- Extremely toxic; no am rita tio a ine signs of poisoning until lethal doaa a ap proached. For em plom e eee Table I Slightly irritating due Aa for Lethane-60 sad to patrslaum die- Lethana4M tillate DDT DDT TDE TDE Methoxyehlor TBB Chlorinated hydrocarbons Hicrotiled powder }*0% wettabla powder D ry technical grade 30% technical grade in >3830 J im a th y i p h th a la ta 30% technical grade in dim ethyl phthalato 1300 D ry technical grade >00 30% technical grade in dim ethyl phthalato 30% ia dimethyl phthalato >3830 >1880 Apparently not absorbed, No effect hence noevidence of toxio- ity No local akin irritation Slight trem ors, anorexia, and emaciation. Complete recovery in 8 to S days Kderst akin irrita Hyperexcitability and eco- tion vulaiona prior to death Fatal outcome delayed 7 days poet exposure Moderate Ida irrita Symptoms of poisoning but tion no deaths; raoofscy in 8 to 6 days Blight akin irritation No vysBptoma of task ity Koderate skin irrita HypenxcitebUlty Indicat tion especially of ing soma absorption abraded area COOSGli DOW 882092 ASSOCIATION OP FOOD AND DRUG OFFICIALS 5 n S T IO B t t i V O B M D lA H O S r TABLE II--Continued AmOXXM ATS LDmK O ./ K O . o iu c A ix m c n tm m c tm cn Chlorinated hydrocarbons--Continued U sdftM Toxaphene Chlordaae Aldrin D ialdria Heptachlor Prolan B u las D ry form 2% ta dimethyl 1% in vanishing eraam baas D ry (n q rJ form 20% ia dimethyl phthalata 20% ia dimethyl P^thaiaU 4% ia dimethyl p h th a le te 4% ia dimethyl phthalete D ry powder 20% ia phthalete U ndiluted Stada U ndiluted grade dimethyl teoh&ical teehnscnl >4000 >ut 0 >4000 <710 <no <iso <150 2000 <7*0 >4000 >4000 Moderate sida irrita- Seven rymptoma, oobtuI- tioa none, ro o m y ia 6 to 1 days No aigntdeant rita tio n No significant local local ir ir- I ; 1 H yperexdtability pro- peering into eonenWoaa end death rite tio a Moderate aida irrita- H yperexdtability, b a t d o tioa deathe; reoorery in 3 to 3 day Moderate liria irrita- Tremora Iaadiac to oon- tioa Tuleioai aad death Seram Ida irritation Tremora, eoaeolaioai aad death No akin irritation Serem tetania type of eon- uliiona aad death No aida irritation L ikaaldria No aida irritation No aida irritation Steam anorexia, hyperexcitability, eoaeulaioaa end death Ax for the dry powder No orideaca oi aida ir- No erideneo a t poae eye- rita tio a tsmio effects Ai lor prolea Ax {or prolan Organic phosphates F a ia th io a D im ethyl parathioa Pamozone OMPA EPN TEPP ASP-47 411 Undiluted technical tra d e U ndiluted technical trade Undiluted technical grade 20% ia water Undiluted technical (rada U ndiluted technical grade Undiluted technical trade U ndiluted technical rade 40-50 300-400 10 <7*0 30-40 3 1 300 No aida irritation Extrema waakaam, (u tro - ' w ith any of theee intastionsl disturbance compounde prior to death Herbicides 2,4-D 15% water aolutlon aa ammonium ealt 13% of freo add ia dimethyl phthalate 13% of act!re in- ttcd ieat in a com mercial formuletioa 1400 >1400 <300 S lith t edema of akin Ataxia aad muaeolar weaknaaa prior to death 81i|ht edema of akin Mild symptoms as sbovs but ao deaths Moderate akin irrita Secern depresin, m aeeaier tion cepecially of paraiysid and death abraded areaa C005G I2 5158 6 ASSOCIATION OF FOOD AND DECO OFFICIALS tursexDE raucvumox TABLE ll-Concluded LAmDOuKnDoC./ATI. DZUULimen rrttnezexffscxi E ^ d o th sl 1%in n t v Hlete bydr- Technical grade aid* powder Iqrb Herbicides--Continued 100 1 Severo aidn irritatioaj Afiortn* ta d hwnoturie specially oi abraded beten death am a in >4000 No local alca offacta No eyntemle affocta Activators a-Propyl 4% in dimethyl boma phthalata Piperonyl 30% in dimethyl butoxido phthalata Yaa Dyka 2M 8% in dimethyl phthalata Sulfoxida U ndiluted technical (rad* Fiptroayl cy- Se tabla m elohex- aaona >37* >1880 >470 > (.0 ml. No akin irritation Ko yvtemio eflecte aotod a t thia doaaca No akin irritation Hyperexcitability and oon- nlaioaa but ao dootho SUsht irritation of aidn' S ich t anorexia and nicht kn Moderate akin irrita- No eyatemio effeeta taon TABLE III Dermal toxicity: Multiple (repeated daily) expoture in rabbit* yssnass Am oznun LDm, MOe/KO. HUltV Plant products and derivatives Rotenone Pyrethrins Allethrin Nicotine Dihydrdrotenone 100-200 200-400 40 100-200 No deaths at 100 mg. during a 21-day daily ex posure period. No survivors after 12 to 14 doses a t 200 mg. No deaths at 200 mg. during a 21-day daily ex- poaure period. No survivors after 8 to 14 doses a t 400 mg. No injury up to 1 gm. per square foot of cloth during a 2 1-day wear test Based on a 2 1-dav test. In a 21-day wear test no survivors after one exposure at 2 gfh. per square foot of cloth; no deaths at the 1 gm. per square foot level No deaths a t 100 mg. during a 21-day daily ex posure period. No survivors after 10 doses at 200 mg. 0 q 0 5 G I3 5159 ASSOCIATION OF FOOD AND DRUG OFFICIALS TABLE III--Continued 7 o nSTtCZBX LADVPitS,OMXOO.U/KTOS. levisr* < Thiocyanates oo 00 Thanite < 1.0 ml. Lethane-60 < 1.0 ml. Lethane-3S4 Lethane-3S4 Spe cial 0 .1 ml. 0.5 ml. No survivors after 4 to 5 doses a t this level; effects of lower doses not determined No survivors after 5 doses at this level; effects of lower doses not determined (Approximately half of the animals died after 8 | doses during a 2 1-day daily exposure period or o CO Chlorinated hydrocarbons DDT 150 TDE 200-400 Methoxychlor TBH Lindane 600 < 200 20-50 Toxapbene Chlordane 40 20-40 Aldrin Dieldrin Heptachlor Prolan <5 <5 <20 400-800 Bulan Parathion - ,L__________ <200 5-15 Based on a 90-day exposure period. Intercurrent infections resulting from lowered resistance brought about by the chronic posioning effects of DDT appeared to be contributing cause of death. No survivors a t 300 mg. after 10 to 14 doses Severe symptoms but no deaths a t 200 mg. during a 90-day exposure period. No survivors after 6 doses at 400 mg. Based on a 90-day exposure period. At 1200 mg. all animals died after 7 to 19 doses No survivors after 5 to 8 doses. Effects of lower doses not determined Severe symptoms but no deaths a t 20 mg. during a 21-day exposure period. No survivors after 2 to 9 doses a t 50 mg. Based on a 90-day exposure period At 20 mg. one animal died after 51 doses during a 90-day exposure period. No survivors a t 40 mg. after 10 to 26 doses No survivors after 4 to 10 doses. Effects of lower doses not determined No survivors after 14 doses. Effect of lower doses not determined No survivors after 14 doses. Effect of lower doses not determined Symptoms but no fatalities at 400 mg. during a 21-day exposure period. No survivors a t 800 mg. after 6 doses No survivors after 3 to 3 doses. Effects of lower doses not determined , Severe symptoms at 5 mg. during a 21-day exposure period. No survivors at 15 mg. after 3 to 5 doses 8 ASSOCIATION OF FOOD AND DRUG OFFICIALS kstxod* TABLE I II--Continued Amozoun ID, HO/K& ipfun Organic phosphates Dimethyl parathion Paraoxone EPN TEPP ASP-47 E-838 25-100 <1 <5 1-2 10-40 Symptoms of poisoning but no deaths at 25 mg. during a 21-day exposure period. No survivors after 2 No survivors after 14 to 15 doses. Effects of lower doses not determined D eath of one animal in the group exposed a t the rate of 1 gm. per square foot of cloth towards the end of a 2 1-day wear test No survivors after 9 to 13 doses. Effects of lower doses not determined Severe symptoms but no deaths a t 1 mg. during a 21-day exposure period. No survivors after 3 doses a t 2 mg. Severe poisoning but no deaths after 40 doses at 10 mg. during a 90-day exposure period. No survivors after 11 to 15 doses a t 40 mg. Herbicides 2,4-D Endothal Maleic hydroxide No toxicity noted after 1 m l./kg. of a commercial product containing 15% of the active ingredient applied daily for 10 doses . :i >40 No significant systemic effects a t this dose during a 21-day exposure period. Higher doses were not tried i- >40 No effects at this dose during a 21-day exposure period. Higher doses were not tried Activators n-Propyl some Piperonyl butoxide Von Dyke 264 Piperonyl cyclo hexanone Death of one animal a t this dose during a 90-day exposure period. Higher doses were not tried Based on a 90-day test Symptoms but no deaths a t this dose during a 90day exposure period Death of one animal a t 100 mg. after 45 doses during a 90-day exposure period. No survivors^ after 4-10 doses at 200 mg. to be slight. Single massive exposures to insecticides which ore liquid at ordinary temperatures, or solutions of solid materials in kerosene or other petroleum distillates, must be considered as dangerous. C005G15 5161 882096 ASSOCIATION OF FOOD AND DBTJG OFFICIALS 9G Repeated daily skin exposure to insecticides, especially in solution, con stitutes a real hazard. As a rule many of the materials in concentrated formulations are skin irritants which serves as a warning of dermal contact. However, in dilute solutions this warning sign is lost and because of the delayed effects of a number of insecticides, serious poisoning m ay occur during the handling and application before the danger of poisoning is realized. t COOSGiG 16 5163 \8 *v - 'vi' 12 5084 .-.v .*':v' & ^ i Mowhsaloal Reseeroh Deportami ',*>#53 THE DOW OBKZCU. COMPAIT 1. -%** o b jM t resulte o r s m zn tm rzoR resta o t CORREHT PRODOCTIOH 2.WDICHLOROPRBROmCSTZC acid a COMPASSO WItH PAST PRODOCTIOH MATERIAL. m m :g m sjwsE Pln'd ;VJWork Bfr To Check Central K t i u n h Index, Bxeevtlve Reeearch Conlttee Texas Pile A Western Central Reseereh Pile H. 1. Borie Brlttaa's Divisimi Attni R. C. Dosser C. A. Hlghblll Medleal Depertaent Attni H. H. Gay Safety Depertaent Atta* MaeCnteheon V - s s - i i. pt. * < i r -- ._! * a* r / - f-f .V.|.-Tr^ m m rer o,ep|cr? A P S i -j i s s l ^ - v * A : ' 3 -- * IhLrh L.G.f.APRn i Aarlonlturai Chealeals Divisimi, 1^*7 Bid. Atta I. V. Brlttoa WflM The skla Irritatine propertles f enrreat prosastica:;.' 2,V-D old ere stallar to those of 2,WD eold preparo! hy tha\ aev aethod latroduoed in 1950. 2 vV-Dlohloropheaax7aoetlo aold Fheaoxyeeetlo soldi 2(Wlehloro, Aoetle soldi 2,Wlshloropheaoxr*t Aeetlo soldi phsnaxy-, 2 #'*-dlehloro-, 0( TMIS KEPOST THKPftOPCKtY. . 'O f . ' v ' v ^ TNI MW CRtM.tU CdHPAHT . -rii 515 4 / psUV *.. r- rt >V a ' J gwg * T23.1V - U . * 1 " ' * 2 , ^ 'm m - '" r.v sS c * / rf}`** A ftv i u i of dermatitis baro jp-.'-iJ: . ooanootlon w ith tho a a a u fa e tu re o f 2 ,e - 0 o eld by 9 Bov I Company, A sa a p le o f c u rra n t p ro d u ctio n a a t s r l a l . - fo r sk in ir r i t a t i o n to o ts fo r comparison w ith p a st production storiai, SfflgMBMS' ' '> ? 1 , Tbs skin i r r i t a t i n e p ro p o rtlas o f orront 2.W-D s o ld aro s i a i l a r to th o se o f 2 V<M> aoiA p roparod b j r t h a aothod introdooed in 1 9 5 0 , -V 2 , Oaap 2,14-d io h le ro p h e n o sy u o e tio a o id i s m o d e ra te ly ` i r r i t a t i n e and stronc so lu tio n s o f tho aold aro s li h tly irritntind t o th e skin* on p roloncod and .re p e a te d s k in o o o ta o t, - v S?**** HI 3 , D e rm a titis w i l l p ro b a b ly r e s u l t i n some o i f t h a , su b s e t a a to r i a l , p a r t i c u l a r l y i f w e t, i s allow ed t o e o n t a o t t h o ,1R sk in repeatedly o r fo r proloncod p erio d s, MATERIAL ._>**;t s i ^ oe V . ' f / Haaet 2,W"Dlohlorophanoxyaeetle aoid Formult Cl H 0 S tructural! Cl o 0 - .C - C - OE S a p i r l o a l i CqH^Oj CIj -: i. . 2 . . t '^ e ' " rf-H* '- B,R,0 . H i e Ho.! T23.1V-U-9 B ,R ,0, X Ho,I 2372-21 Bour s e ! P la n t fr o d u o tlo a Saapio obtained by! B, R, Hoyle ;'-VVi v 0001-7 ' 4 r%** jh. Jpf SS$,':7- T 33.1V -11-9 pm* 3 r. +* rasramra uma When d ry 2 , h- 0 mold w i bandaged re p e a te d ly o n to then 90?.^":* V/-*<,*'"' shaven abdaeon o f a rab b it, fir* days par w tk , far a t o t a l Cf;v 30 a p p lic a tio n s l a 1W day*, v a ry a l i g h t s k in i r r i t a t i o n o f guest ~ * % # 4<^ % j0'$i-;r ' a b l* s ig n ifie s * * r e s u l t e d . kben daap 2.W-0 a a ld aa a ta a ta d l a sa a a n a a a a r , a o d o ra t* Ida I r r i t a t i o n o b a ra e ta rls a d by hypo r aa l a p i * & ' ;4S: aa d a n p a r f l e l a l n a o ro a la r e s u l t e d . ' fcSCVs*'- Repeated a p p lle a tlo n a o f a lOjf aaapanaloa o f th e a a to ria l;< l a to r to th a oar and shaven abdoaan o f a ra b b it prodoaod a lig h t* ; w . a k in - I r r i t a t i o n . Repeated a p p lle a tlo n a ' o f a l.O jf aaaponaloai o f t>r 2 ,W > a e ld l a a a to r t o th a a a r and shavan abdi a o f a r a b b it i-v:^j'*C'AV prodooad vary a lig h t akin I r r ita tio n on tha abdoaan. r* Caaaa o f d a r a a tltla have boon obaerred la eeonaetloa w ith' th o aaan fa ctu r* o f 2 tV ^ieh lo ro p h eao x y ae* tle a a ld l a th e p a s t oa a s t a t e r o f oooaalona. Tha p ra aan t p ro b laa than l a r e a l i t y l a an old problaa. *> - - A review o f tho eonelaaloaa roaehad la toa& eologloal t a ta d la a oa 2.V-D a e ld re p o rte d by V, K. Rove oa 1O -12-J0 (B lo o h aale al Raaaarch Department P lla R oa, T23.lW-U3 and 'j/iA'ifcj '`i t M t2 3 lh * U ^ ) fo llo v a i 1 . " I t would saaa fro a th ese r o s a lta t h a t ' la p u ro 2,U4> l a nor* l i k e l y to produce a s l f n l f l o a a t aa o u a t o f a k l a ` I r r ita tio n than la pur* a a te r la li aad a la o , I t aaaaa th a t a daap p ro d u c t l a nor* l i k e l y to produce I r r i t a t i o n th a n l a a d ry i i i .Sag 2 . "Although n e i t h e r th o a a t e r l a l p re p are d by th o o ld o r th o now e th o d la a s k in s o n a l t l s e r , th e d if f e r e n c e In a b i l i t y to praduoo.1p rla a ry skin i r r i t a t i o n la su b s ta n tia l and tho lik elih o o d Of ik la 5* 1 !') i \iJ? !?> oooi-7.$#a ,,(V.-**U- X- '**. a.-9^;-JoS CT' t*- % f f t .. l.  - ' V'tV-'"- 5 1 >. Z*J ,r<THE DOW CHEMICAL COMPAQ? * WV ' ^ sSribleet ?*p333V RESULTS 0? BARGE FIBBING TOXICOLOGICAL Fil*. T2J.1W-H-7 TESTS ON ^f-DICHLOROPHEHOXZACBTIC ACID Chg. 5862 PREPARED BY A NEW METHOD. Ree'd 3-30-50 Fin'd lf-3-52 Work By R.L. Holling: : "worth V? r</A*V%**** To Check Central Research Index Executive Research Cosmlttee >:#^^pw^3Mler:A :ife^#+jitoaterniCentral Research File y-//-0 P*. B tttcnTs-'iDivision Agricultural-Chemicals Division Attn: J. W. Britton :\ ` "i -i- ' ' ,.. V i. Field Agricultural Chemicals Resss *eaairchh,. 11--11*.77 BT TlMd*g1. ^ :' ..-v'^'$AAttttnn* W. C. Dutton . .; , , :;--2i'r_r.V '.';,V * * - v -: .* r.' ^Agricultural Chemicals Laboratory,:Seal'Beach -/^'^Attni ' J. F. Kagy ''V-V . V v'.' 'Vv> Agricultural Chemicals Research Laboratory Attn* G. E. Lynn - J. S. Johnson Medical Department Attn: JJ. H. Gay Safety Department Attn: S. M. MacCutcheon UNIT INDEX Results of range finding toxicological tests on 2,V-dlchlorophenoxyacetlc acid prepared by a-new method are , presented. Health hazards and precautions for safe handling are given* TOS REPORT IS THE PROPERS OF THE DOW CHEMICAL COMPLY 5169 C0017C4 5n?tf*'c4 j *. *;<*' B3^w i h * * ;, - - * : :.** * i & . '-'i' *"l**J*'4dBMfc - v - is*. A cetic a c id (2,V-<1tohTnrophouoxi)-, 1"5.4 * .-- 11 . IT. -- .s,, * H & . . 'fii A aew n e tte d fo r p rep arin g 2, t ^ !l o h ioru phanayi< /-f.:;J old m s ln tro d eed In 1950 which g ars a w h ite r and o d o rlaaa p ro d o e t. What a re th e ta x ic o lo g le a l s u b je c t n o ta r ia l p rep ared by th e new aethod o f hew do th ey eoapare v ith th o se o f th e o ld e r p re d n o tt. -^ _ soRarera? :7 1. 2,U -D ichlorophenoxyaeetle s o ld la *.* * -tJ aoderate in < # is *s o ral to z lo ity . v-1y f '& iift'* 2 . The fre e a c id and I t s stro n g s o lu tio n s a r e 'i ^ e `m I r r i ta t in g to th e e y e s. Weak so ln tlo n s a re s lig h tly i r r l t a t to the ey es. v . :,*%*** 3 . The d ry a a t s r l a l does n o t pr odnoe s ig n if ic a n t a k ln j I r r ita tio n on repeated a p p lic a tio n to ra b b it a k in , b a t a t i so ln tlo n s prodnoe s lig h t sk in I r r ita tio n . "';y U . 2 (W D iohlorophaao^raoetlo so ld I s n o t abosedi *th e sk in In any appreciable q u a n tity . V i1?'* ** : ,l * * - V v 5. 2.Woiehlorephenosyaoetls said prepared by the aethod la slgnlfleantly loss irritating to rabbit Ida than *i oldtr produet. Tests oarrlsd oat oa hnoans a 14 U Howe oa 10-12.J0 (lloohcaleal losearah o. T23.lW.U-d) hare aeafimed tho oanelaslana laadhol tren* stadias oa rabbits. Together these tests bare shtea that did . . * !i. . V . 5170 00017C5 ^UA^/.L-H II H M i><: .'.>i'A-i\i tfS** t-L:?-> *>1 % M U>C |hT .iMt O rj f u " $r-M j R *'? li * 1 I * a ,, i * |l ,\H *4) i *Vs Kl&V ;t <$ S-:.: y F Bf i ' r U V : ! " *( *A`^; W i! A ' !' ( U . 4 - i i - w r t*#\ ij M6 I RvF;;:*pli| ` C ! ti i* 'i ":i. sI !> i i fr it' B: 7j>ri W rn .,:' :-.r- Eft r-': an ftrtfrttfli - - ?v^:#-Mggr 2 ,W leh la ro p h a n o x y a a a tla M id v t 2Atirfn Xb ` V v o f th a M id l a propylana c ly e o l m i p laaad 1b th a aya a f o - - m r t j I r r ita tio n o h araatarlsad by aarkad p a la v . . . and a o rn a a l d a a tf* ra a n lta d . Khan a 10J( a o lstla B o f t h a a a t ` ln propylana (ly e o l w u ta a ta d ln a a la lla r aaan art a l l f h t ^ f-i. jfigi tra a a la n t aya Ir rita tio n raso ltad lan ad lata aaaM ng o f f aya v ith v a ta r radnead th a aaoont o f aya w g y iw - w tln Irrita tio n Khan th a d ry 2 ,V-d m IA u baadacad ra p M ta rtlj sharan abdonan o f a ra b b lt, f lr a daya p ar vaak, fb r a to ta l a p p lle a tlo n a ln 1U d ay a , no a lc & lfla a n t a k la I r r i t a t i o n I --M B apaatad a p p lle a tlo n a o f a 10 a o ln tlo n o f th a a n to r la l l n e a r b lto l a e a ta ta to th a a a r and aharen abdi i o f a r b l t prodnead alicht akln Irritation oharMtarlsad by allcht aeallnaaa and h a lr lo aa. SttU&i&nslaa Zn th a ak ln I r r i t a t i o n ta a ta eontfaatad, th a ra n a ' a a Vt! arld a n a a th a t 2 ,V -d leh lo rep h a n o iy aaa tle M id n a al th a akln ln any app reelab la d o a s tlty . * "tmTTM 2 ,V -0 leh lo ro p h aao x y aaatla M id and I t a s t are aararaly lr r ita tla g to tha aya. l'-5 \ Tha d ry fr a a M id a p p a ra n tly la n o t tr r lf e a tla c to,. oiM.' ln tM t aklnt b m r a r , prarlooa m k an th la 10-22-50 by T . K. Bona (S la a h a n la a l Raaaarah I o , T23. 1W U -3) h a t ahowi th a danp prodaa ta t a a l l * U y t a th a ak ln an p ro lan cad and ta p a a ta d a a n ta a t, OO 'S* -ff.: 5172 0001797 8 8 3 S f o V ;/ ' _^>V 1' T 23.1V -1 1 -7 h| J 7 th is n o ta r ia l produc e s lig h t sk in i r r i t a t i o n on prolonged J/3?5f ta d re p eated ak in e o a ta a t. A ccordingly, th a ra id good re a so n t a 1 " Z?.;r. H av a th a t prolonged and ra p a a ta d sk in e e n ta e t fay hwnans w ith t t r ^ jpX vr^ daap aald o r I ts strong so lu tio n s e ig h t re s u lt la th e ^ & o f da* i t l t l s . li: >9t*nu 2,V-01ohlorophenexyaeetle so ld I s a o d a ra te ly to rlo sw allow ed. Although i t would ba p o ssib ls fo r a p arso n to ^ * # 3 '* ,; a s u f f l ia n t anouat o f th a a a ta r la l to ba dangerous to U f a , th a ra ; '^ *i I s s lig h t h asard from in g e stio n In o rd in ary In d u s tria l o p e ra tio n s, .b**SVs - PRECAUTIONS TOR SATE HANDLING 't-fi Zt Is stro n g ly recesneaded th a t ays p ro taatlo n ba re -- . qulred ahanarar tha fra a acid o r I ts strong so lu tio n s ara handled, -Pace s h ie ld s , gogglss w ith sld a s h ie ld s , o r -the e q u iv a le n t 111 v ig il .- a ffo rd s u ita b le p ro te c tio n . S u ita b le f a c i l i t i e s fo r w ashing th a 4r ayes w ith w ater should ba re a d ily a v a ila b le . . . ,, I fre c a u tlo n s should ba tah sn to' p rav an t prolonged o r re p eated sk in c o n ta c t w ith 2.^ -d lch lo ro p h sn o x y aeatlc a c id , : it tV- e sp e c ia lly w ith the dasp acid o r I ts strong so lu tio n s, such as n ig h t oacur fro a w earing co n taaln atad clo th in g o r f t f a ilu r e to wash th a sk in soon a f te r c o n ta c t w ith th a n o ta r ia l, ^ ^ 5' lib b e r gloves nay ba su ita b le fo r preventing co n tac t cn th a b ah ts rista . * - 1i IgAT TO DO POP E2P03DHM itj I f 2,b-41shlorophenosyseetle aa ld e e a ta e ts th e e y a s, .fd th ey should ba sahad tharauahir w ith flow ing w ater fo r at least fifte e n a la n te s , and soon as u o salb la. In th a ev en t o f c o n ta c t upon th e p e rso n , earn -a \ c i ...`- v 5173 0001733 ? W $ ? % W & k S.~ : jS/', ` 5 3 a J * - M * '- * o r ahoaa should to w i w d ad t t e s k ia thoroughly ib M ap al t o r . U r t i l i ! i t t t n t t l . lt atela i r r i t a t i o n la ra n t o r l f er p a ia p e r s is ta . C oats * a lo th in g should t e utshed thoroughly b efore ro -u sa , Xf 2,b^leh lo ro p b en caq raeetlo old should t e e * l l * d / .' . .* '."i'*.--^S I tin e sh ould t e Indnood p ro a p tly by tin k lin g S to th ro a t vittoJKT r *.")>. tp'~ / fin g e r o r by c a r ta i 111 as soap so lu tio n o r a a lt su t (2 ta b le sp o o n fo ls o f ta b la s a l t l a a g la ss ot un tar) i l e a l a tta n tlo n th an should te o b tain ed , a t o aaa. V-V ' > *.*vr | v , ? A ,- JL' -' -V* - - .* 'S-.i W'V, 0001739 <2o % I V I t c f ' r i n t n l ( m m A s h n h r M r t o r Fm. h **: !> i:v*u O i i v i a i .k c.7 m i : I ' h i t m .St a t i c V*.|. ; : v j # No. V.'Si CHJ&HCAIJI I^iOTO: A HKl'OHT TO TIJK AiiriOCIATIOX OF FOOD AX3) DltKO OFFICIALS OX CURRENT DiiVKLOI'i.IiOX'i'3 PART II. PESTICIDES S E C T IO N III. SIJI*A C U T E A N D C H R O N IC T O X IC IT Y ARNOLD J. LEHMAN, M.D. C h ie f, D iv isio n o f Pharm acology, U . S . Food and D rug A dm inistration Sections I and II in this series of articles on pesticides dealt with acute and d cn n al toxicity, am i appeared in th e O ctober 1951 and January 1052 issues of th e lh illetin , resp ectively. T h e present article summarizes- th e subacute and chronic toxicity of n num ber of im portant pesticides. Recausc of the tim e and effort involved in long-term studies, th e list is not a s ex tensive as th at found in th e first tw o articles. Table IV summ arizes the subacute and chronic toxicity in rats. I t will be understood from the word in g of the table headings that betw een the low est level w ith groas effects and the highest level w ithout gross effects there were no interm ediate - dosage levels. Som e special effects of the rodenticidcs in rats are presented in T able V , and T ables V I and V II sum m arize the highlights of chronic studies of certain h eavy m etals and chlorinated hydrocarbon insecticides in dogs, flum e c f th ese stud ies are still in progress and therefore certain conclusions in th e present paper m ay bo altered in the light of later d ata, and certain m issing figures v/iil even tu ally be supplied. Id en tity o f th e com pounds w hich appear in Section III will be found in th e first article of tills series.1 S U B A C U T E AND C U ItO X IC T O X IC IT Y IN RATS T h is is sum m arized in T able IV . A ny study of less tiian about a year m ust b e classed as cubacute. A point o f interest in the tabulation is that the re sponse of anim als to chronic ingestion of a pesticide does not follow a set pattern. For cxam nlsj in th e feeding of rotenono there is a good parallelism between levels fed and degree of effect. T h e levels of feeding descend from a value w hich produces effects such a s loss of appetite, retardation of grow th and in som e instances even sym ptom s of poisoning, to a lower value w here no effects are observed, then to an even lo ver value a t which on ly m icroscopic changes in tissues are seen, and finally to a value which pro duces no effects. T here are som e exceptions to the rule that the dietary level, a t w hich effects as enum erated above are produced, is alw ays higher than th e level a t w hich pathologic tissu e changes nia!:o their appearance. 1Ounrterly Hull. Anjn. of F A D 0 :Tk:i::!j of U.f?., XV, No. 4, p. 12J. 47 vj Vj4 ro 00 o f c c c K DOW CC04S5X 5176 TA1JLK IV Subacuto anil Chronic Toxicity in the Rat DIETARYCONC.ZHTIATIOH 10 n l t l T i t UIIUOII PESTICIDI Timeo( Fe^lint In Vietiti Lowest Uvei V/lth Gross Kflccts Highest Uvei Without Ctrr.3 Etlccu Lorrrit Uvei With Tiesuc Dusse* Highest Uvei Without Tissue Dsouz* Natural Products und Derivatives H otenono.................................... P y r e t h r i n s .................................. A llcthrin..................................... R yania......................................... D ih y d r o r o t e n o n o ...................... 104 104 10 10 10 00 0000 10,000 15,000 100 25 .1000 eooo 10,000 25 5 COCO 2 1000 15,000 TLo Metals Copper (as chlorido).............. Mercuric (as a cc ta to )............. Mercuric (as phenyl mor- curie a ce ta te )........................ Selenium (from grain)............ Selenium (as sclcnido)............ Cadmium (as chloride).......... Antimony (os trioxide).......... Barium (as ehlorido).............. 101 101 101 104 101 104 101 101 2500 40 40 3* 10* 45 1000 10,000 1000 10 500 250 10 2.5 10 15* 600* 2COO 0.5 3* 10* 15* 500* 10,000 0 .1 2000 Chlorinated Hydrocarbons D D T ............................................ T D E ............................................. D F D D T ...................................... M cthoxychior............................ T b l l ............................................. A BU ............................................. BB I............................................. D B II............................................ L in d a n o ....................................... T o x a p h e n e .................................. C b l o r d a n c .................................. A ld r in .......................................... D icldrin...................................... Prolan.......................................... B u la n ........................................... 104 101 23 101 101 101 101 101 104 101 104 10 10 40 40 100 400 1000 200 100 100 10* SCO* 100 400 75 50 50 2C0* 1000 50 100* 200* 100 50 50 50 100 25 25* 25* 200* 5 ICO* 200* 500 50 0 10* SOO* ICO 100 2.5* 25* 25* 200* 200* 1 2C0 10 10 50 25 Organic Phosphates Parathi^a.................................... ISPN............................................. A S P -4 7 ......................................... H E T P .......................................... Tricthyi phosphate................. D iethyl phosphate.................. 101 52 52 12 31 20 25 10 100 130 1 ) GO 1G0 GO 180 1000 10,000 5000* GW0 5000 43 CGO 75S576 O * QSGmmoO ASSOCIATION 'JO f FOOD AND J3RUQ OFFICIALS .40 nsncoE TABLE IV--Continued OICTASYCONCXU1ATIUII CCMS 1 IUi.*.1.1 Tot of Fecrtin* in YVc:ls Lswnt Level Will, (jioij Jscli llfchttt Ixvr.i Viilbnt (mi Eflccts Lcrcit Level W.:l, ri5,v* Desuse lliclcu Level V.'i.bo-Jl Tit:i Decer: Dinitro Compounds DN-111.................................... 35 1000 2C0 1000 2 ,4 -D ....................................... 2^4,5 -T ........................................ Mcthyi-1-naphthalene acetic a c i d ...................................... Endothal............. .................. Maleic hydroxide................... Herbicides SO 5000 30 cooo 101 20,000 17 5C0* 17 50,000 I0G0 1CC0 / 1000* 1000* 2500 10,000 20,000 2500 000 2500 50,000 10,000 Activators n-Propyl-isom o.......................... Pipcrouyl butoxide.................. V an D yke 204........................... 17 17 17 6000* 5GC0* COCO* 000* 5C00* 5000* Rodenticides 10S0............................................... 104 25 10 5* A N T U .......................................... 104 100 0 50* W arfarin...................................... 1 to 2 6.25 (death)* Zinc Phosphide......................... 5 100* Miscellaneous Ar a s a n ......................................... Aram itc....................................... P h ygon ........................................ 341 C ............................................ 65 32 52 26 1000 5000 2500 10.0GC 300 1COO 1000 5CG0* 1000 200* 5000* 200 * Lowest level fed. Para'Jiion is an example of this. I t m ay be noted th at no histopathological changes could be dem onstrated in animals fed a diet containing 100 ppm. parathion for 2 years. This concentration is 4 times th a t which produced sym ptom s of poisoning, and 10 times the level a t which no such effects were noted. Other compounds showing similar responses are rr.ethoxychlor and Endothal. Another point of interest is th at the LD-0 of a pesticide is of very little if an y significance for predicting it3 chronic effects. B B II may be cited as a n example. In thi3 instance the LDW of B B II 3 approximately GOOO 0G04G53 5178 5 0 ASSOCIATION OF FOOD AND DRUG OiT7CIAL,l mg/kg, a relatively low order of acute toxicity. However, the chronic in gestion of 13BH leads to gross and microscopic evidence of intoxication a t the low level of 10 ppm. in tho diet, a Irish order of chronic toxicity. A third point is th at tho behavior and general appearance of an animal chronically ingesting a pesticide is a crude endpoint us a measure* of chronic toxicity. This is dem onstrated w ith D D T. A dietary concentration of ICO ppm. is ncccssaiy before evidence of poisoning is' observed, y e t microscopic evidence of liver damage is seen a t 5 ppm. The importance of hisiopathological examination of tissue of cnimals on long-term feeding studies cannot be overemphasized. TOXICITY O F RODENTICIDE3 IN BAIT FORM Table V presents a summ ary of the influence of food on the effectiveness of rodcnticides. These pesticides are usually employed in the form of baited cowman* TABLE V Toxicity of Rodcnticidta LSI, Cf UO./KC. A* tbe C hcninl Is Bait FciTB 10S0 Castrix ANTU Strychnine Thallium sulfate Zin. phosphide Rea squill 1.7 1.7 0 10.2 23 450 3C0 5 10 30 25 (females) 15S (males) 25 450 100-500 (females) 6CQ-SCC0 (males) food, hence, for tho purpose of this experiment various dosage levels of each rodcnticide were incorporated into 4 grams of laboratory diet and these 4-gram portions were offered to starved rats. Animals not promptly con suming tho entire am ount were rejected. Tho eitect of the bait on the oral L D yissum m arised below. A comparison of the values shown th a t the bait reduced tho rodenticidnl effect ot ail compounds except thallium sulfate and zinc phosphide. .War farin was not tested in this manner because its effect is dependent on a con tinuous and prolonged insuit to the coagulating mechanism of the blood and therefore not adaptable for study under tae above conditions. S U B A C U T E T O X IC IT Y O F SO M E H E A V Y M ETA LS IN DOGS The compounds listed in Table VI were fed to the animals 7 days a week, the prescribed dosage being mixed with a :<mall portion of the diet before | | 0004854 ;.................... 5179 i ASSOCIATION* OF FOOD AND DP.UQ OFFICIAL'S SI being administered. The daily dosage was calculated on the basis of the m etal. For the dog on a dry diet 1 mg/kg/d&y represents about 40 parts per million in the total diet. If each of the vulucs in the above tabic is multiplied by 40 an approximate figure will be obtained representing the dietary concentration in parts per million. It should be pointed out that this tablo also indicates dilTcrcnccs in toxic action of substances if given in small quantities of food (dogs) tu.d in the bulk diet (rat3). In addition true individual difTcrcnces may play a part. By making use of some of the data in Tabic IV it is possible to compare the relative susceptibility of dogs and TABLE VI Tolerases of Doga to Some Heavy Metals Administered Orally VITAL DAILYDOSS DI1AT1AX Of ASUlXfvrEATii sm en Arsenic (os trixido) Lend (as acetate) Cadmium (as chloride) Barium (as chloride) Selenium (as sclcnatc) I/**. 3 .IS 0.33 10.0 20.0 2.5 0.5 50 weeks 33 weeks 5t: weeks 5J weeks 4 weeks 0 weeks No gross evidenco of injury Fatal to the animals No gross evidenco of injury No gross evidenco of injury Fatal to the animals No gross evidence of injury rats to subacute poisoning by heavy metals. This may be summarized as follows: KRAI A ftw n e ........................................................................ Tinfl .......................................................................... r .i t i m i i n n .................................................................. H a n u m ........................................................................ S /!nniiim .................................................................... M TS K l KXU2Q.HCf I 3 t DIET Dei Rat 127 less than 13 4C0 soo 20 217 4CC0 45 2030 between 3 and 1U This summvy C P:phasizc3 the f a c t t h a t in nppraising the hazards of pes ticides toxicological studies should include more than one species of animals. CHRONIC TOXICITY OF CHLOHINATET* INSECTICIDES IN DOGS In these experiments the animals were fed the insecticide daily 5 days a week. The insecticide was dissolved in com oil and administered in capsule form, the one exception being an experiment with DDT in which the dry capsuled material was given. The results are summarized in Table VII. u o * -a cc c c GG0435a 5130 DOW?5S58Q 52 ASSOCIATION OP POOD AND DDUQ OFFICIAIS TABLE VII Survival Time o f Dogs Fed Various Daily Dose* of Chlorinated insecticides in Corn Oil . XNSZCTiaOK OACTMSt mnr tta or AtflUAIS mu CCCCASTOUTa SAC1CXAJTSQST LAST oca; b TATSor foavjvwa ANUlAlS DDT Bf/i*. 0* 4 0 Rt) 10 10 0 3 1 isjt 22 ISO TDE 10 0 50 Methoxyebior 200 20 55 0 4 1 600 4 om 105 TBH Lindane Toxaphene Chlordane Aldrin Dieldrin Heptachlor 0 3 15 7 10 0 25 3 10 2 54 0 2 40 2 20 2 64 62 22 1 2 0.5 3 3 7 G 3 1' 35 2 14 1 33 0 2 32 2 25 2 SO 4 39 2 fil 2 24 2 109 0 10 ** 2 10 5 2 o# 23 2 2o 22 1 22 S3 0 .5 1 OA# 14 5 . 2 nA* 13 1 4 3 205 isyt 1 Sacrificed a t 11CO days for autopsy S00 Survivors sacrificed at 11C5 and 1400 daya for autopsy Sacrificed a t 1400 days for autopsy 020 One animal sacrificed a t 050 and two us 1410 days for autopsy 2S Survivors sacrificed a t 1200 and 1550 days for autopsy 48 3 221 Smvivor sacrificed a t 1250 days for autopsy Sacrificed a t 1350 days for autopsy 37 20 03 Cl50 22 173 344 One animal still living a t 277 days and two a t ISO days 17 55 33 SOO 201 Two animals still living a t 275 and ICO days, respectively 21 424 One animal still living a t 455 days Administered in the ary form. 0G04836 5181 ASSOCIATION Off FOOD AND DflUG OFFICIALS 53 If tics h e a c h cf tho relationship tliat 1 nig/fcg/day fer tbs deg on a dry diet represents approximately 0 parts per million in the total diet, the dosages given in Table VII can be expressed in t e n s of parts per million end tho susceptibility of dogs end rats to some of tho chlorinated insecti cides can bo compared on a similar basis. One useful criterion in evaluating the chronic effects cf a substance is the mortality rate of the various levels of feeding after 0 months on the diet. In case of the dog if our choice is limited to th at dosage level at v/hich come animals survived for at l w i G months end compared with similar data for the rat, the following values for the two epscice are obtained: aaxcR cm (U SC ZfTO lLXTT 1A1ZO C f PACTS P2 U ltllO S C IT S S D S T D .-j Rt D D T .......................... - .............................................. t d e ......................................................................................................................... MethosychJor............................................ T .in / f im f l................................................................................................................ T osupheae................................................................ ................................................... Aldrin............................................................. Oisldrin.......................................................... 2000 2CC0 12,GOO 400 400 200 40 20 00 2000 2000 1G0O 1COO 4C0 200 2G0 . Thin summary demonstrates the general tendency that dogs are more cuscsptible to tho effects of poisons than rats, and again brings to mind the necessity for evaluating the pharmacological effects of a substance in more than one species of animals before attempting to assess tho hazards. COO'ii C 5182 5183 DOW881914 Reprinted fn m A iaacunoii or Food t D ra o O m c u u o r T I E United States Vol. XVI. No. 4. October 1952 PrinUd in JSJL CHEM ICALS IN FOODS: A REPO RT TO TH E ASSOCIATION OF FOOD A N D D R U G OFFICLALS ON C U R R E N T DEVELOPM ENTS PART II. PESTICIDES SECTION V. PATHOLOGY ARNOLD J. LEHMAN, M D . C hief, D ivisio n o f Pharm acology, U . S . Food and Drug A dm in istration Sections I, II, III and IV of this series of articles dealt w ith the acute, dermal, subacute and chronic toxicities, and w ith certain biochem ical as pects, of a num ber of im portant pesticides. T he reports were published in the October 1951, and January, April and July 1952, issues of the Quarterly Bulletin, respectively. T h e present report gives the principal gross and histopathological changes as observed in rats following the subacute and chronic ingestion of pesticides. T he pesticides discussed are those listed in T able IV in Section III of the series, and reference should be m ade to that table for the dosage necessary to produce som e, but not necessarily all of the changes m entioned. Id en tity of the com pounds listed in Table X V will b e foun d in th e first a rticle o f th is series1. I t should be em phasized that this tabular sum m ary sim ply cannot go into details, does not consider the acute changes w ith m assive dosages, and that much of the pathological study of pesticides done in the D ivision of Pharm acology is not represented in the table. T w o of the majpr om issions are feeding studies in dogs w ith som e of the com pounds under discussion, and the inunction of them on rabbits. W hen the lesions observed in such studies have been strikingly different than those in the rat feeding tests, they will be briefly m entioned. Generally speaking, the rat organs studied m icroscopically for each of the compounds listed in Table X V include lung, heart, liver, spleen, pancreas, stom ach, sm all intestine, colon, kidney, adrenal, testis, ovary, uterus, thyroid, parathyroid, leg m uscles, leg bones and bone marrow. T he rat has no gall bladder. U rinary bladder, prostate and lym ph nodes were sectioned less frequently than the other structures m entioned. It w ill be relatively obvious from inspection of the table that the liver is the organ m ost fre quently affected, both m icroscopically and grossly, as m ight be expected from its detoxifying functions. On the other hand, although extrahepatic lesions occur less frequently, the sum total of such lesions show s a wide variety, and is an im portant part of the overall picture of pesticide toxicity. 1Quarterly Bull., Assn, of F AO Officials of U. S., XV No. 4, p. 122, (October) 1951. CG05G03 ASSOCIATION OF FOOD AND DRCQ OFFICIALS 127 STGT88MOO BIBLIOGRAPHY Much of the data presented in the five articles of tins series has not been published. However, the background for some of the material will be found in the following refer ences which have been arranged according to subject matter. Acute Toxicity Woodard, G., N elson, A. A. and Calvert, H. 0 .: Acute and Subacute toxicity of DDT (2,2-bis(p-chlorophenyl)-l,ll l-trichloroethane) to laboratory animals. J. Pharmacol. Exptl. Therap., 82,152,1944. Woodard, G. and H agan, . C.: Toxicological Studies on the isomers and mixtures of isomers of benzene hexachloride. Federation Proc., 6, No. 1., P art II., 386,1947. Hagan, . C. and Woodard, G.: Toxicological Properties of hexaethyl tetraphoaphate. Federation Proc., 6, No. 1, P art II, 335, 1947. Haoan, . C. and Woodard, G.: Toxicity of 0,0-diethyl O-p-nitrophenyl thiophosphate (parathion). Federation Proc., 7, No. l 'P a r t 1 ,224, 1948. Dermal Toxicity D raize, J . II., N elson, A. A. and Calvert, H. 0 .: Percutaneous Absorption of DDT in Laboratory animals. J. Pharmacol. xptl. Therap., 82, 159,1944. Chronic Toxicity F itzhugh, 0 . G. and Nelson, A. A.: The chronic oral toxicity of DDT (2,2-Bis (p-chlorophenyl-l,l,l-trichloroethane). J. Pharmacol. Exptl. Therap., 89, 18, 1947. Woodward, G. and Nelson, A. A.: Effects observed in dogs following the prolonged feeding of D D T and its analogues. Federation Proc., 7, No. 1, P art 1 ,266,1948. F itzhugh, 0 . G., N elson, A. A. and F rawlxt, J. P .: The chronic toxicities of tech- nical benzene hexachloride and its isomers. J. Pharmacol. Exptl. Therap., 100, 59,1950. F itzhugh, 0 . G., N elson, A. A-, Lauo, E. P. and K unze, F. M.: Chronic oral tox icities of mercuri-phenyl and mercuric salts. Arch. I n d Hyg. and Occupa tional M ed, 2,433,1950. Biochemistry Woodard, G., Ofner, R. R. and Montgoaiert, C. M .: Accumulation of D D T in the body fat and its appearance in the milk of dogs. Science, 102, 177,1945. Woodard, G. and Ofner, R. R .: Accumulation of DDT in the fat of rata in relation to dietary level and length of feeding., Federation Proc., 2, No. 1., P art II , 215, 1946. Woodard, G., D avidow, B. and Lehman, A. J .: Metabolism of chlorinated hydro carbon insecticides. J . Ind. and Eng. Chem., 40, 711, 1948. Lauo, E. P ., N elson, A. A., F itzhugh, 0 . G. and K unze, F. M .: Liver cell alteration and DDT storage in the fat of the ra t induced by dietary levels of 1-50 ppm. DDT. J . Pharmacol. Exptl. Therap., 98, 268, 1950. K unze, F. M., L auo, E . P. and Prickett, C. S.: Storage of methoxychlor in the fat of the rat. Federation Proc., 9, No. 1., Port I., 293, 1950. K unze, F. M., Lauo, E. P. and Priceett, C. S.: Storage of methoxychlor in the fat of the rat. Proc. Soc. Exp. Biol. <kMed.,76,415,1950. ,/ GGQ5G04 ASSOCIATION OF FOOD AND DRVG OFFICIALS 5186 TABLE XV The principal grot effect and hitlopathological change* in the rat following lubacute and chronic ingettion of pesticide KSTICIDI doss irrten ISTOrATSOLOGICALCHAMOIS Natural Producta and Derivulivca Rotcnone Pyrethrina Allethrin Ryania Di hydrorotenone Growth retardation; increaaed incideuce of hepatic tumora Slight increaso in lcidnoy weight Growth retardation; tremora Growth retardation Growth retardation Peculiar. In survivors, u small number of hepatic cell masses 14 to 1 cm, between hyperplasia and tumor, at 2-10 ppin, but not a t higher levels Preliminary examination indicates th at histological chungcs will be little if any Animals on this short-term experiment not examined histo logically Negative at 16,000 ppm osExperiment in progress; only a few animals received yet Mtala Copper (aa chloride) Mercury (aa mercuric ace* tale) Mercury (a8 phenylmercuric acetate) Selenium (from grain) M ortality (6000 ppm ); slight rough* neaa of liver Growth retardation; kidney enlargement Growth retardation; kidneya cnlarged and granular. Note: the valuea of 40 and 10 given in tho first two columns under diotury concentrations in tuble IV, Sec tion III, should have keen 0.6 and 0.1 respectively M ortality; cirrhosis of liver; hemoperitoneum; ascites; hepatomas Low grade necrotizing changes in liver and kidney; apparently increased incidcnco of lymphosarcoma and leukemia Kidnoy damngo As above but nt lower dosage levels. At highest level, occasional ulceration of cecum GG05oG5 Cirrhosis of liver; hepatomns; slight hyperplasia and hemosidorosis of splenic pulp; slight liyperplnsin of bone marrow; slight focal myocardiul fibrosis; minor rennl changes 6188 M ASSOCIATION OF FOOD AND DRUG OFFICIALS 5187 ---------- *-------------------------- Selenium (as selenide) As above Cadmium^as chloride) Anemiu; bleached teeth Antimony (as trioxide) Barium (as chloride) Growth retardation; alight changes in color and texture of liver Growth retardation As above Hyjicrplasiu of bone marrow and spleen; controlobular de generative changes in liver Necrosis and other less severe degenerative changes in liver Slight degree of barium precipilaliou in urinary tract DDT Mclhoxycblor TBH AB1I BBH DBH Lindane Toxaphcne Chlordane Aldrin Dicldrin Chlorinated Hydrocarbons M ortality (COO ppm); growth retar From all of this groupof chlorinated compounds except Mcthox- dation; tremors; liver enlarge ycldor, the liver shows a more or less similar and characteristic ment. (In dog, jaundice and complex of histological alterations. Tho complex consists of hemorrhages.) ccntrolobular hepatic cell enlargement, with increased oxy- Liver enlargement. (In dog, atrophy philia, peripheral marginalion of basophilic granules, and of adrenal cortex, and fatty liver.) (with formnlin fixation) u tendency to hynlinixution of the Liver enlargement. No bleaching of teeth. Growth retardation; liver tumors. Liver enlargement. M ortality (800 ppm); liver enlarge ment. 800 ppm quickly fatal; liver enlarge ment. Liver enlargement. M ortality (800 ppm); tremors; con vulsions; liver enlargement. Enlargement of liver and (alight) kidney. M ortality (400 ppm); liver enlarge ment; hyperexei(ability. M ortality (100 ppm); convulsions. Mortulity (100 ppm); convulsions. remainder of the cytoplasm. Qualitatively it parallels liver weight. At lovcls such as GOOppm DDT or 200 ppm Chlordano the histological changes are outstanding and unmistakable, but a t low levels tho miniinul changes present require ex perience und careful comparison with controls for detection. At the higher levels of feeding, noil-characteristic changes such as focal necrosis, fatty degeneration, etc., may bo mixed with the characteristic ones. The characteristic changes do not occur in non-rodent animals Mcthoxychlor and to a lesser extent DDT caused slight in creases in the incidence of hepatic cell adenomas. Chlordane caused a slight degreo of adenomatoid hyperplasia of the hepatio cells Organs other thnn the liver are relatively uninvolved with this group of chlorinated insecticides. High levels of ABH, Lin dane, TBH und Chlordano caused slight or moderate in- T6T88 MOO CGQSG06 TADLE XV-- Continued MST1C1DS cross stre e ts HISTOrATIlOLOOICAL CtlAHOSS Chlorinated Hydrocarbons--Continued Prolan Bulun Liver enlargement Liver enlargement creases in the amount of focal nephritis, and TDII caused moderate testicular atrophy It should be noted th at not all of the compounds were fed for 2 years Organic Phosphates Parulhion EPN ASP-47 HETP Tricthyl phosphato Diethyl phosphate M ortality (100 ppm); tremors Some weight retardation a t 180 ppm in females M ortality Ncgiilive Negative Negative Long term animals negative. At maximum tolerated levels for 1 month, only changes of inanition At 180 ppm, in females, zona glomcrulosa of adrenal cortex widened, with foamier cells Negative Animalsfrom this experiment not examined histologically Slight hyaline granular degeneration of renal convoluted tubu lar epithelium, slight hyperplasia of bone marrow, slight in crease in hepatic cell size Negative Dinitro Compounds DN-111 Growth rcturdntion Negative a t 1000 ppm; animals on 6000 ppm not examined 5188 Herbicides 5000 ppm not tolerated; no olTcct Questionable slight hypoplasia of bone marrow. None of the c from 1000 ppm characteristic hepatic cell changes seen with the chlorinated insectieidca _ 5000 ppm not tolcruted; no effect Negative. None of the characteristic heputio cell changes scon from 1000 ppm M ethyl>1-naphthalene acetic Growth retardation acid with the chlorinated insecticides Examination not complote but it apjiears th at tlioro will bo only nl"0,cl," ll'a'___________________________o T S T c g M O f T *: V ? V .* *.... ' -v ASSOCIATION OF FOOD AND DRUG OFFICIALS 5189 Kndolhul Maleic hydratide q-l'm pyl Homo Pqtcrunyl Imloxidu Van l)ykc 2til 10S0 ANTU Wurfurin Zine phosphide Arasan Arumi to Phygon 3D C Growth rolardalion Growtli retardation k Negative oxccpt for slight changes of inanition Negative Aeliva ora All Hlmwed liver nnlargriiifiil of u|>-. All allowed about the Hamo ilegreo of periportal liopalin cell pruxImuU-ly tho samo degree hypertrophy und slight fully chango, and of ruuul tubular pigment of a weur-and-lear typo Hoden licldca Mortality; bleached teeth; enlarged spleen; atrophic testes M ortality (200 ppm); growth retar* dation; spcctuele eyes; thinning. of hair; deformities of legs and feet Multiple hemorrhages Mortality. Liver damage (discolora tion, nutmeg appearance, rough ness of surface) Chronic congestion of spleen; atrophy of testis. Teeth not seotioned Hyperplasia of spleen, bone marrow and thyroid; thickening of spongiosa and thinning of cortex of long bones; hyaline cen* trolobular hepatie cells; calcified renal medullary tubular casts; terminal focal necrosis of stomach mucosa, thinning of adrenal cortex Multiple hemorrhages; questionable slight inflammatory changes in heart and liver F atty degeneration and nucrosis of liver Miscellaneous M ortality (2500 ppm); disorientation; (unsteady on rear logs) Liver tumors and discoloration; growth retardation High m ortality (2500 ppm); growth rolurdution Growth retardation Preliminary examination indicates that histological changes will be slight, except for changes of inanition, in abdominal and thoracic viscera. At 2500 ppm, calcification in brain stem and cerebellum, and dystrophic changes in leg muscles Ilcpntie cell adenomas frequent a t 6000 ppm; hyperplastic changes at 1000 ppm; 200 ppm not yet oxamined Possibly increused incidence of focal nephritis; 2500 ppm uni* mats not examined Unusual. Negativo except for foamy macrophages in lamina propria of villi of small intestine CG0360S 6T6T88MOCJ 132 ASSOCIATION OF FOOD AND DRUG OFFICIALS Lauo, . P. and K cnze, F. M.: Effect of carbon tetrachloride on toxicity and storage of methoxychlor in the rat. Federation Proc., 10, No. 1., P art I.t 318,1051. D atidow, B., Haoan, E. C., and Radohsxi, J . L.: A metabolite of chlordane in tissues of animals. Federation Proc., 10, No. 1., P art I., 291,1951. Lauq, E. P., K unze, F. M. and Pbiceett, C. 8.: The occurrence of DDT in Human V fat and milk. Arch. Ind. Hyg. and Occupational Med., 3, 245,1951. J' D atidow, B. and Fhawlet, J . P .: Tissue distribution, accumulation and elimination of isomers of benzene hexachloride. Proc. Soc. Exp. Biol. Med., 76,780,1951. 'v^ Datidow, B. and Radomski, R. L .: Metabolite of heptachlor, its analysis, storage and toxicity. Federation Proc., 11, No. 1, P art 1 ,336,1952. F hawley, J . P ., Hagan, E . C. and F itzhuoh, 0. Q .:A comparative pharmacological and toxicological study of organic phosphates-anticholinesterase compounds. J. Pharmacol. Exptl. Therap., 105,156,1952. Pathology N elson, A. A., et al.: Histopathological changes following Administration of DDT to several species of animals. Public Health Reports, 59, 1009,1944. N elson, A. A. and Woodard, G.: Severe adrenal cortical atrophy (cytotoxic) and liver damage produced in dogs by feeding 2,2-bis-(parachlorophenyl)-l, 1dichloroethane (DDD or TDE). Arch. Path., 48, 387, 1949. by feeding at low levels, 1 to 100 ppm. Federation Proc., 9, No. 1, P art 1 ,339,1950. {j i '* GG05G03 5190 6TS Harnessing Chemical Sprays to Serve A the SPORTSMAN through new game management practices Since the beginning of time man has looked at new ways o f doing things w ith suspicion, som etim es justifiably. T he American sportsman proved no exception when he first saw highway departments and power companies doing wholesale spraying of highway and utility right-of-ways to control brush with new chemical weedkillers. In many sections of the country, there has been an outburst of vio lent criticism of chemical vegetation control practices. T he spoksemen for the sportsmen have claimed that such spraying destroys gam e, gam e habitat, flowers and natural beauty. W h ile there are certain real hazards connected with chemical weed control, properly used, most of the new id killers can successfully be harnessed to serve the ^ jrtsmen through better game management practices without any significant evils. It is true that all spraying should be done intelligently and under the supervision of trained, competent people. T he manufacturers o f weed control chemicals are doing their level best to see that their materials are judiciously applied. Like the sportsman, they condemn spraying which is done promiscuously and indiscrim inately. The sportsman need have no fear that maintenance men o f highway departments and of America's power, telephone and telegraph companies are not alarmed when they hear reports of promiscuous spraying, because they are alarmed. They have much at stake. Chemical vegetation control means a savings of maintenance costs that adds up to m il lions of dollars. T hese savings are directly reflected in our utility rates in the case of power companies. Railroads and highway departments are effecting similar savings. The American farmer was the first to make the use of chemical weedkillers standard field practice. In the grain states he increased phenomenally his output of small grains and corn by reducing weed competition. For the most part, his application of chemicals in crops has not brought objections from the sportsmen. It has always been essential for farmers to cut brush in pastures and in drainage ditches. Like industrial users of spray materials, he was quick to discover the economies o f chemical controL M ost farmers are anxious to leave cover where it does not interfere with sound farming prac tice. 3 y selective use o f brush killers woody species that beneneither sportsmen nor farmers are bein g removed from fence rows to perm it desirable species to spread or to be planted if they are not present. In this new field o f habitat control by chemicals the sportsman and farmer have surprisingly parallel interests. The program presents the sportsman with a new opportun ity to better his public relations program with the farmer if he makes him self familiar w ith the farmer's problems and understands their relationship to game habitat. The chemicals most widely used for vegetation control are 2,4-D and 2,4,5-T. N either of these chemicals is toxic to man, animals, birds, fish or insects w hen used in accordance w ith recommended practices. Actually, in the case of 2,4-D and 2,4,5-T dosages in ex cess o f those commonly employed have been found to have no adverse effect on animal life. Tests have been conducted at several land grant colleges in which pastures were sprayed with common weedkillers, including 2,4-D and 2,4,5-T without adverse effect on cattle, horses, sheep, pigs, and chickens. In the laboratories of America's chem ical industry and in the land grant colleges and in private testing laboratories these materials have been thoroughly studied by toxicologists. 'Wide experience over m illions o f acres has verified these experimental findings. For ex ample, 2,4-D has been widely used for water hyacinth con trol on the bayous of. Louisiana and in Florida without ad-, verse effects on fish. The hue and cry against indiscriminate spraying without, any doubt started in instances where spray operators on These men of West Penn Power Company crews are spraying a brush killer which is non toxic to wildlife. The relatively small strip of land they spray will be converted to grass by the second season. Over the years it will save the company's customers millions of dollars. Most conservation people recognize that this spray operation represents conversion of habitat rather thari destruction of habitat. 5192 D o w n to Ea rth , Fall, 1952 y highways attempted chemically to eradicate tail brush badly in need of cutting. It is understandable that the sportsman who saw the un pleasant browned appearance that resulted from a few instances of such indiscrim inate and ill advised spraying would w ell b e concerned about the future of w ildlife habitat. Fears commonly expressed in relation to roadside spray ing include the danger of erosion and the destruction of habitat. M ost thinking game management m en w ill agree Railroads have a different problem than utility and telegraph companies in that they must remove brush for several yards from the track and all vegetation near the ties. This is a must for safe operation of our nations railroads. Obviously, grazing on railroad right-of-ways is not safe for animals, wild or domestic. that the presence of brush along the roadside for game cover has been overemphasized. Such heavy cover is hazardous to the motorists, to the game, (w hen you consider fast m ov ing traffic) and it sets up an ideal opportunity for the poacher w ho shoots from car windows. Actually, there w ill still be adequate roadside cover for sm all game. Grasses grow profusely when brush is removed and more sunlight can reach the soil. Grasses are not damaged by 2,4-D or 2,4,5-T. Thick grass sod is one of the best covers for the prevention of erosion. In Okla homa, soil conservationists have found 45% less run-off from a productive pasture previously sprayed to k ill scrub oak than from adjacent m ixed grass-brush land. Dead brush along roadsides after chemical spraying is admittedly unsightly but it is a temporary factor in the process of chemical conversion from brush to grass. Spray crews know now that they must spray brush before it grows too tall or remove it by cutting. Stumps are then sprayed to prevent regrowth. Unless sprayed brush is tall, the in creased growth of grass obliterates much of the dead brush. The control of weed vegetation on right-of-ways is an integral part of maintenance. Proper use of selective herb icides can serve to convert game habitat rather than to destroy it. W here the outlook is conversion of habitat rather than destruction, game management authorities are beginning to see the value of selective herbicides as new tools. In actual practice, some of the utility companies who are criticized by the sportsman when he first sees discolored brush, may in the long run pay for and provide improved habitat. The game specialists w ith whom the subject has been discussed are generally agreed that the grass land devel oped along lines by vegetation control is good for rab bits and deer. 2,4-D and 2,4,5-T are ideal for use in game manage ment because of their very low toxicity to all animal life and the degree to which accurate application can be controlled. For exam ple, it is possible to k ill selectively susceptible species growing with more resistant species by using marg inal concentrations. The Forest Service has used these materials applied w ith hand sprayers to the base o f large hardwood trees to lib erate desirable pine piznrings a t a co st considerably isekrw "that o f hand slashing. These same compounds are used to remove hardwood species from the more resistant Lob lolly pines in the southeast : In many areas under today's improved forest fire protec tion we have a condition in which w ildlife species h i need of grass and low-growing brush are finding uniform forest stands, such as aspen in the lake states, a very unfavorable environm ent This is a recognized fact and, in areas where there are no desirable trees, game managers already have resorted to the use of the axe, controlled fire and even the bulldozer in an effort to open up the forest crown to perm it growth o f low brush and herbaceous material. W hen w e consider costs o f labor and mechanical bulldozing it is apparent that chemicals can com pete very satisfactorily. Judiciously used, chemical sprays can often do an even better and safer job at this lower cost. Consider the case of controlled burning -- for game habitat improvement. Often on poor soils it is several years before any volum e of ground vegetation reappears. In the m eantime w e have an erosion problem and quite possibly we jeopardize our public educational program for forest fire prevention. U sing an aerial application it is possible to kill or injure the overstory of poor trees such as aspen and thus encour age the development of low growing shrubs and herbaceous plants. The plausibility of aerial application is proved by die past spraying of 500,000 acres o f m esquite in the south west at an average cost of approximately $5.00 per acre to improve carrying capacity of rangeland. W e do not wish to im ply that selective herbicides w ill revolutionize game management practices. The need for interspersion of cover type is w ell recognized for such spe cies as sharptail grouse, ruffed grouse, w hitetail deer, cottontail rabbit, and other w ildlife. Chemical sprays are just another tool available to professional game m en in their effort to manipulate environmental factors to speed up production of today's hard hunted species. Like controlled burning and other established practices, indiscrim inate use of herbicides could prove an enemy of w ildlife. Properly employed, they can be useful tools in the hands of game management people. As new herbicides are developed and carefully tested it w ill becom e increasingly easy and profit able to selectively kill worthless plants w hile encouraging the type of cover that may be desired in any given area. Farmers know what chemical maintenance can do to reduce weed population and taxes that are used to support our roads. Every where spray crews go there is widespread interest. This picture was taken in Ohio. Such programs can be conductedwithout injury to game or adjacent crops. Education and cooperation are the keys to success of such programs. 8 5193 Woody Plant Control Along Roads n Shasta National Forest By A lva G. N e u n s , Training Assistant, California Forest and Range Experiment Station1 A mountain road in the Shasta National Forest mowed in 949 and sprayed in 1950. C o n t r o l l in g sprouting brush species in the moun tains with sprays of 2,4-D and 2,4,5-T depends mostly on how the chemical is applied. This has been demonstrated on the Shasta National Forest in California, where a fight is being waged against roadside brush with the battle cry: 100% Coverage -- 100% Control One hundred and eighty miles of brush were sprayed in 1950 and 1951, and emphasizing the importance of total coverage accomplished three things. First -- and most valuable -- it gave something to achieve, a definite goal, so that "green'' crews could be trained quickly and easily. Second, it assured complete kill of all sprouting growth above the ground and a maximum of root kill. Third, control was successful and long lasting enough that retreatment the next year was usually unnecessary. The last is important because of the inaccessibility of most forest roads. Shasta National Forest has a network of 1200 miles of forest roads. They wind their way over steep mountain ous country from an elevation Work closetravel slour of 1000 feet or less to 9000 feet. Some 900 miles of them have brush covered rights-ofway which in the past had to be hand-cut, bulldozed, or mowed with a brush mower every 3 to 5 years to keep the roads from disappearing be neath the brush. In a search for less costly brush - control methods, the California Forest and Range Experiment Station tested the selective herbicides -- 2,4-D and 2,4,5-T -- from 1945 to 1949. Tests were made on plots growing more than 15 troublesome species, including live oaks, deciduous oaks, maples, manzanitas, chinquapin, cherry, snowbrush, and whitethorn. It was found that all of che above ground parts of these species could be completely killed and that the total root kill was high if complete coverage was achieved. Seedlings of all species could be completely killed. Chemical spray carefully applied by research workers on plot-sized road shoulders is one thing, but the same formulation in the hands of the average spray man is quite another. Manpower shortages make it almost impos sible to keep good foremen or spray men for any length of time once they are trained. Consequently, job operations and equipment were simplified as much as practicable. During the 1950 season, then, it was possible to spray 70 miles of previously cut, sprouting roadside brush with a high degree of success and at reasonable cost. The follow ing rules for spraying and the slogan, "100% Coverage -- 100% Control," were based on the experience gained that year and are now used as a basis for all crew training.