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THE TOXICITY OF POLYCHLORINATED POLYCYCLIC COMPOUNDS AND RELATED CHEMICALS Author: Rennie D. Kimbrough* Center for Dilease Control Atlanta, Ca. Referees; M. M. Uarites J. A. Greff Medical Reseated Council MRC Toateolotr Unit Medical Reaaarcd Council Laboratories Canhaltnn, Sumy Eniland I | INTRODUCTION Only (he small group of the polychlorinated polycyclic compounds given in Figures I and 2 will be reviewed in this article. Several years ago when this laboratory was the Atlanta Toxicology Branch of (he Food and Drug Administration, concern was voiced over the possible long-term effects of polychlorinated biphenyls, and we began to study (he (oxicily of Aroclor 1254 and Aroclor 1260. About this lime a follow-up study was performed on a group of workers in New Jersey who were engaged in the production of 2,4,5-T.1 These workers had very severe chloracne and had previously been shown to have porphyria.2 During that same time, a petition was filed with the Food and Drug Administration for a residue level of hexachlorophenc on certain food crops. Hcxachlorophene, as well as 2,4,5-T, is- made from trichlorophenol. although the chemical reactions re quite different. When we checked the available literature on (he toxicity of hexachlurophene we fell that adequate toxicity data by present standards were not available, particularly not if the compound was meant to be ingested. Further more. Larson3 had pointed out that hexachloro phenc was more toxic than commonly acknowl edged. Hexachlurophene and similar chemicals behave differently from such polycyclic polychlurinaled compounds as the chlorinated biphenyls (PCBs). Hexachlurophene in particular is acutely much more toxic than the PCBs and is fairly rapidly eliminated from the body, while the PCBs have a very low acute toxicity, but are very poorly metabolized, particularly those wlih more than four chlorine atoms on the phenyl ring*. The long-term effects are therefore more critical for compounds such as PCBs. The chlorinated dibenzodioxtns and furans, some of which are extremely toxic on an acute basis, may also show persistence and their long-term effects may be equally as important. These observations show that it would be erroneous to make predictions on long-term effects from results obtained In acute toxicity studies. 'Formerly with the Environmental Protection Agency, Chemblee Toxicology Laboratory, 4770 Buford Highway, ftamhiec, Go 90)41. January 1974 449 '",l '" ........................................................ MQNS 069483 * ** * --v_/~1 Polychlorinated itphenyls (PCS'i) III XX X Polychlorinated Terphtrtyls XX XX II Chlorinated Dtbenrofurans Chlertnsted Dfbenxedtoxln Polychlorinated Naphthalenes FIGURE l. The materials shewn in this figure m mixture) of compounds in which several but usually not H of the positions (-X) are chlorine and the real are hydrogen. The properties of the materials depend on the degree of chlorinetion of the mixture. Alt ere insoluble in water but soluble in varying degrees in organic solvents and bi fits. The trede name for the U.S. product (Monsanto) or the chlorinated biphenyls and terphenyls it Arodor. The trade name it followed by the number. For the biphenyls, the (tret 2 digitt of this number are 12, and for the terphenyts, 54 In eaeh Instance the hat 2 digits give (he percentage of chlorine. lit at Cl Cl Cl Cl HtXACHtOhOMCNC MCHUNIOftffNC *, r -M*tfcy lana-hti() .4,4- trtcMenfhenel) l.l* sthyl*ee-b1*14-eMereehenel> SITHIOMX. r,r-TMe-at(.a*ei<MeraeHeweU lit: ci! 1,1,1' trlchlirt* t'tijNlmr^iWyl rtw (IrtcletSH Irtwsan OTJOD) -O--o~c rntTiciCM 1. t' -TMo-bt(*-chleroyHnol) ,3,,a,S'*TetraclilaPOsaM]rlanUtet (ICC) j.I.I'-TrlcMarcsreanlHee (TCft) FIGURE 2. The compound! in this figure are whits crystalline solids with claerly defined melting points above I MFC. They are practically insoluble in water and soiubls in varying amounts in organic advents and in fait. There containing an OK group, all the above excepting 3,4,4'>trkMoroeatban0lda, are soluble in aqueous alkali 444 CRC Critical Rrrirm in Toxicology m WP iv MONS 069484 t)a In fm Hl> llh ell El. V' ea| r The present review is an attempt to correlate various toxic effects produced by the chemicals listed in Figures I and 2 in order to belter understand and appreciate their behavior. The subjects covered in this article have lately enjoyed great interest and a rapidly growing number of scientific reports are available. Since a number of the chemicals discussed produce similar or related effects, their toxicology is discussed together rather than listing each compound sepa rately, which would have led to a great deal of repetition. A detailed discussion of the various chemical reactions, analytical methods for the determina tion of the compounds, is not given. The review has been written with the toxic effects of the various compounds as the focal point and the literature cited serves to illustrate these points. No attempt has been made to present a complete review of the literature in this area. DEVELOPMENT, USAGE, AND OCCURRENCE A description of the synthesis of chlorimtetl biphenyls (PCBs) can be found in Liebig's Annaien as early as 1681.4 Successful production of diphenyl in commercial quantities was not report ed until 1930.* At that time the chlorination of technical biphenyl was also described.4 Biphenyl itself is fungistatic and used as a preservative for citrus fruits.7 Chlorinated biphenyls are very table, and lave been used for protective coittnp, as plastic resins or gums for varnishes, and for waterproofing and fhmeproofing of wood. The excellent dielectric properties of these compounds were noted as early a* 1930. Until the advent of the PCBs. mineral oil was used ai a cooling and dielectric liquid impregnani for high voltage electric apparatus such as transformers and capacitors. Since mineral oil is flammable and flammable gases could also be produced, restric tions for the use and installations of these trans formers had to be set, particularly for indoor installation. When mineral oils were replaced by the Aroclors, (he fire and explosion hazards were eliminated. This was recognized by the National Board of Fire Underwriters and (he National Electric Code was modified.* Use of Aroclors also greatly reduced the physical size of the capacitors.' A recent summary of the varied uses of PCBs hits been provided by Broadhursl.1 n With a peak production of 42,327 short tons of PCBs in 1970, Monsanto is the sole producer of Aroclors (U.S. tradename for PCBs) in the U.S. Yearly PCB production figures for ihe period 1960-1971 were reported by Monsanto.'1 The production increas ed steadily until 1970 and dropped to 20.236 short tons in 1971 due to a voluntary restriction of the use of PCBs by the company. Trade names in other countries include Kanneclor and Santhaterm in Japan, CJophen. Flendor, Pltenuclor, and Sovul in Europe. The polychlorinated capacitor liquids, com monly called askareis, are mixtures of chlorinated biphenyls and chlorinated benzenes. A variety of other industrial applications10 have been found for PCBs. They have served as fluids for hydraulic, gas turbine, and vacuum pumps, as heat transfer fluids, as plasticizers, adhesives, textile coatings, surface coatings in paints and varnishes, as seal ants, and as part of formulations to prepare pressure sensitive records and color copying papers. The extent of the current use of PCBs in printing processes is unknown. Other suggested uses over the years have included catalyst carrier for polymerization of olefins, conversion of water permeable soil to a non permeable state, and combined insecticide and bactericide formulations. Mixtures of chlorinated biphenyls and chlorinated naphthalenes have been employed to insulate electric wires and cables, particularly when they were used in the mining induslry and on warships.1 * Residues of polychlorinated biphenyls have been reported in the environment by a number of authors in different parts of the world. These reports were usually from areas where industrial pollution was responsible for the environmental contamination. Hidden,13 after finding PCBs in marine flsh and mollusks from Scottish waters, noticed that they were generally highest in samples from the Firth of Clyde and he discovered that the PCB contamination probably originated from industrial sewage. The sewage sludge was trans ported from two Glasgow purification works by boats and dumped in deep water six miles south of Garroch Head. The disposal of sewage sludge on land could lead to the introduction of PCB residues into the terrestrial ecosystem by transfer through food chains. So far PCB residues have not been delected in fresh water flsh in Scotland. Polychlorinated biphenyls hive also been found Jammy 1974 447 * HONS 069485 in wildlife samples collected along live Dutch coast and the Rhine Rivci'4 and in fish and seal in different parts of the Baltic Sea along the Swedish coast.1 * Riscbrough et a).14 compared the presence of PCBs with that of DDT and its metabolites in various birds. These authors found very low concentrations of PCBs in wildlife species in Baja, California, Mexico, and the Gulf of California, which arc very remote areas with a high ratio of DDT to PCB of approximately 9 or 10. In sea birds fiom the Pacific, the ratio was between 5 and 10 and at that time, PCBs were not found in eggs of the Adelie penguin from Cape Crozier, Antarctica. Relatively high PCB concentrations were found in San Francisco Bay, Puget Sound, and San Diego Bay and the ratio of DDT to PCBs in wildlife samples from this area was between i and 2. This is again emphasized by the finding of Dustman et al.17 Twelve Alaskan bald eagle eggs had median PCB residues of 1.6S ppm while 11 eggs collected in Maine, Michigan, Minnesota, and Florida showed a median concentration of 9.7 ppm. Zitko1 * found low concentrations of PCBs in different types of fish taken from several lakes and the St. John River System, New Brunswick, Canada, and the Nova Scotia banks. Fish from the Milwaukee River and Lake Michigan in the mid west purl of the U S , where the river enters into the lake, contained varying amounts of PCBs1 * while fish taken from various small lakes in Wisconsin without industrial discharge failed to show PCBs. Large predators from Lake Michigan contained as much as 10 to 25 ppm PCBs on a fresh weight basis expressed as Arodor 12S4 and salmon eggs contained as much as 12 to 17 ppm PCBs on a fresh weight basis. Further studies by Veith3 of the concentration of PCBs in different rivers emptying into Green Bay indicated a trend towards a decrease in PCB concentrations since the voluntary partial ban on PCB sales by Monsanto was imple mented in September of 1970. If PCB discharges are very high a flthkill may result such as the one in Escambia Bay, Florida in 1969.31 In that particular episode the PCBs had entered a plant's effluent through accidental leak age of heat exchange fluid. The PCB found in this case resembled Arodor 1254 most closely. The peak amount found in water of the Escambia River at the outfall amounted to 275 ppb and the peak concentration found in oysters from Escambia Bay was 3.0 ppm. Arodor residues in sediment samples taken near the outfall readied 486 ppm. When the leakage from Ihe plum was corrected, Arodor levels gradually decreased in this area. The wildlife portion of the U.S. National Monitoring Program analyzed starlings, mallard and black ducks, bald eagles, and fish of several species for PCBs. According to Shekel,33 monitor ing for lerphenyls is also done but not for any of the other chlorinated compounds in this report. Research surveys that include fisheating birds, insectivorous birds, ospreys, woodcocks, and cer tain ducks also monitor only for PCBs and for pesticides. Oysters (Cnssostrea vtrpnlca) are a good indicator of PCB and other chlorinated hydrocarbon concentrations in water and a nation al monitoring program in these mollutks in 15 coastal areas has been developed.33 So far. moUusks have only very occasionally contained low levels of PCBs. In summary, the PCB concentrations in water re low or nonexistent in remote lakes and fresh water streams and can be as high as 50 Mg/I (5 ppb I or higher in polluted rivers such as the Hudson River.34 The PCB concentration is usually higher in sediment from these polluted waters because PCBs are only very slightly soluble in water35 Since PCBs are stored in living matter, they arc concentrated in the food chain and the usual "biomagnification" is encountered as we know n from other poorly metabolized and excreted sub stances. The concentration of PCBs in fish, par ticularly (hose in the upper end of the food chain from polluted riven and lakes, is usually estimated in the ppm range and fisheating birds will have somewhat higher levels of PCBs than the fish from the same geographic location.31 Young animals may contain less PCBs than their older counter parts of the same species in a particular location. PCBs are also infrequently encountered in dairy products, meat, fish, and poultry in ready-to-eat human food.3 7,3 * Occasionally, human food and domestic animal feed have been more extensively contaminated with PCBs. In these instances, the contamination could usually be traced to local misuse of transformer fluid, leakage of PCBs from heat exchange fluid, or contamination of recycled paper with PCBs where the paper had been used for food packaging such as cereal boxes.39 The UJS. Food and Drug Administration has recently Kt temporary toierwees for PCBs in certain food products (see Table 1). 441 CRC Critical Rcriewt m Toxicology MOMS 069486 TA1II I- I Twnporary Tofcranre* Exprenad m Ph(i par Million (ppm) fcftiaMialwd bp U>S. Food and Drop Administration (1) Milk (I'm bails) (2) Ouir>- pfndurlt (fill basis) (3) Poultry (liit basil) (4) Kppf (5) romplote and finished animal feeds (M Animal food component* (7) l-islt and shrllfixh (edible portion) (II) Infant and junior food (91 Paper rood-packaging maleriul 2.5 2.5 5.0 0.5 0.2 2.0 5.0 0.2 10.0 Nme: The lolvranec for paper food-packaging material tkall nut apply lo Mich material separated from the park*ed food by a functional barrier impermeable to PCM migraiion. The l ood and Drug Administration will provide upon request the analytical methods it wiU use for enforcing the temporary tolerances. All of these aspects of PCBs in the environment have been the subject of a number of reports, reviews,141 and conferences.91-3 * Terphenyls and chlorinated terphenyls are used ai reaclor coolants. The terphenyls consist of mixtures of ortho-, meta-, and paratcrpitenyls. Chlorinated terphenyls are produced by Monsanto and termed Aroclor, followed by numbers. The first 2 digits of these numbers are 54 and the last 2 indicate the percent of chlorine such as Aroclor 5460. Aroclor series 25 and 44 consist of mixtures of polychlorinated biphenyls and polychlorinated terphenyls.94 According to Zltko et al.,97 poly chlorinated terphenyls have been found in environ mental samples. Polychlorinated terphenyls do not elute from GLC columns under conditions used for PCBs 9 # which may explain why they have only been detected occasionally, but the way they are used probably also precludes their extensive spread in the environment. Chlorinated dibemodioxins and chlorinated dibenzofurant may occur aa contaminants of cyclic chlorinated hydrocarbons. In 1959 Tonvta et al.9* reported on the synthesis of polyhalodibenzo-p-dioxins. According to these authors, chlorophends, when heated to about 200*0 or more under alkaline conditions and pressure, will form chlorodibenzodioxins, as shown below. '-IX, :.0XX' --* a--^XX' + 2 NaO Under similar conditions a dtlorodibcnzofuran may also be formed. H*0 NtCI y Chlorinated dibenzodioxins and chlorinated diben- show a great deal of difference in the type and it sofurans are formed, or can be formed, as contam amount of contamination and different lots of the d inants in (he manufacture of a variety of products same product may vary in composition, making y which use chlorophenols and chlorobenzenes as toxicological evaluations of these products very itirting materials, such as 2,4,5-T, pentachloro- difficult. Rigorous production control and cleanup si plienol, nilrofen, hexa- and pentachlorobenzene. procedures need lo be implemented so that the m and tri- and telrachlorophenoi. Polychlorinated composition of different lots manufactured by the d biphenyls may also be contaminated with chlor tame company becomes more uniform. id inated dibenzofurans.40'41 and chlorinated diben- Villanueva et ai 49 and Jensen and Renberg44 zofurans may result from polychlorinated reported the presence of chlorinated dibenzo-p- biphenyls under UV irradiation.43 The toxicity of dioxin in pentachlorophenol. Hydroxy- these products varies depending on the position nonachlorodiphenyl ether was also found end mb number of chlorines attached to the phenyl referred to as "predtoxin" under the anumptlon rinp. Products from different manufacturers may that It represented a precursor of dioxin. January 1974 449 J MGNS 069467 Y5T According to Johnson et al.,4* the predom inant dioxin in a commercial penlachlorophenol was oclachlorodibcnzodioxin while (race amounts of hcxachlurodibcnzndioxin and no letrachiorodibenzodioxin were found. Since the technical penlachlorophenol was in sumc respects more toxic in animal studies, than the pure product, the production process of penlachlorophenol was changed to minimize the dioxin concentration and a new product has now been registered under the Federal Insecticide Fungicide and Rodenticide Act with a lower dioxin content. Technical 2,4,5-T has also been found to be contaminated with a dioxin. In this instance, unfortunately, the very toxic 2,3,7,8 lelrachlorodibenzodioxin,44-48 was present. The contami nation of the 2.4,5-T with 2,3,7,8-letrachlorodibenzodioxin ranged from < I ppm to is much as 32 ppm over the years. In newly manufactured 2,4,5-T in the U.S. the content of letrachiorodi* ben/op-dioxin luis been substantially reduced and is usually kept below 0.1 ppm. Whether other dioxins or furans have ever been detected in 2.4.5*T in Ihe U.S. has not been reported in the literature. Cliloiinaicd dioxins have also been found in so-called "toxic fat," which produces chick edema disease when fed to chickens. After several years of study by industry and Ihe Food and Drug Administration, Flick et al.4f were able to separate a purified crystalline product which produced chick edema disease. Two years later, Cantrell et al.5 announced that 1,2,3,7,8,9* chJorodibcnzo-p-dioxin was one of the toxic compounds that produced chick edema. The origin of the chlorinated dibenzodioxin in "toxic fat" was not clear. It was suspected that it arose from chlorophenois which were used to preserve hides for the manufacture of leather. Tallows and greases were obtained from these hides51 and a more recent outbreak of chick edema disease was traced to the contamination with chlorophenois of soapstock used a> feed fats. Higginbotham et el.55 showed that chlorophenois and their salts, when heated, undergo condensation reactions and form chlorinated derivatives of dibenzo-p-dioxin; furthermore, chlorinated dioxins and predioxins were originally present in some of the chlorinated phenols. Generally no efforts have been made to determine whether various chlorinated dioxins and furans are present in the environment. However, Baughman and Mcselson55 recently reported the presence of dioxins in fish taken from four locations in Vietnam in Military Region III, which includes Saigon and several provinces to the north.54 The concentrations ranged from I ft i0 814 ppt. ZHko** was not able to demonstrate chlor inated dibenzodioxins and dibenzofurans in aquatic animals from various locations in Canada. Chlorinated naphthalenes may so far have remained unidentified since they interfere with other chlorinated compounds in any mull(residue analysis and are detected only when mass spec trometry Is also performed.54 The vanous germicides given in Figure 2 have been used as antiseptics, deodorants, and particu larly, as far as hexachlorophene is concerned, also as preservatives for various cosmetics, soaps, and lotions. A listing of different products that contained hexachlorophene prior to the new regu lations was given by the American Pharmaceutical Association.57 Registration of bithionoi was cancelled in the U.S.58 because it produced photosensitivity reactions in humans using soaps that contained this product. None of the poly chlorinated polycyclic compounds used as germi cides have been shown to accumulate tn the environment. Hexachlorophene has additional uses as a fungicide on ornamental plants, on conveyor chains as a mildcwstal, in laundry rinses as a mildewstat, in shoes and air filters and assorted industrial uses as a fungislat, and on leather, paper, and textiles.59 It is also registered as a pesticide for use on cucumbers, peppers, and tomatoes.40 and for cotton. Dtchlorophene is used as a mildew preventive on cotton and is recommended as a fungicide and bactericide for the protection of textiles and materials from molds.41 CHEMISTRY: METHODS FOR CHEMICAL DETERMINATION AND PHOTOLYSIS Much of the chemistry of polychlorinated biphenyls has recently been reviewed.31'i* Commercial chlorinated biphenyls are mixtures of biphenyl groups with different numbers of chlorine atoms (Figure I). When hydrogen atoms are replaced by chlorine on the biphenyl ring, a targe number of substitution combinations can arise since the number of chlorine atoms on the molecules can vary and the chlorines can also be 430 CRC Critical Reviews in Ttnkohgy jiu* tesui chJo Clill of Mr Ml varn C005 naie pons con* 124 resr 1 call inst An 27? chk mar a cht mu ad< lab me do CSV ch. an au m< sis m. Ur d> P( in Sll h. u u R d. A HONS 069488 iHachcd to the molecule in different positions, (Mulling in a variety of isomers. Even if the chlorination may be the same, different commer cial batches may vary in the relative concentration of isomers. The PCBs produced in the U.S. by Monsanto fall under the trade name Aroclor. The various Aroclor mixtures have a great number of 1 constituents, and when the biphenyl is chlori nsied, 210 different chemicals are theoretically ' possible. Aroclor 1254, for instance, yielded 69 i constituents while the fractionation of Aroclor | 1242 and 1260 yielded 45 and 78 components, I respectively*4 The chlorinated biphenyls (PCBs) are chemi cally very inert, resistant to corrosive chemicals, insoluble in water, and have a low vapor pressure. Aroclor 1221, for instance, has a boiling point of 278*C. The boiling point increases with increasing chlorination. The chlorinated biphenyls are deterI mined in biological and environmental samples by | i combination of electron capture gas liquid : chromatography and mass spectrometry. Nuclear ; magnetic resonance spectroscopy is used in I addition to the above two procedures in some laboratories. PCBs were first identified in environI mental samples by Jensen in 1966*1 They were detected as interfering peaks on GLC analysis of environmental samples tlut were analyzed for : chlorinated pesticide residues.*1 ( The PCBs interfered with routine pesticide analytes for chlorinated hydrocarbons. Various I authors have therefore published separation | methods44 and Reynolds31 discussed this extenI lively in a review. According to Safe and Hutzinger,49 the use of rnisi spectrometry for structural studies of PCBs is lima ad because the chlorine atoms may be ran domized between the phenyl groups when the PCBs are fragmented by electrons. The only exception (o this ia 2,2'-dichlorobiphenyl. The qusotiiation of polychlorinated biphenyl in environmental samples has met with difficulty since the PCBs represent mixtures. Several workers have compared PCB components in Held samples to Aroclor 12S4 because the sample chroma tograms were most similar to these mixtures.44-44 Rote and Murphy41 quantitated individual peaks of different Arodors by means of a response curve. The eemilogarithmic reletionship of detector response (total peak area/16 ng) to average chlorine content was obtained for each Amelor by the method of least squares. From this relationship the theoretical response of the detector to each chlorinated biphenyl was calcu lated. When this approach was used, the levels of PCBs were lower than when the PCB levels were compared to the Aroclor 1254 standard. Because of these difficulties in quantitation, the PCB levels reported from different laboratories may vary greatly. The various problems encountered in the analytical methodology of polychlorinated biphenyls and their quantitation are also described in a U.S. FDA report which can be obtained upon request from the U.S. Food and Drug Adminis tration in Rockville, Md. The title of the report is A nelyttcal Methodology for Polychlorinated Biphenyls (Feb. 1973). Polychlorinated terphenyls (Figure 1) are not eluted from gas chromatography columns under conditions used for polychlorinated biphenyls.34 By altering their gas chromatograph column, Zitko et al.