Document 6wQeQXDNdGEnzVDRQwmO8ayoR

THE TOXICITY OF POLYCHLORINATED POLYCYCLIC COMPOUNDS AND RELATED CHEMICALS Author: Renate D. Kimbrough* Center for Disease Control Atlanta, Ca. Referees: M. M. Names J, B. Greig Medical Research Council MRC Toxicology Unil Medical Research Council Laboratories Carshaltnn, Surrey England INTRODUCTION Only the small group of the polychlorinated polycyclic compounds given in Figures 1 and 2 will be reviewed in this article. Several years ago when this laboratory was the Atlanta Toxicology Branch of the Food and Drug Administration, concern was voiced over the possible long-term effects of polychlorinated biphenyls, and wc began to study the toxicity of Aroclor 1254 and Aroclor 1260. About this time a follow-up study was performed on a group or 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 Hied with the Food and Drug Administration for a residue level of hcxachlorophene on certain food crops. HcxachJorophcne, as well as 2,4,5-T, is made from Irichlorophenol, although the chemical reactions ire quite different. When we checked the available literature on the toxicity of hexachlorophene we felt 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 hexachlorophenc was more toxic than commonly acknowl edged. Hcxachlorophene and similar chemicals behave differently from such polycyclic polychlorinated compounds as the chlorinated biphenyls (PCBs). Hcxachlorophene 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 with more than four chlorine atoms on the phenyl rings. The long-term effects arc therefore more critical for compounds such as PCBs. The chlorinated dibenzodioxins 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 Lnvironmenlal Protection Agency, Chamblee Toxicology Laboratory, 4770 Buford Highway, Humblee, Ua 30341. January 1974 445 DStf 031843 1 Chlorinated Dlbenzofurans Chlorinated Olbenrodloxtn Polychlorinated Naphthalenes FIGURE 1. The materials shown in this figure are mixtures of compounds in which several but usually not all of the positions (-X) ire chlorine and the rest are hydrogen. The properties of the materials depend on the degree of chlorination of the mixture. All are insoluble in water but soluble in varying degrees in organic solvents and in fats. The trade name for the U.S. product (Monsanto) of the chlorinated biphenyls and terphenyls is Aroclor. The trade name is followed by the number. For the biphenyls, the First 2 digits of this number are 12, and for the terphenyls, 54. In each Instance the last 2 digits give the percentage of chlorine. OH OH s Cl t ct HtXACHlOROPHCNE DICHLORORHENE ?,2'-Hettiyltne-bts(J,4,6-trtch1oropheno1) i ,2'-ftethyltn-b1s(4-cliloro(>h*rM>l) rvCt ct BITHIONOC t,2*-Thlo-bts(4,6-dlchtorephenolI 1 OK 24,4' trfehlore- 2'hydroxy-diphenyl ether (Trlclosin Irt}fsn 0P3O0) OH OH OH 0 Ct C-IW v ci a FtHTICiOR 2,2' -TMo-bIs(4-cMoropher>o1) ct Cl Cl Jt3',4,5'TetricHlorosancyUn1Mde (TCC) 3,4,4'-Trtchlorcerbsnlllde (1CP) FIGURE 2. The compounds in (his figure axe white crystalline solids with clearly defined melting points above 160*0. They arc practically insoluble in water and soluble in varying amounts in organic solvents and in fats. Those containing an OH group, all the above excepting 3,4,4'*trichlorocarbanUkle, are aoluble in aqueous alkali. 446 CRC Critical Reviews in Toxicology pi ""IM" DSW 031844 v;} li'v un cn nu nu or u> ra rc rc In hi! va lit at re US di C\ U' II Cl si :\s w Cl ;k) CO voi CJj f la In I'o HI til el B El P ea STLCOPCB4015806 The present review is an attempt to correlate various toxic effects pioduccd by the chemicals listed in Figures I and 2 in order to better understand and appreciate (heir 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 (he literature cited serves to illustrate these points. No attempt has been made to present a complete review of (he literature in (his area. DEVELOPMENT, USAGE, AND OCCURRENCE A description of the synthesis of chlorinated biphenyls (PCBs) can be found in Liebig's Annalen as early as 1881.4 Successful production of diphenyl in commercial quantities was not report ed until I930.s At that time the chlorination of technical biphenyl was also described. Biphenyl itself is fungistatic and used as a preservative for citrus fruits.7 Chlorinated biphenyls are very stable, and have been used for protective coatings, as plastic resins or gums for varnishes, and for waterproofing and flamcproofing of wood. The excellent dielectric properties of these compounds were noted as early as 1930. Until the advent of the PCBs, mineral oil was used as a cooling and dielectric liquid impregnant 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, the fire and explosion hazards were eliminated. This was recognized by the National Board of Fire Underwriters and the National Electric Code was modified. Use of Aroclors also greatly reduced the physical size of (he capacitors.