- H ow to Sp r a y : ( 1) Cover all leaf and stem surfaces to ground level with spray-- especially the undersides of the leaves. ( 2 ) Travel slowly so that you can see what you are spraying and know when the spray is covering the plant. This usually means walking rather than riding the truck. ( 3 ) Hold the nozzle close to the shrub so that the spraymist will not be blown away or wasted. (4 ) Get down into dense growth using your short boom to pull it apart as you spray. (5 ) Keep pump pressures high enough to ensure spray turbulence and misting. Regrowth likethis is ready for spraying. (6 ) Check back after spraying from time to time to find what degree of coverage you are getting. Rule (6) is very important. The spray-crew foreman should check back continuously to make sure all the growth is being treated. W hen diesel oil or an oil and water emul- 1The California Forest and Range Experiment Station is main tained by the Forest Service, U. S. Department of Agriculture, in cooperation with the University of California, at Berkeley, Calif. "The use of 2,4-D and 2,4,5-T for Brush Control on California Roads and Trails. U. S. Dept, of Agriculture. Forest Service, Berkeley, 1950. 14 5 1 9 5 7 --- sion is used as a carrier for 2,4-D and 2,4,5-T, the spray material can be seen on the leaves and stems of the plants for several hours after the spray is applied. And nothing shows a spray-crew the importance of complete coverage more than to take them back over the job a week or two later when the leaves are drying. The men can see how much is still green when they don't get coverage -- also how dead the plants look when they do. Equipment used on the Shasta National Forest is simple, inexpensive, and readily converted for fire fighting and other purposes. It consists of a stakeside truck geared to travel very slowly and big enough to carry eight, 50-gallon drums--an average day's supply of spray mixture. A pump used in fire fighting that will develop up to 250 lbs. pressure (100 lbs. is minimum operating pressure) is mounted on a rear corner of the truck bed, 2 high-pressure hoses are attached and fitted with a short boom for average spray ing and a longer boom for high banks. Trigger-type shut off valves and solid cone nozzles (30 angle spray, 1.1 GPM at 100 psi discharge) on swivel fittings complete the assem bly. Pumping is done directly from the 50-gallon drums. A low step is mounted on the back of the truck to carry spray men short distances or while spraying light brush. This equipment is easy to use and requires little main tenance; 180 miles were sprayed continuously with no lost time due to equipment failure. Walking speed and trigger control on the nozzles con tribute as much to economy of spray as they do to total coverage. Complete control of the spray is a necessity wherever plant cover varies .in density as it does on moun tain road shoulders. Dense cover always requires contin uous spraying, but medium and light densities do not. Inermittent spraying -- shutting off the nozzle between plants--prevents waste of material on bare ground. Average spray costs in 1950 on 70 miles of road, includ ing labor, equipment and materials, were $28.00 per mile. Brush on all road shoulders had been cut and cleared for visibility and reduced fire hazard, with a brush mower or bulldozer, and the sprouting growth was treated with chemical spray. Some mechanical clearing costs as much as $240.00 per mile. Chemical control can eliminate the need for cutting in the future. Re-spraying will be neces sary from time to time, but costs should decrease as control ecomes well established. To decrease costs, oil and water emulsions were substi tuted for straight diesel oil as a carrier in 1951. Although the average cover was much denser in the area sprayed T h e spray m e n -walk unless there are very high banks to treat. that year, costs on 110 miles of road were $25.00 per mile. As little as 4 gallons of oil per 50 gallons of emulsion were used, but oil was increased as the season advanced to insure better penetration of tougher leaves and stems. During the 1950 field program an attempt was made to vary concentrations of 2,4-D and 2,4,5-T according to whether the predominating woody plant on the site was hard or easy to kill. Since spray material output averaged one 50-gallon drum per mile, it seemed possible to change the formulation in each new barrel to fit the hardest to kill species on the mile ahead. A table was provided from which the foreman could figure the number of quarts of each chemical needed per 50 gallons for a given concen tration. Concentrations ranged from 8000 ppm 2,4,5-T and 4000 ppm 2,4-D to 1000 ppm of each for light retreat ments. In 1951 to further simplify the job, concentrations were standardized at 4000 ppm 2,4,5-T and 2000 ppm 2,4-D. It was found to be eas ier to mix chemical, oil, emul sifier, and water at headquar ters than depend on mixing and changing concentrations on the job. The slight advantage gained in root kill from higher concentration is offset by the loss of efficiency and labor time involved. Spraying can begin as early as May in the lower alti tudes and continue throughout September in the higher altitudes. The season of most rapid plant growth does not occur until late in July in .the high country, so that a long spray season can be planned. Woody plant species, exposure, stage of plant growth, soils, temperatures, moisture and growth rate all change mile by mile on forest roads. These and other factors are thought to influence the root killing power of 2,4-D and 2,4,5-T. But only one influencing factor can be made consistent throughout a mountain road brush-control job. That one factor is coverage, and a well-trained spray-crew and foreman who make total coverage a matter of pride can make the difference between poor control and 100 percent control. V 5196 Down to Earth, Summer, 1952 AH 5197 A+ i Controlling Scrub Aspen W ith BASAL SPRAYS By Jo h n L A r en d , Research Forester Lower Peninsula Forest Research Center, Lake States Forest Experiment Station, East Lansing, Michigan B a s a l SPRAYS, also called "bark and dormant sprays," for controlling undesirable woody plants have been under study by various investigators in M ichigan since 1946*. Ib is paper describes the results of basal spray tests made three years ago which appear to offer a satisfactory method for controlling scrub aspen in northern lower M ichigan. T h b Co n tr o l Pr o b lem : Aspen is a valuable tree species for wood products on good forest sites in the Lake States region. However, on poor sites aspen is usually an inferior grade tree. As a result, forest and game managers frequently need to control scrub aspen to improve w ildlife habitat or establish tree species m ore adaptable to the forest site. W hen aspen trees are cut or girdled mechanically, numerous sprouts and suckers develop from che stumps and roots for distances as far as 100 feet from the parent tree. Consequently, aspen reproduces itself very w ell after cutting. T o date no satisfactory method for reducing the sprouting o f scrub aspen has been reported. Several investigators ** have reported that the season of the year when aspen trees are cut has som e effect on the vigor and number of sprouts and suckers which develop. Sprouts developing from aspen trees cut during the summer months have been found to be less numerous and less vigorous than those developing from trees cut at other seasons o f the year. Aspen sprouts developing from trees cut in the summer, however, have generally been sufficient in number to satisfactorily restock the stand w ith aspen reproduction. Although the density and vigor o f aspen sprouting can be reduced by summer cutting of the parent tree, the reduction is not sufficient for satisfactory controL Ba s a l Spra ys T ested : Starting in February 1950, die Lake States Forest Experi ment Station initiated a number of chemical herbicide tests in northern lower M ichigan designed to control sev eral low value hardwood species, including scrub aspen, oak, and red maple, for pine release purposes. D ifferent methods of chemical control including basal sprays, were applied to these low grade hardwoods in the winter, spring, summer and falL A lso, since aspen sprouting has been found to be somewhat affected by the season o f the year when the parent trees are cut, a special seasonal study was made of scrub aspen treated w ith basal sprays at weekly intervals throughout the entire growing season (M ay through O ctober). The basal sprays tested included esters of 2,4-D (Esteron 44 and Esteron T en-T en), 2,4,5-T (Esteron 2 4 5 ), and 5 0 /5 0 mixtures o f each (Esteron Brush K iller) m ixed w ith Grade 3 diesel oiL Concentrations of 2 and 4 percent by volum e (8 to 16 lbs. o f acid per 100 gals.) were applied to 1At Dow Chemical Company, Midland, Michigan, and Michigan State College, East Lansing, Michigan. ' Zehngraff, P. J. 1946. Season cutting affects aspen sprouting. Lake States Forest Experiment Station Technical Note 230. *Stoeckeler, J. H. 1948. The growth of quaking aspen as affected by soil properties and fire. Jour, of For. 46(10) 727-737. Aspen sprouts and suckers which developed from aspen trees treated with -basal sprays during the dormant and earh growing season. 10 the basal portion of individual trees to heights of 2 and 4 feet. In all cases the basal portion treated was carefully w etted to produce-runoff com pletely around the tree. r E c r s o f B a s a l Sf r a y s o n T o p s o f Sc r u b .As p e n : A ll of these ester formulations o f 2,4-D and 2,4,5-T when m ixed w ith diesel o il at concentrations o f 2 to 4 percent by volum e and applied to die basal portion of aspen in su ffic ie n t q u a n tity are capable of k illin g the top of the tree. T he volum e o f herbicide applied in basal spray work is very important, especially for the larger trees. For example, only slight differences were observed in the rate of top k ill and subsequent sprouting o f aspen treated w ith 2 and 4 percent mixtures of esters of 2,4-D and 2,4,5-T in diesel o il when the basal portion of the trees were sprayed to a height of 4 fe e t Moreover, a 2 percent mixture applied to the basal portion of the trees to a height o f 4 feet is consistendy more effective than a 4 percent m ixture applied to a height o f 2 fe e t H owever, spraying the base o f the tree to a height of 4 feet requires a considerable volume of herbicide compared to a 2 root h eig h t W hen scrub aspen is treated w ith basal sprays (2 to 4 percent mixtures by volum e of 2,4-D and 2,4,5-T esters in diesel o il) during the dormant season, the tops are gen erally dead by the end of the first growing season follow ing the treatm ent The treated trees usually leaf out in the spring, but the foliage starts turning yellow shordy after full leaf developm ent Scrub aspen treated w ith basal sprays during the early grow ing season starts showing the effects o f treatment in about 10 days. However, trees treated in late July and August probably w ill not show the effects o f the basal spray treatment until the follow ing growing season. In other words, during the early grow ing season the tops of aspen trees are more easily killed by basal spray treatment than they are at any other season of the year. D uring the late growing season, and in the fall and w inter months, aspen trees are generally more resis tant to basal sprays. It is during these seasons that 2,4,5-T esters are more effective than 2,4-D esters. Also, a larger volume o f herbicide needs to be applied to the basal portion of the tree during the fall and-winter months than during the early grow ing season. Consequendy, a 3 percent m ix ture by volum e o f Esteron 245 (2,4:5-T in diesel oil applied from d ie ground level to a height o f 2 feet in soaking quan tities is an effective year-around basal spray treatment for woody plant control. E f f e c t s o f B a s a l Sp r a y s o n Sp r o u t in g o f A s p e n : N o sprouts or suckers were found at the end of the first g row in g season from the aspen trees treated w ith basal sprays at different seasons of the year. However, those cut and girdled sprouted prolifically the first year-- the sprouts averaging about 3 feet in height. A t the end of the second growing season suckers were found w ithin 2 to 5 feet of the basal-sprayed aspen, exce p t fo r th ose treated a fte r fu ll leaf d evelo pm en t. T h re e g ro w in g seasons a fte r the basal spray treatments the same sprouting condition prevailed -- no suckers or sprouts were found from aspen treated after full leaf developm ent through September, whereas suckers developed from the aspen sim ilarly treated during the dor mant period and during the early grow ing season before full leaf development. H ie weekly basal spray tests were conducted only on one area of scrub aspen. However, sim ilar basal spray treat ments were also applied to scrub aspen at tw o other loca tions in northern lower M ichigan during the same year at various seasons: ( 1 ) dormant (February); (2 ) early growing season (M ay); (3 ) summer (Ju ly); and (4 ) fall (Septem ber). The sprouting results on the other test areas were the same, L e., no sprouts or suckers were found among the scrub Aspen rrearea in July and only a few among the aspen trees treated in September. However, sprouts were both numerous and vigorous among the trees treated in February and in May. Su m m a r y a n d C o n c l u s io n s : Scrub aspen trees have been killed without subsequent sprouting and suckering for three years in northern lower M ichigan by applying basal sprays during the summer months of late June, July, and August follow ing full leaf developm ent. Aspen similarly treated during the dormant and early growing seasons suckered prolifically beginning the second year after the basal spray treatment. Season of the year during which the scrub aspen is treated with basal spray has considerable effect on the concentra tion and volume of spray applied for an effective top k ill as w ell as subsequent sprouting of the treated trees. A 2 percent mixture (by volum e) of 2,4-D and 2,4,5-T (Esteron Brush K iller) in diesel o il sprayed on the basal Aspen trees killed by basal sprays applied during late June, July and August after full leaf development. No sprouts or suckers have developed for three growing seasons after the trees were killed with basal sprays. (Trees sprayed summer, 1950. Photo taken in September, 1952). portion of the tree to a height of 2 to 4 feet is effective in killing the top of aspen during die growing season. How ever for year-around basal spray treatment, a 3 percent m ix ture of 2,4,5-T (Esteron 245) or 12 lbs. of acid equivalent per 100 gallons; o f Grade 3 diesel oil, applied to die basal portion of the tree to a height of 2 feet is recommended. Basal spraying aspen w ith either o f these herbicide m ix tures during late June, July and August follow ing full leaf developm ent offers a promising method of controlling scrub aspen w ithout subsequent sprouting for at least three years. 11 _ 51 D ow n to TH, Summer, 195 008S .DOWN to EARTH 1 T^ ^ C o p y r ig h t I Q 5 3 by The D o w Chemical Company J-IW A review of agricultural chemical progress volume 9 no 3 winter 1^53 B otanist O liver A . Leonard on the D avis Campus o f th e U niver sity o f C alifornia p oin ts out 2,4-D filled cuts around th e base o f a large live oak tree. T h e etas go an inch or tw o in to th e wood. W oody plants constitute problems on some of the follow ing areas in California: ( 1 ) 2,500,000 acres of commercial timberland that is little more than brush; ( 2 ) 7,500,000 acres of woodland-grass; ( 3 ) 7,300,000 acres of chamise; ( 4 ) 2,400,000 acres of chaparral other than chamise; ( 5 ) 5,000,000 acres o f big sagebrush. "Woody plants, also, are problems on some other areas. A map is presented showing the distribution of some of the. more important areas where brush is a problem in California. There are many groups of people whose welfare is involved in controlling, woody plants. Ranchers are interested in brush control in order both to reduce brush encroachment on cleared rangeland and to reclaim valueless brushland by transforming it into good pasture. They are interested in controlling brush and trees around springs and along water courses in order to have more water available for livestock and domestic purposes. In some areas, there is interest in controlling "woody plants to make more water available for irrigation. Foresters are interested in woody plant control to aid in reforestation. Scotch broom ( Cytisui scoparius) is a serious pest in the forested areas of several Sierra Nevada counties and prevents natural pine reforestation. It is readily killed by one application of 2,4-D (low-volatile ester) applied as a foliage spray in June or July, but seedling problems have not been solved. Chemical Control of Woody Plants in C A LIF O R N IA By Oliver A Leonard, Associate Botanist, University of California, Davis, California Ribes sp. must be controlled in the sugar pine (P irns lambertiana) sites, since many members of this genus are alter nate hosts for the white pine blister rust. Willows along stream banks hinder streambed maintenance and thus make flood control more difficult and complicate the mosquito control problem. Chemical brush control along rights-ofway is becoming more common. Poison oak (Rhus diversiloba) is of personal concern to more people in California than any other woody plant pest. It is controllable with either 2,4-D or 2,4,5-T, although 2,4,5-T is slightly super ior. From one to three applications, spaced at yearly inter vals, have been necessary for a complete controL There is some evidence, too, that acid or amine formulations, plus 1% low toxic spray oil, may result in better control than is obtained with some esters. The Botany Department, University of California, Davis, California is conducting basic research on the chemical con trol of woody plants, including ( 1 ) cuticle penetration of herbicides and ( 2 ) radio-isotope studies. Working with live oak (Quercus wisUzenii) and toyon (Pbotinia arbutifolia), for example, it was determined that 2,4-D moved rapidly out of treated leaves and thence downward in the bark in February when the shoots were still "dormant." Growth of roots during this period related both to the downward flow of materials from the leaves and to the fact that these woody plants are sensitive to hormone sprays at this time of the year. Some general results of experimentation in chemical weed control in various types of woody vegetation are presented. Coastal Sagebrush a n d Coyote Brush Areas: Coastal. sagebrush (Artem isia califomica) and coyote brush (Baccharis pilularis) are readily killed with 2,4-D at rates of 2 to 3 pounds per acre, applied by aircraft. Accord ing to available evidence, results should be more uniformly satisfactory when amine 2,4-D is used. The recommenda tion is 3 pounds acid equivalent per acre in about 8 gallons of water and one gallon of light-medium summer spray o il The favored period for spraying is during April, May and June, but good results have been secured on coyote DOWN TO EARTH * A R eview o f A gricultural Chemical Progress Published by T h e D ow Chem ical Company, M idland, M ichigan, m anufacturers o f agricultural chemicals. Tw enty-one thousand copies distributed to those engaged in agricultural research and instruction in Am erica and abroad Eugene Perrin, Editor. ON THE COVER IN THIS ISSUE C hem ical control o f woody plants in California A sim ple device fo r applying E steron. 245 * E thylene dibrom ide fo r th e control o f nem atodes in th e organic soils o f M ichigan D alapon, a n e w . system ic grass killer introduced fo r control o f industrial vegetation T im ber stand im provem ent on the W . G. Jones State Forest, Conroe, Texas R eport on Venezuelan experim ents on grass control in rice w ith Premerge. 2 See picture legend on P*g* 3 5202 brush up to September 1. Two pounds o f 2,4-D ester (low -volatile), plus 1 gallon of Diesel oil and.8 VS gallons o f water per acre have resulted in kills only slightly in ferior to chose obtained with the amine treatment. Control of mixed coastal brush with one application by aircrafc is not adequate and must be followed by some addi tional spraying, probably with a ground rig. A t present, the iniual spray is with a brush killer using 2 pounds of acid equivalent per acre, plus 1 gallon of Diesel oil and 8 Vi gallons of water, applied by helicopter. The advantages of spraying over other methods of brush control have been outstanding, especially in terms of in creased grass production. Chaparral and Chamise Areas: Because chaparral and chamise occupy so much land in California, considerable study has M en undertaken to determine feasibility of control methods, the pattern of recovery from various treatments and the performance of desirable associated vegetation and subsequent seeding. Chamise and chaparral have frequently been burned in the past, but these areas usually revert rapidly to the origi nal condition because of crown sprouts and seedlings. The sites are frequently almost devoid of grass before being burned and killing the brush is of little value. Grass can be established most readily by burning the brush in the fall and seeding the desired grasses and legumes immediately. Grass competes with the brush sprouts and seedlings and results in the death of many of the latter. A broadcast spray should not be applied until the second spring after the burning and seeding so that ( 1) the legumes will have gone to seed once, building up a reserve of hard seed in the C hem ise urea seeded to perennial grasses. T h e author is standing on th e boundary betw een a sprayed (background ) and unsprayed (foreground) area. T h is is the same general area fro m w hich th e data presented in th e table w ere obtained. D r. Leonard is pointing to the location w here 2,4-D am ine bat been placed in a cut in this interior liv e oak 33 m onths pre viously. T he wood is rotting and stem s have broken over. E ight separate cuts and 16 m l. o f undiluted 2,4-D am ine were used. soil and ( 2 ) sprouts will have developed and most of the brush seeds will have germinated. Good success has been achieved in killing brush sprouts and seedlings developing after a fire on chaparral ana cha mise areas by applying a broadcast spray, consisting of 2 pounds of low volatile 2,4-D per acre in 2 gallons of Diesel oil and 31 Yi gallons of water. March and April appear to be the optimum time for spraying. Such a spray will kill most of the chamise ( Adenostoma fasciculatum), yerba santa ( Eriodictyon californicum) , golden'fleece ( Haplopappus arborescent) , deer-weed (Lotus scoparius), rush rose ( Heliantbemum scoparium), creeping sage (Salvia sonom ensis), and brush seedlings, including chamise, Ceanotbus sp., and Arctostapbylos sp. Seventy percent of the toyon (Pbotim a arbutifolia) may be killed. A partial top kill of interior live oak (Quercus w islizem t), coffeeberry (Rbam nus californica), and redbud (Cercis occidentalis) is se cured. The latter species can be killed by from one to chree applications of a spray containing 4 pounds acid equivalent of a brush killer in one gallon of Diesel oil and 98 gallons of water. Where C eanotbus sp . are abundant, there is some advan tage in using a spray that contains some 2,4,5-T, especially on the larger plants. Aircraft spraying has not been sufficiently successful thus far to be used commercially. Early in these studies, the isopropyl esters were com pared with the mixed propylene glycol butyl ether esters of 2,4-D and 2,4,5-T, when formulated the same. One such test conducted in March 1951 resulted in a 22% and 20% kill of chamise sprouts with 2 pounds of the isopropyl esters of 2,4-D and 2,4,5-T, respectively, and a 78% and 54% kill with the propylene glycol butyl ether esters. The advantages in using some oil in the spray mixture have been demonstrated. There is some evidence to indicate that an oil of low toxicity gives a superior kill to that obtained with Diesel oil on chamise (high volume spraying). There has been an increase in die production of grass on areas that have been sprayed, over that on unsprayed areas. One example of this was on a chamise site. The brush was broken down with a bulldozer in the summer of 1950 and the area burned in October of the same year. Immediately following the burning, the area was seeded by airplane to harding grass (P h a la ris tu b e ro sa ), smilograss (O ry zo p sis m ilia c e a ), tall fescue (F estu ca a ru n d in a c e a ), and rose 5203 D o w n to Earth, W inter, 1953 T his helicopter (.coming in fo r a landing) -was used to spray coyote brush.' .> clover ( T rifolium b irtu m ). Broadcast. spray treatments were applied in 1932, (note table for one example). The quick returns as a result of spraying are indicated. It should be cautioned that the results obtained in seeding this peren nial grass mixture were unusually good. The effect of 2,4-D on chamise sprout kill and on grass production one year after spraying is shown in the table. A broadcast spray was applied on April 17, 1932, using the propylene glycol butyl ether .ester of 2,4-D in 2 gallons f Diesel oil and 37VS gallons of water per acre. Grass production was determined in July 1953. (Coop, study with Charles Carlson, Calif. State Division of Forestry). : 2,4-D per acre lbs. 0 1 2 K ill o f Cbamise sprouts % 0 50 87 D ry w eight o f grass per acre lbs. 639 1378 1771 W oodland-Grass Areas: There is considerable interest in clearing some of the woodland-grass areas. These areas are often burned, but a top kill is usually not obtained, except in live oak thickets or where brush has invaded. 1.Bulldozing is commonly employed, especially where the soil and topography are suitable. After bulldozing and after fire, live oak sprouts develop from underground parts and grow rapidly, so that within a few years die area reverts again to the original state. The present recommendation is to spray the sprouts with a mixture containing 4 lbs. of 2,4-D plus 2,4,5-T (Brush Killer), plus one gallon of Diesel oil plus 98 gallons of water. The spraying should be in the spring and early summer months and repeated until the sprouts, are all killed. Very few sprouts will be alive after the third year. The best kill of live oak sprouts thus far achieved by one application was with a spray containing 2 lbs. of 2,4-D (amine form) in 100 gallons of water plus .1 pint of a sticker spreader. The spray was applied in October of 1950 and by July of 1953, 56% of the stumps had no live sprouts left. The cut-surface method using the amine of 2,4-D is ap plicable to the oak woodland areas. Cuts made with an axe or hatchet should be deep enough to extend well into the sap-wood Trees are sensitive at all times of the year, but less chemical is required to kill trees in the winter and spring than in the summer and falL On Digger pine ( Pinsts sabiniana),. ihe cuts can be 8 inches (center to center) and good results obtained using 2 mL of 2,4-D amine ( 4 lb. acid equivalent) per cut (undiluted). On Blue oak ( Quercus douglasii), the cuts can be 6 inches center to center and again 2 mL per cut should be used Live oak is a vigorous sprouter ana the cuts should be con tinuous for best results. Stumps can be treated the same as the standing trees, although some retreatment will be neces sary; for example, from one to 6 sprouts had appeared on 4 out of 18 live oak stumps that had been treated 2 years previously with the amine of 2,4-D. 2,4,5-T amine ap peared to be rather ineffective, since most of the stumps developed sprouts. Big Sagebrush: Sagebrush ( Artem isia tridentata) is being controlled ef fectively with a mixture containing 2 lbs. of 2,4-D ester, plus V i gallon o f Diesel oil and 9 gallons of water, applied in June. Co n clu sio n s: Many different problems exist on the millions of acres of brush covered land in California. Ch<miraU are being used in many situations and their use will increase since certain jobs can be done better. However, on much of the low-value rangeland, it will be necessary to do a good job more economically than is now possible. This is a challenge to industry as well as to federal and state research and extension agencies. 4 5204 & J: V:? ir?-' < _ v-y. ! w A Simple Device for Applying Esteron 245 By DAVID B. COOK Cooxrox Forest, Stepbentown Center, New York EDITOR'S NOTE: Mr. Cook reports on a home-made variation of basal bark application equipment. Two years' use has convinced him of its practical value. W e are glad to publish his ideas which may prove helpful to others. ESTERON 245 is rapidly making a name for itself as an effective silvicultural tooL Mixed with water, it can be used advantageously as a foliage spray on woody plants up to eight feet talL Trees larger than that, and up to 6 inches in diameter at the ground, can best be treated with a basal spray of ESTERON 245 in oiL This latter technic is particularly useful in the selective deadening of unwanted, unmerchantable stems where a residual stand is to be re tained. It is also a means of treating cut stumps to prevent sprouting. The trade literature suggests that basal sprays be applied with a knapsack or small tank sprayer equipped with a wand bearing a nozzle set to deliver a narrow, solid cone of spray at low pressure. Such equipment is quite expensive. Moreover, it is impractical to use it for anything but "hor mone" sprays because of the difficulty of completely clean ing it, and because "hormones" are effective against certain sensitive plants even in very small amounts. And, in prac tical operation, it is difficult to maintain an even, low pressure and to keep most of the spray on the target. My early experience convinced me that such rigs were too complicated at least for my use; that pressure was un necessary; and that simple gravity flow would be sufficient. It was apparent that a free flow of mixture would give a more thorough soaking effect and would run down onto the thin bark a t the root crown better than would even the coarsest pressure spray. A simple and effective basal sprayer was assembled from a two-gallon lubricating oil can with an air vent punched in the cap and a short pipe tapped into the bottom. For convenience in carry ing, th e can was mounted on a ply wood packboard with rope shoulder straps. T o the can was at tached three feet of Y&" "pure latex tub ing", such as is used for handling blood. (I learned the hard way that ordinary red rubber tubing disin tegrates from contact with kerosene). On this was threaded Basal spray applicator made w ith 2-gallon o il can. a clip-type tubing damp and the end slipped onto a 24" length o f Va" alumi num pipe. Valve and aluminum pipe are mounted in a wooden T h e applicator in use. T h e drenching stream gives the stem a good soaking. handle to give a better hand hold. The whole rig weighs 3V* pounds and can be put together for a little labor and about $1 in cash,- the latex tubing being the most expen sive item. In operation, die tip of the wand is placed against the desired spot on the lower stem, the valve undipped and die thumb pressure released enough to give the desired flow. The bore of the tube is Y&", so the liquid flows out in a solid stream about the size of a match-stick, at no great ressure and with almost no wasteful spatter. Rate of ow can be nicely adjusted by thumb pressure. On trees up to 2" in diameter, the kerosene will spread readily all the way around the stem from a single point of application. Somewhat bigger stems will require some lateral movement of the wand, while those over 4" must be spotted from opposite sides. In moving from tree to tree, the valve can be clipped shut; die small bore of the tube will keep the liquid from running out. W hen not in use, the wana is carried in the dothespin clip on the board, ' leaving the worker with both hands free. This simple rig has several advantages. There being no pressure to maintain, the operator has one hand free and nothing to think about except aim ing the wand. H e has but to shrug his shoulders to find out from the sloshing how much liquid the can still contains. Filling is simply and quickly done. In two seasons of work, it has proved it self a good tool. The formulauon used has been a 4% solution of ESTE RON 245 in kerosene, plus Vi teaspoon of R ed-0 oil dye; when freshly applied, this bright red is conspicuous, espedally on grey birch. W ith this device, I have been able to accurately apply 2 gallons per hour, that being enough to treat about two hundred 3-4" trees. It is not so effident on stems less than Vi" in diameter because of the difficulty of holding the wand tip on such a small target, and letting out so small a quantity of liquid. The drenching stream gives the stem a thorough soaking, which may be one of the factors contributing to my uniformly good success in getting complete kill on a wide variety of northern hard wood species. 5 5205 D o w n to Ea rth , W in ter, 19- S'*"" -- w--n->JTw t.act r TIM BER STA N D IM PROVEM ENT O n the W. G . Jones State Forest, Conroe, Texas B y Charles T . Stbalby, J r.* In many .areas of the south the once pure stands of south ern pines have undergone changes, so that today the under story and in many cases the overstory is a mixture of south ern pines and southern hardwoods. T o help insure and in crease survival, to enable more rapid growth and better development, to increase the maximum financial yields and to remove low valued hardwood competition from the higher valued intolerant pines, various individual treat ments and combinations of treatments have been used in timber stand improvement programs. Several examples of individual treatments are girdling, frilling and felling. A combination treatment could be any of these individual treatments and the addition of a chemical herbicide. . The W . G. Jones State Forest, under the administration of the Texas Forest Service, is dedicated to demonstrational and research use. In an effort to obtain cost records and to set up a project as a demonstration of timber stand improvement and research with 2,4,5-T, a combination treatment of girdling and basal spraying with 2,4,5-T was used on a ninety-nine acre compartment of the forest. The compartment treated was typical of many East Texas forest stands. The pine-hardwood overstory was composed of loblolly and shortleaf pine, several species of red and white oaks, sweet and black gum, mockernut hickory, American elm and dogwood, with pine making up sixtyfive percent and hardwood thirty-five percent of the stand. H ie understory was composed o f seedlings and saplings of hawthorns, bays and yaupon in a seventy percent hard wood-- thirty percent pine--proportion. The initial treatment consisted of girdling all hardwoods, eight inches d. b. h. and larger, which overtopped and/or interfered with established pine trees or reproduction, using the two hack girdling method. After the girdling was com pleted, the compartment was treated using the basal spray method. A ll hardwoods less than eight inches d. b. h. that overtopped and/or interfered with the pine were treated. A two and eight-tenths percent concentration, by volume, was used for the basal spray, by mixing one and one-half gallons of Esteron 245 with fifty-three and one-half gallons of diesel oiL This concentration was selected on the basis of research work conducted by Ray E. Goddard, Assistant Silviculturist of the Texas Forest Service, who found satis fying killing results were obtainable with it, on the basis of preliminary work, and that it was more economical than the higher concentrations. Spraying began in late March, 1952, and was completed in the latter part of April, using local unskilled labor. An inspection or the results of the treatment was made in November, 1952. Table 1 is a summary of the cost study of the individual treatments and of the entire project. The sizes of the trees girdled ranged from eight to eigh teen inches d. b. h., with the average tree about twelve inches. The sizes of the stems sprayed ranged from less than one-half inch to eight inches, with the average about four inches. One gallon of the spray solution treated approximately seventy-five stems. The number of stems treated per acre by girdling ranged from ten to thirty, with the average of about fifteen. The number of stems treated by spraying ranged from seventyfive to one hundred fifty, with the average of about one hundred ten per acre. To check die results of the treatments, a cruise was made of - the compartment in November 1952, dividing the treated stems into four degrees of results: dead, dying, ab normal and not affected. Table 2 is a summary of the results of the treatments, showing the number and percentage of stems treated, the total number of stems and the cost per stem treated. Tables 3 and 4 show the results of the individual girdling and spraying treatments. The project from the standpoint of releasing established pines was very beneficial A total of ten thousand four 12 5 2 0 6 L ooking up a t p in e saplings and dead branches o f overtopping hardwoods. hundred ninety-four trees were released; seven thousand one hundred twenty-eight by spraying and.three thousand three hundred sixty-six by girdling. From the initial observations o f this project it appears that the com bination treatment gave more complete results in timber stand improvement than would either method by itself. Although the costs per acre for the individual treatments were lower than that o f the combination, it only cost an additional one dollar and eighty-nine cents to re lease an additional seven thousand one hundred twentyeight trees by spraying, after the compartment had been girdled. T o girdle or lop this smaller material would have m artially increased the girdling cost, not to- mention the probability o f sprouting, if no chemical was used. To spray the larger trees instead of girdling would increase the TABLE I Summary o f Co st Study T im ber Stand Im pr o v e m e n t Pr o je c t Ite m '-- H ours Cost . C ost/A cre Girdling la b o r ------------Girdling supervision____ Total girdling cost---------- Spraying lab o r----------- -- Spraying supervision-------Diesel oil (220 g a L )----2,4,5-T (6 g a L ) ----------- Total spraying cost_____ Total girdling c o st_____ Total spraying c o s t____ Total cost ___________ 93.0 $ 69.75 42.5 27.90 $ 97.65 $ 0.99 79.0 $ 59-25 32.0 21.76 28.16 78.60 - $187.77 $ 1.89 $ 97.65 187.77 $285.42 ' $ 2.88 spraying cost materially above the cent and one-half per stem, hence higher costs per acre. The results of this project show that over ten thousand pine trees were released at the nominal cost of two dollars eighty-eight cents per acre by using a combination treat ment of girdling all hardwoods over eight inches d. b. h. and spraying all hardwoods under eight inches d. b. h. that were over-topping and/or interfering with established pine reproduction. TABLE II Su m m ary o f Resu lts o f T reatm ents Item 1 Acre 99 Acres Total hardwood stems - _____________ 868 - 85.932 Total hardwood stems treated__________ 136 15,444 ' Percent of hardwood stems treated_:____ 15.6 15.6 Total hardwood stems sprayed__________ " 121 11,979 ; Percent hardwood stems sprayed_______ _ 13.9 13.9- Cost per stem sprayed__________ _____ : $.015 : $.015 Total hardwood stems girdled______ - ___ - 15 ` 1.485 Percent hardwood stems girdled______ - 1.7 1.7 Cost per stem girdled_________________ $.065 $.065 .' TABLE HI Summary o f Spraying T r ea tm en t T im ber Stand Im pr o v em en t P rotect Item Total hardwood stems sprayed__________ Total dead _________________________ Percent d e a d _____ ______ ___________ Total dying _______________ ________ Percent d y in g _______________________ Total abnormal______________________ Percent abnormal ______ '________ ____ Total no effect______________________ Percent no effect_____________________ 1 Acre 121 101 83.5 5 4.1 15 12.4- 0 0 99 Acres 11,979 . 9,999 83.5 495 ` 4.1 1,485 12.4 0 0 1953 Observations o f R esu lts: In the spring of 1953, several weeks after the vegetation had leafed out, an inspection was made of the results, Many o f the treated stems which had been reported as 5207 Dow n to Earth, W in ter, TABLE IV Su m m a r y o f G irdling T r e a t m e n t T imber Stand Im provem ent Pro ject Item ________________________ 1 A ere 99 A crti Total hardwood steins girdled___________ Total d e a d ---------------- 1--------------------- Percent d e a d -------------------------------------Total dying__________________________ Percent d y in g -----------------------------------Total abnormal_______________________ Percent abnorm al_____________________ Total no effect ______________ !________ Percent no effect______________________ 15 14 93.3 0 0 1 6.7 0 0 1,485 1,386 93-3 0 0 99 6.7 0 0 dead the previous fall had leafed out over one-half of the crown surface. A much smaller number had stump sprouted; had partially leafed out in the crown; or had leafed out from the stem. Over fifty percent of die stems treated showed no visible effects of die spraying what soever, and had seemingly recovered. A late summer inspection of the compartment showed chat many of the trees which had seemingly recovered at a low percentage concentration, by volume, of 2,4,5-T is sufficient to kill or seriously affect the overtopping smaller hardwoods in timber stand improvement work in-.south ern pine-hardwood stands. ' -. Sum m a ry : The results show that after eighteen months, over fifty Pine sapling (foreground) , dead sw eet gum ( im m ediately behind p in e ), and dead oak and sweet gum at le ft. Leaves are dead on tree at le ft. TABLE V Com pa riso n o f Spraying Resu lts T im ber Sta n d Im pr o v em en t P r o tect -Ite m _________ - ' PERCENTAGES Pall Spring Sum m er 1952 1 9 5 3 ' 1953 Percentage dead __________ 31.4 52.2 Percentage dying _________ 4.1 1.6 38.2 Percentage abnormal____ __ 12.4 14.0 4.0 Percentage no e ffe c t_______ ____ 0 53-0 5.6 T reating girdled hardwood w ith botai spray. the time of the spring inspection, had died or were dying. Some few had recovered and showed litde visible evidence of any spraying effects. Sweet gum made up over ninety-five percent of the stems which had leafed out and had seemingly recovered in the spring and late summer inspections, and one hundred per cent of the stems which showed no spraying effects in the late summer inspection. The red oaks made up the greatest proportion of the remaining five -percent, with only minor percentages for all other species. The following table shows a comparison of the results of the spraying treatment at the various inspection periods. From the three observations of the results it appears that percent of die treated stems are dead, in many cases already fallen. Another thirty-eight to forty percent should be dead within a year. A portion of these stems may leaf out again next spring, bur this summer's results show that only a relatively few will be able to survive and. re: cover completely. In the final analysis, if the results follow present indica tions, over ninety percent of the overtopping smaH hard woods in southern pine-hardwood stands can be adequately controlled in a timber stand improvement program which is economical and easily accomplished and which produces the desired results. Formerly District Forester, District 6, Texas Forest Service, Ginroe, Texas. At present General Manager and Forester, Indepen dent Pulpwood Producers, Inc., Geveland, Texas. 14 \ 5 2 0 8 - *\> ued. On to r of 13 day3 d those died he e litter ii dustrv as a fodder supplement for beef cattle, thereby improving' meat quality and increasing quantity in less time with less food. R cferrncei 1. S m a ll, J . IC. T h e n o r m a l o f S o u th e a s t e r n F lo ra . Now Y ork: Tubl. by a u th o r (1033). li. F e ert, S. D., a n d F o x , L. E . J . A m . P h a rm . A sso c.. 41, 433 (1052). 3. P in c e s. G,, a n d T him ann. K. V. T h e H orm ones, Vol. 2. New York : A cadem ic Press (1030). 4. W'E lciiE nT , C. K .r a n d Korbig an, S . J . E n d o c n n o Jo i/i/, 5. 741 (1042). 3. Zondek, B r L a n cet, lO, 10 (103G). *. S l e e t ii, R.\ B. A g r ic u ltu r a l D e p t., U n iv e rs ity o f F lo r id a , personal com m unication. 7. A n drew s, F . N\, B eeson, W. M., a n d J o h n so n , F. P . J . A n im a l S c i.r 9 , G77 (1 0 3 0 ). 8. IYeodeb, M . G.. L acteh. U \ M., a n d F oote, P . A . J . A m . Pharm . Assoc., 41, 230 (1052). 9. IIallioax, J . E. J . In d . Eng. Chem ., 1. 206 (1900). 10. P ic k e t t , J. .\L F lo rid a E x p t. S ta . B u ll. IT (1 8 0 0 ). 11. T r en ch , R . B., a n d Abbott, O. D. F lo rid a A g r. E x p t. S ta . Tech. Bull. No. 4J4 (1048). M anuscript received F ebruary 11, 1033. determination as described by Triiog and Meyer (1). Four hours after trratment the sprayed plants showed epinasty and otlier 2.4-D elTeets. Ten hours after treatment many of the leaves had curled, and the plants were becoming recumbent. The next morn ing the plants were somewhat chlorotic and the dis tortion had increased. The following day many of the leaves had developed necrotic spots. One week after treatment the leaves on most sprayed plants had withered and those that adhered were very chlorotic and sickly in appearance. The uprooted plants remained fresh for the first day but after that they deteriorated so rapidly that by the end of the week it was not possible to obtain leaf samples. ; Figure 1 shows the fluctuations of inorganic phos L S 0-5V 3O 9 IV4I9 2.4- D Affects Phosphorus M etabolism A. J. Loustalot, M. P. Morris,. J. Garcia^ and. C. Pagin. F ederal E x p e rim en t S tation- in P u e rto R ico,1 td a y a g u e z . LS0-5S.'ia6 I V 224 A preliminary experiment in which Commelina sp. and Xanthosoma sp. were analyzed 24 hr, and 1 wk after being treated with.2,4-D (2,4-dichlorophenoxy- acetic acid) showed that the percentage of water-solu ble phosphorus in treated plants was consistently LSD-5T.-274 CV 329 higher than in untreated plants. The following ex periment was carried out to obtain additional infor mation on the effect of 2,4-D on phosphorus metabo lism. A prepared field, was divided into 12 plots each H arm t time offer freofmtrtf (Horn) 5 2 x 2 4 f t and planted to a variety of white beans, Blanca Bonita (P.R. 1632).. The experimental design consisted of 3 treatments each replicated 4 times in . randomized'blocks. P ic. I. Levels of inorpm le phosphorus (ppm of dry m at ter) In bean plants analyzed a t various intervals s f te r treat m eat w ith 0.1% solution of sodium 2.4-D. phorns in the leaves, stems, and roots, respectively, of When the plants were about 15 in. high and had treated, check, and uprooted plants. started to set fru it 1 plot in each of the 4 replications The data obtained at each sampling period, ex was sprayed with OJ.% aqueous solution of sodium pressed as parts of inorganic phosphorus per million 2.4- D. The plants in another plot of each replication parts of dry material, was analyzed statistically by were uprooted at the same time and left lying on the the analysis of variance and the least significant dif ground-to die gradually. The third plot in each repli ferences between treatments determined. cation was left as a control. Two rows of unsprnved or Four hours after treatment there was no appreciable undisturbed plants were left as borders around each difference in the amount of inorganic phosphorus in plot. The treatments were started at 6 A.xr., and sam the leaves and roots of treated and check plants, but ples of 100 plants were taken from oneh replication the stems of treated plants had a significantly higher of all treatments at 4,10, 24, and 48 hr and 1 wk after amount than the checks. The uprooted plants had treatment. somewhat less inorganic P than the treated plants in The leaves, stems, and roots were separated, fresh all 3 organs. Ten hours after treatment inorganic P and dry weights obtained, and a composite sample of had dropped in all organs of all treatments except in 300 g of dry powdered tissue from each replication the stems of uprooted plants, where it was somewhat was nnalyzed for inorganic phosphorus. Aliquots of a higher; but in roots, stems, nnd leaves of treated hot water extract of the dry tissues were clarified with plants it was higher and significantly more so in the 0.5 g charcoal and used fo r inorganic phosphorus roots and stems than in the corresponding organs of 1 Ailmlnlalpnsl hy th e O tH re of E xperim ent S tatio n s, Aerl- check plants. The samples taken 24 h r afte r treatment cultural Itew urch A dm inistration. I'SDA. showed a sharp rise in the level of inorganic P in roots November 20, 1933 5210 ,, 4^ - 0009995" 00W2156862 pqpqcI7Mnn tefa- r* items, and leaves of treated plants; the level in the roots and leaves was significantly higher than in the checks. There was also a rise in the inorganic phos phorus content of stems and roots of cheek plants, hut it was less than in treated plants. The leaves of check plants showed no increase as did the leaves o treated plants in this phosphorus fraction, indicating that the most pronounced effect o 2,4-D on phos phorus metabolism occurred in the leaves at this time. Forty-eight hours after treatment the level of in organic P in^leaves, stems, and particularly roots of treated plants was significantly higher than in check plants. In the roots, stems, and leaves of uprooted plants it was about the same as in the check plants. One week after treatment inorganic P in the roots of treated plants had increased significantly and this co incided with a sharp decline in the leaves, indicating that it may have been translocated from the leaves to the roots. There was practically no change in the inor ganic phosphorus fraction in treated stems. Although by this time the level in the leaves and stems of check plants also declined, there was no corresponding in crease in the-check roots as there was in the treated roots. Inorganic P in leaves and stems of treated plants fluctuated in. most instances like that in the check plants, but .this fraction was consistent, and at most sampling dates, except the first, significantly higher in roots, stems, and leaves of treated plants. This experiment provides a clue to the mode of ac tion of 2,4-D, e.g., it may inhibit or interrupt the phosphate metabolism in the plant. These data and the fact that very small amounts of 2,4-D produce drastic effects suggest that 2,4-D may inhibit or poison the enzyme or system responsible for the hydrolysis or synthesis of the high energy phosphates. Reference--- I . T n e n c , a n d M etf.h. In d . E n g . C h em . A n a l E d ., X. I3G (1020). M anuscript received Ju n e IS. 1933. Colorimetric M ethod for D eterm ination o f Aureom ycin, Carbomycin, Erythromycin, and Terramycin in A queous Solution D. Perlman Squibb Institute fo r Medical Research, N ew Brunswick, N ew Jersey Wo have observed that the acid hydrolvzates of Aureomvein ( I ) , carbomycin (2), erythromycin (.?), and Terramycin ( i) react with the arsenomolyhdnte reagent to produce blue colored complexes. The opti cal density of the color formed has been found to he a function of the quantity of antibiotic present. Satis factory results have been obtained with the following procedure. Aliquots containing from 10 to 40 |ig of antibiotic are added to colorimeter tubes and the solution evapo rated to dryness using an air jet. Two milliliters of G-Y II2S 0 4 and 1 ml of arsenomolvbdatc reagent (Nel son's [5] reagent diluted with 2 parts of distilled water) are added. The tubes arc plugged with loose fitting corks and placed in a boiling water bath. A fter a 15-ruin heating period the tubes are cooled to room temperature and the contents diluted with 5 ml of dis tilled water. Color intensity is determined using a photoelectric colorimeter equipped with 660 mp filter. A series of tubes containing known quantities of the Iantibiotic are prepared and treated simultaneously with the tubes containing unknown quantities of the antibiotic. The values obtained with this series of known solution are used to calculate the constants of Beers' law and to standardize the determinations. The sensitivity of the method varies somewhat with the particular antibiotic under consideration. The practical working range for Terramycin and Aureo mycin is from 2 to 40 pg/tube; fo r erythromycin it is 5-S0 pg/tube; and fo r carbomycin it is 10-160 pg/tube. Smaller quantities may he determined if only 1 ml of distilled water is added after the heating period, and micro cells are used to determine the opti cal densities. Apparently the hydrolysis with 6 N acid is necessary to obtain maximum sensitivity of the method, and use of more dilute acid resulted in re duced sensitivity. Only Terramycin will reduce the arsenomolyhdnte reagent without a preliminary hy drolysis, and in this instance the working range has been found to be from 20 to 160 pg/tube. Some of the data collected in analyzing aqueous solutions contain ing known quantities of Terramycin are summarized in Table 1. TABLE I A n a l y sis o p S o lu tio n s to r T e r r a jit c m ' C o n t en t Solution T erram vcin added* pg/m l Terramycin foundt pg/m l Distiiicd water 2% Glucose 2% Starch 0 10 30 100 0 10 30 0 10 30 0 9.7; 9 .9 ; 10.2 29.8; 30.7; 30.7 98.6; 99.7; 101.5 0 9.5 ; 9.7; 10.0 27.7; 30.6; 30.6 0 9.1; 9.7; 9.7 28.8; 29.0; 29.6 T o r m m r c in h y d ro ch lo rid e w.w used in thce h tudlea. A ll an aly ses nro presented in term s of th e free hnse. | A ntibiotic extracted from aqueous solution w ith m ethyl Isotiutvi ketone. This method cannot be applied directly to solutions containing carbohydrates and other substances which react when heated with the arsenomolyhdnte reagent. These four antibiotics may be separated from carbo hydrates by extraction from aqueous solution (pH 7.2) into chloroform, amylacetate, -butanol and methylisobutyl ketone. All the antibiotic has been re covered in the solvent phase when equal volumes of the solvent and aqueous solution have been used. S c ie n c e , Vol. 11S 5211 000999C, 3*1 5212 A^ DOW 321(529 & ro ono Vj>4 o Toxicity of 2,4-Dichlorophenoxyacetic Acid and 2,4,5-Trichlorophenoxyacetic Acid A Report on Their Acute and Chronic Toxicity in Dogs VICTOR A. DRILL, Ph.D., M.D. AND TOMIHARU HIRATZKA, M.D. DETROIT 5213 0002070 D O W 32163U Reprinted from the .1. M. A. .Ireliii-es of Industrial Hygiene aud Occupational Medicine January 1953, Vol. 7, pp. 61-67 Copyright, 1953, by American Medical Association TOXICITY OF 2,4-DICHLOROPHENOXYACETIC ACID AND 2,4,5-TRICHLOROPHENOXYACETIC ACID A Report on T h tir A cute and Chronic Toxicity in Dogs VICTOR A DRILL. M i.D , M.D. AMO TOMIHARU HIRATZKA, M.D. * ocntoiT BO T H 2,4-dichIorophenoxyacetic acid (2,4-D ) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T ) may act as plant hormones and herbicides 1 and are now being fairly widely used for the control of certain types of weeds on farm lands. They are being sprayed from airplanes on many acres of specialized farms and grazing land and are present in trademark preparations sold at retail for home gardeners. W ith the widespread use of these herbicides, possible toxic effects are important, as accidental ingestion may occur or residues may be inadvertently encountered in food. In an earlier report, 2,4-D was mentioned as being nontoxic to animals and man when administered orally.1*'11 However, the injection of large doses of 2,4-D in animals produced symptoms similar to those seen in clinical myotonia.1 More detailed observations were reported by H ill and Carlisle who studied the acute and subacute effects of 2,4-D in various species of animals.* There have been no reports concerning the toxic properties of 2,4,5-T. The present study concerns the acute oral toxicity and chronic oral toxicity of 2,4-D and 2,4,5-T in dogs. EXPERIMENTAL PROCEDURES Adult mongrel dogs of both sexes were used. Most of the animals were housed in the laboratory for a period of two to three months before the study was started. During this control period they were immunized against distemper with the Green vaccine. The dogs were fed a standard stock diet (Friskies) ad libitum. Both 2,4-D and 2,4,5-T were commercial materials, with a purity of 98.5 and 98.9% and freezing points of 132.8 and 150.6 G, respectively. In the acute studies, the calculated dose of 2,4-D or 2,4,5-T was administered as a single oral dose in capsules. The dogs were observed for a period of 14 days, at which time the sur- This study was supported by a grant from the Dow Chemical Company. From the Department of Physiology and Pharmacology and the Department of Pathology, Wayne University College of Medicine. 1. (a) Hildebrand, E. M .: War on Weeds, Science 103:465-468, 1946. (b) van Overbeek, J., and Velez, I .: Use of 2,4-DichIorophenoxyacetic Acid as a Selective Herbicide in the Tropics, Science 103:472-473, 1946. (r) Smith, F. G.; Hamner, G G, and Carlson, R. F .: Control of Ragweed Pollen Production with 2,4-DichIorophenoxyacetic Acid, Science 103:473-474, 1946. (<f) Marth, P. G, and Mitchell, J. W .: 2,4-Dichlorophenoxyacetic Acid as a Differential Herbicide, Bot. Gaz. 106:224-232, 1944. 2. Bucher, N. L. R .: Effects of 2,4-Dichlorophenoxyacetic Acid on Experimental Animals, Proc. Soc. Exper. Biol. & Med. 63:204-205, 1946. 3. Hill, E. V., and Carlisle, H .: Toxicity of 2,4-Dichlorophenoxyacetic Acid for Expcrimental Animals, J. Indust. Hyg. & Toxicol. 29:85-95, 1947. 5214 0002071 \' 2 vivors were autopsied. In Ihe chronic studies, the 2,4-D or 2,4,5-T was administered orally in capsules five days a week over a 13 week period. Each capsule was imbedded in a 4 to 5 gm. piece of commercial canned dog food, which the animals consumed readily, thus avoiding the continued trauma of the stomach tube. During the study the animals were weighed twice a week. Control blood counts were taken before the administration of the drug, on the 30th day, and on the 90th day at the completion of the study. Hemoglobin was determined with a photoelectric colorimeter. Upon death or at the completion of the study, the animals were autopsied. Tissues were taken from the lung, heart, liver, kidney, adrenals, spleen, thyroid, and ovary or testes, fixed in 10% formalin, and stained with hematoxylin and cosin. Table 1.--Effect of Single Oral Doses of 2,4-DicMorophcnoxyacctic Acid and 2,4j-Trichlorophenoxyacctic Acid in Dogs Dot Xo.* IF .... 2 M... SF.... 4 M... 1M... 4 F.... 7 F.... 8 F-- 9F.... 10F.... U F.... 12 M... 13M... 14 M... 15 F.... 15F.... 17F.... 18 F.... 19 M... 20 M... 21F.... M... Dos. M*./K*. 250 250 100 100 so LWosse,igKhgt. 2,4'D --0.1 -0.8 --1.0 -2.0 --0.4 --2.5 --2.0 --1.7 --2.4 0.0 +0.1 +0.1 2,4,5-T --1.1 --1.4 --(U --1.0 --1.0 -0.4 +0.3 --LS +0.1 --0M Symptoms* + ++ +++ ++ ++ + 0 + 0 0 0 0 + + 0 0 0 0 0 0 0 0 Death Dayt 2 3 8 7 4 8 0 9 s s s s 3 3 S 7 8 8 S 3 8 8 Mortality /t 3/3 2/4 0/3 1/1 1/1 1/4 0/4 *F In this column meant lemsle: M, mat*. t Column Indicates general severity ot symptoms cs discussed In text. 0 means no chance from normal. ; S in this column means survived It day test period. HESULTS I. A cute Oral Toxicity.-- (a ) M ortality: The effect of single oral doses of 2,4-D or 2,4,5-T on survival for 14 days is shown in Table 1. The deaths were delayed and occurred two to nine days after the compounds were administered. The oral L .D .5o of 2,4-D was approximately 100 mg. per kilogram of body weight; for 2,4,5-T it was in the range of 100 m g./kg. or higher. ( b ) Body W eight: The large oral doses of 2,4-D or 2,4,5-T produced a decrease in body weight (Table 1 ). Such animals developed various degrees of anorexia, and those that died refused even canned dog food toward the end of their survival. * 4. Evelyn, K. A .: A Stabilized Photoelectric Colorimeter with Light Filters, J. Biol. Chem. U S :63-75, 1936. O O CO ro 03 CO I i I mi: oftc we. leg. ap ant ar qui not In : sta: par in r pair up tox: of obst 14 . liini wer of a in ti ( lung: 2,4,1 dogs and hepc erat: such whic I of 2, The admi dogs ( aniir. begar 5215 0002072 U W yj U, 3 ifed orally 4 to 5 gm. .-oiding the ed twice a e 30th day, ed with a imals were tyroid, and (c ) Sym ptom s: In dogs that died the effect produced by 2,4-D varied from a mild ataxia and stiffness in the hind legs to a definite myotonia. Initial signs were often noted six hours after the oral administration of 2,4-D. At this time the animals were more quiet than normal, and a slight ataxia was occasionally present. The hind legs were always affected first; the forelegs later or not at all. There was usually a progressive increase in spasm in the hind legs associated with increasing ataxia, and some dogs spontaneously extended the hind limbs in a spastic movement lasting a few seconds. The knee jerk was either normal or hyperactive. and M ortality i/J 3/3 V* In the early stages of acute poisoning with 2,4-D, the animals were usually quiet. If the animal was lifted and made to walk, the spasm and ataxia were clearly noticed, but after it had walked for a while, the spasm and ataxia tended to decrease. In the later stages of intoxication, the animal, when placed on its feet, was unable to stand and would then sit or lie in an awkward position with spastic limbs marked t particularly by spasm of the hind legs. Other dogs stumbled and rolled on the floor in attempts to right themselves. An occasional dog showed evidence of irritation or pain when the skin over the back of the neck was grasped. Some dogs, when held f up by the shoulders, would show extension and crossing of the hind limbs. As the toxic effects of the drug progressed, the dogs eventually refused the various types of food offered. Occasionally, sneezing, rubbing of the eyes, and diarrhea were observed, but not vomiting. 0/3 Such signs were also seen in one dog that survived a lower dose of 2,4-D for 14 days (Table 1 ). In this instance the changes were relatively mild and were limited to spasm of the. hind legs and a slight ataxia. 1/1 Marked effects were not observed in the dogs receiving 2,4,5-T. The changes 1/1 were limited to a slight or moderate stiffness in the hind legs, with the development 11* of ataxia in the two dogs receiving the highest doses. N o symptoms were evident in the one dog that died after a single dose of 100 m g./kg. (Table 1). 0/4 .Tom normal. a of 2,4-D e delayed T he oral :ight; for oduced a egrees of d of their r ( d ) P athology: Changes observed were limited to the gastrointestinal tract and lungs. Four dogs (1 , 2, 4, and 6 ) receiving 2,4-D and two dogs (13 and 17) fed 2.4.5- T showed mild to diffuse redness of the mucosa in the small intestine. In some dogs pneumonia was present. Histologically, the above dogs showed some necrosis and inflammation of the intestinal mucosa. One dog (1 3 ) had a moderate, diffuse hepatic necrosis. Two dogs (13 and 16) had a mild degree of renal tubular degen eration. In most dogs that died the findings were limited to nonspecific changes, such as hepatic congestion. In many cases death was. apparently due to pneumonia, which followed the development of anorexia, weight loss, and myotonia. I I . Chronic O ral Toxicity.