*7 were able to detect polychlorinated terphenyls. Chlorinated naphthalenes (Figure I) elute at the tame time and cause interference with the determination of chlorinated biphenyls and chlori nated hydrocarbon pesticides in general, unless mass spectrometry it performed to establish the identity of the various chemicals involved.54 The methods presently available for the deter mination of tricMoracarbenUide and other germi cides (Figure 2) are not very well developed. Graber et al.44 outlined a thin layer chroma tography method for the determination of these chemicals in soap and very briefly mentioned other methods that have been employed in the past. Several methods for the determination of chlor inated dibenzodioxins and chlorinated dibenzo furans have been described. The goal of these methods is two-fold. First of all, quite a number of technical polychlorinated organic compounds may contain one or several of these compounds''0-73 as contaminants and methods have been developed to determine them in various technical products. Other methods <(ealt primarily with toxic fats where many attempts were made to isolate and identify the compounds responsible for chick edema disease.73 Some of these methods were described in the papers that will be cited in connection with the description of chick edema disease in this article. When preparing the various chlorinated dibenzodioxins and dibenzofurans for toxicity studies in January 1974 4SI Ob?'**9 HONS order to obtain pure products, methods also need to be available for their separation and identifi cation.14 One of the problems with tetrachlorodibcnzodioxin as well as tetrachlorodibenzofuran is their severe toxicity in the microgram range which will probably not result in an accumulation within the ppm in biological tissues, for instance, for residue analysis. Baughman and Meselson"* have recently described a method for the determination of these materials, within the ppt range. //exac/i/orophene is made from trichlorophenol. A process for its production was patented by Gump.15 Additional methods for the produc tion and purification for hexschlorophene have been published and are cited in Chemical Abxtracts. Recently several methods74-1 9 have been developed for the determination of small amounts of hexachlorophene in blood and biological tissues. Some methods were based on extraction of hexachlorophene with ether or ethanol or a mixture of ether and ethanol. An acetyl or methyl derivative was formed and the hexachlorophene was determined with electron capture &it chroma tography.19 In an earlier method described by Bachmann and Shcllar,** simple benzene extraction was used and no derivative of hexaclilorophcne was made, which led to very variable results. The various advantages and disadvantages of these methods were discussed by Ulsamer.19 In earlier work my co-workers used ether for extraction and prepared a dimethyl ether deriv ative of hexachlorophene. * Recovery with this method was only about 75% and we have recently altered our method of extraction to obtain better recovery.*3 A method developed by KabacofTet al.1* for the determination of hexachlorophene is, according to (he authors, also suitable for deter mination of dichiorophene and Gutenmann and Uska* have described a method for the determin ation of hexachlorophene in several agricultural products. The production of hexachlorophene from (richiorophenol proceeds at low temperature and under acidic conditions which makes the produc tion of a chlorinated dibenzodioxin as an impurity highly unlikely. Since chemicals that are found in the environ ment are exposed to sunlight, the effect of UV light on the compounds under discussion has been jvtn considerable attention. Hustert and Korte*4 synihcti/cd a few PCB isomers, n;um.l\. 2,4.6,2'.4',6'-hexachlorobiphenyl, 2.4.5.2'.4'.i<hexachlorobiphenyl. 2.4.2\4'-ietrachlnrobiphctiyl 2,5,2',5'tetrachlorobiphenyl, and irradiated tlie various isomers with UV light. A mercury vjp*n lamp with quartz filters was used ss a light source The UV spectrum of the synthetized chlorinated biphenyls only shows significant absorption below 300 nm. When these compounds were irradiated in a solvent such as hexane, acetone, methanol, or mixture of methanol and water, dechlorination and polymerization of the compounds wax observed. When the PCB isomers were irradiated in perfluorinated dimethylcyclohexanc. an inert medium, isomerization, and chlorination dechlor* ination were observed. These experiments indicate that, under environmental conditions, photolysis products may result with a lower chlorine content but small amounts of higher chlorinaied biphenyls may also occur in low concentrations. Safe and Hutzinger*5 reported that the irradiation of 2,4.6,2,.4\6'-hexachlorobiphenyl at 310 nm resulted in compounds of lower chlorine ctmicni. such as di-, trk tetra*. and peniachlorobiplicnvls In these investigations hexane and methanol were used as solvents. It is possible that now comp*.... .. arc formed by loss of chlorine. Rcarr.iuccnu ni jiul condensation may lead to chlmobiplicm In m environmental samples that arc not present m commercial PCB mixtures. The 24-hr irradiation <>i 3,3',4,4'tetrachlorobi phenyl, 2,2'.i>.<-'tetrachlorobiphenyl, 2,2 ,$,$ -letrachlorobiphenyl dissolved in hexane resulted in about 703 loss oi the starting material, while with 2,2'.4,4'.5.5'hexachlorobiphenyl, only 3.8% remained and less than 1% of 2,2',3,3',4,4',5,5'*octachlorubiphcnyl remained. The irradiation of Aroclor 1254 iit hexane resulted in a change of the composition of the Aroclor mixture. The gas chromatogram obtained from the irradiated Aroclor showed shorter retention times than the standard Aroclor 1254. The irradiation of Aroclor (254 *n * dioxine water mixture also containing sodium bicarbonate did not lead to the formation of chlorodibenzofurans or hydroxychlorobiphcnyls. The compounds that were observed corresponded to PCB molecules to which water was added and a more polar "carboxylic" Kid fraction. Irradiation of Aroclor as a thin film with a frequent addition of water led to newly formed hydroxylaicd compounds and a more polar "carboxylic" fraction. The results obtained from irradiating 492 CMC Critical Rrtfmi tot Toxkofogy M0NS 069490 Ivl. ho nm |ow in f lion M in jert lorale kfiit |yii Lrndf |nm yls. TTC lldl i ml in in i of 6rnyl of .5'leu y* in i of am ved ;lor ia um of yl*. Jed 4a ion ion led lie" ini PCUs therefore dc (tended greatly on the solvents used ami ihc pH of iftc solution in addition lo Ihc Itglu source used und nwy result in dechiurinalion, formation of polymers, hydroxyiation, and carboxylic products."* Exposure of various chlorinated dibenzodioxins lo summer sunlight or fluorescent UV light with an intensity of roughly 100 pw/cml in the presence of organic solvents such as methanol led to rapid photolysis of 2,3,7,8-tetrachloro- and 2,7-dichlorodihenzodioxin. while 1,2,3,4,6,7,8,9-octachiorodibenzo-p-dioxm produced a series of chlorinated dioxins with uniformly decreasing chlorine content. Some evi dence was also found that reactions other than reductive dechlorination occurred.*7 Wren 2,3,1 .B-tcWachloiodibcnzndioxin was spread on a glass plate in a methanol solution and the meth anol was allowed lo dry, quantitative recovery of the dioxin was possible even after 14 days of \JV irradiation. Similar results were obtained when 2.3.7,8-(ctrachlorodibczodioxin was dissolved in methanol, put on a gluts plate, covered with soil, and irradiated.17 These results demonstrated that under favorable environmental conditions, when ; organic hydrogen donors are present, photolysis will lake place. Hare surfaces of soil, concrete, or , water are not effective inducers of photolysis. ' Chlorinated dibenzodioxins could at times prove to be quite persistent. Partial dechlorination may also occur with chlorinated diphenyls or the higher Jilminatcd dibenzodioxins and may result in less chlorinated compounds. The possible long lasting presence of (he dioxins is also emphasized by Kearney et al.,M who recovered 56 and 63% of originally applied tcirachlorodibenzodioxln after one year in Hagerstown and Lakeland, Md. soils. Tcirachloroselicylanilitie and similar compounds split off chlorine atoms when exposed lo sunlight. This is briefly mentioned In connec ; tion with photosensitivity reactions of the skin. Shaffer et al.*f irradiated hexachiorophene in absolute ethanol with UV light and obtained a number of dechlorinated bisphenols resulting from loss of chlorine in (he ortho and para positions teUtivt to the hydroxy group. GENERAL TOXICITY AND EFFECT ON REPRODUCTION Diphenyl itself, without arty chlorine atoms, is a fungistatic agent which is used as a preservative for citrus fruits, and is usually impregnated into the wrapping paper. The protected citrus fruit contains a certain amount of biphenyl residue. In different countries the amount allowed as a residue on citms frutt varies between 70 to 110 ppm of biphenyl. Booth et al.* found t reversible nephrotoxic effect of biphenyl on (he rat kidney when rats were fed 0.5% or 1% (10,000 ppm biphenyl in the diet). None of these effects was observed on diets containing 0.1% (1000 ppm biphenyl). Deichmann et al.#l showed that pro longed exposure to air containing diphenyl in high concentrations of 5,40, and 300 mg/m3 produced Uver and kidney injury as well as broncho pulmonary lesions in mice and rats. In general, diphenyl has always been considered to be relatively nontoxic. In 1969, In Finland, a man who had been intensively exposed to diphenyl for II yean became 11) and died.*7 Diphenyl impreg nated wrapping paper had been manufactured in Finland for about 15 years In the plant where the man was employed. The equipment used in the impregnating process was cleaned 5 to 10 times a year with tri- or tetrachloroethylene. Tire cause of death of the worker in this particular report was not satisfactorily explained and neither were the complaints or 3) other workers engaged in the same factory. Since chlorinated ethylene com pounds were uwd for cleaning, it should be established whether organic solvents containing halogens could lead to the production of toxic substances when they are brought in contact with diphenyls. If this proves to be the esse, then care rfiould be taken that this situation is avoided. The KBs are also not very toxic when given as a single or a few repeated doses to birds snd mammals. However, the juvenile pink shrimp is very sensitive to PCBs in water, while trout and catfish can tolerate higher doses; insects are also not very sensitive to PCBs. Pin flsh are not quite as sensitive to a single dose, but will die when exposed for 14 to 45 days to 5 ppb of Aroclor 1254 7 J PCBs ire of a low order of toxicity to rats and rabbits when given as a tingle dose. The scute oral toxicity decreases in rats with increasing chlorine content, while this is not as obvious when the dermal toxicity is tested in rabbits.31 This may be due to poorer absorption of the higher chlorinated compounds. In studies with adult Sherman strain rats, we found that the oral U)Se for Aroclor 1254 and 1260 wal somewhere In the Jsinisry 1974 MGNS 069491 range of 4 fa 10 g. These two compounds proved to be more toxic in weanling rats. The single oral LD, for weanlings was 129$ mg/kg, and 1315 mg/kg for Aroclor 1254 and 1260, respectively, and the lowest lethal doses were 1000 mg/kg for Aroclor 1254, and 1200 mg/kg for Aroclor 1260. The intravenous LD*0 for Aroclor 1254 in adult female rats was 358 mg/kg.*4 A number of short-term studies have been reported where the PCBs were given to animals over a period of one or two weeks. When Ibe PCBs were given in this fashion they were somewhat more toxic than when they were given in a single dose. These studies are reviewed in great detail by Nelson el al.,J and will not be reported here since they do not give a great deal of additional information. Vos and Kocntan9* studied the toxicity of three PCII preparations in chickens, Phenoclor DD6. a French product, Clophcn A(0, a German product, and Aroclor 1260, a product from Monsanto in the U S. The birds were fed 400 ppm of PCBs in their diets for 60 days; the Phenoclor and Clophcn were much more toxic than Aroclor. The birds fed the two European samples died between 12 and 60 days after onset of exposure. The Imdings at autopsy consisted of hydro* pericardium, ascites, subcutaneous edema, and liver necrosis. Only 3 of 20 birds fed Aroclor 1260 died during this time with hydropericardium. None of the Aroclor fed birds showed liver necrosis. Wiih the help of mass spectrometric and microcoulometric analyses, the authors9' determined that the two European samples were contaminated with (etra- and pentachlorodibenzofurans. As an additional contaminant, chlorinated naphthalenes were found. Dermal toxicity studies with the three com* pounds, Clophcn, Phenoclor, and Aroclor 1260, in rabbits99 after two days led to reddening of the skin where the materials were applied. The skin reaction was more pronounced after one week, desquamation of the external epidermal layers of the skin wu observed, the hair grew at a reduced rate, the skin became thickened, and prominent transverse wrinkles developed. The test animals showed a gradual lots of weight, and one of the rabbits given Phenoclor and three of those given Ctophen died. All but one rabbit showed fluores cence of the liver under UV light, and microscopic changes were also observed in Ihe livers and the treated skin. Vos and Notenboom-Ram'7 compared the dermal toxicity of the isomer 2,4,5,2',4',5'. hexachlorobiphenyl with that of a PCD mixture (Aroclor 1260) in rabbits. Their dermal application resulted in early microscopic skin lesions in ihc Aroclor group. The lesions in the 2,4,5,2'.4'.5'hexachlorohiphenyl group appeared later and were less severe. Both experimental groups showed liver changes. The liver damage was more pronounced in the animals that had been exposed to hexachloro biphenyl than in those that had been exposed to the Aroclor. In inhalation studies with Aroclor 1242 and 1254, rats, mice, rabbits, guinea pigs, and one cat were exposed to PCB vapors five days a week fur several weeks. The concentration of Aroclor 1242 vapor was 1.90 to 8.63 u%/\ and no i/I effects were observed. The exposure of animals to the vapor of Aroclor 1254 in the concentration of 5.4 or 1.5 pg/1 produced enlarged livers in rats. An inter current respiratory infection in some control .in well as experimental rats made it difficult w interpret the results.99 Since it is now known that the chronic toxicity of PCBs is a more important parameter in establishing their effects than short term exposures, further studies with lower level , over longer periods of time would give mote meaningful information. Fortunately, most of the compounds under discussion have a low vapor pressure so that exposure by inhalation probably does not present a great problem. A number of reproduction studies m various species have been conducted with PCBs; usually, embryo toxicity wu observed hut there were no malformations. Heath et a!.99 were unable to demonstrate an effect on npnxluction with diet ary levels of 25 and 50 ppm of Aroclor 1254 m mallards and bobwhites. An increase in eggshell cracking or reduction in shell thickness was also not observed while DDE, which was used as a positive control, induced significant shell thinning and cracking and a reduction in hatching successes in mallards but not in bobwhite. In field studies with brown pelican shells, it was found that thinning could be best correlated to DDE residues rather than dieldrin or PCBs This finding indicates, according to Blus et al ,100 the PCBs are not responsible for eggshell thinning in most birds following environmental exposure. Peakall1*1 fed ringdoves 10 ppm Aroclor 1254 454 CRC Critical Rtvlewt to Toxtcototty HONS 069*92 the llie .5'lure ilon the '.5*. ere iver d in iro* 110 and cut fur 242 vere r of 1.5 met* il as I to that rtani wrl tfVtflh now r ihc apoi ably HOUR tally, e no If to diet* 54 in phcll i alao at a nning cesses it was ad to .This * the ing in 1254 foi 6 months He injected others with 160 mg/kg intiapcritoufaily for 4 days, and evaluated the ashed eggshell weights or these birds. He found no difference in egphelt thickness between the experimental and the control groups. In a pheasant reproduction study with Aroclor 1254.'**09 a significantly greater difference in the number of eggs (hat were pipped but not hatched was found in the group of hens that received 50 mg of PCBs weekly. Egg production and hutchehilily was lower In this group, the survival of chickens that did hatch at six weeks of age was reduced, and the ones that did survive were significantly lighter than the controls. Aroclor 1242 at 10 ppm or 100 ppm and Arodot 1254 at 100 ppm in the diets of chickens did reduce egg production and hatchability and caused thin eggshells, while Aroclor 1242 at I ppm, Aroclor 1254 at 10 ppm, and Aroclor 1260 at 100 ppm did not affect reproduction in chickens.1 fl4 In a rat reproduction study, decreas ed survival of pups at a dietary level of 100 ppm of either Aroclor 1242 or 1254 was observed. The results obtained from the reproduction studies in birds particularly seem to Indicate that Ihc lower chlorinated biphenyls affect reproduction more than the higher chlorinated biphenyls, and il is very important to determine whether the lower culminated biphenyls that have been suggested as replacements for presently employed chlorinated biphenyls have an effect on reproduction. In another rat reproduction study94 the lowest dose of Aroclor 1254 that affected reproduction was 20 ppm (1.5 mg/kg/day) which manifested itself in a decreased number of litters and lew pups per litter. Neither 1 ppm nor 5 ppm of Aroclor 1254 had an effect on reproduction in the Sherman strain rat; however, an exposure of the dams to the Arodorc at 5 ppm or higher increased the liver lo body weight ratios in weanling rats of both sexes. At 1 ppm the increase in liver weight was observed only in Fla and Fib weanling male rats. The dietary levels of 100, 20. and 5 ppm of Aroclor 1260 did not affect reproduction; how ever, the liver-to-body weight ratio of 21 -day-old pups was increased at all exposure levels. Mink are highly susceptible to the toxic effects of PCBs. A daily dietary intake of 30 ppm resulted in death in about 6 months. Exposure to 5 ppm Aroclor 1254 in the daily diet severely effected reproduction.'05 Unfortunately, it is often very difficult to compare the various dietary levels that affect different species, since the food consumption may vary decisively from one animal species to another and the daily food Intake in g/kg body wt. is often not included in the scientific report. Some mice strains, for instance, will be exposed to as much of a given substance that is present in the diet at the concentration of 300 ppm as rats on a diet containing 600 ppm or more. With some species it is of course difficult or almost impossible to measure the food consumption, but a greater effort in establishing these very basic facts would be helpful for the interpretation of toxic effects in different species. Not very much is known at present about the toxicity of terphenyfs or chlorinated terphenyh These materials have not been studied to any great extent. The few studies that are available only describe very specific effects such as microsomal enzyme induction in the liver and give no overall evaluation of their toxicity. The most toxic of the various chlorinated dibcnxodioxins and dibentofumnt is 2,3,7,8-tetrachlorodibcnzodloxin which, in the German litera ture. is sometimes referred to as 2,3,6,7-tetrachlorodtbenzodioxin. Single oral LDS0 values are female rats: 44.7 jig/kg; male rats: 22.5 /ag/kg; male guinea pigs: 0.72 jig/kg: rabbits: <30 ug/kg; and dogs: between 30 and 300 #ig/kg.'* It is possible that young animals are more susceptible to the toxic effects of this compound than older snimals and LDS0 values should therefore also be determined m weanling animals. One of the characteristics of this compound was that it caused delayed death, sometimes is delayed u 40 days after a single dose. During the period of intoxica tion, the snimals lost a great deal of weight.' On the other hand, 2.0 g/kg of dichlorodibenzo-p-dioxin and 4.0 g/kg body weight octachlorodibenzo-p-dioxin were not lethal to rats. Hexa-, hepta-, and trichlorodibenzo-p-dioxin were more toxic.' Schulz'07 in earlier experiments found that oral doses of 20 to 50 jig/kg/body weight of 2,3,7,fl>tetrachJorodibenzodioxjn resulted in fatal liver necrosis in rabbits. More recently Milnes10* reported (hat 10/ag/kgwas lethal to rabbits. The 2,3,7,8-tetrachlorodibenzodioxin hat a variety of specific toxic effects. Whether these effects vary in different species is presently not well defined. With the interest in this and the other related compounds, more information on January 1974 459 HONS 069493 ihcir toxicity probably will soon become available. TJk* pnrblems inhcrenl in studying these com pounds are (he unavailability of some of (hem, the fact that they may not be completely pure, particularly the hex- and hepta- pieparations, and of course with respect to the tetrachlorodlbeiuodioxin, its extreme toxicity, which makes it difficult to handle. In contrast to PCBs, teratogenic as well as fciotoxic effects have been reported for 2,3.7,8tetrachlorodibcnxodioxin. Sparschu et al.'*' gave 0. 0.03, 0.125, 0.5, 2.0, and 8.0 jig/kg/day of 2.3.7,8-tclrachlorodibenzo-p-dioxin to pregnant ruts on days <> to 15 of pregnancy. With the 0.03 pg/kg dosage level, an effect on the fetus was not observed, but doses from 0.125 Mg/kg on up resulted in fetal mortality, early and late resorp tions. and fetal intestinal hemorrhage. Khera and Ruddick'10 gave doses ranging from 16 to 0.125 jrg/kg !'> Wislar nits on days 6 to 15 of pregnancy. Tltcsc author* also observed pronounced fetotoxicity at all dosage levels except 0.125 fig/kg. The affected fetuses showed cerebral und intestinal hemorrhage as well as subcutaneous edema at autopsy. The offspring of mothers given 0 5 and I /jg/kg of the tctrachlorodibenzo-p-dioxin gained less weight during their suckling period and their survival to weaning was reduced. This could have been caused by the intrauterine effect of (he dioxin or by excretion of (he dioxin in the milk of the dams given the material during pregnancy. Courtney and Moore11' observed cleft palates in 3 strains of mice given 3 Mg/kg TCDD subcutane ously from days 6 to 15 of pregnancy and an increased incidence of kidney anomalies. Hydronephrotic kidneys were also produced in mouse pups that were nursed by dams treated with TCDD during pregnancy or at time of parturition.113 The 2,3,7,8-telrachlorodibenzo-p-dioxin Is extremely toxic in the chick embryo assay.5 * In the rat, hexachiorodibenzodioxin caused fetotoxic effects at I and 10 jig/kg and teratogenic effects at doses of 100 Mg/kg.1* Harris et al.