* A recent summary of (he varied uses of PCBs has been provided by Broadhurst.1 n With a peak production of 42,527 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 the period 1960-1971 were reported by Monsanto.11 The production increas ed steadily until 1970 and dropped to 20,236 short tons in 1971 due to a voluntary restriction of (he use of PCBs by the company. Trade names in other countries include Kanneclor and Santhaterm in Japan, CJophcn, FJenclor, Phenocloi, and Sovul in Europe. The polychlorinated capacitor liquids, com monly called askarels, 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 coalings 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 (he 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 industry and on warships.12 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. Holden,13 after finding PCBs in marine fish 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 (he introduction of PCB residues into the terrestrial ecosystem by transfer through food chains. So far PCB residues have not been detected in fresh water fish in Scotland. Polychlorinated biphenyls have also been found January 1974 447 > m DSW 031845 STLCOPCB4015807 in wildlife samples collected along the l)u I eh coasl and tlie Rhine River14 and in fish and seal in different parts of the Baltic Sea along the Swedish coast.1 s Riscbioogh et al.16 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 1 and 2. This is again emphasized by the findings of Dustman et al.17 Twelve Alaskan bald eagle eggs had median PCB residues of 1.65 ppm while 11 eggs collected in Maine, Michigan, Minnesota, and Florida showed a median concentration of 9.7 ppm. Zitko* * 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 part of the U.S., where the river enters into the lake, contained varying amounts of PCBs'9 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 Aroclor 1254 and salmon eggs contained as much as 12 to 17 ppm PCBs on a fresh weight basis. Further studies by Veith2? 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 fishkill may result such as the one in Escambia Bay, Florida in 1969.21 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 Aroclor 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. Aroclor residues in sediment samples taken near the outfall readied 486 ppm. When the leakage from the plain was corrected, Aroclor 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 Stickel,22 monitor ing for lerphenyls is also done but not for any of the other chlorinated compounds in this report. Research surveys that include fishealing birds, insectivorous birds, ospreys, woodcocks, and cer tain ducks also monitor only for PCBs and for pesticides. Oysters (Crassostrea virginica) are a good indicator of PCB and other chlorinated hydrocarbon concentrations in water and a nation al monitoring program in these mollusks in 15 coastal areas has been developed.23 So far, mollusks have only very occasionally contained low levels of PCBs. In summary, the PCB concentrations in water are low or nonexistent in remote lakes and fresh water streams and can be as high as 50 pg/l (5 ppb) or higher in polluted rivers such as the Hudson River.24 The PCB concentration is usually higher in sediment from these polluted waters because PCBs are only very slightly soluble in water.25 Since PCBs are stored in living matter, they .tic concentrated in the food chain and the usual "biomagnification" is encountered as wc know it from other poorly metabolized and excreted sub stances. The concentration of PCBs in fish, par ticularly those in the upper end of the food chain from polluted rivers 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.26 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.27,28 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.29 The U.S. Food and Drug Administration has recently set temporary tolerances for PCBs in certain food products (see Table 1). 