-- (a ) M ortality: All dogs receiving 2, 5, or 10 mg. of 2,4-D or 2,4,5-T per kilogram of body weight survived the 90-day test period. The dogs receiving 20 m g./kg. of these compounds died during the study. The administration of 20 mg./kg. in divided doses twice a day produced death in both dogs receiving 2,4,5-T and in one dog receiving 2,4-D (Table 2 ). ( b ) Body W eigh t: The only significant change in body weight occurred in the animals that failed to survive the study (Table 2 ). Loss of weight in these dogs began 7 to 12 days before the death of the dog. s, J. Biol. (c ) General Sym ptom s: Dogs tliat survived the oral administration of 2,4-D or 2.4.5- T for 90 days were free of any symptoms. 5216 002073 4 The three dogs that died while receiving the highest dose of 2,4-D showed signs differing somewhat from those observed in the acute studies. The dogs were quiet, weaker, and less responsive than normal. Muscle tonus was higher in the hind legs, particularly on passive extension. The hind legs were held more stiffly than usual when walking, and a slight ataxia was present. During the last two to three days of their survival two of the dogs showed difficulty in chewing or swallowing and eventually even refused a small bolus of canned dog food (usually readily con sumed). There was also some oozing of blood from the gums and buccal mucosa. T able 2.--Body Weight and Survival of Dogs F.ed 2,4-Dichlorophenoxyacetie Acid and 2,4S-TrichtoTophcnaxyacetic Acid for 90 days Dor Xo.' 23F.................. 24 F.................. 25X................. 28F.................. 27 X................. 28F.................. 29X................. X ................. 33 X................. 32 X................. 33F................. 34 X................. 35X................. 38F.................. 37X................. 38F.................. 39X................. 40F.................. 41 X................. 42 F............... .. 43X................. 44 F.................. 43X................. 46F.................. 47F.................. 43X................. Dow, Mr./Kf. Initial, K>. 2,4-D 14. 93 8.8 15.2 M 12.0 14.7 11.8 174 13.0 9J 124 13.4 2.4>T 74 124 114 34 84 124 123 13.0 93 114 10.5 14.9 10.9 Welrht x- Flual, Kf. 18.4 10.9 94 13.3 93 124 14J 114 17.3 13.7 54 10.1 9.7 7.7 14.5 12.7 10.0 8.4 124 U4 12.2 93 94 7J 12.7 U our.. K. +3.7 +1.8 +2.7 -1.4 0.0 +04 +04 +0-3 +0.8 +0.7 --1.9 --24 --3.7 +0.4 +3.4 +1-4 +14 --04 +04 --04 --04 -04 --to -34 --24 --2.7 * F In thii column menu female: X. mala. t Daily dose divided and administered one-bait In a. m. and one-balf In p. m. 1S In this column meana survived 90 day test period. ' Death Dart S s s s s s s s s 49 13 29 S 8 a s s s s s 49 75 11 59 In dogs that died while receiving 2,4,5-T, the prominent effects were weakness,' slight stiffness in the hind legs, difficulty in swallowing food, and, in one dog, bleeding from the gums. (d ) Blood Count: The administration of 2,4-D or 2,4,5-T did not have any significant effect on the hemoglobin, red cell count, or total white cell count of animals that survived or died during the study. The differential blood count remained normal in the surviving animals. In three animals that died a terminal fall in the percentage of lymphocytes was observed (Table 3 ). ( e ) Organ W eights: There was no significant change in the weight of the thyroid gland, adrenal gland, heart, liver, or kidney in animals that survived the 90-day period of study. Iirtw o of the throe dogs that died during the administration a O of 2 was ( redn dog the : ' 03 ro T ab: 0 3 Dor S t 03 0 3 23F........... ! 24F........... ) 33F........... 37X............ 33X. 33F.. 31 X. 35 X. 43X. 48F. 47F. 43X. F to tbl* t Dally dost To conserve (3 did nc and ! This : death demur early : 5217 0002074 wed signs ere quiet, hind legs, han usual hree days wing and ;dily con1 nutcosa. -tcid and D eath D ajt ! 8` 8 8 8 8 s s 49 18 zs 8 8 8 8 .g 8 8 8 11 59 eakness, )ne dog. have any count of rem ained ill in the it of the .ived the listration s of 2,4-D, there was a slight increase in heart and kidney weight. A sim ilar change was noted in two of the four dogs that died as a result of 2,4,5-T. ( / ) Gross P athology: T w o dogs receiving 2,4-D (26 and 34) showed areas of redness in the duodenum. W ith the adm inistration of 2,4,5-T there developed in dog 42 a diffusely reddened duodenum and jejunum , while in dog 48, autopsied on the 59th day, a generalized icterus was demonstrated. c C T able 3.-- E ffect o f 2,4-D and 2,4J - T on H em oglobin, T otal Blood, and D ifferential C ounts cc tc Do* No.* Dom . U *./K *. Day H b. Om. % BBC, UIL WBC Differential Count ai ' N eut.. Lym ph., M on., B as., EosId., D eath % % % % % Day o c C ontrol D o n a t ........ < 0 13J0 5.72 17X00 66 a 00 6 a 13.82 5.93 15X00 61 a 0 0 7 ,, 90 11.70 0.09 11X50 n 27 1 0 0 a r .................. i a t ........ 0 11X0 9.01 10X00 a 11 9 0 0 SO 14.71 6.66 13,150 n ii 70 1 a 14.SJ 9.19 7X00 79 a 0 0 0 a. 0 9 .a 9.03 15X00 67 28 1 0 10 10.79 5.63 11X00 56 1 1 0 1 ,, 12.74 XZ 14.700 n 18 1 0 0 17 M................. 0 11*9 5.93 14X00 55 39 1 0 4 a 13.31 5.78 14,900 a a 407 90 11.99 9.47 9 ,4 a 4 51 1 0 0 - 14-D S U ................. a t 0 10.41 5.01 11X00 61 17 5 0 4 10 11.44 606 15,400 66 a 4 04 90 n _ a (J5 m a TO a 0 0 8 1 . ....... at 0 11.19 5.5 14X50 54 41 i 01 a 14.49 5X9 9,7a 47 tt 4 9 9 49 14J 9 5.67 15X00 79 14 7 0 0 49 MU........ 0 lia m is x a a 44 4 0 1 19 ISM................. a 9 14.90 147 11,150 79 11 i 0 12 a 14X9 1M 9XS a 17 4 0 i a 49 U........ 1.4X-T 0 1194 I S 13,4a n U 0 10 .a 194 199 15,450 77 U 9 0 7 49 U 1X7 X a 91 41 0 0a 49 T _....... 0 1149 7.10 19X50 a a 1 0 a 14.M I S 11X00 n a 0 0 1 79 1 47 r ......... - 49 U................. 0 12.44 i n nxa a a 4 0 1 11 0 1149 9X1 ixa n 44 0 1 mm a US in 14X50 n 19 4 0 0 a 11.97 114 7X79 M 9i0 0 a ~~ *1* to th li eolum a maana fem ale: U . m ale. t Dally dose divided u d edralnlstertd one-belf In *. m. end one-belt la p. m. To comerve iptce, only date on control dots end tbe hl*hest dole* a n (Iren. ( g ) Microscopic C hanges: The heart, lungs, thyroid, adrenal, ovary, or testes did not demonstrate any significant changes. Three of the dogs receiving 2,4,5-T and two receiving 2,4-D showed an occasional area of focal necrosis in the liver. T his finding, however, was not related to either the dose of the compounds or the death of any animal (Table 4 ) and was judged to be of no significance. T he duo denum of dogs 26, 34, 42, and 48 showed varying degrees of hyperemia and some early infiltration of cells in the mucosa. T he subinucosa and serosa were norm al. 0002075 5218 6 A slight increase in the number of casts in kidney sections was noted in some dogs (Table 4 ) , but again this was unrelated to dosage and was of doubtful significance. COMMENT In the acute studies, single large oral doses of 2,4-D administered to dogs may produce signs characteristic of clinical myotonia. This effect of 2,4-D was similar to that observed after various experimental species had received injections.1** In addition, some dogs showed signs of irritation and pain when the skin of the back of the neck was grasped, which may indicate meningeal irritation. The extension and crossing of the hind limbs, when lifted by the shoulders, may point to a spinal of T able 4.-- H istological Changes in L iver and K idney o f D ogs F ed 2,4-D or 2 ,4 J -T fo r 90 D ays Doc No. 23............................. 24............................. 25............................. 26............................. 27............................. 26............................. ............................. 30............................. 31............................. 31............................. 33............................. 35............................. 36............................. 37............................. 38............................. 39............................. 40............................. 41............ .......... 42............................. 43............................. 44............................. 45............................. 46............................. 48................... ......... D o. M f./K c. Son Son t 1 5 5 10 10 10 so 20 so Non* Non* S s s s 10 10 10 20 so so Llvtr D eath D ar C once* tlo n S.4-D 0 0 0 0 1+ *+ 0 0 1+ 0 49 1+ ss s+ Focal X ecroit* 0 0 0 0 . 1+ 0 0 0 0 0 1+ 0 S.4.4-T ,, M .. 49 7# so 0 0 0 0 0 0 + 0 1+ 1+ 1+ *+ 0 0 0 0 0 1+* 0 0 0 1+ 1+ 0 * Al*o a l + fibroil*. T Iuuc* e n d e d on bast* o t 0, 1 + , S + , t+, and *+ . Ca*t* 0 0 0 1+ 0 1+ 0 0 0 0 0 0 Tab. Deem. 0 0 0 0 0 1+ 0 0 0 0 0 00 0 1+ 0 0 00 *+ 1+ 1+ 0 00 00 00 1+ 00 central lesion. Other' effects occasionally observed were sneezing, rubbing of the eyes, and diarrhea. The effects of 2,4,5-T, previously studied, were not as severe, being limited to stiffness in the hind legs and ataxia. The higher doses of either compound produced anorexia and weight loss. Although initial effects may be seen six hours after the compounds are given, the syndrome developed slowly in the dogs, and death was delayed, occurring several days after the administration of the compounds. In the chronic studies, the dogs survived doses of 2, 5, or 10 mg. of 2,4-D or 2,4,5-T per kilogram of body weight without apparent ill effects. However, the daily oral administration of 20 m g./kg. of 2,4-D produced death in three of four dogs within 18 to 49 days. This delayed death may indicate a cumulative effect of 2,4-D . The signs observed in animals dying from the 20 m g./kg. dose of 2,4-D a O Z ZP to Ci CO C" diffe tratii ness, All f and sliglr dose WitI E cant of th 2,4,5 perip leucc dogs days, perip L occas chror. signii 2,4,5organ t: appre produ oral L mg. p spastic A! 2,4,5-' sympt T1 I died, ; study. gums, swallo in the De to patl 5219 0002076 UOW 321636 fI 7 some dogs ignificance. i differed somewhat from those seen in the acute studies. With the chronic adminis I tration, the animals exhibited chiefly a stiffness of the hind legs and ataxia, weak ness, difficulty in chewing or swallowing, and occasionally bleeding from the gums. All four dogs which received 20 mg./kg. of 2,4,5-T succumbed between the 11th dogs may ras similar ions.1'* In the back of and the 75th day of the study. Toxic effects in these animals consistently included slight muscle spasm and difficulty in swallowing food. Thus, the chronic oral toxic dose for 2,4,5-T in dogs seems to be in the same range'as that obtained with 2,4-D. With both drugs, there was a terminal loss in body weight. ension and Dogs surviving the chronic doses of 2,4-D or 2,4,5-T did not show any signifi 1 spinal or cant change in hemoglobin, total blood counts, or differential blood count. In three of the dogs (64. 57, and 105) which died while receiving 20 mg./kg. of 2,4-D or fo r 90 Days 2.4.5- T, there was a definite decrease in the percentage of lymphocytes in the peripheral blood. Hill and Carlisle * have observed a fall in the polymorphonuclear l-u--t-j-------- --> Tub. Dcsn. leucocyte count in one dog and a decrease in the per cent of lymphocytes in two dogs to which 50 mg./kg. of 2,4-D was administered intravenously each day for six days. In mice which received chronic injections there was no alteration in the 0 peripheral blood picture.1 0 0 Large doses of 2,4-D administered intravenously or, in the present study, orally 0 0 occasionally produced some necrosis in the liver. However, the administration of 1+ chronic oral doses produced only a slight and inconsistent focal necrosis of doubtful 0 0 significance (Table 4). Deaths during the chronic oral administration of 2,4-D or < 2.4.5-T were not correlated with significant lesions in the liver, kidney, or other 0 0 organs examined. 0 SUMHABY The acute oral L.D.M of 2,4-dichlorophenoxyacetic acid (2,4-D) in dogs was s 0 approximately 100 mg. per kilogram of body weight. Doses in this range or higher 0 produced a definite myotonia accompanied by anorexia and weight loss. The acute 0 0 oral L.D.so for 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was in the range of 100 1+ mg. per kilogram or higher. Such toxic doses produced only signs of a mild 0 0 spasticity. 0 0 All dogs survived the oral feeding of 2, 5, or 10 mg. per kilogram of 2,4-D or 0 2.4.5- T 5 days a week for a period of 90 days. These doses did not produce any 0 symptoms or changes in body weight, organ weights, or blood count. Three of the four dogs receiving repeated doses of 20 mg. per kilogram of 2,4-D t died, and all four dogs receiving 20 mg. per kilogram of 2,4,5-T died during the ng of the study. Toxicity to 2,4-D at the dosage level was accompanied by bleeding from the as severe, gums, necrotic changes in the buccal mucosa, and some difficulty in chewing and s of either i swallowing, but there was only little evidence of clinical myotonia. A terminal fall ay be seen in the percentage of lymphocytes was observed in three of the animals. ivly in the Death during the repeated administration of 2,4-D or 2,4,5-T was not related tion of the to pathological changes in the liver, kidney, or other organs examined. f 2,4-D or wever, the ce of four e effect of : of 2,4-D Printed and Published in the United States of America 0002077 5221 I 4' V. '/ *i& - , v. ; ` I-? f. ,*'y SUSPECTED POISONING O F DOGS FROM EATING GRASSES TREATED WITH 2,4-D D . L.-Baker, O.Y.M., F. K. Ramsay, D.Y.M., M.A., and E. P. Sylvester, B.S., M.S., Ph.O. Iowa State College Ames, Iowa Frequently dogs are admitted to the vet erinary clinic with the history th at the owner feels that the animal has been poisoned from the effects of having con sumed grass treated with 2,4-D. I t has been commonly observed th at dogs, on oc casion, will eat various quantities of grass. We were unable to find any information in the literature concerning field cases of the toxicity of 2,4-D-treated grasses in dogs. Hill and Carlisle1 have noted that, by intravenous daily administration of from 25 to 200 mg. of 2,4-D per Kg. of body weight, effects ranging from sub acute intoxications to death may be pro duced. They further noted that dogs in jected with 2,4-D showed a considerable susceptibility to the development of liver damage. Other work concerning 2,4-D poisoning in various animals has been done by other workers.3'* Since there is so little information available on 2,4-D poisoning in dogs, in which the 2,4-D was consumed on treated grasses or administered per os, the following trials were conducted. Individual, heavy stands of lush grow ing bluegrass, orchard grass, timothy, and quack grass were sprayed with the water emulsion ester (butyl ester 4 Ib./gal.) form of 2,4-D, a t the rate of 4 pounds per acre. These are the most common grasses that dogs are most likely to eat. The spraying was a t least twice as heavy as ordinarily recommended and sufficient to cause foliar damage to the grasses. The spraying was done on June 18, 1952, and the grasses were harvested in four sepa rate lots (112 Gm. each) 48 hours later, on June 20, 1952. These grasses were chopped in small pieces and mixed in pro- ' Dr. Baker Is aaociato protestor of veterinary med icine. Dr. Bamsey is associate' professor of veterinary pathology in the Division of Veterinary Medicine and Dr. Sylvester is extension professor of botany and plant pathology- K* n * portioS S R W y j y with fresh horsemeat and dog meal. This mixture was fed to two healthy one-year-old mon grel dogs, weighing 20 to 25 pounds each, in .equal .amounts, in three consecutive feedings, tw o on June 20 a t morning and evening meals, and the last one a t the morning meal on June 21. Apparently the palatnfoHity of the food was not altered appreciably, as the dogs immediately con sumed all of the food. The dogs were observed but no change in attitude or signs of any ill effects could be detected a t any time within 96 hours following the first meal. On June 24, both animals appeared to be in excellent health, and a subsequent trial was instigated. Each animal was given 5 cc. of the same ester concentrate of 2,4-D orally in a gelatin capsule. This dosage represents approximately 500 mg. of 2,4-D per Kg. I t is noteworthy that this was a single dose given per os. No clinical deleterious effects were observed a t any time within 96 hours following this oral administration. On June 28, one dog was sacrificed. Necropsy examination of this animal failed to reveal any macroscopic lesions. The other animal, under daily observation, remained in apparently excellent health for the fol lowing 82 days, a t which time these experi ments were considered concluded. We have not arrived a t any definite con clusions with this meager information. However, in most instances of suspected 2,4-D poisoning entered a t th is clinic, some other cause has been definitely estab lished. We are not cognizant of a proved field case of 2,4-D poisoning in dogs having occurred in this area. References 1. Hm. E. V- and CaxUalo. H.: Ind. Hyg. &Toxicol, s e (1947) 33-93. 2. Ennis. W. B.; Thompson. H. S - and Smith. H. B.: Tributyi phosphate as a solvent for pnparins concentrated and oil solutions of 2.4-D acid and similar substances. Science te e (1946) 473. 3. Grigsby. B. B . and Curtson. B. 1\: Soma effects of herbicides on pasture and grazing livestock. Mich. Agr. Exper. Sta. Quar. Bun. aa (1850) 378. 4. Bildebrand. S. M.: War on creeds. Science te e (19) 463. 3. Bildebrand. S. H.: HerhlcMal action of 2.4-D on the erater hyacinth (Xichontte craasipes) Science 103 (1943) 477. 6. Smith. T. O.i Hammer. C. I*, and Carisnn. B. 7.: Control of ragweed- pollen- with 2.4-D. Sdaswo te a (19) 473. i/ t lV e [ 4 i ) 194 The North Americas Veteriaeries [fas2 Clini Sodi 400 H. W. Monroe, i F. E. Ec! Detroit, l Intrav tended c recent : been eir agents anesthes: the oldei every br inductior cedures. The a constant! thetic in met the anestheti proaches paper is in dogs i To ap anestheti low toxic cated, an side reac desirable investiga clinical ii Material i Since J administc hospital, action, dc period of sodium w the very about equ was used Dr. Knlrk stofi of th Parke. Davi. Thimyl March. 1953 JIW JJ 0012187 5222 5223 i CH EM ICA LS ARE EFFEC TIV E /or Woody Plant Control in the South By A. H. W alker, Extension Range Specialist, Texas A and M College System, College Station, Texas Editor's N o te: The use of chemicals to increase forage and livestock produc ation by the control of undesirable brush on range land and pas tures is relatively new and challenging field. The author is among the first to conduct an extension program which has brought such information to ranchers. W oody plant contpal-is-one of the biggest problems faced by the farmer, ranchman and timber producer in the South today. There are about 240 million acres in the United States infested with undesirable brush and nearly half of this, or 108 million acres are in the fourteen southern states. W ith this brush under control, how much would it add to livestock producers' income?-- possibly one-third of a billion dollars annually. Undesirable trees and brush use up water and minerals which should be available for forage growth for increased livestock production. Some water resources information recently compiled by the Experiment Station in Texas illustrates this point. These data show that the annual average precipitation amounts to 361 million acre feet of which 131 m illion acre feet is lost to brush and weed growth, 44 m illion acre feet runs-off and only 30 million acre feet is used by desirable plant growth of all kinds. Actually, brush and weeds use one third o f all precipitation received. W e hear a lot about un-used run-off and find in Texas that three times as much water is utilized by worthless brush and weeds as runs off down our rivers. Actually, brush and weeds use four times as much water as all desirable plant growth-- this includes all cultivated crops as well as range and pas ture grasses. W e have a potential of four times the pres ent production in Texas if this water could be properly utilized. Lack of adequate water is not such a problem in all states but neither is die brush problem so immense. Texas has some 63 million acres infested. Sometimes the brush infestation is so heavy that live stock handling on the range is difficult. Often this brush grows on the most productive land. Some brush con trolled areas have produced five times as much grass as untreated ones the year following treatment even under drouth conditions. Average production increase following woody plant control usually varies from 20 to 50 per cent, depending upon rainfall, site and management practices. This means more pounds of meat per acre and increased efficiency o f production. In timber areas, hardwoods often hold back or crowd out desirable pine trees. Control of these hardwoods increases the quantity and quality of for age production and also makes livestock handling easier In forest areas the release of desirable timber growth i: both practical and profitable. Proper management following brush control is ven important. Control of woody plants so that an operator car continue to overgraze is folly. Brush control is just ont phase of a pasture management program just as building a tank or constructing a fence. For permanent benefits tc result to a pasture, certain range management practice such as proper stocking, deferred or rotation grazing ant in some cases reseeding and fertilization must be followec W e are primarily concerned with chemical control c woody plants here and it does offer the most promise of an one method. Mechanical equipment such as bulldozer: anchor chains, brush and weed cutters and root plows ofte: have a place in brush control and maintenance work. Some times a combination of mechanical and chemical method, w ill give the best control per dollar spent. Controlled burr, ing in timber areas to increase grazing can be utilized L some cases. N o one method of brush control can be recoir Top Left: Pott oak and blackjack oak aerially sprayed in May 1952, shows 63 percent root kill 18 months following treatment. For age production has been materially increased. (Texas Agri. Expt. Sta. photo). Lower Left: Cut-off stumps treated with 2.4J-T in diesel oil did not sprout. Good native grass made a quick come-back. Right: The author examines the trunks of post oak trees basal sprayed with 2,4J-T in oil two years be fore. On six inch trees and larger, frill treatment is cheaper and is very effective. 10 5224 M 2,4,5-T ds to vi.makiii*"' JM 'OKM ATIOX OX I I e UUICIUES AND A S KVAI.UATIOS OK T llK IB U s e (J^J receiving ,4/ hT were slight mustele spasm, and difficulty in swallowing. llisrnlogicnl studies re* vealed mild change* in the liver and kidney*. The A nt experimental toxieolocieal work eondneted on farm animal* wan that of Mitritoli and TABLE 1-- Matorial* Studied* Csmmon a a m a C h rm ic s l n am e 3 .4 - D 3 .4 ,5 - T M CP 3 ,4 - D ic h la ra p h m x ra c r(i< a d d 3 ,4 .3 - T rt c h la r o p k m a x jr a c X l* a d d S - M s th rl- 4 - e h la ra p h ra a s ra c M i a d d Silvas 4 - C h la ( te to la x )ra < * < le a d d 3 - ( 3 .4 .3 - T ric h te re p h a n a s r ) - p ia p to n ic a c id * lh M a n W ife s h a m ic a l* M i ia h a rb id d a l fo n a ila tte a . eo-workcr.* Thews investigator pastured sheep and eons on folia go sprayed with more than rcrommended amounts of 2,4-D without effect. Thoy also fed a loetating cow SJi Gm. of 2,4-D daily for 10(1 days without ill effects. Milk from the cow did not contain a demonstrable quantity of 2.4*1). No effects were observed in the ealf to which thia milk wan fed. nor was there eny 2,4-1) found in the serum of the ealf. The serum of the cow, however, contained 8.4 p.pjn. of 2.4-D though none waa found in the liver, kidney, or fatty tissues. Early ia IPSO, flrigshy and Farwell* published the results of experiments with 2,4-D and 2,4,5-T involving domestic livestock. In thia study, alfalfa wai sprayed with various herbieidal formulations including three different 2,4-D preparations and on* 2.4,3-T preparation. Livestock including horse*, daily and beef cattle, sheep, swine, and chickens were immediately pastured ia the freehly treated areas. In summary of thia work, the authors stated, " Result! of thia experiment indicate that none of the herbicide* used had any serious physio logical effect upon the livestock involved. They also indicate th a t none of the livestock preferred any of the sprayed arena to those th at were unsprayed; however, with the lots sprayed with 2,4-D herbicide, the livestock grazed the areas sprayed almost as woll aa the nasprayed. 8inec the rates used were two to four times greater than recommended dotage, it seems th a t the farm use of these materials for pasture weed control is a rea sonably safe procedure." Mattsiala St u m d Commercial herbicides generally ran lie described as formulations of active ingredients. The active ingredient usually appears ns a salt which ia solu ble ia water or as an ester which ia soluble in oiL Formulations may lie designed for use as water solutions, w ater emulsions, oil solutions, oil emul sions with water, or dusts, depending upon the use for which they are intended. Basically, most fo r mulations contain, besides the active iagrrdieata. a dispersant, a solvent, a wetting agent, and per ilsps a diluent. These a n usually considered to be inort ingredients although thoy may have a pro found inducnes upon herbieidal effectiveness. Ia the studies reported herein, toxieologirai data a n reported not only fo r the basic aetivc ingre dients aad their ehemieal derivatives (table 1) bnt also for the Aniihed formulatioas as they are marketed* (table 2). Oual A D M unsnunost o r S u r a u Domes Procedure.--The toxicity of the various herbi eidal materials aad formulations wfaea adminis tered ia single oral doses to various speeics of animals baa been determined. All rata, guinea pigs, and rabbits need were young adult animals from the stoek 'colonies of this laboratory. The mien w e n young adults obtained from Garworth Farms. The ehieka used were New Hampshire Beds obtained from a commercial hatchery when about 3 days of ago aad were about 3 weeks of age when treated. The teat awterials were administered by iatulwtion unless otherwise indicated. Aqueous solutions were used wherever possible. Olive oil or corn oil solutions w en used where water was not appro priate. Surviving animals were observed until re covery waa certain, usually about two weeks. Remit.--The resulta of the acute oral studies are summarized in tibie* 3 and 4. I t s U a d e -sa sM d d ra g a lis te d ia ta b le 3 s a d ia th e te s t a n re d a c ts e t T h e D e w C h e m ic a l ( V , M id la n d . K ic k . TABLE 3--H erbieidal Form ulations Studied T n d sa ia s Aativaiaeredienss ( ) 3 .4 - D e w w * d k ille r (fo rm a la 4 0 ) E s t e r a * 3 4 S (e ld f e r a n ls t ia a ) E ster e 3 4 5 ( pr w s t fo r m a l* lia s ) E s te re te n -te a B ra s h k ille r SO -M B ra s h k ilte r T E s t e ra 7 (u a s d ia * 0 s n ia tia a a a ljr ) E s t e rn a 7 S E (a s a d i s s ilh a r *a a s ia t ic a a r w a te r s m a ls ia a ) E s ts r o a b ra s h k ille r (a id fo rm 1 stte a ) E s te rn a b ra s h k ilt e r (p ir n a t fo n o la ite a ) E a rn s (w a s caUad H -1 0 7 1 ) D a w M C F s m in a w a a d k ille r 4 5 .0 A lk a a a la m ia a s a lte W 1 .4 - 0 44.0 Imarapjrl etter al 3,4-D 3 3 .3 T s a p ra p y l s ta r o t 3 .4 ,5 - T ; 1 3 .1 M lz c d a m p i r a t r r s a l 3 ,4 .5 - T 5 .3 M a n te , d i- , u ip r a p iim s (b r a s i h a t e i r t h a r a te ra a l 3 .4 ,5 - T 7 0 .3 M a n te , tr ip ra p jr la a s ( ly e s l b e te l a th a r a tte ra a l 3 ,4 - D 3 7 2 Matei s t e n a l 3 .4 - D : I l i Sauri s a u ra a l 3 ,4 > T SM Batyt estere al 3,43-T < 3 7 .1 Im p r o p r i s s ta ra o l 3 ,4 - D ; 3 t .4 a - B a t e i s ta ra a l 3 ,4 - D M J Is a p r a p il ste r i a 1 3 ,4 - D ; M J B s t y i s t e s i* 1 3 .4 - lb 3 5 .5 la s p ta p irl s t e n a l 3 ,4 - D ; 3 4 .4 Ia a p ra p y t a m s rs a l 3 .4 ,5 - T 13 4 J M a n te , d i- , tr ip ro p jrls a a ( t e s a i b a te t th a r m ta rs a l 3 ,4 - D i 3 3 .0 M a n te , 41-, tr ip r a p r ia a a (t e c a i b a te t c ib a r m u r a 1 3 ,4 .5 - T 6 4 .5 M a n te , di-, tr lp ro p rt e a * ( ir n a l h o te l U b a r ta ta ra a l *11v n 5 5 .1 A lk a a a la a u n a s a lto s i M C P .5225 lVi`00287.0 , 508765 624 Y. lv. Kowe ano T. A. IIvjia Am. J. Vt. Ko . U fTtim 1044 In nil cases where the (lata were suffi ciently extensive to allow statistical analy sis, the l.d.3, values with their 19/20 confi dence limits were determined by the method of Litchfield and Wilcoxon.T In all other eases, the Ld.*, values were esti mated and the range between the dose al lowing survival of all animals treated and the dose causing all aiiimulx to die is given. F o r comparative purposes, some of the vai- TABLE 3-- Sum m ary of A cute O ral T oxicity of V arious Basio Horbicldal M atarais Material SpfHn Ldue (19/30ennddaaca Iboita) Btx Vrhirie (mt/kt-l 3,4-0 (3,4-Dlchlarnphtnoxracaiicacid) * 3.4-D, Ihttaltaiint aalta . ' : . ' 3,4-D, n4ha Mit 3,4-0, ieepreprt atar 3,4-D. a M betri ton 3,4-D. cae-. 41-, tripreyrleae(treatbotri thertetare 3,4,5-T (3.4,5-TrichlonpheoesraeetieuU) . 3,4,5-T, ieepreprteater 3.4.5-T, auxedbotri aten 3,4,S-T, alni Miri teten MCP (4-eklere-e-tatoxraeaticaciderS-Metkri- 4-cklsrephtnareeetiaodd) MCP, etaiooioU caves, (3(S.4.3-triehleropheaeBy) pnpieote aid) flea, wivedbotrieetan (Ovos. Bese-,di-,trlproprleocfijad botri etheratan Rat M Oliveeil 375 (303-404) Mice M Oliveil MR (313-434) Guie* pift MaadP Oliveeil 440 (397-443) Chicha MeodP Olivaeil Mi (354-417) Rane Do* (4) Captata 100 (35-350) . Rane* Chiche (1) Water -- (340-704) (dea(eoa add equlv- aient beala) Beta Reta (3) T Water Water . tos (410-1.003) 440 iT--I--- Ceinro pipe M Water 4SI (417-737) anise* pipe (3) Water 1.000 ---, Mire (3) Water 374 --- -Rabbita (3) Water 400 Reta MendP Oliveeil 700 (549-401) Guin pia M Olivedi 440 (441-471) Mice M Oliveta 441 (304-744) . Chiche MaadP Olivaoil 1,430 (1.137-1.749) Reta F Carota 430 (330-944) Oaete pife T CeroeQ 444 (404-1.IM) >Rabbita F Cernea 434 (353-713) Ranre Mire F Cernea 713 (400-1,040) Cblrke MendP Undiluted 3,000 115904,940) Ru P Cunta 170 (410-4401 Rete Mice Quinte pin Chiche De*e(4) (Rata /Ouiueopige .Mice Ret Rabbit Mice Scinco pige Rota Rot Rat Onion pife Kat ~Bat Rabbita Chicha Rot OeioMpifa Mica Chicha Rabbit M OliveeO 100 (301-440) M Oliveta *49 (345-419) MendP Oliaceli 341 (107-473) MaadP OliveeO >10 (311-454) Raute CapeeIt 100 (40-340) MendP OUvoeU 494 (430-594) P OliveeU 440 (343-447) P OliveeO MI (190-799) f Cernea a i (919-739) Rente M Cenali 713 (5OO-1.000) F CaneO 940 (74-1,313) Rente P CeneO t u (404-1,000) Rente P OUveett 740 (400-1.000) Reefe M Cenca 700 (400-1,000) Reefe M Water LSOO (1,000-3,000) Kenia M Water L300 (430-1,000) MaadP ContU 440 (500-740) Rente 7 Canafl 900 (350-1.000) Rente P Undiluted 750 (S0O-1.00O) MandP Con til UM (707-3.0M) P CeneO 431 (473-414) Ranen K CeneO * 1,340 (500-3.000) Rente P Con eil 1,410 (1.000-3.0M) MaadP Candi LIM (447-1,470) P Undiluted 410 (410-1.070) o O 5228 lisi.J.V kt.I(k. O c n u i i 1(1.14 IXKOICM ATION OS IlU IIICII> ES ANO AN KVA J.PA TIO X OK T lIK JK UK fi-. n<*s givcii in flic lite ra tu re a rc included in characterized by depress! growl It rale, table 3. excessive mortality, slightly increased liver Toxic symptoms generally observed in weights, and slight cloudy swelling of the animals made ill with this type of com* liver. pound include loss of appetite, loss of Those on the two higher diets were de weight, depression, roughness of coat, gen stroyed after twelve days as they were not eral tenseness, and muscular weakness par eating aud were rapidi)' lwiug weight. Ex ticularly of the posterior -uarters. Post amination revealed increased liver and mortem findings usually include irritation kidney weights and slight pathological of the stomach of small animals and of the changes in these organa. abomasum of rum inants, m inor evidence of Experiment 3, Chick*.--Procedure.--Twelve New liver and kidney injury, and in some in Hampshire Bed chicks weighing from 210 to 340 stances congestion of the lungs. Om. each were divided into four groups of 3 chicks each and maintained for aerea days on Ooal AminnsTXATioN* o r S n u n s Dosxs o r diets containing 0 (controls), 300, 1,000, and 3,000 2.4- 0 to B ats a n s Ch ic k s p-pjn. of 2,4-D. Experiment 1, S a lt. -- Procedure. -- M a tc h e d groups of 5 or 0 ynung adult female rata from tho stock colony of this laboratory wore fed 2,4-D five times s week for four weeks by intubation. Results.--The only adverse effects ap p ar ent grossly were a redaction in food intake and a retarded growth rate in the birds fed the diet containing 3,000 p.p.m. of 2,4-D. The dosages employed wero 0.0 (controls), 3.0, No histopathological examinations were 10.0, 30.0, 100.0, and 300.0 mg./kg. adiniuisterod made. as aliquots of olivo oil solutions emulsified in about 2 mL of 3 to 10 per cent aqueous gum srabic solu Osai. A sso n sn u io M to St u m tion. The controls received appropriate doses of Purpom.--Since essentially all of the toxicolog olive oiL ical information oa 2,4-D aud 2,4,3-T type herbi Result*.--The control animals and those cides was obtained using small animals and since animals receiving 3.0, 10.0, and 30.0 m g./ kg. of 2,4-1) showed no adverse effects as judged by gross appearance and behavior, mortality, growth, hematological values, blood urea-nitrogen concentrations, organ weights, and gross and microscopic exami nation of the tissues. the practical application of these materials in-, voIves exposure also to large aninuiis, experiments wero undertakes to ascertain if extrapolation o f the data obtained oa the laboratory animale to largo animals was justified. Esteroa Ernsk Killer.--Sineo e s t e r o a b r a s h killer (formulation of esters of 2,4-D ami 2,4,3-T) is perhaps the widest used of tho products dis Hats receiving 100.0 m g./kg. showed cussed herein, it was ehosea for study. varying degrees of gastrointestinal irrita In tho experiments outlined in tho following tion, slight cloudy swelling in the liver, and a depressed growth rate. Those receiving 300.0 mg./kg. failed rapidly and died. Se vere gastrointestinal irritation was die principal adverse efFcct observed. Experiment 3, B u t i.P r o c e d u r e . -- M a tc h e d groups of 3 young adult female rata from the stock colonies of this laboratory were placed on diets containing 0 (control), 100, 300, 1,000, 3,000, and 10.000 p.pju. (parts per million by weight) of paragraphs, administrations wero all by intuba tion. Treated animals wero constantly under ob servation and tho ordinary clinical olwerrations were routinely made. In general, only punitive observations are reported. Experim ent 1.--A steer weighing 291 kg. was given a single done of 1,000 m g./kg. No perceptible symptoms of toxicity were observed. Experim ent 2.--A steer weighing 293 kg. 2.4- D. was given 1,000 mg./kg. of estenui brush Results.--The controls and those animals killer on each of three successive days. receiving the diets containing 100 and 300 General depression, decreased food and wa p.p.m. of 2,4-1) fo r 113 days showed no ad ter intake, and decreased rumen m otility vene effects as judged by food consump .were observed following the th ird dose. tion, growth, general appearance, m ortal These symptoms became increasingly se ity, blood nrea-uitrogen concentrations, he vere until the steer died on the tin n day matological examinations, organ weights, following the lost dose, i Death was undra- and gross and microscopic examination of inatie; no convulsions, struggling, or signs the tissues. of pain were observed at any time. Nec Those on the diet containing 1,000 p.pjn. ropsy revealed the following: Bumcn con for 113 days suffered slight adverse effects tents were dry and .-:..ellcd strongly of the 5227 OUW508767. 62 V. K. Howe axd T. A. IIymas AM. J. V*KT. UIS. H W 134 cstcruu: tile uboinastuu was impacted and the coni cuts of the intestines were entirely fluid: the mesenteric vessels were congested and the spleen was dark and shrunken. Experiment 3.--A steer weighing 295 kg. wax given 300 mg./kg. on each of two con* scent ive (lavs. On the third day, the animal was off feed and nnueu motility had essentially ceased. On the fourth day, the steer appeared perfectly normal and there were no discernible after-effects. Experim ent 4.--Another steer weighing 336 kg. was given 500 m g./kg. on each of three successive days. No toxic symptoms were observerd and the animal remained on full feed. Experim ent 5.--A steer weighing 230 kg. was given 100 mg./kg. of esteron brush killer on each of fifteen consecutive days without any outward appearance of ad verse effects. The animal was killed for study forty-eight hours following the last dose. Gross examination of the internal or gana revealed only slight petechial hemor rhage in the. duodenum and a mild diifuse irritation in the abomasum. Ilistological examination of tissues from the lung, heart, spleen, adrenuls. pancreas, thyroid, thymus, bladder, and lymph nodes revealed no ab normalities. Sections from the liver re vealed small areas of focal hemorrhagic necrosis surrounded by areas of fatty de generation. In the kidneys, very slight in terstitial edema and congestion in the me dulla and cortieal medullary region were observed. Although gross examination of the gastrointestinal tract revealed mild ir- ritatiou in the duodenum and abomasum, sections of these organs failed to indicate any changes of significance. Kuron ( II -1078).--Because of the cur rent interest in derivatives of chlorinated plienoxypropiouie acid, one of the more promising formulations, kuron (table 1). was included in the work with eattie. A steer weighing 230 kg. was given 100 m g./ kg. of kuron on each of fifteen consecutive days. No adverse effects were noted when judged by general appearance, behavior, and weight gain. The animal was killed for examination forty-eight hours following the last dose. Gross examination of the in- TABLE 4-- Sum m ary of Aeuto Oral T oxicity of V arious HcrbicM al F orm ulations (Dow) Valeria! Spati* Max Velici* lAee (10/3U ronSdeae* liait) (acJkc.) 3.4-Umr m i kfllar 3.4*13* c H kinrr (fa m u li 40) Satana 44 E rta la * 34S (a U ) Sam oa S4S (m v ) Sam oa i*a*Ua Bniafe killer SO-SO Kraal kUlarT Hruak kBarTO Brah km* TOE Salam i Woafc kUar (a U ) S a u n a k n u k k iU e r (s a w ) Colata pia Bau Hata Bau Baia Rat RaU Cuiaaa pica Mica Rabbiu Ckicka Rat Calata pio* S in Rabbiu Ckicka KaU -- Rat Rat Rata (aiata pia Coina pia Rabbiu Click SUar f Water F Water X Olir*) r Esulala* io ater X lilla* all r (llia* eli F Rauiaiaa io ater T KaniUian ia alar T Cera ai) r Undllntrd S a n a r Undiluird F KanUino In nter y Kmuiaiaa In votar y mire ail X Undiluted U nod K Undiluted r C an oil K rnrn nil X Kmulnim in nier 31 end F V a n oil X Corn nil t F l 'ora nil V and T Cara oil X and F Tarn ail CadilMtd 3.MO <1.000-3.000) Rane* MO (700*1,000) Raaao SO (300*1.000) Kaaaa I.D O ft (300-3.000) Rana* 4041 (600*1.000) TOO (660-600) I.O TO (700*1,060) 1.160 (30*1,630) 00 (634*1.070) Raaa* 1.430 (600*3.000) 4.0011 (3.700*6.900) 1 .20 0 (763*1.660) 1.410 (676*3.300) 1.230 (036*1.630) 4 9 (604-1,190) s,mN) (1.330*3.940) Itaaa* 730 (M O .I.IM 0 ) Kara* 3 0 0 (3 6 0 -I.IH H I) Rsire 1.000 (300-3,000) 4 M (00.960) I.3 3 U (1,040*1.430) 1.400 (1.390*1.640) M O (7 9 0 -1 .1 6 0 ) Rana* 3,000 (1.000-3.000) Graaur than 1,000 5228 0002323- I xwmwiatiox ox Iltam ciu ts axd ax Evall-atiox ok T a g Use 27 s< ti*nial organs revealed no abnormalities. stm t! the similarity in susceptibility of Histological examination of tissues from cattle and small laboratory animal* to this tlio lung, heart, liver, spleen, pancreas, type of herbicidal materials. I t has also thymus, bladder, lymph nodes, rumen. ret been demonstrated that cattle are distinctly iculum, omasum, and abomasum revealed more tolerant of this type material than no. abnormalities. Very* slight interstitial dogs. edema and congestion was- observed in the The following conclusions with regard m edulla and cortical m edullary regions of to cattle seem justified: the kiduey, but since this has frequently 1) A daily dose of 30 m g ./k g probably been observed in untreated animals, its can be tolerated for prolonged periods significance is questionable. without adverse effect. 2) A daily dose of 100 m g./kg. would Discussion' not be expected to cause any ill effects un Siyitificauce of Experimental Result*.-- Study of the data presented in table 3 in dicate* that the various forms of 2,4-1) and 2,4,5-T all fall in the same range of toxicity for rata, mice, guinea pigs, and rabbits. The dog appears to be somewhat more suscep tible to these materials than the other spe cies studied, and chicks appear to be more tolerant. I t is also apparent that MCP (2-incthyl-4-chlorophenoxy'acetic acid), silvex (2-|2,4,5-trichlorophenoxy] propionic acid), and their derivatives are less toxic acutely thau the corresponding derivatives of 2,4-D and 2,4,5-T. The l.d.M values for 2,4-D and 2,4,5-T and their common deriva tives are iu the range of 300 to 1,000 m g./ kg. fo r the rat, mouse, guinea pig, and rab bit, whereas for II CP and silvex, the corre sponding values range from 600 to 1,400 m g .A g . The data presented in table 4 show th at the acute oral toxicity of the commercial formulations, in general, tends to be pro portional to that which might be expected from their content of active ingredients. Thus, it appears that the " inert ingre dients" present in these formulations arc less toxic than the active agents. F u rth er more, the " inerts" do not appear to exert a potentiating effect upon the toxicity of the active materials. For the commercial formulations studied, the Ld.M values for rata, mice, gninea pigs, rabbits, and cattle range from 500 to 2,000 m g ./k g .; for chicks the range is from 2,000 to 4,000 m g./kg. The results of repeated oral administra tions indicate th at 2,4-D and 2,4,5-T can be less exposures were continued fo r a week or longer. However, if exposures were con tinued, some liver and kidney injury, aud perhaps some gastrointestinal irritation could occur. Death or serious illness would not be anticipated. 3) A daily dose of 500 m g./kg., if coutinned for several day's, could result iu serious effects. A single dose of 500 m g ./ kg. would not be likely to cause serious effects. 4) A single dose of 1,000 m g./kg. m ay or may not cause illness. Repeated doses of this size are very likely to cause death. Evaluation of Direct H atard to Live* stock.--In actual practice, esteron brush killer (a formulation of esters of 2,4-D anil 2,4,5-T) aud similar form ulations arc not ordinarily used on areas containing appre ciable forage at a rate greater than 2 quarts per acre or 47 mg./sq. f t Assuming that off of the herbicide ap plied at this dosage rate is deposited ou edible forage, an animal weighing 350 kg. (770 lb.) would have to consume all the forage on 744 sq. f t r f treated area to ac quire a dose of 100 m g./kg. of the herbi cidal formulation. Such a dose could be tolerated daily for a number of days with out any serious effects. Based on the same assumption, it would be necessary for the animal to graze completely an area ten times as large, or 7,440 sq. f t (0.17 acres) in order to acquire an acutely toxic dose (approximately 1,000 m g./kg.). Since these assumptions are based upon complete availability* to livestock of all the OUO IUO c tolerated without adverse effects in doses herbicide applied, an obviously impassible only slightly smaller than those which cause condition, it is apparent that there is little, toxic effects when given only ouce. This if any, direct hazard to livestock or wild fact demonstrates that these materials have life foragiug areas treated with heabicides a low degree of chronicity. of the type described herein. This has been The studies with cattle have demon- confirmed by the extensive use of these ma- 5229 0002824 G2S V. K. Howe and T. A. IIymas Am. J. Vkt. i: k. UCTIWM 1354 Icrials over a number of years without any bility but, so far, it has not been estab proved eases of adverse effects. lished ns a fact. - Eraluntion o f Indirect Hazard to Live In evaluating the hazard associated with stock.--There has been much discussion of the forementioned possibilities, Dr. Wil the possibility th at spraying with these lard in 1951 ** stated as follows: " In the herbicides might cause some plants to be* first place, literally dozens of carefully come toxic, toxic plants to become more controlled feediug experiments, both with toxic, and ordinarily noupalatable plants 2,4-D and 2,4,5-T directly and with vege to become palatable. -Considerable investi tation sprayed with them, have uniformly gation of these possibilities has resulted in and without exception failed to show any the general conclusion th at from a practi poisonous effects on auy kind of livestock. cal standpoint these suppositions were more " In the second place, literally millions of fanciful than factual. acres of pastures with livestock on them In 1950, W illard u of Ohio State Univer have been sprayed in the last six or seven sity and A gricultural Experim ent Station years without injury to livestock. I am sure . pointed out that the spraying of certain there'is no experimenter here who has not types of plants with 2,4-D increased their used 2,4-D in occupied pastures. In our nitrate content to hazardous levels. state, one company over a period of four The leaves of sugar beets accidentally years has sprayed 60,000 miles of roadside ' sprayed with 2,4-D were reported to have in twenty-six counties. D uring that time, caused the poisoning of some livestock. he has had only five reports of suspected However, siuce 2,4-D is never knowingly poisonings, none of them substantiated, 2 used on sugar beets, this docs not appear of swine, 2 of cattle, 1 of sheep. A company to present a serious practical hazard. Other in Pennsylvania, which has sprayed 10,000 weeds such as lambsquarter, pigweed, and acres of right-of-ways per year, reports smartweed have also been reported as con ju st 4 cases, only 1 of which was not com taining increased quantities of n itra te after pletely cleared up as due to other causes, treatm ent with 2,4-D. Since livestock do in five years of work." not ordinarily cat these species, it would La 1953, Dr. S. N. P e r tig 1* of Cornell seem that the hazard presented by the ac- University summarized the situation re .. cumulation of nitrate in these plants is garding poisoning as follows, " There are ; slight. The lack of reported cases of nitrate no known cases of actual herbieidal poison ' / poisoning would seem to substantiate this ing from field application of presently used supposition. h e rb ic id e s marketed as nonpoisonous.* Wild cherry is recognized as a hazardous There is no sound toxicological evidence plant to have in grazing areas. Couch* available of the presently publicized ni hits suggested th at the w ilting of wild trate-phenol and nitrite poisoning as a cherry leaves results in the hydrolysis of common cause of death. True, nitrate and cyanogenic glucosidcs to form free hydro- nitrite poisoning has occurred in cattle, : cyanic acid (IICN ) and, therefore, th at cut sheep, and swine from eating plants grown wild cherry is much more hazardous to live on high nitrate soils or from other feed stock than the growing plants. By analogy, sources. None of these, however, have been it has been postulated th at wild cherry definitely traced or shown to be caused by leaves wilted as a result of treatm ent with herbieidal treatm en t All the alleged cases 2,4-D and 2,4,5-T herbicides likewise might of herbieidal poisoniug of livestock and be more toxic than growing leaves. Bar wildlife that have been definitely diag ren s and Lynn * and Grigsby and Boll,1* nosed have bceu caused by one of the fol working, independently, d e m o n s tra te d lowing: (1) lead, (2) arsenic, (3) hard dearly that the IICN eontent of wild ware disease, (4) poisouous plants, (5) old chcrxy leaves sprayed with estcron brush age, (6) parasites, (7) drowning, (8) poor killer was actually lower than the content marksmanship, (9) contaminated food, and .msprayed leaves. (10) injection or oral dosage of some medi Dr. W illard also has called to attention cine or drug. In all cases that have been the possibility th at treatm ent w ith 2,4-D carefully surveyed, even though the herbi- type herbicides might make certain natu rally poisonous plants more palatable or even attractive to livestock. This is a possi- * D r . Turtle' cUaufteatfea S t a rt h U M aurkrta4 M M -Iw iM m u ladaSaa rash aularisla aa Ibaaa Aiaaaaard la tfcia paprr. 5230 0002825 f* M A U uct u i * "' I nkjiimatiox on I I ehbicides and an Evaluation o r Th km Lad lad eide has been associated with the trouble, acress to spray tanks or other containers of it has in no case been directly or indirectly the mutcrials. related to the deatlis reported." R ifin n cu . S ummary and Conclusions 1 Bucher, K uqr R.: KITerta ed M'OirUanpImi- j 1 j The acute oral l.d.j, values for 2,4-D, \ 2,4,5-T, and their various derivatives com* monly used in hcrbicidal preparations fall in. the range of 300 to 1,000 m g./kg. for exyaertic Acid on Rxprrimeatal AcimaU. P w . Sm. KxptL SM . u d Med, H , (1940): 304-303. 'HID. Kdiria C , end Cxrltxie. Harold: Toxicity ad 3.4- DtcbleroplMaexyacetic Arid for Kxprrimraul Aaimala. J. Iaduxt. Hy. aad Toxicol, <f, (1947) : 93-43. DJero. Mrlria K , aad Nartbea, Hmry T.: fcjfrcix . rats, mice, guinea pigs, an d rabbits. Gen ed 3.4-Olchlecophcioxjcceric Add ea Chicha. Srieacc. erally, dogs are more susceptible and chicks 190, (1949) : 479-400. Drill, victor A , and Hirattka, Temikaro: V n ir ilf od are more tolerant of this type of material. 3,4.DicMotaphxaoxyacelio Add and !.4.S-1Wktan|d<- On a weight basis, the toxicity to cattle aexyaeeiic Add. A Report aa Their Acate aad (hrenie Texidty ia D e n . Arch. la d ed . Hy*. aad O cp. Mrd, .-appears to be quite sim ilar to the toxicity 7. (1993) : 41-47. Ito ordinary laboratory animals. 2) The acute oral l.d.M values for MltchcO, 3. W , Hodfaea, & X , aad Gaetjenm, C. K.: comTckoltaerraempkeaexeadxyot aeardoc Animale Add. J. to Teed Caatalainc 3,4-Ul* Aaim. S d , 4, (194S) : 334. mercial form ulations of 2,4-D and 2,4,5-T type materials arc approximately propor- 333. Grifxby, B. H , aad TarwaO, X. D.: Seme XSectx ed Uerhieidee ea Paatara aad ea Ciada Llrextack. Mich- ' tional to th eir content of active ingredient. iaa Aerie. Zxper. Static Qaaik Ball, it, (1990): . . The " inert ingredients" do not appear to 974.999. MtchSald. 3. T , J r , aad Wilaaaaa. P.: A Simpliled ; contribute m aterially to the oral toxicity Mathsd ad Bralasda Doao-XSact Xaparimcan J. Phar- of the 3) formulations. Data available indicate tliat the maad. aad Kxper. Therap , 44, (1949) : 99-113. 2,4-D Caaek. Jomm P.: Pciceaia that Pradaco Hydncyaaie Add. od Uteotoek by Ploato U A D A Lexlet H , . and 2,4,5-T type herbicides have a low 194. chronicity. * Lyan. O. X , aad Barreal, K. C.: The Hydracyaaio Acid (KCN) Coxtoat od Wild Cherry Leerae Sprayed 4) M C P ( 2 -m e t h y 1-4 -cklorophenoxyW- ith a Brook Killer Ceataiaia Lav Taladla Xatrn at . acetic acid) and silvex (2-[2,4,5-trichloro- 3.4- D aad 3,4,9-T. Proa. Sixth Aaa. Meet. XertheaaC Wred Ceotrot Cent, (1993) : 331-333. phenoxy] propionic acid) an d their herbi- Gricxhy. B. H , aad Ball, a D-: Soma U m f cidal derivatives appear to be slightly less - toxic than the corresponding 2,4-D and 2.4,5-T type m aterials. 5) The hazard to livestock and wildlife associated with the use as recommended of .herbicides containing 2,4-D, 2,4,5-T, MCP, - and silvex is negligible. I t should be rccognixed, however, th at toxic amounts of these materials can be obtained if animals have Herbiddal Spraya aa tka Hydroeyaaia Add Oeateal of Lmtao ad Wild Blade Cherry (Pnum* d em ia s XArhart;. P n c . Sixth Aaa. Moat. Kortheart. Weed CMtrd Cead.. (1933): 337-330. u Willard, C. 3.'. Iadiroct Xffeete ad Harkleldre. Prac. Srreath Aaa. Meet. Keith Ceotral Weed Coatrat Cead. (1990): 110-113. WlHard. C. J.: The gtataa ed Rarkiddal P ilmaiaa. Pro. Kickth Aaa. Meet. Karth CMtral Weed Caarml Cead. (1931): *4-49. Parti, B S .: HarMddal Peieeaia ed IJeeeUek. Sappl. Pro*. Soreath Aaa. MeeA Hartheu. Weed Con tra Cead, (1M 4): 44-47. C - Or o C -V - 2 0002S26 31 5232 3\ DOWN to EARTH j T ^ Copyright 1954 by The Dow Chemical Company / J f A review of agricultural chemical progress volume 10 no 2 fall 1954 up w m A/'A 1`, \ i =U \\i! |!i" < V*s > .'* , * r^, p-x -V, * * * - -. -fc> - r i i > . F r ( - -J -T >,* -r -x,r v.v*v-iiAv Vr.TJv^- # ? Vi N^I^7* *> V r > V ^ c f .* ":& T l % - KILLING SMALL UNDESIRABLE HARDWOODS by u ofthe Co rn ell to o l By R ay E. Goddard, Assistant Silviculturist, Texas Forest Service, A lto, Texas Applying 2,4J - T in D iesel o il to hardwood with Cornell tool. The device is heavy, much like a spud, and permits the injec tion of the chemical in cuts in the basal area. Results of a small scale study, established in July and August, 1952, on the W . Goodrich Jones State Forest near Conroe, Texas, indicate that 2,4,5-T applied with a Cornell tree poisoning tool may be an effective method of releasing young pines from competing undesirable hardwoods. Plots, one-tenth acre in size, were set up to compare the effectiveness of 2,4,5-T ester applied as a basal spray and with a Cornell tooL The propylene glycol butyl ether ester was used in each case. A 2Vi per cent solution of 2,4,5-T, by volume, in diesel oil (10 lb. acid equivalent per 100 gal lons of spray), was applied by the basal, spray method to all hardwoods on two plots in July and August, 1952. Solu tions of 10 and 20 per cent 2,4,5-T in Diesel oil (10 and 20 percent 2,4,5-T, 40 and 80 lb. acid equivalent per 100 gal lons of spray) were applied with the Cornell tool to trees in two plots in July. In August, one additional plot was treated with a 10 per cent solution in Diesel oil and one plot was treated with a 10 per cent emulsion in water, both applied with the Cornell tooL As both application methods are primarily useful in the treatment of small trees, the plots were located in a stand dominated by sapling size hardwoods. However, the few larger hardwoods found on the plots were treated by the various methods being tested. Species predominating were redgum, blackgum and southern red oak. Smaller numbers of post, white knd water oaks, yaupon, holly, sassa fras, tree huckleberry,'red mulberry and red haw were .also found on the plots. W ith basal spray applications, the bark of treated trees was wet to the point of run-off from the root collar to TABLE I Vo lu m b o f Ch em ica l So l u t io n s a n d Average Siz e o f T rees T reated by Basal Spray and Cornell Tool M ethods Method of Application Coacen- ' tration (b y volume) P erC en t D iluent Average Average volume used diameter of per tree* treated trees m l inches Basal spray Cornell tool Cornell tool Cornell tool 2Vl 10 10 20 Diesel oil water Diesel oil Diesel oil 200 8 11 11 3.7 2.6 3.2 3-3 * Aretwge determined from the total amount need per p lo t approximately one foot above the ground. Cornell tool cuts were spaced approximately one inch aparr around the tree base. Table I indicates average diameter of treated trees and the average volume of each solution used per tree. In September, 1953, a little over one year after treat ments were applied, 100 per cent of the hardwoods 10 inches in diameter and smaller treated with 20 per cent 2,4,5-T by the Cornell tool method were dead. Of these, only 1.6 per cent have resprouted. The 10 per cent 2,4,5-T in Diesel oil treatment was also effective. A total of 88 per cent of hardwoods under 10 inches in diameter were dead.. Hardwood mortality was less and sprouting more pro lific in the plot where die 10 per cent 2,4,5-T water emul sion was applied with the Cornell tooL Trees treated with 2,4^-T by die basal spray method usually die more slowly than when fresh wounds are treated. On die basal spray plots, 53 per cent of the hardwoods un der ten inches in diameter were dead one year after treat ment. By die start of the second spring after treatment, 63 per cent were dead. Many of the trees still surviving at that time were in a weakened condition and had been drastically reduced in living crown. ; .. The results of these treatments are summarized by species groups in Table U. As only two larger trees have died as a result of any of these treatments, only those less than ten inches in diameter have been included in these data. Ta b l e ~IL'~-Percentage o f D ead H ardwoods 10 In c h e s d. b. h . and un d er T w o Years a fter T rea tm en t w it h _____________________. Basal Spray Cornell T ool Spe cies Basal Spray T reatm ents 216% . 2.4.3-T in Dieiel OU Number o f trees reeled Per cont For cont of trots of trots dtod sprouting - 10% 2.4.3-T in IVeter Numbor of trots trostod Percent Forconi of reel of trots deed sprouting Co r n e l l To o l T reatm ents 10% 2.4.3-T m Jieiel Oil Numbor For cout Percent of trots of trots of trees trootod iood tprouting 20% 2.4.3-T in Dieiel Oil Number For cont Percent o f tree! . of trots of reel reeled dood sprouting Redgum Bladegum Oaks* Yaupon Sc Holly Miscellaneous* All species 70 17 14 6 11 117 63 . 0 41 0 57 12 100 0 90 0 63 2 49 49 15 33 57 83 5 60 3 100 129 61 4 20 23 0 0 17 49 88 20 90 40 .88 7 100 4 75 120 88 0 47 100 0 0 10 100 10 3 ' 5..... "1 0 0 0 0 1 100 0 0 1 100 0 1 64 100 2 1 Includes mostly southern red oak with a few post, white and water oaks. *10010363 sassafras, wax myrtle, tree huckleberry, sumac, mulberry and. red haw. 5 5234 D ow n to E arth, F all 19. 