`13 gave female rats daily doses of 0.1, 1, or 10 Mg/kg TCDD for 31 days. Rats given 10 Mg/kg lost weight and most of them were moribund within 3 weeks. Weight gain was also less at the I Mg/kg dietary level. The rats receiving 0.1 Mg/kg were not affected. In experiments with guinea pigs, 9 of 10 animals died after a single dose of 3 Mg/kg TCDD, while guinea pip receiving! tingle dose of 1 Mg/kg pined leas weight. All guinea pip receiving I Mg/kg/week died within 24 to 32 days am) . weekly dose of 0.2 Mg/kg depressed their weight Of the various animal species tested, the guinea pig seemed to be most susceptible to (he toxic effects of TCDD. Reduced thymus aid spleen weight* were observed in rats that received a single dose of 25 Mg/kg TCDD. The rats that died at the various dosage levels showed massive hemorrhages into the heart, liver, brain, adrenal glands, and gastrointesti nal tract. Necrosis and ulceration of the glandular part of the stomach were observed. Occasionally organized thrombi were noted and the liver reveal ed necrosis of parenchymal cells. Subiethal doses of TCDD produced microscopic changes in liver, kidneys, and thyroid. In the kidneys the collecting tubules were primarily affected. Doses of 0.1 Pft^kg/day for 31 days, a single dose of 5 pg/kg. and multiple weekly doses of 1.0 and 0.2 Mg/kg did not produce changes in rats discernible with the light microscope. Toxicity studies were conducted in our labora tory with dichlorophene14 Tire oral LDsn for adult female rats when (he material was pvcti in peanut oil by stomach tube was 1,660 mg/kg and in males it was 1,500 mg/kg. The only signs of toxicity observed in these rats were diarrhea ami some depression. When 200 mg/kg was applied daily in propylene glycol to the skin of a clipped area of the back of male adult rats as a 12 5. solution, local irritation of the skin with some ulceration resulted but there was no systemic toxicity. The acute intravenous LDS0 in adult male rats was 16.8 mg/kg when it was given m j saline lecithin suspension. In a (wo pneration rut reproduction study, a dietary level as high as 1.000 ppm (50 mg/kg/day) had no effect. Gross and microscopic examination of the various organs including the brains of the parents as well a* t he offspring did not reveal any morphological changes that could be related to dichlorophene exposure These finding? indicated that dichlorophene was not very toxic to rati. The great difference between the oral and the intravenous LD<,, suggested either poor absorption or rapid break down of the material in the gastrointestinal tract. Martins*1 lists the acute LDSo for guineu pigs as 1,250 mg/kg and for dog?, 2,000 mg/kg. Rats ted for 90 days on a diet containing 2,000 ppm showed no evidence of toxicity. The acute toxicity of hexachbropltcnv is much greater and varies in different animei species and is 45* CKC Crilktl Rrrir** to Toxfrabuy m HONS 069494 alio dependent on (he mule of administration and the solvent in which it is given. Gump11 * lists the acute oral LD*n in mice as 168 mg/kg and cites LOiu values reported by other investigators rang ing from 80 mjt/kg to 215 mg/kg. Dogs and sheep were more susceptible to the toxic effects of hexachlorophene. The interest in the effect of Itexaclrinrophcne on sheep in particular stemmed from the fact that it is effective against liver dukes' '* and advocated for its anthelmintic properties. This even led to the treatment of cliiidren with orally administered hexachlorophene in China.111 Intravenously administered hexachlorophene in rats resulted in an LDS0 value of 9.1 mg/kg and in rabbits of 8.5 mg/kg. More recently we found that the oral LDS0 (single dose) for adult male ruts was 66 mg/kg and for adult female rats 56 mg/kg: for weanlings it was 120 mg/kg when the material was given in peanut oil by stomuch tube to Sherman strain rats. The acute intravenous LDSo was 7.5 mg/kg in adult male rats.1 * * Nakaue ct al."9 reported similar oral LDS0 values in Wislar rats. The single oral LDio for sheep and cattle lies between 30 and 60 mg/kg.*30 Single dermal LDt0 values have not been reported in the literature. We found in rats*1 * that single applications of 600 mg/kg hexachloro phene in 95% ethyl alcohol solution caused death m l of 10 rats. Dermal application of 24 and 48 mg/kg/day for 30 days in propylene glycol or in a detergent as a 3%. solution caused ulceration of the skin and affected the white matter of the central nervous system in at least half of the rats. A dose of 12 mg/kg/day as a 1.5% solution caused only mild erythema and desquamation of the skin of rats. No effect on the central nervous system was observed. Whether a single dose given in either propylene glycol or a detergent would be more toxic than one given in ethyl alcohol was not established. The signs of acute hexachlorophene poisoning following a single oral dose varied with the age of the rats.11* Adult rats suffered from severe depression and diarrhea, but showed few signs of leg weakness. Weanlings, on the other hand, developed posterior leg weakness as well as de pression. The leg weakness became more apparent in the adults when they were given repeated doses of hexachlorophene. Death was usually preceded by convulsions in rats dosed intravenously. In rats killed with hexachlorophene, rigor mortis developed very rapidly and was very pronounced. Body temperature elevation following hexachloro phene poisoning has also been recorded in rats.1 *9 Both changes, the pronounced rigor mortis and the temperature elevation, may be related to the fact that hexachlorophene uncouples oxidative phos phorylation. Additional symptoms of hexachloro phene poisoning particularly in other species are discussed under neurotoxicity. Nakaue et at.1*9 fed 400 ppm hexachloro phene (28.9 mg/kg/day) to Wistar rats. They developed leg weakness within 5 days, diarrhea, emaciation, and they died in 7 to 10 days. No deaths occurred in Ihe rati that were fed 200 ppm (aboul 13 mg/kg/day)for 16weeks. In a two generation rat reproduction study which we conducted in our laboratory, we found a reduced survival In the offspring of the Ft generation fed 100 ppm (11.8 to 5.5 mg/kg/day) In their diel for 54 and 166 days. This was statistically significant in the second breeding of the F| generation. In the second generation the litters were slightly smaller, but the survival to weaning was not significantly reduced.118 A dietary level of 20 ppm (2.3 to 1.1 mg/kg) did not influence reproduction. Thorpe131 reported that the oral administration of hexachlorophene to male rats as well as single doses of 25 and 50 mg/kg hexachlorophene to sheep produced de generation of spermatogenic cells; muitinucleated cells were observed within the tubules of the testes. The doses given by Thorpe were higher than the ones we used In the reproduction study, and microscopic examination of the testes of the rats used in our reproduction study did not reveal any abnormalities. Toxic doses of hexachlorophene given to the dam during pregnancy may result in malformations of the offspring. Thus far, two studies have been reported in the literature; both studies were conducted in rail.113'133 It should be determined whether malformations can also be produced in other species and with lower dotes before any conclusion can be drawn from these findings. A number of human poisoning cases have been reported in the literature and some of these were fatal. Most of the poisoning cases in humans have either been caused by accidental ingestion, because a hexachlorophene detergent solution waa mistaken for milk of magnesia, or by the applica tion of hexachlorophene to large areas of burned or otherwise damaged skin.",,,34 Larson3 was January 1974 4S7 HONS 069495 ihe first to recognize that hunted patients developed convulsions and other toxic symptoms al ter repeated exposure to hexachlorophene which was applied to hunted areas of (lie skin. The incidence of convulsions in burned patients decreased when the use of hexachlorophene was discontinued.',s Eleven cases have been reported13 * 12 7 in which a single dose of 3% hexachlorophene deter gent solution had been taken orally. Gastro intestinal symptoms including nausea and vomiting were observed in ten of these cases; the eleventh case, a child who drank about 250 mg/kg hexa* chlomphcne, became comatose and died. One additional death occurred in this group, however, tliis was moat likely due to other causes. A 1 7-day-old infant was accidentally given daily oral doses of 37 mg/kg hexachlorophene for 7 days. On the third day the child started to have loose stools. On the fourth day of hexachlorophene administra tion the child developed spasms of the extremities with twitching of the face, sucking movements of the lips, and lateral shaking movements of the head. It was also noted in the case report that the child refused to take the medicine, there was drooling and frequent vomiting, and it is not quite clear whether the child received 37 mg/kg/day. The child survived. A week after the Uexachlorophene suspension was discontinued, persistent spastic flexion of the fingers, hands, and arms was still observed. After a month the hands were slightly flexed at the wrists but were not spastic. Horizontal nystagmus was observed but cleared later. The child was discharged when it was two months and three weeks old. At this point it had shown great improvement and when checked at six months (he child was healthy looking.111 PUapil mentioned in hit discussion a number of other unpublished cases, some of which were reported to the National Clearing House for Poison Control Centers. Among these was the case of en infant who by mistake got a spoonful of PhiioHex in each feeding bottle for approximately two weeks, when it suddenly died. Chung et *1.' '1 treated a total of 105 patients 5 to 15 yean old with hexachlorophene for chlonorchiasis sinensis infestation. Sixty of these patients received I dose of 20 mg/kg, 32 received 2 doses of 20 mg/kg/day, 8 patients received 3 daily doses of 20 mg/kg, and 5 patients received 20 mg/kg body weight on the first day and 10 mg/kg on the second day. The toxicity reactions observed in these patients were mainly confined lu the central nervous system and to the gash". intestinal tract. The group that received 3 doses of 20 mg/kg/day had the most severe reaction. One patient in this group became comatose on the fourth day with loss of light reflex and positive pyramidal signs. Fundus examination of the eyes revealed mild papillary edema. A low grade fever was also observed. Complete recovery occurred within three days. One half to two thirds of the patients had diarrhea within 1 to 2 hr alter medication and in some cases a marked depressive effect on the central nervous system was also observed. An additional case of poisoning after oral ingestion was reported by Korlof ami Winsten117 in Scandinavia and two fairly recent cases with fatal outcome occurred in the U.S.' Herter131 was probably the first to report a human poisoning case caused by the dermal absorption of hexachlorophene. Because of a language barrier, the mother of a newborn infant applied 3% hexachlorophene as a lotion after the infant's hath without rinsing the baby. After four days, excoriations appeared on the child's face and buttocks, the infant developed convulsions, his face twitched and his extremities jerked. A roving nystagmus wss present. This child recovered with in 16 days after exposure to hexachlorophene had been discontinued. In addition to the two cases already mentioned in which poisoning occurred because of oral Ingestion, Muiiick139 also reported four cases children who died following the treatment for bums In two instances and the treatment for severe ichUuosis with a 3%hexachlorophene bath. Larson3 reported six burned patients who developed central nervous system symptoms following the dermal exposure to hexachloro phene. All six patients recovered. Korlof and Winsten111 reported two cases of poisoning following the dermal application of hexachloro phene. Both patients were fairly extensively burned, and developed central and peripheral nerve damage as well is some kidney impairment. Lockhart133 mentioned six deaths that were reported to the FDA caused by the topical application of hexachlorophene on burned patients. In all of thae cases the skin was damaged, probably enhancing absorption of hexa chlorophene, and although skin does represent a barrier, it is not impermeable - a fact we rend to forget at times. Damaged or abnormal skin may be 45t CPC Critic*! Rcriewt At Toxicology MOMS 069496 ( (0 hoSOf Dn* the live ryes pver rred (he fter live also fter and rent i > ri a mal if a fant the Tour and hit ving withhad >ned oral nfur for lath, wlio lomt loro* and ming ioro* Ively hcral nent. ware ipieil imed waa hexa* ant a nd to syba nunc permeable. and the skin of prematures, newborns, ami children in general may have absorption characteristics other than (hose of adult human beings, another factor that ri often neglected. Recently11'>'134 41 deaths due to hexacltlorophenc poisoning have been reported in France. Tire deaths occurred in infants who were treated with a powder to which 6% hexachlorophene had been added by mistake. This powder wa mainly applied in the diaper area where the skin became inflamed, this as well as the diaper which served as an occlusive dressing, probably en hanced absorption. These cases wilt not be made public until litigation problems have been settled. Little published information ri available on the toxicity of the germicides in Figure 2 aside from hexacltlorophenc and dichiorophene. The acute oral LD*o for tricloson (TCC) in mice and rats ri about 4,000 mg/kg.1 ,s The acute subcutaneous toxicity for rats was 14,700 mg/kg and the acute dermal toxicity was about 9.3 g in rabbits. Concentrations of t to 5% solutions caused eye irritation in rabbits. Daily oral doses of TCC up to 1.000 mg/kg/day for 4 weeks did not produce an effect in rats. Wlven 2.5 and 5% suspensions in gum arabic were applied dermally to rats for 4 weeks, the animals did not gain as much weight. Un published material cited in the minutes of the OTC (Over the Counter Drugs) Panel listed a "no effect" level as 170 mg/kg/day in rats and for a 90-day feeding study in rabbits, 138 mg/kg/day. In baboons a daily oral dose of 100 mg/kg/day did not produce a toxic reaction. The minutes alao mentioned some work where lower doses showed an effect on rats and dogs. Since some of these results are conflicting, further studies are indicated and should be performed with products used as ingredients in soaps; a chemical analysis of these products is necessary to determine whether im purities might be responsible for varying results. Chlorosniline as a result of healing during manu facture could be present as such an impurity. Of the anilines, according to Hamblin,194 only p- and iiKhloroaniline may cause methemoglobinemia if absorbed through the skin. The o- and m-chloroanUine msy cause liver and kidney damage. Tridosan itself ri also absorbed through the skin and absorption is greater when applied to damaged skin. SPECIAL TOXIC EFFECTS Skin Reactions Chloracne This occupational skin disease can be produced by a number of chemical compounds.137 Poly chlorinated biphenyls as well at the chlorinated naphthalenes, a few isomers of the chlorinated dlbenzodioxins, and chlorinated dibenzofurans are all able to produce chloracne in a certain pro portion of the human population. Oily skin and large pores seem to predispose lo the disease while the opposite ri true for smooth, tender skin and it ri possible that young children might be less susceptible because of the nature of their skin. Chloracne has also occurred in workers engaged in (he production of 2,4,5-T1. During the indus trial production of trlchlorophenol a sudden rise in temperature within the reactor has occasionally resulted in accidents. In one instance this exo thermic reaction caused an explosion.104 When the rise in temperature occurred, 2,3,7,8-letrachlorodibenzodioxin was formed and severe out breaks of chloracne were observed among the workers.'371 The term chloracne was first used by Herxheimer in 1899,'9 4 who thought it was caused by free chlorine generated in certain factories. Wauer in I91819* and Tcleky in 1927140 suggested the term "Pernakrankheit." Another term sometimes used is halogcnwaxacne. As the word chloracne implies, part of the lesion of this occupational skin disease resembles adolescent acne; however, it is generally more severe and the distribution of the lesions is not consistent with adolescent acne, although it may be superimposed on adolescent acne. Chloracne is described in the literature as consisting of the formation of comedones with or without cysts and pustules. The follicular orifices are filled with sebaceous and keratinous material. Melanosis and a secondary inflammatory reaction may also exist. In addition to this description, skin changes occur that have been discussed less frequently in the more recent literature. These skin changes are usually referred to as cable rash or cable itch. Cable rash has been observed in workers who have insulated cables with various mixtures of chlorin ated naphthalenes and chlorinated biphenyls and in workers handling these cables.141 It has been observed also in factories (hat make con denser*.'4 3,14 3 According to Braun,143 a few of January 1974 HONS 069497 I (he cases with manifest chloracne started with changes in the skin of the face resembling photo sensitivity and the bearers of these lesions suffered severe punt us in the areas of the skin lesions. This lesion termed cable rash occurred predominantly on the cheeks but also on the skin of the arms. The outcome of cable rash varied; it could either disappear or it could develop into full-blown chloracne. In addition to the pustules and comedones of typical chloracne, brownish keratinization of the skin was observed in some patients. Braun142 pointed out that the workers, particularly those with poor hygiene, transmitted the disease to their spouses and children through direct contact. Many of the occupational cases of chloracne that have been reported in the literature have been reviewed previously.1 37,1 44 Jones and Alden145 reported one of the first outbreaks of chloracne in a company that was engaged in the manufacture of chlorinated biphenyls. They described one typical case and mentioned 24 men working on the manufacture of chlorinated biphenyls; 23 had had an acneform eruption on the face and body. Particularly during the )930s and J940s large outbreaks of chloracne occurred in a number of factories. Much discussion has been devoted in the literature to the problem of whether the chloracne was produced by external contact alone or whether it was caused by systemic absorption, particularly the inhalation of vapors. One question asked was whether internal administration could cause chloracne, and if so whit part it played in any particular case. As we know now from outbreaks of poisoning due to the consumption of rice oil that was contaminated with polychlor inated biphenyls in Japan (see under "Yusho"), chloracne can be produced by the systemic absorp tion of chlorinated biphenyls. The experiments by Shelley and Kligman144 and Plewig147 showed that the skin lesions could also be produced by applying the chloracnegenic compound to the skin. After occupational chloracne manifested itself, the patients alio complained of systemic effects such is loss of appetite, nausea, edema of the face and hands, abdominal pain, vomiting, and burning and soreness of (he eyes. Particularly with (he chlorinated naphthalenes, hepatotoxic effects have also been observed. Application of chemicals to (he skin can lead to (heir absorption and cause systemic toxicity. Whether a systemic toxic diva results will depend on the skin surface aica covered, the amount of the toxic substance applied, and the ability of the skin to absorb n If dermal absorption is poor, s single applicuium will usually not be very toxic. However, f the ompound is not easily metabolized and exuded, repeated dermal application may result in suffi cient accumulation in the body to cause systemic toxicity. Crowe144 has pointed out that chloracne due to chlorinated naphthalenes has decreased because these compounds have been used less since the Second World War. However, Weber in 1`W4* reported an outbreak of chloracne in a company engaged in the production of electric coils ihat were coaled with a mixture of chlorinated naph. thalencs with the tradename "Nibtenwax." Detailed examination by several German Invest igators indicated that the capacity of 2.4.5* trichlorophenol as well as 2.4,5-T(2,4.5-tnchlomphenoxyacetic acid)1 44,1 ## to produce chloracne was due to contamination with 2,3,7,8-tctraehlorodibenzofuran or 2,3,7,8>tetrachlorodibenzodioxin. Schwartz et a!.iSI indicated that the must potent chloracne producing agents were the chlor inated naphthalenes, chlorodiphenyis. and diiorodiphenyloxides. Persons whu worked with chlorinated naphthalenes usually developed .mic after a month or more of exposure. Light microscopic observations made of i he chloracne lesions of humans varied somewhat, depending primarily on the length of time the lesions had existed at the time of examination The earliest change encountered consisted of acamholic widening of the external root sheath nt the hair follicle. Above a layer of basal cells <> to 10 layers of large round cells were observed, followed by a layer of 2 to 3 cel) rows of granular cells. In later skin biopsies, folding of the hasjl layer occurred and comedo formation was observed, resulting in dilatation of the follicle and atrophy of the epithelium surrounding the com edo. The contents of (he follicles consisted predominantly of keratinous material rather ilun sebum. The sebaceous glands also underwent changes. Within the alveoh of the glands ihe basophil cells increased in number and basui ceil hyperplasia was noted. The ducts of the sebaceous glands were filled with a solid mass of basophil epithelial cells. The sebaceous glands gradually 440 CRC Critical Review* to Toxicology p 069*98 HOHS I disappeared and in later aecretiom were only be investigated in chloracne patients who manifest tft recognized as solid protrusions of Ihc comedo hyperpigmentalion. ea will. Aside from atrophy of Ihc sebaceous glands, In none of the human case reports on chloracne ce hypeipbsiii has also been described and many large have the mucous membranes been studied to any If tebaccous glands forming huge cysts filled pri great extent. Vaginal smears in women and sputa, ill marily with Keratinous material have been corneal, and nasal scrapings may give helpful it- observed. Hyperkeratosis and acanthosis of the information in this respect. Since in animals d, n surrounding epidermis bus usually accompanied exposed to polychlorinated polycyclic compounds ic these lesions. Foreign body granuioinala which are low vitamin A levels have been observed in the thought to result from rupture of the follicular liver, it is possible that the compounds may afTect cysts have been found in the skin.147,151 In the vitamin A absorption and storage, and adequate M tie Japanese poisoning incident (Yuslm), the predom utilization in the liver and elsewhere in the body. W inant skin lesion was marked hyperkeratosis of the If the lesion is produced by inadequate utilization it apidermis, cystic dilatation of the hair follicles, of vitamin A, then administration of vitamin A per and an increase in the melanin pigment in the basal se may not necesurily alter the course of the *y i cells of the epidermis. Foreign body granulomats disease. Protein deficiency may also mimic hypo- ii- were also observed. The contents of the cysts vtlaminosis A in that it affects transport of vitamin consisted partially of keratin. In a stillborn child A. The liver stores 90% of the available vitamin A it' the hyperkeratosis was even more pronounced, and theae stores may be affected in liver disease. ,5* accompanied by atrophy of the epidermis; cystic The microscopic appearance of the skin lesions i>- dilation of hair follicles was observed, especially in resembles in some respects observations made in lie the skin of the head. According to earlier skin of patients suffering from vitamin A >r- reports15 * some differences exist in the micro deficiency. In vitamin A deficiency, hyperkeratosis II- scopic changes observed in the skin following of the hair follicles is observed. Multiple firm exposure to the various chlorinated cyclic com papules may develop as a result of the keratin pounds (hat can produce the lesion. It is. of plugs in the sebaceous glands. Dryness and >11 1- course, possible that these histopathologic vari scaliness as well as furunculosis of the stun may ations arc in reality only differences that exist also be present. In contrast, night