Document 59JVpNxvpaxrBoKo79bpK12R

tt* 1 P iu iiu L u o l DI O TES T .Po.k(*\q.Iy::tiiiJ; PiCu 1E 10 f R O N TAG t ROAD N O R Tu nR O oi;. Il l i n o i s 00062 A a t A C O t J C 119 humdnc m*)ot f S e p t e m b e r 9 1968 M r. E lm er P . W heeler Monsanto Company 800 N. L indbergh Blvd. St. Louis, M issouri 63166 D ear M r. W heeler: I am subm itting a collection of protocols for studies w ith A ro clo rs. The studies proposed, with p ric e s, a re as follows: 1. Rat T is s u e Study A roclor 1 2levels A rodov II 2levels A ro c lo r ?TJ ? i ^\*^|r Toxaphcnc 2levels C o n tro ls T o ta l $2,700 Additional refe ren c e m ate ria ls could be included fo r $600 p e r m a te r ia l (2 le v e ls ). This p rice Is exclusive of an aly ses. If you choose for us to do all or p a rt of the i.nr.Jytical w ork, we could w ork on a cost plus basis o r we could p ro vide you with a quotation after we have run through the m ethod. Wc found no re a lly u se fu l in fo rm a tio n in our scan of the l ite r a tu r e on the metabolism of chlorinated pesticides. I feel that we can get much inform a tion from analysis for tissu e residues and from study of u rine and fcccs collected from treated an im als: Jt is not possible to quote for such studies in the absence of fore-know ledge of findings. This, could be se t up on a cost plus basis with provision to review after the expenditure of a defined sum . One should be able to at le a st define the problem on a budget of $5,000. NEV 0 3 1 1 0 9 780647 M**< E lm er P . lVh e e le r S e p t e m b e r 9 1968 ' Page 2 II. Chlchcn Toxicity, Reproduction and Meat and E gg Residue Study T hree A ro d o rs with common controls E x clu siv e of i.'.*.V/Lical c o sts $15,000 ILt. M allard Duck Study T hree Aroclovs with common controls E x clu siv e of anr.ly t Ic a l.c o sts $5,000 IV. 3-G cnoration Reproduction Study in Rats T hree A roclors with common controls $35,000 V. 2 -Y e a r Chronic Toxicity Study in Rats T hree A roclors with common controls $57,500 V I. 2 - Y ear C hronic T oxicity Study in Dors Th ree A roclors with common controls VII. Subacute Fish .Toxicity Study We have not done such a study but we have b u ilt one apparatus which allows investigation of as m any as five concentrations of one m a te ria l plus controls sim ultaneously fo r extended p erio d s of tim e, startin g with fingerlings and continuing through a reproduction cycle if this is desired. If-one w anted only to m easu re the accum ulation of test m aterials over a 90-day period, the cost, using 5 concentrations, would be ap proxim ately $1,500 per species for one m ateria l, exclusive of analyses. Extending the study through a reproduction cycle would cost an addition al $3,500. In any of these stu d ies, m a te ria l (urine, feces or tissu e s) could be retained for analysis to determ ine species differences relative to accum ulation and m etabolism . NEV 0 3 1 H 0 r 780648 * M r. E lm e r P . Who cio r S eptem ber 9 1968 Page 3 The cum ulative cost of the defined studios, exclusive of analytical costs and fish s tu d ie s, am ounts to $200,200. As D r. Cal and rn h a s told y o u / billin g s could l>c a rra n g e d to accom m odate to your budgetary needs. Your com m ents on this p ro g ra m w ill be w elcom e and should fu rth e r per sonal discussions seem d esirab le, we will be glad to m eet with you cither h ere or in St. L ouis. Sincorcly yours Otic E. F anchcr, P h. D D irector one. NEV 0 3 1 1 1 1 r 780649 9 f& tiki l I l i o - K 1*10 lOAD NORTHKROOi:. ILLINOIS 6 0 062 . ARIA C C O I Jt: mtPttoMc }<;oio 9nc. PROTOCOLS FOR MONSANTO COMPANY AROCLOR STUDIES NEV 0 3 1 1 1 2 780650 \I PROTOCOL FOR . MONSANTO COMPANY CHICKEN TOXICITY, REPRODUCTION AND RESIDUE STUDY ON AROCLOR The object oi the study w ill be to in v estig ate the g e n e ra l toxic e ffects of the test m aLerial on young adult m ale and fem ale chickens with special