448 CRC Critical Reviewt in Toxicology DS 031846 STLCOPCB4015808 TAIIl I- I Temporary Tolerances Expressed as Parts per Million (ppm) Established by U.S. Food and Drug Administration (1) Milk (I'al basis) (2) Hairy products (I'al basis) (3) Poultry (lat busis) (4) Epps (5) Complete and finished animal feeds (hi Animal Iced components (7) I ish and shellfish (edible portion) (R) Infant and junior food (9) Puper food-packaging material 2.5 2.5 5.0 0.5 0.2 2.0 5.0 0.2 10.0 Nmc: The tolerance for paper food-packaging material shall nol apply lo such material separated from the packaged food by a functional barrier impermeable to PCB mipration. Tire l ood and Drup Administration will provide upon request the analytical methods it will use for enforcing llte temporary tolerances. mixtures of ortho-, meta-, and paraterphenyis. 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.3* According to Zitko et al.,37 poly chlorinated terphenyls have been found in environ mental samples. Polychlorinated terphenyls do not elute from GLC columns under conditions used for PCBs,38 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 dibenzodioxins and chlorinated dibenzofumns may occur as contaminants of cyclic chlorinated hydrocarbons. In 1959 Tomila et al.3* reported on the t All of these aspects of PCBs in the environment synthesis of polyhalodibenzo-p-dioxins. According have been the subject of a number of reports, to these authors, chlorophenols, when heated to l reviews,30 and conferences.3 1-3 * about 200C or more under alkaline conditions r Terphenyls and chlorinated tcrphcnyls are used and pressure, will form chlorodibenzodioxins, as as reactor coolants. The terphenyls consist of shown below. i. t! ONa Cl a. t CL CL Cl NaO' + 2 NaO Clw Under similar conditions a chlorodibcnzofuran n; may also be formed. d: e o, n Cl. s c,--c^a' HjO + NaCI y Chlorinated dibenzodioxins and chlorinated diben- show a great deal of difference in the type and it zofuians arc 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 le Marling materials, sucli as 2,4,5-T, penlachloro- difficult. Rigorous production control and cleanup |al phenol, nilrofen, hexa- and pentachlorobenzene, procedures need to be implemented so that the tm and Iri- and (elrachlorophenol. Polychlorinated composition of different lots manufactured by the :d biphenyls may also be contaminated with chlor same company becomes more uniform. ;d inated dibenzofurans,40'41 and chlorinated diben Villanueva et al.43 and Jensen and Renberg44 he zofurans may result from polychlorinated reported the presence of chlorinated dibenzo-p- iy biphenyls under UV irradiation.43 The toxicity of dioxin in pentachlorophenol. Hydroxyjd these products varies depending on the position nonachlorodiphenyl ether was also found and and number of chlorines attached to the phenyl referred to as "predioxin" under the assumption rinp. Products from different manufacturers may that It represented a precursor of dioxin. January 1974 449 DSW 031847 According lo Johnson ct al.,45 the predom inant dioxin in a commercial pcnluchlorophcnol was oclachlorodibcnzodioxin while (race amounts of hcxachlorodibenzodioxin and no tetrachlorodibenzodioxin were found. Since the technical pcniacltloropltcnol was in some respects more toxic in animal studies, than the pure product, the production process of pentachlorophenol 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 lo be contaminated with a dioxin. In this instance, unfortunately, (he very toxic 2,3,7,8 tetrachlorodibenzodioxin,46-4 8 was present. The contami nation of (he 2,4,5-T with 2,3,7,8-letrachlorodibenzodioxin ranged from < I ppm to as much as 32 ppm over the years. In newly manufactured 2,4,5-T in the U.S. (he content of (etrachlorodiben/.o-p-dioxin has been substantially reduced and is usually kept below 0.1 ppm. Whelher other dioxins or furans have ever been detected in 2,4,5-T irt the U.S. has not been reported in the literature. Chlorinated dioxins liaYc also been found in so-called "toxic fat," which produces chick edema disease when fed lo chickens. After several years of study by industry and the Food and Drug Administration, Flick et al.49 were able lo separate a purified crystalline product which pioduccd chick edema disease. Two years later, Cantrell ct al.50 announced that 1,2,3,7,8,9clilorodibcnzo-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 chlorophcnols 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 lo the contamination with chlorophenols of soapstock used as feed