blindness has 0 th between an early, a well-developed, and a not been reported as a symptom in patients ne subsiding lesion. - suffering from chloracne. Unfortunately, chlor Many theories have been advanced to explain acne outbreaks are often not well investigated and, he the development of chloracne. None is satis because of problems with litigation, accounts are factory. The concept of mechanical plugging of often only published many years after the out it. he the hair follicles on skin exposure to polychlor break has occurred if they are published at all. in. inated polycyclic compounds is not supported by Chloracne is a very persistent skin disease and the fact that chloracne develops following in the Japanese outbreak of poisoning with chloroof of ingestion of these compounds. It is possible that diphenyls, the skin changes were still present in a to the skin lesions are caused by a toxic effect on the number of patients three years after exposure to *ii i epithelium of the skin and its appendages which is the chlorodiphenyls had been discontinued. Whet lar manifested in proliferation and loss of normal needs to be investigated it whether this persistence function of the cell. Since (he chemicals are lipid of the skin disease is caused only by the fact that Ml **t soluble (hey could occur in a more concentrated once the lesion has developed it regresses very hum in sebum and therefore affect sebaceous slowly, or whether it may be sustained by the nd ni- glands more than sweat glands or Mlivary glands. chloracne-producing chemicals which have been ed The reports on the Yuslio outbreak suggest that absorbed and stored in the body, particularly the lun proliferation of the epithelium of the mucous adipose tissue, and are only very gradually ml membranes may also occur. The increased pigmen excreted. In patients with chloracne, adipose tissue he tation of the skin and mucous membranes may biopsies should be analyzed for the presence of the :ell indicate a functional disturbance resulting in offending polycyclic polychlorinated compounds. Hit increased melanin production. Hyperpigmentalion If adipose tissue is unobtainable, the sebum of the hit it also observed when the adrenals sre damaged, as ear canal or the content of the skin lesions may illy in Addison's disease, and adtenal function should represent a good substitute. January 1974 461 069499 HONS Apparently there is no known effective treat ment for chloracnc and the best control measure It prevention. The manufacture of chlorinated hydrocarbons and the coating of wires and con* densors with insulating material* containing them should be done in totally enclosed areas so that the fumes of the insulating substance# and of the solvents if any are used do not come in contact with (he workers. The workers should be provided with clean overalls and underclothes daily. These should be laundered at the plant and cleaned in such a way that no chlorinated hydrocarbons remain on them. Shower baths after work should be compulsory and supervised and there should be sufficient shower facilities provided so that the workers are not unduly delayed after work. Special synthetic wetting agents'41 seem to remove the material from the skin more effectively than ordinary soaps. The experimental induction of chloracne in men has also been accomplished with 2 appli cations of 10 Mg 2,3,7,8-tetrachlorodibenzo-pdioxin107 and with Halowax 1014, pentachloronaphthalene, and hexachloronaphlhalene.'4 7 Experimental work in animals has shown that most species do not develop the same type of dermatitis. The rabbit's skin,11 * particularly the inner aspect of the ear lobe, provides a good test medium for the development of acneform derma titis. The rabbits, in addition to the development of hyperkeratosis of the ear, will also show x hepalotoxic effect.' * Microscopic examination of the changes in the rabbit ear were described by Jones and Krizek.1 ss Jnagami and Koga114 were able to produce hyperkeratosis in hairless mice that were fed rice oil contaminated with Kaneclor 400. These mice also showed hepatomegaly Microscopic examination of the skin revealed cystic dilation and hyperkeraloaia of hair follicles and sweat glands. Hairless mice may be another suitable experimental animal for testing the effect of these chemicals on the skin. X-ttisease in Animals ChJoracne-type lesions do not usually occur in other species except for the rabbit and the hairless mouse, already mentioned. However, in 1947 Olafson1 * 5 described a disease in cattle which was designated as X-Uiteue or hyperkeratosis. Symptomatology included excessive lacrimation, diarrhea, polyuria, marked salivation, and dlachargs from the nostrils. Tha animals developed a chronic cough, poor sppetite, numerous red maculae in (he buccal mucosa; in addition, hyper keratosis of the skin developed. The skin was hard, and Fissured particularly across the withers and sides of the neck. Many wart-like proliferations on the lips, tongue, and hard palate were also observ ed. The abomasum was gresiJy inflamed, swollen, and edematous with many superficial ulcers.1 54 In the cows that had X-disease, a low vitamin A plasma level was also observed, which again indi cates that vitamin A may play a role in the etiology of these various disease entities The disease was first thought to represent a virus infection and other factors were also suspect ed. After numerous investigations it was finally established that X-disease could be produced in cattle by the ingestion of hi^ily chlorinated naphthalenes, and also by petroleum products such at crankcase oil. Bell'*7 tested the ability of the various compounds of the chlorinated naphthalene group to produce X-disease. He demonstrated in his studies that dichtorinaied and trichiorinated naphthalenes did not result in X-diaeaae, while tetrachloronaphthalene had an effect and the higher chlorinated naphthalenes such as peniachloronaphthaiene. hexadtioronaphthalene, heptachloronaphthalenc. and octachioronaphthalene caused severe disease. Octachloronaphthalene was less toxic than hc\aand heptachloronaphthalenc. WagenerM* repott ed (he occurrence of hyperkeratosis in caitle m Germany between 1946 and 1948- The caiiM- of this outbreak was traced to a wood preservunx c which was used in postwar Germany. The author proved experimentally that animals housed in cabins painted with the wood preservative develop ed typical X-disease. The poisonous ingredient in the wood preservative appeared only in certain lots of this product and latar lots did not contain it. The poisonous ingredient was never identified by chemical analysis. Animal experiments were conducted wiili the toxic ingredients of rice bran oil (hat produced Yusho in Japan. These toxic substances consisted of a mixture of chlorinated biphenyls containing 48% chlorine, with a trace of 0.01% of naphtha lenes.' *' When mice were exposed to them lor 3 to 4 months, they showed eczematous changes-of the skin around the eyelids, erosion, ulceration and perforation of the ear laps, loss of hair, and erosion and ulceration of the ikin around the nack, forekp, and sides of the chest. The authors 442 OtCOMeet JteWIrwv (ft Tbxtcolaw HONS 069500 ...iicvi that. ai Ihe end of tlic experiment. some >jV>- .lump's in the skin simitar lo Ihose in the iuui. , (>>}vnnKiiiuJ mice were teen in some of the and , i-oniiol mice also, which makes il difficult to Ml (Kl \ interpret llicir findings. itCfV- | in l`>57 a disease dial will he discussed in ollen, pouter detail later in this article (recurred in a large ** In 1 number of chickens. 11 was termed chick edema lilt A indi- t the jiiraac and was found to be caused by certain unde fit. Allen ami Catstcns,r,f> fed this substance tu monkeys and found that, among other changes which will he discussed lalcr. the monkeys cnl a ipectlnally *d in Inatcd hIucii Illy of developed a generalized alopecia and subcutaneous edema one to two months before death. School tie rt il.1 `1 produced hyperkeratosis of the skin in isls by feeding them hcxachloronaphthalcnc. Young swine'*1 and sheep'*3 did not develop ikin Icmoiir as a result of exposure to chlorinated niphlhtilcncs. Inaicd }. Ha id and tit in id an aknes hkmv and (wane, hex* eporltile in use of rvative author wd in rvcloplent in tin Iota tain it. FhtiUHiMtlat / Dermatitis and Irritation ofthe Skin As was pointed out earlier, the hyperpigmenta- \m observed in patients with chloracnc as well as the reaction described as cable rash could also be j die result of a photosensitive reaction. This photo| Kimiivc reaction is the predominant lesion in the j skin produced by a number of germicides, namely, bitliiunoi (Figure 2), tetrachlorosalicylanilide (TCSA). and a number of other halogenated phenolic compounds including fentidor. Patients who develop pholodermstitis complain initially of a burning sensation, and this is followed by aching A rash develops which in (he beginning is erythematous and associated with varying degrees of edema. As it subsides, pigmentation is observ ed.1 M Wilkinson'45 was the first one to describe cues where patients developed a photo allergy which was due to the incorporation of TCSA into popular soaps. He observed thaf the outbreak of fled by dermatitis was confined lo the areas that usually were exposed lo light. Most of the cases reported ith the (mm England were transient and as soon as aduced exposure to the compound was discontinued the ntilted reaction subsided. Jillson and Baughman1*6 and taining Jillson,1*7 on (he other hand, reported that their iphthan for 3 rates were more persistent. Willis and Kligman'*" demonstrated that the nges of photocomact allergy of (he skin with these various eration compounds was simply a contact sensitization. dr, and The reason that allergic reactions of the skin nd the i developed only after exposure to the sun was due author! i to the photodecomposiiion of the various products to which the skin had been exposed. If various brominated and chlorinated salicylanllides were irradiated in vitro in an appropriate solvent, tire resulting breakdown products caused an aller gic reaction on contact, withoui exposure to UV light. These studies were performed because of information provided in earlier studies by Coxon et al.,'*9 who had irradiated brominated and chlorinated salicylanllides and found that these compounds gave off halogens. With 3,5 substituted salicylanilides, the halogen substituted on the 3 position was lost. Telrachlomsalicylanilide photo sensitized subjects reacted to 3'. 4', 5-trichlorosalicylanilide and to 3', 4'dlchiorotalicylanilide. A reaction to nonhalogenated salicyianilklc was not observed. Halogens on the anilide ring were un affected by UV radiation, while the halogens on the salicyl ring were replaced stepwise by hydro gen upon irradiation. Among the patients who developed photosensi tivity lo tetrachJorosaiicylanilide reported by Wilkinson,1** a group of patients in a shop of a factory was also included. This same outbreak was reported by Jones,'70 who found (hat among 106 employees who used (he same soap very frequent ly, 31% were affected by a dermatitis due to contact with tetrachlorosalicylanilidc present in the soap. Additional cases of photodermatitis due to the exposure to tetrachJorosaiicylanilide were reported by Cilnan et al.,171 who pointed out that pronounced edema of the eyelids was also observ ed when involvement was severe. Some cases were persistent or developed acute relapses on further exposure to sunlight without further exposure to the offending chemical. Frenk'73 reported an additional nine cases of acute contact dermatitis of the hands due to tetrachlorosalicylanillde. Epstein17* described a photoallergic reaction in two patients to tribromosaiicylanilide (TBS). With patch tests these patients showed a cross reaction to TCSA. A similar cross reaction pattern to a number of halogenated lallcylaniiides was also observed by Otmundsen114 in patients who had developed photodermatitis following exposure lo tribromotaiicylanilide. Because of the photosensitivity reactions and the cross reactions that were also found with bithionol, which is sitralar in structure to tetrachiorotalicylanilide (Figure 2), the U.S. Food and Drug Administration, on October 24, 1967, with drew all new drug applications of all drop that January 1974 HONS 069501 I contained bitluonol. In (lie notice of withdrawal of uppmval in the Federal Register, the Commissioner of (he Food and Drug Administra tion pointed out that blthionol may in some instances cause j very persistent pholosensitization ami sevete dermatitis may occur with exposure to sunlight without further contact with the sensitiz ing articles, and that bithionol may produce cross pholosensitizalion with other commonly used chemicals such as certain lialogcnated salicylanilidcs and hcxachlorophenc. Hcxachlorophcnc preparations may also cause irritation of the skin if these preparations are used frequently. A true allergic reaction to hexachloro* phene is apparently rare.'15 The more severe reactions were usually confined to the skin of the scrotum and consisted of a primary irritant contact dermatitis.176 '77 The cases reported by Baker cl ol.17* also included a 33-monlh-old Caucasian female who developed a rash around the buttocks, vulva, and upper thighs following the use of a hcxachlorophenc preparation in her bath water. We observed severe skin irritation and ulcera tion in rats that were exposed dermally to a 3% hexachlorophene solution in propylene glycol or a detergent. The skin reaction was not as severe when the hexachlorophene concentration was reduced to 1.5%.'19 Boutwell et al.*19 reported considerable skin irritation in mice that had S mg/mouse applied twice weekly for 21 weeks. Harber et al.119 were able to induce contact photosensitivity with TCSA or TBS. These authors also found that cross contact sensitivity and photosensitivity to hexachlorophene existed in guinea pip with primary photosensitivity to TCSA and TBS. Photosensitivity to trichlorocarbanilide (TCC) was not produced in guinea pigs. The guinea pig may represent s suitable snimal species to study contact and photosensitivity of these and related compounds. After exposure to the photosensitizing agents has been discontinued, humans may still be sensitive to sunlight, a fact that needs further study. To a certain extent this could be due to a persistence of the chemical compound on or in the skin. Dichlorophtne used as an antimicrobial preserv ative may also cause contact dermatitis. Cross sensitivity to hexachlorophene was not demonstrated.190 464 CMC CrHieel Merlews In Toxicology Chick Edema Simpson et al.'91 and Sanger et al.'dcstnb ed a new highly fatal disease in chickens. The fmi outbreak occurred in 1957 in Georgia. Uigr numbers of birds from approximately three weeks of age to adult laying hens were affected. Clinical signs consisted of dyspnea, reduced body weight gain, stunting, subcutaneous edema, paleness, and sudden death. In young chickens, gasping was the first noticeable sign. This was followed by i waddling, unsteady gait. Grots inspection of the birds revealed a pale heart, fluid was noticed in the pericardial sac, and the liven were pale, mottled. and hud an irregular granular surface. In advanced stages of the disease the chickens had luip distended abdomens that were filled with fluid Ecchymotic hemorrhages were present in the skin beneath the wings, on the legs, and over (he Q bone in a few chickens. The kidneys were pale and swollen. Following the first outbreak, no addition al cases were reported until late summer and early fall of 19S7. when extensive losses of chickens and turkeys occurred throu^iout the southeastern pan of the U.S- Suspicion soon developed that losses were caused by the consumption of rations containing certain lots of animal fat.1*' The animal fats contained tallows and greases obtained it by-products from hides preserved with chlorophenols prior to leather manufacture.51 This material was excluded from commercial poultry rations and no additional cases occurred where animal fats were responsible. Simpson c: al.191 came to the conclusion that the edema m the chickens was the result of vascular changes that seemed to be the primary lesion. The vascular lesion, or endoiheliosis, consisted of proliferation and hypertrophy of the endothelial cells of arterioles and small arteries in most tissues. Pronounced proliferative changes in endothelium of the glomerular capillaries were observed, but no significant changes were noticed in the tubulai epithelium of the kidney. Necrosis of the parenchymal cells of the liver was also noted. In some reports it was pointed out that exposure to toxic fat resulted in a decrease of the total serum protein and a shift on the albumin u> globulin ratio with a decrease in the albumin fir-tion. This could, of course, lead to edema formation, particularly when combined with ni.ilfunctioning glomeruli; capillaries. It has also been suggested that increased permeability of the eardiovascular bed contributed to the edema forma- , n ,, u , ci p c, \s |, m J a ht ly ih u in u p* * th cl re re th lit .it ;c al a to bi sh Iv fa 2 u ec c< lh ic i.i m e>., nbt u ch *ONS 069502 ribfirst irpe ' fkfc 1 tea) | Ight , and llte ya the the led. iced * uid. akin 0 and ion* irly and pari wies con* imal 1u loro* olhei studies Uic scrum protein ^^Yniralion did not differ from that of the ViHtirola* *4'' "* and increased permeability of the ;jrdiovasctilar bed was advanced as (lie sole cause the fluid accumulation. In addition, pulmonary cdina was observed in diseased chickens and perivascular lymphocytic infiltration as well as edema of the cardiac muscle with interstitial limphocytlc Infiltration has been noted.1** Button microscopic examination of the cardiac muscle revealed degeneration and loss of mito chondria.1 14 it is possible that the heart may play t primary role in the development of chick edema, but a hepatorenal syndrome could be the under lying cause as well. Studies that investigate the development of the lesion more closely are needed to elucidate these problems. The type of diet also influenced the development of the disease. It could be brought on more rapidly witli a semipurified diet than with a natural grain ration; mdium chloride in (lie diet was a prerequisite for the development of the syndrome.' *1 If low rations of (lie toxic fat were fed to chickens they did not develop chick edema but ('production and hatchability were drastically reduced. Toxic fat was not the only product that caused the chick edema syndrome. Chlorinated biphenyls rcial rred n et ta in caused the disease in chickens188 and in Bengalese {Indies.2* A mixture of pentachioronaphthalene end hexachloronaphthalenc fed to chickens also resulted in chick edema disease.189 Cantrell et ngot i).lVa announced in 1967 (hat one of the toxic :uiar compounds capable of producing chick edema itton i of Pro- found in toxic fat was 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin. Then Higgenbotham et al.8: showed that the chick edema factor was produced n of by two principal compounds isolated from toxic i no bular aren* fats: 2,3.7-trichlorodibenzo-p-dioxin. and 2,3,7,8-lctracliluiudibenzo-p-dioxin. They also suggested fats and fatty acids that contain commercial chlorophenols as possible sources of that contamination. When crude fats and tallows are f the healed to produce fatty acids, chlorophcnol in to until! Aetna melbeen t carwma* residues may be converted to a chick edema factor. Whether these last three compounds mentioned are the only ones that produce chick edema disease is questionable unless the chlori nated naphthalenes as well us chlorinated biphenyls that produced chick edema were also omtaminaled with them. Another outbreak of chick edema diaeaae occurred In 1969.81 In this incident millions of birds in North Carolina were involved. The outbreak was traced to a vegetable oil refinery where feed fat became contaminated with "chlorophenols.'' The company formulated the chlorophenols as antimicrobial water treat ment products. An underground pipeline from the '`pesticide'* plant led to (raps used to collect by-product fatty acids held as feed fats. Flick et al.188 have recently shown that pyro lytic products of 2.3,4,6-te(rachlorophenol were toxic to chicks and produced chick edema if SO ppb were fed for 21 days. The product fed consisted of S3% hexa-. 45% heptu-, and 25% octachloro-p-dibenzodioxin. The pyrolytic product of pentachlorophenol consisted of a mixture of 5% hexa-, 62% hepta-, and 33% octachlorodibenzo-p-dioxin that produced mild edema and increased mortality when fed a diet containing I ppm (200 jug total ingestion) of the material for 21 days. When a mixture of tri- and tetrachlorodibenzo-p-dioxin was fed to the animals at a dietary concentration of 0.01 ppm (a calcula ted intake level of 1.9 #xg), the birds developed edema and 83% of them died. Further studies with pure compounds are necessary to confirm these findings. The effect of PCBs on the chicken was again demonstrated when PCBs that were used in the U.S. as heat transfer fluids in food processing plants contaminated large quantities of Osh meal that was processed for animal feed in a plant in Wilmington. North Carolina.'91 From about April 1971 until July 1971, PCBs leaked into (he Osh meal and 16 thousand tons of contaminated Osh meal were distributed to more than 60 companies in 10 states. One of the purchasers of the Osh meal. The Holly Farms, die nation's largest poultry producer, had to slaughter 77,000 fowls after discovering contamination of the chickens with PCBs. Some of the levels found in the adipose tissue of the animals were as high as 40 ppm. The Holly Farms first discovered that the chicken meal was contaminated because the hatchubiiily of their eggs was drastically reduced to 18% of normal. in February and March of 1968 an outbreak of chick edema also occurred in Japan, where more than 400,000 chickens reportedly died. The disease was traced to the contamination of animal feed with Kancchior 400, a Japanese PCB. The chick edema disease occurred simultaneously with an outbreak of polychlorinated biphenyl poisoning In people that was due to the contamination of January 1974 HONS 069503 rice brail oil. also contaminated with Kunechlor 400 and produced by (he same company.1 *2 Metcalfe19,1 suggested dial chick edema factor could he minimized if fleshings grease from hides preserved with technical penlachlorophenols con taining chlorinated dibenzodioxins were no longer used for food products. It was also pointed out that the use of glue emulsions containing chloroplicnols in rendering operations is another possible source of the toxic factor. Whether elimination of these sources from animal food products will prevent chick edema epidemics remains to be seen, since polychlorinated biphenyls that may be con taminated with chlorinated dibensofurans have also produced chick edema disease. Yusho During the summer of 1968, 13 cases of chioracne were observed in the western part of Japan, centering around Fukuoka prefecture. The incidence of the disease seemed to be familial and it became apparent that all patients had used the same brand of cooking oil (canned Kanemi rice oil). These findings were picked up by the news papers and a strong demand was voiced to elucidate the cause. Therefore, a Yusho study group was established by Kyushu University.192 The term Yusho stands for rice oil disease. After extensive investigations it was found that several illnesses occurred in a number of households that had used common cans of rice bran oil. When S total number of 89 males and 100 females that suffered from Yusho were examined, a variety of symptoms were found in these patients. The incidence of the different symptoms that varied a great deal is given in Table 2. The epidemic outbreak was finally, sfter very extensive investigation, pinpointed to Kanemi oil, manufac tured on February 5, 1968. Chemical analysis of this oil established that it was contaminated with 2,000 ppm of Kanechlor 400, a chlorobiphenyl that contains 48% chlorine. It was also demonstra ted that most of the components of the chlorobiphenyls were stored in sdipose tissue of the patients for quite a long time and passed through the placenta into the fetus of a few women who happened to be pregnant.194 The amount of oil necessary to produce disease was found to be at least 2.5 I. The younger the patient, the higher the morbidity rate with a smaller total amount of ingested oil. The average mount of chlorobiphenyl consumed by individual patients was calculated to be a total of 2 g.