em phasis upon hatchability of eggs and viability of offspring. In addition sam ples of m eat and eggs will be analyzed for test m a te ria l resid u es (cither by M onsanto or by B io -T e st). S ixteen m ale and 80 fem a le 12-w eek old w hite L eg h o rn ch ick en s w ill be selected for the study. These anim als w ill be equally divided into one rmitvA 1 i rwi fViwa^n I*^e^rri*fton*c"* TVa* fV**wa^awi aTMa*** "a ,.g|P " Va*^ Ivn graded dietary levels of the te s t m a te ria l. Dosing w ill begin Im m ediately and will continue through the egg collection period. The chickens in the control and test groups w ill be w eighed weekly and observations for m o rtality , reactions and general food disappearance ' w ill be m ade periodically during the study. . As each group begins laying (at 17 to 18 w eeks o f'a g e), the eggs w ill be collected daily and egg weights recorded. When the egg weights a re co n sisten tly betw een 50 and 60 g ra m s , and at le a s t 20 p e r cent of the hens a re laying, the daily egg production w ill be collected and placed in an Incuhnlor tra y . Egg collection w ill be continued for 28 days fro m collection NEV 0 3 1 1 1 3 780651 M 13 3 11 U U L] Day One o r until at l e a s t 100 e ggs p e r g io u p a r c a v a il a b le fo r h a tc h in g . P u r lug the sto rag e perio d (not to exceed five days fol; any group of eggs)# the eggs w ill be lurried tw ice daily. The eggs w ill be placed in an incubator m aintained at 99 to 100F with a wet bulb reading of B5F. The egg production for an additional week will be collected for analysis. Total egg production and egg weights will be recorded. At the end of the egg collection period, one-half of the .chickens in each group will be sacrificed, dc/eathcred and quick-frozen. The c a r casses and the eggs retained for analysis will be shipped to Monsanto ot will be retained for analysis at B io-T est. The rem aining chickens w ill be placed on a 30-day reco v ery regim en 1 during which lim e the test m a te ria l w ill be rem oved fro m the te st d iets, i'.ggs w ill be collected once each w eek. A t the end 01 this p e rio d , the r e m aining chickens w ill be sa crifice d and handled in the sam e m anner as those at the end of the test feeding p erio d . The c a rc a sse s and eggs w ill again be * retained for analysis. T he chicks fro m the f i r s t g e n e ra tio n w ill be o b se rv e d fo r 3.0 days post-hatching. ... KJP:DMS 9-9-6?. NEV 03111A r 780652 L PROTOCOL FOR MONSANTO COMPANY DUCK TOXICJTY AND RESIDUE STUDY ON AROCLOR f The object of the study w ill be to Investigate the g e n eral toxic effects of the test m ate ria l on young adult m ale and fem ale m allard ducks and to determ ine residue levels in specific.organs and whole c a rc a s s . Twenty m ale and twenty fem ale young adult m allard ducks w ill be selected for the study. T hese anim als will be equally divided into one control and th ree te s t gro u p s. The th ree te s t groups w ill co rresp o n d to graded dietary levels of the test m aterial. r*r*Vt/* r .i n r. V .e .ir>. i K. o r r t n t r r i ' r.r*r? o i o s( r r n n r , w i l l V 'v A r r l i p f M '^ r V l v p r* rT observations for m o rtality , reactions and general food disappearance w ill be made periodically during the study. At the end of a 30-day feeding period, one-half of the anim als in each group will be sa c rific e d , defeath cred and subjected to a g ro ss pathological *exam ination. The liv er, kidneys and sam ples of skeletal m uscle and fat * will be rem oved from each bird and packaged separately. T hese tissues * will be quick-frozen and retained for analysis for the te s t m ateria l The rem aining ducks w ill be placed on a 30-day recovery regim en during which tim e the te s t m a te r ia l w ill be rem oved fro m the test* dic*ts. NEV 0 3 1 1 1 5 780653 A1 the end of th is p e ri oil, the: rem ain in g d u ih