fats. Higginbotham el al.5' showed that chlorophcnols and their salts, when healed, 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 arc present in the environment. However, Baughman and Mcselson53 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 18 lo 814 ppt. Zitko55 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 multiresidue analysis and are detected only when mass spec trometry Is also performed.5* The various germicides given in Figure 2 have been used as antiseptics, deodorants, and particu larly, as fat 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 bithionol was cancelled in the U.S.58 because it produced photosensitivity reactions in humans using soaps that contained this product. None of the polycldorinated polycyclic compounds used as germi cides have been shown to accumulate in lire environment. Hexachlorophene has additional uses as a fungicide on ornamental plants, on conveyor chains as a mildewstat, in laundry rinses us a mildewstat, in shoes and air filters and assorted industrial uses as a fungistat, and on leather, paper, and textiles.59 It is also registered as a pesticide for use on cucumbers, peppers, and tomatoes,60 and for cotton. Dichlorophene 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.61 CHEMISTRY: METHODS FOR CHEMICAL DETERMINATION AND PHOTOLYSIS Much of the chemistry of polychlorinated biphenyls has recently been reviewed.33'34 Commercial chlorinated biphenyls are mixtures of biphenyl groups with different numbers of chlorine atoms (Figure l). When hydrogen atoms are replaced by chlorine on the biphenyl ring, a large number of substitution combinations can arise since the number of chlorine atoms on the molecules can vary and the chlorines can also be a l Mi rcsul chin cial of i' Mon varn con? naic poss con1 124 resp call insc ' An 27 f chi mii a chi mu ad lab mi diCM ch ;m au m si' m lii dP( ex in si h. n h B u d a A 4J0 CRC Criticel Reviews In Toxicology DSW 031848 STLCOPCB4015810 altachcd to the molecule in different positions, resulting in a variety of isomers. Even if (he chlorination may be tire 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 constituents, and when the biphenyl is chlori nated, 210 different chemicals are theoretically possible. Aroclor 1254, for instance, yielded 69 constituents while the fractionation of Aroclor 1242 and 1260 yielded 45 and 78 components, 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 278C. The boiling point increases with increasing chlorination. The chlorinated biphenyls are deter mined in biological and environmental samples by i combination of electron capture gas liquid chrumatography and mass spectrometry. Nuclear magnetic resonance spectroscopy is used in addition to the above two procedures in some laboratories. PCBs were first identified in environ mental samples by Jensen in 1966.6 2 They were delccicd as in lei feting peaks on GLC analysis of environmental samples that were analyzed for chlorinated pesticide residues.** The PCBs interfered with routine pesticide analyses for chlorinated hydrocarbons. Various authors have therefore published separation methods*4 and Reynolds3* discussed this exten sively in a review. According to Safe and Hutzinger,*5 the use of mass spectrometry for structural studies of PCBs is limi ed because (he chlorine atoms may be ran domized between the phenyl groups when the PCBs are fragmented by electrons. The only exception to this is 2,2,-dichlorobiphenyl. The quantitation of polychlorinated biphenyl in environmental samples has met with difficulty since the PCBs represent mixtures. Several workers have compared PCB components in field samples to Aroclor 1254 because the sample chroma tograms were most similar to these mixtures.**'6* Rote and Murphy** quantitated individual peaks of different Aroclors by means of a response curve. The semilogarithmic relationship of detector response (total peak area/16 ng) to image chlorine content was obtained for each Aroclor 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 Analytical Methodology for Polychlorinated Biphenyls (Feb. 1973). Polychlorinated terphenyls (Figure 1) are not eluted from gas chromatography columns under condiiions used for polychlorinated biphenyls.3* By altering their gas chromatograph column, Zitko et al.37 were able to detect polychlorinated ter phenyls. Chlorinated naphthalenes (Figure I) elute at the same time and cause interference with the determination of chlorinated biphenyls and chlori nated hydrocarbon pesticides in general, unless mass spectrometry is performed to establish the identity of (he various chemicals involved.5* The methods presently available for the deter mination of trichlorocarbanilide and other germi cides (Figure 2) are not very well developed. Graber et al.