``M (Various reports written on this subject gm different accounts of what actually was consumed and the amount can at best only be estimated / When the rice bran oil was heated umici reduced pressure, Kanechlor leaked from the old pipes in which holes were subsequently discovered The Yusho outbreak spread not only ovci Fukuoka, Japan, but also over 20 other prefectures in the western part of Japan. A total of 1,057 poisoning cases resulted according to the lalest tabulation (August 1971, Ministry of Welfare).1,4 The most common initial symptom experienced by 136 patients with Yusho was Increased eye discharge and swelling of the upper eyelids (38.3%) followed by acneform erupt urn. follicular accentuation (33.1%), and pigmentation of the skin (9.6%). Itching of the skin and `'stiffening" of the soles of the feet and the palms of the hands were also observed. Pigmented mucus membranes were noticed in many of the patients, a small percentage of them developed jaundice many complained of a feeling of weakness and headaches; occasionally vomiting, diarrhea, and fever also occurred. Various neurological symptoms manifested themselves such as transient visual disturbances, numbness and spasms m ilklimbs, and hearing difficulties. Most patients showed acnelike skin eruptions that resembled ii>.chioracne observed after occupational exposure m chlorinated naphthalenes or chlorinated biphenyls. When the age and sex specific incidence rates were analyzed it was found that a significant sex difference was not present, but s lower risk for both males and females in the age groups over (>0 years was noted. The proportion of severe eases among those age 13 to 29 was significantly larger than that of other age groups. During the Yusho epidemic, 11 women with Yusho and 2 wives whose husbands had Yusho. but who themselves did not show the disease, delivered 10 livebom snd 2 stillborn babies. Nine of these babies had unusually greyish, dark brown stained skin, and dark pigmentation of the gingiva and nails was noted in five of them. Most infants also had heavy eye discharge. Histological exami nation of a stillborn fetus showed a marked hyperkeratosis and atrophy of the epidermis .iml cystic dilation of hair follicles, especially oi Uiom of the head. A marked increase of mol.min pigment in the basal calls of the epidermis was also noted. Most fetuses that were bom were smaller 444 atCCrtHed/tevirwt in Tbxkohtf HONS 069504 give lined >lcd.) mder c old ered. over , : ( j [ j oilier | Isi of i * the t of plom WttS ipper Miort, slion end lahm tucus tenu; idicc, k snd . and | OglCul ilien t i) l he iietm i J Ihc are to any Is. were t MX k for >er 60 CSKI larger i with 'usho, iscasc. . Nine tirown gingiva nfsnts ;xsmilarked is and those lelsnin si also mailer tabu: 2 Symplon* (fompliinU) ot hlimS (S9 malM, 100 Ptmiln, u of October 31, 1968)* Symptomi Males * Females % ttlackeninp of nail* Ittock *pnt* in nil pore* sweating in palm* . Acndike ikin eruption* Red spot* on limb* Itching - Ounce it) ikin color Swcllinp of hand* and feet Hardening of backs and hands Pigmentation of mucous membranes Sebum (gum accretion in eyes) Hyperemia of mucous membranes in ayes Temporary failing of eyesight Jaundice Swelling of upper eyelids Scnac of weakness Nundinal* of hands and feet I'cvcr Hearing difficulty Spasms of hands and feet Headuches Vomiting Diarrhea 83.1 64.0 $0 6 87.6 20.2 42.7 73.3 20.2 24.7 56.2 88.8 70.8 36.2 11.2 71.9 58.4 32.6 16.9 18.0 7.9 30.3 23.6 19.1 75.0 56.0 55.0 82.0 16 0 52.0 72.0 41.0 29.0 47.0 83.0 71.0 55.0 11.0 74.0 52.0 39.0 19.0 19.0 8.0 39.0 28.0 17.0 *Rcprinicd with permission from kuratsune el al.*** than Ihc national standards and four of them were small for gestational age babies. As they grew older their skin gradually became lighter over a period of three months. Okunnira and Katsuki19 7 studied a group of 24 patients whose main complaint had been acneform skin eruptions and who had been diagnosed as definitely having Yusho. Eighteen of these were adults and six were children. Irrespective of the seriousness of the disease in all patients, the initial symptoms were eye discharge associated with edema of the eyelids, defects in vision, generalized fatigue, anorexiu followed by comedo and acneform eruptions of the face, and pigmentation snd flattening of Ihc nails. The principal subjective symptoms were fatigue, numbness of the lower limbs, and intolerance for sake, which is a Japanese alcoholic beverage made from fermented rice. Three of twelve females had an irregular menstrual cycle. Seven of nine patients with serious disease showed a low grade fmr. Moat laboratory testa conducted on these patients were normal, including serum enzyme values. A slight elevation in alkaline phosphatase was observed in over half of the seriously ill groups. An increase in the globulin fraction of the serum protein was noted with a concomitant reduction of the albumin fraction, but the total serum proteins were within the normal range. These same 24 patients had abnormally elevated serum tri glycerides ranging from 200 to 600 mg %. In 12 of 24 cases, while the total serum cholesterol remained unchanged, phospholipids tended to be somewhat lower.19* Preliminary animal experi ments in rabbits conducted by the same authors showed that lipemia was consistently induced within several days. The levels returned to their previously normal values several weeks after expo sure had been discontinued. The aerum concentration of triglyceride is usually elevated in humans in glycogen storage disease (Von Gierke's disease). An Increase in plasma triglyceride concentration is also observed in essential hyperglyceridemm, an apparent eon- January 1974 467 HONS 069505 i pctut.il anomaly. Is^omial byperpl) ccridcmia has well as extensive hemorrhages into various orgam 8' Invit ol'fccivt'it in sihliitgs of a number of families, including (he adrenals. Reduced thymus and 41 t (mi ilie imxic of trautmissum is not known. The spleen weights were also observed In rats (hat I* course of this disease is generally benign and is received a single dose of 25 jig/kg TCDD and in UV consistent with a normal life span.1 *9 Observation mice the cell mediated immunity was found to be rej of the familial cases shows that the mere elevation suppressed.103 The various findings listed here do of triglyceride does noi in itself increase the risk of not conclusively prove that the immune response arteriosclerosis. is specifically altered by these compounds as w Another interesting fact in (his poisoning encounter it following radiation exposure or epidemic was that out of 2J eases with obvious Yuxlto. (0 showed symptoms or signs of u sensory neuropathy that included numbness, pain and hypoe.sihesia, and. in addition. I case also slmwcd iiflcxia.J^1 this effect on the sensory nerves would not be immediately obvious in animal experiments and probably should be investigated further. Samples of sputa and adipose tissue collected from patients who suffered from Yuslio contained chlorinated biphenyls on chemical analysis.'*4 The concentration in adipose tissue was much therapy with alkylating agents. In most studies relatively high doses of the chemical compounds were given, resulting in general toxicity accompanied by weight loss. Weight loss in itself may lead to atrophy of the spleen. In humans maintained on a low caloric intake with negative nitrogen balance, a general reduction oflymphoid tissue results which is not specifically related to the immune response and vitamin A deficiency it also accompanied by a reduction in lymphatic tissue. In this respect it is also of interest (hat when monkeys were fed `'toxic fat" containing clu ity fra{ (Po lire silt1 nun stin Hen (AL scu higher than in sputa. Recently Kuratsune et al.* ** reported on the present status of the patients who suffered from Yusho. Many of the patients were, still suffering from the illness. When the findings made in IS9 patients in 1970 were compared with those observed in 1969, it was found that half of the patients had improved clinically, while the remaining half showed no such improvement and more than 10% of the patients had become worse. polycyclic polychlorinated hydrocarbons. The germinal centers in the lymph nodes and spleen as well as islands of hematopoietic cells in bone marrow were extremely sparse.** This is more extensively discussed in connection with chick edema. The fact that a combination of duck hepatitis virus and PCBs is more toxic to 10day-old ducklings than the virus alone suggests a reduced immune response but could also merely represent an additive damaging effect on the liver, 3CUI boil subs 1; wor1 acid lad ll urn pity Immunosuppression made more susceptible to infection by the toxic Several reports have indicated that PCBs and effects of PCBs. So far no convincing evidence of i possibly some of (he chlorinated dibenzodioxins "wasting disease" ts a result of exposure to re: and furans may alter the Immune response in animals. Atrophy of the thymus and reduced total white count were observed in rabbits following the dermal exposure to PCB fractions containing tetraand pentachlorodibenzofuran ** In fish, exposure chlorinated polycyclic compounds has been reported in rodents or any other species in the literature. Atrophy of the thymus in newborn rodents reduces the capacity of these animals to produce 73 of ilrsl and to Aroclor 1254 for 14 to 45 days led to serum antibodies, resulting in wasting disease, also emaciation and funguslike akin lesions.*5 However, no attempt was made to isolate referred to as runt disease or homologous disease. Such animals are then incapable of rejecting not organisms from these lesions. When 10-day-old mallard ducklings were fed diets containing PCBs foreign skin grafts,304 which probably represents the moat convincing evidence for homologous as (Aroclor 1254) and were subsequently injected disease. A shift in the immune response could interperltoneally with a duck hepatitis virus, influence the development and growth of cancer significantly more ducklings died than when they received the virus alone.303 In guinea pigs, and alter the defense mechanism in infections. In order to prove that PCBs as well as chlorinated diet subietha! dosage levels of 2,3,7,8-tetrachlorodi- dibenzodioxins and furans do indeed Influence the benzodioxin produced severe atrophy of the immune response, the effect of these substances thymus with destruction of lymphocytes, on newborn animals of several spacies should be lymphoid depletion in spleen and lymph nodes, u tested with adequate parameters such as akin 461 CKC Critic*! Aevfews to Toskvhfy MONS 069506 fans and that d in obe e do IHIM i we or diei itnds Icily isclf MM Hive liold d lo yi* tttic that ning The m at tone nore hick luck 10lls irely iver, oxic e of i lo teen (he lenu juce also rate. :tin| lenti gout uuld nccr s. In ated i (he (ices d be kin grafting and the measuring of (lie development of antibodies. Whether the oilier chlorinated polycylic compounds in this erticle else effect the immune response has not been suggested or reported. Biochemical Effects and Chemical Toxicology Porphyria Porphyria cutanea tarda in humans is an acquired defect in hepatic porphyrin metabolism, characterized by uroporphorinuria, photosensitiv ity is manifested by blisters, and mechanical fragility of the skin. The hepatic porphyria (Porphyria cutania tarda) which is responsible for the increase in porphyrins and the skin photosen* tilivity can be produced experimentally by a number of drugs: all of these have the ability to stimulate the activity of the initial enzyme in tire heme synthesis 5-aminolevulinic acid synthetase (ALA). In addition lo this acquired porphyria, an acute intermittent porphyria erthropoietica end an scute intermittent porphyria hepatica, which are both hereditary, exist together with s number of subgroups which will not be considered here. In 1964, Bleiberg et si.1 reported studies of 29 workers in a 2.4-D (2.4-dlclUorophenoxyacetic acid) and a 2,4,5-T (trichlorophenoxyacetic acid) factory. Many of the workers had chloracne and II had abnormal excretion of urinary uroporphyrins. Many of the workers with uroporphyrinuria had hirsutism, hyperpigmentation, and increased skin fragility. Liver dysfunction was noted in two hospitalized patients. Poland et al.` reinvestigated the workers of the same plant studied by Bleiberg et al.5 They studied a total of 73 male employees; citloracne was found in 80% of the workers. In this study six years after the first report, clinical porphyria was not observed snd only one worker had persistent uroporphy* rinurla. In the Japanese poisoning epidemic (Yuslio), increased uroporphyrin excretion was not reported. Apparently polychlorinated biphenyls, at well as tetrachlorodibenxodioxins, produce hepatic porphyria in animals. Experimental hepatic porphyria was observed in Sherman strain rats when the rats were exposed to Aroclor 1254 in the diet. The porphyria resembled hexacldorobenzene poisoning and human porphyria cutanea tarda.2 * When female rats were fed 100 ppm Aroclor 1254 ia the diet (approximately 7.9 mg/kg/dty) they became porphyric after a delay of about 2 to 4 months. At this time the urinary porphyrin excretion increased rapidly. Uroporphyrin excretion was elevated to a maximum of 550-fold, prophobilinogen 27-fold, and 6-aminolevulinic acid 18-fold. The major porphyrins found in urine from Aroclor treated rate were 8-carboxyporphy rin (73%) and 7-carboxyporphyrin (16%). Substantial increases In amounts of 5 and 6 carboxcylic porphyrins were also observed. A 10-fold increase in the excretion of coproporphy* rins was observed, but the increase in uroporphyrin excretion was 1,400-fold. In control rat urines, coproporphyrin (81%) represented the major potphyrin fraction. A good correlation between urinary porphyrins and liver porphyrins was also observed. Only a trace of porphyrin was found in liver samples in control rats. When feeding rats 100 ppm Aroclor 1254, 6-aminolevulinic acid synthetase was not increased in the first month of exposure. After this time 6-aminolevulinic acid synthetase was elevated 3to 6-fo)d in porphyric rats but was normal in nonporphyric Aroclor treated rats. Cytochrome P-450 and microsomal heme were also increased throughout the study but the increase was maximal in one week despite the absence of an increase of the rate limiting enzyme in heme synthesis 5-aminolevulinic acid synthetase. When a large single dose of Aroclor 1254 (500 mg/kg) was given, a 4-fold induction of 6-aminolevulinic acid synthetase could be demonstrated in the liver 5 hr after dosing the animals. These results suggested that 6-aminolevulinic add synthetase induction occurred when porphyria had developed if lower levels of Aroclor 1254 were fed over a period of time, but when high doses of Aroclor were given it was induced immediately. Vos and Koeman05 reported that chemical porphyria was produced by the two European PCB products, Phenoclor DP6 and Clophen A60, as well as the American product, Aroclor 1260. The porphyrogenic action was observed in chickens, quag, and rats. It his not been established so far whether only certain isomers of the polychlori* natsd biphenyls or contamination with a chlorinated dibenzofuran is responsible for the production of hepatic porphyria cutanea tarda. Poland and Clover204 used fertile chicken eggs at a developmental stage of 15 to 20 days and injected them with 2S m! of the Haiogtnatad dibenzo-p-dioxint. They found that 2,3,7,8-tetrachlorodlbenzo-p-dtoxin produced doat-related Janaary 1974 I MONS 069507 increase in ALA synthetase activity. The lowest dose tested, 4.66 xlO*11 mol/egg (I.S ng) doubled the enzyme activity. These results were statistically significant. Studies with other halogenated dibenzo-p* dioxins gave the following results: the 2,3,7-trichloro and 2,3,6-tribromo isomers are potent inducers of ALA synthetase while 2,3 -d i ch loro and 2,7-dichloro, 2,8-dlchloro, 1.3.6,Melracltloro, and 1,2,3,4-tetrachloro Isomers all failed to Induce ALA synthetase at doses up to 2.S mg/egg. Of the limited number of halogenated dibenzo-p-dioxins that have been tested for fetal toxicity and the ability to produce chtomcnc, those that are toxic at low doses also induce ALA synthetase. Whether some of the polyneurotic symptoms reported in Yusho and also in severe exposure to technical 2,4,5*T or technical trichlorophenol that were heavily contaminated with letrachlorodibenzodioxin are caused by an effect on the porphyrin metabolism merits investigation since neurotoxic effects have been reported in acute intermittent porphyria.107 The dioxins that induce ALA synthetase in the liver of (lie chick embryo may also induce it in the mammalian system. Woods100 was not able to induce 5-aminolevulinic acid synthetase (ALA synthetase) in the liver of male rats up to 30 days after oral doses of 2,3,7,8-telrachlorodibenzodioxin. Guinea pigs and mice also did not show an increase of ALA synthetase following the admini stration of 2,3,7,8-tetrachlorodibenzodioxin. How* ever, it was possible to demonstate an increase in ALA synthetase and uroporphyrins in the liver in C$ black male mice given 25 Mg/kg/week for 4 weeks.100 It it possible that ALA synthetase induction varies in time with age and sex and may show species variation. Induction in mammals may require longer dosing, since, for instance, porphyria and induction of ALA synthetase with moderate doses of Aroclor 1254 did not develop until 2 to 4 months after dosing was started.30' Female animals and the embryo may be more susceptible titan adult male animals. Effect nn Other finzymes and on Vitamin A A number of investigators have shown that the poiychiorinatcd biphenyls induce liver microsomal enzymes.110 **11 Norback and Allen111 correlated an increase of hepatic microsomal enzyme activity with smooth endoplasmic reticu lum proliferation. Litters! et al.ni studied the effect of four PCD mixtures, namely. Antlor 1242, 1248, 1254, and 1260. The animals woe fed the material for 4 weeks and dietary levels employed were 500, 50. 5 and 0.5 ppm Ai lusher dietary levels an Increase in liver weights was observed. Elevation of triglycerides in the liver seen st 500 ppm with maximum effect occulting at approximately 50% chlorine content An increase in cytochrome P-450 was observed at the 50 and 500 ppm dietary levels of all PCB mixtures. An Induction of lire pentobarbital hydroxyiation and an increase in methylase activity were observed at the higher dietary levels. Induction of pentobarbital hydroxyiation increased with Increasing chlorine content of the PCBs. Nitro reductase activity was markedly stimulated with Increasing doses of PCB and the induefitm was seen at levels as low as 0.5 ppm for all mixtures. It is difficult to evaluate the enzyme induction at these low dietary levels since the control animal food can be contaminated at times with PCBs. ' Two types of inducers of drug-metabolizing enzymes of the liver have been reported: one group to which phenobarbilsl belongs resulted in increased P-450 content of the liver and induction of a variety of pathways including benzpyrene hydroxylase and demethylation ofethyimorphinc. The polycytic hydrocarbons such as 3,4-benzpyrene belong to the second group, which resulted in the formation of an abnormal .ytochrome. P-448, and increased benzpyrene hydroxyiation, but not ethyimorphinc donc.liv lation. Alvares et al.111 reported that PCB treat ment produced an increase in cytochrome P-448, an increase in benzpyrene hydroxyiation, and N-demethylation. Since the PCBs represent mixtures, it is possible that the type of induction may depend upon the isomers present. The various effects of PCBs on liver microsomal enzymes can be blocked by the priot administration of actmomycin D. When the effect of a number of PCBs on the beef heart mitochrondrial enzyme system was studied, it was found that they inhibited NADUoxidase, succinoxidase, and cytochrome oxidase activity. It has not been established whether (hesc same effects will occur in vivo. Benthc et al.:14 induced liver microsomal enzymes by intraperitnneal injection of a single dose of Aroclor 1248 or 1232. Most of the microaomal enzymes have been studied in the rat, but they are affected in other species is well. Alien et el.1M reported liver 470 OtC Critical Reviews in Toxtcohgy MOMS 069500 r IW I ff :ls 01 ;u m Mg \n he n. on re of til ro(h I8t II II III n| ne In on ne ic. si cli 10ne lautIK. nd ut on >ut un 10* on rat Di ne IK l ro tor icn ier vtr hypertrophy aflei feeding polychlorinated biphenyls (Arodor F248) or a polychlorinated iriphrnyl (Arodor 5460) for three monlhs to primates. The increase In liver size was attributed lo the increase in smooth endoplasmic reticulum and induction of microsomal enzymes was observed. Proof of the induction of microsomal enzymes in birds is the finding made by Uncer and Pfeakall31* (hat livers obtained from American keslrels exposed lo either Arodor 1254 or Arodor 1262 showed an increase in the in vilro breakdown of oestradiol lo a more polar metabolile. This effect on sex hormones is also emphasized by Platnnow et al.717 Arodor 1254 given lo Yorkshire boars reduced the urinary excretion of gonadal steroids. Both the polychlorinated biphenyls as well as the chlorinated naphthalenes affect the vitamin A content of (lie liver. This was particularly studied with (he chlorinated naphthalenes since hyperker atosis and other abnormal keratin formation of the skin is often accompanied by vitamin A deficiency. Low vitamin A plasma levels were observed in cattle with X-disease and young swine.'>*'167 A decrease in vitamin A storage in the liver of Japanese quail and rats that were fed Arodor 1242 was also demonstrated.71R Since vitamin A deficiency affects reproduction, this aspect should be further investigated to determine whether the effect of PCBs on reproduction is secondary to vitamin A deficiency.7'9 tcobiclion and Johnstone7 7 0 studied the effect of various PCD isomers on the hepatic enzyme system. Biphenyl itself did not cause induction of hepatic drug metabolizing enzymes and neither did monochlorobiphenyl (4-CI). Substitution of chloro groups at the three and four positions of the diand (elrachlorobiphcnyls caused induction of microsomal monooxygenases. Induction of micro* soma) monooxygenases occurred also when hexaand octachlorobiphenyis were given to the rats. Nitroreductase and carboxylesterase activities were not affected while all compounds produced a marked induction of the tulfobromophlhalein conjugating system. Terphettyls as well as chlori nated terphcnyls increased the pentobarbital meta bolism in the rat with corresponding increase in smooth endoplasmic reticulum which suggested microsomal enzyme induction.7 7',7 7 7 Lucler el al.5 71 reported that a single oral dose of 5 or 25 vg/kg body weight of tetmcMorodfbenxodioxin induced cytochrome P-450 signifi cantly in male rats for a period of up to 28 days. Cytochrome b-S content was also increased. Hydroxylation of aniline was induced and aminopyrine demethylation rates were decreased. Micro somal proline was increased by approximately 15%. The induction of UDP glucuronyltransferase was moat striking Oxidative phosphorylation rates in rat liver mitochondria using succinate as a substrate were unchanged. The enzyme induction was observed as early as one day - the peak induction was reached between three and ten day* after treatment. The enzyme induction was accom panied by an increase in smooth endoplasmic reticulum and a mild increase in rough endo plasmic reticulum.114 These changes were first observed three days after the injection of the telrachlorodibenzodioxin. Thus, enlargement of the liver during exposure to various chlorinated compounds is accompanied by an increase in smooth endoplasmic reticulum. Furthermore, if "inclusions" or "hyaline bodies" are seen within the cytoplasm with the light microscope, concentrically arranged membranes that often surround lipid vacuoles can be seen with the electron microscope (Figure 6). Hexachbrophene, on the other hand, does not increase the size of the liver."