s w ill be s a c rific e d and handled in the sam e m an n e r a s those at the end of the te s t feeding p e rio d . R JP :D \!S 9-9-68 NEV 0 3 1 1 1 6 r-----------780654 `*i * `PROTOCOL FOR MONSANTO COMPANY THREE-GENERATION REPRODUCTION STUDY OF AROCLOR * IN ALBINO RATS I, G eneral A th re e -g e n e ra tio n rep ro d u ctio n study w ill be conducted w ith S p rag u e-D aw ley d e riv e d albino r a t s . A to ta l of 96 w e an lin g s (32 m a le s and 64 fem ales) w ill be equ ally subdivided into th re e te s t g ro u p s and one control group. The th re e te s t groups w ill co rresp o n d to the feeding of th re e graded d ie ta ry lev els of the te st m a te ria l. II. F irs t G eneration A. Parental Animals * . 1. D ie ts and F e e d in g A ll diets w ill be prep ared in the c e n tra l diet room of the la b o ra to rie s. T he b a sic ra tio n w ith which a ll d iets a rc p re p a re d Is a . standard, pulverized ra t stock diet*. The diets for any given te st group w ill be p re p a red by adding a calcu lated w eight of the te s t m a te ria l to a % pre-weighed portion of the stock diet. T he am o u n t o f food allo tted p er w eek to each r a t Is to be sufficient for ad libitum feeding. H ow ever, checks w ill be m ade daily to e n su re th a t the food j a r s a rc not em pty. NEV 0 3 1 1 1 7 Purina Rat Chow, R alston Purina Company; St. L ouis, M issouri. 780655 f c h f A t o l 111 U - i fc b l JGtowtoUti, tec. . l, 9 Z . Tlociy W eights pncl Weight C ains * Tho body weight of each ra t in every group will be d eter m ined and reco rd ed initially, and weekly th ereafter until the anim als a r e 100 days old, '. 3. M ortality, Reactions and G eneral O bservations M ortality and abnorm al behavioral reactions will be recorded dally. The anim als also will be observed for fertility , length of gestation and lactation perform ance, 4. M ating P rocedure W hen th e a n im a ls r e a c h 100 days of a g e, each m ale W ill be random ly m ated with two fem ales from the sam e group. A fter sue* ccssful copulation, determ ined by the presence of a copulation plug or blood In the vagina, the m ale w ill be rem oved from the fem ales and returned to h is o rig in al cage. Any m ale failing to copulate within two cstrus cycles of the fem ale will be replaced by another m ale of the sam e group. H ow ever, no m ore than two m ales w ill be used per fem ale during a given breeding cycle. R ecords w ill be kept of the num ber of successful copulations observed, the num ber of e stru s cycles req u ired to obtain a m ating, and the num ber of resulting pregnancies," T hese data'w ill be . Utilized to calculate m ating and fertility indices. T h e F l a l i t t e r s obtained w ill be w eaned at 21 days p o st partum , Tho fem ales w ill then be givon a 10-day re s t period and again m ated, the above procedure being repeated to obtain F ib weanlings, NEV 0 3 1 1 1 0 * "W . 4 *-*li'V*l* 780656 l' 1 v ' 5. Pathologic Studies A lter tho second HUcr has been weaned (following approx im a tely 33 w eeks on te s t), 8 m a le and 8 fem ale a n im als fro m each group will be sacrificed and gro ss pathologic observations will be m ade. Organ weights will be taken on the liv e r, kidneys, spleen, gonads, h e art, b rain and any organs which appear abnorm al. A com plete set of tissu es will be rem oved and fixed in a ten per cent form alin solution. * In addition, m icroscopic exam inations will be conducted upon five m ales and five fem ales from both the control and the high te s t groups. If abnorm alities are noted, the affected organs of the lower te s t group anim als will also be examined, NEV 0 3 1 1 1 9 780657 A The following lis mice and organs will bo exam ined: T issues and Organs Examined G rossly H eart Trachea Lungs L iv er Pancreas Esophagus Stom ach Intestinal tract Spleen Lymph nodes Kidneys U rinary bladder T este s O varies P ro