*9 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 dibenzofurans 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 compounds10-7 2 as contaminants and methods have been developed to determine them in various technical products. Other methods dealt 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 diben zodioxins and dibenzofurans for toxicity studies in January 1974 J451 DSW 031849 STLCOPCB4015811 order lo obtain pure products, methods also need lo be available for their separation and identifi cation.14 One of the problems with tetrachlorodibcnzo- dioxin as well as tetrachlorodilienzofuran 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 Mcselson53 have recently described a method for the determination of these materials, within the ppt range. Hexachlorophene is made from trichlorophenol. A process for its production was patented by Gump.7* Additional methods for the produc tion and purification for hexachlorophene have been published and are cited in Chemical Abstracts. Recently several methods76'79 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 gas chroma tography.79 In an earlier method described by Bachmann and Shcllar.*0 simple benzene extraction was used and no derivative of hexachlorophcne was made, which led to very variable results. The various advantages and disadvantages of these methods were discussed by Ulsamer.79 In earlier work my co-workers used ether for extraction and prepared a dimethyl ether deriv ative of hexachlorophene.*1 Recovery with this method was only about 75% and we have recently altered our method of extraction lo obtain belter recovery.*5 A method developed by Kabacoff et a!.78 for the determination of hexachlorophene is, according lo the authors, also suitable for deter mination of dicldorophene and Gutenmann and Usk83 have described a method for the determin ation of hexachlorophene in several agricultural products. The production of hexachlorophene from trichlorophenol 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 given considerable attention. Hustert and Korte*4 synthetized a few PCB isomers, namdy. 2,4,6,2',4',6'-hcxachlorobiphcnyl, 2.4.5.2'.4'.<>' hexachlorobiphcnyl, 2.4.2\4'-lelrachl<>robi|)licnyl. 2,5,2',5'-tctrachlorobiphenyl, and irradiated the various isomers with UV light. A mercury vjpur lamp with quartz fillers was used as 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 a mixture of methanol and water, dechlorination and polymerization of the compounds was observed. When the PCB isomers were irradiated in perfiuorinaled 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 chlorinated biphenyls may also occur in low concentrations. Safe and Hutzinger*5 reported that the irradiation of 2,4,6,2',4',6'-hexachlorobiphcnyl at 310 nm resulted in compounds of lower chlorine conicnl. such as di-, tri-, Ictra-. and pcntachlorobiplicnyls In these investigations hexane and mctliaiml uat used as solvents. It is possible that now compounds arc formed by loss of chlorine. Rcarrangcmcni and condensation may lead to chlorohiphem la in environmental samples that arc not present in commercial PCB mixtures. The 24-hr irradiation of 3,3',4,4'-tetrachlorobiphenyl, 2,2' .o telrachlorobiphenyl, 2,2',5,5'-tetrachlorobiphcnyl dissolved in hexane resulted in about 7091 loss ot the starling material, while with 2,2',4,4'.5,5'hexachlorobiphcnyl, only 3.8% remained and less than 1% of 2,2\3,3,,4,4',5,5,-octachlorobiphenvl remained. The irradiation of Arodor 1254 in hexane resulted in a change of the composition of the Aroclor mixture. The gas chromatogram obtained from the irradiated Arodor showed shorter retention limes than the standard Arodor 1254. The irradiation of Arodor I2S4 in a dioxane water mixture also containing sodium bicarbonate did not lead to the formation of chlorodibenzofurans or hydroxychlorobipltcnyls The compounds that were observed corresponded to PCB molecules to which water was added and a more polar "carboxylic" acid 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 432 CRC Critical Rrvicns in Toxicology DSW 031850 STLCOPCB4015812 PCBs thcicforc depended greatly