* The livers show no ultrastructuril changes and probably microsomal enzymes are not induced. Whether other compounds listed in Figure 2 affect liver microsomal enzymes is not known. Hexachiorophene has an effect on a variety of other enzyme systems than those mentioned thus far. Cammer and Moore775 and Caldwell et al.776 have shown that hexachlorophene uncoupled oxidative phorphorylation, increased adenosine triphos phatase activity, but did not affect the activities of electron transport enzymes when compared to controls. Gould el al.77 7 demonstrated that hexa chlorophene inhibited succinoxidase activity of rat heart, liver, and kidney preparations. According to unpublished material cited by Gluck,77* hexa chlorophene is conjugated to the glucuronide conjugate which is excreted in bile. The proportion of conjugated hexachlorophene to unconjugated hexachlorophene in blood may be more significant in determining the toxicity of hexachlorophene than the hexachlorophene blood levels as they are presently determined. Methemogbbinemia In the early 1960s outbreaks of methemoglob- January 1974 471 HGNS 069509 incmias were reported from hospital nur series. *1 ' I10 One out break involved a total of 18 infants with o level of methemogtobin from 4.9 to 30% of (he total hemoglobin. An epidemiological investigation traced the outbreak to 3,4,4'trichlorocarhanilide (TCC) that was used as a 2% solution in a rinse in the laundering process, after which the laundry was neutralized with an acid and then dried in an autoclave. Twelve of the eighteen cases in this report were prematures who were probably longer exposed to the offending chemical than term infants since their hospital stay is usually longer. It is also possible that chemicals penetrate more easily through the skin of prema* lures. In another outbreak, methemoglobinemia occurred among a group or curtodial patients in an institution. These patients were given soap suds enemas with boiled soap that contained 2% TCC.131 It is possible that by heating TCC a breakdown product occurred which produced the methemo globinemia. P-chloroaniline and 3,4-dichlotoaniUne have been suggested at breakdown products. None of the other compounds discussed in this article have thus far been shown to produce methemo globinemia. Storage, Distribution, Metabolism, and Excretion As we progress further in our study on the distribution, metabolism, excretion, and absorption of compounds such as chlorinated biphenyls, chlorinated terphenyls, the chlorinated dibenzodioxins, and chlorinated naphthalenes, we will probably find that since they are lipid soluble they are stored in quite the same way at the more studied persistent pesticides such as DDT and dieidrin. From the evidence presently available on the chlorinated biphenyls it can be assumed that the turnover of these compounds in the body, particularly of those that have a high chlorine content, may be slower than some of the persistent pesticides. The turnover rate will vary with (he degree of chlorination. Hexachlorophene, on the other hand, is fairly rapidly excreted and presently no information is available on bilhionol and related compounds that have a germicidal effect. All of these compounds are not very well absorbed by the gastrointestinal tract or the skin. Analysis of various tiaiues of rats given a single dose of the PCB Aroclor I25413* showed the highest concentration of PCBs in adlpoee tissue and the least in plasma. Brain, liver, and kidney had levels of (he same order of magnitude but were lower. If, for instance, 1,600 mg/kg body weight of Aroclor I2S4 were given by stomach tube, 24 hr later the mean concentration m adipose tissue was 1,146 ppm. The concentrations observed in individual animals varied greatly. Similar levels were found in adipose tissue when 3,200 mg/kg of ArOclor 1260 were given by stomach tube to adult female tats. The levels of Aroclor 1.254 found in the brain showed a mean of 138 ppm and the values observed for Aroclor IJ60 were 145 ppm. All of the values were calculated on a wet weight basis. The same concentrations in ppm were found with both compounds even though only half of the amount of Aroclor 1254 was given. This is probably due to the fact that only a certain amount of Aroclor is absorbed at a given time. When 100 ppm of Aroclor !254 was fed to male weanling rats for 58 days the mean concen tration of Aroclor derived materials found in the feces varied roughly between 11 and 35 ppm. After the animals had been removed from the experimental diet for a week, the excretion in the feces was 8 ppm and after 16 days' recovery an average of 2.5 ppm was found in the feces. PCBs at the concentration of about 2 ppm were still observed in the feces 24 days after the animals were removed from the experimental diets. During the entire study the amount of PCB derived material recovered from the urine remained below I ppm. While PCBs were fed to the rats, the gas chromatogram obtained from Ihe feces was superimpotable on the one obtained from the standard Arodor 1254 that was fed to the rats. Once the animals were no longer fed Aroclor 1254 the number of peaks observed in the gas chroma tograms that resulted from Ihe analysis of the fecal material decreased. Fewer peaks were also observed in the gas chromatograms obtained for the tissues and from urine. During the 58-day feeding period, a gradual increase in the concen tration of the PCB derivatives was observed in tissues. The highest concentration was found in adipose tissue; after 58 days it reached mean values of about 500 ppm. The concentration in the liver only amounted to a mean of 12 ppm at that time. Following a recovery period of 71 days when the animals were fed plain chow, the concentration of the PCB derived material in adlpoee tissue had not decreased appreciably and 472 CRC Crilket Reviews in ToMkohgy HONS 069510 Kill (bowed values in (he neighborhood of about 400 ppm and 4.7 ppm was found in (he liver- The concentration in the brain never reached very high proportions; it ranged from about 2 to 4 ppm. Following a 7 Way recovery period the amount in the brain was still about 2 ppm. When Aroclor 1254 wu fed for 240 days at the dietary level of 100 ppm, the concentration of (he PCB derived meterial observed in adipose tissue had reached values of about 1,000 ppm. If, on the other hand, 100 ppm of DDT is fed to male rati for 2 years, the concentration In adipose tissue is about 9S ppm.*" In a separate study where 500 ppm of Aroclor I2S4 was fed to male rats for 6 months and the exposure (o PCB containing diets was subsequently discontinued for an additional 10 months, the concentration of the PCBs ranged from 924 to 1,688 ppm in adipoae tissue with an arithmetic mean of 1,192 ppm. The concen trations in the liver ranged from 17.3 ppm to 26.24 ppm with an arithmetic mean of 22.65 p,Lprmjr. .1)4 Further studies are necessary to determine whether PCBs are stored in tissues to a certain point when a steady state is reached and the concentration does not increase further. The given concentration at which this equilibrium is reached in adipose tissue would vary with the amount fed to the animals and the type of compound. The effect of starvation or illness when the amount of adipose tissue in the mammalian body is suddenly reduced also needs to be investigated further. Would this lead to increased excretion of the material or to redistribution or both? Dahlgren et el.*3* who determined PCB tissue levels in pheasants, found that a concentration of 300 to 400 ppm of the PCB derived material was usually found in the brains of birds that had died, while live birds that were fed the same amount of PCBs had much lower concentrations in their brain tissue. Studies in bobwhite quail showed that relative peak heights and chromatograms for the tissue extracts were similar to the Aroclor standard after partitioning with acetonitrile-hexane when the birds were given a single capsule of Aroclor and their carcasses were analyzed soon after exposure. This suggests absorption of all components of Aroclor. The gastrointestinsl tract, liver, skin, wings, and feet had been removed prior to analysis. When bobwhite quail were fed Aroclor 1254 for 14 days and then allowed to recover, the ps chromatograms showed a gradual reduction in the number of peaks and after 42 days on a control diet only 4 major PCB components remain, which suggested selective excretion and/or metabolism of some of the components of the Aroclor tested.*3* In a reproduction study, Heath et il.** analysed 2 second-year eggs of mallards fed 25 ppm Aroclor 1254 in their diet for 2 years and found 56 and 33 ppm PCB by wet weight. The gas chromatogram showed a pronounced dimin ution of the early peaks when it was compared to the standard, suggesting again a selective reduction of the components with lower chlorine concen trations. This is also evident in environmental temples. Lower orpnisms such as mussels and Hah contained a higher concentration of PCBs with lower chlorination than birds.11 In mammals, excretion of PCBs in milk and transplacental passage to also observed. Feeding cows 200 mg/day of Aroclor 1254 for 60 days** resulted in higher PCB levels in milk fat than in body fat. At 30 days' exposure, 45.6 ppm was found in milk fat and 21.9 ppm in body fat;at 60 days* exposure, 66.7 ppm waa found in milk fat and 44 ppm in body fat. However, once exposure to PCBs was discontinued, the concentration in milk fat sank below that of body fat. Transplacental passage of PCBs also occurred.*37**3* Sherman strain rats were given Aroclor 1254 in peanut oil by stomach tube during days 7 to 15 of gestation. A mean of 20.60 ppm PCB derived material was found in their milk when 10 mg/kg/day was given to the dams during pregnancy, while those given 50 mg/kg/day excreted a mean of 66.34 ppm in their milk on a wet weight basis. Fetuses taken by Caesarean section on day 20 of pregnancy contained a mean of 0.63 ppm and 1.38 ppm of PCBs, respectively. With a fivefold difference In dosage, the concen tration In fetuses showed a twofold difference and in milk a threefold difference.*3 * The hyperpigmented babies observed in the Yusho incidents in Japan represent additional circumstantial evidence of transplacental passage of PCBs. Polychlorinated biphenyls have also been de tected in human adipose tissue. Biros et al.*3* analyzed two human adipose tissue samples which contained 200 and 600 ppm PCBs ranging from pentachlorobiphenyl to decachlorobiphenyl and Price and Welch*40 found 100 ppm PCBs in another human adipose tissue sample. According January 1974 473 HONS 069511 i> rKV ami WVhh."' II to iM I In* pi*m*il population in the U.N. contain I ppm or more Pl'Bs in their adipose tissue. Among IVB sources ilicy discussed (lie food chain ami house dus( which, from residences in southwestern Michigan, contained up to I HO ppm ITUs. Polychlorinated biphenyls were found in .11% of 637 samples of human adipose tissue (ha! were collected from the general population of Ihc IJ.S.. as purl of the Human Monitoring Survey. Of the samples ana* ly/cd. 5% contained more than 2 ppm.24' PCBs have also been found in human adipose tissues of the general population of Scandinavia143 and Japan,244 ,43 and in human milk and human adipose tissue of the general population of Germany.243 In general, low concentrations of PCBs were stored in adipose tissue in a certain proportion of the general populalion. This is probably more prevalent in the highly industrial ized countries and almost nonexistent in under developed countries unless most of the food was imported from polluted areas. These low levels of PCBs are harmless, as far as we know at the moment; however, the occasional levels found in the 100 to 1,000 ppm range give rise to concern. It would be of interest to establish whetlter average adipose tissue levels increase with age and whether those in certain professions such as painters, printers, textile workers, workers in plastic fac tories, and those engaged in the manufacture of transformers and capacitors had higher PCB- levels than the general population. Reports on the absorption, storage, and metab olism of chlorinated naphthalenes, terphenyls, dichlorophene, bilhionols, fenticlor, triclosan, and tetrachloroialicylanilide are aimoat nonexistent in the literature. Some unpublished results on some of the compounds used as germicides are men tioned in the summary minutes of the Over the Counter Drug Panel on antimicrobials. Copies can be obtained by writing to Dr. Mary Bruch, Bureau of Drugs. Rockville, Md. Approximately 9% of triclosan, alio called Irgasan DP300 and CH 3565 (2,4.4'-trichloro-2'- hydroxydiphcnyl ether). Is absorbed when it is applied dermally in a soap solution. The halflife of the chemical was 20 hr when applied dermally and 10 hr when given intravenously. Humans excrete 65% in urine and 20% in feces after intravenous injection. It is excreted either as a free compound or as a glucuronide. In dogs nearly 100% of the injected material Is recovered as lltc glucuronide or sulfate m mm,' and fcccs over a 5-day period. It has been shown that TCC is absorbed through Ihc skin. Tissue levels of the chlorinated nrjihatvl Aroclor 5460 were determined in codfish.24'1 In this study it was found that Arocior 5460 w;c apparently poorly absorbed from the gastrointesti nal tract; H was stored in all tissues that were analyzed. The highest concentration was found in the liver and followinga single dose of I g some of it was still present in (he codfish after 70 days. an indication of slow excretion. The authors pointed out that with the method used to determine the chlorinated terphenyls, it would be difficult to distinguish chlorinated biphenyls from chlorinated terphenyls. A comparison of the chromatograms of the standard with those obtained from chlorin ated terphenyls extracted from tissues revealed more of (he earlier peaks in material obtained from tissues and fewer of the later peaks. A variation in the intensity of the peaks was also observed. There seems to be a change in the chlorinated terphenyls within Ihc living organism, however, part of this could also have been intro duced as an artefact by the analytical methods or by differential absorption from the gastrointestinal tract. Calves born to cows with X-disease developed hyperkeratosis and it was found that higliU chlorinated naphthalenes were excreted in milk.*54 This suggests that chlorinated naphtha lenes may well follow similar storage and excretion patterns as PCBs. Chlorinated dibenzodioxins and furans arc probably also stored in tissues of animals and men who are exposed to them, but again not much information Is presently available. Firestone ei al.347 fed the untaponifiable fraction isolated from "toxic fat" or 3% "toxic fat'' itself to young cockerels. The "toxic fat" contained chiorodibenzodioxins with 2 to 8 chlorine atoms. When the excreta were analyzed it was found that a certain percentage of the hexachlorodibenzndioxins had been absorbed from the gastrointest inal tract, while all of (he octachlorodtbcn/odioxins remained in the gastrointestinal tract, ami only very little of the heplachlorodiben/.odioNin was absorbed. Because of the presence of inter fering components, the chick tissues and excreta could not be examined for the lower chlorinated dioxins. In the organs, the highest concentration was found in the liver and an appreciable amount 474 CRC Critical Reviews tn Toxicology *QNS 069512 wax also found in lhe hone aiul skin, while the mgaiut contained vety few dioxins. Some rwjefuv for a ptmiblc metabolism of Itexa- and licptachlorodibcnzodioxin by the chick was also observed. Zilko and Wildish34* fed di-, trh. (etra-, and ociachlorodibcnzofuran lo fish and detected only nctuchlorodibeiuofurau in muscle and gut of dead fish. One problem with metabolism, storage, and distribution studies of chlorinated dibenzodioxins and furuns is lhal not all of the com pounds are readily available and, if they are available, any kmg-icnn feeding studies in large animals are expensive. The very toxic dioxin compounds cm be given only at such low leveit that they escape detection easily. In our labora tory we detected a chlorinated dibenzofuran in the urine of rata fed Aroclor 1254. We found this same chlorinated dibenzofuran in the Aroclor ilseir*0 as a contaminant. Williams el al.344 fed rats a total of 22.7 and 120.7 jig of octachloiodibcn/ndioxin. They re covered a great deal of the compound In the feces. Very small amounts were recovered in the liver at both dosage levels and in adipose tissue at the higher dosage level. Further studies are needed to establish the absorption, excretion, and storage of these compounds, and (heir persistence. Studies with labeled material may eventually give us some information on their metabolism. Piper et al.3s* give a single oral dose of C14 labeled 2,3,7,8tetrachlorodibcnzo-p-dioxin to rats and found that about half of the radioactive material was re covered within 21 days in the feces and only 13% In urine and 3% in expired air. Most of the remaining radioactive material was found in the liver. In another study3Si 100 jig of Cl3*octachlorodibenzodioxin was given daily to rats by stomach tube for 21 days. Less than 0.3% of the material was absorbed. Subsequently the rats received a control diet for 7 weeks and still showed 20% of the absorbed material found at 21 days in their tissues. The in vivo metabolites of the various com pounds under discussion have in most instances not been identified. Block and Cornish3 3 3 studied the urinary excretion of biphenyl and 4* chlorobiphenyl in rabbits. Biphenytgiucosiduronic acid and 4-hydroxybiphenyl were isolated from urine of rabbits fed biphenyls. The rabbits fed the 4 c h 1 or ob i p h e n y I excreted 4-(pchlorophenyl)-phenol and 4-chlorobiphenyl glu- cosiduronide. Twice as much 4-chlorobiphenyl as biphenyl was excreted as the glucnsiduronic acid derivative. It is possible that other low chlorinated biphenyls and naphthalenes are excreted in urine in a similar manner; whether they would be detected by present gas chromatographic methods would have to be checked by combining the identification of radio labeled material with gps chromatography. Hutzinger et al.3*3 found evidence that some chlorinated biphenyls are hydroxylated by some species. Apparently trout is not able to hydroxylate 4-chloroblphenyl, 4,4'-dichloroblphcnyl, and 2,2',3,5'-te!rachlorobipheny], while the rat and the pigeon both formed monohydroxychiorobiphenyl from 4-chlorobiphenyl. In addition, the rat also produced dihydrochlorobiphenyl. Large quantities of unchanged material were excreted by the rat when 4,4'-dichlorobiphenyl and 2,2',5,5'* tetrachlorobiphenyl were given to them and only a monohydroxyderivative was identified in their urine. Pigeons followed a similar pattern but no dihydrochlorobiphenyl was observed in the bird excreta fed 4-chloroblphenyl. No hydroxy metab olites could be identified in the excreta of the three species when they were given 2,2',4,4'$,5'hexachlorobiphenyl. No evidence of reductive dechlorination was observed in any species. Only very few studies have been conducted with radiolabeled hexachbrophene to study its absorption, distribution, and metabolism. Wit and Van Genderen354 gave rats and rabbits hexachlorophcne-C14 (2,2'-methyiene-Ci 4 bis(3,4,6trichlorophenol). Complete recovery of radio activity in excreta after a single dose of 50 or 15 mg/kg of radioactive hexachlorophene was ob served in rabbits within 5 days. Roughly one third was excreted in urine and feces, respectively, as unchanged hexachlorophene and the remainder was excreted in the feces at unidentified metab olites. The metabolites could not be extracted from the feces with ethanol. The biological halflife calculated from the experimental results was found to be approximately 17.8 hr. In rats, about two thirds of the radioactivity was recovered in feces within seven days and only very little in urine. A complete recovery of radioactivity was not obtained in these studies within seven days. Five cows given a single dose of 15 mg/kg body weight of hexachlorophene excreted about half to two thirds in their feces within five days. Very little was excreted in urine and none in milk. January 1974 475 HONS 069513 Metabolites weir ihyI identified and tissue levels were 1101 determined in this study. The authors riled ;< study hy Kok,353 who did determine k'VK'hloioptinio in various organs of rats. (.'.inoil et al.i5h studied the absorption of C-14 labeled Ucxaddwophene on (Ik tail skin of the rat. Hexachlorophene in a dclctgcnl solution was absorbed by the intact skin of the tail at (lie average rate of 1.7 pg/cm* of exposed surface ic:i/Ih. Immediately alter the infliction of second dcgicc burns, the absorption rate increased 2.5 times that of the normal skin surface. After 24 hr the absorption rate dropped to normal or sub* normal levels. Immediately after traumatizing the tail, the absorption rate of hexachlorophene was even greater than following the infliction of a burn wound. Manowit/. and Johnston337 found that hexa chlorophene was deposited on the skin when used in various formulations and could be extracted from the skin by soaking the'exposed areas (hands and forearms) in alcolrol. The amount recovered increased in proportion to the amount applied. These authors were unable to confirm earlier investigations which suggested that the quantity of hexachlorophene retained by the skin reached a plateau level after a number of washings and remained relatively constant thereafter. Arms were washed and rinsed every 20 min. When extractions with alcohol were made on different arms after different numbers of washings, it was found that the amount of hexachlorophene recovered in creased with the number of washings and no plateau was reached. A greater amount of hexa chlorophene was also deposited on the skin with increasing length of time of a single washing. Since we now know that hexachlorophene is also ab sorbed from the intact skin and some of it is probably also lost from the surface of the skin, the time interval between the washings plays a role in the amount of hexachlorophene found to accumu late on the skin. Also of interest is that bathing water containing 4 mg/1 of hexachlorophene de posited approximately the same amount of the chemical on the skin as a single washing with a 2% hexachlorophene soap. Since hexachlorophene was used very exten sively and often unknowingly by the general population, wc investigated whether it was present in the blood of a few random samples of the gcncrul population. Wc found that the arithmetic mean concentration of hexachtorophene in adults was 0.028 ppm Oig/g) of whole blood of a total of 14 samples. In infants who were washed with hexachlorophene in the nursery the amount of hexachlorophene found in whole blood after the infants hud anywhere from 2 to 14 washes averaged O.IW ppm (pg/g whole blood). Blood obtained from the cord at the time of birth contained an arithmetic mean of 0.022 ppm hexachlorophene.15 4 *3 * * In the rats that were used in a reproduction study and were fed 100 ppm hexachlorophene in the diet for 258 days, the hexachlorophene blood levels averaged 1.21 ppm, while the blood levels of rats fed 500 ppm of hexachlorophene for 55 days who had severe symptoms of poisoning amounted to 8.5 ppm.1 **.a60 At the time we reported these blood levels, our recovery rate was only about 75%. AJI blood levels reported at that time were determined in whole blood. (It is assumed at the moment that blood levels are equivalent to half the amount of that found when plasma is ana lyzed.) A study undertaken at the request of Sterling Laboratories133 illustrated that newborn rhesus monkeys following total body bathing with a 3% detergent solution for I week had blood levels of about 1.5pg/m) (ppm). In subsequent months a plateau effect was observed and at 90 days the blood level was equivalent to l .l jig/mi. These monkeys showed status spongiosus of (Ik white matter of the brain, this indicates that at least in the monkey, blood levels of about 1.5 pg/ml whole blood were consistent with fiuid accumulation in the myelin sheath of the central nervous system. Lockhart133 cited unpublished studies by Gluck, who found that hexachlorophene blood levels in term infants after wishing with a 3% hexachlorophene detergent solution were some what higher than those reported by us with a mean concentration of 0.345 ppm 0%/g whole blood). This difference could have been due to a differ ence in washing procedure and a better recovery rate in the analytical method in Gluck's study, or both. > Premature infants apparently absorbed hexa chlorophene more readily. In IS premature infants that were washed with a 3% hexachlorophene detergent solution, the average blood concen tration amounted to 0.475 ppm(jig/g).341 Abbott et al.343 using a different washing regimen found a mean hexachlorophene blood concentration 12 to 18 hr after the initial wah of 0.154 ppm (jrg/g i | j CK m 7"jc MQNS 069516 .of iih of the Hcs txi rlli pm ion In iod of W led ew t)Ul ere I Ilf ulf nsof nm 'ilh tod ent 90 iu(. the at 1.5 uid l ral by DOd 3% me* ean *). Ter nary . or exi* mu lerw can* jolt und i 12 */s whole bUnnl) amt on the 7 to *>ih day of life. 24 to 4N hr a liei the second or third application of hi.rhere. the r.w * M.