sta te Sem inal vesicles U icrus P ituitary gland Adrenal glands Salivary glands Thyroid glands Skeletal m uscle Bone Peripheral nerve Eyes B rain Thymus Optic nerves Aorta ' Spinal cord T issues and Organs E xam ined M icro sco p ically * H eart (right and left vcnLriclcs) Trachea Lungs L iv e r Pancreas Esophagus Stomach (cardiac, fundlc and pyloric regions) Sm all intestine (duodenum, jejunum and ileum ) Caecum Colon Spleen. Lymph node (cervical and m esenteric) Kidney U rinary bladder T estis Ovary P ro sta te Sem inal vesicles U terus P itu itary gland A drenal gland Salivary gland (subm axillary) Thyroid gland Parathyroid gland Bone m arrow (sternum and fem ur) P eripheral nerve (sciatic) B rain (cerebrum , cerebellum and pons) Spinal cord (three levels) P o st-m o rtem anim als will be examined in the sam e m anner hut organ weights will not be recorded. Throughout gross and m icroscopic exam ination, particular attention will he paid to the rep roductive o rg an s. NEW 0 3 1 1 2 0 780658 I & 1 O * i fc b I -- --- ------------------- B , P ro geny 1, Body Weigh t Data Pups w ill be weighed at weaning. . 2, M ortaHty, Reactions and G eneral O bservations All pups w ill be examined for physical abnorm alities at b irth and the num ber of viable and stillborn m em bers of each litte r recorded. Records of survival at periodic Intervals during the lacta tion period will be m aintained and a final exam ination for physical abnorm alities w ill be made at the weaning of each litte r. 3. Survival Data 4_ Survival indices w ill be calcxilated for vario u s points 1 during the lactation period. On the fifth day of lactation. U tters of g re a te r than 10 pups w ill be re d u c e d to th a t n u m b e r. 4. Pathologic Studies A gross internal pathologic exam ination w ill be made upon any pup appearing abnorm al. No such exam inations w ill be m ade upon progeny which appear norm al. * , * III. Second and Third G enerations 1" --~ -- * . The procedures followed for the second and third generations will be Identical to those described for the firs t generation except that the parental anim als for the F2 generation in each group will be selected from F ib weanlings of that group. P arents for the F3 generation will be R elucted !ro m F2\i litte r s . Ngy 0 3 1 1 2 1 780659 / In addition, ton m ale and ten fem aie pups from the F31> l it t e r s of each te s t group and the c o n tro l group w ill bo s a c rific e d a t w eaning (21 days post-partum ) and subjected to complete gross and m icroscopic exam ination. IV. R ep o rts A complete form al report will be subm itted upon com pletion of oach generation. > ^ n h ri Cl !1 I PROTOCOL FOR I MONSANTO COMPANY 1 W O -Y E A R CHRONIC ORAL TOXICITY OF AROCLOR IN ALBINO RATS I. Outline of Investigation A. T ype p.nd Length! T w o-Y ear C hronic O ral T o x icity B. Animal Species T ested: Charlos R iver Strain Albino Rats C. O rganisation: Group C o n tro l T est! T e st II T est III N um ber of Animals M ale Fem ale 30 30 30 30 30 30 30 30 D ietary Level of T est M aterial iPPr0 None A dm inistered Low Level M iddle Level High Level D. M eans of A dm inistration: V oluntary oral ingestion; ad libitum, feeding of diets containing various levels of te st m aterial. II, P aram eters To Be Investigated A. Body W eights All a n im als w ill be w eighed w eekly d u rin g the f ir s t th re e m onths of the study and m onthly for the rem ain d er of the tw o -y e a r te s t p erio d . NEV 0 3 1 1 2 3 * * *v *- v `f *; 780661 (/< l i; 1U * n o i s. )j. Pood Consumption W eekly food consum ption w ill be reco rd ed for th e f ir s t th re e months of the investigation, Thereafter monthly spot checks will be m ade. C. M ortality and Reactions Cheeks for m ortality and untoward behavioral reactions will b e conducted daily* , * D , Hcm atologic Studies| C linical Blood C hem istry Studies and Urine Analyses * The studies listed below will be conducted on five ra ts of each, sex from the C ontrol Croup and T est Group III at the beginning of the study end a fte r 3, 6, 9, 12, 18 and 24 m onths of feeding: 1. H em atology: H e m ato crit value, hem oglobin co n cen tratio n , erythrocyte count and total and differential leukocyte counts. 