on the solvents used and the pH of the solution in addition to the light source used and may result in dechlorination, formation of polymers, hydroxylation, and carboxylic products."6 Exposure of various chlorinated dibenzodioxins to summer sunlight or fluorescent UV light with an intensity of roughly 100 gw/cm1 in the presence of organic solvents such as methanol led to rapid photolysis of 2,3,7,8-le(rachloro- and 2,7-dichlorodibenzodioxin. while 1,2,3.4,6,7,8,9-octachlorodibenzo-p-dioxin produced a series of chlorinated dioxins with uniformly decreasing chlorine conlcnl. Some evi dence was also found that reactions other than reductive dechlorination occurred.87 When 2.3.7.8- tctrachlorodibcnzodioxin was spread on a glass plate in a methanol solution and the meth anol was allowed to dry, quantitative recovery of the dioxin was possible even after 14 days of UV irradiation. Similar results were obtained when 2.3.7.8- tciruchlomdibcnzodioxin was dissolved in methanol, put on a glass plate, covered with soil, and irradiated,81 These results demonstrated that under favorable environmental conditions, when organic hydrogen donors are present, photolysis will lake place. Bare surfaces of soil, concrete, or water arc 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 chlorinated dibenzodioxins and may result in less chlorinated compounds. The possible long lasting presence of the dioxins is also emphasized by Kearney et al.,88 who recovered 56 and 63% of originally applied tetrachlorodibenzodioxin after one year in Hagerstown and Lakeland, Md. soils. Tel rachlnrnsalicylanilide and similar compounds split off chlorine atoms when exposed to sunlight. This is briefly mentioned in connec tion will) photosensitivity reactions of the skin. Shaffer el al.89 irradiated hexachlorophene in absolute ethanol with UV light and obtained a number of dechlorinated bisphcnols resulting from loss of chlorine in the ortho and para positions relative to the hydroxy group, GENERAL TOXICITY AND EFFECT ON REPRODUCTION Diphenyl Itself, without any 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 b residue on citrus fruit varies between 70 to 110 ppm of biphenyl. Booth et al.90 found a reversible nephrotoxic effect of biphenyl on the 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.91 showed that pro longed exposure to air containing diphenyl in high concentrations of 5,40, and 300 mg/m3 produced liver and kidney injury as well as broncho pulmonary lesions in mice and rats. In genera), diphenyl has always been considered to be relatively nontoxic. In 1969, in Finland, a man who had been intensively exposed to diphenyl for II years became ill and died." 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 tetrachloroethylcne. The cause of death of the worker in this particular report was not satisfactorily explained and neither were the complaints of 31 other workers engaged in the same factory. Since chlorinated ethylene com pounds were used 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 case, then care should be taken that this situation is avoided. The PCBs art also not very toxic when given as a single or a few repeated doses to birds and 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 fish are not quite as sensitive to a single dose, but will die when exposed for 14 to 45 days to 5 ppb of Aroclor 1254.93 PCBs are of a low order of toxicity to rats and rabbits when given as a single dose. The acute oral toxicity decreases in rats with increasing chlorine content, while this Is not as obvious when the dermal toxicity is tested in rabbits.33 This may be due to poorer absorption of the higher chlorinated compounds. In studies with adult Sherman strain rats, we found that the oral LDS0 for Aroclor 1254 and 1260 was somewhere in the January 1974 453 r DSW 031851 range of 4 to 10 g. Tliesc two compounds proved to he more toxic in weanling rats. The single oral LDS0 for weanlings was 1295 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 LDS0 for Aroclor 1254 in adult female rats was 358 mg/kg.9* 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 the 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 et al.JI and will not be reported here since they do not give a great deal of additional information. Vos and Kocman95 studied the toxicity of three PCB preparations in chickens, Phenoclor PB6, a French product, Clophcn A60, 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 (he (wo Kuropean samples died between 1 2 and 60 days after onset of exposure. The findings 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. Willi the help of mass spectrometric and microcoulometric analyses, the authors9* determined that (he two European samples were contaminated with tetra- and penlachlorodibenzofurans. As an additional contaminant, chlorinated naphthalenes were found. Dermal toxicity studies with the three com pounds, Clophen, Phenoclor, and Aroclor 1260, in rabbits96 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 was observed, the hair grew at a reduced rate, the skin became thickened, and prominent transverse wrinkles developed. The test animals showed a gradual loss of weight, and one of the rabbits given Phenoclor and three of those given Clophen died. All but one rabbit showed fluores cence of the liver under UV light, and microscopic changes were also observed in (he livers and (lie treated skin. Vos and Notenboom-Ram9 7 compared the dermal toxicity of the isomer 2,4,5,2',4',5'hexachlorobiphenyl with that of a PCB mixture (Aroclor I 260) in rabbits. Their dermal application resulted in early microscopic skin lesions in the Aroclor group. The lesions in (he 2,4,5,2',4'.5'hexachlorobiphenyl 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 hexachlorobiphenyl than in those that had been exposed to the Aroclor. In inhalation studies with Aroclor 1 242 and 1254, rats, mice, rabbits, guinea pigs, and one cal were exposed to PCB vapors five days a week for several weeks. The concentration of Aroclor 1242 vapor was 1.90 to 8.63 pg/1 and no ill effects were observed. The exposure of animals to the vapor of Aroclor 1254 in the concentration of 5.4 or 1.5 fig/1 produced enlarged livers in rats. An inter current respiratory infection in some control .is well as experimental rats made it difficult to interpret the results.98 Since ft is now known that the chronic toxicity of PCBs is a more important parameter in establishing (heir effects than short term exposures, further studies with lower level, over longer periods of time would give mine 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 in various species have been conducted with PCBs. usually, embryo toxicity was observed but (here were no malformations. Heath et al." were unable to demonstrate an effect on reproduction with diet ary levels of 25 and 50 ppm of Aroclor 1254 in 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 corielated to DDE residues rather than dieldrin or PCBs This finding indicates, according to Blus et al.,100 tire PCBs are not responsible for eggshell thinning in most birds following environmental exposure. Pcakall101 fed ringdoves 10 ppm Aroclor 1254 454 CKC Critical Reviews In Toxicology mm DSW 031852 STLCOPCB4015814 llic the '.5'ui re lion (lie 5'. 'erc iver d in (HO d (o and cal for m: verc >r of 1.5 uieril as I to that i uni liorlL'VW.S ,nore f lire apot sably rious rally, ic no Ic to did54 in gsltcll s also as a nning 'cesses it was ;ed to . This >0 the ling in r 1254 for 6 months He injected others with 160 mg/kg intrapcritoncally for 4 days, and evaluated the aslted eggshell weights of these birds. He found no difference in eggshell thickness between the ex|)crimcntal and the control groups. In a pheasant reproduction study with Aroclor 1254,'01,103 a significantly greater difference in the number of eggs that were pipped but not hatched was found in tltc group of hens that received 50 mg of PCBs weekly. Egg production and hatchabiiity was lower in this group, the survival of chickens that did hatch a( six weeks of age was reduced, and (he ones that did survive were significantly lighter than the controls. Aroclor 1242 at 10 ppm or 100 ppm and Aroclor 1254 at 100 ppm in the diets of chickens did reduce egg production and hatchabiiity 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.'04 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 the lower chlorinated biphenyls affect reproduction more than the higher chlorinated biphenyls, and it is very important to determine whether the lower chlorinated biphenyls 1 hut have been suggested as replacements for presently employed chlorinated biphenyls have an effect on reproduction. In another rat reproduction study9'1 the lowest dose of Aroclor 1254 that affected reproduction was 20 ppm (1.5 mg/kg/day) wliich manifested itself in a decreased number of litters and less 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 