\\J !eve> .vr.'-. -.tcd .rtv.r-; uusmt m k\*ul blood of U> inlJiiis w js 0.013 ppm Oig/g). When hexachiorophene was applied only on the day of delivery, the hcxachlorophene blood levels gradu ally decreased from a mean of 0.145 ppm on the lint day to a mean of 0.057 ppm on the ninth day, following an increase to 0.194 ppm on the fourth day. The authors suggested that premature infants may absorb more hexachiorophene through their skin since in one infant 29 weeks of gestation and weighing 1.42 kg the blood level was 1.17 ppm 24 hr after the initial wash and 0.655 ppm at 48 hours. Aider el al.*43 reported mean hexachiorophene blood levels of 0.18 ppm in infants who were dusted with a powder containing 0.33% hexachiorophene. Adults using hexachiorophene preparations also absorbed it via the skin.1" The blood levels obtained in adults that exposed their total body surface to a 3% hexachiorophene preparation twice daily for 60 days, 5 min each time, showed blood levels of approximately 0.68 ppm (jig/ml whole blood) with a range of 0.25 to 1.08 ppm (pg/inl whole blood). If only hand washing was employed for a period of 28 days, 4 times daily, a mean blood level of 0.07 ppm was reached and in a face hand washing study the mean blood level after 28 days was 0.196 pg/ml. Mood levels obtained in a patient with burns who died between 24 and 48 hr after (he last hexachiorophene application were 2.2 ppm (pg/g whole blood). The blood levels that have been reported all indicaie that following dermal hexa chiorophene exposure the chemical can be found in blood, and from information through animal studies as well as some human experience, levela of approximately 1.5 to 2 ppm (jig/g) in whole blood in humans are presently considered to represent toxic levels if the analysis for blood was performed with the methods mentioned elsewhere in this article. Probably a better way of aasessing a toxic dole would be to measure the amount of hexachlorophsne in the brain, but for obvious reasons that is not possible. Female rats fed hexachiorophene at the rate of 300 ppm (25 mg/kg body weighl/day) for 55 days with very definite lesions had about 4 ppm hexachiorophene in their brain and 3.91 ppm in the liver. Squirrel monkeys given 5 mg/kg body wcighl/tlay of hexachiorophene lor .IX days showed a mean hcxachlomphcnc vomcniiaiton in ;).* I*:;-. -, 0 3s pjvn and .3 n\uv ii,t tkAachlotophctu' 1'om.vntuhoii ppm/'*'* The recovery rule in this study was only 75%. These squirrel monkeys had no overt clinics) signs of toxicity, but showed mild status ipongkmis of the white matter of the brain. Electroencephalo- graphic changes had also been observed in these primates. Ulsamer et al.*4* found hexachioro phene brain levels of about I ppm in newborn rats with signs of central nervous system toxicity, in I suspected case of human hexachiorophene poisoning in a child, the hexachiorophene brain level was about 2.2 ppm.14 1 Adult rats dying after the administration of a single dose of hexachioro phene showed concentrations of 6 to 9 ppm of hexachiorophene in the brain.244 Some variation of the concentration of hexachiorophene in the brain which an be related to neurotoxtc symptoms must be expected in different species and perhaps also with age and sex. Whether the fluid accumulation which may occur in the myelin sheaths after repeated exposure to hexachioro phene would alter hexachiorophene brain levels has thus far not been reported. With present methods, we are probably only determining free hexachiorophene while the conju gated portion remains undetected. The toxicity of hexachiorophene will vary with the ability of the organism to conjugate, metabolize, and excrete this material. We found in rati that hexachiorophene is excreted in milk of dams fed hexachiorophene at the rate of 20 and 100 ppm in the diet (2.3-1.1 mg/kg/day and 11.8-5.5 mg/kg/day, respectively, for 241 days). Electron capture, gas-liquid chroma tographic determinations revealed levels of 0.07 and 0.33 ppm in the milk. Since hexachiorophene did not teem to accumulate to a great extent similar values would probably have been obtained, had the levels In milk in these animals been measured earlier. Although we now have some information on the absorption and distribution as well as excretion of hexachiorophene, the possible storage of the material as well as its metabolism needs further investigation. Since hexachiorophene was found in cord blood of infants, it an be assumed that transplacental passage also occurred. Morphologic Effect on the Uver Some of the chlorinated naphthalenes, cMori* Janwry 1974 477 I M0NS 069515 natcil dihcnzodioxins, chlorinated dlbcnzoftirans, the chlorinated biphenyls, as well as the chlori nated terphenyls have an effect on the liver in various species, including humans. Bennett et ill.5*1 compared the toxicity of differently chlori nated naphthalenes and a biphenyl that contained 65% chlorine. Tri-chloronaphlhalene apparently was less hepalotoxic than the higher chlorinated compounds and the chlorinated diphenyl was the most toxic of all. The studies were done in rats and the exposure was by feeding as well as inhalation. The findings deacribed in the liver consisted of liver necrosis, fat accumulation, and the presence of hyaline bodies in the cytoplasm of the liver ceils. A marked deposit of finely divided granular yellow or yellow-brown pigment was observed in KupfTer cells, particularly in those animals that had been exposed to the chlorinated biphenyls. This pigment apparently did not stain characteristically for either hemosiderin or hemofuchsin. Of couree, all liven were enlarged macroscopically; in addition, these authors described proliferative changes in occasional bile ducts, particularly after feeding mixtures of chlori nated naphthalenes and chlorinated biphenyls. The number of bile ducts was increased in certain areas and the epithelial cells lining the bile ducts showed mitotic figures. In inhalation studies with low concentrations of PCBs which consisted of either a 16-hr daily exposure to average concentrations of 0.57 mg/m3, or to 8 hr daily with an average concen tration of 0.93 mg/mJ, neither group of rats appeared ill. The periods of exposure varied from 37 to 143 days. When the animals were sacrificed the livers were pale or slightly yellow and some what mottled. Microscopic examination of the livers- revealed similar changes as had been observed after feeding the material, and an increased vacuolation of the liver cells. Micro scopic evidence of recovery was not observed in the livers of rats that had been exposed to the chlorinated diphenyl for a total of 10S days and were removed from exposure for 2 months. Similar findings were made by Miller1* * who studied (he effect of a PCB which contained approximately 42% chlorine. This author observed varying degrees of liver damage after subcutaneous injection as well as oral ingestion of the toxic substance in guinea pip, rail, and rabbits. The liver damage consisted of fatty degeneration and atrophy of the central lobular cells. In the rat, hyaline bodies within the cytoplasm of the liver cells were also noted. Most of the liver damage was found in the guines pig, less in the rabbit, and least in the rat. Nishizumi,3** who studied (he effects of a Japanese biphenyl Kaneclor 400 with 40'^ chlorine in monkeys and mice, described enlarge ment of liver, fatty changes, granular cytoplasm of the hepatocytes, increases in the size of KupfTer cells and hepatocytes, and a brown pigment in some of these cells. He also studied the livers under the electron microscope and found an Increase in smooth endoplasmic reticulum, vari ation in the appearance of mitochondria, and an increase in the number of microbodies In addition, "myelin figures" were observed in the cytoplasm of hepatocytes. Vos and Koeman" observed liver necrosis in chickens that were dosed with European PCBs, while a Monsanto product with 60% chlorine did not produce this effect. Increased amounts of iron were demonstrated with Perl'i iron slain in the liven of all chickens exposed to the European as well as the American PCB sample. Vos and Beems44 studied the PCB-induced lesions in the liver of rabbits, following dermal exposure to Aroclor 1260, Phenoclor, and Clophen. The micro scopic changes observed in the liver included fatty degeneration, focal necrosis, centrolohular liver cell atrophy, ceroid pigment in Kupffer colls, periportal fibrosis, and cytoplasmic hyaline degen eration. The feeding of Aroclor 5460, a chlorinated terphenyl, Arodor 1254, or a chlorinated dibenzop-dioxin, not further specified in the article, led to liver hypertrophy of Sprague-Dawley rats within eight days to three weeks. Ultrastructural alter ations were similar in the three groups and consisted of numerous multilayered, concentric membrane arrays and proliferated smooth endo plasmic reticulum. In many instances these mem brane arrays surrounded lipid droplets.170 Rahims given 300 mg of Aroclor 1242 and 1254 once a week for 14 weeks developed enlarged livers and the ones given Aroclor 1254 showed mkizonal necrosis of the hepatic lobules. The livers of rabbits fed Aroclor 1221 in a similar fashion did not show any histologic changes.171 We conducted studies to compare the toxicity of Aroclor 1254 and Aroclor 1260 in Sherman strain rats. The acute effect of these two mixtures on the liver was negligible. If rats were given a 47$ OtC cmktl Renew$ to Toxkvkxy MOMS 069516 > - V v*. I, V> , '-; v... - / S * n * *v , V- r^w..,^.^ *-1 At ' ' 1 l'IGURl-1 3. Section of liver of u rat Ted Arocior 1260 Tor 90 days Thii figure Utuitratei mitotic fipuic and a hyitorcliromaiK- mirioua (anowr) H I E X 500. single dose of 10.000 mg/kg in peanut oil by tlomadi lube, and were sacrificed the next day, the Uvci wus essentially normal microscopically. Dates of 1.000 ppm (72 mg/kg) of Arocior 1260 for .1 months produced only an increase in mitotic figures (Figure 3) and enlarged hepstocytes. When groups of rats were fed dietary levels of 20. 100. $00, and 1,000 ppm of Arocior 1260 and 20,100, and 500 ppm Arocior 1254 for 8 months, the liver changes were pronounced.273 The hepstocytes were enlarged in many of (he rats of the experi mental groups and lipid accumulation, foamy cytoplasm, and a brown pigment which stained partially positive for hemosiderin were observed in the livers. The pigment was primarily observed in macruphages and Kupffer cells. Inclusions (Figure 4) that stained slightly more eosinophilic than the surrounding cytoplasm were present in the cells. These inclusions within the cytoplasm have been described for many compounds such as mirex273 (dodecachloro-octahydro-l ,3,4-metheno-2Hcyclobuta[cd] penlalene), DDT2 74 , 1 ,1,1 -(trichloro-2,2-bi*(p-cWorophenyl) ethane dieidrin2 75 , 1,2,3,4,l 0,10-hexachloro-6,7-epoxy-1,4,4a5,6,7,7,Ba*oclahydro-l ,4-endo-exo-5,8- di me I ha nonaphthalene, and piperonyl bu l ox I de2 74 (a-(2(-butoxyethoxy)ethoxy)-4,5methytenedioxy-2-propyllohiene). They are identi cal to the so-called hyaline bodies that were described in the earlier literature by Bennett el al.341 and Miller.261 Aside from these general changes, extensive, grayish-white, firm, glistening areas were noticed in the livers of a number of the experimental animals, particularly at the high dietary levels. Microscopic examination of these grayish-white areas showed that the hepatic parenchyma had been replaced by glandular pale staining epithilial cells that formed ducts and were surrounded by proliferating fibrous (issue (Figure 5). Larger lesions often had extensive fibrosis and also contained collagen and the ducts that were formed by epithilial pale staining cells were markedly dilated and contained necrotic debris or mucus. The lesion has been classified as adenofibrosis for the time being. It has been described in detail by Edwards and White274 who observed it in rats that were fed butter yellow (p-dimethyiamino-azobenzene). These lesions enlarge peripherally by the formation of new pendular structures. Hepatic January 1974 HONS 069517 FIGURE 4. Section of liver from ret fed Arodor 1254. A duster of ittciunom u premm in the cytoplasm of one hcpafucyte (arrow). Usually iodivklual cells contain only one of (hex formations at a time. ToluMinr blue stain of msraphiss embedded material X 300. tissue may be (rapped between the glands of the peripheral area of adenofibrosis. The older central parts of (he lesions sometimes contain pigmented avascular collagenized stroma with atrophic epi thelial cells lining dilated ducts that appear atrophic and dead. The glands of actively growing adenofibrosis show atypical epithelium with large pleomorphic nuclei. The epithelium can occasion ally become stratified. Parts of the lesion may be quite extensive, while in addition, in many areas of (lie same liver small fod of adenofibrosis exist simultaneously In dose relationship to portal areas. It has therefore been assumed that these lesions arise from bile ducts, but they can also be primarily confined to the region of the hepatic veins. The absence of bile ducts around hepatic veins led to the conclusion277 that a precursor, other than bile ducts, existed from which adenofibrosis developed. However some investigators of the lesions have assumed that it represented an atypical bile duct proliferation, alio referred to as cholangiofibrosis.278 In addition to this lesion we also observed small dusters of glandular cells within areas of adenofibrosis n well as adjacent to blood vessels that were surrounded by relatively normal appearing hepatic parenchyma with turn ing characteristics of salivary 0and tissue. These dusters of cells very closely resembled panocatic tissue.3 79 The distinction between adenofilnosis and adenocarcinoma ia difficult. The glands .ire quite atypical in adenofibrosis but it may be helpful to remember that in adenofibrosis, in contrast to adenocarcinoma, the irregularity observed is limited and duplicated with consider able exactness from lesion to lesion. Mucus is always present in adenofibrosis, but not always in adenocarcinoma. The stroma of adenofibrosis is concentrically arranged, young and cellular at ilic periphery and old and hyalinized towards the center of the fesion.277 Of course a liver showing adenofibrosis may also contain carcinomatous lesions. So far conclusive evidence is lacking in ihe literature that adenofibrosis pves rise to carci nomas. However, it has been found to coexist with carcinoma of the liver in rats and some investi gators feel that the lesion is in fact a precursor of cancer.220 Choline deficient rats apparently, in addition to other changes in the liver, also develop 480 CRC Crittcei Revtrwt bi Toxicology HOMS 069518 I l-U;UKi: 5. Section illuKirnic* an area of adenoflbroife of the liver of rat fed Arovhsr 1254. Non* lUc r|>ilMiul cell* forming imatl duct*. The duett contain cellular debri* and are nirrounded by fibroin* which can become quite pronounced, H A K X 125. rely tin* tew Hlic Mil ire be > in rity tier* i it t in I it (he the ving lout the rci* with esii>r of In lop adenofibrosis.2"1 However, these ruts were fed peanut meal and loymcal and therefore (lie possi bility exists that the experimental diet was con* laminaied with a inycoloxin. Bcmict el al.367 also described the lesion (adenofibrosis) in rats when a mixture of chlorinated naphthalenes and chlorodiphcnyls was fed to litem. A critical review of the hnlopathogencsis of this lesion was made by Slrwyii and Snell.377 Ullroslructurai changes of (he livers of animals exposed to (lie Aroclor consisted of an increase in smooth endoplasmic reticulum and atypical mitochondria. Many lipid vacuoles were observed, pailitulaily at Use Itigher dietary levels. They were it times surrounded by concentrically arranged membranes. Tlsc "Inclusions" or "hyaline" bodies observed in the cytoplasm with (he light micro scope correspond to these formations when they are examined under (Ire electron microscope. Their ultrastnictural appearance has led to their being teferred to as "linger prints" (Figure 6). The epithelial component of the adenofibrosis outlined consisted of cuboidal or columnar cells with the fret surface lined by microvilli, granular cytoplasm with many ribosomes, and a few endoplasmic membranes. Some of the celh contained a great deal of mucus and resembled goblet cells. Tononiumcnts and terminal bars were also observed. These various observations suggested that the epithelial component of adenofibrosis had all the morphological characteristics of bile duct epithelium. Chickens with chick edema disease and cattle with the X-disease, already mentioned, usually showed changes In the liver which consisted predominantly of liver cell necrosis and fat ac cumulation. The hepatotoxic effect of chlorinated naphthalenes has also been described in rats.36 7 In a number of reports on the occupational occurrence of chloracne In workers that were exposed to chlorinated naphthalenes, chlorinated diphenyls, or 2,4,5-T. and technical irichlorophenol, reference is made to the hepatotoxic effect of these compounds. This hepatotoxic effect can manifest itself only in a decreased tolerance for alcohol; it may also develop into hepatic deficiency with acute yellow atrophy and jaundice. Apparently the effect on the liver was January 1974 MGNS 069519 FIGURE 6. Electron microftraph of a portion of the cytoplasm of a hepaiocyte. The Mction ihowi concentrically arranged arrays or membranes which surround vacuoles contaMng lipid. Lead citrate, uranyl acetate X 34,200. particularly bad following exposure to halo-waxes, which are chlorinated naphthalenes.2*1 Cotter382 reported seven cases of pentachlorinated naphthalene poisoning in workers who were engaged in manufacturing wire cable during World War II for the navy. Pour of the workers develop ed jaundice and two died. Microscopic examina tion of the Uver of the two workers that died showed complete loss of liver oells in some areas, the centrolobular areas were hemorrhagic, and prominent bile duct proliferation was seen in the periphery. Another group of compounds that have at least some representatives that cause a hepatotoxic effect are the chlorinated dibenzodioxins and chlorinated dibenzofurans. Single oral doses of 1 to 10 Mg/kg 2,3,7,8tetmchlorodibenxodioxin given to rabbits produc ed s hepatotoxic effect. At the higher dotage level the animals died and the lower doses caused serious Uver damage.1** Schulz1*1 had reported earlier that single oral doses of 20-50 Mg/kg body weight resulted in fatal liver necrosis in rabbits. * Weanling rabbits were treated with 2,3,7,8tetrachlorodibenzo-p-dioxin by applying a dose of 1.67 pg daily to the inner aspect of both ears for three days. The total dose given was 7.0S jig/kg body weight. When these rabbits were sacrificed -18 days following the last application of the dioxin, their liven were significantly larger than those of the controls. Microscopic examination of the liven showed enlarged hepatocytes. Some of the hepatocytes were multinucleated. The cyto- plasm * foamy or vacuolated. A light brown pigment in some liver cells as well as Kupffer ceils was also observed. Inclusions were seen in the cytoplasm in some liver calls, slight interstitial fibrosis was present, and two of three rabbit livers examined showed foci of necrosis that were surrounded by flbreato.3*4 ' ; j > j ! j ! | j Studies with "toxic fat,'* the cause of chick edema disease, showed that it had a necrotizing i 482 OtC Crittnl RtHrwt to ToMkokgy 06952 Clio.! on the livoi in chickens. As we now know, rite toxic upen is rcsjMinwblc for duck edema disease are chlorinated dibcu/.o-p-<Jioxin. Toxic foi* ai*o altered liver morphology in Macacca niulaita monkeys.3"* Hcpatocyies were enlarged, multinuclcatcd and focal necrosis in the centralobular /.one was observed. Many liver cells were vacuolated and stained positive for neutral fat. In our studies of rats given hcxach/orophcne, we did not observe any liver pathology with the light microscope and no change with the electron microscope at the dosage levels that we employed.'1* Thorpe*11 on the other hand, observed periportal fatty changes in liven of sheep given three or four doses of 50 mg/kg hexachlorophene and histochemical changes were also observ ed in lltc hcpatocyies. Pugh and Crowley3"4 also observed hcpalotoxicity in sheep after giving them hexacUlmophene. Of the other germicides discussed in this article, only Iriclosan (Irgasan DP-300) (2,4,4-trichloro- 2'-hydroxydiphenyl ether) has been reported to have a toxic effect on the liver at a dose of 125 mg/kg and enlarged lho liver of rats at a dose of 25 mg/kg. according to tire summary minutes of the OTC Panel. U.S. Food and Drug Administration. It was not stated whether these were single or repealed doses. Oral administration of Iriclosan to beagle dogs produced jaundice and severe liver damage was observed. The effect that these com pounds have on the liver will have to be studied further. / Tire accumulation of fat in the liver indicates that cither fat transport or fat metabolism is impaired; if the insult to the liver is removed, the liver gradually recovers unless the lesion has become irreversible. The increase in smooth endo plasmic reticulum as well as the "incJusions" will also disappear after a certain amount of time if exposure to the material it discontinued.3"7 The significance of the increase in smooth endoplasmic reticulum and the hyaline bodies with a concomit ant rise in liver microsomal enzyme activity is presently not understood. These changes are con sidered adaptive and beneficial, but in addition to metabolizing unwanted chemicals at a higher rale, the stimulated liver also metabolites some medica tions more rapidly.31" as well as substances produced by the body such as steroids. Whether the inclusions within the cytoplasm represent the so-called inactive smooth endoplasmic reticulum which is deposited in the form of arrays of membranes within (lie ceil is mil known. The fact that smooth endoplasmic reticulum may become hypoactive was first postulated by HutteTei et al.3** Fibrosis of (he liver as well as necrosis and the adenofibrosis observed after PCD exposure in rats should definitely be considered a very aerious lesion. Usually adenofibrosis occurs concomitantly with hepatomas or hepatocarcinomas in rodent livers. A recent publication by Kitmira and Baba3"** showed that hepatomas can be induced in nts with Kanechlor 400. Chronic feeding studies may show that other PCBs will also induce hepatomas In rodents. Neurotoxicity In some of the reports on chloracne and also in the Yusho incident, peripheral neuropathy is mentioned as well as psychic alterations*,3** as part of the symptomatology of poisoning, but in most reports not much emphasis was put on it. In the Yusho epidemic Muni and Kuroiwa3** per formed more detailed examinations in 21 cases admitted consecutively to Kuyushu University Hospital in the northern part of Japan. Seven of the patients complained of numbness or pain in the peripheral portion of their extremities and in five cases, hypoesthesia, hypalgesia, and thermo hypesthesia were noted. Slowing as well as reduc tion of the sensory nerve conduction velocity in the radial and sural nerve was observed in a number of these patients. Headache and peripheral nervous system symptoms were also reported in a poisoning episode which occurred in s Finnish company where paper was impregnated with biphenyl.