3 a. 3 a_ 5235 KERBCIDE DETERMINATION APPENDIX V I I Determination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Grain and Seed ROLAND P. MARQUARDT and E. N. LUCE The Dow Chemical Co., Midland, Mich. The 2,4-D acid is extracted from a dry sample o f m eal with an acidified ether-chloroform solvent. By further treatment, including a chromatographic separation, interfering m aterials are removed. The color produced by the reaction of 2,4-D acid with chromo tropic acid in concentrated sulfuric acid is measured on a suitable spectrophotometer. As little as 5 y of 2,4-D acid .in q 200-gram sample can be detected. T __Jrw\ j r~ : i-Z /tk lie use o f 2,4-dichlorophenoxy- a c e t ic acid (2,4-D acid) as a weed killer on a variety of crops has made it * 4 * i **** trated sulfuric acid with 800 mi. of water and cool the dilute acid to room tem perature; then dissolve 50 grains of 2,4-D acid from the filtrate once with a solution of 200 ml. of water and 50 ml. of 5V sodium hydroxide and twice with a desirable to develop an analytical stannous chloride, SnQj.2HjO, in the solution of 200 ml. of water and 10 ml. of method capable of detecting minute acid. 5V sodium hydroxide. Shake well each quantities of 2,4-D acid which may be picked up by the plant. The possibility of translocation of the 2,4-D acid must also be determined. The method of Marquardt and Luce (-/) was not adaptable directly to this problem, and the procedure of Gordon 2,4-Dichlorophenoxyacctic acid, stand ard solution. Dissolve 0.500 gram of 2,4-D acid in chloroform and dilute to 500 ml. Dilute a 10.0-ml. aliquot to 100 ml. with chloroform. Dilute a 10.0-ml. aliquot of this second solution to 100 ml. with chloroform. The final solution time to ensure complete extractions. Discard the organic layer and wash the combined aqueous extracts three times with 100-ml. portions of technical grade chloroform. Acidify the aqueous solution with 50 ml. of concentrated hydrochloric acid. and Bcroza (o) did not ofier a means of contains 0.01 mg. of 2,4-D acid per ml. Extract the 2,4-D acid from the solution separation from the substrata or indicate the possibility of detecting only trace nritirs. A method of extraction and separation of the 2,4-D acid was developed and the colorimetric method of Freed (2) further refined to give the accuracy demanded for this problem. R aagentt Chloroform, technical grade. Ether, U.S.P. grade. Acetic acid, glacial. Sodium hydroxide, approximately 52V. Hydrochloric acid, concentrated. So dium hydroxide, approximately 0.5V. Phosphotungstic acid. Dissolve 40 grams of phosphotungstic acid (approxi mately PjO,.24WO.25HjO) in water and dilute the solution to 100 ml. Hytlo Super-Cel, a Cclite product, diatomaceous silica, made by JohnsManville. Absolute methanol in U.S.P. chloro form, 3.0 and 7.0% by volume (see sec tion on chromatographic separation of 2,4-D acid). Buffered extraction solution, pH 6.85. Dissolve 10.0 grains of dibasic sodium phosphate, NajIIPO.7HiO, and 10.0 grams of monobasic sodium phosphate, NaHjPOi.HjO, in water and dilute to exaedy 1.0 liter. Chloroform, analytical reagent grade. Ihromotropic acid. Dissolve 0.10 gram of the sodium salt of chromotropic acid (l,H-dihydroxynaphthalene-3,6-disulfonic acid) in 100 ml. of concentrated sulfuric acid (95.5%). Sulfuric acid-stannous chloride solu tion, dilute. Mix 200 ml. of concen- Apparatus The tube for the chromatographic column is a glass tube, 17 mm. in inside diameter and 50 cm. long, one end of which is constricted and attached to a smaller glass tube, 5 mm. in inside diam eter and 10 cm. long. A suitable apparatus for regulating air pressure and an interval timer are also required. A Coleman spectrophotometer, Model 11, equipped with a PC-4 filter and 5cm. cuvettes, was used in this investiga tion. Any photometer measuring light transmittance at 565 m u should be suit able. Proemdurn Grind the grain or seed to a fine meal to pass through a No. 10 sieve. In this work a No. 1 Wiley mill was used. Place a 200-gram sample of the meal in a 2-liter glass-stoppered bottle, and add 500 ml. of technical grade chloro form, 500 ml. of ether, and 10 ml. of acetic acid. Stopper the bottle, fasten ing the stopper with Okonite tape. Place the bottle and contents in a shaking machine for 1 hour. Use a 2-liter filtering flask, a Bchner funnel 16 cm. in diameter, and What man's No. 1 or similar grade filter paper. Rinse the bottle by adding 50 ml. of technical grade chloroform and 50 mi. of ether and wash the insoluble material with the rinsing solution. Discard the insoluble materiaL Using a 2-liter separatory funnel, extract the with 125 ml. of ether. Repeat the ex traction with 100 ml. of ether and com bine the extracts. Discard the aqueous solution. Filter the ether solution, using What man's No. l" or equivalent grade filter paper, into a 250-mi. separatory funnel. Extract the 2,4-D acid from the filtered solution once with 50 ml. of 0.5V sodium hydroxide and twice with 25-mi. portions of 0.5V sodium hydroxide. Combine the caustic extracts. Pour the caustic solution into the 250ml. separatory funnel and wash it three times with 10-mi. portions of chloroform, discarding the washings. Acidify the solution with 10 ml. of concentrated hydrochloric add and add 5 ml. of phosphotungstic add solution. Extract the 2,4-D acid with three 10-ml. portions of chloroform and combine the extracts. Prepare the column for the chromato graphic separation by placing a plug of glass wool in the bottom of the tube at the constriction. Add Hyflo Super-Cel and settle it in the tube by bouncing the tube gently on a large rubber stopper. Continue adding more Super-Cel and bouncing the tube gently until the SuperCel is packed in the tube for a distance of 20 cm. Filter the chloroform solution into the tube, using No. 1 Whatman's or eq u iv a lent grade filter paper. Apply air pres sure at the top of the tube to push the solution into the Super-Cel. Using proper air pressure, percolate 150 ml. of the 3.0% methanol in chloro form solution through the column at the rate of 150 ml. per hour, followed ^ '^ 5 ^ g 3 * g Q Tn n n It Reprinted from AGRICULTURAL AND FOOD CHEMISTRY, Vol. 3, No. 1, Page 61, January 1955 nnm o mended exclusively. Operators should study their particu lar conditions in die light of available information to de termine the most profitable methods to follow. ost chemical research work on woody plant control u.. range or pasture land should be guided by these funda mental objectives: (1 ) development of effective treatment, (2 ) cost and possible net return and (3) range manage ment practices necessary for continued benefits. Some fed eral and state research men who have contributed materi ally to chemical brush control in the South are: C E. Fisher at Spur, Texas on mesquite; Dave Savage and Pat Mcllvain at Woodward, Oklahoma on sagebrush and Shinnery oak; Fred Peevy and John Cassady on Louisiana oaks; L E. Chaiken on hardwoods in North Carolina; Harry Elwell on oak brush at Guthrie, Oklahoma; and R. A. Darrow and Wayne McCulIy on post and blackjack oaks, prickly pear, whitebrush and huisache in Texas. This work has guided further research and demonstrations in the South. Chemicals used in brush control work may be selective such as 2,4-D, 2,4,5-T and silvex or nonselective such as ammonium sulfamate, arsenic and kerosene. Fundamentals of the ultimate in a woody plant control chemical are: (1) nonpoisonous to man or livestock; (2 ) effective under a wide range of conditions; (3 ) cheap; (4) easy to apply and to handle; (5 ) not damaging to grass or cultivated crops. Present chemicals meet some of these requirements but not all of them. Chemicals may be applied by broadcast methods or by individual tree or plant treatment. Tractor or jeep drawn power spray equipment can be used on low growing brush or following mechanical control work. Aerial application provides economical and rapid coverage of large brush areas. Such broadcast methods have certain limitations. At the- ;ent time, most chemicals applied when the brush is in . foliage, control only certain kinds of brush and must be used very carefully in susceptible crop areas. Sprays may be applied to individual plants with hand operated or cattle sprayers. Such equipment is suitable for foliage treatment of scattered individual seedlings or small brush and for frill, basal and stump treatment. Extensive brush control research conducted at the U. S. Southern Great Plains Field Station at Woodward, Oklahoma shows that three-fourths of the sand sage brush can be killed with one proper aerial application of 2,4-D. Forage and beef production is increased from 50 to 75 percent if grazing is light or summer deferment is prac ticed following treatment. The cost of sagebrush spray ing on experimental areas has been repaid completely the first year following treatment. More than one million acres of mesquite have been aeri ally sprayed in Texas during the past 3 years with Yi -Va lb. of 2,4,5-T (as low-volatile ester) per acre in diesel oil at a cost of $3.00 to $3.50 per acre. Top kills will average around 90 percent and root kills 40 percent. Mesquite is the biggest one species problem in Texas with a dense stand on 35 million acres and scattered stands on another 15 million acres. It is a prolific sprouter from the bud zone. Stage of growth, moisture condition, size of trees and m an, agement following treatment largely determine the bene fits derived from spraying. Grass growth and beef produc tion have been materially increased on all areas sprayed and the writer has yet to visit an area on which mesquite sprayi " has not paid for itself. erial control of post oak and blackjack oak has been tried on a limited scale. It appears practical and economi cal (Figure 1). Two pounds of 2,4,5-T add equivalent per ii acre (as low volatile ester) should be applied after plants have reached full leaf in the spring. The cost will be about $8.00 per acre but these oak areas have more favorable rain fall and will produce more forage than many mesquite areas. It should be emphasized that for both mesquite and oak, control rather than eradication is die result. Additional sprayings will be necesary to control regrowth. Can all kinds or upland and bottomland hardwoods be aerially con trolled economically? There is a need for more research on this problem. Aerial application is die cheapest method known with the limitation that acreages must be large and no susceptible crops are dose by. Possibly hardwoods may be controlled by aerial applica tion of 2,4,5-T, silvex, or MCP in timber areas. Earlier re search indicated chemicals would kill pines but later work makes researchers wonder. These possibilities should be fully explored. Chemicals may be applied to the foliage of individual trees, to the cut-off stump, trunk base of standing trees or in cups or frills made in the bark 2,4,5-T ester and ammon ium sulfamate are the most widely used and accepted chem icals. In Texas and Oklahoma, 2,4,5-T has generally given cheaper and more effective results with less labor. Stump treatment using 2,4,5-T ester diluted in diesel oil or kerosene gives good control of sprouting for most spe cies (Figure 2). Trunk base treatment using 2,4,5-T in oil gives good results on trees under six inches in diameter but some species require twice the concentration of chem ical as others (Figure 3). Ammonium sulfamate crystals in cups spaced not over six inches apart and close to the ground or applied in water solution to frills usually con trols sprouting on oaks. A solution of 2,4,5-T in oil ap plied in single overlapping axe cuts shows much promise of cheap, effective control of all kinds of trees. The value of any program is dependent upon its accep tance by the people. Extension woody .plant control in Texas has been presented through county agent training meetings, method demonstrations in counties, newspaper, magazine and radio stories and TV demonstrations. In 1952, working with the county agents, the writer conducted brush control meetings or demonstrations in 46 different counties. In 25 counties, 33 field demonstrations were set up using 2,4,5-T and ammonium sulfamate on stumps, trunks and in frills. These demonstrations were perman ently marked with the kinds of chemical used and method of treatment. Last year, 40 brush control meetings and 30 field demonstrations in 24 different counties were held. For the past two years, this gives a total of 86 brush con trol meetings, 63 field demonstrations in 49 different coun ties and 150 hardwoods demonstrations conducted by county agricultural agents and this specialise As a result of an evaluation of these field demonstrations together with experiment station results, a circular, "More Grass from Controlling Hardwoods with Chemicals" C-330, has been prepared for the guidance of Texas farmers and ranchmen and is available from the Texas Agricultural Ex tension Service. A Challenge Whereas brush control is the biggest problem facing the farmer, ranchman and timber producer in many southern areas, it also presents a very great opportunity. In busi ness 3 or 4 percent interest is considered a fair investment while the application of present woody plant control infor mation can result in 20 to 50 per cent increase in desirable production and 100 per cent gain is not uncommon when coupled with proper range, pasture and timber management. 5237 D o w n to Earth, Summer 1954 3o 3o 5238 C -T - * m Summary o f Toxicological Information on 2,4-D and 2,4,5-T Type Herbicides and an Evaluation of the Hazards to Livestock Associated with Their Use V. JC ROWE, M.S.. and T. A. HYMAS, D.V.M. Midland, Michigan T h e rs E o r herbicide* fo r tiie control of cnee in potency Itween crude and purified projw- undesirable vegetation lias been practiced rations, or lictween tiui sodium or ammonium salts. for many years. Some of the older mate* riaU commonly used are chlorates, arsenicals, compounds of borou. certain oils, and even table salt. While many of these mate* rials are still being used for this purpose, Deaths from large dose* were believed due to ven tricular fibrillation; if death wan delayed, myoto nia. stiffness of extremities, ataxia, paralysis, aad cornu were olamrvedr In Kulmeutr studies, the wine investigators ol>served intoxication in dogs after six daily intra research over the past decade has revealed venous injections of 22 GnuAg.; rata were fed a a family of new herbicidal materials. They diet containing 1.000 parts per million (p.p.ra.) of are the chlorinated phenoxvacetic acids and 2.4- D for a month without harmful effects; guinea closely associated compounds -of which pigs tolerate*! ten doses of 100 m gA g- over a 2.4- D (2,4-dichlorophenoxyucetic acid) and twelrc-dny jieriod; aad the inhalation of the so 2.4,5-T (2,4,5-trichIorophenoxyacetic acid) are prototypes. Early in the development of these new dium w it as a dost failed to eanso systemie effects in guinea pigs. The main evidences of ehronie in toxication oliserved in rats were visceral conges tion an*l edematous kidneys with degenerative herbicides, studies were undertaken by The ehnngcs ia the tubules; only dogs exhibited hepatic Dow Chemical Company to determine their damage with central degeneration aad congestion. toxicity to warm-blooded animals. I t is the Ia 104, Bjorn and N'orthcn' reported the re purpose of this paper to make the acquired sults of studies conducted on ehieks with an alka-. toxicological information a v a ila b le to uolamine w it of 2,4-D. Tho acute oral lethal rang others. was found to be 380 to 762 mg.Ag. When given repeatedly (12 doses ia 28 days), 28 mgAg-/doso Unaumnuc Review waa without effect, while 280 mg-Ag./dose caused depression of growth. These authors express no Uucherl ia 1046 wan among tlx* Ant to report concern about the likelihood of tosic effects ia the reanlU of experiment* with mail anirnul* using ehirkeus under ordinary conditions of nao and 2.4- D. Temporary myotonia lasting from eight to point ont that at a spraying rate of 1 lb. of 2,4-D twenty-four hours or more following a single in per acre (a normal rate of application) a ehiekea jection of ISO to 250 mg./fcg. wa* observed' in weighing 1 kg. would have to consume all the miee. rats, rabbits, and dags. Repented injections 2.4- D applied on 72 sq. ft. within a day or two to o f mailer amounts, 50 to 100 mg.Ag./day to mire ubtuin a lethal dose. for ninoty days, failed to elicit either n character Ia 1P55, Drill and llirutska' reported the results istic chronic syndrome or n striking histological of acute and ehronie oral toxicity studieo on 2,4*1) picture. Miee undergoing this treatment became and 2,4,5-T with dogs. These men found that the pregnant and bore apparently normal litters. Re- acute oral l.du> values wera about 100 mg-Ag. far IK-atcd injections of 2,4-D did not alter the rate of each of the material*. At this dosage level, 2,4-D growth of twu transplantable mouse sarcomas. produced definite myotonia accompanied by anor In 1047, Hill and Carlisle* published the results exia and weight low while 2,4,5-T produced only of toxicological studies involving various prepara signs of mild spasticity. Both 2,4-D and 2,4,5-T tions of 2,4-D. In sente oral studies, they found were fed five times a week for ninety days at the L i s for miee to be 375 mgykg.; for rata, G68 dosage levels of 2, 5, sod 10 mgVkg. without ad utg-Ag.; for rabbits, MOOmgykg.; and for guinea verse effects. At a dosage level of 20 m itA a , pigs, 1,000 mgVkg. The hugest dose administered Imtli materials caused serious effects; 3 of 4 dogs to monkeys without serious after-effeeta was 214 on the 2.4-T> died while oil of tho 4 dogs on 2,4,5-T mg-Ag.; 428 mg-Ag. roused nausea, vomiting, died. The animals receiving tho 2,4-0 exhibited lethargy, musel# incoordination, aad head drop. stiffuem of the hind legs, difficulty in swallowing, These workers obserrsd that all species m eted bleeding of the gums, ueerorie ehangeo in the similarly and that there was no significant differ- lmerul mueow, and mild liver and kidney changes. Vrotm the Biochemical Besrarrh Dpartant (Bowel aad the Acricultural Chrsikal Rasesreh Laboratory A significant decrease la the number of blood lyntphoeytes was observed terminally ia 3 of the 4 (Kjioss), The Dow Chemical Co^ MMIaad. )(irh. animals. The toxic symptoms noted in the animals [822] DOW 5087F3 V 5239 : 0002319 ^ 1* ml. oi 7.11% methanol in chloroform. Discard llir clllucnt. Then percolate 15 ml. of 7.0% meth anol in dilornforiu through ilic column at the same rate, collecting the cfllucnt in a 250-inJ. beaker. Pour the diluent into a 250-ml. separa tory funnel. Extract the 2,4-D acid from the solution with three 25-mi. por tions of buffered extraction solution and combine the extracts. Return the extract solution to the separatory funnel and wash it three times with 10-ml. portions of analytical reagent grade dtloroform. Discard the wash ings. Acidify the solution with 2 ml. of con centrated hydrochloric acid and extract the 2,4-D acid with three 10-inl. portions of analytical reagent grade dtloroform. Combine the extracts in a 30-ml. beaker. Decant the chloroform solution from the globules of water adhering to the beaker into another clean, dry 30-ml. beaker. Gently evaporate the chloro form by use of a steam bath (test tube clamps may he used to hold the beakers d rrp in the steam bath to facilitate the evaporation). Evaporate just to dry ness and remove the beaker immediately. Cool. Add 5.0 ml. of chrnmwmpic acid re agent to the residue in the beaker and swirl to make a homogeneous solution. Place the lieakcr and contents in an oven set at 150s =fc 2* C. for exactly 10.0 minutes (use interval timer). Cool the solution to room temperature (winepurple colored if 2,4-D is present). Pour the solution with stirring into about 30 ml. of sulfuric acid-stannous chloride solution, rinsing the beaker with a few milliliters of the solution. Stan timing with the interval timer and cool the solution to approximately room tem perature, using a cold water bath. After abuut 5 minutrs, pour the solu tion into a 50-inl. volumetric flask and make tu volume with sulfuric acid- Figvre 1. Recovery of 2,4-D odd from wheat moot two stannous chloride, solution. Mix well and (her through a Whatman's No. 42 or equivalent grade of filter paper. After 30 to 45 minutes, determine the per cent transmhiancy of the filtrate at 565 ma, using 5-cin. cuvettes with water as the reference liquid set at 100%. Read the milligrams of 2,4-D acid contained by the sample front a graph made front the data obtained by analysis of untreated meal to which known quan tities of 2,4-D acid had been added. Calculation Calculate the parts per million of 2,4-D acid present in the sample as follows: Mg. of 2,4-D acid X 1000 grains of sample p.p.m. of 2,4-D add Preparation of Graph Add 0.0, 1.0, 5.0, and 10.0 ml. of the standard solution containing 0.01 mg. of 2.4- D acid per ml. to 200-gram samples of untreated meal, and determine 2,4-D acid according to the procedure. Using the data obtained, construct a suitable graph. Data obtained by the authors on wheat meal are shown in 1 able I. Reference data on known amounts of 2.4- D acid were obtained by pipetting 0, 1.0, 5.0, and 10.0 ml. of the standard solution containing 0.01 mg. of 2,4-D ad d per ml. into four 50-ml. beakers and gently evaporating the chloroform by use of a steam bath. Then 5.0 ml. of chromotropic add reagent were added and the color was developed according to the procedure. Results are given in Table II. The data from Tables I and II are shown graphically in Figure 1. Com parison of the two sets of data shows that the recovery of 2,4-D acid, although not complete, is adequate to detect and esti mate trace quantities of the acid in wheat meal. The over-all loss is corrected in the preparation of the graph. Graphs made for meals of barley', flax, dried peas, and oats were similar to the one prepared for wheat meal. For oats, it was fouQd expedient to use 100-gram samples. The analytical data found on the samples of grain and seed listed above correlate very well with the information available in each case. Chromatographic Separation of 2,4 -0 A cid The developing solution and the elut ing solution used with the Hyflo SuperCel for the chromatographic separation of the 2,4-D acid should be carefully standardized in order to make a proper separation. The adsorptive strength of Hyflo Super-Cel was found to vary somewhat Table 1. Transm ittancy V alues for 2,4-D Acid in W heat Meal ,4 -0 Acid, Mg. % rroflsmrtfoftcy Nil (blank) 0.01 0.05 0.10 89.0 79.8 58.8 42.0 DOW 509189 Tabla II. Transmittancy Values for 2,4-D Acid* 3 ,4 -0 Add, Mg. Nil (blank) 0.01 0.05 0.10 % TrorumiHoocy 97.1 86.2 53.7 27.8 from batch to batch. Therefore, a uni form batch of Super-Cel, in adequate supply for standardization of the chro matographic separation and for use in many subsequent separations, should be set aside. The presence of water was found to affect the elution strength of the solu tions used in the chromatographic sepa ration. Since the water content in tech nical methanol may vary from drum to drum, several gallons of methanol from a selected drum should be stored and used for standardizing the solutions and for the duplication of these solutions there after. In this investigation, methanol containing 0.03% water was used. It is probably not necessary to store chloroform, as variation in quality from various lots appeared to have no signifi cant effect on the elution strengths of the solutions. Prepare the column for the chromato graphic separation by packing the tube with Super-Ce] for a distance of 20 cm. Pour about 30 ml. of chloroform con taining 0.10 mg. of 2,4-D arid into the tube. Apply air pressure at the top of the tul>e to force the solution into the Super-Cel. Percolate 150 ml. of 3.0% methanol in chloroform solution through die column at the rate of 150 ml. per hour, followed by 25 ml. of 7.0% chloroform in meth anol. Discard the effluent. Then pass 50 m>. of 7.0% methanol in chloroform through the column at the same rate and save the liquid in a 100-ml. beaker. Repeat this tlirce times with the similar portions of the eluting sulution. Test for 2,4-D acid in each beaker as follows: Evaporate the liquid by use of a steatn bath, add 10 ml. of chromotropic acid reagent, and swiri to make a uni form solution with any residue. Piece the beaker and contents in an oven set at 150 2s C. for 10 minutes. Then examine the solutions for the wine-purple color which indicates the presence of 2,4- D acid. For a good chromatographic separa tion of the 2,4-D acid, results shoulHd bHe:flO_ ,,0 _ri ,1 06I60S MOa 1. At must, a faint w ine-purple color in the first beaker, indicating a trace of 2,4-D acid. 2. A deep wine-purple color in the icco n d beaker, indicating inot of the original 0.10 mg. of 2 ,4 -0 acid. 3. A pale wine-purple color in the third akcr, indicating only a sm all portion of m e original 0.10 mg. of 2,4-D acid. 4. N o w ine-purple color noticeable in the fourth beaker, indicating the absence of 2,4-D acid. The fourth 50-tnl. portion of 7.0% methanol in chloroform is not run through the tube during a regular analy sis. It is done here for testing the chromatographic separation to make sure that all of the 2,4-D acid is eluted with the first 150 ml. of 7.0% methanol in chloroform. If the results do not show a good sepa ration of the 2,4-D acid, the amount of methanol in the two solutions of chloro form should be adjusted. Discussion Phosphotungstic acid is used to sepa- ' rate proteins when the 2,4-D acid is ex tracted with chloroform from the acidi fied aqueous solution. The buffered extraction solution sepa rates the 2,4-D acid from some of the small amount of acidic material remain ing with the 2,4-D acid after the chro matographic separation. After the final separation of the 2,4-D acid, a very small amount of acidic material from the grain sample may be present, which will give an amber color when healed with chromotmpic acid reagent. The stannous chloride in the dilute sulfuric acid bleaches the amber color, but under the conditions of the experiment it docs not bleach the winepurple color. Though the solution from an untreated grain sample obtained by analysis is not water-white, the per cent transmittancy is high and constant. The color reaction of 2,4-D acid with chromotropic acid is not quite specific. Formaldehyde will produce the same wine-purple color (7). According to Freed (2), phenoxyacetic acid and its derivatives will give the same color, perhaps because formaldehyde is a de composition product. The procedure can probably be used for phenoxyacetic acid and many of its derivatives besides 2,4-D acid, although the solutions of methanol in chloroform may have to be modified to make a proper chromatographic separation of the desired compound. The procedure as given has been used to determine 4chloro-o-toloxyacetic acid (MCI* acid). Although this procedure was developed principally for the determination of 2,4D acid in grain and seed, it can probably *be used for other agricultural products. Fresh vegetables such as peas should be dried by suitable means and the analysis made on a meal of the dried sample. It is possible to detect less than 0.05 p.p.m. of 2,4-D acid in samples by this analytical procedure. Interferences by other compounds were not experienced as, under the conditions of the procedure, the color test is specific for aryloxyacedc acids. LHorxdurw CHod (1) Bricker, C. E., and Johnson, H. R., Ind. Eng. C km ., Anal. E d., 17, 4003 (1945). (2) Freed, V. H., Scitnct, 107, 98-9 (1948). (3) Gordon, N., and Beroza, M., Anal. C km ., 24, 1968 (1952). (4) Marquardt, R. P., and Luce, E. N., Ibid., 23, 1484-6 (1951). R a tia td fo r rttruw Ja m m y 20, 1954. Accoptod S n tm b tr 8 , 1954. a 0002832 33 5242 w m * T H STATUS Of ? $ T 1 C 1 D S M O e f t T H M tL L tA *mNomM r o THE. F e & ftA i poop,OlJJG 4 c o i e r i e G- E Lynn, The Dow Chemical Company A C -r Although representatives of industry and Government have tried to clarify the status of pesticide chemicals under at the present time and on which there seems to be con siderable confusion. die Miller Amendment (Public Law 518, 83rd Congress) to the Federal Food, Drug and Cosmetic Act, there are still some misunderstandings regarding recommendations that can be made for the coming season. Mr. L S. Hitchner, Executive Secretary of the National Agricultural Chemi cals Association, speaking on the subject at The Confer ence on Problems Involved in the Study, Evaluation and Application o f Pesticides with Special Reference to Safety of Use, Washington, D. G, October 19, 1955, said in pan: "Under present Federal legislation, industry is required to supply two basic types of data before sale in inter state commerce. 1. To the United States Department of Agriculture, information and evidence showing the effectiveness of the material and of particular interest at this time the pesticide residue remaining on a crop when used in accordance with label directions. This information, in due course, reaches, where a residue remains, the Food and Drug Administration. 2. To the Food and Drug Administration, the toxicity daca required by them in order to determine a tolerance, if one is required. In establishing a tolerance the amount of residue remaining is considered. "The residue data is obtained from work done by com panies in their own laboratories; from held operations; from 1. Some products are exempt from the provisions of the Law because they are not considered poisonous or dele terious. Example: Sulphur. 2. Products which have no tolerance established. This category would include materials which leave no residue and, therefore, require no tolerance. Example: 2,4-D on small grains. . 3. An exemption from a tolerance. Food and Drug may exempt materials from a tolerance because of their safe characteristics. Examples: Pyrethrum and Copper. 4. A product may have a tolerance established in so many -parts per million. Example: DDT, which has a seven parts per million tolerance. 5. A zero tolerance. A zero tolerance would apply when a material can be used but only under such conditions as result in no residue when the crop is marketed. Example: Mercury. A zero tolerance is often confused with "no tolerance." 6. Certain provisions of the Act may be extended until July 22, 1956. "Those interested in finding out where a product stands in the six categories listed above should write to the manufacturer." the work of Land Grant Colleges, both on cooperative proj ects and independently; from private research institutions and from expert research organizations doing work in toxi cology and medicine. "The result of this program means that adequate infor mation on residues is deposited by the company to reason ably assure, that when used as directed, residues, if any, will be within the tolerance determined by the Food and Drug Administration. Under this program the grower is reasonably assured that when used as directed there is no excess residue and the public likewise is assured there is no undue hazard to the public health. Status o f D o w Agricultural Ch em ica ls All current labels for Dow herbicides, insecticides, fumigants and fungicides have been cleared by the Pesticide Regulation Section of the United States Department of Agriculture. In those cases in the following text where extensions are indicated it is probable that tolerances or exemptions will be established before the law becomes fully effective on July 22, 1956, and that current label directions will continue to be in effect. "As a practical matter, growers and others will have to ' depend primarily on the label to assure there is no excess residue. All instructions on the label, including directions H erbicides 1. Baron (2-(2,4,5-trichlorophenoxy)ethyl 2,2-dichloropropionate). Not presently used on food crops, therefore, for use, timing of applications and other conditions must no tolerance is necessary. be explicitly followed." 2. Brush Killer T, Brush Killer 50-50, Esteron Brush Mr. Hitchner also pointed our that there were, "six cate gories in which the residue tolerance problem can fall Killer, Esteron 245, and Kuron (2,4-D, 2,4,5-T and silvex [2,4,5-trichlorophenoxypropionic acid] brush killers). Not 8 5243 used on food crops, with the possible exception that some might be sprayed on pastures in areas immediately around brush. No residue problem exists (see 3) and no toler ances are necessary. 3. All 2,4-D and 2,4,5-T weed killers. Data have been presented to the ILS.D-A. that show no residue problem exists on small grain or in milk. In addition, the F.DA. has agreed that there is no residue problem in meat. No residue tolerance is necessary. 4. Dow MGP Amine Weed Killer, (4-chIoro-o-toIoxyacetic acid, or 2-methyl-4-chlorophenoxyacetic acid, amine salt). The character and use of die chemical are similar to 2,4-D. It is the opinion of qualified experts in the field that no residue problem exists and that no tolerance will be necessary. An Industry-National Agricultural Chemi cals Association Committee is now conducting analyti cal investigations. 5. Dalapon, Sodium Salt 83%, (sodium 2,2-dichloropropionate). Data have been presented to the U.S.D.A. that show no residue is present in sugar cane at harvest. With respect to pre-planting applications, dalapon is destroyed in warm, moist soil within several weeks after application (see D o w n to E arth, 11 (2 ):2 (1955) ). 6. Dow General Weed Killer, Premerge and Dow Selective Weed Killer, (formulations based on dinitro-orec-butylphenol). Dinitro-o-rec-butylphenol has a zero tol erance. Dow has presented data to the U.S.D.A. that show no residues are present in the harvested crops when the products are used in accordance with label directions. These have been cleared by the U.S.D.A. It should be noted that in pre-harvest treatment of seed crops with Dow General Weed Killer, the label warns that the forage is notj o be used as animal food, since such forage may contain DN residue. 7. Sodium TCA 90%, (sodium trichloroacetate). Data have been submitted to die U.S.D.A. that show no resi dues are present in sugar beets, cabbage and tomatoes when used in accordance with label recommendations. In view of the sugar cane data on dalapon, it is unlikely that residues will occur in this crop. Data are now being obtained. It is not anticipated that residue tolerance will be necessary for TCA. Insecticides 8. Arsenare of Lead. A residue tolerance of 7 ppm of combined lead has been set on the raw agricultural com modities that appear on the Dow labeL 9. DDT. A residue tolerance of 7 ppm has been set on a wide variety of raw agricultural commodities appearing on the Dow labeL An extension has been granted for meat until March 1, 1956. 10. DN-Dry Mix No. 1 (dinitro-o-cydohexylphenol). A residue tolerance of 1 ppm on citrus has been set. 11. DN-Dry Mix No. 2 (dinitro-o-cresol). A zero tolerance has been set. No residues from stria dormant application. 12. DN-289 (dinitro-o-xec-butylphenol, triethanolamine salt). Zero tolerance. No residues from strict dormant application, or when applied after harvest. 13. DN-111 (dinitro-o-cyclohexylphenol, dicydohexylamine salt). A 1 ppm tolerance has been set on apples, apricots, beans, blackberries, black-eyed peas, celery, cherries, citrus, grapes, loganberries, neaarines, peaches, pears, plums, quinces, raspberries and strawberries. 14. Lindane, BHG Tolerances for a large number of, raw agricultural commodities have been set as follows: l i n d a n e ............................. 10 ppm benzene hexachloride . . 5 ppm An extension has been granted for lindane for several additional items until January 22, 1956 and on BHC in meat until March 1, 1956. 15. Lime Sulfur, Sulfur. The residues left by these pestiddes are exempted from a tolerance. 16. Ovotran Wettable (p-chlorophenyl p-chlorobenzenesulfonate). An extension has been granted until March 1, 1956 for citrus. A petition has been submined for tol erance in and on dtrus, apples, pears, peaches, plums and prunes. 17. Parathion. A tolerance of 1 ppm has been set for a wide range of fruits and vegetables appearing on the Dow labeL An extension has been granted to January 22, 1956 for field CTops, forage aops, hops and olives. 18. Systox. A tolerance of 0.75 ppm has been set for apples, broccoli, brussels sprouts, cabbage, cauliflower, muskmelons, oranges, pears, potatoes, strawberries and walnuts. 19- Dowfume 75, Dowfume EB-5, Dowfume EB-15, Grain Fumigant (80-20 Mixture). All grain fumigants con taining carbon tetrachloride, carbon disulfide, ethylene dichloride, and ethylene dibromide have been extended until March 1, 1956. Dow and other companies have been doing analytical work with the Food and Drug Administra tion to obtain the necessary data to establish tolerances or exemptions. 20. Methyl Bromide. An extension has been granted until January 22, 1956 for certain raw agricultural com modities. Dow has filed a petition asking for tolerances on apples, pears, quinces, onions, tomatoes, eggplants, beets, beans, cocoa beans, wheat, rice, rye, oats, barley, com, grain sorghum, sweet potatoes, alfalfa hay, peanuts, potatoes, pecans, turnips, rutabagas and cottonseed. 21. Dowfume W-85, Dowfume W-40, Garden Dowfume ( ethylene dibromide soil fumigants). An extension has been granted until January 22, 1956. Dow has filed a petition asking for tolerances on: Lima beans, com, strawberries, sugar beets, asparagus, lettuce, sweet potatoes, parsnips, potatoes, turnips, rutabagas, carrots, celery and cottonseed. (Tobacco was not requested since it is not a food). Addi tional data are being obtained for tolerance in other aops. Fungicides 22. Lime Sulfur, Sulfur (See item 15). 23. Ferradow (ferbam). A tolerance of 7 ppm has been set for a wide variety of fruits and vegetables appearing on the Dow labeL Other Agricultural Chemicals Nematocides, defoliants, and plant growth regulators are not defined as economic poisons by the Federal Inseaicide, Fungicide, and Rodenticide A a. They are not registered by die U. S. D. A. and tolerances cannot be set under the Miller Amendment. Tolerances of 1 ppm naphthaleneacetic acid (App-L-Set) and 5 ppm for 2,4-D were s a on certain fruits as a result of the 1950 hearings. Data are being obtained on Color-Set (2,4,5-trichlorophenoxypropionic acid) to determine if residues -are present at harvest. Magron (Dow magnesium chlorate defoliant) has been investigated and no residues of magnesium chlor ate have been found on rice or beans defoliated with this produa. 9 5244 D o w n to Earth, Winter 195 34 5245 i T 1...A.- Containing 6.15 Pounds per FOR THE Ac||ve Ingredient: 2,4,5-Trtchlorophenoxyacetlc Acid, Propylene Glycol ( C | l l , | ) Ip c ( l l , <0 j l Buryi Ether E s t e r s __________ _____________________ 2,4,5-Trichlorophenoayacellc Acid Equivalent 42.5? Inert In g re d ie n ts____________________________________________________________________ 5.3 34.7% Esteron 2 4 J it recommended for use in controlling herboceous and woody plants grow ing in rangeland, pastures, fence rows, ditch banks, farmyards and right-of-ways. It is cffectise in controlling certain 2 ,4 -0 resistant plants such as ash, brombles, ground chefty, hawthorn, horse nettle, maple, mesquitc, oak, osage oronge, palmetto, poison ivy, prickly pear cactus, salmonbcrry, wild blackberry, wild rose and certain species of Ribcs, as well as many other woody ond herboceous species. D IR EC T IO N S PREPARIN G THE S P R A Y : Add holf the required amount ol water or oil to the spray lank, then odd the E.tcrnn 245 with oqilalion, and finally the bolancc of the water or oil with continued oqilolipn. W A R N IN G : II Esteron 245 is to be used In preparing straight oil mi lures, la not let voter get into the Esteron 245 itself nor into lire fin ished m ivlqic. N O T E: Esteron 2 15 ferms on emulsion-- not a solution - in water, and the Esteron 245 lends to separate out on slandinq. Provide agitation to prevent such separation and ensure uniformity of spray mixture. For Trealinq Smeli A rc a i: One lahlcspoonlul cf Eslcron 245 in l ' j gotico ol oil or voler is a|-.v''ximatrly eguivalent to onc quoti in 100 galloni. FO LIA G E T R E A T M EN T : Spray woody growth up to 6 or 8 fuel toll alter foliage is well developed, using o d r e n c h i n g sproy containing 3 quarts of Eslcron 245 per 100 gallons ol safer. Toller brush can be sprayed successfully, although in many cases basal bark or stump treatment is prcfcroble. Poison Ivy, most brambles and some other species may be controlled bv using 2 quarts per 100 gallons of water. Coverage should ha 'omplele, ond oil ports of the plants, Including foliage, shoot stems ond bark, should be wet with the spray, Bed results usually w ill be obtained from applications made scon o iler maximum lalioge development in the spring. W ith good growing conditions ond adequate soil moisture, applications may l,e mode up to 2 or 3 weeks behrre nor mal Irost dale. Less effective control may result during hot, dry sveolher when deep soil moisture is deficient. Power equipment with pressures up lo 250 pounds will aid en to.. CD /? 5 7 Gallon of Powerful, Low Volatilty Esters of 2,1,5-T 2,4,5-T Acid Equivnl CONTROL OF MANY SPECIES OF TREES, BRUSH AND BROAD-LEAVED W In ob taining satisfactory spray co verag e. Repeot ap p licatio n s m ay be necessary os new grow th develops, but a single treatm ent in any one yeor is usuolly su fficien t. N O T E : M any brood-leaved w eeds ore controlled by this application. BASAL BARK TREATM EN TS Brush and small trees con he controlled by spraying the basal parts of brush stems and tree trunks to a height of 12 to 15 inches from the ground line. Use a solution of 4 gollons of Esteron 2-15 in 96 gallons I I pint in 3 gallons! of diesel oil, fuel oil or kerosene. W ith certain species, 2 qollons of Esteron 2d5 In 98 gollons of diesel oil, fuel oil or kerosene is effective. Knapsack or power equipment moy be used, but complete wetting of the indicated orco is necessary, par ticularly at the ground line. This means spraying until run-dawn or run-off to the ground line Is noticeable. Old or rough bark requires mare sproy volume than young or smooth bark. Low pressures are deslroble. Apply ol any time, including the winter months. Often dcloyed response and killing can be expected. Treated brush or tree-, preferably should not be cut for o period of one year following application. STUM P T R E A T M E N T : W here qrowlh is more thon 6 to 8 lert toll, cut it clme the ground ond spray the slumps and stubs with -I gallons of Esteron 215 in 96 g:l'< ns tor I pint In 3 qoll-nst of diesel oil, fuel oil or kerosene, mixed llmtoughly. thoroughly all exposed bark, as well os cut surfaces. This means spraying until run down or run-off la the ground line is noticeable. Old or rough hark requires mote spray volume than young or smooth bark. Apply at any lime, including t h e w i n t e r months, prcfcrobly to ficshly-cut stumps. Best results ore usually obtained on stumps two inches across or larger. 'F R ILL " T R E A T M E N T : For large trees, make a single-hack girdle or " frill" cf ^.ot. lopping axe cuts completely around the Iree as close lo the ground ns possible. T>*-1 1 the injured area with a mixture of 2 gollons of Eslcton 2-15 in 100 gallons I 1; pint in 3 gallons) of diesel oil, fuel oil or kerosene. A IR F L A N E A P P LIC A T IO N S: To control mesqulte, use I to I 1 1 pints o f Esteron 2 15 per ocre In 3 gollons of woter ond 1 gollon of w atcr-clcor diesel ht-l. Apply 50 to 80 dttys o iler lirsl leaves appeor. Do not treat If tlrculh hos prevented heavy frli-g c growth. For post oak and btock|ack oak, use 2 quarts of Esteron 2-15 per acre. M is this either with 3 to `I gollons of wolcr and I gollon of dtescl cil. or with 3 la d gallons of diesel oil. Apply oiler foliaqc Is fully developed. N O TE: Soil mnislu-c must be odequale for normol growth. Rc-trcot os accessory In succeeding years. Cl o Do vr LVt |Im:' te n , vci D- In ! ,1,.. r* in p lw '1 L-rr i I1 c,p pt 9 S 6 T 2 0 N11 9Z0GCH r m I  OvU. I. *.* + wjoa.n0., .A rmw q|UJ,i s ',. ''., *V.* ?*`n^V'v-'v* .?*J',,Vt:' v{['. ' . *>: ):.' , 1\ iiA /krb 'tkid U Ju , 4/i.' / ',.' 'V !t`il. irf<rtiMfcii<diiil.*UVdL^'WVi* .,,jl w *Jwi>twJl. i I- 1. J *'> 7S M08SSN MULTl-USl U S A ? ! WSSSS) a m lotilty' Esters of 2,4,5-T 2,4,5-T Acid Equivalent 4 Pounds per Gallon >n I* 7 E CONTROL QF M ANY SPECIES OF TREES, BRUSH AND BROAD-LEAVED WEEDS I x j l4 Jin oblolnino satisfactory spray coverage. Repeal applications may be necessary as new L L .growth develops, but a single treatment In ony one year is usuolly sufficient, N O TE: W ARNING! * Do not apply Esteron 245 directly to, or otherwise permi.t.'it to come into contact svith Mony broad-leaved weeds are controlled by this application. vegetables, flowers, grapes, fruit trees, ornomcntols, cotton or other desirable plants BA SAL BARK T R E A T M EN T : Brush and small trees can be controlled by spraying the basal ports of brush stems and tree trunks to a height of 12 la 15 Inches from the ground line. Use a solution of -t gallons of Esteron 2-15 In 9 6 gallons 11 pint In 3 gallons I of diesel oil, fuel oil or kerosene. W ith certain species, 2 gallons of Esteron 2-15 in 98 gallons of diesel oil, fuel oil or kerosene is effective. Knopsack or power equipment may be used, but complete wetting of the Indicated area Is necessary, par ticularly at |he ground line. This meant spraying until run-dawn or run-aff to the which are sensitive to 2 ,4,5-T , ond do not permit spray mists containing it to drift onto them, since even minute quantities of the spray may cause severe injury during bath growing and dormant periods. (Coarse sprays are less likely to d rill, t Accordingly, applications by airplane, ground rigs and hand dispensers should be carried out only when there Is no hasard from drift. Do not apply by airplane in the vicinity of cot ton, grapes ar other desirable 2 ,4 ,5 -T susceptible vegetation. At higher temperatures vaporization may cause in|ury to susceptible plants growing nearby. ground line It goliceabte. Old or rough bark requires mare spray volume than young Do not use on lawns of creeping grosses, such as bent, except for spot spraying, nor on or smooth berk. Low pressures are desirable. Apply at any time, Including the winter freshly seeded turf until grass hos become well established. (Most legumes are usuolly m -o lli'. Often delayed response and killing can be expected. Treated brush or trees preh-'ddy should not be cut for o period of one yeor following application. damaged or killed.l Do not contaminate Irrigation ditches or water used for domest'C purposes. Do not store near fertilizers, seeds, insecticides or fungicides. To avoid STUM P T R E A T M EN T : Where growth is more than 6 to 8 feet tall, cut it close to the ground and spray the stumps ond stubs with -1 gollons cf Esteron 2-15 in 96 gollons . _ tor I pint in 3 notionsi of diesel oil, fuel oil or kerosene, mixed thoroughly. W et injury t4 desirable plants, do not store, handle or opply other agricultural chemicals with Ihti same containers or equipment used with Esteron 24 5. Local conditions moy affect the use of herbicides. Stole agricultural authorities in . jlt'or^ugbly ql| exposed bark, as well as cut surfaces. This means spraying until run- many stales issue to immandations to fit local conditions. ' 'd o w n ar run-aff to the ground line Is noticeable. Old or rough bark requires mare spray volume than young ar Smooth bark. Apply at any lime, including the winter Be sure that use ol this product conforms to all applicable regulations. months, preferably to freshly-cut stumps. Best results ore usuolly obtained on stumps two inches dcross or larger. CAUTION! MAY CAUSE SKIN IRRITATION Avoid Contact with Eyes, Skin and Clothing " F R IL L " T R E A T M EN T : For large trees, make a single-hack girdle or "frill" of over lapping axe cuts completely around the tree as close to the ground as possible. Treol the ln|urcd orro with a mixture of 2 gollons of Esteron 2-15 in ICO gollons ( * i pint In 3 gallons I of diesel oil, fuel oil or kerosene. N O T IC E Seller makes no worronty of ony kind, express or implied, concerning the use of ibis product.. Buyer assumes all risk of use or hondling, whether In accordance with direc tions or not. A IR PLA N E A P P LIC A T IO N S: To control metquite, use I to I ! i pints of Esteron 245 per acre in 3 gallons of woter and I gallon of water-clear diesel fuel. Apply 50 to U. S. Patents No. 2.390.941; 2 ,3 9 6 .5 1 3 ; 2,4 7 2 ,3 4 7 and 2,562,855 80 doys after first leaves appear. Do not treot If drculh has prevented heovy foliage growth. For post oak and blackjack oofc, use 2 quarts of Esteron 245 per acre. Mix THE DOW CHEMICAl COr.UWflY ihls cither with 3 la 4 gallons ol water ond I gallon of diesel oil, or with 3 to. 4 gallons M IDLAND, M ICHIGAN ol diesel oil. Apply alter foliage Is fully developed. N O TE: Soil moisture must be adequate for normal growth. R c-lreal os necessary in succeeding ycors. * * M IDLAND DIVISION 5247 966120NH ( 7 n n P T I fJjflfl 3* 5248 increased number of diskings between spraying and seed ing resulted in a slightly lower stand of trefoil plants. This was not apparent at the Southeastern Ohio test. In other irds, a lesser amount of tillage on the 7-day interval ._rt a firmer seedbed which meant less coverage of seeds and earlier germination. Current tests in Ohio are directed at determining the minimum amount of tillage necessary with herbicide treatment. TABLE II P ercent Stand o f Kentucky Bluegrass in T illed and Un -tillbd Areas Rem aining in th e Fall o f 1936 Following H erbicide T reatments Made on __________ October 20, 1955 and April 19, 1936_________ Harbicida Rnta lb*/A Acid f manient fa ll Applied_______ Spring Applied TilingaN o*tilinga Tilinga lio*tilinga D alap o n -1 0 ________ Dalapon- 5 _______ Dalapon - 2 0 _______ Dalapon - 1 0 _______ Trace Trace 75% Trace 35% Trace 45% Trace ATA- 4 ____________ Trace ATA- 2 ____________ ATA - 8 ____________ Trace ATA- 4 ________________ 90% Trace 60% Trace 30% 10% Table II summarizes data taken in a field test at the U.S.D.A. Soil and Water Conservation Research Station at Coshocton, Ohio. Duplicate plots were laid out in the fall of 1953 for spraying to compare fall and spring treatments. Estimates of stand of bluegrass in the 1955 fall and 1956 spring treated areas were made in the fall of 1956. The main point brought out in these data is the effect of tillage on percent of bluegrass kill, both fall and spring. The tillage implement used in this case was a disk harrow. All tillage plots were disked thoroughly 5-10 days after spray ing and fall sprayed plots were disked again in the spring prior to seeding. The bluegrass kill was almost complete on fall and spring sprayed tillage plots. The heavier fall herbicide rates without any tillage also resulted in almost complete kill of bluegrass. The importance of tillage in conjunction with spring applied chemicals is quite appar ent even at heavier Tates. The stand of trefoil obtained by band seeding in this test was variable and inconclusive but indications are that satisfactory stands of trefoil can be established with fall applied herbicide and no cultivation. On these fall-treated, no-tillage plots the trefoil stand was estimated to be from 50 to 80 percent. Again, winter survival will affect appraisals of results. In conclusion these data show the possibilities of using herbicides in seedbed preparation for birdsfoot trefoil. Proper use of available materials will suppress competing vegetation sufficiently to permit better stands of trefoil with a minimum of cultivation. Dalapon can be used effectively as either a spring or fall treatment It lends itself readily to spring application because of its low toxicity to birdsfoot trefoil seedlings. On the other hand ATA is probably best applied only in the fall if spring seeding is intended. There is need for further research to determine: (1) The minimum amount of tillage necessary, (2) The usefulness of the new pasture renovation seeding equip ment that is on the market, (3) Minimum amount of chemical to use under various conditions. REFERENCES (1) Sprague, M. A., The substitution of chemicals for tillage in pasture renovation, Agron. Jour. 44:405-409, 1952. (2) Kates, Allan H., M. A. Sprague and R. J. Aldrich, Promis ing chemicals for seedbed preparation, Down To Earth, Vol. 10, No. 4, 1955 (Pages 10 and 11). RESIDUE TOLERANCES FOR DOW A G R IC U LTU R A L CHEM ICALS By G. E. Ly n n *, The Dow Chemical Company It is now well recognized that pesticidal chemicals must be used in agriculture to prevent crop and animal losses during production and storage. Pesticidal chemicals, such as insecticides and weed killers, have become a necessary part of the farming economy and take their place in importance along with the tractor and other modern farm ing tools. Pesticides can be used safely and without hazard to the consumer of the food crop. Although some pesti cides may leave trace residues on food crops, it has been shown by extensive toxicological investigations that the small amounts of most pesticides that appear in food can be consumed without any adverse effect on the health of man or animals. Under these circumstances the Miller Amendment to the Federal Food, Drug and Cosmetic Act provides a means by which adequate residue toler ances on raw agricultural commodities can be established to permit the use of the pesticides. The Dow Chemical Company has obtained the necessary data to establish many such tolerances for its agricultural chemical products. Additional data are being obtained to expand the list of commodities and crops on which both old and new pesticides may be used. Many pesticidal chemicals are used in ways which do not result in a residue. Application early in the growing season, or use at low dosage rates often eliminates the residue problem. Numerical tolerances are not necessary under these circumstances. These chemicals are, however, subject to registration and ample proof of the lack of resi due must be submitted to the United States Department of Agriculture before a registration is granted. For example, Premergef, containing dinitro-o-sec butylphenol, is recom mended for a variety of pre- and postemergence herbicidal uses. Each use has been investigated thoroughly not only for effectiveness on weeds and safety on crops but also for possible crop residue. When used as directed on the label, there is no crop residue problem. Users of agricultural chemicals are urged to follow the directions that appear on the labels. Any other use, or deoarture from label (Continued next page) 'Midland, Michigan. t Premerge is a trademark of The Dow Chemical Company. 5249 D o w n t o \ E a r t h , Spring, 1957 . i recommendations, may result in poor performance and residue problems not covered by tolerances. Some chemicals, used in agriculture, are not classified as pesticides by the Federal Law and thus cannot be regis tered. Dow agricultural chemicals in this category are Magron, Colorset, App-l-set, anhydrous ammonia and the soil fumigants, Fumazone, Telone and Dorlone. The Dow Chemical Company has carefully investigated these chem cals and when each is employed in accordance with got agricultural practice, as directed on the label, no pubi health hazard will result. The following information summarizes the status many registered Dow Agricultural Chemical products as January 15, 1957. RESIDUE TO LERAN CE STATUS FUMIGANTS D ow A gricultural Product Dowfume * EB-5 and Dowfume EB-15 Dowfume 75 Dowfume 80-20 Dowfume C Dowfume 1; Dowfume G Dowfume H Dowfume J Verrifume* Serafume* Methyl Bromide A ctive Ingredients ethylene dibromide ethylene dichloride carbon tetrachloride ethylene dichloride carbon tetrachloride carbon tetrachloride carbon disulfide carbon tetrachloride carbon disulfide ' ethylene dibromide carbon tetrachloride ethylene dichloride ethylene dibromide sulfur dioxide ethylene dichloride carbon tetrachloride ethylene dibromide carbon tetrachloride carbon disulfide sulfur dioxide ethylene dichloride carbon tetrachloride ethylene dibromide carbon tetrachloride carbon disulfide carbon tetrachloride ethylene dibromide ethylene dichloride carbon disulfide methyl bromide Dowfume MC-2 Dowfume W-85 methyl bromide chloropicrin ethylene dibromide Picfume* chloropicrin Trademarks of The Dow Chemical Company Tolerances Carbon tetrachloride, ethylene dichloride, carbon disulfide, and the orga bromide residues from ethylene dibromide are exempted from the requ ment of a tolerance when used as a fumigant for the following grains: barley, corn, oats, popcorn, rice, rye, sorghum (milo) and wheat. Tolerances of 50 ppm are established for residues of inorganic bromi (calculated as Br) in or on the following grains that have been fumiga with ethylene dibromide: barley, corn, oats, popcorn, rice, rye, sorghum (milo) and wheat. Sulfur dioxide in amounts used does not constitute a residue problem. The tolerances for residues of inorganic bromides (calculated as Br) or on raw agricultural commodities which have been fumigated with met bromide are as follows: (a) 5 ppm in or on apples, pears, quinces. (b) 20 ppm in or on eggplants, onions, tomatoes. (c) 30 ppm in or on beets, rutabagas, turnips. (d) 50 ppm in or on alfalfa, hay, barley, brans, green brans, lima brans, snap beans, corn, grain sorghum (milo), oats, rice, rye, wheat. (e) 75 ppm in or on potatoes, sweet potatoes. (f) 200 ppm in or on cottonseed. When used in accordance with directions as a plant bed treatment and w' transplants are moved to soil not treated with this fumigant, no detear residues are probable in or on a food crop. The tolerance for residues of inorganic bromide (calculated as Br) in or raw agricultural commodities grown on soil treated with ethylene dibrorr are as follows: (a) 5 ppm in or on lima brans, strawberries. (b) 10 ppm in or on asparagus, cauliflower. (c) 25 ppm in or on cotton. (d) 50 ppm in or on sweet potatoes. (e) 75 ppm in or on carrots (with or without tops), parsnios. Leaves no residues on food crops when used as a soil fumigant in accord: with Dow label instruaions. 10 5250 *% HERBICIDES rt'tw A gricultural P roduct A ctive Ingredient! Tolerances Dowpon* -dalapon, sodium salt (2,2-dichloropropionic add, sodium salt) Tolerances established for 5 ppm in or on sugar bens and 35 ppm in or on cottonseed (calculated as 2,2-dichloropropionic add). No residues result from early pre-planting treatments when used in accordance with Dow label instructions. No residues result from use on sugar cane. Dow Sodium TCA 90% trichloroacetic add, sodium salt When used in accordance with Dow label recommendations, no residue in or on a food crop will result. Premerge* Dow Selective Weed Killer Dow General Weed Killer tmeron* 44 dinitro-o-sec butylphenol, alkanolamine salts dinitro-o-sec-butylphenol, ammonium salt dinitro-o-sec-butyiphenoi, 2,4-dichlorophenoxyacedc add, isopropyl ester Zero tolerances established. When used in accordance with Dow label recommendations, no residues in or on a food crop will result. tirteron Ten Ten 4Estexon 99 2,4-dichlorophenoxyacetic add, propylene glycol butyl ether esters Esreron 76E 2.4-dichlorophenoxyacetic add isopropyl ester and 2.4-dichlorophenoxyacetic acid, butvl esters Ccteron Brush Killer 2,4-dichlorophenoxyacetic add, propylene glycol butyl ether esters, and 2,4,5-trichlorophenoxyacedc add, propylene glycol butyl ether esters Csteron 245 Reddon* 2,4,5-trichlorophenoxyacetic add, propylene glycol butyl ether esters flow 2,4,5-T Amine :ed Killer 2,4,5-trichlorophenoxyacetic add. triethylamine salt i\.uron* 2 (2,4,5-trichlorophenoxy) propionic add, propylene glycol butyl ether esters 2,4-Dow Weed Killer, formula 40 Home Use Weed Killer 2,4-dichlorophenoxyacetic acid, alkanolamine salts Dow MCP Amine Weed Killer 2-methyl-4-chlorophenoxyacetic add. alkanolamine salts * Trademarks of The Dow Chemicai Compnj When used in accordance with Dow label recommendations, no residue in or on a food crop will resu lt (Note: A tolerance of 5 ppm 2,4-dichlorophenoxyacetic acid has been established in or on apples, dtrus fruits, pears, and quinces.) -- INSECTICIDES D ow A gricultural Product A ctive Ingredients T o le ra n c e i DN-Dry Mix* No. 1 dinitro-o-cydohexylphenol A residue tolerance of 1 ppm is established in or on citrus fruits. DN-11I* dinitro-o-cydohexyiphenol, dicydohexyl amine salt A residue tolerance of 1 ppm is established in or on apples, apricots, beans, blackberries, black-eyed peas, celery, cherries, citrus fruits, grapes, logan berries, nectarines, peaches, pears, plums, (fresh prunes), quince, and raspberries. DN-Dry Mix* No. 2 dinitro-0-cresol Zero tolerance established. When used in accordance with Dow label recom mendations, no residue in or on a food crop will result. DN-289* dinitro-o-sec. butylphenol, tri-ethanolamine salt Zero tolerance established. When used in accordance with Dow label recommendations, no residue in or on a food crop will result. Ovotran* Wettable ovex (p-chlorophenyl p-chloro- Residue tolerances are established as follows: benzenesulfonate) (a) 3 ppm in or on apples, peaches, pears, and plums (prunes). (b) 5 ppm in or on grapefruit, lemons, oranges, and tangerines. Dowicide* A Sodium-o-phenylphenate A tolerance of 10 ppm is established for residues of sodium o-phenylphenate (calculated as o-phenylphenol) in or on each of the following citrus fruits: oranges, lemons, limes, grapefruit, tangerines, rangelos, citrus citron, kumquar. A tolerance of 5 ppm is established for residues of sodium o-phenylphenate (calculated as o-phenylphenol), in or on apples and pears. * Trademarks of The Dow Chemical Company ii 5251 D o w n to Earth, Spring, 1957 5252 A CD file no. HG-51 V TOXICITY OF "HERBICIDES TO DOMESTIC ANIMALS by T. A. Hymas, DVM Agricultural Chemical Research THE DOW CHEMICAL COMPANY Mid land, Michigan May 29, 1958 In reviewing the paper that I am about to present I believe the title should more appropriately be "The Lack of Toxicity of Herbicides to Domestic Animals" rather than "Toxicity of Herbicides to Domestic Animals." The use of herbicides for the control of undesirable vegetation has been practiced for many years, some of the older materials that have been used are the chlorates, arsenlcals, boron compounds, certain oils, and even table salt. While some of these materials are still being used on occasion for this purpose, research over the past 14 years has revealed new herbi cide materials that are superior to the older materials and in some instances are less toxic to animals and also man. The herbicides that I wish to dis cuss at this time are organic chemical compounds that act 3ystemically when applied to vegetation. They have a selective toxicity to specific liants and are currently being used throughout the world for the control undesirable vegetation. I am going to confine my remarks to several of the 2,4-D type materials which can be classed as chlorinated phenoxy-acetic acid compounds, and to a relatively newcomer to the field of selective herbicides, dalapon, known chemically as 2,2-dichloropropionic acid. To begin, I would like to give some comparative data that has been ob tained by numerous investigators, to compare the toxicity of these herbicides when given orally to different species of animals. In T a b l e No. 1 the ac u t e oral L D c q values for several species of animals fed the most widely used chloropnenoxy-acetic acid compounds is given. These are 2,4-D (2,4-dlchlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichloro- phenoxy-acetic acid), and silvex which is 2-(2,4,5-trichlorophenoxy) propionic acid. The chlorinated phenoxyacetic and propionic acid compounds are very similar in chemical make-up, they are also similar in their toxicity to animals as one might expect. Dogs appear to be the most sus ceptible species to the chlorinated phenoxy acetic and propionic acid materials. Where there are two sets of figures given in the table they represent a range of values to cover the pure acids, also esters and salts of the compounds, sex differences, and work by different investigators. While, the basic acid has not b e e n greatly altered when formulated, as an ester or salt, the foiroulation may only slightly alter the biological ctivity of the toxicant as measured by herbicidal activity and toxicity ico animals. 5253 The value given for cattle was obtained using a mixture of 2,4-D and 2r%,4it,5c -Tm. 0003361 Tl 'H -2- Becauae of the similarity of toxicity of 2,4-D and 2,4,5-T to laboratory animals and the wide use of the combination of these herbicides in .commercial products, it was decided to use a combination of the two materials (Esteron* Brush Killer) containing approximately equal parts of 2,4-D and 2,4,5-T as esters, to study the acute oral toxicity of the materials in cattle. t The cattle used for these studies were dairy cattle of mixed breeding weighing fr o m 250 to 336 kilograms. Table No. 2 is a summary of the work accomplished. Experiment # 1 : A steer weighing 291 Kg. was given a single oral dose of 1,000 mg/Kg acid equivalent of Esteron Brush Killer, sufficient herbicide to prepare 11 gallons of spray at the highest level recommended, 4 qts/100 gal. of spray, or 1 6 .6 gallons at the usual recommended rate of 3 qts/100 gal. of spray. This dosage had no observable effect on the steer. A second steer weighing 295 Kg. was given 1,000 mg/Kg acid equivalent of this same mixture of 2,4-D and 2,4,5-T, as Esteron Brush Killer, on each of three successive days. The quantity of herbicide given would treat 1/2 acre of heavy brushy, woody, or weedy area at the recommended use level. The day following the third dose a general depression was noted, the animal was off feed and lethargic. The general attitude and behavior became progressively worse until the animal died on the third day following l the last dose. Death was undramatlc. The steer evidenced no pain, no struggling, or signs of violence. Upon autopsy it was noted that the rumen had a strong odor that is characteristic of the 2,4-D type materials. The reticulum, rumen, omasum, abomasum and fore-part of the duodenum were abnormally dry and Impacted. Rumen stasis was evident. Beyond the duodenum the G. I. tract contents were fluid. No other gross abnormalities were evident. A third steer weighing 295 Kg. was given 500 mg/Kg on each of two consecu tive days. Oh the third day the animal was off feed and rumen motility had essentially ceased. Otherwise the animal appeared normal. On the fourth day, this steer appeared normal and there were no discernable after effects. A fourth animal weighing 336 Kg. was given 500 mg/Kg on each of three successive days and no toxic symptoms were observed.The quantity of herbicide given would make 2 7-5 gallons of spray at the recommended con- . centration of 4 lbs. acid equivalent per 100 gallons of spray. The animal remained on full feed without showing effects of any kind. < A fifth steer weighing 250 Kg. was then given 100 mg/Kg of the 2,4-D, 2,4,5-T mixture on 15 consecutive days and observed carefully. There were no outward appearances of adverse effects. The animal was necropsled 48 hours following the last dosage. Gross examination of the internal organs revealed only slight petechial hemorrhage in the duodenum and a very mild diffuse irritation in the abomasum. Histological examination of U tissues revealed very mild pathology in the liver and kidneys. The liver contained small areas of focol hemorrhagic necrosis surrounded by areas * Trademark of The Dow Chemical Company 5254 0003362 I' of fatty degeneration. In the kidneys a slight interstitial adema and congestion of the cortical medullary regions was observed. The amount of 2,4-D and 2,4,5-T that was ingested by this steer was 0.8 lb., which is sufficient to make 20.5 gallons of spray to treat dense or hard to kill foliage or herbage. It would be Impossible for one or more bovine to consume all of the toxi- c cant applied in 2 0 to 30 gallons of spray even if they were confined in c an area where all vegetation was treated. Some toxicant drips or is c sprayed on the ground and m uch vegetation that is sprayed is of an unpalatable nature being brush, weeds or the like. I do not believe the chlorinated phenoxy acetic acid herbicides constitute any hazard whatsoeve: to cattle If they are handled with reasonable care and used according to recommendations of the manufacturer. I would now like to consider dalapon (2,2-dichloropropionic acid) for a few minutes. Slide # 3 gives the acute oral toxicities of dalapon In several species of animals. In eva l u a t i n g these figures it is well to r e m ember that the LDc q value for sodium chloride (table salt) is 2,500 to 3,500 mg/Kg In laboratory animals. The figure reported for dogs, an LDc q of greater than ^ j 1,000 mg/Kg, is the largest dosage that dogs would retain without emesis. We have found that quite often it is very difficult to determine acute oral toxicities in dogs because of their ability to regurgitate such materials and our inability to control this reflex even with the use of morphine, various sedatives, tranquilizers, etc., which, of course, we are always reluctant to use because of the effect of the anti-emetic Itself. I would like to say at this point that where we have had an opportunity to compare the acute oral toxicity values of herbicide materials In small animals with those obtained using cattle, the two values are usually close. If real differences exist, to date, in our work, cattle have tolerated the larger doses, perhaps because of the bacterial flora in the rumen and their ability to decompose organic materials. We attempted to attain acute oral LDeQ values for dalapon In cattle but, as can be seen in slide #4, when 4,000 mg/Kg, approximately 3*5 lbs. of the chemical was "pumped" into a steer at one time, 5-5 lbs. was "pumped" into another steer over a ten day period and an equal amount on a weight basis "pumped" into a suckling calf, and the worst reaction seen was a transient digestive upset and, I might add, a very effective grass killing Job in our grass pasture where urination occurred following dosing. We gave up trying to kill cattle with dalapon as it appeared they would need to be drowned in it. Histopathological studies of tissues from the animals that were sacrificed revealed no abnormalities. We concluded that dalapon was not a hazard on farms, rights-of-way, in ditches, or farm ponds, or under any conditions of practical use. This conclusion has been sub- \.y stantiated by several years use in this country and other countries without a single substantiated case of poisoning being brought to our attention. And, we believe if poisoning cases had occurred we would know y&lHl336about them. Being associated with a chemical company fo r several r ' 5255 4- - I know that where chemicals are used and any abnormality is observed, the first.thoug h t is u s u a l l y -- the chemical did it. Any legitimate chemical manufacturers has always expended considerable time and effort to deter mine the hazards of having chemicals used where men and animals are likelyr- to be exposed. 