2. C linical Blood C hem istry: Blood u re a nitrogen concentration (BUN), scru m alkaline phosphatase activ ity (SAP), blood glucose concentration and scrum glutam ic-pyruvic tra n s am inase activity (SGPT). 3* U rin e A n a ly se s: A lbum in c o n ce n tra tio n , g lu c o se c o n c e n tra - .* tion, m icroscopic elem ents exam ination, p!I determ ination and specific gravity. The above lis te d s tu d ie s w ill be conducted on a n im a ls In the `"V . lower dose group:: if significant findings appear among high dose anim als. NV 031124 780662 r" t i I i / . u i ' J >< k * ^ i* v i P U K, P ath o lo gic Studies 1. G r o s s . . Com plete gross' autopsies will be conducted upon all post m ortem anim als unless precluded by post-m ortem autolysis, all anim als sacrificed in e x tre m is! five m ales and five fem ales from each group sacrificed after three, months of testing and upon all anim als surviving 24 m onths of testin g . At the tim e of each autopsy, re p re se n ta tiv e tissu e s and organs w ill be tak en and fixed in 10 p er cent fo rm alin so lu tio n . A bsolute organ w eights w ill be reco rd ed and org an - body * w eight ratio s and organ - b ra in w eight ratio s w ill be calcu lated and su b jected to s ta tis tic a l a n aly sis, v iz. A nalysis of V ariance a n d /o r " t" - te s ts . The follow ing o rg an s w ill be Included: liv e r, kidneys, sp leen , gonads, h e a rt, b rain and any o th er organ appearing ab n o rm al upon g ro s s exarnlna tio n . 2, M icroscopic M icroscopic exam inations will be conducted upon tissu es and organs tak en fro m se lec te d an im als sa c rific e d In e x tre m is, and five m ales and five fem ales from the control and highest dietary dose, groups * sa c rific e d a fte r th re e m onths and 24 m onths of te stin g . The tis s u e s and o rg an s *to be exam ined w ill include: h e a rt, liv e r , lung, p a n c re a s , s to m ach (card:?., fundus and pylorus), sm all intestine (duodenum, jejunum and ileum ), caecum , colon, spleen, lymph node, kidney, u rin ary bladder, testes, ovary, prostate, u teru s, pituitary gland, adrenal gland, salivary *I * NEV 0 3 1 1 2 5 I 780663 F.lnnd (su b m a x illary ), th y ro id gland, p a ra th y ro id gland, sk e le ta l m u sc le , bone m arrow , peripheral nerve, trachea, spinal cord, eye, optic nerve and braiii (cerebrum , cerebellum and pons). Any significant tissu e or organ changes at the high dose level would also be exam ined at the lower lev els. F. Reports In te rim sta tu s r e p o rts w ill be subm itted a fte r 3, 12 and 18 . ' .months of testing. T hese rep o rts w ill contain pertinent data collected to that date, At the conclusion of the study, th ree copies of a final form al report will be subm itted. ' '-9-5-68 NEV 0 3 1 1 2 6 780664  PROTOCOL* FOR MONSANTO COMPANY TWO-YEAR CHRONIC ORAL TOXICITY OP AROCLOR IN BEAGLE DOGS I. Outline of E xperim ent A, Tyne and Length; Tw o-Y ear Chronic O ral Toxicity B. Animal Species T ested : P ure-B red Beagle Dogs C, Test M aterial: A roclor D. O rg an izatio n : T h re e T e st C roups and One C' o n tro l G roup 1* -Number of D ogs/G roup: E ight {four m a le s and to u r fe m a le s) L* ? T stymie* --* -- - -- - -- . . - - - - PiVrtnn n Wfinft T est Group I - Low level T est Group II - Middle level T e st Group III - High level *1 3. M eans of A dm inistration: Voluntary o ral ingestion; ad libitum t feeding of te s t m ateria l in diet. II, P aram eters To Be Investigated A. Body W eights / ** . '* All anim als w ill be weighed weekly. - B*. Food C onsum ption I s W eekly food consum ption w ill be reco rd ed . C. M ortality and R eactions M ortality and untow ard behavioral reactions will be recorded daily. NEV 0 3 1 1 2 7 1), II om a tul o gic Slug) o a , V>\oo cl Ch cm i st r y Sin d!c s and Uv inc Ana.ly r c s The stu d ie s lis te d below w ill be conducted upon a ll dogs p r io r ro the Inception of the te s t, a fte r 1, 3, 6, 12 and 18 m onths of te s tin g and just p rio r to its conclusion. 