*1 The only chemical of the group of polychlorin ated, polycyclic compounds discussed in this report that has a predominant central neurotoxic effect in animals as well as humans is hexachlorophene. Adult female rats that were fed 500 ppm hexachlorophene in the diet for 14 weeks (about 30 to 18 mg/kg body weight/day) developed leg weakness after 12 to 19 days of exposure. This leg weakness which was only evident in the hindlegs progressed to paralysis within 3 to 5 weeks.3*1 At autopsy the brains of the exposed rats were enlarged; they weighed an average of 2.6 g while the control brains weighed an average of 1.99 g. Microscopic examination of the brains revealed normal gray matter throughout the brain and the spinal cord In the few instances that the spinal January 1974 413 FIGURE 7. Electron micrograph from the white matter of the brain of a rat, illustrating status spongiosus. Note ilic Lijuempty appearing vacuoles lined by myelin (MY * myelin, A axon), load citrate, uranyl acetate X 34,200. cord was examined. The while matter on the contrary seemed widened and showed many cystic spaces that appeared empty and were lined with strands of material that stained positive for myelin. The vacuoles were very numerous and varied greatly in size giving the tissue a spongy appearance. The brains in these studies were usually fixed in either Bouin's solution or buffered formalin for light microscopic study. Brain tissue, particularly that of rodents, may normally show small vacuoles that are considered to represent an artefact.3*3 These vacuoles are smaller, usually very uniform, and at times contain a homogeneous pink staining material. They are usually separated by a wider area of normal appearing white matter than the vacuoles in status spongiosus. Brains that show (his artefact do not weigh significantly more than those (hat do not show it. Electron microscopic examination of brains of rats given hexachlorophene showed that the vacuolation in Ihs white matter of the brain revealed large vacuoles lined by myelin. The axons dial were present appeared usually to be quite normal, occasionally strands of myelin had separated and traversed the vacuoles. The vacuoles were other wise empty except for some electron dense granu lar material (Figure 7). No other alterations wcic observed in the while matter. This lesion is mil unique for hexachlorophene; other chemicals that have produced status spongiosus as we have called the lesion include triethyltin,3*3 isoniazid,2*4 the halogenated benzanillde, 2-aceloxy-4-chlom-.f.5diiodobenzanilide,3*1 and rafoxanide.1*'' which is the 3,S-diiodo-3'-chloro-4'-(p-chloro-phcnoxy) sallcylanilide. Triethyitin seemed to produce the lesion at a much lower dietary level and mine rapidly than the other chemicals mentioned, p.utkularfy hexachlorophene. In an additional study, after feeding rats 500 ppm hexachlorophene in the diet for 10 weeks and then discontinuing the exposure to hexachlomphene, function began to return in the hind quarters of paralysed rats after 2 weeks and wiilun 4*4 CKC Crtlkml Mrrtev in TbjrJrofacr MOWS 069522 6 weeks the ruts had almost completely recovered 3% hexachlorophene detergent solution developed clinically. However, microscopic examination of a diffuse status spongiosus of the white milter of the btains of these rats showed that a few cystic the brain.30* Santolucito364'**' was able to ipuces were still present 12 weeks after the produce the vacuolation of the white matter, the exposure lo hexachlorophene had been discon so-called status spongiosus, by injecting squirrel tinued. In weanling rats, but not In adult rats, the monkeys daily subcutaneously with S mg hexa* brain lesion and symptoms of paralysis could be chJorophenc/kg of body weight for 37 days. When produced with a single oral dose of 100 mg/kg of monkeys were fed hexachlorophene they did not body weight given by stomach tube in peanut develop the brain lesion but showed electro- oil.11 * Part of the reason for being able to encephalographic changes which need further produce the brain lesion in (he weanling rats with investigation. The monkeys- that showed the a single dose may be related lo the fact that the vacuolation also demonstrated electroencephalo- weanling was able to survive a higher single dose graphic changes. Sheep have apparently also shown than the adult rat. Other species ihul have ulso shown paralysis status spongiosus of (he white matter according to Hale and Reid.33 Lampert et al.33 were able to and other neurological deficits after exposure lo hexachlorophene are rabbits,1*'' pigs.1** and cats.3*' Sheep become blind according to Udall and Malone130 Hanig et al.3M recorded the neurological deficit* produced in cat* in more detail. Early symptoms consisted of lassitude, weakness and ataxia of the hindlegs, impaired righting reflex, patellar hyperreflexia which later developed into hyporeflexia. urinary retention, and eventually complete flaccid paralysis. The cranial nerve functions remained intact except produce the lesion in mice as well as rats. These authors reported that the morphological changes were also observed in frozen sections of brains quenched in liquid nitrogen. Trypan blue injected Intraperitoneally did not stain the brain, an indica tion that the blood brain barrier was intact as far as trypan blue was concerned. The brain changes consistent with status spongi osus due to hexachlorophene exposure have also been described in humans, particularly children. Mullick130 examined the brains of six human pietciinitially. In these cats an increase in cysternal cases that died from hexachlorophene poisoning. cerebralspinal fluid pressure was observed. The two adults in this group did not show In our first studies wc continued exposure lo vacuolation of the white matter of the brain. They hexachlorophene for a period of 14 weeks because had died less than 48 hr after exposure to we had designed the experiment to conduct an approximate 90-day feeding study. It is not hexachlorophene. The four children (two with congenital ichthyosis, two with burns) had at least necessary (o feed rats for (his length of time. As three or more days of exposure to hexichioro- wc determined later, microscopic changes can be phene baths in which a 3% hexachlorophene observed in rats after a feeding period of two preparation was used. The final concentration of weeks when the animals were exposed to levels hexachlorophene in the bath water was not high enough lo produce the lesion. Some vacuoia- known. AH four children showed status spongiosus lion can already be observed 3 lo 4 days after of (he white matter. The brain weight given in one onset of exposure to hexachlorophene in some of the cases, a 12-day-old child, was 42S g. The rm. When lower dietary levels were fed in a normal brain weight for a child that age is 382 g. s. reproduction study,"* the dietary level of 20 Shuman et al.3*4 in a retrospective blind study h ppm (24-0.9 mg/kg body weight/day) had no were able lo demonstrate that the incidence of t) effect on the brain. At the dietary level of 100 status spongiosus in the brain stem reticular ie ppm (11.8-4.6 mg/kg body weight/day) focal formation in infants who had died from a variety re areas of vacuolatiun of the white matter of the of causes could be correlated to hexachlorophene r- brain were observed in some of the rats, but they exposure. The authors found 21 cases in a series of were never extensive. The rats in these lower 250 autopsies of children. Of these cases, 18 were K> dietary levels did not show any clinical neurologi premature infants weighing less than 1.400 g and id cal symptoms. were of less than 30 weeks' gestation. All but 2 of o- The brain lesion observed in rats was also the cases had 3 or more total washes with 3% d daacribed in monkeys following the exposure to hexachlorophene. In the group of cases that were in hexochiurophene. Newborn monkeys washed with studied were a number of stillborns who did not January |*74 MOWS 069523 \Uuw ilu- li 'ion jiuI of course had no* had dermal ................... .. c\[himoc. Shuman el *1- IM, .miIhv cases of Lclicrer Siwe's disease m which they had observed status spongiosus of In- klure warier. Each of ihe children with l.em iei Siwe's disease had had pronounced expo se lo liexadiloropftene because of (he skin lesions which had been vigorously scrubbed with a detergent containing the chemical. The various oi'scivjiumt show that, in patients with abnormal skin as well as the skin of the premature, hexa chlorophene may be absorbed in sufficient amounts to cause status spongiosus. The fact that Mood levels in premature infants washed with hexachJomphcne tend to be higher than in mature infants also substantiates these findings. Almost identical observations were made by Powell e( at.3 05 In premature infants weighing less than t,400 g. 7 of 13 that had 4 or more exposures to hexachlorophene washes showed status spongjosus of flic myelinated tracts of the brain stem. If this group was further subdivided according to the number of exposures, 6 of 9 infants that had 9 or mure exposures to hexa- chJorophcnc washes showed the lesion, and l of 4 that was washed 4 to 8 times. Four stillborn* and 13 infants washed 0 to 3 times with hexachioro- phene and weighing less than I,400 g did not show the spongy change. Electron microscopic examina tion showed that the spongy change observed in the premature infants was due to a split in the myelin sheaths. In August of 1972 the press reported a number of deaths of infants in France that occurred because a baby powder to which hexachlorophene had accidentally been added at a concentration of 6% was used on the infants. The final death toll amounted to 41 infants and young children30* (New Vork News, Dec. 13, 1972). Detailed accounts hove not been published of this incident because of litigation problems. As we pointed out earlier, under acute toxicity, the higher the con centration of hexachlorophene is in a preparation applied to (he skin, the more likely it is that skin damage will result. Skin damage also occurred in the French incident. In addition to this, the powder was probably used in many instances where some diaper rash already existed and was then well covered with a diaper, which may have contributed to increased absorption in this area. The brains and spinal cord of a few Infants from the French poisoning outbreak that were examined microscopically showed status spongitv sus of the white matter.30* These various accounts show that status spungjosus can also be produced in humans and probably represents a nonspecific reaction which can be elicited by a number ofchemicals. The lesion status spongiosus of the white matter as far as we now know teems to represent a specific or restricted type of brain edema where Ihe fluid accumulation is confined to the inside of the myelin sheath and myelin is not actually damaged or at least breakdown products of myelin have not been observed. The term status spungiosut or spongy change has been used as a descriptive term and a spongy change of the gray matter may also occur in certain diseases. Status spongiosus together with other morphological changes can be observed in the brain in a variety of many etiologically different diaeases and the reader is referred to KJatzo,307 Ardonalo and Lamperl,10* and a recent editorial in the British Medical Journal.30* From clinically observed poisoning cases as well as animal experiments it is evident that the brain lesion is to a great extent reversible. However, in our own studies as well as those by Lampert el al.,101 microscopic changes In rodents did not completely regress and the fact (hat sheep do not recover their eyesight after hexachlorophene poisoning110 indicates that repair may not always be complete. No reasonable explanation exists at the moment to indicate why certain chemicals produce this very specific edema confined to certain structures of the centra) nervous system. Gastrointestinal Lesions Only two types of compounds under discunion have produced lesions in the gastrointestinal tract. We observed in our study with Sheiman strain rats diet high single oral doses of 3 00 mg/Vg Aroclor 1254 and 1260 or more caused ulceration of the gastric and duodenal mucosa (Figure 8). Ulcers in the GI tract were not observed in the long-term feeding studies with 500 ppm or less. No other changes in the gastrointestinal tract were observed Ringer et al.,'0* in their reproduction study with mink, observed that Aroclor 1254 produced hem orrhage in the gastrointestinal tract of the off spring. Allen and Norback1 '* observed hyperplasia and dysplasia of the gastric mucosa in rhesus monkeys. Six of these rhesus monkeys were fed 4M CKC Cri(k*t Reviews in Toxkohty MGftS 069524 FIGURE 8. Section or the glandular portion of the stomach of a rat given a single dose of 3,000 mg/kg of Aroclor 1254. This figure illustrates the focal lost of mucna and the Infiltration by inflammatory round cells. H A E X 50. 300 ppm Aroclor 1248 and six were given 5,000 ppm Aroclor 5460, a polychlorinated triphenyi. The glandular formations in this lesion were highly atypical; penetration of the muacularia mucosae and invasion of the submucosa by (he mucosal epithelium were observed. Allen and Carstens29* reported an effect of toxic fat on the gastric mucosa in 18 of 27 monkeys, Mocaca mulatto, which consisted of hypertrophy of the gastric mucosa and small gastric ulcers within the mucosa. The toxic fat used in these studies, as we now know, contained chlorinated dibenzodtoxins. It has also been observed that hemorrhaging occurred into the gastrointestinal tract of rat fetuses who were exposed to 2,3,7,8-tetrachlorodibento-o-dioxin in utero. It was not stated in the reports whether the gastrointestinal hemorrhages were caused by ulceration of the mucosa.'10,111 Ulceration of the glandular portion of the adult rat stomach was also observed.''1 PatJems with chloracne frequently complained of lots of appetite, nausea, vomiting, and abdom inal pain, and Goldmann1 *1 described a worker with a follicular dermatitis who died six months after exposure to 2,3,7,8-telrachlorodibensodioxin. At autopsy a ruptured duodenal ulcer and a gastric ulcer were found. Whether any relation ship existed between the ulcers and the exposure to the poisonous chemical was not mentioned in the paper. It should be established if these gastric lesions also occur at tower dietary levels and whether the contamination of the chemicals with various chlor inated dibenzodioxinsand furans is responsible for them. Valuable information might be obtained from retrospective studies of workers following occupational expoture. Whether the ulcers are caused by direct effect of the chemical on the mucosa of the gastrointestinal tract or whether stress plays a primary role in their etiology similar to ulcers that may occur in burned patients for instance (Curling's ulcer) is not clear. COMMENTS The acute toxicity of compounds such as the chlorinated biphenyls, the chlorinated naphthatenet, and the chlorinated terphenyls it very low. January 1974 417 HONS 069525 On ihc oilier hand, I hey arc poorly broken down in (he environment and (he metabolism of the biphenyls with more then four chlorine atoms teems to be negligible. Because of these character* istics, Ihe use, wherever possible, of chlorinated biphenyls that consist of mixtures of compounds with one to four chlorines has been proposed. Neither Ihe chlorinated naphthalenes nor the chlorinated terphenyls have so far been shown to present a pollution problem. Whether the various disease entities reviewed in this article are produced by all the different products mentioned in connection with them is not established with certainty. For instance, some of the compounds that produce chloracne or chick edema have been shown to be contaminated with various chlorinated dibenaodioxins and chlor inated dlbenzofursns. The possibility remains that the compounds illustrated In Figure I are contami nated with these chemicals and the products themselves are not really responsible for the observed symptoms. On the other hand, a number of structurally unrelated compounds may produce the same disease entity. Some of the chlorinated dibenaodioxins and the chlorinated dibenzofuTans are highly toxic. The most toxic is 2,3,7,8-tetrachlorodibenzodioxins, while the dibenzodioxins without any chlorine and those with eight chlorines are much less toxic. The difference in toxicity of these compounds may be partly caused by the fact that they are wry poorly dissolved and absorbed. Intravenous toxicity data are thus far not available on most of these compounds. We also do not know at present if the porphyria which develops after prolonged exposure to a number of technical compounds such as hexa* chlorobenzene, chlorinated biphenyls, chlorinated dibenzodloxtn, and 2,4,5-T is In many Instances due to the contamination of these products with chlorinated dibenzodioxins; of course it may be possible that one or more chlorinated dibenzofursns also produce porphyria. It has alto not been established whether all compounds that produce porphyria in animals will do so in humans as well. In general, the exposure of the various popu lation groups to chlorinated biphenyls and other cyclic chlorinated hydrocarbons is at too low a range to induce porphyria. Whether this can also be said for occupational exposure is not known. When production conditions and general hygiene of the workers are poor, porphyria cutanea tarda can apparently occur.3 It is also not known ifiltc combined effect of different porphyria inducing agents is cumulative. Whether the photosensitivity observed in humans with some of the germicides discussed in this paper is related to an efTcct on the porphyrin metabolism was not evident from the published literature. So far the mechanism that causes chick edema, chloracne, X-disease, and liver disease hat not been satisfactorily demonstrated. Chick edema disease haa been thought to be caused by proliferation of the vascular endothelium. This might explain tlie fluid accumulation observed in diseased animals, but so far the sequence of events that led to this disease is not really clear and factors other than the embarrassment of the vascular system may play a role in its etiology such as a toxic myocarditis, decrease in serum proteins, and renal failure. More detailed study of the pathology of the various disease entities is needed for a bet ter understanding of the development of the various lesions mentioned. The chlorinated polycyclic hydrocarbons seem to alter the immune response of certain animal species and perhaps also of people. These very important findings need to be pursued further. Thus far it has not been established beyond doubi whether this is caused by a general debilitating effect on the animal of which atrophy of the lymphoendotheliai system may be one manifes tation, or whether this is a specific alteration, as we know it from the effects of radiation and expoaure to certain alkylating agents. In this respect not enough attention has been paid to the function of the adrenal, and several of the observed symptoms in humans such as the hyper pigmentation suggest hypofunction of the adrenal cortex. Depending on Ihe information obtained from these studies, a tolerance level of S ppm in some food products as set by the U.S. Food and Drug Administration may have to be revised particularly since no long-term low level feeding study has been reported in animals. Many questions have recently been raised about the use of germicides in various toiletries, common soaps, etc. The objection to their use in the specific case of hexachiorophene is its neurotoxic effect, that can also occur in humans, particularly if the skin is defective In some way. Of course, limited localized exposure, such as handwashing or treatment of a small wound, will not produce neurotoxicity. 4M ac CHiktl tH Toxkoicty 069526 *ONS In infant nurseries in hospitals, one of the greatest problems is (be constant threat of a staphylococcus infection of epidemic proportions. In order to prevent outbreaks of sittphyloccocus infections, a routine was developed in many hospitals In (lie U.S. and elsewhere in (he early 1060`s where infants were washed once daily with a .1% hexachlorophene detergent solution, and in addition Ihc diaper area was washed several times a day. Tlte detergent was subsequently rinsed off more or leu vigorously, depending upon the philosophy of tlte different nurses. In some hos pitals the detergent was directly applied to the infenl, In others it was first diluted in water. Since the neuroloxic effect of hcxachlorophene has become known, many hospitals have changed their policies on the use of hcxachlorophene. Another drawback to this routine, in addition to a possible neuroloxic effect, is that hexachlorophene keeps not only tlte staphylococcus of certain phage types from growing on the skin, hut reduces all Gram-positive bacteria.'11 which if highly unde sirable and may lead to disease. Recently Evans et si.*1' have pointed out that a marked annual variation exists in the prevalence of several bac teria in the nares as well as the umbilical cord of newborns. A decline of staphylococcus aureus was observed in the fad; the prevalence of ttscficrichia coli and streptococcus also varied. Staphylococcus epidermis and Enterobacter organisms did not show this fluctuation. Any studies of efficacy undertaken in a clinical setting should take these normal fluctuations into account and it is possible (hat good general housekeeping practices are ultimately more effective in preventing bacterial disease in newborns than various germicides. Unfortunately, breaks in lechnic do occur which in many hospitals makes the use of germicides on infants desirable. The information reviewed in this article shows lht the toxic effects of the polycyclic polychlor inated compounds are complex. The mere know ledge of an LDjo value in an animal species docs nol give us a great deal of information about the toxicity of a compound. Toxicity or toxic effects are a relative concept and such household items as table salt, vitamins, pepper, and ally! propyl disulfide volatilizing from onions to name a few are also toxic. Nevertheless, we have to learn not to use chemicals indiscriminately, without knowing a great deal about them, particularly their long-range effects, when they are persistent and poorly metabolized. The Indiscriminate use of lliese products and discharge into the environment invariably lead to their restriction or ban, which is accompanied by a great deal of emotionalism, distorted facts, and often great economic loss. Prudence, better judgment, and a critical approach to the usage of chemicals and drugs, particularly an evaluation of whether in specific situations they are necessary or nol. may help us avoid these situations in the future. Particular attention should be paid to avoiding unnecessary exposure of children to chemicals that we know little about. Children may react differently from adults and as far as long-term exposure is concerned, they would be (he most vulnerable. ACKNOWLEDGMENT My sincere thanks go to my colleagues and former co-workers who have made this review possible, particularly to Dr*. R. E. Zehr, J. E. Suggs, V. W. Burse, R. E. Jennings, R. E. Under, V. E. Sedlak, J. A. Goldstein, E. C. VIHaneueva, and T, B. Gaines for letting me incorporate some of the unpublished results Into this manuscript. REFERENCES 1. Man4, A. ? Smith, D., Metier, G,, and ftmkk, P., A health survey of workers in a 2,4-D end 2,4.5-T plant. Arch. Environ. Hetilh, 22, 316,1971. 2. Bteibwg, J., WaUen, M., Brodkin, R., and Applebaum, T. L., Industrially acquired porphyria, Arch Dermeiol., 89, 793,1964. 3. Larson, 0. L., Studies show hexachlorophene causes burn syndrome,/ Am. Motp. Anoc., 42,63,1968. 4. Schmidt, H. and Schulz, G., Uber Diphenylbasen, Amuiett, 207,338, 1881. 5. riming. C. H., Physical characteristics and commercial possibilities of chlorinated diphenyl, Ind. Eng. Otem., 22, II BO. 1930. 6. 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