2 A great deal of work and effort always goes into toxicology work on the product also the preparation of labels and literature to inform the consumlng public of the limitations, handling hazards and possible hazards that may be encountered when the chemical is used. ^ ^ ^ Dalapon is being widely used to control undesirable grass species such a s ^ Johnsongras s , quackgrass, and is also being used to control the d e s l r a b l e Q species of grass that are growing in undesirable locations. It is also finding wide usage to control cattails and marsh grasses that so commonly choke drainage ditches, waterways, farm ponds, wildlife refuges, etc. The active material may be applied at a rate of from 1.5 to 40 lbs. per acre d e pending on the type of problem being attacked. When the material is prepared as a spray up to 20 lbs. per 100 gallons, a 1,000# cow would have to drink at least 15 gallons of the undiluted most concentrated spray mix to evidence the slightest symptoms of intoxication. And she could probably survive even a dose of double this quantity or 30 gallons of the 20 lbs', per 100 gallon spray mix. If a 1,000# cow had access to a spray tank con taining the usual and most frequent concentration of dalapon, 10-15 lbs. per 100 gallons, she would need to drink 33 bo 50 gallons of the spray mix to evidence the slightest intoxication. It Is interesting, to note that since the advent of the 2,4 - D herbicides, over 225 million lbs. of the herbicide have been used. This would be sufficient to treat some 565 million acres of land at a practical use level of 0.4 lbs. acid equivalent per acre. To date, to our knowledge, there has not been one single substantiated case of poisoning due to the herbicide. As you gentlemen might guess, there have been numerous allega tions and there have been a number of cases of reported poisonings that have occurred following application of herbicides, but in every case that has been followed up and a diagnosis was possible, the herbicide has been exonerated. There has been much discussion of the possibility that spraying with herbicides might cause some plants to become toxic and other plants that are ordinarily non-palatable to become palatable. Considerable investiga tion of these possibilities has resulted in the general conclusion th^t from a practical standpoint these suppositions are more fanciful than factual. To date the only effect on herbage that has been proven that might be harmful to livestock is in the case of certain plants that have been sprayed with 2,4-D or similar materials. The nitrate content can be in creased. F o r instance, in the case of sugar beets, accidentally sprayed with 2,4-D, the nitrate content of the leaves is actually Increased and it is possible that the feeding of sugar beet tops treated with 2,4-D could cause nitrate poisoning. Of course, the only time 2,4-D would be applied to sugar beets is by accident, as the material is not recommended for use in sugar beet production and has never been applied for thinning purposes. It has been reported that other plants, such as lambsquarters, pigweed, ' 5256 0003364 4 t I OPSom and smartweed will increase their nitrate content after treatment with 2,4-D. Since livestock do not ordinarily eat these species, it would not s e e m to be a praqtical problem, although it is possible that if animals were starved to eating such plants and the plants were sprayed with 2,4-D that nitrate poisoning could occur. It might be pointed out that environ mental and mechanical factors can cause as significant changes in nitrate level as herbicides. C c. The question has been raised many times: Will wild cherry present a special problem when sprayed with 2,4-D type herbicides? It has been rather conclusively proven by Grigsby and Baugh, also Grigsby and Blakeslee at the Michigan State University, also by Barrons and Lynn of The Dow Chemica Company that wild cherry does not increase in HCN content when it is treated with 2,4-D. This has been proven by chemical methods and further substantiated by practical type tests. In a test conducted by Grigsby and Bla k e s l e e of the M i c h i g a n State University and L. L. C o u l t e r of The Dow . Chemical Company, sheep were pastured on wild cherry that had been sprayed. Immediately prior to the time the sheep were turned into the brushy pasture, two quartersectlons of the pasture including the wild cherry plants were sprayed with a mixture of 2,4-D and 2,4,5-T, the sheep graced the wild cherry plants-- not only at the time they were turned into the pasture but for several days following, and until the vegetation actually turned yellow and brown, and was so markedly affected by the herbicide that it was no -^longer palatable. At no time was there any adverse effects noted in these Jsheep, and at no time was there evidence that the treatment of the herbage affected the normal habits or the normal functions of the sheep that grazed on It. Th e r e was no preference shown f o r either treated, or untreated plants. In all of the alleged cases of herbicldal poisoning that have been definitely diagnosed, the diagnosis has been such causes as: shipping fever, leptospirosis, anaplasmosis, arsenic, poisonous plants, lead, poor marks manship (hot lead), parasites, hardware disease, etc. It should be born in mind that many types of weeds and foliage have in the past and will continue to cause poisoning whether they are sprayed or whether they are not sprayed wi t h herbicides. It is a l s o known that various members of the;`sudan grass and sorghum family have caused difficulties in the past and undoubtedly will cause them in the future. Here, again, losses occurred prior to the use of modern herbicides and-will continue despite herbicide usage. Some workers have felt that these systemic herbicides may improve the palatablllty of normally unpalatable poisonous plants to a degree where animals will graze them. However, in actual grazing trials on desirable grasses and legumes, livestock have never shown preference for the treated foliage. In fact, where there has been a preference shown, it has always been for the untreated plants, and until it can be proven that the palatablllty of any plants is actually increased by the use of herbicides, we should keep an open mind. I have prepared a table (Slide #5) to show the minimum and maximum spray concentration of the products used and the minimum quantities of spray a `-'1,000# cow would need to drink, or sprayed area she would have to consume all of the vegetation from (assuming all spray is retained on the vegetation) to evidence even mild intoxication from ingestion of the herbicide. 5257 000336  \ 9 n pqni11 -6- You should remember that the more concentrated the spray, the heavier and more unpalatable the vegetation to be sprayed. I have brought along a few slides to give you a better Idea of Just how these materials work and where you might expect to see them used. t c Table #1 Animal Species Mice Rats Rabbits Guinea pigs Dogs Chicks Cattle ACUTE ORAL TOXICITIES OF 2,4-D a n d r e l a t e d c o m p o u n d s LD50 (MsAg) 2,4-D 375-713 375-805 424-800 469-1000 100 540-2000 >1000* 2,4,5-T 389-551 481-500 712 381-750 100 310 Sllvex 1410 600-621 750-819 1200-1250 --. 1190 % * Mixture of equal parts 2,4-D and 2,4,5-T 5258 000336 uunuO JU dIJ i * S' Sex and Wt. In Kgs. S-291 S-295 S-295 S-336 S-250 7- - Table #2 TOXICITY OP A MIXTURE OP 2,4-D, 2,4,5-T GIVEN ORALLY TO CATTLE Dosage Mg/Kg. 1000 1000/da. for 3 da. 500/da. for 2 da. 500/da. for 3 da. 100/da. for 15 da. Observations No effect Fatal Moderate transient toxicity No effect Grossly no effect. Microscopically, liver & kidney mildly affected. Table #3 ACUTE ORAL TOXICITY OP DALAPON (Sodium Salt) Animal Species Mice Rata Guinea pigs Rabbits V Dogs Chicks Cattle L D c jQ Values >4,000 6 ,600-8 ,10 0 3,400 3,400 >1,000 5,700 >4,000 5259 oo<m$? * 1809QM0(I Slide #4 TOXICITY OP DALAPON (Sodium Salt) GIVEN ORALLY TO CATTLE Sex and Wt. in Kgs. S-336 S-290 S-337 S-394 P*-252 S*-59 Dosage MgAg_ 2,000. 3,000 3,500 4,000 1000/da. for 10 da. 1000/da. for 10 da. Observations No effect Mild transient toxicity Mild transient toxicity Mild transient toxicity Moderate toxic symptoms No effect * Necropsied for histopathology studies Herbicide 2,4-D and 2,4,5-T Dalapon (Sodium salt) Slide #5 Spray Con centration lbs/100 gal. 0.25 4.0 1000# Cow May Become Intox- Icated if She Ingests Spray Con- . All Spray from centrate Sprayed Area (Gallons) (Sa. feet) 400 174,240 25 10,890 1.5 20.0 333 143,748 25 5,445 5260 0003368 31 52S1 3T FORESTRY LIBRARY JUN 2 7 : . UNIVERSITY OF CALIFORNIA BERKELEY eing more susceptible to the than Scotch (P. sylvestris t red (P- resinosa Ait.) pine, though environmental condicannot be rigidly controlled e field during and after aptions, spraying should be done er optimum conditions of teni a e and light intensity to in minimum seedling damage. Literature Cited ` SSIt t , F. M. 1951. Use of petroleum roducts as selective herbicides in uthern pine seedbeds. Jour. Pores- 19: 773-775. u so n , E. J. 1949. The use of oil Lrays for the control of weeds in Coniferous nurseries--1948 supple ment. Mimeog. by N. Y. State Con servation Dept., Albany, N. Y. 3. F aulkner, B. 1952. Notes on nursery irrigation and on chemical weed con trol practices in the U. S. A. and Canada. Forestry 25(2): 126-134. 4. L aCroix, J. D., and A. T. Guard. 1956. Morphological and histological modifications of pine seedlings in duced by petroleum naphtha. Canad. Jour. Botany 34(4) : 621-627. 5. Stoeckzler, J. H. 1949. Control of weeds in conifer nurseries by mineral spirits. Lake States Forest Expt. Sta. Paper No. 17. J. D. L aCroix an d A. T. Guard Respectively, Department of Biology, University of Detroit, Detroit, Mich.; Department of Biological Sciences, Purdue University, Lafayette, Ind. 5 percent of the sprayed trees sprouted. Relative effectiveness of the treatments as site preparations is revealed by survival of planted loblolly pine seedlings. In Novem ber 1954, after a very dry summer, loblolly survival was 53 percent on the plots where silvicide was ap plied on stumps, 55 percent where stumps were left untreated, and 83 percent where hardwoods had re ceived basal spray. The silivicide had no adverse effect on the pine seedlings under either treatment, even when planting followed its ap plication by only a few hours. However, none of the three methods afforded an adequate " one shot" release treatment for Texas conditions. A f t e r t h r e e growing seasons, two of which were very dry, pine survival on the two cut treatments had declined to 45 and 27 percent for the areas with asal Spray with 2,4,5-T for Winter Hardwood Control and without silvicide, respectively, while on the basal-spray area it In East Texas was down to 64 percent. Many of Removal of competing hard"oods is essential to re-establishent of pine on many dry sites, erial spraying of silvicides or and-c-learing with heavy machin ery promises to be useful on ex tensive operations. For owners of mail tracts, however, hand methds that can be applied at planting .time are most acceptable. iSearch for such a method at the ustin Experimental Forest in east Texas revealed that for Decem ber application, and, presumably throughout the dormant season, 2K 5-T is more effective when aplied as a basal spray than when painted on cut stumps. ~ Triplicate sets of plots were treated and planted to loblolly pine .(Pinvs taeda L.) in December 1953, near the beginning of the nor mal planting season. On one set of butyl ether ester) in 16 gallons of No. 2 diesel oil, a concentration of 23.5 pounds of acid per hundred gallons. In both treatments the solution was applied to the point of runoff. Of the original stand of 13-yearold mixed hardwoods, 20 sweetgum (Liquidambar styracifi.ua L.) trees or stumps on each plot were ob served for three years. By April 1954, 92 percent of the stumps without silvicide had sprouted, and this increased to 100 percent by the following November. The 2, 4, 5-T solution on cut stumps de layed but did not reduce sprout ing : 37 percent of the stumps sprouted by April, 90 percent by November, and 97 percent by April 1955. Untreated stumps produced significantly larger and slightly more sprouts than stumps with the pines needed release--on the cut areas from hardwood sprouts, and on the basal-spray areas from invading herbs and vines and from the expanded crowns of the few surviving hardwoods. Had adequate release been given at the end of the second year, 80 percent of the pines on the basal-spray areas might have survived, as compared with less than 50 percent on the other treatments. The poor showing of 2, 4, 5-T solution on cut stumps appears to be related to time of application. In tests made during the growing sea son even more dilute solutions on cut stumps have permanently in hibited sprouting. For applications concurrent with planting, which must be done during the dormant season, 2, 4, 5-T is apparently best applied as a basal spray. plots all hardwoods were basal- silvicide, but by the third growing J ames R. D avis sprayed, on a second set all hard woods were cut close to the ground, a&d on the third the hardwoods Were similarly cut and the stumps Painted with silvicide. The silvicide season sprouts on both treatments had outgrown the surviving pine seedlings. Ninety-three percent of the basal-sprayed tr e e s e v e n t u a l l y Southern Forest Experiment Station,1 Forest Service, U. S. Department of Agriculture l-r the first and third treatments was a mixture of 1 gallon of Dow , steron 245 '(a propylene glycol died, though more than half of them retained their leaves through out the 'first growing season.1' Only 'East Texas Research Center, main tained in cooperation with Stephen F. Austin State College, Nacogdoches, T"" 5263 31a 5264 \ > V Krprinlrd from the J ournal o r F orestry, Vol. 57, No. C, June 105 Concentrated or Diluted 2,45-T as a Supplement to Girdling? John J. Stransky T h is article reports on tests in stalled to determine 'whether fallstrength 2,4,5-T concentrate, ap plied in a fine spray, would control sprouting and hasten crown kill of hardwoods girdled by ax or ma chine. A n earlier study evaluated the effect of 2,4,5-T on cut stumps {2). A supplementary chemical treat ment is needed where vigorously sprouting hardwoods are girdled to release small seedlings, or where, as in east Texas, sprout control is essential to conserve limited mois ture (4,5). An effective concentrate would reduce the bulk of liquid enough so that the silvicide could be carried and applied by the ax man or operator of the girdling ma chine, thus eliminating the heavy transportation costs and separate T a r author is on the staff of the South ern Forest Experiment Station, Forest Service, 17. S. Department of Agriculture, E ast Texas Research Center, maintained in cooperation with the Stephen F . Aus tin S tate College, Nacogdoches, Texas. application required with standard dilutions. D esig n 1 Post oak (Quercus steUata Wangenh.) and sweetgum (Liquidambar ttyracif.ua L.) were chosen as test species because of their preval ence as weed trees on upland pine sites and because both sprout prolifically when girdled. In some areas sweetgum has also shown a ten dency to produce enough callus growth to bridge the shallow cut made by the power girdler. The study was conducted on up land pine-hardwood sites within the Stephen F. Austin Experimental Forest, near Nacogdoches, Texas. Soils are sandy loams overlying clay, and fairly representative of the better Upper Coastal Plain sites in east Texas. Study trees were of two d.b.h. classes. 3.6 to 5.5 inches and 5.6 to 7.5 inches. All had single `This study was designed and partially installed by T. A. Harrington. stems and were judged free of un derground connection with their neighbors. Girdling was either by ax, in which case a standard double-hack girdle was cut, or by the " Little Beaver" power girdler, produced by the Haynes Manufacturing Company Livingston, Texas. This gasoline-powered machine employs a flexible shaft to power a cutting head which produces a smooth, rounded incision about % inch wide. Properly used, it cuts through the bark and well into the sapwood, completely severing the cambium. The chemical was a commercial preparation of 2.4,5-T, Dow Esteron 245, containing 42.5 percent propylene glycol ether ester by weight (4 pounds acid equivalent per gallon). The dilute solution was made up with Diesel oil, in propor tion of 1 to 50, and was painted on the trees with a brush. The concen trate was applied to both ax and machine girdles from a plastic 5265 J o k e 1959 4*>>'i1 squeeze bottle which dispensed a very fine spray. Both dosages were applied in such quantity as to give approximately equal amounts of %4,5-T acid to each tree. The main installations, in April 1955, subjected sweetgums and post oaks to ax and power girdling with out chemical, with dilute chemical, and with the concentrate. On each of the two species the six treatments were randomly applied, so that ten trees in each of the two size classes received each treatment. There were three replications of the treatment pattern. Since other workers (3, 6) have found seasonal differences in re sponse to silvicides, parts of the ex periment were replicated in Janu ary, July, and November 1955. Five sweetgum trees were treated per size-treatment group. Treatments were limited to power girdling with dilute, concentrated, and no chem ical. There were three replications each season, as in the main study. The amount of concentrate or equivalent applied in each treat ment is shown in Table 1. All trees were inspected one and two full growing seasons after be ing treated. Crown condition, num ber of sprouts per tree, and length of dominant sprout were criteria for determining treatment effec tiveness. This report is based on the second year's data. Method of Girdling Without chemicals, ax girdling killed crowns of 90 percent of treat ed trees, significantly more than girdling with the machine, which killed 75 percent (Table 2). The difference was greatest in post oak; there tops of all ax-girdled trees died within two years. With chem icals, the two girdling methods were substantially equal, both in top-kill, which consistently exceeded 90 per cent, and in percent of completely dead trees, which varied with con centration. All tops still alive after two years were damaged, and are expected to die eventually except in the few cases where the girdle has been bridged by callus tissue, or where the tree is sustained by root grafts. T a b l e 1.-- Co n c e n t r a t io n oh E q u iv a l e n t P er T r e a t m e n t Season of installation Species 2,4,5-T concentrate per tree As concentrate As dilute solution W inter -Spring Summer Fall Sweetgum Sweetgum and post oak Sweetgum Sweetgum Ce -2.2' .9* 1.0 1.0 Ce 1.0 1.0 1.0 "Thinned 50-50 with Diesel oil to facilitate application in low temperature. This light dilution produced resulta comparable to the full-strength concentrate and was not distinguished in the analysis. Table 2.--Average P ercent o r Sweetoum and P ost Oak Trees Crown-Killed and Completely Dead Two T ears A rr e s Treatment in April 1955 Treatment Crown-killed with and without sprouts Crown-killed without sprouts No chemical Power g ird le ----------------------------------- , Ax g ir d le ___ !--------------------------------- 75 90 Mean -- ------------------------------------------------- 85 Percent1 40 46 42 Concentrated 2,4,5-T Power g ir d le _____________________________89 Ax girdle ..... ------- 96 M w ii _______ ______________________ 99 55 57 56 Dilute 2,4,5-T Power girdle .---------------------------------At girdle Mean 99 100 100 87 86 _______________ 87 "Mean values weighted by are sin transformation. The difference between the two gir dling methods, therefore, is believed to reflect promptness rather than completeness of kill. With both methods sprouting was excessive without chemicals. Chemicals A ll chemical treatments gave a highly significantly better topkill than simple girdling. Both concen trated and dilute 2,4,5-T were ef fective, killing the crowns of more than 97 percent of the treated trees. While there was little difference between concentrated and dilute acid in top-kill efficiency, the con centrate proved to be a poor sprout inhibitor. In the April tests, the average percentage of completely dead trees was not high enough for the concentrate to be significantly better than simple girdling; in the seasonal test with sweetgum the complete kill percentages were identical (39 percent) for both of these treatments (Fig. 1). The dilute solution, with a complete kill of 87 percent on both species after spring application, and an average of 56 percent on sweetgum in the seasonal test, was significantly more effective, and provided acceptable sprout control. Of the trees that remained alive below the girdle, those that received dilute acid applications developed fewest sprouts. Results with the concentrate were only a little better than those from simple girdling. The height of the dominant sprout follows a similar pattern, averag ing about 0.8 foot shorter with di lute 2,4,5-T than with the other two treatments. The superior effectiveness of the dilute solution suggests that the Diesel oil rather than the 2,4,5-T might be the effective agent. Studies by Campbell and Peevy (1), how ever, as well as informal tests in Texas, indicate that Diesel oil alone has little sprout-inhibiting quality. It appears rather that effective quantities of 2,4,5-T are translo cated into tissues below the girdle only when diluted. This parallels experience with 2,4,5-T on cut stumps, where large quantities of dilute chemical were most effective (2). Apparently the rather viscous concentrate is not readily absorbed into the wood, while the thin Dieseloil solution penetrates much more readily. 5266 * 434 J ournal of F orestry CROWN KILLED: NO 2,4,5-T WITH SPROUTS E3 WITHOUT SPROUTS CONCENTRATED 2,4,5-T DILUTE 2,4,5-T EE) F lo. L.--Crown kill and sprouting of girdled sweetgums, by season and treatm ent (average of ax and machine girdling). Season, Species, Tree Size In the sweetgum tests, spring treatments were most effective in both top-kill and inhibition of sprouts. Treatments in sommer were better than those in fall and winter (Fig. 1 ). Similarly, on trees not completely dead, spronts were fewest and smallest after spring treatment While the differences between seasons were not statisti cally significant, they are consistent with those reported by other work ers (5, ff). Without chemicals about 75 per cent of the post oaks sprouted after girdling as compared with about 40 percent of the sweetgums. The dif ference was not statistically sig nificant, and is inconsistent with considerable experience which rates sweetgnm the more difficult species to controL The tests were confined to a rath- er narrow range of diameters--3.6 to 7.5 inches d.b.h.--because these size classes have generally sprouted most vigorously after girdling. Within this range, cLbJh. of tree was significant only in the percent of sweetgums top-killed in the sea sonal test. Here smaller trees were top-killed more effectively than large ones. Differences were largest where no chemical was applied. Conclusions A x girdling was significantly bet ter than power girdling only in topkill without chemical treatments. With chemicals there was no dif ference between ax and power gir dling. Top-kill differences reflect promptness rather than eventual completeness of kill, since remain ing tops are moribund. A ll treatments involving 2,4,5-T resulted in significantly higher top- kill than girdling alone. Concen trated and dilute 2,4,5-T killed crowns equally well; the concen trate, however, proved to be a poor sprout inhibitor, while the dilute solution was very effective. There was no significant differ ence between the response of sweetgum and post oak. Within the limited diameter range of the tests, size of tree did not affect results significantly. Dilute 2,4,5-T was most effective in reducing sprouting when applied in early spring, least effective in the dormant season. It is evident that the saving in material costs and transportation of dilutent envisaged in the design of this study cannot be fully achieved by application of undi luted 2,4,5-T. Where promptness of top kill is a primary considera tion, application of full-strength 2,4.5-T in cambium girdles may prove usefuL Where both prompt kill and inhibition of sprouts are required, the chemical should be ap plied in dilute form. Further tests are needed to deter mine whether some intermediate concentration of 2,4,5-T can be ef fective without being too bulky for use with a power girdler. Literature Cited 1. Campbell, B. S., and I t o A. P ervi. 1950. Chemical control of undesirable southern hardwoods. Jour. Range Mgt. 3:118-124. 2. Davis, J . B. 1958. Diluted 2.4,5-T more lethal than undiluted in east Texas. Jour. Forestry 56:516. 3. Grano, Charles X 1958. Besponse of southern red oak to seasonal appli cations of 2,4,5-T. Jonr. Forestry 56: 140-141. __ 4. M n m r , A. L 1956.. W hat gives with girdling t Southern Lumberman 193(2417) :214-215. 5. P eeyt, P eed A. 1956. Methods for controlling hardwoods. Fornata and People 6(3) :22-25, 34-35. 6. Shipman, B. D. 1958. Effect of sea son of treatment on girdling and chemical control of oak and sweetgnm. Jour. Forestry 56:33-35. 3S 5268 3$ SUBJECT) OR COMPOUND). AUTHOR)! T-C N ' THE DOW CHEM ICAL COM PANY BIOCHEMICAL RESEARCH LABORATORY JOURNAL ABSTRACT ^ y VT^c'r K~ - ( ( 0-t>) f./O Z G J - 4 Ps5 C-n (> h a J l .L u . I j . f t . A . f & h ')' 0 3 CO cn ABSTRACT 6cm^Cc* o ^ M - * * * , t'V M ~ /if * --V --- -------- 1 -7 0 U ___ > z j u u . , /q + .X -9* u ^ _ 7- /3 ' /-/- A i^-r //. e^ . . u * c -r'~ f Uk ^ i z t * - /* * - ? : vty. ^ i '* r ~ > ? ? ^ ' y' LABORATORY! 8T1 T y fc s fC . DATE: " < e - j i Piiirto ii u. s. a. t-g r S - 0009999 ROUTE TOt 4 e0 W WfjTC -rffeO. .i ar~ fc e .i.- 5269 ^ m . <iS.HL FILE : 5270 C. A. Apffel*: The Cytostatic Action of Certain Auxins; Prelimi nary Review [Action cytostatiquede certaines auxines; s Compte rendu prliminaire]. a- Presse Medicale, 67, (6), 207-209(1959). --o vO Si o <3 en tu Pi eo o *Tangiers RECEIVED JUN 05 1974 REGISTRATION f Translated from French by the Ralph McElroy Co., Custom Division., 2102 Rio Grande, Austin, Texas 78705 USA 0007450 ' 5271 . Code: 101-4141-2 THE CYTOSTATIC ACTION OF CERTAIN AUXINS; PRELIMINARY REVIEW * 0. W a r b u r g h a s p o s t u l a t e d t h a t a c a n c e r o u s c e l l c a n n o t s u r vive as such if its deficit of r e spiratory energy is c o v e r e d by a s u f f i c i e n t a u g m e n t a t i o n o f f e r m e n t a t i v e g l y c o l y s i s [1]. I n 1 9 S 4 > I a t t e m p t e d to g e t a c y t o s t a t i c e f f e c t u s i n g m o n o i o d o a c e t a t e o f soda which reduced the level of fermentative glycolysis wi t h o u t i n f l u e n c i n g r e s p i r a t o r y f e r m e n t [2]. T h e r e s u l t w a s d i a m e t r i c a l l y o p p o s e d to the a n t i c i p a t e d effect a n d the s t i m u l a t i o n w h i c h it gave spar k e d my interest in auxins and phy tohormoyies which are a n t a g o n i s t i c t o i o d o a c e t i c a c i d [3]. Fundamental principles Optimal doses of auxins like indoleacetic acid and 2,4-dichloro phenoxyacetic acid can increase oxygen consumption of vegetal tissue from 20 to 60% [4]. This respiration increase accompanies a carbon hydrate depletion. Strong concentrations inhibit growth and respir ation in the same way as fermentative glycolysis [5]; but r e s p i r a tion can "still be.kept elevated at those doses which inhibit growth. Auxin activity is thwarted by reagents of -SH groups such as io d o acetic acid. Thus auxins act through intermediary sulfhydryl en zyme groups, most particularly those dehydrogena -S of the Krebs cycle. Interaction with the -SH group of coenzyr.e A should be forseen as possible. Auxin action resembles coenzyme action. It par ticipates in most phosphorylation systems and contributes to the aerobic transport of phosphates [6]. In plants, auxins are bound to fraction I vegetable proteins. This fraction has a phosphatase activity and can, along with animal phosphatases,rcatalyze trans phosphorylations [7]. In stimulating concentrations, auxins p r o mote water absorption by vegetable cells, absorption which depends on an oxidative metabolism and very particularly -^n oxidative phosphorylations [8]. Moreover, these substances provoke in tis sues a considerably diminished potassium rate which is elevated in ,, 5272 0007451 f Page 2 malign tumors. But their most remarkable property is to influence the decisive work of the nucleic acid content of cells. DMA and RNA rates increase or abate as a function of auxin c o n c e n t r a t i o n [9}. r Finally, auxins are antagonistic to such cell division factors as adenine and kinetin [1]. Particularly interesting is the action of phytohormones on the tissues of cultured vegetable tumors ("crown galls") . Weak concentrations totally lack a stimulating action [11]. On the other hand, the grov/th of tumorous sunflower and periwinkle t i s sues lessens in the presence of weak concentrations of indoleacetic acid, indolebutyric acid and 1-naphthaleneacetic acid. More eleva-4 ted concentrations (1 x 10 ) totally inhibit this growth. Such concentrations also stimulate normal sunflower tissue [12]. A t -4 1 x 10 , indoleacetic acid stops the wet weight increase of t u morous tissues and tissues "accustomed" to scorrcnera [13]. The 2,4-D inhibits tumorous sunflower tissue at concentrations of * 13 *5 1 x 10 J , and tumorous tobacco tissue from 1 x 10 only. Auxin action in plants increases as they grow more rapidly; young tissues are more susceptible than old ones. In one and the same plant, the less mature and less differentiated cells are the most responsive. . Struckmeyer and his collaborators [14] have histologically o b served transformations of tumorous sunflower tissues under the i n fluence of auxins. Weak concentrations have not, on the whole, modified wet weight but have diminished meristem cells per surface u-nit and have increased scalariforn vessels. A t strong c o n c e n t r a tions, the number of cells of all types greatly diminished and the wet weight fell impressively. Proof In 'spite of these facts, auxins have been neglected in the search for cytostatic substances, no doubt for the following rea sons- The mere name "growth substances", implying a growth stimu lant, may be one of the causes of their omission. But such desig nations are deceptive, since auxins can, according "to their c o n centration, also stimulate as well as inhibit growth of vegetable 527:b o o ? 4 5 2 liUl/V I Page 3 tissue, and since weak concentrations, again, inhibit tumorous tissue. Besides, auxins have attracted to themselves the reputation of being carcinogenic. Previously the formation of a callus at the surface level appeared suspect, even though it was really a benign hyperplasia. Gautheret [15] advanced a m o s t serious a r g u ment: From a carrot tissue which he had cultivated for many years in a cultured medium increased with 0.1 mg/ indoleacetic acid, Gautheret isolated a cellular strain able to continue to gr o w and proliferate without any additional indoleacetic acid. This new tissue, which he called "habituated tissue", was friable and trans lucid, consequently morphologically modified. Grafted onto a 4 healthy plant, it became a true tumor [16j . This transformation of "habituated" tissue, apparently cancerous tissue, was generally attributed to a u x i n 's influence. It is acceptable today to doubt this type of perception. In effect, Sanford and his collaborators, starting from a single mesenchymal cell, successfully isolated at the end of twenty months a cellular strain ("clone") which, in 97% of the cases, instead of forming sarcomas, inoculated the cells of healthy mice [17] . Other authors likewise have obser v e d i n v i t r o such a transformation of normal mammal cells into cancerous cells [18 The appearance of malignity lasted from four months up to four years. Goldblatt and Cameron attributed the malign appearance to an anaerobiase. In none of the other cases was it possible to determine the cause of a given carcinogenic agent. In fact, Sanford came to the fallowing conclusion: all cell strains, cultivated in a hetero logous medium for a sufficiently long time, finally acquired the property of giving birth to either sarcomas or carcinomas when these cells have inoculated animals of the species from which the culture originated. In other w o r d s ,*every tissue, culture is event ually carcinogenic. Two other facts have provoked the thought that phytohormones can be carcinogenic: a) the B a c t e r i u m t u n e f a c i s n s , agent of v e g e table tumors, produces indoleacetic acid in an artificial medium; b) vegetable tumors contain more auxin than corresponding healthy Page 4 tissue. However, indoleacetic acid production by 3. i u m e f a c i s n s reaches at most 125 nicrograms per liter [19], when 10-30 million micrograms is necessary to artificially provoke a gall [20]. Other bacteria also produce auxins in an artificial medium; but they never instigate the formation of tumors [21]. The high auxin rate verified in vegetable tumors is due to the fact that because of their incomplete oxidations, they consist of more free auxin and less bound auxin in protein fractions. The auxinprotein connection depends in effect on normal oxidations and 1 the presence of adenosinetriphesphate [22]. Another reason for eliminating auxins from the list of actual cytostatics is the fact that these are substances whose action is strictly limited to vegetable metabolism. However, past and pres ent publications report on work proving their influence in animal metabolism. We recall the discovery of the regular presence of auxin a and indoleacetic acid in human urine [23] whi c h was a t t r i buted to the action of intestinal bacteria. In 1949, Raoul and Marnay verified a growth stimulation in young rats under the e f fect of w e a k doses of indoleacetic acid [24] . Zambotti and De B e r n ard threw into relief indoleacetic acid's influence on respiration and glycolysis in'animal cells [25] . Finally the works of Louis have shown an impressive parallel between indoleacetic acid's effect on the cultured tissues of vegetables as well as animals. In a n a e r obic conditions, weak concentrations have been stimulants and strong concentrations have been inhibitors to cultured cardiac cells of chicken embryos. In aerobiosis, only inhibition by very strong doses was observed. Indoleacetic acid distinctly works on nuclear division; increasing concentrations severely diminish the number of mitoses [26] . Thus, I have led up to the search for whethet auxins, whose cytostatic effect on "crown galls" has previously been pointed out, jT can as well inhibit animal and human tumors. 0007454 > 5275 Page 5 Toxicity Given the massive use of auxins in agriculture, their toxicity has been tested several times. Six grams of 2,4-dichlorophenoxyacetic acid was administered to cows for three and a half months without provoking the least symptoms of intoxication. A research worker has taken 0.5 grams of the same substance for twenty-cne days without disturbance. In spite of this, I verified one more instance of 2,4-D tox icity, this time in the form of ethyl ester, among rabbits. At first, a dose of 0.01 g/kg of weight was injected intramuscularly. After five days, the dose was increased to 0.02 and, after another five days, to 0.05 g/kg. The experiment was stopped at the twen tieth day. The appetite and vivacity of the animals were not at all impaired as the doses were raised. As a continuation, I parenterally administered auxin to animals and humans in average daily doses of 3 to 4 mg/kg for very long periods, without ever observing toxic phenomena or undesirable reactions. A patient re- m ceived, for one hundred forty days, an ethyl ester of 2,4-D, then an isopropyl ester of 2,4,5-trichloropher.oxyacetic acid in daily intramuscular doses of 0.10 to 0.30 grams without presenting symptoms of intoxication. These products were employed in an olive oil solution. Intragluteal injections caused neither burns m nor aches and were easily reabsorbed. During the course of a year, the hundreds of injections never provoked an abscess. ***. Concerning administration p e r os, it should be noted that daily quantities of 0.30 g and more (divided into doses of 0.10 g) soon released a laxative effect. Other subjects, particularly those given 2,4-D and 2,4,5-T esters, experienced a disagreeable gout. The fact ought to be emphasized''that hematopoietic organs and the blood formi ula v/as not influenced. The rat e s of chole s t e r ol, urea, and glucose in blood were never augmented. Only a slight hypoglycemia was occasionally verified. Arterial pressure was at ho time raised during the treatment. We are dealing with a group 0007455 5276 Page 6 of very weakly toxic substances, not having, in opposition to the antimetaboliies and nitrogen mustard derivatives, any effect on hematopoiesis. After not having ever exceeded daily doses of 0.45 g from October 1956 until October 1957, I decided to use daily quantities of 1.25 to 2.50 g (once at 1.25 to 1.50 or twice at 1.0 to 1.25 g intramuscularly). Even at these very elevated doses, the injected substance was painless and easily reabsorbed; on the other hand, certain disturbances soon appeared. They consisted of a lassitude, vparticularly in the legs, a heightened thirst, vertigo, and, in two cases, a slight confusion. The last two symptoms did not ap pear at the most elevated doses. The patients exhibited then a certain inebriety. Twice I noted diarrhea, and once, vomiting, A few days after the interruption of treatment, the toxic symptoms disappeared completely. As a notable secondary effect during treatment with these elevated doses, arterial pressure markedly diminished. Drill has pointed out among mammals the following toxic symp toms: weakness, diarrhea, weight loss, and, in certain species, a curious state of a myotonic response to quinine [27]. Preliminary analyses The initial analyses were attempted with incoleacetic acid and the sodium salt of 2,4-dichlorophenoxyacetic acid. The first was mixed with equimolecular quantities of piperazine, rendered thus hydrosoluble, and injected intravenously in a 1 to 2% solution at a rate of 0.02 to 0.10 g/day. The second, hydrosoluble naturally, was administered in the same fashion in a dose of 0.10 g/day. Five cases (a lymphogranulomatosis, i.e., Hodgkin's disease, a m e t a s t a tic carcinoma mastitoides, a lymphosarcoma, a multiple metastatic stomach cancer, and an adenbcarcinoma of the prostate with postoperatory mtastass) were thus treated for twenty-five days with out either amelioration or aggravation. Neither of the two sub stances provoked the appearance of toxic symptoms. The injections were painless, non-hardening, and did not release, s e c o nda r y ^ i ^ c t i o n s 000745 Page 7 These analyses seem to confirm the inactivity of auxins in humans in contradiction to the qualified observations on the c u l tured tissues of animal origin. One can ask if, at a very elevated' human blood pH (pH of human blood: 7.3 to 7.4; pH of vegetable t i s sues:: 5. 5 to 6.0), auxins have not been dissociated to the poi n t of losing their activity. Effectively, Audus has been able to verify that a weak dissociation of 2,4-D is linked to an elevated toxicity for plants. Already at a pH of 3.28, 50% of the molecules were dissociated. Audus admits that 2,4-D works uniquely as a nonvdissociative molecule [231. Among the derivatives of indole a c e t i c acid and phenoxyacetic acid, the alkaline salts and the amides are ionized in the same style as the acids; only the ester and nitrile molecules and the corresponding aldehydes continue to be nondissociative. Analysis is thus followed up for these latter categories, principally with the esters. Analyses with non-icr.icable auxins Derivatives employed: Fr o m October 15, 1956 to October 11, 1957, auxins w e r e a d m i n istered in daily doses of less than 0.45 g . .1 successively e m ployed the ethyl ester of 2,4-dicnlorophenoxyacetic acid, and the isopropyl ester of 2,4,5-trichlorophenoxyacetic acid. The first exists on the market in the form of a brown liquid, soluble in any proportion of vegetable oil. Even at the strongest concentrations (50%), the solutions stay clear and resistant to sterilization. The second appears in the form of a light brown crystal composed of a compact mass- that ought to be melted at 8 0 to 90 to be able to dissolve in olive oil. Mixed in cold oil, the substance immed iately recrystallizes; but after reheating (sterilization), the solution stays stable, at .least up to 15%. By October 1957, the impression became clear that a daily dose of 0.10 to 0.20 g of these esters could not bring about in -7 humans conditions which corresponded to a concentration of 1 x 0 .to 1 x 10 because such concentrations were the minimum rate necessary to obtain total inhibition in a vegetable tumor culture. i 5278 0007407 I MM " 6061C Page 8 One must in reality administer doses near 1.0 g at least and, since it is important that the rate in the interior level be m a i n tained above 1 mg % for the w h o l e day, p e r dicrr. q uantities of 1.50 to 2.50 appear appropriate. However, the administration of such quantities is not possible wit h 25 to 50% solutions or w i t h iso propyl esters of 2,4,5-T given its limited solubility. I am ob liged thus to return to 2,4-D ethyl ester; but I have r u n up 4 against the toxic troubles I indicated before. I am forced then to find more active derivatives that allow a d m i n i s t r a t i o n 'of ^smaller quantities. The ethyl and methyl esters of 2,4,5-tri- . chlorophenoxyacetic acid fulfill these conditions. Phytobiological tests fixed at first an activity five times greater than that of 2.4- D ethyl ester. These white and compact needle crystals do not have a limited solubility in olive oil. Instead of the projected 30% solution, a 20% solution wa s prepared wi t h the addition of 5% acetonitrile. The substance recrystallized however and it had to be reheated in a boiler to redissolve it before each injection. Above 30 the solution was stable. None of the experimental data, phytobiological or otherwise, has been published concerning the 2.4.5- T methyl ester. One knox*/s nevertheless that the esters of phytohormones inhibited germination and budding more as the radi cal alcohols of these esters allowed the number of carbon atoms more ^ riance. The white 2,4,5-T crystals of methyl ester are again less soluble than those of ethyl ester. The recrystalliza tion of a prepared 10% solution required reheating in a boiler be fore injection. The fear that instigated the use of the methyl ester was now confirmed. The methyl 2,4,5-trichlorophenoxyacetate could be administered parentarally in doses of 0.85 g per day for six weeks without provoking the least apparition of trouble. r The effects of the two primary derivatives e m p l o y e d (.ethyl 2.4- dichloropheno^yacetate and isopropyl 2,4,5-trichlorophenoxyacetate tate) we r e g r a c s o rr.odo the same and the second did not exceed by a bit .ne first in efficiency. The ethyl and meth y l esters of 2.4.5- trichloronnonoxyacctic acid proved to have a cytostatic activity superior to that of the isopropyl ester. This precedence 5279 0007458 I Page 9 inspired confidence in methyl ester whose tolerance was perfect. Successfully ameliorating its solubility will allow highly ele vated doses to be given. Clinical experience: Of the 41 cases treated, 5 were animals. The auxins used had a particularly marked effect on benign turners. In four cases of fibroadenoma of the prostrate, the troubles (retention, dysuria, pains, and stuttering and precipivtant urination) disappeared during the first week. At the end of ten to fiften days, hypertrophy had retrcceeded in such a manner that the treatment could stop on the fifteenth day. Efficacy was also remarkable on a recently appeared diffuse adenoma of the right lobe of the thyroid. After fifteen injections of 0.15 g of 2,-4,5-T isopropyl ester, a goiter, as large as a prune, disappeared. A fibremyema of the uterus retrcceeded, during twenty days cf treat ment, from the size cf a grapefruit to that of a detectable pain curve. On- cancerous tumors of reduced m a l i g n i t y and with o u t d e t e c t able metastases, efficacy was equally remarkable; there was always amelioration and, -in three cases, recovery (one facial fibroepithelioma in a donkey and two epitheliomas of the type u l c u s r o d e n s in a dog and a cat). 4 In cases of average malignity, there was a clinical recovery (tumor at the point of cholecystic parting in a 55 year old woman wi'th enormous metas t a s e s of the mesen t e r i c ganglia), ameliorations, momentary halts, or a slackening of the process occurred. The cases of great mali g n i t y (for example pulmonary cancer of the P a n coast type) or preagonic malignity reacted hardly or not at all. It is noted that at doses less than 0.45 g, the esters of 2,4-D and 2,4,5-T ^ere perfectly well supported by infants. A seven year old boy, afflicted with a malign lymphogranulomatosis, received a daily dose of 0.10 g for three months without any unde sirable secondary reaction. 5280 0007459 Page 10 Among the treated cases were mammary adenocarcinomas, bron chogenic carcinomas, rectal cancers, epitheliomas, a lymphoma, a lymphosarcoma with multiple tumors, etc., but no leukemia. Radiotherapy a little (less than three weeks) prior to auxin treatment increased tumor sensitivity in these lymphosarcomas. A remarkable effect was observed in a man affected with a broncho genic carcinoma who, a few days before treatment with 2,4-D ethyl ester, had received 1500 r in six sessions. A fifty-eight year old woman, suffering from an inoperable cancer of the uterus, re acted in such an extremely favorable manner at that time to 2,4-D ester treatment that she then received a total dose of 11,480 r.' After 48 injections starting on December 24, 1956, she remains r clinically healed. Radiotherapy and treatment with esters of auxins, applied simultaneously, seem to have a reciprocal potential. A proof was furnished b y a 56 year old woman afflicted with a lymphosarcoma with multiple tumors. A single treatment of 2,4-D ethyl ester gave an amelioration; but the patient had developed a resistance and did not react to a second series. On April 4, 1957, treatment was begun again, this time with 2,4,5-T isopropyl ester at a rate of 0.10 g/day intramuscularly, simultaneous with X-ray radiation. Although the patient received in all, as of May 12, 1957, only 2100 r (fifteen sessions of 125-150 r under 175 kw.with 1 mm Cu and 1 mm Al, distributed across four areas, two cervical and two axillary), the gangliary tumors nearly totally retroceded. This r ^ u l t , for a second radiotherapy and considering the given dosage, was unforseen. In another case, also treated simultaneously with X-rays and a 2,4,3-T isopropyl ester, a total dose of 2250 r spread over twenty-two days sufficed to completely dissipate Hodgkin's tumors. This also was a question of radiotherapy repeated for the second time. In a third case, where the efficacy of esters of auxins seemed to h6ve been exhausted, a simultaneous radiotherapy resulted in new and palpable progress. In an osteosarcoma of the thigh, on the other hand, simultaneous radi.._ion was a disastrous influence and had to be stopped. > 52810007460. t n # h 6 / Page 11 The efficacy of esters of auxins was at a maximum for the first two weeks; then began to decline; but stayed still notable for one of two weeks. After a month, there was hardly any pro gress at all. With 2,4,5-T isopropyl ester, efficacy diminished perhaps a little faster. It seems thus to develop a certain re sistance. The efficacy of esters of auxins regularly increased through simultaneous administration of riboflavin p e r cs and intravenously. Note that the auxins employed were contaminated by polychloro-. phenol. These latter considerably augmented the action of auxinoxidases by whi c h furthermore they induced or amplified formation [29 It is possible that these polychlorophenols are in part responsible for the resistance provoked above. As with 2,4-dinitrophenol, they disconnect respiration, properly called oxidative phosphorylations, and prevent the formation of ATP. However, the commercial esters of auxins of which I availed myself continually bring to mind iso mers such as 2,6-D and 2, 4 , 6-T. These isomers inhibit the action of active auxins. Also, in the clinical analyses which have been attempted in a Parisian hospital, we exerted ourselves to utilize substances carefully purified by recrvstallization in the appro- * priate solvents. .We further hope to be able to test, on an index of raised activity, other auxins which I was not able to prepare in sufficient quantities. Discussion In 1953, Silberberger and Skoog [9] proved that auxins like indoleacetic acid work in a direct fashion on the amount of nucleic acids in vegetable tissues and that growth depends to a large ex tent on this relation. The amount of ribonucleic acid and deoxy ribonucleic acid in a vegetable tissue cultivated i n v i t r o begins to increase at th^ moment when increasing quantities of indole acetic acid are added to the culture medium. For deoxyribonucleic acid, this increase is optimal at 0.014 mg/1; for r ibonucleic acid, it is only optimal at 3.3 mg/.. Exceeding the optimal doses of indoleacetic acid diminishes the amount of nucleic acids. At > 5282 0007461 Page 12 90 mg/1, it ought to be lower than that of the controls and finish by reaching-- 50% and less towards 270 mg/&. Increase and decrease of this amount parallels the fluctuations in wet weight. We re call here S t r u c k m e y e r 's observations [14] on the tissue cultures of vegetable tumors. He remarked on a considerable drop in the wet weight under the influence of strong concentrations of auxins. We also recall here that 2,4-D ethyl ester is more active than indoleacetic acid and that tumorous tissue is only sensitive to the inhibitory effect of phytohermones. We can thus suppose that at elevated concentrations of certain auxins, the tumor's amount of ribo- and especially deoxyribonucleic acids can be reduced. This is particularly important if one bears in mind the decisive role that ribo- and especially deoxyribonucleic acids play in the divi sion of nuclei and cells. Fro m 1953 to 1956, Miller [30], sta r t ing with a preparation of deoxyribonucleic acid from herring sperm, successfully isolated a highly cytokinetic substance, whose pres ence was impartially verified in analogous preparations from the thymus of a calf and in yeast extract. This factor, which he called kinetin, was capable of releasing cellular division in a concentration of one yg/Jl. It turned out to be a puric derivative, 6-furfurylaminopurine. Fresh preparations of deoxyribonucleic acid did not exhibit kinetin action; this only appeared after abandoning the preparation for months or after a sojourn of sixty minutes in an autoclave at 125. The kinetin seems to be a p r o duct of deoxyribonucleic acid disintegration and it is correct to svippose that its presence in tissues is strictly d ependent on the amount of deoxyribonucleic acid. It thus seems justified to pro pose the hypothesis according to which the cytostatic action of an auxin is explained by the rarefaction of cell division factors such as kinetin. From the work of Skjpog and his school [31] , it follows that, of t$ie two elements of v egetable growth, elong a t i o n and cellular division, the first depends on having all of the auxin concentration; the second, on the factors such as kinetin or an integral part of this 'last, adenine. A modifi c a t i o n between the proportions of adenine and auxin favoring the first expresses ( 5283 o o o v isr*;* a n r ^ I **f I Page 13 itself by an increased DMA content; a modification favoring auxin finds its expression in an elevation of the RNA content. This leads us to propose the following equation: Adenine (kinetin)/auxin = DMA/RUA = division/elongation. Skoog and Tsui have shown that elevated adenine concentrations and weak auxin concentrations favor budding and that on the con trary budding was inhibited by elevated auxin and weak adenine con centrations [32]. This antagonism between the factors of cellular division such as adenine (which can be potentialized by pyrimi- dines like guanine, thymine, and cytosine) and kinetin on the one ' hand, and the factors of elongation such as indoleacetic acid on the other hand, is of a very peculiar nature. In fact, kinetin is only active in the presence of traces of auxin [33]; inversely, it seems that auxins need the presence of adenine to have an effect on cellular elongation [34]. Still, a great preponderance of the one can stifle the characteristic effect of the other [35]. It is perhaps daring to transpose into the domain of animal 1 biology facts discovered in that of phytobiology; for the moment however, these latter alone can help us understand the effect of auxins on tumors in humans and animals. Summary In weak concentrations, auxins accelerate the catabolism of carbon hydrates, activate oxidative phosphylorations, increase oxygen consumption in tissues and stimulate growth. In elevated concentrations, they complete the depletion of carbon hydrates, obstruct anaerobic glucolysis, reduce the rate of potassium in tissues, and inhibit growth. Auxins ^regulate the rate of nucleic acids and are antagonistic to factors of cellular division such as kinetin or -6-furfurylaminopurine. Their inhibitory action on growth is remarkably selective. It is most evident for certain vegetable species than fwx others and, in all cases, stronger as the tissue in which it operates is younger and less differentiated. 1 52840007463 Page 14 The utilization of auxins as synthetic herbicides is based on these properties. Tissues of vegetable tumors are more sensitive to auxin's inhibitory action than corresponding normal tissues. Auxins operate, although to a lesser degree, on animal meta bolism. The author has undertaken to utilize certain auxins for the selective inhibition of benign and malignant tumors in humans and animals. Auxins in ionizable form prove to be inactive. On the contrary, non-dissociable derivatives (esters of alcohols) manifest considerable cytostatic action. The ethyl ester of 2,4dichlorophsnoxyacetic acid, the isopropyl ester of 2,4,5-trichloro phenoxyacetic.acid, and especially the methyl ester of the same acid are particularly active. The anti-tumor effect of these aux ins is reinforced by riboflavin. Radiotherapy concomitant or slightly preceding (less than three weeks) treatment with auxins considerably reinforces the cytostatic action of this last, even when the dose of rays is subliminary [sic]. The cited esters of auxins have a very slight toxicity and never depress hematopoeisis Their hyperglycemic effect was confirmed. They were prefer ably administered in an oily solution by intramuscular injections of 0.50 to 1.0 g/day. Rests of four days can be intercalated b e tween successive series of fifteen days. The esters of auxins utilized must be purified of any contam ination by the corresponding polychlorophenols and*neighboring isomers, whether inactive or antagonistic. Acknowledgments Among the treated cases, there were five animals. I direct m y thanks to Dr. E. C. Preston (The People's D ispensary for Sick Animals, Tangiers) for having permitted the treatment of them. I equally thank him for the efficient* help and enlightened counsel that he so often squandered on me. However, I have to express my gratitude to Messieurs the Pr. H. V e l d s t r a and H. Linser at the same time as to the Rhone Poulenc Laboratories, and Phili s-Roxan for the substances which they freely consented to put at my dis posal. 5 2 85 000?4 6 4 _ Page 15 It o c i [1] O. Warburg, N a t u r w i s s c n s c h . , 1, 483 (1954); 2, 401 (1955). [2] E. Lundsgaard, Am. Rev. B i o c h e m . , 1_, 377 (1938); K. V. Thimann a n d W. D. Bonner, Am. J. Botany, 36_, 214 (1949) . [3] J. Berger, P. Smith and G. S. Avery, Jr., Am. J. Botany, 3 3 , 601 (1946). [4] G. S. Avery, Jr., Plant Growth Substances, p. 105, F. Skoog, Univ. Wiscons. Press, Madison (1951). -[5] F. G. Smith, Plant Physiol., 23, -70 (1948). V [6] J. Bonner, Am. J. Botany, 36, 323, 429 (1949). C [7] B. Axelrod, J. Biol. C h e m . , 1 7 2 , 1 (1948). ' [81 D. P. Hackett and K. V. Thimann, Proc. Nat. Acad. Sci., 3 8 , 770 (1952); J. Bonner, R. S. Bandurski and A. Millerd, Physiol. Plantarum, 6, 511 (1953). [9] J. S i l b e r b e r g e r , Jr. and F. Skoog, Science, 1 1 3 , 443 (1953). [10] F. Skoog and C. Tsui, Plant Growth Substances, p. 263, Univ. Wiscons. Press, Madison (1951). [11] R. S. De Ropp, Am. J. Botany, 3, 248 (1947); R. J. Gautheret, C. R. Acad. Sci., 22, 1728 (1950). [12] R. S.^ De Ropp, Am. J. Botany, 3, 53 (1947) . [13] R. J. Gautheret, C. R. Soc. Biol., i2, 774 (1948). [14] B. E. Struckmeyer, A. C. Hildebrandt and A. J*. Riker, Am. J. Botany, 36, 491 (1949). [15] R. J. Gautheret, Bull. Soc. Chim. Biol., 2, 13 (1942). [115] G. Camus and R. J. Gautheret, C. R. Acad. Sci., 2 2 6 , 744 (1948). [17] K. K. Sanford-, G. D. Likely and W. R. Earle, J. Nat. Cancer I n s t . , 15, (2), 215 (1954). [18] H. Gold b l a t t and G. Cameron, J. Exper. M e d . , 7, 525 (1953); W. R. Earle and A. Nettleship, J*. Nat. Cancer Inst., , 213 (1943); G. O. Gey, M. K. Gey, W. M. Firor and W. 0. Self, A c t a Unio. Intern, contra Cancrum, 6, 706 (1945); K. K. Sanford, W. R. Earle, . Shelton, E. L. Schilling, E. M. Duchesne, G. D. L i k e l y and M. M. Becker, J. Nat. Cancer Inst., LI, 351 (1550). [-19] S. B. Locke, A. J. Riker and B. M. Duggar, Acir. Res., 59, 519, 535 (1939). " .5286 0007465 .\ Page 16 [20] A. J. Riker, II. Berch and B. M. Duggar, J. Agr. R e s . , 6 3 , 395 (1941). [ 21] S. B. Locke, A. J. Riker and B. II. Duggar, J. Agr. Res., 9 t 519, 535 (1939). [ 22] S. M. Siegel and A. W. Galston, Proc. Nat. Acad. Sci. Wash. _39, 1111 (1953). [23] F. K o e g l , A. J. Haagen-Smit and H. Erxleben , Z. Physiol. Chem., 228, 90 (1934). [ 2 4 ^ Y. Raoul and Ch. Marnay, Bull. Soc. Chim. Biol., 31, 289 (1949) [25] B. De Bernard, Boll. Soc. Ital. Biol. S p e r . , , 81 (1952); V. Zambo'tti and B. De Bernard, Boll. Soc. Ital. Biol. Sper.', 28, 928 (1952). [26] R. Louis, Zeitschr. f. Zellfschg., 41, 385 (1955). [27] V. A. Drill, Pharmakology in Medicine, 60/7, McGraw-Hill Boo k Co., New-York, Toronto, London (1954). L. J. A u d u s , N e w Phytologist, 8, 97 (1949). [29] P. L. Goldacre, A. W. Galston and R. L. Weintraud, Arch. Biochem. Biophys., 3, 358 (1953). [30] C. O. Miller, Proc. Soc. Exp. Biol, and Med., 3, 561 (1953); C. 0. Miller and F. Skoog, m. J. Botany, 0, 768 (1953) ; C. or Miller,. F. Skoog, F. S. Okumura, M. H. Von Saltza and F. M. Strong, J. Am. Chem. Soc., 7, 1375 (1956). [31] F. Skoog, Brookhaven Symposia in Biology, , 400 (1954) . [32] F. Skoog and C. Tsui, Plant Growth Substances, p. 263, Univ. Wiscons. Press, Madison (1951). [33] C. O. Miller, F. Skoog, M. H. Von Saltza and F. M. Strong, J. Am. Chem. Soc., 77, 1392 (1955). [34] C. O. Miller, Proc. Soc. Exp. Biol, and Med., 3, 561 (1953). [35] F. Skoog and C. 0. Miller, Symposia Soc. Exptl. Biol., 11, 118, Cambridge Univ. Press (1957*). ( 000746*. 5287 5288 H-o  5291 EF SERVICE Journal o f the W eed Society o f A m erica 5292 BRIEF PAPERS Iso-octyl Ester of 2,4,5-T in Hardwood Control C arter B . G ibbs1 Da v is an d D u k e (2) in 1955 reported su ccessfu l u se o f a 36 lb ah g solution o f the propylene glycol butyl ether ester o f 2 ,4 ,5 -T a p p lie d by'`m ean s o f a tree in jecto r.- T h is to o l is essen tially a m o d i fication o f the C ornell tool described by C ope and Spaeth (3) in 1931, and subsequently tested w ith am m onium sulfam ate (I, 5) and 2 ,4 ,5 -T (4, 6, 7). Since the iso-octyl ester o f 2 ,4 ,5 -T is generally available at low er cost than the previously tested propylene glycol butyl ether ester, a study to assess its effectiveness was installed in M arch 1957 by the Nacogdoches Research Center in cooperation w ith the Sam H ous ton N ational Forest. Each o f four concentrations of the chem ical, 40, 20, 13.3, and 8 lb ahg in d iesel oil, was ap p lied to 30 sweetgum s (L iquidam bar styraciflua L.) and 30 oaks (Quereus stellnta W angenh., Q. m arilandica M uenchh., and Q. falcata M ichx.). Trees were of two diam eter classes, 0.6-3.5 inches and 3.6-6.5 inches. A total of 480 trees w ere thus injected-- all in circum basal wounds, on e per inch o f estim ated tree diam eter. Since an aim o f the study was to exam ine the effectiveness o f various concentrations under field conditions, no special measures were taken to assure exactly uniform applications of the silvicide. Each treated tree was inspected for crown condition and sprouting in M ay and O ctober 1957 and in N ovem ber 1958. A t the first inspection, three m onths after treatm ent, crown dam age was visible only on trees treated w ith the higher concentrations. B y the end o f th e first g ro w in g season the m ost effective treatm ent, 40 lb ahg, had top-killed 100 percent of the sm all trees and 93 per cent o f the large trees. T op -k ill was slower on sw eetgum than on oaks. N o treated trees sprouted during the first grow ing season. R esults at the end of the second grow ing season are shown in T ab le 1 as percents corresponding to the m ean angle o f equal inform ation (arcsin \J % transform ation). T h e difference betw een 100 percent top-kill for 40 lb ahg and 75 percent for 20 lb ahg was h igh ly significant. T h e 64 p ercent top-kill w ith 13.3 lb ahg was sig nificantly m ore effective than 33 percent w ith the 8 lb ahg. D if ferences in com plete kill w ere sim ilarly significant. Differences in both top-kill and com plete k ill between oaks and sweetgum were significant, an indication that sweetgum was harder to k ill than the oak species tested. T h ere w ere no significant differences in either top-kill or com plete 'Nacogdoches Research Center, maintained at Nacogdoches, Texas, coop eratively by Stephen F. Austin State College and Southern Forest Experiment Station,' Forest Service, U. S. Department of Agriculture. 