1, H em atology: H e m a to c rit, hem oglobin, o ry lh ro c y te count, and both total and differential leukocyte counts. Z% Blood C h e m istry: Blood u re a n itro g en , blood glucose, se ru m alkaline phosphatase, serum glutam ic-pyruvic transam inase and serum glutam ic-oxalacctic transam inase. 3, U rine A nalyses: Albumin, glucose, pH, m icroscopic elem ents and specific gravity. \Nl E . P ath o lo g ic Studies 1 G ross Pathologic Studies Com plete g ro ss autopsies will be conducted upon anim als sacrificed in extrem is and upon all post-m ortem anim als during the inves tigation. At the conclusion of the 24-m onth study, all surviving dog9 w ill bo sacrificed and exam ined. At the lim e of each sacrifice, a com plete set of tissu es will be rem oved from each dog and preserv ed in ten per cent form alin solution for histopathologic exam ination. Also at the tim e of each sacrifice, the weight of the liver, kidneys, spleen, gonads, thyroid, * adrenals, pituitary and h e a rt will he recorded, ^ H is to patholo%ic S tudies H istopathologic exam inations will be conducted on tissu es taken from anim als sa crifice d in e x tre m is, from p o st-m o rte m anim als NEV 0 3 1 1 2 8 i 780666 ami fro m a ll a n im als s a c rific e d a fte r 24 m onth? of feeding. The tis s u e s to be included in the examination; a re as follows: lung, h e art, liv er, pancreas, m esen teric lym ph node, stom ach (cardia, fundus and pylorus), sm all intestine (duodenum, jejunum and ileum ), colon, caecum , kidney, adrenal gland, u rin ary bladder, testes, ovaries, p ro state, thyroid gland, subm axillary salivary gland, parathyroid gland, u teru s, brain (cerebrum , corcbcllum and pons), bone and bone m arrow (fem ur), and skolctal m uscle (gastrocnom ius). F. R eports i In te rim s ta tu s r e p o r ts w ill bo su b m itted a fte r 3, IE and 18 m onths of testing, These rep o rts will contain pertinent data collected.to that date. At the conclusion of the study, three copies of a final form al report will be subm itted. MLK:PS!i 9-5-68 MEV 0 3 1 1 2 9 r 780667 PROTOCOL FOR i, MONSANTO COMPANY *. A TISSUE RESIDUE STUDY IN ALJNO RATS WITH AROCLORS I. Outline of E xperim ent i A. Type and Length: T issu e Residue Study; d u ratio n 30 days B. A nim al S pecies T e ste d : M ale Albino R ats (approxim ately 100 g) *C. O rganization of G roups: ` Group N um ber of Anim als M ale Fem ale D ietary Concentration in ppm C o n tro l 2 15 15 None 15 15 Low do se le v e l 15 1C t t: ~ t j -- . D. T est M ate;ial A d m inistration Diets w ill be p re p a re d fresh weekly by m ixing the calculated quantity of lest m aterial with a standard pulverized rat ration. Food will be allowed ad libitum . II. P a ra m e te rs to bo Studied * Feed Consumption * * Feed consum ption w ill be determ ined weekly for each anim al. B. Reactions Observation.*: fo r untow ard behavioral rea ctio n s w ill be m ade. NEV 0 3 1 1 3 0 r 780668 L . *1 P l f M P !M IV / ** C . G ro jU l io)ciy Upon sa c iific e , organa and tissu es w ill be exam ined grossly for pathologic a lte ra tio n s, . 111. Sam ple C o llectio n At the end of 30 clays all an im als will be sa c rific e d and sam p les of m uscle, fat, liv er and kidney w ill be taken from each anim al. Sam ples of each tissue from each anim al will be frozen and w ill eith er be shipped to Monsanto for analysis o r analyses w ill be done by B io -T e st. OEF:DMS 9-9-66 ** v ' NEV 031131 780669