'Manufactured by Reuel Little, Madill, Oklahoma. 462 Conctntraiion (lb ahg) 40. . 20. . 13.3. S.. k ill between the tw o tree sm trees sp ro u tin g (in c lu d in g t. greater for th e sm aller size T h e effectiveness o f iso-oct oaks and gums in east Texc ester o f 2 ,4 ,5 - T ca n b e bas> W h ere o n ly oak s are to b e ci o f the iso-octyl ester m ay be s species w ith sim ilar resistance concentration w ill be requir Lnx 1. B rasincto.v , J. J. Poisoning < Fanner 9(6):5. 1950. 2. D avis, J. R,, and D uke. V . B . ( man 191(2393): 171-112. 1955. 3. Co pe, J. A., and Spaeth, J. N . Jour. Forestry 29:775-783. 19' 4. Goddard, R. E. R illing small ui tool. Down to Earth (Dow Ch 5. G rako, C. X . Effectiveness o f . Lumberman 185(2316):44. 46, 4 6. Jokela, J. J., and Lorenz. R. W. nating cull trees from woodlanu 1955. 7. Smith, J. L. Tests o f injected che sas mountains. Proc South. IV v u 5293 * 3 d Control ise o f a 36 lb ahg ester of 2,4 ,5 -T ssentially a m odiid Spaeth (3) in fam ate (1, 5) and vailable at low er nitvl ether ester, arch 1957 by the i the Sam H ousof the chem ical, to 30 sweetgum s us stellata W anchv.). T rees w ere : s. A to ta l o f rounds, one per effectiveness of special measures of the silvidde. and sprouting in ent, crown dam concentrations, ctive treatm en t, rees and 93 per:etgum than on w ing season. ii are sh o w n in angle of equal :eren ce b etw een r 20 lb ahg was lb ahg was sig8 lb ahg. D if. Differences in sweetgum were to k ill than the <ill o r co m p le te ics, Texas, coop7orest Experiment V i % r i- s 2 S' :. * ! G ibbs : I so-o c t y l E ster 463 T a b le 1. Top-kill and complete kill two growing seasons after injection with 2,4,5-T. C oncentration (lb ahg) 4 0 .............................................. 2 0 .............................................. 1 3 . 3 ........................................... s .............................................. T i m top-killed, pet Oak 100 92 69 39 S w eetgum 100 54 55 27 B oth 100 75 64 33 T rees killed, no sprouts, pet O ak 100 82 64 35 Sw eetgum 100 50 54 23 B oth 100 67 59 29 k ill b etw een the two tree size-classes, though the total percentage of trees sp rou ting (inclu din g those w ith live tops) was significantly greater fo r the sm aller size class. T h e effectiveness o f iso-octyl ester o f 2 ,4 ,5 -T w hen injected into oaks and gum s in east T exas means that choice of a low -volatile ester o f 2 ,4 ,5 -T can be based on relative costs o f the chem ical. W here o n ly oaks are to be controlled, the 20 lb ah g concentration o f the iso-octyl ester m ay be satisfactory. W here sw eetgum or other species w ith sim ilar resistance to silvicides are to be treated, a higher concentration w ill be required. L iterature C ited 1. B rasingtox, J. J. Poisoning scrub oaks with the Cornell tool. Forest Fanner 9(6):5. 1950. 2. D avis, J. R,, and D uke. W. B. Quick, Bunyan, the needle! South. Lumber man 191(2393): 171-172. 1955. 3. Co pe, J. A., and Spaeth, J. N. T he killing of trees with sodium arsenite. Jour. Forestry 29:775-783. 1931. 4. Goddard, R. E. Killing small undesirable hardwoods by use of the Cornell tool. Down to Earth (Dow Chem. Co.) 10(2):5. 1954. 5. G rako, C. X. Effectiveness o f Ainmate in controlling hardwoods. South. Lumberman 185(2316):44. 46, 48, 50. 1952. 6. J okela, J. J., and Lorenz, R. W. A comparison of three methods of elim i nating cull trees from woodlands with 2,4,5-T. Jour. Forestry 53:901-904. 1955. 7. Sm it h , J. L. Tests of injected chemicals for hardwood control in the Arkan sas mountains. Proc. South. Weed Conf. 12:123-125. 1959. 43 gg 43 V .1DOM 21 5 6 8 5 5 Industrial Hygiene Digeit April. 1962 anomalies la tha electroencephalogram (EEC), consisting of bllattral synchronous theta-wave activity and occasional bilataral synchronous spike and wave complaxas, baliavad to ba as sociatad with brain stem injury. Tbasa spaciiic disturbaacaa in tha EEC may parsist for soma tima after apparent clinical recovery, but they almost always disappear spontaneously, after discontinuation of exposure. Systematic survey of a large group of exposed workers may occur without preceding or subsequent convulsions and even without any'clinical symptoms or signs. Periodical electroencephalographic examination is a valuable method for the detection of early subclinical intoxication. Moreover, if suspect symptoms suddenly appear during the interval between periodic examinations, particularly if myoclonia occurs, an additional EEC should immediately be taken. Appearance in the EEC of the abovetnentioned specific disturb ances is an indication for removal of the worker from further exposure. Clinical recovery after convulsions has always been complete. With adequate precautions it is possible to pre vent intoxications among workers who have daily contact with the insecticides over a period of years. -- Cond. from authors' summary 354 Nervous System Effects of a Chemical Herbicide. I. Desi, et al. Arch. Environmental Health 4, 95-102 (Jan. 1962). '** After the parenteral administration of 2 ,4 -0 a reversible inhibition of cerebral electrical activity was observed in the acute experiments, and in chronic experiments the same was present to a gradually increasing degree. Toxic electroencephalogram signs were developing. According to conditioned-reflex experiments, the higher nervous activity suffered severe damage. The changes are probably produced by the 2 ,4 -0 molecule itself and not by some of its degeneration products. The point of attack seems to be the reticular formation. The changes manifest themselves as early as 24 hours after exposure. The changes found in animal experiments suggest the need of caution in the use of 2 ,4 -0 . Increased protection and . special neurological examination of workmen in contact with 2 ,4 -0 is necessary. -- Authors' summary INDUSTRIAL DUST$ 355 Maple-Bark Disease. Pneumonitis Due to Coniosporium Corticale. O. A. Emanuel, B. R. Lawton and F. J. WenaeL New Engl. J. Med. 266, 333-337 (Feb. 15, 1962). The clinical impression that some types of pneumonitis may be caused by individual hypersensitivity to molds or their products has received increasing attention in recent years. The exact role of molds, dust and individual susceptibility, however, is not fully understood. "Farmer's lung" is thought to be caused by a reaction of this type, but bagassosis and "mush room-grower* s disease" may also be related to such a phenomenon. The authors present a case of pneumonitis due to Coniosporium corticals. The spores were demonstrated micro scopically from tissue obtained by lung biopsy, and cultures yielded a fungus identical to the mycelial phase of C. corticals. To the knowledge of the authors this is the first case since Tewey and his co-workers (J. Am. Med.Assn. 99, 453-459 (1932)) reported their 35 cases in 1932, and the first demonstration of a diffuse type of interstitial pneumonitis with granuloma formation due to a reaction to a specific fungus spore. / 356 Istradarmal Sensitivity Testing in Man with a Purified Vaccine for Q Fever. O. B. Lackman, et al. Am. J. Public Health 52, 87-93 (Jan. 1962). 5296 A standard product for use in a quantitative intradermal test to determine sensi tivity against C. burnetii has been described. The importance of utilising organisms in Phase Zfor preparing vaccines has been emphasised. Recommended modifications in the intradermal test include use of 0.02 complement-fixing unit as the skin-test dose and utilisation of indura tion as the most reliable indicator of sensitivity. Finally, the authors suggest wider use of the intradermal test as a pre-vaccination procedure and in epidemiological studies to determine the prevalence of infection of man by C. burnetii. -- Authors' suxsfik^0 9 9 3 ? it. 5297 V olum e 711 r .-I . . . 196i . . . 6294 . . 41-ii . . 570 . . . 598r . . . 766 1 1 3 ,325 . . . 536* . . . 94< lv . . . . 768( ..... 3614 ___ 474* . . . 3614 . . . 693f __ 364 ___ 227* ... 1 . . . 293f . . . 9901 . . . 762 . . . 609 . . . . 90$ . 990? ... sssi . . . 41436 . . . 566* . . . 5051 . . . 443i 134* 1961 _ -a . . . 790* im 4 fc 3GG'6 - / U .H ENDOCRINOLOGY Vo lu m e 71 JU L Y , 1962 Copyright () 1962 by Lht Endocrino Society N umber 1 Some Effects of 2,4-Dichlorophenoxyacetic Acid on T hyroid Function in the Rat: Effects on Iodine Accumulation1 WARNER H. FLORSHEIM a n d SHIRLEY M. VELCOFF . H.cioisotope Service, U. S. Veterans Hospital, Long Beach, California, and Uniuersity of California M-cical School, Los Angeles, California THE STUDY of the action of drugs upon thyroid function has been a fruitful method of elucidating the de tails of thyroid metabolism. Several years ago, Sds and Kertai (1 ), in the rturse of a study of tyrosine antagonists, noted that 2,4-dichlorophenoxyacetic avid `2,4-D), a commonly used weedivide, had some rather unusual effects upon thyroid function in the rat. They showed th at administration of the drug in nontoxic doses for several days led to a marked increase in the thyroidal radioiodine uptake without any effect upon thyroid weight or the fraction of radio iodine that becomes protein-bound. We decided to investigate further the mech anisms underlying these effects of 2,4-D on thyroid function. M aterials and Methods The 2,4-D used was Eastm an "white !ab*r' m aterial purified b y D a tt a 's (2) Received September 15,1960. Aided in p art by Research Grants H-20J3 and A-4455 from the USPHS. _*Presented a t the 4th International Goitre Conference, London, England, July, 1960. A BSTRA CT. 2,4-Dicblorophenoxyacetic a d d , a compound widely used as a weediride, markedly increases I m uptake by ra t thyroid in oiuo. Measurements of a number of thyroid function criteria, including thyroid cell height, rate of discharge of I 111 from the gland, and serum and pituitary TSH levels, indicate that this effect does not involve the pituitary thyrotrophic hormone but represents a direct effect on the peripheral iodide pool. This was confirmed by isotope equilibrium analysis of the serum iodide level. The effect can be dem onstrated only in rats in which the thyroid functions normally, and does not occur in hypophysectomized or iodine-depleted ani mals. m ethod. I t was dissolved in 0.15 M sodium bicarbonate solution and administered by daily sc injection. M ale Sprague-Dawley rats were housed in a room kept at 24 C and fed W ayne Lab Blox (iodine content: 2.0 pg/g) and tap water. Thyroid function studies were carried out as described previously (3). Iodine was determined by a m odification o f B o d a n sk y 's w et ashing tech n iq u e (4). M c K en zie's m ouse assay (5) w as used to assay thyrotropin in rat pituitaries and in con centrates o f pooled rat serum prepared by B ates's percolation m ethod (6). A nalysis o f thyroidal iodinated am ino acids w as carried out by the ion exchange m ethod o f G alton and Pitt-K ivers (7) after digestion w ith 5298 FLORSHEIM AND VELCOFF Volum e 71 J Inly, 1962 T able 1. Effects of 2,4-D on thyroid function parameters Test Rats per group Controls 2,4-D-treated Significance i of difference - (P) 24-hr I m Uptake Serum PBI Thyroid Weight Thyroidal Total Iodine 24-hr Conversion R atio T :S Thyroid Cell Height T J of Thyroidal I U1 .9 10 9 9 6 6 6 6 4 .3 9 -`-0.30%* 3 .2 + 0 .1 Mg'/100 ml 9.41 0.44 mg 78 3 mg/100 g 46 3% 31 +2 13.0+0.8 m 5.4 0.3 days 6.40 0.23% 2 .7 0.1 Mg/100 ml 9.52 0.33 mg 79 3 mg/100 g 44 3% 48 5 12.6 0.2 m 5.3 0.3 days .001 .002 -- -- -- .02 -- -- * : -1 * Standard error of the mean. A pancreatin (California Corporation for Bio chem ical Research) in pH 8.2 borate buffer. Only the fractions that could be eluted with aqueous acetic acid were analyzed. Serum iodide levels were measured by the isotope equilibrium technique as described by Simon and Morel (8). R ats were kept on iodine-free water and the Rem ington diet (N utritional Biochem icals Carp.), contain in g on ly 16 Mg io d in e/k g , for 26 d a y s. D uring th e entire treatm ent period, they received by daily sc injection 1.0 cc o f a solution con taining, on the first day o f the experiment, 0 .8 pc I 111 and a k n ow n q u an tity (either 5 or 10 pg) o f stable iodide. D uring the last 7 days o f th e regim en, h a lf th e rats received SO m g / kg body weight o f 2,4-D , while the rest re ceived the bicarbonate vehicle only. After autopsy, 3 cc of serum from each rat was treated w ith 5 vol o f 10% trichloroacetic acid, the TCA precipitate was washed once w ith TCA, and its radioactivity was deter mined in a well-type scintillation counter. T o the combined TCA supem ates, 5 m g o f car rier iodide was added and the iodide was precipitated with an excess o f palladium chloride. T he palladium iodide precipitate was washed with w ater and n-butanol and then dissolved in concentrated ammonium hydroxide and quantitatively transferred to planchettes for counting of the contained radioactivity w ith a gas-flow Geiger counter. T h e conversion factors for calculating iodide content from the rad ioactivity data were de--# rived by assaying aliquots o f the injection 4 solution for radioiodine and iodine content S R ecovery stu d ies showed th a t iodide re--^ covery w as q u a n tita tiv e and th a t a n y traces-* o f thyroxine carried down w ith th e palladium^ iodide precipitate were elim inated readily by ^ washing the precipitate once w ith n-butanol g before planchetting. j R esu lts We first established a dose of 2,4-D i that would consistently duplicate the-r findings of Sos and Kertai (1 ). At 80 mg/kg per day, the animals continued to gain weight almost normally, and there'? was no effect upon thyroid, pituitary, ! adrenal or testicular weight even when# 2,4-D administration was continued for - several weeks. A t 100 mg/kg, both t thyroid and body weight decreased, and - at higher doses paralysis occurred. Table, 1 summarizes some of the effects upon thyroid function parameters. The effect i of the serum PBI, which appears to bee caused by changes in serum and tissue binding of thyroxine, will be discussed.* fully in a subsequent paper. J 2,4-D had no apparent effect on thy-i Table 2. 2,4-D effect on 24-hour thyroidal I 111 uptake after hypophysectomy and iodine depletion Hypophysectomized Iodine Depleted R ats per group 8 6 Controls 0.43 0.04%* 14.9 1.0% 2,4-D-treated 0.33 0.05% 14.8 1.2% * Standard error of the mean. T in T :iNil! -------* Standard roidal I m i rats, and i means o f i w eeks there m 11,1 u p t: Since thi thyroidal . serum P B I in v estig a te! ney histolc was no inc and the se was unafft lion. Iodic serum ino. hours after did n ot d iff trol levels. ' are oth er ci found th a t pletely u ne thyroid cell m ice w ere neither w a iodine from kept on a g w hile bein; developm ei term inal v radioiodide w;fs n o t e le I'itu ita r y T K e n z ie 's a s 2.4-D . Seru ured on ly 'era fro m 1 6). W e fou; units (m U ) treated and D O W 680483 J. 'oum e 7 1 ' ^zincarce i:zrence P. V :fj :a w ere d e-< e in je c tio n ^ ne co n ten t. 4 io d id e r e -j| m v traces.# - nailadimn^ r e a d ily by-^j . n -b u tan o l 2,4-D* i the* i . A t SO.* i:in ued to # and th e r e p ir o ita r y ,.# van when- :inued for* >.z. both* ?.sed, and# id. Table 5 -crs upon# 'he effect-4 u s to beii nd tissue^ discussed . on thy-J ated J ;? ? c 1* -t 1962 E F F E C T S O F 2,4 -D O N T H Y R O ID F U N C T IO N T able 3. Effect of 2,4-D on goitrogenesis due to 10 days of propylthiouracil feeding Controls 2.4- D- tre a te d Thvroid Weight T :S Ratio Number of Rats 25.5 0.9* mg 157 15 6 25.8 0 .6 mg 205 31 6 Standard error of the mean. roirial I u*uptake in hypophysectomized rars. and in rats depleted of iodine by means of the Remington diet for two weeks there also was no effect of the drug m I :i uptake by the thyroid (Table 2). Since the association of an increased thyroidal I 1*1 uptake with a lowered *rrum PBI is found in nephrosis, we investigated this possibility, but kid ney histology remained normal, there was no increased urinary protein loss, and the serum electrophoretic pattern was unaffected by 2,4-D administra tion. Iodide clearance, as judged by .rum inorganic iodide-Im levels 24 hours after tracer administration, also ciid not differ significantly from the con trol levels. When we attempted to meas ure other criteria of thyroid function, we f--und that many of these remained com pletely unchanged (Table 1 ). Thus, the thyroid cell height and hiotslogic appear ance were not affected by 2,4-D, and neither was the rate of loss of radioi<>dine from the gland. When rats were kept on a goitrogenic diet for two weeks while being given 2,4-D daily, goiter development was unaffected and the terminal value of the thyroid:serum radioiodide concentration (T:S) ratio was not elevated significantly (Table 3). i'ituitary TSH stores measured by Mc Kenzie's assay (5) were unaffected by 2.4-D. Serum TSH levels could be meas ured only after concentrating pooled -*ra from 12 rats by Bates's procedure 0 . We found levels of about 0.025 milliunits 'mU)/cc of serum in both 2,4-Dtrested and control rats (Table 4). The iodinated amino acids of the thyroid glands of treated and control rats were analyzed 24 hours after radio iodine administration. Using the ionexchange chromatographic system of Galton and Pitt-Rivers (7) on pancreatin 1 digests, we obtained the results shown in Table 5. Total iodine analyses were per formed on aliquots of the thyroxine frac tion and the pooled iodotyrosine eluates, and the percentage of thyroxine among the total iodinated amino acids also was found to be unaffected by 2,4-D. The results of the determination of the serum iodide level and PB I by the isotope equilibrium technique are shown in Table 6. To confirm the magnitude of the serum iodide levels found, we calculated the difference between the serum total iodine and the serum protein-bound iodine levels in 11 control rats by chem ical assay. This difference averaged 1.0 0.1 (standard error) /ig/100 ml, in good agreement with the levels cal culated from radioactivity data in the animals in the experiment of Table 6 th at were on the same iodine intake. T able 4. P itu ita ry an d serum thyrotropin assays Pituitary TSH Content; Controls (5) 93 15* mU per gland 2.4-D-Treated (5) 10S 20 mU per gland Serum Levels; Controls 2.4- D-Trcated 0.028m U /cc 0.024mU/cc * Standard error of the mean. 5300 0003469 4 FLORSHEIM AND VELCOFF Volum e 7j 4 .July, 1962 T able 5. Analysis of thyroidal iodinated amino acids Controls Number of rats 12 24-hr I:" uptake 6.9 0.8% Serum PBI 3 .9 0 .1 0g /10 O ml 24-hr conversion ratio 30 2% I " 1 Distribution: Thyroglobulin 4.9 0.4% loaotyrosines 62.6 2.4% Iodothyronines 10.6 0 .8 % I ,B Distribution: Iodothvronines as fraction of total iodinated amino acids 19.4 0.7% 2,4-D-treated 12 10 .2 1 .0% 3 .2 0 .1 #g'100 ml 26 2 % 4.2 0.1%, 62.9 1.9% 11.7+0.97# 2 1.0 0 .8 % Significance s of difference 4 (F) ; < .0 2 < .0 0 1 t : .' 2 - 4 :& -> 1 > .i i D iscu ssio n control rats with regard to the distribu-4 2,4-D has rather unusual effects on tion of stable iodine and of radioiodine 4 thyroid radioiodine uptake and serum among thyroglobulin, iodinated ty ro -i PBI, the two most commonly used sines, and tri- and tetra-iodothyronine.-4j criteria of thyroid function. I t is one of We did not determine whether or not the $ the very few compounds known th at iodide content differs, since this fraction * -4elevates I '}1 uptake. The only previously was not separated from partial hydroly- described situations in which similar re* s products and iodine bound to in-+t suits are obtained are nephrosis and soluble cellular debris. i iodine deficiency. In both of these there is The results of the 24*hour radioiodine * evidence of increased thyrotropin secre- uptake test, therefore, seem to conflict a tion from the pituitary, whereas we with a battery of other thyroid function 4 could show no evidence of this after tests. I t appears th a t the enhancement * 2,4-D administration. Pituitary and of thyroidal I >Muptake must be caused * serum thyrotropin assays support the by a change in the specific activity of f evidence afforded by the unchanged serum iodide due to a lesser dilution of thyroidal I m release rate and by the : the injected carrier-free radioiodide, i histologic appearance of the glands and The isotope equilibrium experiments at % the epithelial cell height. In our hands, both the 5 and the 10 fig per ra t per day 5 the index of precision of McKenzie's iodine intake level showed clear depres- - assay has averaged about .32, which sion of the serum iodide level. T he likeli- i allows for a large margin of uncertainty hood of radiation damage to the thyroid in the results. With the low levels of TSH in these experiments is negligible, since i found in serum, which we estimate at the total radiation dose received by the i about 0.025 mU/cc, we could use only glands was well below the 3,000 r five mice per point, and this severely threshold below which radiation damage -3 limited the accuracy of the assay. The has not been demonstrated (10). The k TSH level we find in the serum of our iodide levels found were of the same A rats is much lower than th at which has order of magnitude as those calculated 4 been reported by D 'Angelo, using the from the difference between chemically \ stasis tadpole assay (9). determined serum total iodine and the i In the present study, we found no PBI, which is not in agreement with differences between 2,4-D-treated and the work of Gerbaulet and collaborators * 11), who ! scrum to 1 value calci 1*131 diffre derbaulet'. tains iodid iodide as tl pound m a\ It is of i isotope equ tarried oufrom radio: and signifie termined o: cal analys The avera similar find Middleswo: this discret It does not to achieve the 26-day I'itt-Rivers calculating ing equalit; iodinated rr the tumov. iodine after that 23 da: 97l"c of eq tions, since 4.0 to 4.6 Simon (15), technique i Experiment 1 : Strum Iodide l*Ul Chemical PB I Experiment 2: Scrum Iodide 1'BI Chemical PBI * Standar* DOW 680485 j Volume 71 $1 raiacance '4 d:a*rence <.02 <.001 t i . * ? > .l 3 listribu- 3 ioiodine -v i tyro- 4 Tonine. not the Taction ydrolyto in- ators E F F E C T S O F 2,4-D ON T H Y R O ID FU N C TIO N .i . who believe iodide levels in human ^rum to be about one twentieth of the 7 :`.ue calculated from the total iodinej>HI difference. However, it appears that (ie-rbaulet's assumption th at saliva con* :.iins iodide in equilibrium with plasma ;.,.dide as the only iodine-containing compuund may not be valid (12,13). It is of some interest that, in all the isotope equilibrium experiments we have carried out, the PBI level calculated from radioactivity data was consistently .ir.d significantly lower than the PBI de termined on the same samples by cheroiva! analysis using TCA precipitation. The average difference was 35%. A -imilar finding has been reported by Van Middlesworth (14). The significance of this discrepancy is under investigation. It does not appear to be due to a failure to achieve isotopic equilibrium during the 26-day experimental period. Using Hit-Rivers and Hall's (10) method of calculating the period needed for achiev ing equality of specific activity of all iodinated materials in the animals from the turnover rate of thyroidal radioiodine after a single tracer dose, we found that 23 days should suffice to achieve &:; of equilibrium under our condi tions, since the half-life observed was 4.0 to 4.6 days. However, Morel and Simon 115), using a much more laborious technique for determining the attain ment of equilibrium, required 32 days to achieve 95% equilibration in rats th a t were considerably older than the ones used in Pitt-Rivers' and our studies. In experiments to be reported elsewhere, we have administered the long-lived isotope I ,Mfor 35 days and still observed highly significant differences between the PB I calculated from radioactivity data and the chemically determined PBI, which were of the same magnitude as those re corded in Table 6. For these reasons, we believe that isotopic equilibrium was , attained in our experiments and th a t other explanations must be sought for the discrepancies between the P B I values obtained by the alternate pro cedures. In any case, the determination of serum inorganic iodide levels, which was the object of these experiments, should certainly be valid since inorganic iodide equilibrates much more rapidly than the organically bound iodine. Thus 2,4-D appears to be one of the few agents other than iodide itself th a t has been shown to alter the serum iodide pool specifically. Our results again emphasize the necessity of using a large number of thyroid function tests when exploring thyroid-pituitary interrela tionships, since many peripheral factors, few of which have been explored ade quately, can invalidate several of the commonly used tests. T able 6. Isotope equilibrium determination of serum iodide and PB I Controls 2,4-D-treatcd Experim ent l : 14 rats p e r group on d a ily done o f S tig iodide Strjjn Iodide 0.78 0.03* pg/100 ml 0 53 0.03 Mg/100 ml P 3I 1.92 0 .1 0 pg/10 0 ml 0 87 0.08 >ig/100 ml Chemical P B I 3 .3 0 .1 ig/10 0 ml 1 7 0.1 Mg/100 ml Experim ent 2 : 14 ruts p e r group on d a ily dose o f 10 ng iodide .Serum Iodide 1.48 0 .0 8 pg/100 mi 0 92 0.04 *g/100 ml PBI 1.92 0 .0 7 jig/10 0 ml 1 35 0.09 pg/lOO ml Chemical P B I 3 .2 0 .1 ^g/10 0 ml 1 9 0.1 ig/100 ml Significance of difference (P) .005 <.001 <.001 <.001 <.001 <.001 * Standard error of the mean. 0 1 05 o00 * 00 05 f 5302 0003471 hw :.32?S? a FLORSHEIM A N D VELCOFF Volum e 71 R eferences 1. Sds, J . and P . Kertai, Acta Physiol. Acad. Sci. Hung. 14: 367, 1958. 2. Datia, S. K. and G. Banerjee, J. Indian Chtm. Soc. 3 1 : 397,1954. 3. Florsheira, W. H ., Endocrinology 62: 783, 1956. 4. Bodansky, O., R . S. Benua and G. Pen* nachia, Amer. J . Clin. Path. 30: 375, 1956. 5. McKenzie, J. M ., Endocrinology 63: 372, 1958. 6. Bates, R. W., M. M. Garrison and T . B. Howard, Endocrinology 65: 7,1959. 7. Gaiton, V. A. and R . Pitt-R iven, Biochem. J . 72:310,1959. 8. Simon, C. and F. Morel, Int. J . Appi. Radial. 8: 35, 1960. 9. D'Angelo, S. A. and R. E . Tramo, Ann. N . Y. Acad. Sci. 72: 239,1958. 10. Pitt-Rivers, R. and J. E . Rail, Endocrinology 68 : 309, 1961. 11. Gerbaulet, K., W, Fittig and W. Maurer, Klin. Wschr. 38:474,1960. 12. Nicolas, G., Schweiz. Monatsschr. Zakn- heilk. 70:633,1960. 13. Ceia, M., J . M. Sobral and W . Clode, Art}. Pat. 3 1 : 411, 1959. 14. Van Middlesworth, L. and A . P . Intoccia, Metabolism 6 :1 , 1957. 15. Morel, F . and C. Simon, C. R. Soc. Biol. 151:1106,1957. il -jj 41 o a| 11 j| ? O o00 CO <1 Iodide to Epir NORM/ Harrison i University HPHE : A berg, lease of thyrotro creased thyroid re-exami from stu by the In these release ' thyroid ing adm epineph: mental i plasma vein anc these v; publishe were ini duced i examine port th< associat TSH. Ii . epineph prompt thyroid - M ale ; in weigh Rcceiv *Prese Universi: M innesoi * Prese Stanford Alto, Cal 5303 0OO34|I2 Poes -5*h 12162 UOM 1 71415 s Trans. 8b - f ftecord MID. CODEN Cl Lille Medical V. 7(10) 1049-51 (1962) Clinical Facts POLYNEURITIS AFTER USING A NEED KILLER: CONTAINING ACID 2-4-D. By Philippe Foissac - Gegoux, Annie Lelievre, Bernard Basin and Pierre Warot. B y 1958, one of us (*) had already drawn attention on the risks Involved with the more and the more frequent use of chemi cal products in agriculture: two observations were considered at the beginning of this work which concerned the dangers de rived from handling organics derived from phosphorus, powerful insecticides; one of these observations concerned an agricul tural worker who handled, many months before the installation of a polyradiculorephritis syndrome weed killers, sodium and 003930 5305 (L W 1 714156 potassium salts of 2- methyl -4-.chlorophenoxyacetic acid; for several reasons, we had eliminated in this case the toxic origin of the neurological troubles..., but we ignored at that time that these products could have a toxic effect. In truth, the cases of intoxication due to weed killers are rare and are especially known by toxicologists and doctors; but it is feared that they m a y multiply: for almost all.the substances destined to stop the development of weeds are dan gerous to man. Mineral compounds were being used such as chlo rates and sulphuric acid: these were three times more incon venient with an action often little selective, hence a global destruction of grasses and cereals, of a delicate use (risks of explosion and fire) and of a sure toxicity (burns-methemoglo- binemy). The "vegetable hormones", the last commers, make up an undisputed progress. This name is given to chemical bodies whose action is comparable to that of phytohormones elaborated by the plant itself. In small doses, they in fact stimulate the growth, of young vegetable cells; but in the large doses that they are used they cause "a crazy exaltation of the man i festations of growth, a disorderly activity which drives the most deverse monstruositles, the intense Consumption of reserves and finally the death of the grass". Uiese "hormones" are oxyacetic derivations of the benzine nucleus and of the naphtalene nucleus of the phenyl, naphtyl and indolacetic, propionic and butylic acids as well as some of th e i r derivatives. One of the m o s t wide l y used is 2-*J- dichlorophenoxyacetic acid (Ac. 2-^-D.) whose action powerful and 5306 i ' n ' 'i 1 selective, Is not, however, without Inconvenience as the following observation proves. Observation: G . .. Gerard, 52 ye a r s old, farmer, was referred to the Neurology Clinic on July 30, 1962 by Doctor Chuffart for sen sory and motor problems of the lower limbs. This man, without any previous pathological problem, who Is not an alcoholic, dates the beginning of his troubles precisely on June 2, 19 6 2 . On that date he felt, suddenly, an acute pain throughout the entire right orbital and periorbital region becoming weaker the next day making room for (burning sensation) paresthesra and numbness in the same area. Also on June 3rd , he noticed a (decreased sensitivity) hypoesthesia in the right leg. On June 16, two weeks.later, the right leg recovered a normal sensitivity but the numbness was now felt on the left leg; besides, the lower left limb is now "weaker" and cannot move except with the aid of a cane. The problems remained about the same untlll July 30 when he entered the service. He now felt a persistant hypoesthesia in the right eye area, with hypoethesia of the cornea, the patient now felt some pain in the same area. The subject is uncertain of his lower left limb where a alight reduction -global- of the segmentary force is noticed. There is no other trophic vaso-motor trouble. The knee reflexes are rather sharp while the ankle reflexes are suppressed. The lower left limb suffers from a global 5 3 0 7 GC0^ 3 2 DOW 1 714158 hypoethesia only deep sensitivity was maintained. The neurological examination of the upper limbs is strictly negative. Besides the general condition is excellent. There is no albuminuria. The blood formula reveals an eosinophilia (8# o n A u g 2 and 9# Aug 30). Two electrophoretic blood tests, several weeks apart show a slight decrease in albumina (43 then 46 grams) and-an increase of alpha -2- globulines (13 then 15 grams per liter). The renal biological exam, the galactosuria provoked, the reactions of floculatlon do not show any abnormalities. The electrocardiogram is normal. So is the backside of the eye and the E.E.G. The electrodiagnosis shows the following results: ------ p r o o f of s t i m u l a t i o n of Aug 2, 1962: Integrity of the upper limbs; in the lower limbs there.is no evident weakness but slight problems of excitability (galvanic hyperexcitability galvanic and faradic hypoexcitability of the extensor of the left b i g toe ----- h igh c h r o n a x l e s of the extensors o f the left big toe) witness however a slight neurogenic seizure (Doctor Spy); ------- e l e c t romyography of the two frontal tibia on A u g u s t 4, 1962 (Doctor Ramez): trace of neurogenic seizure of the two muscles examined. The patient leaves the clinic on September 8, 1962: during hosp i t a l i z a t i o n he was submitted to a daily injection o f 200mg of vitamin B1 and 250mg. of vitamin B6 ; besides 5>000 gammes of hydrocobalamine were administered every three days. The facial aches p r a c t i c a l l y disappeared; the deficiency of sensitivity in 5308 DOW 1 714159 the face and the lower left limb was notably Improved* the motor deficiency disappeared and the patient was able to walk normally; the areflexia achillelne persists, there is no visi ble atrophy. There are still electromyographic signs of neurogenic seizure of the right frontal tibia (the left side was not examined). On the contrary the electrodiagnostic of stimula tion :of August 28 are absolutely normal. The aetiology of this polyneuritis of the lower limbs with irritative and deficient right trigeminus was suggested to us by the patient himself who was able to relate the begin ning of his trouble while killing grass. Made June 1st and the last days of May 1962. This operation consisted of a spraying of two different solutions but both having a base of acid 2 - 4 -D, containing 235 and 410 grams per liters. It was carried out with a tractor which tows the spraying device and which is in a cabin, open in the back; it was very windy when the operations took place and the wind beat the toxic substance in the cabin. These exceptional circumstances, the long exposure to the toxic substance, we had to admit that it was a polyneuritis due to 2-4-D acid. Besides, this observation is not alone in the litterature that includes a certain number of facts of intoxication by these substances, notably in the case of farmers: some hours after inhalation of the toxic substance there is generally vomiting, 5309 c c O *J !j 1 * DOW 1 7141G0 6 then subjective sensitive troubles of, the type, o f .paresthesis or even pain, then motor troubles, the paralysis of the four limbs or the lower limbs. This sensitive - motor polyneuritis ' inconstantly associated with an albuminuria and an inter~ o c o l i t e board pic t u r e chart description ------ is usually irre versible or very slowly and very partially reversible. The toxicity of these substances is equally proven in the case of intoxication of workers at a vegetable hormone factory: Die penetration of the toxic substance through inhalation, but also through the skin; the workers often complained of somnolence-, of the lower limbs feeling heavy, gastralgia, loss of appetite, hypersialosis, as well as hypersensitivity of hearing: all of these troubles are, generally, transient. Finally, experimen tally, 2-4-D acid administered intravenously or even orally can be fatal: thi3 happens suddenly, either in a neurological stage in the form of a coma with hypertonic of the limbs, or ventricular fibrillation with relatively large doses, but varying according to the animal tested (about 3C0mg to one gram). To our knowledge, there was no acute intoxication. Bibliography Assouly M.- Selective weed killers and growth substances. Tecnical survey. Pathological effect on man during the manu facture of the ester of 2-4-D. Arch. Ma i . Prof., 1951 1, 2 6 . G o l s t e i n N. P . J o n e s P.H. and Brown J.R. --Polyneuritis after exposure to an ester of 2-^-D acid. DOW 1 714161 I * * H i l l E.V. a n d C a r l i s l e A. ------ T o x i u i t y o i 2-*l-D. for experimental animals. Jour. Indust. Hyg. and Toxic, 197^, 29, 2, 85-95. Lhoste I. - C h e m i c a l weed killers. O . R . S . T . O . M . , Paris, 1958. Footnote p. 1 (*) P. Nayrac, M. Fontan, P. Warot, J. Lescut and J. Delahousse: L i l l e Medical, 1958, 3* 3 161-16U. 5311 0C0333G i-IU M B b lC A L t - n - iy - 9t 175 cu ra i'} : /off.j-/ ( n c r ) l'O L Y X K X 7.77'/; A P I t K s r s A U K D I X m i s //A7.7M.\T : L '. U 'I D K *.'r /h par l'IiilipiN* KiiL'%'.r-*'Kairx Annie L i-3.ikykk. l!<-ni;inl M\six et l'iorre Wtittvr. DOW 1 714162 - D j -ii lr*S. I*un d 'e n tr e n ou s t.*) a v a it a ttir l 'a tte n tio n -sur le s risqu e q u e com p o rta it l 'u tili sation d e plu en plu frequente en agriculture de produit chim ique : deux observations taient l'o r ig in e d e c e tr a v a il d a n s leiiu el l 'a ce cn t a v a it t ml su r le d an gers do la m anipulation des drivs organiques du phosphore, insecticide puis sants ; l'u n e de ces observations concernait un ouvrier agricole ayant m anipul, plusieurs m ois avant rin sta lla tio u d*un syndrom e de polyrudiculonvrite. des dsherbants, sels sodique et potassique de l'ueidc 2-m thyl-4-ohloroihnoxyactiquo ; pour plusieurs raison, nous avion s lim in d an s eu eas I origine toxique de troubles neurologique..., m ais n ous iu n o rio n s l' p o q u e pie ces p r o d u its p o u vaient avoir une action toxique. A vrai dire, les eas d'intoxieatiou pur des dsherbants sont rares et sont surtout connus de toxicologu es et d e s m d ecins d u tra v a il : m ais il est c r a in d r e q u 'il n e se m u ltip lie n t : c a r p resq u e touti > les substances destines entraver le dve lop p em en t d e s m a u v a ises horl>es son t d a n g e reu se s l>onr l'h om m e. O n u tilisa it n a g u re de com p oss m inraux parm i lesquels les chlorates et l'acide sulfurique : ceux-ci avaient le triple inconvnient d 'u n e a c tio n s o u v e n t jm-u s le c tiv e , d 'o u n e d es tru ctio n g lo b a le d e s h erb es et d es cra les, d 'u n em ploi dlicat (risques d 'explosion et d'incendie) -( d 'u n e to x ie ir c e r ta in e (b r lu res m th m o g lo b i n m ie). I.es h o rm o n es vg ta it1 , d ern ire venues, (instituent un progrs indiscutable. Ou donne ce nom des corps chim iques dont l'action est com parable (die des phytohorm one lalxnccs pur la p la n te elle-m m e. A doses faib les, elles stim u lent en effet la croissance des cellu les vgtale jeu n es ; m ai a u x dose forte au x q u elles on le emploie, elles provoquent une exaltation folle des m anifestations de croissance, nue activit dsor donne qui entrane l'apparition de m onstruosits les plus d iverses, la consom m ation intense des rserves et finalem ent la mort au stade herbac . (*) P. N avra', i! . F oxta . P. Warot. J. Lkhiut et J. Du . vkocssk : /.;/! Mi-tlicul. 1958. X 3. 161-164. Lille Mdical. 3" Srie. Anne 1962. Tome VII. N" 10 t'e s horm ones sont des drivs oxynctiqucs d u noyau benzne et du noyau napbtaiciic des acides pbcnyl, nnphlyi et iiidoluctiquc. propionique et hutyliqiic. ainsi que de certains de (cuis d riv s. 1, ' o.; des p lu s em ploys est l'a c id e 2-4d iclilo ro p h n o x y actiq u c (A c. "2-4-D.i dont l'.iction, p u issa n te et slective, n 'est c e p e n d a n t pas litucc de tout inconvnient ainsi que le prouve l'observa tion suivante. O B S ER V A T IO N : 0 ,, Cuvant. 52 ans. agriculteur, est adress U (.'Unique Neurologique te 30 juillet 1962. par 1 Docteur C n rrro tT pour des rrnuldt-s senxitifs ;( moteurs de* memlnv.s infrieurs. Cet homme. sans anti-relents put hologiques notables, qui n'est pas thylique, situe trs prcisment le dbut de ses troubles au 2 Juin 1962. H ressentit ce jour-ln. brusquement. une douleur vive duns toute la rgion orbitaire et priorbitaire droite, s'attnuant ds le leiulemain (tour taire place 4 des paresthsies et une a-.n-stiisie ncctqiaiit le mn:>- t -rritolre. la* 2 juin galement, il note l'apparition d'une Itvpoestiisie de la jambe droite. Ia> 16 juin, soit deux semaines ans le dbut. la jambe droite a rcupr une sensibilit nor: ale mais l'anesthsie, qui persirto & lit face, a gagn la jambe gauche ; d'autre part. le membre infrieur gauche est - plus faible a et la marche nVst possible qu'avec l'aide d'une canne. Los troubles restent sensiblement identiques Jusqu'au 30 juiller. date de l'entre dans te service. On constate alors la ix-rsistaner d'une liypovsilisie dans le terr.toire de ('ophtalmique droit, avee hytioesrlisie mrneunc ; le malade accu.<e encore dans ce mme territoire quel ques pisodes douloureux trs i-uxsag.-rs et d'int- tisit' modre. Le sujet se dit incertain de sou nnml.iv infrieur gauche au niveau duquel ou constate une lg...- dimi nution -- globale -- de la force srgmotitain*. li n'existe aucun trouble trophique, aucun trouble vaso-moteur. Les rliexrs roMilietts sont plutt vifs tandis que les uchillens sont abolis. Le membre infrieur gauche est le sige d'une hypoe sdisle globale, neule la se nsibilit profonde tant conserve. !<19 531 03937 L V u n r a n cu i -logique ilm uit-gibnn su p rie u rs vle strictem en t n g atif. D'autr- (-art. l'tat gn ral e st e x c e lle n t. I l n'jr a >.>as d 'a lb u m in u rie . U i lorm ui-.angtiine r v le u n e o s in o p h ilie <S' 2 aot .-t 9 , le 30 a o t). Deux e x a m e n s l-rli-np|inr*iitue>. sa n g u in s . & p lu s ie u r s s e m a in e s d 'in t e r v a lle . mont t i nt tin* hait** lg re de l'a lb u m in e (13 put. 46 g ra m m e s) h une au g m e n ta tio n les alphu-2-glohulint-a (13 p u is l g ra m m e s par litre). L f b ila n b ioln iiiq ui1 r n a l, la u aln rtoxiirn - prvH|ue, les ractio n s il ilnciilatinti ne m ontrant pas d'anom alie*. L ' le c tro c a rrilo R ra n im e p s i n o rm a l. 11 pn pat de m m e du fond dVcii et dp l'E .E .G . L'lecirm lia g n o stic donne les renseignem ents suivan ts : -- p re u v e dp s tim u la tio n du 3 aot 1!*62 : in t g rit dp* m em bres su p rie u rs ; au x m em bres in f rie u rs, il n'y a (tas de ra ctio n de ralentissem ent vidente m a is les lgers trouilles de l'excitabilit (hyperexcitabilit g a lv a n iq u e h y p o e x cita b ilit g alvan iq u e et farad iq u e le l'exten seu r propre du Rros o rteil gauche - ebronuxies leves des extensi tirs du sro s orteil c a n d ie ) tntoiRnent cependant d'une ltcre a tte in te ueurognc (D o cteu r t jr x ) : -- iertrom yogm phie des deux jam bire antrieurs du 4 a o t 1963 ( D o r t e u r I I a m i x ) : tra c d 'a tte in te neuroRne di*a d eu x m u s c le s e x p lo r s. L e m a la d e q u itte la c lin iq u e le S sep tem b re 1963 : d urant l'hospitalisation il a t soum is une injection q u o tid ien n e de 2nn niK. de v ita m in e B 1 et 2.'0 m g.. de vitam ine B 6 ; en outre.' l'tiydrocobalam lne lui a t a d m in is t r e & la dose de 5.utt R a n im a s tous les* tr o is jou rs. L e s algies faciales ont pratiquem ent d isp aru ; les troubles sen sitifs d ficitaires de la face et du m embre in f rie u r gauche se sont notablem ent attnus ; le dficit m oteur a d isp aru et la m arch e est redevenue n o rm a le; Parflexie achillenne iiersiste ; aucune atrophie ne s'est d e ssin e . 11 p e rs is t e d es s ig n e s le ctro m y o g ra p h iq u e s d'a tte in te neurogne du jam h ie r a n trieu r droit (l'exam en n'a pas t pratiqu du ct gauche). P a r contre, les rponses 1 l' lectro d iag n o stic de stim u la tio n , en date du 28 aot, sont absolum ent norm ales. L 'tio lo R ie de cette p o ly n v rite des m em bres in t rie u rs avec atteinte irritu tiv e et dficitaire triKm ellnire droite n o u s a t su g g r e p a r le m a la d e lui-m m e q u i n 'a pas m anqu de fa ire le rapprochem ent en tre le dbut de ses tro u b le s e t u n e o p ra tio n de d sherb ag e e ffectu e te 1 " Ju in et les d e rn ie rs jo u rs de m ai 1963. Celle-ci a co nsist en une p u lvrisatio n de deux solutio ns diff re n te s m a is toutes d eu x & base d'acide 2-4-D. et en co n te n a n t 235 et 410 g ra m m e s p a r litre . E l l e s 'e st effec tue g rce un tra cte u r qui rem orque le p u lvrisateur et q ui est surm ont d'une cabine, ouverte l'arrire ; or, les o pratio n s se sont droul--* par vent violent et ce lu i-ci ra b a tta it d a n s la ca b in e le nuug-- le su b sta n ce toxique. 1050 C e s circonstances exceptionnelles, la longue dure d'exposition au toxique, nous ont fait adni-ttre qu'il s'agissait vraisem blablem ent d'une polynvrite due 5 l'acide 2-i-D. A it reste, eeite nlisi-rvatiuti n Y-d pus isole ilnu s la l i t t r a t u r e ip ti m m p o r t o u n c e r t a i n tn m il.ro le fa its d 'in to x icatio n p a r ecs sub stances, notam m ent e lle * d e s a g r ic u lt e u r s : ipn-lipp-s h e u re s p r is l'in h a la tio n ilu to xiq u e s u rv ie n n e n t lia h itiu ili-m oni les v o m is s e m e n t s , p u is 1rs t r o u b le s s e n s it i f s s u b j e c t i f s OH t y p tle p a r e s t h s ie s o u m m o h* i lo u le u r s . p u is le s t r o u b le s m o t e u r s , l'a t t e in t ' p n r a ly t j t p i c jm>u v a n t in t re s s e r b-s p iatro in i-n ih rcx nu h's membre; ^ in f r ie u r s . iV t t c |M ilynvrit* M -nsitivo -n io triee -- ^ in r p n s t u n im c n t nssoeii* un* n ib u m i n u r i e ( u n t a b le a u d 'e n t r o c o lit e -- s- r v le h a b it u e lle m e n t ^ i r r v e r s i b l e ,u t r s le n t e m e n t e t t r s p a r t ie lle m e n t rversib le. I.a itixieif 1* s stilista n ees est galem ent prouvs' par b-s cas l'in to x ica tio n ch ez le s ouvriers ra v a illa n t la fab rication les b orm oues vgtales: la |MMitrutiM lu to x h tu e s' ferait p ar iubalati'in. m ais au ssi par vi* -ulanc ; b-s o u v rie rs se plai g n en t sou ven t le som n olen ce, tle se n sa tio n s le lurl*ur <l*s m em bres in f rieu rs, d e g a stra lg ie* . l'ano re x ie e t riiy p ersia lo rrh e. a in si pie d `un<* liyjM-rse n sih ilit le l'o u e : to u s *es trou b les s u it , lans In rgi*, fugiHi's. K nlin. xprim'iitab'mi`n t. l'aeide 'J-4-IX. alm inistr par voie in tr a v ein e u se ni mme par voie orale, mut en tran er lu mort : celle-ci survient. soit dans un tableau neurologiipic sous fo rm e d e com a a v ec h y p e rto n ie les ex trm its. so it p a r Hlirillation v en tric u la ire . *t ce p our des doses relativem ent leves, m ais videm m ent varialles su iv a n t l'a n im a l en expri-nce (de l'o rtlrc de 300 m g. un gramme en line p rise). A notre con naissance. il n'a pas t effectu d 'intoxication subaigu. ta B IB LIO G R A P H IE A x m ic i.y M. -- D sh e rb a n te s le c tifs et su b sta n ce s d* croissance. A peru technique. E ffe t pathologique sur l'hom m e au coure de la fab ricatio n de l'ester du 2-4-D. .1r e h . 3 M . P m f ,, 1951. 1. 26. Go i.s i k i x N .P .. J*SKj P .H . an d B k iiw s J .R . -- P o ly n v rite a p r s e x p o sitio n un e s te r de l'acid e 2-4-D. I n .Irr/. .l/if. /*r/,, i9 6 0 , p. 384 (a n a ly s e ). H tt.i. E V . and C a s i. i .*i.k A . -- T o x lc it y of 2-4-D. for experi mental anim ais. J o u r, lu ilim t. H uo. un J Tnxir. 1947. S. 2, 85-95. L hostk I. -- I.?x P a r is . 1958. * r h niif/H' i. 0 It-S.T-fi.M-. L i l l e M dical. 3" S r ie . A n n e 1962. To m e V I I , N" 10 5313 0003938 itm M>.v s a <; c i D i V i n i C . -- S u r l'in to x ic a tio n alxui* p ar un dfeii'-rban t. 1'aciile 2-l-D. C o n tribu tio n ctlu iq u e. f ' t l i 'i M n lic tt. 1361. | | . 6. :S0-!S5. Ti*.\ Al.S. -- -Ac -suj*-t it** ^herbtm r;lectifs. A n h. X'tl. 1951. 1. 2C-d. t lu. . ii:k> M .K. and Ni:im.\ ll.T. -- Effe-ts of 2-l-lich!' -i. p!n-no.\yai:**ilc acid on chicks a`ci> 194$. i73-4S'i. Les -mpto-s. < ucricuitOTA d*-s dsbrrbanlT slicilis l <ls n:Kiil;u'isa'r>ui vgtation. A yriculttm - y n u t \ ' t i \ i \ 1356. 19. 176. 45-3*. DOW \ 714164 m e x -s u i t e ,i ai e m sd s M oxorym uEM :* de l a d il t e . r x cas de Pierre W'akot. -bn-ipies IH x v x . Ren Lj-xuik et Michel Duuuiihs. L on gtem p s con sid re com m e l 'a p a n a g e p resqu e e x c lu s if le l'a n im a l, Jr in fe c tio n list ric n n o sem b le m* m a n ifeste r ch ez ("homme a v ec u n e frq u en ce rue : d'aprs Bi_VA^yr et SnuiEU, TO cas seule- nient le listriose lutmuVnc avaient t rapportes avant tandis pu* .`liai nouveaux eus l'taient K* lilti lilT. \ Sans lotit- I* ruiun>stic\ bactriologique est-il fait plus souvent pt'autrcfolsY o u passe souvent *t lu diagnostic ImetrioIieaSque paire que l'on Varie trop vite. sus l'ap p ellatiu V d c facilit de eoutaiiiiiiants diphtriniorphcs ilc-Ttau-illes ('nuit positif isls d'hetiiocitltures, le liqUiHe cphaloraeliidien ou le tout autiv produit e t \ i u i vint l'autlienthiues listt ia ( L . u 'i :y w o x s i k ) . \ l a i s il est trs vraisetulilalile aussi qui- la diffusion, \eeim nue, le nuf.-eij.iti dans le rgne animal oiilflune la niulliplieatioti les transmissions acvidentellcai l'homme. Il est rare pie le eliu ieie u fasse le d ia g n o stic le list rio se i-t en d em a n d e eo n lir m a tio n a u bio logiste : rien l'tonnant cela si l'on considre qu'il s'agit .1'une infectiou de connaissance rcente et dont la frquence est d ailleu rs toute rela tiv e: la littra tu re fran aise n e eom portait en effet, en 19."f). u u e i l o b serv a tio n s. D 'a u tr e p art, les listrios-s se p rsen te n t so u s d es a sp e c ts e |in iq u e s e x tir m e m e n t livcts : l'im p o r ta m e sta tist p ie le S i:i.ini:kii. [ x n ta n t s u r les .`144 ea s p u b lic s laits le inonde en l!)(. relve USD ta ts scp tiru p yoh cm iip n -s lu iiiiuvi-au-u. 100 m cn iiig itv s p u r u lc u le s avis* ou sa n s e n c p h a lite , 41 form es sa n g u in e s ty p e le inonotiui-lose iiifeetietise. Kl e o n ju m tiv ite s g ra n u lo m ateu ses. le s a u tr e s -as i*neeruuut les lo ca li sa tio n s v isc r a les liverv-s : <-iulo*ai`d lte s. m yoearlit-s. liu 'lrites, -le... Tout au plus pi'ut-ou parfois souponner l 'ori* gin* listcrieune d'un tat septicopyohiniiuc du nouveau-n. ou l'une mningite purulente de IVnrant m de l 'adulte, parce qu'il s'ugit l des manifestations les plus frequentes de l'infection. .Mais, le plus souvent, le mrite du liagnostie revient au seul baetriologistc... et ce n'est pl'a posieriori, lorstpie l> germe est identifi, jue le elinieien mettra l'accent sur (uel.ptes nuances smii>l>gi<pios pii auraient lu faire voquer lu listriose, tuais dont la signification lui avait vita pp. ("est ainsi pie les cltoses se sont puss':es luns l'oltservation que nous rapiHtrtons d'une mnin gite purulente de l'a.lullc, inquitante IVinhli1 par l `intensit les troubles de la conscience et lu drglement netint-vgiatif : s* fut mit: sur prise d'apprendre pic le germe ivsi*oiisablv en tait listria monocytognc. KnsERVATIOX. patho-aK . L o n . 60 an s. m ca n icien , s a n s an tcd en t loK iiiuiN oeiab l', p rsente, le 17-3-1962. tr s b ru talem en t, avecv e rs 12 h e u re s, un tat in fe ctie u x d 'a llu re g rip p a le , fivre 3 \ co urb ures, m a la ise g n ral, q u i l'oblige s 'a lite r. C \ troubles s 'intensifient les Jo u rs su iv a n ts tandis q u'appai\lssen t cphales de plus en plus intenses t v o m isse m e n ts! L e 22 m a rs `\ co nstate une ra ille u r tnniiute et n u iiaiie 'st a ii -:V la C lin iq u e N-urologique. I l est ad m is au dbut de l\ ir s -n d d i. L 'u n l'entre nous cons tate a lo rs un s y n d r o n \ m ning fra n c , un ta t d'obnu bilation intellectuelle ^ e c lgre ag itation psycho am o trice : la tem p rature ew 39*5 : le m alaie un hoinet pci-niniu-nt ; IV xam eh neurologique ne rvle aiu-un s ic n e p a riic u lie r. L e liquide rach id ien se rvle L il l e .Mdical. S r ie . A im e 13i2. T o m e V i t . X " ! 5314 lOfl 0003939 5315 ir. J . PhjritL 1M, Pathophysiologia An*-:*- PhyrioL, l Arch. 1 . Physiol. -LX u l , A .: A cu peti) 19,287 (1961)jj* > NEW EVIDENCE CONCERNING THE NERVOUS SITE OF ACTION OF A CHEMICAL HERBICIDE CAUSING PROFESSIONAL INTOXICATION By I. D ia l, J . S6s cod I. N ik o u t s INSTITUT*or FAXHOFHY3IOLOCY, USKAL OWITMOnT. StJDAPCSr (Received February 10, 1962) Tba effect of 2,4-dkhlorophenoxy-acetie ad d on tba electroencephalogram ha* been studied in thyroidectom isedand thyroxine-fubitituted cats.Adeerea*e of frequency and an increase of am plitude have been found to result as early as 24 hours after tba onset o t treatm ent. After 5 day, 2 to 3 Ha, 180 to 190 ftV, big, slow, toxic waves pro* dominated. Desynchronisation in response to stim ulation of the reticular formation was significantly shortened, or even extinguished. The prim ary site of action of DCPA is thought to bo in the oerebral cortex. The disturbances of thyroid and cardiac activity were ascribed to lesions in various subcortical centres. The noxious effects of 2,4-dichIorophenoxy-acetic add have been under detailed study at our Institute [5, 6, 13, 14], in view of their significance in labour hygiene. Dichlorophenoxy-acetie add (in the following DCPA) is a ted killer used in increasing amounts mainly in corn fields. It has been shown by us th at chronic treatm ent with DCPA caused very dow, 2 to 3 Ha, high amplitude waves to appear on the electroencephalogram, and poisoning with the substance induced disturbances [S, 6] in. thyroid fraction [13]. As changes in thyroid function are known to produce eleetroeneephalo* paphic changes [8], the question arose whether DCPA damaged the nervous ayitem directly, or the changes observed in the EEG were due to an action a thyroid function. This problem has been investigated in the present experiments. Si 1 ** i A tu tal of 15 c ita , 11 esperimenti! sud 4 contro!*, weighiag L S t * U k |i ero a n d , ffcs ttectricsl aedvity of th centrai aervous System was studied la eneh animai by maona ri 2 frontal aad 2 oceipital orticai built-ia eketrodee, as watt aa bipolar sabaortieul abetrodee yrissd hi th maaoacephattc rotieular formatimi. To placo th eortkal locaoda*, opeaags { s a in diaaMter had beau cut la thcalvarium and tho stivar alactrodas w on ploeod on tao j^ a . To iasart th subcordcal eleetrodaa, aa opaning 4 mm la diamatar was mde, th dura m inristifi tho silver eloetrodes aa d asod in a class tuba wero iaaertod iute tho rotieular for- i by maana of a sterootaetle apparata*. The coordinatee were detarudaad ocoorttag to gs--vlcornAi [9] A itar i : tho' riectrodss a fibria sponge waa placed la the drill boia, I*haarittr of the deep was eontroQed' hiatolagiraily aitar tba experiment. 35' 74 L DtU, J. SdS saS L 1UK0LR3 Tk Iwtw J ii v m w w t r i in place w ith acrylate. A fter EEC tracings had basn recorded from the otherwise intact cats, they wets jeeted to thyroidectom y under aseptic conditions. Sahsaqnantly, the controls wars not treats* in any way. The experim ental 1 wore given 100 pg/kg thyroxine (Hoffman*--La Rack*! daily. Wo than stadiad for 10 days tha experim ental group to determine w hether changes a the EEC would occur. No appreciable changes having been noted, from the 11th day oa u* experim ental animals were given 100 mg/kg of DCPA daily. Tha m aterial was obtained aft** recrystallising three tim es the commercial preparation. Using a 4-channel EEC apparatus, tha spoutaneona electrical activity of tha eoetss and reticular form ation was m ooned daily. We also stadiad the changes in tha duration of cortical desynchronisation in rsspesw to th o electrical stim ulation of the reticular form ation. Tho apparatus need for stim ulation em itted rectangular impulses, w ith tha paramstn* V and 1-5 V, frequency 300 Ha, duration 3 msec. T otal duration of stim ulation was 5 *** Considering th a t tho stim ulations w en applied once a day, no adaptation could infleeo* th a resp onses according to d ata in tha literature [2, 4]. The cardiac rata was determined daily. Result The results. obtained m ay be outlined aa follows. In the control groop th e pro-thyroidectomy avenge frequency of 6 to 7 Ha decreased 24 hoot* after thyroidectomy and remained a t a low level throughout. In tho experimental group no appreciable change in frequency ws* < iw noted after thyroidectomy. On the 10th day after thyroidectomy the frequency ranged from 5-5 to i i H i, aa compared with the pre-thyroidectomy value* of 6 to 7 Ha. On the other hand, frequency decreased 24hours following the administra tion of DCPA. and after 5 days it was as low aa 2 or 3 Ha (Fig. 1). .13 1 Fig. 2. Changes in the frequency of spontaneous cerebral electrical activity in the eontrd and the experim ental group Arrow: thyreideetom y. Double arrow: onset of DCPA treatm ent 5317 0006317  momna s n or action or a num i 75 , ihey were mb* Mata not treats* n n - U M ,J ether cbaagM tr1 |t 11th dmy ho. ,(obtained titer- Similar change* were in the amplitude of the wave* (Fig. 2). In the control group the 60 to 70fiV amplitude* slightlyiacreasedafter thyroidectomy, then remained a t the 90 to 100 /tV level throughout. In the experimental animal* the initial value* were the tame and 10 day* efttr thyroidectomy they were till at the 60 ftV level. Uthe peremeten ilation wee 5 tee. acould inflneaee < DOW 1249364 control group' sated 24 hour* frequency tn t] f the frequencya lectomy valuesj thetdminiatra^ Fig. 1). tivity ia the etinent Fit.2. Change* ia tba am plitude of pontaneoua earahral tU ctrieal a c tin tr, Sign* a ia Fig. 1 O rdiaata: am plitude ia /V. Ahadaa: day* Twenty-four hour* after the onset of DCPA treatment the amplitude hneoed parallely with the decrease of frequency. On the third day the average amplitude was 100 /jV, and by the fifth day it reached value* around 180 la 190 In the G tracings of the controls (Fig. 3), 3 days after thyroidectomy lin t was a significant slowing down (Fig. 3/II), as related to the initial value {fig, 3/1). The rhythm was still slower 11 disys (Fig. 3/111) and 15 days (Fig. jfpT} following thyroidectomy. l( In the experimental animal*- (Fig. 4) there was practically no EEG ,jnge after thyroidectomy (Fig. 4/II), as compared with the pre-thyroidectomy :Bsdag* (Fig. 4/1).Twenty-four hours following the onset of DCPAadministra* :jjgg frequency decreased and the amplitude increased a t sites (Fig. 4/IU ). | flax 5 days the tracings displayed exclusively big, slow,- toxic wave* (Fig. 4/IV). The duration of desynchronisation in response to stimulation of the formation showed characteristic changes (Fig. 5, Fig. 6); the value |(gfrmmd before thyroidectomy was taken to he 100 per cent, later deviations item calculated on th a t basis. ,0006318 , 5318 7 x. otu. j. ads a t l mxoLiTs la the control group the duration of desynchronisation rapidly decreased after thyroidectomy From the 6th i i y on no desynchronization resulted response to stimulation with 0.2 Y. From the first day on the duration of desynchronisation elicited by stimulation with 1.5 V was markedly reduced* lX u m ss) as fi 2-7 7-0 m s-9 /w F{f. 3. EEC r f control gronp. Diagram: cita and eonnaxion batwcan dectrodas. Nuiw--b* No. of r g ia dectrodas. FR : reticolar form ation load L P rio r to thyroideetom y, aorm ai a ctirity . IL 3 days a itar thyroideetomy. Storia* Increata e f ampUtode, dyirhythm ic corra. IO . 11 days aitar thyroidoctomy, and IV. days a ita r thyroideetom y: slowiag and dysrhythm ia slightly iacreaaad In the experimental group desynchronisation time was reduced by 16 per cent on stimulation a t 0 2 Y 10 days after thyroidectomy. The duration m desynchronisation decreased rapidly on DCPA administration, after 24 hours by 50 per cent already, and on the fifth day no desynchronisation at all resulted on stimulation at 0.2 Y. E nvois s m o r A ction o r a chkmical h cubicio 77 Tea days after thyroidectomy the desynchronisation elicited by 1.5 V ^imnUtioa was reduced by 13 per cent. The duration ,of desynchronisation rspidly decreased in response to DCPA, to 45 per.*ent-by the Sth day. r DOW 1 249366 7-3 Ffta-a 3-2 B 2- 7 7-3 FBB-9 t--/****--*. wAAAwyVAsn % ,,iV y , >n 3- 2 tv 2- 7 7-3 ra e-a 3- 2 Fig . 4. EEG of experim ental prm p. Si$ns u in F lf. 3 r N otasi activity before thyroidectom y. IL Normal activity 10 days after thyroidectom y in 24 boars after onset of DCPA treatm ent. Slowing and a t sites increase of am plitude. [Y,5days after onset of DCPA treatm ent. Exdnaively big, alow, toxic delta w ares are risible Changes have been noted in the cardiac rate, too (Fig. 7). In tbe control poop thyroidectomy waa followed by a gradual decrease, in the experimental paap the heart rate decreased by 5 per cent in 10 days after thyroidectomy, following the onset of DCPA treatm ent the decrease was 25 per cent after the Hist day and on the 5th day it was 36 per cent. C006S2.0 : 5320 K 78 l o t s . u d i a i i m xou n \ \ * \ ttpatmmM 02 V m ini 02V 13V 13V Accord; ^Identical in t i(n d ia anixna j.' The apf definitely in ^together witi marked | f ? J * 5 7 J 0 JOjjff 12 H IS Fig. S. C k tu it la tlM do tation o f to rd o l daayaehroaisatioa a f a r lactrieal adwdrd** of tk * M k u v form ation. Graphical representation. D eration M a n thyroid******* 100 M se a t. Dtffaranca from this g trsa la par eant. Arrow: thyroidectomy. D"** arrow: oaaot of 0C ?A traatm ant r: *] i 1 m 1X)* I |J h . .'H 'I'll, i.'i r Fig . 6. Changes la th s deration of w r i n l deaynchroniaatioa foOowiag adm olatioa of tb* rooealar - fbrm atioa :w ith 13 V. Arrow: tho C ut, low-ampiitnde deaynehroonati** aoaaaa aad tho baaal aetM ty reappear. ......... L : Experim ental groap. II: Control groan. 1/1, _ J,,. M o n thyroidectom y. 1/2: Ton day* a f a r thyroidectom y. Doaynchum liatioa tim e hardly ahortaaod. 1/3: S days a fa r aaaat DCPA troatm eat. D uration of dm yachraaiiation strongly reduced. H/2: $ day*, n/3 1 day*, a f a r thyroidaetom y. Darmtioa of daayachreaiaatloa strongly radacad Compa the dixninnti In the lowing of fro `iJ w tr fit we literatur During ht the exper l |. The cfa treatm ent pi Thu, i with intact The ch M severe as ' 0006521 5321 nacrous m or action or a cbodcal a a m ia s s 79 D iscussion According to the above data, the EEG cbangea induced by DCFA w en iitnticsl in thyroidectomised animals maintained with thyroid sobstitationi in -""1 with intact thyroid [5, 6]. The appearance of 2 to 3 H*, over 100 ftV, big, alow waves (which are definitely in the pathological zone) is indicative of a severe toxic state, and together with the shortening of the duration of desynchronisation it points is a marked reduction of central nervous excitability. D o n 249368 Comparison with controls has made it clear th at the slowing down and d i diminution of excitability were not due to thyroidectomy. hi the-control group thyroidectomy was immediately followed by a ||tW>g offrequency, an increase of amplitude and a diminution of excitability. Q m fit well into the pattern of the EEG in hypothyroidism, as reported in literature [8]. ' During the 10 days of thyroid substitution these changes did not occur gthe experimental animals, except for a slight, not significant slowing down. The changes clearly observable as soon as 24 hours after starting DCPA mitui--* prove th a t they had been produced exclusively by that treatm ent. Thus, it may be assumed th at the EEG changes noted in the animals jjtb intact thyroid were not due to alterations in thyroid function. The changes in the reticular formation, as determined by EEG, were just g severe ss those in th e cortex. The cortical desynchronisation in response 0G06G22 r ! 5322 <f 80 L DtSZ. J. SOS aai L RKOUIS to the stim ulation of the reticular formation was shortened in durmtioo, i disappeared altogether. According to data-in the literature [1, 7 ,1 1 ,1 2 ], when stimuli espaU* : of eliciting- cortical desynchronisation lose this ability, it is usually due to a [ functional block in the reticular formation. Thus, our findings may be expl*^ \ by a damage to the structures of the reticular formation. Accordingly DGFA seems to produce lesions at the cortical and **fr* cortical level in the central nervous system. I t may be assumed that the nertoai site of action is the primary one, as the nervous symptoms appear soce* j than the symptoms indicative o f a disturbance of thyroid function. It be surmised th at subcortical centres other than the reticular formation ** also damaged, and their lesion may explain the disturbance of thyroid funeaen* Lesion to subcortical centres may also be responsible for the redact* in heart rate. Under th e experimental conditions employed it cannot be ruled * completely th a t in DCPA poisoning the organism's thyroxine require*#"1 increases and thus what we have on hand is a relative hypothyroidism. I t can he brought up against this view among others that the 15 days, of total thyroidectomy in the control group produced much slighter change# than did the few days of DCPA treatm ent in the experimental group. I t cannot be ruled out with certainty, either, that the EEC patter" was influenced b y changes in the function of other organs. Nevertheless, on the basis of the results obtained it is believed that DCPA acts primarily on the central nervous system, paralysing its function. Tk* functional disorders in different organs are merely secondary to the ceatr* nervous effect. LXTEBATUBE L A ssa m , A , A ssa m . M. G: J. ta p . Thee, l i t , 76 (1954). 2. Bbsmt, 0 .t J. PhyaJoL (Paris) 33, 153 (1*58). 3. B oasis, i t HoaksvIiMSM 4, 10 (1953). i B aastav, P. B* B ar, B. J.: h.dia.NnropkyrioL13, 97 (1951). _ 5. D in , L, 94s, J,, Otass, J,, SOu, F -,-----u--s--e-,--V---.i--A--r-e-f-c-.--m---v-i-r-o-n--m---.-H---l-th--.--A- _ 4. DAm, L, 94s, J* Ouaa, J,, S flts, F,, M issus, V.i Ef4aaa4ftadom6ay 4,43 (1945). 7. F a s n o ,J . D., Vnasaano, 1L, M asocn, H. Ateh. N ovel. PsyeUat. (CHa.)49,H\ (1953). L June, H.I Haadbeek dar iaa. Had.: Bd. V. (Nevologis L) 1214. 9priaar...BBaarrff*s 15* 9. KovAcn, A t A kMriad orvostodoniay viaa4U H iaafsi. Akadfaniai Had*, pwts 19S7# YoL & St. 10o H u m ' P. Go, MztgduTj . V .; Bot. Gm . IK , 224. (1944). 1L K oaosa, G^.Maoooir, E. W.i Eloetrooaeaph. eUa. NsvopkyrioL 1, 455 (1949). 12. Pdaaslss, J^ .9ouoen, L: KbfeL Onroatod. 13, 613 (1961). 13. 34s, J,, K sarai, P.: AeU physioL knag. (Bndapost) 14, 367 (1958). 14. 94s, J,, D ial, L, Kaarar, P , O tJM , J.i Medical Congress Sopcoo, I960. H14s DAsi, Jdssef Sda, iionn N txours, Orvostudomdnyi Egyetem Kdrdlettaiii Intdzete, Budapest, D C , Hdgyes Endt# u. 9. 0G06323 5323 3 62 5 0 r w ^ u ^ n c n * w u u c; JOURNAL COOEa LS HOTOCOPY REQUEST FOR JO U R N A L ARTICLES PLEASE LIST ONLY ONE ARTICLE PER SHEET. 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V>72* U iU tdlT tl]] Q SOURCE: m l * ARTICLE KNOT IN G U S H . Da r*a M il-a*#t#a<JarW 0 YES 0 NO 'COPYRIGHT RESTRICTIONS PROHIBIT PHOTOCOPYING. ^ ^ ^ --mmmmmmwm^ mm^ ^ mM H ^ M iM INDICATE REASON FOR PHOTOCOPY REQUEST: ^ ^ research 0 OTHER (SpacUr). OGbp-4 5325 5326 0003129 DOW 526671 I . TOXICOLOGICAL PROPERTIES OP TORDON* Biochemical Research LabojcafcEX The Dow Chemical Compan y __ C O N T A C T S Midland, Michigan T f i X A U L S E C i & L i T O jSX JJ aa nn uu aa rr yy 15, ip, 1i99603j COOTNHFIEDREWNITSIEAL INFORMATION SUMMARY AND CONCLUSIONS t h b D O W CHEMICAL COMPANY Acute oral toxicity studies were conducted on several species of a n i m a l s . In each case the material was f e d b y single dose' Intuba tion as a suspension or slurry In corn oil. A summary of the acute oral toxicity data appears below: Animal 100 Survival g.Ag. 100 Fatal g.Ag. LD,, 50 g.Ag. Chick (M) 3.98 7.95 approx. 6 Mouse (P) 1.00 7.95 between 2 and 4 Rabbit (Mixed sex) 1.0 3.98 approx. 2 Cavy (P) 2 3.98 approx. 3 Rat (F) -- --- 8.2 - range (6.3-10.7) g . A g body weight** ** Calculated by the Well Modification of the Method of Thompson. Undiluted Tordon when applied directly to the conjunctival sac of the laboratory rabbit produced slight to moderate redness of the conjunctiva which subsided between 48 hours and 1 week. The material was essentially nonirritating to the cornea. Tordon was applied to the shaved, abraded and Intact rabbit belly on a repeated, prolonged, confined basis, (continuous contact under a bandage for 11 days to Intact skin and 3 days to abraded skin). Under this conditions of rather drastic exposure, the material produced essentially no skin. Irritation. Tordon was studied for skin absorption properties b y applying It w i t h Dowanol D P M In graduated doses to rabbit skin under an Impervious cuff. Judging by weight gain and general appearance of the animals, Tordon Is not absorbed In acutely toxic amounts. 5327 # M a m o friT* ^ a M a w m B4 - 0003130 C- 14 SUMMARY OF TOXICOLOGICAL DATA Acute Oral Toxicity Animal Preparation Fed Dose 1f g . A g . ) No. Died No. Fed Response-Remarks Chick Chick Capsules Capsules 3.98 7.95 0/2 3/3 Animals died overnlte. Mouse Mouse Mouse Mouse 20 suspension In corn oil 20# suspension In corn oil 20# suspension in corn oil 20# suspension In c o m oil 1.0 2.0 3.98 7.95 0/2 1/2 1/2 2/2 Animal died 2 days after feeding. Animal died within 2 hours after feeding. Same as above. Rabbit Rabbit Rabbit 39.8# slurry in c o m oil 39.8# slurry In c o m oil 3 9 -856 slurry In c o m oil 1.0 2.0 3.98 0/2 Animals appeared nor mal during and after feeding. 1/2 Animal died 3 days after feeding. 2/2 Same as above. Cavy Cavy 20# suspension In c o m oil 20# suspension In c o m oil 2.0 3.98 0/2 Animals appeared normal during and after feeding. 2/2 One animal died overnlte the other three days later* Rat Rat Rat Rat 39.8 slurry in c o m oil 6.3 39.856 slurry In c o m oil 39.856 slurry In c o m oil 7.95 10.0 39.856 slurry In corn oil 12.6 2/5 1/5 V5 5/5 One animal died 4 days after feeding the other 8 days. Animal died 3 days after feeding. . One animal died 3 days after feeding the other 10 days. Animals okay In one hour but had diarrhea the following day. * Animal was fed 1/2 dose in the morning and 5328 0003131 C- 15 526673 Eye Contact - Rabbit Material Treatment Undiluted Unwashed Undiluted Washed with water No. of Animals 3 3 Response-Remarks Very slightly painful. Slight to moderate conjunctival red ness subsided in 48 hours to one week. Washing enhanced recovery in 2 of the 3 animals tested. o O Skin Contact - Rabbit Material Condition of Skin Undiluted Intact Undiluted Abraded No. of APP1 * 9 3 Site Belly Belly No. of Animals 3 3 Response-Remarks No irritation observed Essentially no irri tation observed. Heal ing appeared to be normal in all cases. Skin Contact Absorption - Rabbit (Cuff Technique) Material Duration of Dose No. Died Exposure (g./kg.) No. Exposed Response-Remarks Undiluted 24 hours 1.0 0/2 Material colored skin reddish-brown, otherwise animals appeared normal during and after exposure. Undiluted 24 hours 2.0 0/2 Same as above. Undiluted 24 hours 3.98 0/3 Exposure was made to Intact skin. Response was essen tially the same as above. Undiluted 24 hours 3.98 0/3 Abrasions were made the full length of the body 1-2 centlmenters apart. Response was-essentially the same as above. NOTE: Material was weighed out for each rabbit and slurried with a small amount (8-9 cc.)of Dowanol DPM (Dipropylene glycol methyl ether). 5329 000313? HI 5330 A!. HEALTH . ' -i .'; / , eiertroItol. Ofic. an.l TreatImrrtici'le In* Health 16 :JJ3, A.: The Efirc of Rat to Health 18:263, nine >n Man, 1*151. J. R.: Un indorcs de un do. Bol. Ofic. mmuniention to P., and Cappel, ratory Animals, Dieldrin Poison:tee on Toxicol- H .: Study of ddcs Gilordan, Industr. Hyg. C : Estudio de ' dieldrin y in en humanos, . 1957. femorandum on h Service, U.S. n and Welfare, ~l - i**v 95 ' Nervous System Effects of a Chemical Herbicide I. DESI. M.D. PROF. J. SOS, M.D. J. OLASZ, M.D. F. SULE AND V. MARKUS BUDAPEST, HUNGARY Thousands o f new compounds are being encou n tered in m an 's env iro nm ent ail over the world. T he biological effects of changes in the chemical environm ent are beginning to appear. Newly synthesized substances are causing different pathological processes. It is necessary, therefore, to become acquainted w ith the dam aging effects of new sub stances on the hum an organism in order to devise protection against them. T he chemical herbicides are one o f a group of the increasingly used com pounds.' Among them, 2,4-dichIorophcnoxy acetic acid (h ereafter designated 2,4-D ) plays an im portant rple. T his compound was synthe sized* in 1944 by Z im m erm an, and its Submitted for |Hillirutiaii June 20, 1%1. From the Institute of Pathophysiology (Direc to r: Prof. J. So*) of the University Medical Sellout, Budapest, Hungary. utilization as a chemical herbicide was pro posed by Mitchell and M arth .' In H ungary it is called D ikonirt and has been marie use of to an increasing degree since 1952.1 Its molecule is sim ilar to that o f tryosinc. It exerts an effect similar to plant hormone. It causes disproportionate grow th and quick death of certain plants. It has a selective effect on different plant species. Among the monocotylcdonous domesticated plants, 2.4- D kills the dicotyledonous w eeds.1-1* In 1946 H ild eb ran d 8 w as of the opinion that peroral 2,4-D had no pathogenic effect on either experimental animals or man. However, not much later B ucher 4 and Hill T found myotonia, m otor disorders, paralyses in the extrem ities, and gastrointestinal symp toms such as vom iting and diarrhea in experimental animals treated with this com pound. O thers observed leukopenia,3 ataxia a n d c o m a 10 a fte r ex perim ental in trap eritoneal adm inistration of large doses of 2.4- D. M ost recently G oldstein6 reported on 3 patients in whom grave sensory and motor symptoms, paresthesias, paralysis, and se vere pain appeared due to the percutaneous effect of 2,4-D. Recovery w as only partial even after years. In H u n g a ry , B o r d a s 1-3 dealt w ith the problem. H e found weight loss and symp toms like those mentioned above in his animals but could not dem onstrate any ob jective changes in w orkers -spraying 2,4 D. " S os et al. n *12 have been ex am in in g the effect of 2,4-D on thyroid function using radioiodine and increased activity ha> been detected. H owever, none of these studies has dealt w ith the problem o f w hether or not 2,4-D has a dam aging effect on the function of the central nervous system. T he present ex periments undertook to study this question. M ethods Thirty-two white male rajs weighing about 240-250 gui., 5 cats, and 2 dogs were used in these experiments. The electrical activity of the central nervous system was examined hy the use of electroencephalograms in acute and chronic 101 D O W 1511217 0 . * < M I 5331 0007167 % J I , I I 4 % *; > -i< . i.- r.t I ';i * ) ij > I 9 A R C H IV E S OF E S V IR O S M E S T A L H E A L T H c\pi**iife<. Twn frontal ami 2 occipital chvtrodc were pl.ieol I the terehral cortex, ami a hii>olar *id>cnrtic:il electrode wa* placed on the reticular format ion. In the acute experiment* the aninialwcrc auc'thcti/cd with 40 mg. <>l pcntuharhilal (Nembutal) per kilogram of holy weight. ami the elcctrolo were placed on the cortex ami into the MiU'ortex after removal of calvaria. In the chronic experiment* the electrode* were lixed to the top of the skull with dental cement and acrilate, and the electroencephalogram* were taken on unancMhrtizcd animals moving without limitatioit. The `pontaneous electrical activity of the animal's hrain as well as response measured in cerebral electric activity to sound and electrical stimuli were recorded. A whistle lasting for 5 sec. was used as the sound stimulus. For the electrical stimulation a s|uare-wave impulse generator was used. The parameters of the square waves were: voltage 1 volt, frequency 100 per second, duration of im pulse 8 msec. The entire duration of the stimu lation was 5 sec. To examine higher nervous activity, conditionedanxiety-reflex experiments were performed with the method elaborated in our Institute.11 Electric shock was applied as the unconditioned and light a* the conditioned stimulus. 2,4-D furnished by r.ord.i- wax administered in its pure form. In the acute experiment* a dose of 200 mg. per kilo gram of l>ody weight was administered intraperi- tou<".dly on a single wcasion. The same amount was given daily in the chronic experiments until the death of the animal*. In the experiments in which cortical application wa* used, some crystalline granule* of the drug were placed upon the exposed cerebral cortex. Tyro-ine was used for the con trols. The brain and spinal marrow of the animals were examined histologically at the end of the experiments. cn Experim ental R esults H* It is well known that if a norm al anim al ^ under complete rest undergoes stim ulation, q q the frequency of cerebral electric activity will immediately rise and the am plitude decrease with resumption of the original activity some time after cessation of the stim u lation. T h is phenom enon is the so-called "desynchronization." A ny chemically active agent which induces the inhibition of cere* f /W e r . --------------- -- I ! ' /v '' Fig. 1.--EEG curve of sleeping ra t Sound stimulus was applied at arrow. A , prior to treat ment. B, thirty min. after injecting 200 mg. per kilogram of body weight of 2,4-D; no desynchronization. C, after 60 minutes; de- B 'l / synchronization appears due to sound stimulus. The drawing of the head shows the location and /^vftSA connection of each electrode. The numbers shown are the number of electrodes. c HERVOUS 0t*ichrmmm- I bral functi of nerve desynchror duration o abolishes i sensory ini neurons ac creases the also a stat other stim I n th e fi? effect of rats was < Fig. 3.--I (left) and t on the cortf electrodes 1electrodcs 1on the sub: afterwards: decrease in electrode* 1 change wlia; utes; norm.-, sumed; form formation. 102 VoL 4, Jan., m 2 Disi rl al. 5332 ' Vf UUW 1511219 f ll.TU ' -I'c .<>- 1 ri- <t was 11 the liicli talline t-XCtl * ci-*n- -limnU t the tiittul ition, tivity iitude al actim ucallcd active cere* Sound treat* 0 mg. D; no -; demuliis. m and imbers . SERYO U S SYSTEM EFFECTS OF HERBICIDE. M nfryw<<i Um 0%m b ** 97 Fiir. 2 .--Effect of electric stimulation on the duration of desynchronization. The abscissa shows time elapsed since the injection of 2,4-D, expressed in minutes. The ordinate shows the duration of desynchronization in sec onds. bral function and decrease in the functiuit of nerve cells slows the spontaneous and desynchronization frequency, shortens the duration of desynchronization, and possibly abolishes it completely. E very chemical or sensory influence causing excitem ent of the neurons accelerates the frequency and de creases the am plitude. D esynchronization is also a state of excitement after one or an other stimulus. In the first series of experim ents the acute effect of the injection of 2,4-D into 8 rats was observed. In untreated animals desynchronization m anifested by response to the sound stimulus could no longer be elicited 10-15 m in. a fte r injection of the drug. T his inhibition lasted 60 min. and was followed by a norm al desynchronization in response to the sound stimulus (Fig. 1). A sim ilar change could be observed due to electrical stimulation of the reticular form a tion (Fig. 2 ). In untreated animals a desynchronization lasting 40 sec. occurred in response to the stimulus. A fter the injec tion o f 2,4-D the duration of desynchroni- 4>-pc Acute Fitr. 3.--EEC effect of 2.4-D (left) anil tyrosine (right) placet! on the cortex: 2,4-D between the electrodes 1-4; tyrosine between the Hcctrodcs 1-2 . //, prior to placing on the substances. B, 25 minutes afterwards; with 2,4-D a marked decrease in amplitude between the electrodes 1-4; with tyrosine no change whatever. C, after 40 min utes; normal electric activity re sumed ; form. rrt. indicates reticular formation. 1 <, i t i .k . * .S' * ; r. M 'P < .I I . . I. 1 ''>fj Disi ft at. t -- -- nr I It 5333 0007169 'Si. $ / 1 I I..\ l I. ' i I l jt I .1 : * .1 ir> i.ii Yv ; I 98 ARCHIVES OE ENVIRONMENTAL HEALTH 7 :i(ii>n a fte r electrical >limulus liecame shorter, and, on the .iserage, at the twentyfilth m inute after injection, dcsynchrouization w as abolished. A fte r the lapse o f 50 min. the resi*onsc could he elicited again. and at the seventieth m inute it reached the d u ra tion characteristic of the period prior to injection of the drug. In the next series of acute experim ents perform ed on S rats and 1 cat, some g ran ules o f crystalline 2,4-D and, in the controls, crystalline granules of tyrosine were placed directly on the cortex. In response to 2,4-D the E E G curves 10-15 min. a fte r application of the d rug showed a m arked decrease in am plitude and an increase in frequency. T he change dem onstrated itself only locally between the 2 electrodes between which the 2,4-D had been placed. A t other sites of the cerebral cortex and in the reticular form ation there w as no change in electrical activity. The am plitude resum ed its original size afte r 33 m in. on the average. T yrosine, applied in the controls, induced no change (Fig. 3 ). In the next expnm ental scries chronic expei imeiiis weie pel formed on S rats, 4 eats, and 2 dogs. D uring the chronic experiments the fre quency of spontaneous electric activity showed a steady slowing. Instead of the waves having an average frequency of 7 per second as prior to treatment, there were large slow ...ives of 2 -per-secund frequency on the tilth day after injection. T he quick waves appearing with desynchronization slowed dow n from 12.5 to 4.5 p e r second by the fourth day. Also the duration of desynchronization was moderated day after day. l 'rio r to treatm ent it averaged 32 sec. A fte r the lapse o f 24 hr. the duration w as only 21.2 sec., a 34.2% decrease. O n the second day of treatment the duration of desynchronization was 14 sec., a 54.5% d e crease. O n the third day the duration of desynchronization was 6 sec., an 81.2% de crease. O n the fourth day this duration was only 4.1 sec., a 91.4% decrease. O n the fifth day there was no desynchronization at all in response to the sound stim ulus (F igs. 4 and 5). 'CHRONIC CAI B. >r; ;> Ooyt Fig. 4.--A, slowing of spontaneous and desynchronization iref|uciicy in chronic experiment; abscissa is number of days; ordinata is fmiticncy per second. II, decrease in the duration of desynchronization. On tiic fifth day no doynchroui/.atMiii is shown at all. Abscissa shows num ber of days. Ordinata shows duration of doynchrom/.atiuii in seconds. Arrow marks onset of treatment. ^ 101 Voi. 4. Jan., 1062 NERVOUS Si r t r. 1 Bwf *i.*i' w ;;c {+-**,**' i ! *1t } M t *> Ijf* I V vi \ i L-4L-- OX Jt o too ai * a 0 * 2 Fig. 6.--; O tti et al. 0007170 5334 I 17 ST.h't'V U S S V S T I ' . M l . l / l . l l s Of- l l l . HI UCI Pf i c Ww'Vf. ' 4 99 A ,Vl,M l ^ 'ivy. itlfW; *l\ ,. v Vi,,-'1^r wjU'''v'*/ *-- '- w ^ - j V ' , ,v " '/^W v ^ ? A ^ v j ' . - r . v V / ;"/ J \ ! ^/'l I Z jV'i --* w/_~''u--' 1V.J/ j*tf- j-1l; 5 - I:.I<ctriHticcpIuluurani of c:i( -iibjrrt d to chronic trrntmrnt. 1it 'imi.ilii'ii- nrc as in tin- former c.i'f-i. Arrow mark- -mind stim ulus. ./. st-ntam<m-. activity prior to treatment. normal wave o f 7- pcr-second frii|ii<;nry. I I , dcsyncltront/.tliiii inanitcslim: it-rlf tliiR to sound stimulus, prior to treat ment; duration: 32 see. The entire duration cannot be marked on the diagram. Frequency is 12.5 i>cr second. C, spontaneous activity on the fourth day of treatment; large, slow waves of 2 -pcr-ccnnd fre quency. D, desynchronization due to sound stimulus on the fourth day of treatment, significant shortening in duration. Frequency is 6 per second. Double arrow marks end of desynchronization. [ h^/'i \ I *> * -- i"',r ft ^. I -- i____-- i-- I-- I- I-- I-- I I I . . I, , < I * .5 * I .t 0 r ' 7 CONDITIONED REFLEXES Manbar cf camtorad nfkmc 4 i * a 0 a s i 0 ~ Y I a 3 * J T VC h a rt at tra rtm a n t Fig. 6.--Number of responses prcipitable with conditioned stimuli in chronic experiment. Dili el ai. 105 53 0007171 UUW 1511221 . s* .v' i 100 i Carace par n w U arc i u r n s o r l m t k o x m l n t a l iila ltu NERVOUS rats was 2 incut. The sixth day. H isto lo g grey or w ci animals. A m yelinizati M portion o m ey er's st showed dc dal tract dach bund lx :i * i. rats were examined in relation to the effect conditioned-reflex response could be elicited o f 2,4-D on higher nervous activity, using at all (F ig. 6 ). a conditioned-anxiety-reflex method. T he rats in the chronic experim ents had T he response to conditioned stimuli of daily determ inations of cardiac output. It l.-? ex perim ental anim als u n d e r continuous decreased by 6 % on the first day and 35.5% '.< ! . treatm ent with 2,4-D w as decreased day by the sixth day (F ig. 7 ). I v. by day despite reinforcem ents given with O ther sym ptom s m anifesting themselves the unconditioned stim ulus every day. The were found to correspond to those mentioned decrease in the num ber o f conditioned-reflex in the literature. These alterations, however, responses was 21% by 24 hr., 58% by the were seen only on the third o r fourth day I It second day, 63% by th e th ird day, an d 79% o f treatm en t. T h e average w eight loss o f the Fig. 8.--Dorsal seg il ment of the spinal mar i-J row of rat subjected to chronic treatment (high magnification) ; demye- linization detectable. 106 Voi. 4. Jan.. 1963 In eval necessary when 2,4toneally f: cortical a; experim ent ascertain v the m etab organism > 2.4- D c brought at quency an in the cou cells. T yro chem ically, ever. T hui specific. Si: w hen place very likely and n o t sor precipitatec the d ru g 's 2.4- D a exerted an acute EEG ished the r cortex and this blockii min. after min., it ma; due to 2,4-i stream, par with a d c 2,4-D, non: Even on onset o f th n f t i et aL 5336 0007172 r it. jflisILTIi 9 ttyt fifth day, no m id be elicited tpcrim ents had liac output. It day and 35.5 % ing themselves sc mentioned tions, however, o r fourth day ight loss of the st-r: vv O rJ -v t i .* ' . s ' yV,*' I'ot. 4. Jan., 1962 S r .K l'OUS SVSTF.M EFFECTS OF ULRMCtUH 101 rats was 23% in the cour>e of the experi ment. T he anim als generally died on the sixth day. H istology failed to reveal changes in the grey or white m atter of the brain of the animals. A myelopathy manifested as dem yeiinizatiou was detectable in the dorsal portion of the spinal cord. W ith Spielm cy cr's stain in g m ethod the nerve libers showed dem yelinization partly in the pyram idal tract an d p a rtly in the Goll an d I'.urdach bundles (F ig. 8 ). in frqueney and a marked decrease in the duration of desynchronization in the chronic E E G experim ents. j!y the fourth day the characteristic large, slow, toxic waves ap|>varcd on the electroencephalogram . O n the fifth day no desynchronization manifested itself due to the constantly increasing toxi cosis. O n the basis o f all these finding-, it is very' likely that 2,4-D accum ulates in the organism and in the cerebral tissue and causes a constantly m ore severe paralysis in the function of the latter. Comment Frpm the conditioned-reflex exam inations it is obvious that 2,4-D also damages the In evaluating th ese ex p erim ents it is higher n ervous activity. T h is effect takes necessary to separate the E E G changes place as early as 24 hr. a fte r exposure and when 2,4-D was adm inistered intraperi- by 48 hr. is rather pronounced. By the fifth toneally from those occurring a fte r direct day all the conditioned reflexes cease. cortical application. T he latter series of It was impossible to perform conditioned- experim ents w ere p erform ed in o rd e r to reflex and E E G studies in one and the same ascertain w hether 2,4-D itself or some of t animal. H ow ever, the results obtained in the metabolized products formed in the organism caused the E E G changes. the 2 groups o f animals are parallel. A fter 24 hr. there was a 34.2% decrease in the 2.4- D crystals placed on the cortex duration of desynchronization and a 2 1 % brought about a reversible increase in fre decrease in the conditioned-reflex responses. quency and decrease in amplitude obvious O n the fifth day there is no desynchroniza in the course o f excitation of the cortical tion in response to stimulation, and the con cells. Tyrosine, which is very close to 2,4-D ditioned reflexes cease completely. chemically, did not cause any change w hat ever. T hus the action m ust be considered T he question of the point of attack may specific. Since 2,4-D also produced changes now be raised. I t is known that the intactness when placed directly on the cortex, it is o f not only the cortex but also o f the very likely that the 2,4-D molecule itself, reticular form ation in the mesencephalon is and not some of its decomposition products, necessary fo r both normal desynchroniza precipitated the pathological symptoms after tion and conditioned-reflex responses. the drug's introduction into the organism. Electroencephalograms showed the same 2.4- D adm inistered in tr a p e r ito n c a lly changes both in the cortex and in the retic exerted an effect o f a different type. In the ular form ation. Separate changes referring acute E E G experim ents it reversibly abol only' to lesions o f the cerebral cortex w ere ished the norm al desynchronization of the co rtex .and o f th e re tic u la r form ation. Since this blocking began on the average of 10 min. after the injection and was over in 60 m in., it m ay 1 assu m ed that the effect was due to 2,4-D on cerebral cells via the blood stream, paralyzing normal function. Eater, with a decrease in the concentration o f 2,4-D, normal function was restored. not dem onstrable. Desynchronization and conditioned-reflex responses decreased and disappeared simultaneously. These data per mit the assum ption that intraperitoneal ad m inistration o f 2,4-D prim arily damages the reticular form ation. T hen, the lesions induced in this region paralyze the function of the cerebral cortex. Even on the first and second day utter H istology failed to dem onstrate any onset of the treatm ent there was a slowing changes either in the cortex o r in the sub- Ofsi ft at. 107 r if I *f Cl . . io C P r 'i \ 5337 0007173 * i i i KiV I t: 102 .iK cniri-.s o r e m i u o x m e x t a l h e a l t h v u tlk al regions, allowing the conclusion th.it T h e change- found in animal cxiierim ents n o obvious m orphological change is p r o srtggc-1 the m v d <f caution in the u -c of duced in the n erv e cells by 2,4-1). O bviou-ly 2.4-1). luciva-cd protection and s o c ia l th e m etabolism an d biological functions of n m ro lo g iral exam ination of w orkm en in th e nerve cells a re dam aged by 2.4-D. T h e cufilact with 2,4 1) is necessary. possibility o f subm ieroscopic changes in the structure cannot be excluded. The histological changes m anifesting Prof. I. S'"--. In-limit' <>f Pathophysiology, Univer.-ily M*;ili.il St I11..I l'.tul.t|t.'-li lliigycs E.-u 9, P.ihI;i|k-i IN. 11lint;.try. 1 themselves in the region of the spinal cord m ay l>c responsible fo r the paralyses o f the REFERENCES extrem ities. 1. Honl.t-, S .: F.xamin.itioii of the Occupational O n the b asis o f the d ata obtained in Toxic Effect of Plant Protective Agents in Hun- anim al ex p erim en ts it m ust be assum ed th at gary, Dissertation, P.mla|>cst, I960. human subjects exposed to the action of 2. Rordas, S .: Szcrdahelyi. J., and Sziza, M.: Occupational Toxic Significance of Recent Plant 2 .4 - D also m ay suffer via sm aller doseP,rotective Agents in Hungary" I. Experimental m ild er disturb an ces in function o f the nerv Toxicologic Studies, Munkavcddcni, 4:14, 1958. o u s system an d o f h igher nervous activity J. Bortlas, S .; Kanyo, B.; Nagy, M.t and Weber, respectively. Functional changes in the nervous system dem onstrated themselves after 24 hr., other pathologic symptoms becoming obvious only T .: II. Workshop Exposure Studies, Munkavedelem, 4:19, 1958. 4. Bucher, N. L. R.: Effect of 2,4-Dicldorophcnoxyacctic Acid on Experimental Animals, Proc. Soc. Exp. Biol. Med. 63:204, 1946. on the th ird o r fo u rth experim ental day. 5. Drill, V. A , and Hiratzka, T .: Toxicity of It is possible that, in laborers w orking with 2.4- D in whom routine methods fail dem onstrate pathologic symptoms, E E G studies m ay call attention to early changes. 2.4- Diclilorophcnoxyacctic Acid and 2,4.5-TritcHohvlogr.op7h:6c1n.ox1y9a5c3c. tic Acid, A.M.A. Arch. Industr. 6 . Goldstein, N. P .; Jones, P. H., and Brown, J. R .: Peripheral Neuropathy after Exposure to I t is d esirable th e re fo re th a t w orkm en en an Ester of Dichlorophenoxyacetic Acid, J.A.M.A. gaged in sp ray in g 2,4-D be protected from 171:1306. 1959. ; .4 , exposure to the poisonous drug and that increased attention be paid to the detection o f early signs o f toxicity. Sum m ary A fter the parenteral administration of 2.4- D a reversible inhibition of cerebral electrical activity was observed in the acute experim ents, and in chronic experim ents the same was present to a gradually in creasing degree. Toxic E E G signs were developing. A ccording to conditioned-reflex experim ents, the higher nervous activity suffered severe damage. The changes are probably produced by the 2,4-D molecule itself and not by some of its degeneration products. T he point of attack seems to be the reticular form ation. The changes mani fest themselves as early as 24 hr. after exposure. * 7. Hill, E. V., and Carlisle, H .: Toxicity of 2.4- Dichlorophcnoxyneetie Acid, for Experimental Animals, J. Industr. Hyg. Toxicol. 29:85, 1947. j 8. Hildebrand, E.M .: War on Weeds, Science 103:465, 1946. 9. Marth. P. C., and Mitchell, J. W .: 2.4Dichlorophenoxyacctic Arid as a Differential Herhiciilc. Birth. Gaz. 106:224. 1944. 10. Sollmanii. T. H.: Manual of Pharmacology and its Applications to Therapeutics and Toxicol ogy, 8th Ed.. Philadelphia, W. B. Saunders Companv, 1957. j 11. S6 s. J,, ami Kcrtai, P .: Effect of Dichlorophcnoxyacctic Acid upon the I 131 Uptake of the Thyroid, Acta Physiol. Acad. Sci. Hungary 14:367,'l95R. 12. Siis, J.; De-i, l.: Kcrtai, P.. ami Olasz, J .: The Neuroendocrine Effects of Tyro-inc-Analogue Coni|xxinds in Auiiu:il Exvprinicnts, Medical Con gress, Sopron, 1960, Lecture, unpiihlishcd (Lata. 13. Weis*. K.; Vaietiszky, Ss and Weisz, P.: Method of Estalilishinciit of Conditioned Anxiety Keflex in Rat, Kiserl. Orvosttul. 5:1, 1953. DOW 1511224 1 Pollut 10S 5338 0007174 5339 -- DOW 1 J - , -------------C--.-3- 509619 5340 f~ l- on -CJ o I R * t * i * t ( t m m t h * J O U R N A L a t th m A m e r U a n V o t f r i n o r y Alecflcal A s to t/v f f o n , V o l. 1 4 3 , N m . 4 , A v g . 1 5 . 1 9 4 5 , p p . 3 9 S 3 9 9 a DOW 509620 Chronic Toxicity cf 2,4-D Aiksnclamine Saits to Cattle J. .S'. Palmer, D.V.M., M.P.H. A vaii^uile uata in d icate th e low toxicity of chlorinated phcnoxvacctic acid com pounds to c a ttle .' *' These conclusions are based on results derived from direct oral dosage of cattle or from exposing cattle to pastures sprayed with various hcrbicidal concentrations. The hazard is negligible when herbicides arc applied in concentra tions th a t effectively control dicotyledonous plants. M any different, hcrbicidal compounds have been introduced on the m arket, but 2,4-D w as th e first of its general type to be accepted. I t has been used in vast am ounts fo r controlling annual weeds in cereal crops and pastures and also for weeds and brush in tu r f and ranges. Re search data on other animal species indi cate th at it is relatively more toxic than other compounds.1 The likelihood of cattle gaining access to intoxicating am ounts of this herbicide in sprayed areas, spray tanks, and storage containers prompted this study. Materials and Methods O 6 yrnrlinx 5trrrs uriphins 300 to 10il 11>. u*rtj initially. 5 won* IIoUtcin*Fii(*'ian>, anti ] was a IlcioOml oivMt. The stooi* wort* foil a mnint`ti;uirt.< ration t of a prolfiti roncontiatc r:ili inmii- in" amt a comlmiuluMi of Uo;-;ui anti alfalfa hay oat h eioitiii". Tlu: talion wn* h)mii weight Jnu was itoiortnl ill liio |tin*'i|>7tU a* well a* in |lt rnn* Irnl. WVi^htft won Irtoiinilird wroklv. 'I ho 1.M) tM ht't hiritit roiilaiiiO(| (tV e alkatiola mill* sail.* Tin* daily !*<. on a l?rrw Tot!io>]<>!,.ir<l I>\< Aniin.il D i-tiiv Aftl K r M f f ! i D iv is io n , A;: i c u l t m *1 KYm-h h Ii S(tviu>, U SD A , K r im ilc , T rt.r.. Tin* %tfiler t h .t n k s lit l-boiftr-Vv rm p *y rts W . Rec* t u f , T. S f t o t , anil I t. J o i n s in m ii i. t ii n .: ti*s Mmly. Tlv |r<t h tfln rid f was a rtiin m m t.il prt.;lml. lalullt! W rril KiMrr, 40,'* p r in tm 'id b y lit.- l)*iw C l t c m i r a l C<., M hI1.i u !. M i d i regimen, was calculated at a rale oi 50, 100. 200, ami 250 ing./k". nf body weight. Tlic daily dose oi llir liijuid herbicide varied from 12.5 to 70.5 Cm. and was arlinini.-tcrcd with a do-c syringe, using water as the vehicle. Each steer was restrained in a squeeze chute to assme accurate dosing. One steer each was treated with 50, 100, and 200 mg./kg. and 2 steers with 250 mg./kg. An untreated steer was maintained as a control. Results One steer treated initially w ith 70.46 Gm. (250 m g./kg.) was eliminated from the study. A chronic respiratory infection developed, considered as incubating before the test w ith the focal point in the pharyngeal region. The herbicide treatm ent probably acted as an irrita tin g factor and set up a fulm inating condition. The char acteristic signs of labored respiration and w eight loss w ere observed. The steer was treated after 1 weeks with appropriate therapy and then sold a fte r regaining w eight. Also, pneum onia unexpectedly de veloped in the control steer, but antibiotic therapy brought quick recovery. A direct relationship was noticed be tween the quantity of daily intake, the ac cumulated total, and the length of the treatm en t period. W ith Ihc increase in daily dose, less time was required to pro duce signs of poisotjing so that the total amount ingested was decreased (Table 1). T he s te e r treated w ith 50 mg. I;g. was given 112 daily tre a tm e n ts (5 per week) ra n g in g from 12.5 to lit.7 (,'m. Ih irin g this lime, the sleet's weight increased from to fiti:'. 111. T he tre a tm e n t period extended from Ju n e 25 to l>i-c. !. 1062, w ith an approxim ate averag e of 4.5 treat- men Is weekly. When the stu d y ended, this 398 J.A.V.M.A., Vol. 143, No. 4 5341 000287S I c TACLE I-- Chronic Toxicity of 2,4-D AH'onolominc Soft to Cottle---*Rclationihip of Dcily Oor-c to Ac cumulated Doicgc Daily dose Sucr lasi. Nu. (niR./lig.) 212 SO 213 100 2U 21) 290 21 200 21!/ 250 Given daily 5 days a Variation (Gm.) 12.5-19.7 25.9*35.7 70.3 54.2*58.7 51.8*53.3 No. treatment** To produce imoxi* carimi Total 112 rn v m n rIS i 34 44 15 20 Accum ulated dolale (Gm.) To produce intoxi* cation Total 2546.4 1*29.3 261C.0 __ 1419.fi 714.3 * 70.5 1958.6 1049.1 Results Unaffected Poisoned Disqualified Poisoned Poisoned DOW 509621 il steer hud no appnront signs of intoxication. pound. The doses w ere based on m illigram s C attle given doses of 100, 200, and 250 of 2,4-D p er kilogram of body w eight and m g ./k g . h ad sig n s of poisoning a f te r 8G, w ere ad m in istered by dose sy rin g e, w ith 3-J, an d 15 tre a tm e n t days, respectively. w a te r as th e vehicle. Doses w ore given The more heavily dosed steers were daily 5 days a week. treated to term ination of the experiment. Signs of poisoning were noticed in the The ste e r given 100 m g ./k g . had to be m ore heavily dosed steers (250 m g ./k g .) m edicated a f te r it developed a digestive a f t e r 15 tre a tm e n ts, in c o n tra st to 86 tr e a t disorder characterized by extrem e tym m ents at 100 m g./kg. A dose of 50 m g./kg. pany. Two days w ere required to correct had no apparent effect on 1 steer a fte r 1 1 2 the condition, although anorexia was doses. A direct relationship was thus de never observed. Rumen atony from accu tected between the relative quantity of mulated herbicide was apparently the pre daily intake to the accumulated total dosage disposing factor; the steer was removed and the length of the treatm ent period. from the experim ent and made an uncom I t was concluded th a t this herbicide has plicated recovery. a low degree of chronic toxicity, apparent P rim ary signs of intoxication in steers ly because it is not readily stored in bovine given 200 and 250 m g./kg. of the com tissues. However, it is possible for cattle to pound were development of dry cracked ingpst enough to produce m orbidity and muzzles and ulcerated mucous membranes, m ortality. w ith epistaxis on routine restrain t. A clear, viscid nasal discharge, associated w ith a fetid odor, was noticed. Progressive Rafarancoi apathy and depression were apparent. One steer given 250 m g./kg. had m oder ate tym pany associated w ith mclena. Ne cropsy revealed severe hem orrhages in the large intestine and pulmonary congestion. The steer given 200 m g./kg. had muscular weakness in the hindijuarters and a stag gering gait, d ro ss pathologic changes Rock. W. H., Itiniis, W., .lame. T_, and William?. M. C : Rc*nlts nf ed ili" lleiliicidc-Ticalcd Pianti lo Calie? and Slut*|i. J.A.Y.M.A., 1311, (March 15, ] % 1 1 : 320.3I. 5Crafls. A. .S.: The Chcmisliv ami Mode of Action nf 1(-'Incide*. Iiilcr-rimrc l'nhlislirrc, New York, N. Y.. ]%1. i(ritf*liy..T. li., and liv id i, K. 0.: Suine EfTcct? nf llriliii idi-- mi Pa-litri* ami un dra/iii" I.ivestoek. Micidiali Agi ir. Kxprr. Sia. Qnail. Unii. '2. (19301: noticed at necropsy were inflammatory swelling of the kidneys and a pneumonic condition considered to be the immediate .".:i:-:i!:r>. *Mii. dirli. J. \T,, ]Inil^'.*ti, R. mnd ('articii-, C. Tolerauto of Kami AniniaU I" Kred Contain- ing 2.11lit Moi ii| I n . i f riti* Arid. J. \ iiin. Sci.. cause of death. 5. (Pilii.l : Z `liad. i.ir. I?. 1).: \Uanri-s in Veit*i iinly Srirnrc, Vid. I. 11..- To>icily of Ili-cv lieidi - an- 1 j lerlnritlc. Sum m ary and Conclusion lo hive*11k., Arailoniu: Pic-a Ine., NV\\ Y.nk, N.Y.. fl9.'i.,;i : 2('> m*(* D aily oral doses of all.anokim ine salts `Iti.n .*, v. K.. ami T\itiu*>. T. A. Summnrv of of 2 , 1-dichlorophorioxyacrtie acid ( 2 , 1-D > w ere given to 5 yearling stecra to de Toxirul o/irul iiifurinrfion on 2 ,b l) ami 2. ;.r.-T Typo ilrilthidr* ;alni an alii.iti<11 of the limititi? to 1.w -tt.1. Ao'iat*-i| uitli *! !lift! V* Ain. J. Vet. L te rm in e th e chronic pixiciiy of |h is eom- ib - ., i'., ( O ri., io :,!) ; i. August 15, 1963 399 0002879 U i, Jf i O NJ O 4ii Vjj --- -V; Toxicity of 2,4Dichlorophenoxyacetic Acid for Man and Dog JOHN K. SKABIKV. MO t NEW ORLEANS ro/i; O ^>5 3 000630! H a 3 ir ?H 4 In November, 1959, Goidstein. Jones. and B row n *1 reported three cases o t peripheral neuropathy following exposure to an ester of dichlorophenoxyacetic a d d (2,4-D ). Tlie first patient had spilled 60 ml of a 1 0 C^ solu tion of an ester of 2,4-D on his forearms without washing it off. The patient felt unduly fatigued on the evening of the same dav, and within the next ten davs he developed nausea, yomiting, and 20-pound weight loss. A pproxim ately two months later he had a sim ilar ex j su re on his legs, ami he developed nausea, vomiting, and diarrh ea d u rin g the subsequent five days. O ne week a fte r the second exposure, he com plained of numbness and aching of the digits of all extrem ities. T h ere was desquam ation of the skin of the palm.- and soles, and within six weeks a fte r the second exj>o>ure he had well-developed neuropathy. *1 he second patient had a less determ inable am ount of exp.osure by dermal 'vetting with kui'm it:! for -uhlic.atir.t; Aug 10, 1002, i m res-nr of Medicine. Pq'-.irtnent of Mrlicute. I.i.nlji.ir.a Slate t.'iiivcr-uy School of Medicine. an ester of 2.4-D. Shortly after exposure, she noted swelling and aching of the feet and legs, which persisted for approxim ately I'/i weeks. A pproxim ately one year later she had a sim ilar exposure to the herbicide with recurrence of dinilar symptoms. However, she later developed anorexia, loss of 20 pounds in weight, and severe pain in her iegs with painful swelling of the metacarpal joints of both hands. A pproxim ately five months a fte r her second exposure she had a riaccid paraparesis. The third patieiir reported had been spray ing with a solution of an ester of 2,4-D. During this activity his sleeves and trouser legs had been wetted with the herbicide, and some of the spray might have been inhaled. On the following day, he noted malaise, headache, nausea, and vomiting. Vertigo was severe on the secuml day following -praying. Paresthesias in the extrem ities and p.in m the legs appeared within fo u r or five days, followed by tw itching of the muscles in the caives ami am is. Kascvulations became generalized. T here were m> neurological or dee ' hit: I ot p ID S that ;>or: the cinc tion 'fin agre that 'he prep. was .n'et: mgr li to 2 .lair iu e r dichi re norrr XI. :hera no ti the t the ; know cocci t'/CUc that cours- were `her.'1.' *.*niy rs: ^JSi'l. TJctic C^i.id ., list, kept pare;- o.ch: 8:. DOVI 1392107 a. ce sf ll 2:n *:i\ .e he t ** JIU tv \ p r.' n t ? ^? 1 :vr f-t I 'T ^ J r` M ".5>; `` V? * - I .* `.V .< -VTjHf * * ' *5 ' f /r's , v exposure, M ie fe e t a n d .u iately 2 y 4 ^iter h e h a d lucide w ith However, of 20 i her legs >;sl jo in ts ' : c months *.i-: a Ilaccid *V r\- , : vm Spray- r of 2,4-D. . ;:.'l tro u se r jrr.icide. ami Tii inhale*!. a-d malaise, *rti-o was 'I'rt-ying. pain in : . \ e iltlVS, in the . became / .If or ,i ' ; ?i > J.i n TOXICITY 203 electromyographic changes, hut his symp total of 464 mg of sodium 2,4-D in 28 toms persisted. In a subsequent com m ent 5 on this report of leripheral neuropathy following exjiosure injections over 37 days, of which 2S8 ing were given intravenously. O n a weight basis, the injections am ounted to 1.17 m g /k g of to an ester of 2,4-D , GoWlwater points out body weight. D og 2 w as given 389 mg in 24 that these a re the first such cases to be re- injections over 32 days, of which 261 mg l*>rted and that one w ould have to know were given intravenously. O n a weight basis, the com position o f the vehicles, solvents, and this am ounted to 3.2 m g /k g of body weight emulsifiers used in the com m ercial p rep ara per injection. D og 3 received 3S9 m g in 24 tion before incrim inating 2,4-D as the injections over 32 days, o t which 277 mg principal toxic com pound. G oldstein ct al were given intravenously. T he dose per agreed w ith G oldw ater's com m ent and stated injection was 2 .6 m g /k g o f body weight. that in one instance they w ere able to obtain Dog 1 was autopsied 202 days a fte r the the label from the container of the 2,4-D first injection of 2 ,4 -D ; dog 2 was autopsied preparation. O n this label, the preparation on the 86th Jay following the initiation of uas stated to contain " 2,4-dichloruphenoxy- treatm ent; and dog 3 was autop-ied on the acetic acid, isopropyl ester 44c/c and inert ingredients 56% ." Because of these reported cases of toxicity to 2.4-D , and the recent study of a man claiming disability of a different type subse quent to spraying w ater hyacinths with a dichlorophenoxyacetic compound, I decided 85th day. All dogs had gained substantial weight during the experim ental period. All dogs showed evidence of visceral histoplas mosis at autopsy, but there were no gross or microscopic lesions in the liver, spleen, lungs, kidneys, or adrenals other than those due to histoplasmosis. No neurological tis to rep o rt certain therapeutic trials w ith plant hormones carried out in 1948 and 1949. My interests in plant hormones were therapeutic rather than toxicologic, and since no therapeutic efficacy was dem onstrated in the trials, no attem pt was made to publish the results. In 1948-1949, there was no known therapeutic agent for disseminated coccidioidomycosis, histoplasmosis, or cryp tococcosis. It appeared remotely possible that the plant hormones might alter the course of these fungous infections. Since we were unable to find any evidence of therapeutic effectiveness in the literature, and only meager reports of animal toxicity, I first screened a m ixture of indole-3-acetic acid, indole-3-propionic acid, a-naphthaiene- sues were examined. A moribund patient with disseminated coccidioidomycosis was selected for intra muscular injection of auxins. This patient received a total of 40 m g of the sodium salt of 2,4-1), 3.3 mg of indole-3-propionic acid, 3.3 mg of indole-3-butyric acid, and 3.3 m g of a-napiuhaleneacetic acid during a period of four days. Death occurred on the fifth day. The patient exhibited no unusual symp toms during treatm ent, and the autopsy findings were only those of generalized coc cidioidomycosis and hypoxia. In A pril, 1949, I decided to treat a 4Syear-old male patient with disseminated coccidioidomycosis with auxins to the limit of tolerance. It was obvious that the patient icetic acid, and 2.4-dichlorophenoxyacetic would die w ithin the n ex t several weeks acid in guinea pigs infected with Cnccidloidcs im m itis. No therapeutic effect was noted. Six female mongrel dogs, of approxi mately one year of age. were infected with flsto tUisma caf'sidamm. Three dogs were kept as controls, and three were treated pnrenterally with the sodium salt of 2,4dichlorophenoxyacetic acid. O nly the treated dogs are of interest here. Dog 1 received a unless the course of the disease could be modified. T he details o f treatm ent appear in Table 1. Intravenous im'iidon of 2 gm of 2,4-D produced no changes in his physical examination and pro voked no complaints from the patient. When 3,600 me of 2,4 - 0 were riven intravenously dum p a period of two hours, a reaction appeared. T. .trine the latter part of the infusion, the patient heemre very (piiet, .'emisliit'roiis, and exmhited fihr.uarj 5345 0G06906 / J / \ i : J .3 > 4 ~3| 3 392108 204 ARCHIVES OF !'/RO.VHEX TAL HEALTH T a b l e l.-- Administration af Auxins to Man D ue Routeof Administration 3/ 4 / 3 / 3/49 3 / #/49 3 / 7/49 3 / 9/49 3/10/49 3/11/43 3/13/49 3/13/49 3/1V49 3/19/49 3/21/49 3/23/49 3/24/49 3/23/49 3/28/49 3/2S/49 3/30/49 4/ 1/49 4/ 2/49 4 / 4/49 4 / 3/49 4/ i/49 IM IM IM IV IM IV IV IV IV rv IV IV IV IV IV IV IV IV IV IV IV IV rv 2.4-D, M I 2 1 160 24 120 120 240 440 400 MO MO soo soo soo 900 SOO yo soo soo soo 2.000 ZjOO Indoi*4-utyrt Acid, M ( 0.M l.M IJ2 c.a l.M \Z 2 13-2 33 33 33 33 3J 33 33 33 33 33 a e n k Acid, M | React** 0.64 l.M 132 6.94 l.M 13.2 13.2 Local buruJcf rrocsino added--ooo Nooa Nooc Non* Nona Nona Nona N<ma Nona Nona Nona Nona Nona Nona Non Non None Nona Nona Nona Sea text * A-- : & *' .V:S s` '*p -Vd) - 'kl- - V. V' , ,t'.-** . *'} '- T ne2, -D waaadministered at the sodium tait. All intravenous medications were administered lu t.OCQml of Ste destrose in wst# movements about his mouth and in both hands and forearms. The fibriilary twitchings persisted for several hours. Stupor deepened to the point where he could not be roused by sound hut did respond to painful stimuli. Stupor was accompanied by hvporede.xia at the knees, ankles, and biceps tendons. Hyporeriexia persisted for 2d hours. Urinary in continence appeared and persisted for 24 hours. There was no change in pulse rate or volume, or respiratory rate o r depth. Seven hours after the termination of the infusion, the patient could be aroused verbally but quickly lapsed into what ap- T able 2.--AcuU Toxicity of 2,4-D for Animais AnUnsl and No. L'acd Mouse Approximately 4J0 Number not stated Rat Approximately ISO Guinea pif Approximately 12S Rabbit Approximately 70 Don 4 M : t i s doe^ga 2 Monkey 1 Chicle S In each {roup Route ot Admin. ( Stomach tuba \ Intrapericormd Sabcutuaeous ( Stomach tub \ Intraperitcneal / Stomach tube \ Intraperuoneal f Intraprrllooral | Intravenous \ Stomacn tub* Oral Oral Intrapcrisooeal Oral Doso-Toxicity, M z/K{ LD* 1.0 it LD i. * L D ,. * L D ..* L D ..* L D ..' L D i. * LD*. L D ,. ' L D ,. 323 373 2*0 C66 C6 i.ooo fc`6 400 MJQ SOO ICO LD. t LD. ' 510 42S Kef. No. 4 4 3 4 4 4 4 4 4 4 & & 4 ld.: i.i)... ; : ?o G 6 Admiaater*ni an ;tit* s j IC of t h r i v e n \i b u i y l e.-trr. fisu re* c i k u tiih il u* 2 .I .P . j u it - p -x\ a ti!m;tr.w!am:3r v/ 2.4*1.). 0006907 , 5346 /V; AtiV. / W I L HEALTH . -s- \j . \.*fr ii f -T 'i- j --A- .. j. Urinary inIor 24 hours, or volume, or :urs after the tient could be ::to what ap- .u r ! ' i.i V. `a* < * - >J-D TOXICITY 203 Tabi.e 3.--Subacute Toxicity of 2,4-D for inimats Animal to d R outt o( No. Used Admin. U<M-Toxl<ity. M t/K c/D t llet No. Dogs 2 2 s X Intraven ously 3J0 X IL D i.. 100 X - LU 0 X (l died, 1 sjcriflud very 111) IS x <1(sacrificed very ill) 4 4 4 4 penred to be deep sleep. When he was aroused, there appeared to be lack of recognition of familiar visitors and apathy. Eighteen hours after receiving die hormone, he talked spontaneously and recog nized everyone. Twenty-four hours after the in fusion he was oriented, eating well, but still lethargic and complaining of profound muscular weakness. Psychomotor retardation was evident. Within 48 hours he had returned to his prereaction status in sofar as could be observed. There was no further evidence of neurologic or muscular change in the subsequent two weeks of his life. His maximum temperature on April 6, 1949, was 103.8 F (39.9 C) and was 104.4 (40.2 C) on April 7. For 48 hours after the intravenous infusion of 3.6 gm of 2,4-D his temperature did not exceed 101 F (38.3 C). He did not have his usual daily rigor. His temperature remained between 1022 and I02.fi F (30 0 to 39.2 C) maximum for six more lays and then resumed its previous Ievl of ap proximately 104 F (40 0 C) daily. On the night of his reaction, the spinal lluid was under normal pressure, contained no cells, and the glucose and chloride concentrations were normal. Unfortunately, the protein content was not deter mined. Approximately 14 hours aiter the last in fusion of 2.4-D, the I loud urea nitrogen was 56 mgCS', the fasting blood glucose 33 mgC, and the carbon dioxide combining power "*3 vol'V The blocd urra nitrogen was 12 mg% on April 12, 1949. There was no alteration in the clinical course of the patient following discontinuation of treatment. The patient died 17 days after the last infusion of 2,4-D. Autopsy was performed 416 hours after death. The gross and microscopic findings were typical of disseminated coccidioidomycosis and cachexia. The heart, pancreas, adrenals, and striated muscle taken from the diaphragm were normal grossly and microscopically. The liver weighed 1,720 gm and was light brown in color. Congestion was present Microscopically, the lobular pattern was well preserved. The central veins and their radiating sinusoids were slightly congested. Throughout the parenchyma were nu merous focal areas of necrosis which were composed primarily of a granulomatous tissue infiltrated by lymphocytes and polymorphonuclear leukocytes. Several giant ceils were seen in some of tiie focal Animal and No. Used Rat i 7 t S o rt Sor S S o rt S or t S o rt Doj 2 2 3 4 Mice Number not stated Chicks S in each croup Tabi.z 4.--Chronir Toxicity of 2,4-D for Animals Route o( Admin. Dose-Tcslcity - In feed Oral (intubation) Oral Parenteral Oral 100 incA c o( reed/da X 21, then 1,1.00 m iA c of leed/da X 10--no ellects 100 m * A l of fetd/da X 30--no effects 400 m t / k t ot !eeil/<U X 30--no ffec'.s I m j/ l i f S X/wk X 4 wk--no effects 10 meA C 3 x /w k X * * t - n o effects 30 m cA c S X/wk X * wk--no effrtts ino ir.g/Vg 5 X/wk X 4 wk--d*pr<*sed growth, gj&troinieaiiaal Irri- ration. cloudy jwellini o f lieer 3T43 mg/kg 5 X/wk X * wfc--L D u# 2 m cA z/d* X 40--I.D 6 mg/kg/Ua X 10 m gAC/ds X VO--L D i 3] m cA c/fla--L U II (da IS, da IS, da 4) 1/3-l/S LD ii/d a XII-W--L D . 14 I.D ii/ila retards erowtlt O.IS m cA c 3 X wk X 4 wk I J mr/Xc 3 X wk X 4 we IS m * /k c3 X wk X w t 1 1 j ISO m cA c 3 X w|s X 4 wk j rJSO mg/kg produces slight retardation of growth) r.e( No. 4 4 4 9 9 9 9 9 5 3 5 3 C * Mica receiving ont-l ::nl L D u per day lrtvc becomo pregni*nt u d imrn* apparently gQ g 5? Scobury .. 5347 . yJ* . . J -- .^ -^ 4 l1 1 i: I* n iT .. i. i \ i. I l 1 I ! fv* ` 206 ARCHII T.S OF /Xl'IROXMFXTAL ilF.Al.ZH granulomas. and a rare CMcidioid/s immitis rnulil he found within the giant cells. These changes were considered due to coccidioidomycosis. The right kidney weighed 180 cm and left kidney 1/0 gm. The capsule stripped with ease from each kidney. Cortical and medullary striations were dis tinct. Except for marked congestion of the capillary bod throughout, especially at the iwripliery of the cortex anu in the glomeruli, there were no micro scopic changes other than those due to numerous granulomatous lesions containing Coccidioidal imtitis. D ogs exhibit the same general symptoms with marked spasm of the hind limbs. In addition, they may show sneezing, rubbing of the eyes, diarrhea, and vomiting. Those living long enough refused all fo o d .-"1"3 T w o m onkeys given 1 gm an d 1.5 gm of sodium 2,4-D, respectively, by the intraperitoneal route exhibited nausea and vomiting initially. The gastrointestinal symptoms w ere followed by stiffness of the legs, with \j ' , W ;/ The brain weighed 1,270 gin. The entire brain anil coverings were normal grossly except for the leptomeninges over the brain stem which were opaque. A pressure cone was seen involving the cerebellum. Multiple sections from the cerebral hemispheres, brain stem, and cerebellum were ex- some muscular incoordination, lethargy, hanging of the head, and ptosis o f the eyelids.4 The symptoms first appeared about four hours after injection and persisted for about 48 hours before lessening in intensity. ./ ;y,, .*' aminrd. Xo microscopic abnormalities were de A t the end o f five days, recovery was com- . tected. plete. ' ` r' C om m ent T he patient reported here had recei ved 12,712 mg of sodium 2,4-D parenteraily prior r ' T he acute, subacute, and chronic toxicity to the intravenous dose which provoked a o f 2,4-D fo r laboratory anim als is sum reaction. Consequently, one cannot state that :-\ m arized in T ables 2, 3, and 4. A n extensive the changes produced by 3.60*1 m g o f sodium tabulation o f the oral toxicity o f the 2,4-D 2.4- D intravenously were en tirely related to and 2,4.5-T type herbicides m anufactured by this single dose. Despite this, there was a the D ow Chemical Com pany appears in the considerable sim ilarity between the reaction >'t article by Rowe and H y rra s .0 and this article produced in the hum an patient an d that pro- I should be consulted by those interested in duced in the two monkeys. H ow ever, on a possible toxicity to livestock. weight basis, the dose adm inistered to our T w o monkeys were given 2,4-D intraper- patient intravenously was approxim ately 66 itoncally.4 but only the larg er dose is re m g /k g o f body w eight w hereas the m axim um corded in Table 2. intraperitoneal Jose given to the monkeys W hen guinea pigs were exposed to either was 428 m g/kg of body weight. a wet cloud of the sodium salt of 2,4-D in a Although the m ajority of studies of acute spraying chamber or to a dry cloud of the toxicity of the 2,4-dichlorophenoxyacetic crude acid, none of the animals exhibited any compounds are in dose agreem ent, the num symptoms and there was no gross evidence ber of animals tested by some authors was nf lung irritation.4 smail, and some disagreements appear which T he symptoms of acute toxicity in mice, cannot be resolved on the basis o f the data rabbits, guinea pigs, and rats are essentially presented. F o r exam ple, the acute I .D-,.. for the sam e.-'1-4 Some anim als die suddenly, ap oral adm inistration o f 2,4-D to dogs was p aren tly from ventricu lar fibrillation. T hose 100 m g /k g according to D rill and H ira tz k a s that do not die suddenly develop stiffness based on the study of four animals. O ther of the extremities, incoordination, lethargy, w orkers' gave an amount of the butyl ester stupor, coma, and death. T ru e myotonia of 2.4-D calculated to contain 500 m g of occurs and is sim ilar to that produced by 2.4- D per kilogram to each of two dogs w ith cevadine fv eratrin e;.'- 7 H ill and C arlisle4 out observing any deleterious effects w 'thin state that the low er extrem ities become p a ra *>6 hours. Clue of the tw o dogs was autopsied lyzed, and till authors agree that m yotonia is and showed no evidence of ar.v m acroscopic m ore evident in the low er ex trem ities than lesions. T he other animal rem ained in ap- in tile upper. 0 0 0 6 9 0 xcellent health for the following ' m l> 5348"' . vP ri - - - y i 'E.lt.TH 'h ' _nipt<>ms ce nbs. In ,, rubbing St ! T h o se :ll', ; 1.5 grn a\ te intra- I vomit- :e ( j mptotns -) ml t-fgs, with , ,ftharjjy, the eyetV d about sred fo r . tensitv. is com* iv.ved 12,je Jy p rio r i n . voiced a ra. tare th a t ^ sou urn v.,, lated to c was a reaction .,-h.nt pro per. on a :1 to our ately 66 r .taxi.-mim . : U'lnkeys r of acute , 'Xyacetic ,) : he uum; i"i' was . which 'he data A fur " e-' was r.uzka 3 w*; O ther ester mg of . - with' within ,.:i!..p'ied i "'Cupic in aj>, ""wing V . ; ]{ - .* '1 1I rj `. , 1 I / 11 1I I1 1 i11 DOW 1392111 n TOXICITY 207 s j days. One would hesitate to cunciude ft'iuii these data th at the butyl ester is less t..\ic than 2,4-dichlorophenoxyacetic acid. The lowest dosage administered intrave nously to dogs (25 m g /k g ) over a period of ,-ix days was toxic,4 producing decubitus ulcers o f the extrem ities and necrotizing le sions about the m outh. A low er lim it for -ubacute toxicity in dogs has not been estab lished. According to D rill anti H iratzka * the largest dose of 2,4-D which could be given i>rally to dogs fo r extended periods w as 10 nig/kg of body weight. Commercial herbicidal preparations of the chlorinated phenoxvacetic acids and associ ated com pounds are usually form ulations of .oe o r m ore active ingredients with solvents, dispersing and wetting agents, and some times co-solvents. These may contain as active ingredients either the acids or their -alts, esters, or amines. T he exact composi tion o f liquid form ulations is o ften not stated in labeling. A lthough Rowe and H y m a s 9 -tated that the acute oral toxicity of the commercial form ulations of the Dow Chemi cal Com pany tended to be projiortional to '.hut expected from their content of active in gredients and that the "inert ingredients" did not appear to exert a potentiating effect upon toxicity, H ill and C arlisle4 found that the toxicity of 2,4-dichloronhenoxyncetic acid dissolved in tributylphosphate and oil was much greater than the toxicity of 2,4-D .done. T he increase in toxicity was due to the tributyiphosphate-oii m ixture itself. The sim e authors perform ed similar studies with A"-butyl alcohol-oil solutions of 2,4-D , and 'Cited "A lthough not conclusive, these ex periments indicated that X-butyl alcohol-oil concentrations of 2,4-D are at least of the sam e o rd e r o f to x ic ity '"as the tributylphosphate-oil solutions." These findings by Hill and C arlisle lend strength to G oldw ater's criticism 3 of the report by Goldstein.1 B ucher,3 w ithout indicating the num ber of animals studied, stated that the histopathulogy produced by 2,4-D in acutely in toxicated mice consisted of wide dilatation of the blood vessels of lungs, liver, and kid- neys. h r noted moderate atrophy of the liver in one chronically treated dog. There was no significant variation in the peripheral blood of chronically treated mice from that observed in controls with respect to hemo globin. red ar.d white blood cell concentra tions, and differential counts. H ill anti C arlisle4 found thac rats and guinea pigs dying of massive doses of 2,4-D consistently exhibited congestion of the vis cera and enlarged swollen kidneys. M icro scopically, there was massive cloudy swelling of the proximal convoluted tubules with cast form ation. The glomeruli and blood vessels were unaffected. Occasional animals showed slight patchy pulmonary edema and alveolar hemorrhages. Rabbits that received injec tions of one fourth of the L D 50 parenterally fo r from 4 to 13 days showed slight-tom oderate degenerative changes in the p roxi mal convoluted tubules. Scattered petechial hemorrhages were present in the lungs of a few animals. Three to seven daily injections of one half of the L D ;<, produced a signifi cant decrease in hemoglobin in fo u r o f six rabbits. The renal changes were sim ilar to those observed in the rabbits receiving one quarter of an I.D-U fur a longer period of time. in addition to the development of bleeding and necrosis of the gums and decubitus ulcers, dogs also exhibit evidence of liver damage more frequently than other animals studied.3'3 T h e histopathology varied from occasional areas of focal necrosis in animals subjected to chronic oral toxicity studies to extensive necrobiosis throughout the liver in one dog 4 given two daily injections of 200 m g /k g of the sodium salt o f 2,4-D. F o r the most part, tings succumbing to massive doses of 2,4-D showed centrolc.bular degeneration, atrophy anti lysis o f the parenchym al cells about the central veins o f the lobules, and congestion and dilatation of the paracentral sinusoitls.4 Dogs also showed cloudy swell ing of the renal tubular e p ith e liu m , anti one dog receiving a m assive do>e 4 showed actual tubular epithelial necrosis. Lymphoid necrosis was observed in the acutely poisoned 97 5V-'nii-v o b o u sio 5349 1* i i l: i it<: j t i. 1* I, (>'. I I .g S S g g g /*$S * * ,vr^*s.v< * ,*.***7 -i? ..T.-...>Tv-'V' ; % '*-?V-.'Jr5'5 392112 -- -08 ARCHIVES OF ENVIRONMENTAL HEALTH.'?'] dogs, an d some o f them showed term inal tically on the neurosurgical sendee o f two " lym phopenia in the peripheral blood. hospitals in the management of stuporous References to human toxicity are meaner. and comatose patients. The oral dose for . /,] H ild e b ra n d 10 m ade the sim ple statem ent man averaged 400 to 1,200 m g in 24 hours, " Some investigators have consumed the btu doses of 4 gm have been given without chemical and suffered no ill effects." As- reaction. T he ester was given intravenously souly 11 stated that w orkers employed in the in doses of 250 to 500 m g at the rate of about ; fabrication o f an ester o f 2,4-dichlorophen- 250 mg in five m inutes. T h e authors state v '_. oxyacetic acid developed symptoms of som that this material had beer, given for from nolence, anorexia and gastralgia, increased eight days to six m onths without a single salivation, a sw eet taste in the m outh, a reaction. No inform ation is given concern- v-? sensation o f drunkenness, heaviness of the ing the possible herbicidal activity of this -V . legs, and hyperacusis. W hether these symp chlorophenoxyacetic acid ester. 'J. toms were related to the 2,4-dichlorophen- . .* uxyacetic acid o r to the chemicals used in Sum m ary / the process o f esterification is not known. C ertainly a sw eet taste in the m outh with T hree dogs with experim ental histoplas- ' V- r sialarrhea suggests the symptoms were re mosis were screened for chronic parenteral lated to the chemicals used in esterification. toxicity of sodium 2,4-dichlorophenoxyacetic H yperacusis was not noted in either of the acid. The dogs were given parenteral sodium p! two patients reported here who received 2,4-D at the rate of 1.17 m g/kg of body parenteral 2,4-D. Mitchell, H odgson, and weight, 3.2 m g/kg of body weight, and 2.6 ->. G aetiens 12 state in a footnote "D r. E. J. m g /k g of body weight p er injection. T re a t A. K rau s of the U niversity o f Chicago reported ment extended between 32 ami 37 Jays. In .. y on January 26, 1946, that a hum an (adult this dosage, there was no evidence of chronic 2,4 > i male") had taken daily by m outh, 5G0 mgm. of toxicity. purified 2,4-D either after the midday or One patient was given a total of 40 mg of t r . evening meals during a period of 2 1 days the sodium salt of 2.4-D. 3.3 mg of indole-3- 2.4- w ithout experiencing ill effects." propionic acid, 3.3 mg o f indole-3-butyric iUs It would seem logical that chemical modifi cation of the chlorinated phenoxyacetic acids might well result in altered pharmacological acid, and 3.3 mg of a-naphthaleneacetic acid intramuscularly during a period of four days. Xo toxicity was observed. action and toxicity. An interesting example A second patient was given a total of 369 of this is the dimethvlaminoethyl ester of mg o f irdoIe-3-butyrie acid, 33.3 mg of - /'-chlorophenoxyacetic acid. T h is ester was naphthaleneacetic acid, arid 12,712 mg of the produced by Thuillier, Rumpf, and Thuillier.la T h e ester has an L D ;i, to r mice of 330 m g/kg when given intravenously and sodium salt of 2,4-dichlorophenoxyacetic acid parenteraliy during a period of 34 days without-observable toxicity. Most of this an I.D ja o f 845 m g /k g when given intra- m aterial was given intravenously. T w o days peritoneally to dogs. Chronic toxicity studies later. 3.6C0 mg o f sodium 2,4-D were given in rats indicated that 300 m g /k g could be intravenously and produced definite clinical given for m ore than five m onths w ithout signs o f toxicity. T he p atient recovered alteration of the blood cell count o r of the clinically' from this toxic episode w ithin -'S organ systems. H ow ever, it ap|teared to act hours, and showed no fu rth e r evidence of as a central nervous system stimulant and was capable of raising a lowered arterial blood pressure. There was no evidence of a myotonic action. Coirault, Pournre. neurologic o r m uscular change in the sub sequent two weeks of life. Death was due to disseminated coccidioidomycosis. The autopsy fimlings are described. Damasio. R ouit, Deligne. David, and Tnlai- A single intravenous dose of 2.000 mg of rach 14 reported using this ester therapeu- sodium 2.4-1) did not produce clmical evi- 5350 rm./ TT69000 EAI.TH. V Ot two .uporous .. lose fo r 'V *, :4 hours, > * ; . without venously of about 'V >rs state ror from a single concern/ . o f this '\Vf 'J'i. . .*< :areiiteral xyacetic i* il sodium uf botiy . and 2 .6 n. T reatJays In i ch .ic i* ' ... 49 mg of indole-3-3-butyric .ce'.ic acid /lur days. >< i<f 369 ng of <Tm-: o f the ' 'xyacetic : 34 ..'ays : of this i wo da vs ''-re g i\e n ii- clinical vcC'ivcred tiin 48 >-::ce fit :l:c sllb was due -- . The 111!" 'f 1 evi- : j -d t o x i c i t y 2C9 donee o f toxicity in the second patient, 7. Eyzaguirre. C ; Folk, B. P. ; Zierler, K. L.; whereas 3,600 mg was definitely toxic. A total of 16,312 m g of sodium 2,4-D was administered to the second patient. T here and Lilienthal, J. L., Jr. : Experimental Myotonia and Repetitive Phenomena: The Veratrinic Effects of 2,4-Diciilorophenoxyacetic Acid in tlie Rat. Amer J Physiol 153:69-77, 1948. were no necropsy findings w hich could be 8. Baker, D. L.; Ramsey, F. K.; and Sylwester, attributed to toxicity o f 2,4-dichlorophen- E. P. : Suspected Poisoning of Dogs From Eating oxyacetic acid. John H. Seabury, MD, Department of Medicine, 5 hool of Medicine, Louisiana State University, . - . l Tulane Ave, New Orleans 12, La. Grasses Treated With 2.4-D, N Amer Vet 34:194, 1953. 9. Rowe, V. K.. and Hymas, T. A. : Summary of Toxicological Information on 2,4-D and 2,4,5-T Type Herbicides and an Evaluation of the Hazards to Livestock Associated With Their Use, .Amer J Vet Res 15 622-629, 1954. REFERENCES 10. Hildebrand, E. M. : War on Weeds, Science 1. Goldstein, N. P .; Jones, P. H .; aud Brown, J. R.: Peripheral Neuropathy After Exposure to an Ester of Dichloroplieroxyacctic Acid, JAMA 171:1306-1309, 1959. 2. Goldwater, L. J . : Peripheral Neuropathy, JAMA 173:87, 1960. 3. Bucher, N. L. R.: Effects of 2,4-Dichlorolihenoxyacetic Acid on Experimental Animals, Proc 103:465-468, 1946. 11. Assouly, M. : Dsherbants slectifs et sub stances de croissance; Aperu Technique; Effet pathologique sur l'homme au cours de la fabrication de l'ester eu 2-4D, Arch Mal Prof 12:25-30, 1951. 12. Mitchell, J. W. ; Hodgson, R. E ; and Gaejens, C. F.: Tolerance of Farm Animais to Feed Containing 2,4-Uichlorcplier.oxyacetic Acid, J Ani mal Sci 5 :226-232, 1946. Soc Exp Biol Med 63:204-205, 1946. 13. Thuillier, J.; Rumpf, P. ; and Thuillier, G.: 4. Hill, E. Z,, ar.d Carlisle, H .: Toxicity of [Derivatives of Acid Regulators of "Plant Growth: 2.4- Dichlorophenoxyacetic Acid for ExperimenIt.alPharmacologic Properties of the Dimethylamino- Animals, J Industr Hyg 29 :S5-95, 1947. ethyl Ester of p-Chlorophenoxyacetic Acid (235 5. Drill, V. A., and Hiratzka, T .: Toxicity of ANP) I, C R Soc Biol (Paris) 133:1914-1918, 1959. 2.4- OichIorophenoxyaeetic Acid and 2,4,5-Trichlo-14. Coirault, R. ; Pourpre. H. ; Damasio, R. ; rnphenoxyacetic Acid, AMA Arch Industr Hyg Rouif, G. ; Deligne, P. ; David, M.; and Taiairacli, 7:61-67, 1953. J. : [The Treatment of Consciousness Disorders in 6. Bjorn, M. K., and Northern H. T .: Effects of Neurosurgery by the Dimethylamincethyl F.ster of 2.4- Dichlorophenoxyacetic Arid on Clicks, Scienpc-eChloropiienoxyacetic Acid (ANP-235)], Presse 108:479-460, 1948. Med 68:215-216, 1960. I II-' Uj !: i .! :i i ,i ! .. ! }r 55 ? r -41. In countries where public health has been longest in effective operation, and where natality has responded to the new pattern of living, the demographic structure has undergone profound change, with a shift of population to older groups. Thus over-iiopulation and ageing are new challenges no less demanding than some of the infectious diseases which are passing. Moreover, where public health has succeeded emphasis has shitted from infectious to degenerative disease, now equally prevalent; some due to real increase, but mostly a manifestation of demographic changes resulting in a higher proportion of old people. New hazards also have arisen as the pattern of life changes with "development" ; carcinoma of the lung can be related to excessive smoking and smoke-laden atmospheres; suicide, alcoholism, and supposeciy stress diseases are directly or indirectly nervous phenomena, the response of the human to the strains of a highly organized society. There is still much barring the way to Utopia. C. P haser EfcncxiifGTOX, in Hobson, W .: The Theory and Practice of Public Health, London: Oxford University Press, l'>6 l, p. 7. .I V. S t i l b u r y 5351 -Jo So O 6 s fi. m ' TOXICOLOGY OF HERBICIDES SV. DALGAARD-MIKKELSEN a n d EMIL POULSEN Department of Pharmacology and Toxicology, Royal Veterinary and Agricultural College, Copenhagen, Denmark Tails or Contnt I. Introduction........................................................... 235 II. Inorganic herbicide*................................................................................................... 236 A. Arsenite................................................................................................................... 226 B. Sodium chlorate...................................................................................................... 227 C. Sulphuric acid...........................................................................................................227 III. Organic herbicides......................................................................................................... 227 A. Chlorinated phenoxy-acids......................................................................................227 B. Chlorinated aliphatic acid* and their sodium salts..............................................231 C. Carbamates and ally! alcohol..................................................................................234 D. Substituted urea*..................................................................................................... 236 E. Triasines................................................................................. 238 F. Substituted phenol*..................................................................................................340 G. Miscellaneous organic herbicides............................................................................ 241 1. Tributyl phosphorotrithioats (D E F )................................................................ 241 2. Maleic hydrazide................................................................................................. 243 3. EndothaJ sodium................................................................................................. 243 . 4. Diquat (Regione)................................................................................................ 244 5. Aminotriazole...................................................................................................; 244 IV. Conclusions.....................................................................................................................245 I. INTRODUCTION In their endeavours to raise productivity, agriculturists have made use of chemicals to a steadily increasing extent. For the purpose of controlling injurious insects, fungi, weeds, etc., a great number of pesticides has been developed, among which the insecticides, especially the organophosphatc cholinesterase inhibitors, have attracted considerable toxicological interest. Relatively little has been written about herbicides, t.e., compounds which have been found useful for weed control. Many of the toxicological data underlying assessments of the risk involved by using them in practice originate from confidential, non-published reports placed at the disposal of the authorities concerned. Such data have not been included in the present survey. Of a number of compounds, the toxicology of which has l>ccn elucidated in relation to their use for purposes other than as herbicides, only a brief account will be given with references to relevant literature. Regarding the practical use of herbicides, reference is made to handbook literature (70, 80, 119, 120). Weedkillers usually are classified into two main groups, selective and non-selective. The selective substances can be used on crops without damaging the cultivated plants, whereas the non-selective types kill all vegetation. Substances of the latter group may therefore be used also for destroying potato haulm and for desiccation of green parts of plants which may unduly delay harvesting, in which case they are often called desiccants or de foliants. The herbicides are grouped, according to their mode of distribution in 225 5353 OOW517G99 226 DALGAAUl>-M!KKKI.SEN AND FOtTLSEN the weeds, into contact herbicides, which are active only at the site of applica tion, translocated herbicides, which arc distributed throughout the whole plant from exposed parts of foliage or roots, and Tcsidual herbicides, which are spread on or in the soil and arc effective mainly against germinating seeds. For the present survey of the toxicology of the herbicides, a classification according to chemical configuration has been chosen. Inasmuch as common names, abbreviations, and registered trade marks * are used indiscriminately in the literature, we have, as far as possible, included the common names recom mended for pesticides by the British Standards Institution, marked (*), names approved by the British Weed Control Council (f), and those approved by the Weed Society of America (}). n. INORGANIC HERBICIDES Prior to the Second World War, mainly inorganic compounds were used for chemical weed control. However, as their actions are not very selective and they are often very persistent in the soil, so that damage to cultivated crops has been difficult to avoid, they have been replaced by organic compounds to a steadily increasing extent. Yet, various familiar inorganic compounds are still used as herbicides, e.g., calcium cyanamide, cupric sulphate, ferrous sulphate, mercurous chloride, potassium cyanate, and sodium tetraborate (borax). The toxicological data on these are well known from the handbook literature. The same is true of arsenites, sodium chlorate, and sulphuric acid, but these compounds will nevertheless by described briefly, because their use as herbicides has introduced special problems of toxicity. A. Arsenites Arsenites are used as non-sclcctive herbicides, especially for destruction of potato haulm, as aqueous solutions of potassium-ortho-arsenite (K*As*Oi) and sodium ortho-arsenite (NaA8Oj). The preparations may also contain metaarsenite and pyro-arsenite alkali salts. In rats, the LD50 for alkali arsenites has been found to be 70 mg/kg when administered by mouth and 150 mg/kg when applied dermnlly (32). In domestic animals (40) and man (105) the toxicity is considerably higher, fatalities having occurred in the horse, cow, and man after oral ingestion of 2 to 10 mg/kg. The solubility, preparation, and purity have a considerable influence on the toxicity of arsenic compounds (48); the toxic properties, actions on the animal organism, manifestations of poisoning, etc., have been excellently summarized recently (108). The use of onenites as weedkillers has occasioned an extensive series of acute, often fatal cases of poisoning in domestic animals, especially cattle, which have eaten contaminated crops or residues of sprays (40). Concentrations toxic to man have been found in milk from cows fed on crops contaminated by arsenites (61). Cases of direct human poisoning have also been observed. An instance has been described where one woman died and four others fell ill after drinking water from a reservoir which had been contaminated through a leaking f>ump valve in the o O 3 cn M o 5354 0002881 DOW 517701 TOXICOLOGY OF HERDICIOES 227 tank of an apparatus for spraying arscnitc weedkiller. The drinking-water contained 180 p.p.m . (p a rts p er million) of As (7, 8 ). T his instance of poisoning in Great. B ritain resulted in a voluntary agreem ent lwtwccn industry and the authorities for discontinuing the use of arscnitc herbicides (32). B . Sodium chlorate Sodium chlorate and other chlorates arc extensively used for killing all vegeta tion of farm yards, railway tracks, roadsides, etc., as well as for destruction of potato haulm. Chlorates possess no particularly great acute toxicity, the LD50 for sodium chlorate adm inistered by m outh to rats having been stated to be 1200 m g/kg (32). N evertheless, oral ingestion of both sodium chlorate and potassium chlorate, used in oral hygiene, has caused numerous coses of poisoning in m an (21, 33, 105). In dom estic anim als, too, cases of acute poisoning have been ob served after oral ingestion of chlorates (40). T he mechanism of the poisoning-- mcthcmoglobin production and its consequences--has been well studied (21, 33, 100). A special problem is th a t of the high inflam m ability of sodium chlorate. This can be reduced in commercial preparations by adm ixture with calcium chloride or sodium chloride, but after dissolution in w ater and spraying, dried residues may become ignited. In Denm ark, a tractor driver whose clothes had become impregnated with sodium chlorate during spraying, died of severe burns after they were ignited by a red-hot exhaust pipe. C. Sulphuric acid Sulphuric acid, which in certain countries, e.g., G reat B ritain, is commonly used for potato haulm destruction, is often applied in high concentrations. Accidents with severe skin burns and eye burns m ay therefore occur (32). In halation of sulphuric acid m ay also exert a toxic effect, as indicated by studies on guinea pigs (3, 4) and on volunteer hum an subjects (5). n i . ORGANIC HERBICIDES A . Chlorinated phenoxy-acide Chlorinated compounds of phenoxy-acetic acid, propionic acid, and butyric acid, h av e been very extensively used w ithin th e p a st 15 years os herbicides under the common nam e of "horm one weedkillers." They stim ulate parts of susceptible plants, particularly w ithin the group of dicotyledons, to excessive, uncontrolled growth, which causes the plants to die (109). The m ost im portant compounds, which in the forms of water-soluble salts or lipoid-soluble esters constitute the active components of commercial preparations, are listed in Table 1 , where the LD 50 values in rats are also given. In addition to the com pounds listed in the table, p-chlorophenoxyacetic acid (4-CPA), sodium 2-(2,4(iichlorophcnoxy)ethyl sulphate (2,4-DES-sodium , Scsone, SES), and y-(2 ,4 ,5 trichlorophenoxy)butyric acid (2,4,5-T B , 4-(2,4,5-T B )) are used as herbicides of this type. 5355 0002832 228 DALGAAItD-MIKKKLSEN AND POUL8EN TABLE 1 Chlorinated phenoxy-acidt o O o-si ro 5356 00028G3 TOXICOLOGY OF HERBICIDES 229 T he nciilo toxicity following oral adm inistration to a num ber of experimental anim als is m oderate. T h e LD50 as a rule is of the order of 300 to 700 m g per kilogram for the species examined (89), excepting dogs, which seem to be more susceptible, where the LD50 for both (2,4-dichlorophcnoxy)acetic acid (2,4-D ) and 2,4,f>-T has been found to be 100 m gA g (27). Experim ents with oral adm inistration of various salts and esters of 2,4-D as pure chemicals and as commercial preparations showed no significant difference in toxicity to a num ber of small anim als from th a t of the free 2,4-D acid (53,89). Short-term studies on rats revealed no signs of reduced intake of food or inhibition of growth in response to adm ixture of 2 ,4-D a t 400 p.p.m . in the fodder for 30 days o r 1000 p.p.m . for 14 days. Subcutaneous injection of 50 to 100 m g 2,4-D per kg to mice daily for 90 days had no dem onstrable effect on the general condition, fertility, or tissue histology (53). In Table 2 arc recorded the results of feeding phcnoxy-compounds to larger animals. Except for the rather consider able toxic effect on sheep of 2 ,4 ,5 -T P , adm inistered in the form of propylene glycol butyl ether esters, these experiments showed th a t the large herbivores seem to tolerate prolonged ingestion of phenoxy-compounds to the same extent as rats. In dogs, on the other hand, these compounds were found to have a pro nounced toxic action : 20 m gA g given for 2 to 3 weeks produced severe, fatal poisoning. Parenteral and oral adm inistration of toxic doses of 2,4-D to experimental animals brings about a characteristic complex of signs and sym ptom s (18,27,53), which has been studied particularly in dogs. A fter a few hours the animals display a disinclination to m ove. T his passiveness is gradually aggravated, and a picture of myotonia develops with rigidity of the skeletal muscles and ataxia. The condition may improve transitorily with movement. In severe cases the anim als show progressive apathy, depression, and m uscular weakness, especially of the hindlegs, with periodic, clonic spasms, and, finally, coma. T he m uscular signs are accompanied by marked anorexia; frequently, irritation of the nose and eyes is indicated by scratching reactions. F urther, bleeding from the nose and m outh m ay occur, as well os diarrhea with blood-stained stools. Local irritation of the alim entary tract often causes vomiting. However, this sign m ay be absent, even after oral adm inistration to dogs (27). A utopsy m ay reveal necrotic ulcers of the oral mucosa and signs of irritation with histologically dem onstrable inflam m atory changes and necrosis of the small intestinal mucosa, as well as focal necrosis in the liver (27, 53) and degeneration of the renal tubules. W hen given by m outh to dogs, even in fatal cases 2 ,4 ,5-T has produced only weak signs in the forms of ataxia and stiff movements of the hindlegs (27). As for the hum an response, a report is available (9, 75) of a m an who in a self-experiment consumed 500 mg of 2,4-D daily for 3 weeks, with no perceptible effect. A ease of acute fatal poisoning has been reported from D enm ark, where a young farm worker com m itted suicide by oral ingestion of not less than 500 mg of 2,4-D . W hen found, the body showed signs of having been subject to violent convulsions before the occurrence of death. The changes post mortem were unspecific hyperem ia of the lungs, liver, and brain (78). DOW 517703 5357 0002SC4 OW517704 VV 230 DALGAARn-MIKKELSF.N AND POOL8EN TABLE 2 Chlorinated phr.noxy-aeitia; xhart-term itludit in dog, ekeep, and etna -- Animal Specie* Daily Dost. As Body Wright Xnult Ref. No. MCPA 2,4-D -- Cow Sheep Dog Dog 30 Tolerated without detectable symptoms during (24) 21 days 100 Tolerated without detectable eymptome during (85) 35 days 2-10 Tolerated without detectable eymptome during (27) 90 days 20 3 of 4 animals died, 18th to 49th day (27) 2,4,5-T 2,4,5-TP Sheep Dog Dog Sheep 100 Tolerated without detectable eymptome during (85) 35 days 2-10 Tolerated without detectable symptoms during (27) 90 days 20 4 of 4 animals died 11th to 76th day (27) 100 Lethal after 11 doses (85) The action of ingested phcnoxy-acid herbicides on muscular function, which is reminiscent of that following administration of halogenated acetic acid com pounds (22, 23), suggests an interference with carbohydrate metabolism. Two cases of a transitory diabetiform condition observed in spraying personnel following work with chlorinated phcnoxy-acid herbicides point in the same direction. However, hyperglycemia and glycosuria could not be reproduced with certainty in rabbits; only one out of live animals responded in this way in ex ploratory experiments, where the doses ranged from 125 to 500 mg/kg and the period of administration from fi to 50 days (68). The results of a long .series of investigations into the toxicity to domestic animals and game of herbicidal preparations and treated crops suggested that acute poisoning caused by consumption of crops from sprayed fields is unlikely to occur (16,24,46,75,87), excepting that "grass-eating" dogs may be poisoned by newly sprayed lawns. Human beings and domestic animals are. presumably liable to poisoning only by oml ingestion of highly concentrated preparations or spray solutions. A special problem in connection with the use of chlorinated phcnoxy-acid compounds is the tendency of the preparations--even in extremely low concen trations--to impart to water, milk, and other nutrients a very persistent chlorophenol-like odor and taste. Spilling of highly concentrated preparations close to wells or water courses has in several instances, by percolation through the strata, given a disagreeable taste to the water for long periods afterwards. Administra tion of 2,4-D to dairy cattle does not seem to result in excretion of Biologically demonstrable amounts in the milk (75). 5358 00028G5 3 TOXICOLOGY OF HERBICIDES 231 Very few toxicological d a ta arc available regarding other chlorinated aromatic acids, introduced alone, o r in com bination as pre-emergence and selective herbi cides for agricultural crops. 2,3,6-Trichlorophenyl-acctic acid (fenaej), 2 ,3 ,6 trichlorobenzoic acid (2 ,3 ,6 -T B A tt, TBA , TCB) and 3-amino-2,5-dichlorobenzoic acid (amibenfl are used for these purposes. T he LD50 of 2,3,6-T B A a fte r a single oral dose to ra ts was sta te d to be w ithin th e range of 700 to 1500 m g/kg (32). B . Chlorinated aliphatic acids and their sodium salts The chlorinated aliphatic acids possess more general phytotoxic properties. Some of these arc used, therefore, for defoliation and desiccation of cultivated plants prior to m echanical harvesting. The m ost im portant compounds are: sodium trichloro-acetatc (T C A -ft sodium), 6odium monochloro-acctate (SMCA), and sodium -dichloropropionate (D aJapon-sodium *tt), as well as the combined compound, 2-(2,4,5-trichlorophenoxy)-ethyl-,2',2'-dichloropropionate (Er- bonft). j Studios on the acute toxicity of sodium trichloro-acetate have shown th a t this substance is not very toxic (118), th eL D 5 0 after oral adm inistration being 3320 m g/kg for rats and 4970 m g/kg for mice. T he animals quickly go into an ones- thesia-like state, which lasts for 36 hours, to be succeeded by coma and death, or by awakening and survival. Sodium trichloro-acetate has a far less local- irritating action than the free acid, which can corrode the skin and mucous membranes. Sodium monochloro-acetale (SMCA) is considerably more toxic. Its action on bacteria and in the anim al organism is reminiscent of th a t of other monohalogen- substituted acetic acid compounds, such as monoiodo- and monobromacetic acid, the enzyme-inhibiting and bacteriostatic properties of which have been thoroughly studied (vide 22). T he LD50 after oral adm inistration has been found to be 76 m g/kg for rats, and 80 m g/kg for guinea pigs (118), and of the same order for geese (20), whereas for mice the toxicity is somewhat lower, th e LD50 being 255 m g /k g (118). I n th e sm all experim ental anim als ap a th y an d loss of weight were noticed, and in the fatal cases death occurred within 3 days. The fatal oral dose for young c attle was found to lie w ithin the range of 100 to 150 m g/kg. Colicky-like restlessness and incoordination were observed, developing in the course of 4 to 5 hours into universal fascicular tw itchings, gnashing of the teeth, anxiety, dyspnea, and tachycardia. Com a and death followed after 9 hours (23). T his picture, which is probably referable to blocking of oxidative m etabolic processes, is rem iniscent of th a t seen in pigs (22) and dogs (6) poisoned with monobromacetic acid. The gross a n d microscopic findings a t autopsy were likewise identical with those described fo r monobrom acetic ocid-poisoncd pigs (22). Sodium 2 ,2-dichloropropionate (D alapon) h as become of p articu lar interest owing to its hcrbicidal action on m onocotyledons, such os grasses, in relatively small quantities, therefore being useful as a selective herbicide in certain crops of cultivated plants. D alapon is absorbed by plants and then translocated. On this DOW517705 5353 0002SG6 l o <o <o o u oo o -vj TABLE 3 Carbamate -- m SOAATS/AOQ DALQAAIID-MIKKEL8EN AND POULSEN ( i , S-lH chloroallyl-di-isopropyl tkiolcarbam ate 3-Ckloroaltyl-NN -dietkyl-dithiocarbamate Sodium N-metkyl dilkio-carbamate dikydrale 3,3-DimetkylUlrakydro-t ,3,3 ,tH-tkiadiatine-t- tkion* en CO oe oo CI 0002 T O X IC O LO G Y O F H E R B IC ID E S CH, \ CH /\ CH, \ CH, \ CH / CH, O N - C - S - C H , --CC IC IIC l Avadcx 4 CH,-CH, S NN - - S --CH.-CCI'.CH, / CH,-CH, COfiC.tt Vegadex* 8 CH,--NH--<5 --S--N 8MDC,} methamaodium, Vapam* S-CH , / B -C \ N -CH , w CH, / CH, DM TT.t Mylona* 395 (119) 850 (119) 830 (29) 500 (119) LOLLlittpQ DOW 517703 2 3 4 DALGAAKD-MIKKELSEN AND POULSEN account, prolonged feeding experiments have been conducted with a view to assessing the possible risk involved by ingestion of residues in vegetables. The toxicity of the substance has l>ccn studied from several aspects through a com prehensive series of experiments (81). Administration of single doses to a number of small experimental animals has given LD50 values for rats, mice, guinea pigs, rabbits, and chickens within the range of 4000 to 9000 mg/kg. Two young cattle survived 1000 mg/kg given by mouth daily for 10 days. Beyond transitory symptoms in one of these (anorexia, diarrhea, indisposition), no signs of a toxic action were found by clinical or pathological examination. After oral administra tion of 15,50, and 100 mg/kg daily to dogs for one year, blood and urine analyses, as well as liver function, tests and histological examination of tissues revealed no signs of a toxic action, beyond an increased weight of the kidneys following the dose of 100 mg/kg. Examination of tissue specimens from the dogs showed up to 78 p.p.m. of Dalapon in kidney and liver tissues (81). Feeding of rats with Dalapon for 2 years in doses of 100,300, and 1000 p.p.m. in the fodder, corresponding to about 5, 15, and 50 mg/kg daily, was tolerated with no signs of a toxic action, apart from a minor increase in kidney weight on the largest dose. In these experiments with 1000 p.pjn., 10 to 30 p.p.m. of Dala pon was found on chemical analysis of the liver and kidney, and 20 p.p.m. in milk. Thus, cumulation docs not take place. In rats, reproduction and lactation proved to be uninfluenced through three generations with daily administration of 300 to 3000 p.p.m. of Dalapon in the fodder (81). The local-irritating action of Dalapon has been studied on rabbits, the skin of which was exposed daily for 10 days to a 10% aqueous solution. No more than signs of a mild, transitory irritation was seen (81). C. Carbamates and ayl alcohol Within this group of herbicides, which, as shown in Table 3, comprises carba mates, thiocarbamatcs, and dithiocorbamatcs, is a number of compounds which are used especially as pre-emergence or selective herbicides on certain crops. Propham and chlorpropham have also been used to prevent potatoes intended for consumption from sprouting while stored. The toxicity of these compounds with prolonged ingestion is therefore of particular interest. Propham (0-/sopropyl N-phcnyl carbamate, IPC). Various investigations have shown th at carbamate estera possess a carcinogenic action. Thus intraperi toneal injection of isopropyl carbamate raised the frequency of lung tumors in a strain of mice from 17 to 90% (06). The idea of inquiring into a possible carcino genic action of Propham, which splits off aniline by acid hydrolysis, therefore suggested itself. However, with prolonged oral, intramuscular, and intrapleural administration of Propham to rats and mice, no signs of a carcinogenic action were found (56). This is in agreement with the results of experiments on groups of four rats each, given 400,800, and 1600 p.p.m., respectively, daily in the fodder for 3 months. No signs were demonstrable here of a toxic action on the general condition, growth, or fertility, and no pathological changes were seen a t autopsy or on histological examination of the tissues (101). 5362 00028G9 TOXICOLOGY OF HEIIIUCIOF.S 235 Experiments with the chemically closely related Chltrrpropham (tsopropyl-N[3-c.hlorophcnylJ carbamate, CII'C) have shown it to have a very low acute toxicity to rats and rabbits, LD50 by oral administration having l)ccn found to l>c of the order of 5000 mg per kg for both species (115). In feeding experi ments on male rats given 310 to 20,000 p.p.m. for 90 days, no effect was observed on growth. Doses of 1250 p.p.m. and higher produced an increase in weight of their livers, without histological changes being demonstrable (116). Jn the light of the above investigations, the observation of the development of skin tumors following painting of the back skin of Propham- and Chlorprophamfed rats with croton oil is of considerable theoretical interest (34). However, comprehensive feeding experiments (67) with administration of 2000 p.p.m. through 2 years to rats and 1 year to dogs revealed no carcinogenic effect. Feeding a t 20,000 p.p.m. provoked in both species signs of a toxic action, which was manifested in rats by retarded growth, increased mortality of the male animals, and increased liver and kidney weights, though without demonstrable histological changes. In dogs, retarded growth, increased weights of liver, kidney, and spleen, and splenic congestion were noted (67). Barbanc (4-chloro-2-butynyl-N-[3-chlorophenyl]carbamate) displays a some what greater acute toxicity than the Propham compounds, the LD30 after oral administration having been found to be 600 mg per kg for the rat and rabbit, and 2-10 mg/kg for the guinea pig. Dermal application of 1600 mg/kg over a period of 24 hours caused no deaths among rats. Oral administration of 9, 19.5, and 37 mg/kg daily for 22 days produced no toxic reaction, whereas 75 mg/kg over the same period effected loss of weight. Feeding experiments with rats showed no toxic action of 150 p.p.m. for 18 months (36). Barbane is a potent skin-sensitizing agent in man, in whom allergic reaction with rash develops at subsequent contact. Protection against cutaneous contact is therefore necessary during its use. Plastic (polyvinyl chloride) seems to be a more suitable protective material than rubber (36). SMDC (sodium N-mcthyl dithiocarbamate dihydrate, metham-sodium) has a special sphere of application, being used for killing weed seeds, soil nematodes, and the like by so-called soil sterilization. When applied in the soil it liberates gaseous methyltsoihiocyanaUs, the active substance, which is also available commer cially as an aqueous solution (Trapcx) for similar purposes. The LD50 of SMDC by oral administration to rats has been stated to be 820 mg/kg (29). The toxicity of mcihylisothiocyanate is considerably higher, the LD50 being 97 mg/kg (82). The main toxicological interest attaches, however, to the pronounced locally irritating action of these substances on the skin and mucous membranes, espe cially those of the respiratory organs, as well as the possibility of absorption of toxic amounts by these routes. The LD50 of SMDC after dermal application to rabbits has been found to be 800 mg/kg (29), whereas daily rubbing of methylisothiocyanate in 10% ethanol for 9 weeks into rabbit ears gave no more than a weak reaction (82). AUyl alcohol. This unsaturated alcohol has the same range of application as SMDC and methylisothiocyanate; it is applied in solution to the soil, where its DOW517709 5363 000289 DOW 517710 23C DALGAARD-MfKKELSEX AND POULSEN action is strong, but of short duration, so that cultivated plants can bo sown about two weeks later and grow without the interference of weed seedlings. Allyl alcohol has a strong locally irritating action and can be absorbed through the alimentary tract, the lungs, and intact skin. Extensive investigations of its toxicity have been made on experimental animals. The early literature, to which Miessner (74) contributed significantly by his investigations from 1891, has been summarized thoroughly (79). The toxicity and mechanism of poisoning have been studied in the United States within recent years (30, 65, 94, 95). LD50 values after oral administmtion to the mouse, rat, and rabbit ranged from 50 to 100 mg/kg, and after percutaneous administration to rabbits, from 45 to 90 mg/kg. Some time after the administration the animals were found apathetic, but not anesthetized, as they reacted strongly to pain stimuli. Lacrimation was seen, and often also increased secretion by salivary and gastroenteric glands, increased motility of the intestines, dyspnea due to pulmonary effusion, loss of weight, hemoconccntration, and vasodilatation. Autopsy revealed congestion of the organs as well as periportal necrosis (30,65). Local application to rabbit skin produced only transitory signs of irritation. Instillation into the eyes of rabbits resulted after 1 hour in redness and swelling, which were intensified in the course of 24 hours. In some instances keratitis was seen, but the condition returned to normal within a week (30). In acute inhalation experiments on rats, half of the exposed animals died in the course of 1 hour a t an air concentration of 1060 p.p.m.; 165 p.p.m. for 4 hours or 76 p.p.m. for 8 hours gave the same result. Experimental volunteers exposed to air concentrations of 0.78 to 25 p.p.m. for 5 minutes one to three times weekly over a period of 50 days, developed no signs of pulmonary dis comfort or affection of the CNS. The subjects could barely detect 0.78 p.p.m., while irritation of the nasal mucosa was first noticed a t 6.25 to 12.5 p.p.m., and eye irritation at 25 p.p.m. (30). Acrolein (acrylic aldehyde), the aldehyde corresponding to allyl alcohol, has been used for control of water plants and algae by introduction under water. The substance has the disadvantage of being highly toxic to fish, but this seems to be compensated for by its quick elimination. The LD50 after single 'subcu taneous administration is 30 mg/kg for mice and 50 mg/kg for rats. Moderate anesthesia occurs, as well as a few convulsive fits and dyspnea, especially in mice. Autopsy with histological examination of tissues has revealed pulmonary edema, chiefly perivascularly and hyperemia, slight fatty degeneration of the liver, and focal inflammatory processes in the kidney. At poisoning after inhala tion, where 0.3 p.p.m. for 30 minutes has a lethal effect, the histological changes-- edema, hyperemia, and epithelial cell degeneration--are confined to the lungs (95). D. Substituted ureas The most important compounds belonging to this group of herbicides have been listed in Table 4, in which it is shown also that these compounds have a low acute toxicity when administered by mouth to rats. For 3j(p-chlorophenyl)-l, 1- 5364 0 0 0 2 8 Si TOXICOLOGY OF HEKDICIDKS TABLE 4 Subetituted ureae 237 DOW 51771 dimcthylurca (Monuron) the approximate lethal dose by single oral administration to guinea pigs and rats has been set a t 670 and 1500 mg/kg, respectively (28). To the same group belong also N-butyl-N'(3,4-dichlorophenyl)-N-methylurea (W huronft), 1 ,3 - d i(2 ,2 ,2 - trichloro-1 - hydroxycthyl)urca (Dichloral urea, D< '(JJ, DU), and N-cyclooctyl-NW-dimethylurea (OMU). The sulwtituted urea derivatives destroy numerous species of plants, and their activity persists for a long time. They ore used in large doses for eradication of all vegetation of farmyards, railway tracks, roadsides, etc. They may also be used in much lower concentrations on cultivated crops, such as asparagus. Owing to the long persistence of these substances, residues may be present in harvested crops. In feeding experiments on rats given Monuron at 25, 250, and 2500 p.p.m. for 2 years, no effect was observed on survival or frequency of tumors, as compared with control groups and in the colony of normal rats. The conclusion was drawn that "there was no indication that Monuron is canccrogenic" (54). In the group given 2500 p.p.m. the mole rats were seen to lose weight after only one month of feeding. During the experiment mild anemia was also found, and a t the end the 5365 0002892 OOW517712 238 DALGAAIMi-MIKKELSEN AND POULSEN weights of the liver and spleen had increased. On histological examination of tissue specimens none of the groups presented changes attributable to an action of Monuron. In experiments on dogs, which received in their diet 2.5,12.5, and 25 mg Monuron per kg for one year, no signs of toxicity were seen during the ex periment, and a t autopsy with histological examination of tissue specimens no changes were noticed that seemed to originate from feeding with Monuron (54). Bats given 500 p.p.m. of Fenuron in the diet for 90 days tolerated this dose with no signs of poisoning (91). Diuron was likewise tolerated a t 50 p.p.m. While 5000 p.p.m. of this compound for 90 days caused no deaths, but loss of weight, a fall in the number of red blood corpuscles, and pathological changes in the spleen were noted (28). Preliminary experiments using Monuron and Diuron on guinea pigs have shown no signs of allergic skin sensitization. Neither have such reactions been observed in experimental subjects or factory employees working with these com pounds (28). E . T riazinea The announcement of the phytotoxic and plant-growth-regulating properties of a series of aminotriazines (41), especially the 2-chloro-2,4-bis(ethylamino)1,3,5-triazine compound, Simazine, initiated the synthesis of a great number of herbicidal derivatives. The two most commonly employed of these are shown in Table 5, whereas the compounds known under the names of: Propazine}(2-chloro- Caapaud TABLE5 Triazinti Fenaala Cnwwna LADbafJm0P.iasHitaeKtnn/kt-f M. Ne. tie* to Rett S-CUoro-4,8-bit (tlkylamino)-l ,S,S- trianru Cl I V H \H NN -l A-l/ / \/ CHtCHi N \ CHt--CHa Sima*MWt t MOO (42) t-Chloro-4-ethylamino S-isopropylaminot,3,S-lriatin* H CH, \ 4 H ^C / / CH, Cl A in l sum! \H AV '''C H i--CH, _____ 1750 * (43) 5366 0002803 TOXICOLOGY OF HERBICIDES TABLE 6 239 DOW 517713 4,6-Ws[tsopropylaminoj-l ,3,5-triazine), Trictazine (2-chloro-4-diethylamino6-cthyIamino-l,3,5-triazine), IpazineJ (Isodiazinc, 2-chloro-4-diethylamino-6wopropylamino-1,3,5-triazine), Simetoncf (2 ,4-5w[ethylamino]-6-methoxy1,3,5 - triazine), PromctoncJ (2,4 - 6ts[t$opropylamino] - 6 - methoxy - 1,3,5triazine), and Atratoncf (2-ethyiamino-4-wopropyIamino-6-mcthoxy-l ,3 ,5-triazinc), are still being used mainly experimentally. These compounds are almost insoluble and persist for a long time in the soil. They have been used mainly for eradication of all vegetation as well as for local wcodkilling under fruit trees and shrubs. Certain species of plants, e.g., asparagus. and maize or com, arc, however, resistant to the hcrbicidal action of the triazincs. l'hoir use on areas with these cultivated plants is therefore practicable but involves a risk of residues in crops for consumption. Studies on the toxicity of Simazine (42) showed that the LD50 after oral ad ministration to mice, rats, rabbits, chickens, and pigeons in all cases exceeded '*000 mg/kg, whereas all rats survived daily doses of 2500 mg pure Simazine per kg for 4 weeks, and 1250 mg of a commercial preparation per kg. R ats fed for two ycara on a diet containing 1,10, and 100 p.pjn. of Simazine revealed no signs of 5367 0002894- DOW 517711 240 DAI.GAAHD-MIKKELSKN AND POULSEN toxicity (40a), and screening test* for various acute actions, such os spasmoiysis on the isolated intestine and analgesia in mice, did not demonstrate significant activity (40a). The LD50 of Atrnziue (`J-chlnro-4-ethylamino-6-tsopropylamino-l ,3,5triazinc) (43) after a single oral administration was found to be 1750 mg/kg for mice and 3080 mg/kg for rats, while a daily dose of 400 mg/kg through 6 weeks proved fatal to half of the test rats. F. Substituted phenols Dinitrophenols. The checkered history of the dinitrophenols also includes a chapter on their applicability as herbicides. In agriculture these compounds are used also as insecticides, ncaricidcs, and ovicides, for potato haulm destruction, pre-harvest desiccation of leguminous seed crops and apple-thinning sprays. The chances of contact are therefore great, and their users are often poisoned when protective measures are disregarded. Three derivatives are commonly used: DNOC (DNC, 2-methyl-4,6-dinitrophenol), Dinoscb (DNPB, 2-sec.butyl-4,6-dinitrophenol), and Dincx (DNOCIIP, 2-cyclohexyl-4 ,f>-dinitrophenol). The chemical configurations and LD50's for rats after oral administration are shown in Table 6. The mechanism of toxic action which is based on the uncoupling of oxidative phosphorylation, has been studied in detail; a survey has been given in this journal (17). A great num ber of toxicological data have been given in Handbook of Toxicology (77), and poisoning of man and domestic animals has been reported in recent surveys (12, 32, 40, 72, 80, 88). Clinical accounts are available from Belgium (52) and Great Britain (15), among others. Up to 1950 seven British cases had been re ported of fatal poisoning with DNOC used as a weedkiller. Countermeasures against poisoning (14) have since been satisfactory in Britain, no fatal accidents having occurred aftertheir introduction (12,32). Peniaehlorphenol. Like the dinitrophenols, pentochlorphcnol is an effective uncoupler of oxidative phosphorylation. The mechanism of toxic action has been studied in experiments with molluscan and mammalian tissues as well as on en zymes (110, 111, 112, 113). It seems to be due to inhibition of the intracellular transfer of energy-rich phosphates as well os their synthesis. In agreement with this, the symptoms of intoxication include acceleration of respiration, hyperpy rexia, hyperglycemia, glycosuria, and promptly occurring motor weakness. As in cases of dinitrophenol poisoning, rigor mortis sets in early and is very pro nounced (26, 45, 64, 71). The acute toxicity of pentachlorphenol as well as that of its sodium salt have been studied by oral, subcutaneous, and dermal administration of single doses to rats and rabbits (2G, 64, 71, 102), and by single subcutaneous injections into dogs (71). The LD50's and the minimum lethal doses were found to vary considerably, ranging from 27 to 550 mg/kg. The highest toxicity was noted for pentachlor phenol dissolved in mineral oil (fuel oil) given by mouth to rats, and the lowest for the sodium salt administered in the same way to rabbits. After dermal appli cation, mineral oil solutions arc likewise more toxic than solutions in vegetable 0002895 TOXICOLOGY OF IIKliniClDKS 241 oil iiul in wilier (64). Furthermore, the local reaction, which may manifest itself l>y changes from cryllicma to grave dermatitis, is stronger with mineral oil as vehicle ((>1,71). The results of short-term studies on a manlier of animal species suggest a certain degree of cumulative action, especially after subcutaneous injection (71); signs of such have also been seen after oral administration to rats and eats (26). In experiments on calves, on the other hand, cumulative action has not been ob served (51). From the pract ical use of pentachlorphenol, this compound is well known as a skin irritant (12); in addition, toxic amounts may be absorbed through the skin. In relation to its use os a timber-preservative, human coses of poisoning have occurred, some of which have been fatal (73,107). Sodium pcntachlorphenate in aqueous solution with mineral oil added, used as a herbicide in pineapple plantations in Australia, has been described as the cause of poisoning of nine workers, five of whom died. In one, who died after 21 hours, pentachlorphenol was demonstrated by chemical analysis of tissue speci mens in quantities of 2 to 14.5 mg per 100 g tissue (45). In cattle, coses of poisoning have been described which ran a fatal course within 24 hours of oral ingestion of 5% pentachlorphenol in kerosene (97). Mild signs and symptoms of poisoning in sheep and cattle have followed 25 mg/kg (85). G. Miscellaneous organic herbicides 1. Tributyl phosphorotrithioate (DEF). Organophosphates have become very extensively used os insecticides within agriculture and horticulture. A compre hensive literature is available on the toxicity of these compounds, related to their cholinesterase-inhibiting action (55). In studying DKF, which is used as a de foliant in cotton fields, attention has been focused on the relatioaship between the toxicity and the inhibiting action on the cholinesterase activity in different organ tissues (76). The LD50 after oral administration to rats is 325 mg/kg (Tabic 7). With daily intraperitoneal injections of 50 mg/kg into rats for up to 60 days no signs of poisoning were observed, although after sacrifice cholinesterase of brain tissue was found to be 14 % of the average in control groups. Injection of 100 mg/kg was lethal after 5 to 30 days. In acute poisoning experiments de pression and inactivity were seen after one hour. These symptoms persisted for 2-1 to 48 hours, unless irritability or manifestation of pain or tremor was produced by external stimuli. The depression developed in some eases into muscular weak ness with rigidity. Not till 2 to 0 hours after injection did the animals also display the usual signs of anticholinesterase poisoning: profuse urination, salivation, and lacrimation, and respiratory impairment increasing to respiratory paralysis and death (7G). The cholinesterase activity of the red blood cells and brain was often poorly related to the. cholinergic signs, often with freedom from any signs or symptoms despite a low activity level (76). Having licon observed also in cases of poisoning with other organic phosphorus compounds (39, 63), this fact does not militate against the hypothesis of cholinesterase inhibition os an etiological fac tor in the mechanism of poisoning. The somewhat differing manifestations of poisoning, and the failure of prophylactic and therapeutic effects of enormous o O CJl r-* Cl 0002896 ( C fla p u d 8,8,8 -trib u tyl phoiphorotrithioal ,3,3,8-Utrabydro-S,8-diozopyridatine Ditodium 7-ozabicyclo-(3,3,1 )heptant-t,3-di earbozylate, Diaodiura 3,ft-andoxohexahydrophthUta en co Oo O o rj co <) 9,t0-dihydro-8a, lOa-diazoniaphmantkrtn* dibromidi, l.J'-etbylene-a.a'-dipyridylium dibromida 3-amino-t ,3,4-lriatol NBBtOOd Q N V NHSTiaUMlW-iUlVVDaVU TABLE 7 M iecellaneout organic herbicide Fwmttli B(.LD50, mffAfbr C ouM or Abbrcvltld Names Perotal Admia* So. auiioo to Hsu C H ,--CH--C H ,--S CH,--CH,--C H . - 8 - P CH.--CH ,--C H ,-B / A 01 C ch1'^\*1h CH NH V0i H / H tC ^ I ^ C H --COON 1o 1 H,C 1 CH--COON v ^H HC--CH HC--CH HC^ NC -C / ^C H , 2Br' llc--N ^ \ +--CH H^C--cil, HN-- N hA A--NH, V DBF 325 Maleic kydrazide, MH( 4000 Endothal-odium1t 80 D iiu o l/tt Region* 400-440 Amitrole.t ATA, amtxol, 25000 mitol (76) 03) (32) (83) (90) STiATSMOa TOXICOLOGY OF HEIU1I0IDK8 243 doses of atropine (70) suggest, however, that other facto also contribute to wards t he toxic action of DKF. 2. Maleic hydrazidc (1,2,3,6-tctrahydro-3,6-dioxopyridnzine) is used for spraying on roadsides, hedges, and the like, to facilitate and reduce the work with cutting, etc. The growth-inhibiting properties are utilized further by spraying the compound on root crops 2 to 4 works before harvesting, as this greatly reduces the tendency to sprouting during storage. In experiments on a number of small animals, the acute and chronic toxicities of this, compound were found to be low. Thus, for instance, the LD50 after oral administration to rats was 4000 mg/kg, while prolonged feeding experiments on rats with 10,000 p.p.m. of maleic hydrazide in the fodder showed no signs of a toxic action within the normal life span (13). The demonstration of the fact that in Vicii faba roots maleic hydrazide not only inhibited the growth of cells, but also caused breakage of the heterochromatin in the chromosomes (25) aroused a great interest in ascertaining whether animal cells might also be susceptible to this action, or whether it is specifically directed against plant cells. This is im portant, because the ability of substances to produce chromosome breakage may be accompanied by carcinogenesis. In thorough investigations, comprising weekly subcutaneous injections into mice and rats of 500 mg/kg for 100 weeks, as well as feeding experiments over the same period with 10,000 p.p.m. of maleic hydrazide in the diet, no signs of a carcinogenic action were demonstrable. Tests for a possi ble co-carcinogcnic action on application together with croton oil to mouse sk in revealed no greater tumorigenic action than th at of the oil alone. Implantation of maleic hydrazide pellets in the bladder of mice did not give rise to tumor forma tion, and administration of 125 mg of the compound per kg to tumour-affected rats had no effect on the tumour growth or cell mitoses. In experiments with tissue cultures of mouse epidermis and guinea-pig skin, no effect was seen on cell divi sion, mitosis, or tissue respiration (13). The fate of maleic hydrazide has been studied in rabbits. After oral administra tion of 100 mg/kg, the rabbits excreted 43 to 62 % unchanged in the urine in 48 houra. Coupling products were not detected, and the fate of the remaining 40% of the administered amount could not be determined (13). 3. Endothal-sodium (disodium 3,6-endoxohexahydrophthalate), which has strong plant-damaging properties, is used particularly as a defoliant and desiccant in leguminous crops, and has also been tried as a pre-emergence herbi cide, e.g., for sugar beets. The compound, which is closely related to cantharidin, is very toxic. After oral administration to rats, the LD50 has been found to be 80 mg/kg (32), whereas the lethal dose by intravenous injection into rabbits and dogs is of the order of 5 to 10 m g/kg (44). Shortly after administration, scratch ing of the nose was observed, and in the dogs, intermittent retching and vomiting also occurred for 2 houra, followed by death or recovery. In the rabbits, head shaking and irregular, greatly accelerated respiration were noticed (98). For both animal species, death was attributed to respiratory failure. In experiments with isolated ventricles of frog hearts and rabbit atria functional depression was seen in response to a 1 in 20,000 solution of Endothal-sodium (44). Hence, cardiac de- DOW 517717 5371 0002SS8 244 DAI.(AAW)-MIKKELSR.V AND POULREN c prcssion has been supposed to l)c tlm cause of death. However, intravenous in jection of Fndolhal into anesthetized dogs, atropinized os well as non-atropinized, caused a fall in blood pressure and respiratory failure, whereas changes of the oo ECG were first noticed in association with severe respiratory impairment in the terminal phase. While cats reacted in the same way as dogs, although with a less $ 517713 pronounced fall in blood pressure, domestic fowls were found to be insensitive to doses four times larger than those which were active on dogs and cats (98). The effects of Endothal both in vitro and in vivo were found to be characterized by manifesting themselves only after a long latency (98). This points towards a metabolite as the proper active factor. Application of a 10 to 20% aqueous solution to the skin produced erosions which developed into necrosis, in some cases resulting in death owing to cutaneous absorption (44). The LD-50 after dermal application to rats was 750 mg/kg (32). 4 Diquat (Reglone). Diquat (9,10-dihydro-8a,10a-diozoniaphenanthrene dibromide) has been proposed as an agent to produce desiccation and defoliation prior to harvesting, destruction of potato haulm, and as a pre-emergence and a total herbicide. The LD50 after oral administration to rats was 400 to 440 mg/kg, whereas the LD50 after subcutaneous injection was 20 mg/kg, with death occurring in the course of 5 to 7 days. At autopsy, the intestinal canal, especially the cecum, was distended; histological examination revealed signs of gastrointestinal irritation, and in the lungs, thickened alveolar walls, particularly after large doses (83). Intrapcritoneal injection of 500 mg/kg to rats produced cyanosis and convul sions, culminating in death after 2 hours. Subcutaneous injection of 1 mg/kg into rats for 3 weeks caused no Rigns of toxicity. Similarly, feeding experiments to rats over 14 months with .500 p.p.m. in the diet disclosed no carcinogenic or toxic action (83). Following oral administration of CM-labclIcd Diquat, 90 to 97% of the amount of activity given was recovered in the feces in the course of 48 hours (83). This, correlated with the difference in toxicity after oral and parenteral ad ministration, suggests minimal absorption from the gastrointestinal canal. 5. Aminotruuole (3-amino-1 ,2 ,4-triazole) is hcrbicidolly active against nu merous species of plants. After absorption, it is translocated through the root as well as through the foliage. I t breaks down rather quickly in the soil, and the exposed plants become chlorotic, as aminotriazole seems to interfere y ith chloro phyll production (47). In animal experiments, aminotriazole showed a low acute toxicity, the LD.50 after oral administration having been found to be 14,700 mg/kg for mice (114) and about 25,000 m gAg for rats (90). No signs of poisoning were seen after in travenous injection of 17.50 mg/kg to a cat (90), of 1600 mg/kg to mice (90), or 1200 mg/kg to a dog (114). A single intraperitoneal dose of 4000 mg/kg was tolerated by mice, whereas 21 doses of 1000 mg/kg each to rats, distributed over 45 days, produced an increase of the thyroid weightof 328% in males and 410% in females, while at the same time the growth and intake of food were found to bo normal (90). In feeding experiments on rats extending over 68 weeks, ino effect was seen on the growth or intake of food a t 10 and 50 p.p.m. in the diet, as compared with O u t tv 0002899 TOXICOLOGY OF HERBICIDES 245 controls, whereas 100 p.p.m. caused a decrease in growth and food intake in male rats during the last few experimental weeks. However, in male rate given 50 p.p.m. the thyroid Income enlarged after 13 weeks (114). Investigations into the effect- on the thyroids of rats of 2 years of feeding with aminotriazolc am stated to have given the following results: In the control group, there was found one ease of cystic follicle with papillary changes, while among the animals given 10 p.p.m. one out of ten examined presented adenoma; further, after 50 p.p.m. two out of 15 examined had adenoma and one apparently adenocarcinoma. Hats given 500 p.p.m. of aminotriazolc in the diet for 17 weeks, which were then retained on a diet free of the compound for 2 weeks prior to sacrifice, appeared to have normal thyroids a t the time of sacrifice (62). The observat ion that rats fed with 100 p.p.m. of the compound for 2 years "developed a significant number of thyroid adenomas and adenocarcinomas," as well os the demonstration of residues of aminotriazolc in marketed cranberries (31), caused prohibition of sale of cranberries and cranberry products of the 1958 and 1959 crops from certain parts of the United States. Furthermore, it resulted in an official announcement from the Secretary of Health, Education, and Wel fare that the reason for the prohibition was "possible contamination by a chemi cal weedkiller, Aminotriazole, which causes cancer in the thyroid of rats" (37). In the ensuing discussion, doubts were raised as to whether the marked anti thyroid action of aminotriazole can justly be characterized as carcinogenic (10, 62). The mechanism of the antithyroid action of aminotriazole seems, on the basis of experiments on rats, to be identical with that of thiouracil derivatives (la, 62). The absorption of I m by the thyroid is depressed in both rats (la) and humans (10), because its incorporation as organically bound iodine is obstructed (la). Following injection of aminotriazole, a reversible inhibition has been noticed of the catalase activity in thyroid tissue (la) as well as in kidney and liver tissues (49, 50, 104). However, to obtain a definite inhibition of the catalase activity, larger doses of aminotriazole are required than are necessary to depress the ab sorption of I m by the thyroid (la). Hence, inhibition of catalase activity can hardly account for the antithyroid action. Studies, in vitro, using purified catalase preparations from liver and red blood corpuscles, have under special experimental conditions shown irreversible inhibition (69). However, the results of experi ments with thyroid tissue suggest that inhibition of thyroid peroxidase is the essential factor (2), a hypothesis that is borne out by the results of experiments with peroxidase from milk and vegetable tissue, which are likewise inhibited by aminotriazolc (19). I t should be added, however, that, the question has not yet been clarified, since enzymes that are active in the purine metabolism of bac teria arc also inhibited, in these cases reversibly by aminotriazole; this observa tion is utilized experimentally (84,117). IV . C O N C LU SIO N S Apart from the fact that most information regarding the toxicology of the lierhicidcs is os yet rather fragmentary--a situation which will be considerably improved when reports from the biological laboratories of industry become avail- DOW 517719 000 2 9 0 0 DOW517729 246 DALGAARD-MIKKELSEN AND FOULSEN able to the public--it is important that attention not be confined to the toxicity of the original compounds. Their influence on plant metabolism might, for in stance, block synthetic chains, leading to the accumulation of endogenous toxic products in vegetables. In this respect it has been shown that 2,4-D, applied in subtoxic amounts to such plants as sugar beets, increases the concentration of nitrate to twenty times the normal level. Consequently, feeding with the foliage involves a risk of poisoning (tw). Attention has also been focused on the increase of cyanide in cyanogenic plants exposed to the action of herbicides (103). As to the toxicity of metabolites of the herbicides themselves, which are formed in the soil and plants after their distribution, little is known. The problem has been touched in connection with investigations into the breakdown of herbicides by the aid of the micro-organisms in the soil. In particular,'the decomposition of the chlorinated phenoxy-ncids and the chlorinated aliphatic acids by soil bac teria and fungi has been submitted to extensive studies (vide 11, 57, 58, 59, 60, 93,121). I t appears that the breakdown of these compounds is almost complete, involving dehalogenation (57-60) and hydroxylation of the side-chain (93). Therefore,^persistent deleterious effect on the microflora of the soil need not be feared after use of the great majority of these organic herbicides (38). This is probably due to the fact that in most cultivated soils the populations of bacteria and fungi are so rich that there will always be found a t least a few individuals that con adapt to almost any substance presented to thepij These will synthesize new specific enzyme systems using the foreign substance as substrate. The re sistant strains of micro-organisms will therefore multiply excessively, until the "substrate" has been decomposed (11) YIn general, the biotransformation of herbicides in soil does not seem to lead to the production of more potent compounds7(ll, 93, 121). Certain inorganic herbicides, e.g., chlorate, may, however, have a deleterious influence on bacterial nitrification in the soil. By reduction of chlorate, hypochlorite may be produced, which has a very strong inhibitory ac tion on the growth of the soil bacteria (1). Pentachlorphenol may have a harmful effect, especially on the soil fungi; various emulsifying and surface-active agents in the hcrbicidal preparat ions may also interfere with the soil flora (38). . The fates of the herbicides in plants have as yet been scarcely elucidated, but intensive studies are being carried on to clarify biotransformation in plants. The explanation of selective phytotoxicity has been founded on these studies;'Uccisive progress was gained when it was discovered that the herbicidolly inactive 4-(2,4-dichlorophenoxy) butyric acid is converted by beta-oxidation to the active 2,4-dichlorophenoxyncctic acid in plants where specific beta-oxidase systems are present. These plants arc harmed while species not possessing the specific en zymes are resistant (109). Conversely, the triazinc derivative, Simazin, in non sensitive plants is degradated by the presence of different factors such as catalase, peroxidase, and polyphenols to substances without phytotoxicity (46a)J Great difficulties in analytical procedures have to be solved before the fates of Jrerbicides in plants are clarified ^92). However,-there is reason to expect th a f c more rational basis will eventually be available, both for the study of the metabolism of herbicides in the animal organism, and for the planning of experimental in- 5374 0002901 TOXICOLOGY OF HERBICIDES 247 vcstigatious of the toxicity of the original compounds as well as of their metabo lites, A knowledge of persistent metabolites in crops for consumption especially will lie of very great importance in the study of long-term actions, including carcinogenic effects (35). REFERENCES 1. Aaxno.B.:OntheBiechariiom of thetnxio actionalohlertlea aadoamoraUtedoaholi rumoa youngwheat piaale. KiinxL LaotbrllAmk. 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