Aroclors at 5 ppm or higher increased the liver-lo-body weight ratios in weanling rats of both sexes. At I 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 affected reproduction.1 05 Unfortunately, it ii 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 terphcnyls or chlorinated terphcnyls. These materials have not been studied to any great extent. The few studies (hat 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 dibenzodioxins and dibenzofurans is 2,3,7,8-letrachlorodibcnzodioxin which, in the German litera ture, is sometimes referred to as 2,3,6,7-tetrachlorodibenzodioxin. Single oral LDS0 values are female rats: 44.7 /rg/kg; male rats: 22.5 fig/kg; male guinea pigs: 0.72 /rg/kg: rabbits: < 30 /rg/kg; and dogs: between 30 and 300 /rg/kg.106 It is possible (hat young animals are more susceptible to the toxic effects of this compound than older animals and LDS0 values should therefore also be determined in weanling animals. One of the characteristics of this compound was that it caused delayed death, sometimes as delayed as 40 days after a single dose. During the period of intoxica tion, the animals lost a great deal of weight.106 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.106 Schulz107 in earlier experiments found that oral doses of 20 to 50 /ig/kg/body weight of 2,3,7,8-tetrachJorodibenzodioxin resulted in fatal liver necrosis in rabbits. More recently Milnes108 reported (hat 10/tg/kg was lethal to rabbits. The 2,3,7,8-tetrachlorodibenzodioxin has a variety of specific toxic effects. Whether these effects vary in different species is presently not well defined. With (he interest in this and the other related compounds, more information on January 1974 455 GSW 031853 STLCOPCB4015815 their toxicity probably will soon become available. The problems inherent in studying these com pounds are the unavailability of some of them, the fact that they may not be completely pure, particularly the hex- and hepta- preparations, and of course with respect to the telrachlorodlbenzodioxin, its extreme toxicity, which makes it difficult to handle. In contiast to RCBs, teratogenic as well as feloloxic effects have been reported for 2,3,7,8tetrachlorodibcn/.odioxin. Sparschu et al.109 gave 0. 0.03, 0.125, 0.5, 2.0, and 8.0 /rg/kg/day of 2,3.7,8-tctrachlorodibenzo-p-dioxin to pregnant rats on days 6 to 15 of pregnancy. With (he 0.03 pg/kg dosage level, an effect on the fetus was not observed, but doses from 0.125 pg/kg on up resulted in fetal mortality, early and late resorp tions. and fetal intestinal hemorrhage. Khera and Roddick1 10 gave doses ranging from 16 to 0.125 pg/kg to Wistar rats on days 6 to 15 of pregnancy. These authors also observed pronounced fetotoxicity at all dosage levels except 0.125 pg/kg. The affected fetuses showed cerebral and intestinal hemorrhage as well as subcutaneous edema at autopsy, The offspring of mothers given 0.5 and I pg/kg of the Ictrachlorodibenzo-p-dioxin gained less weight during (heir suckling period and their survival to weaning was reduced. This could have been caused by the intrauterine effect of the dioxin or by excretion of the dioxin in the milk of the dams given the materia) during pregnancy. Courtney and Moore111 observed cleft palates in 3 strains of mice given 3 pg/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.1 11 The 2,3,7,8-tetrachlorodibenzo-p-dioxin is extremely toxic in the chick embryo assay.52 In the rat, hexachlorodibenzodioxin caused fetoloxic effects at 1 and 10 pg/kg and teratogenic effects at doses of 100 pg/kg.106 Harris et a!.113 gave female rats daily doses of 0.1, 1, or 10 pg/kg TCDD for 3) days. Rats given 10 pg/kg lost weight and most of them were moribund within 3 weeks. Weight gain was also less at the 1 pg/kg dietary level. The rats receiving 0.1 pg/kg were not affected. In experiments with guinea pigs, 9 of 10 animals died after a single dose of 3 pg/kg TCDD, while guinea pigs receiving a single dose of 1 pg/kg gained less weight- Ail guinea pig? receiving 1 pg/kg/week died within 24 to 32 days ami a weekly dose of 0.2 pg/kg depressed their weight. Of the various animal species tested, the guinea pig seemed to be most susceptible to the toxic effects of TCDD. Reduced thymus and spleen weights were observed in rats that received a single dose of 25 pg/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. Sublethal doses of TCDD produced microscopic changes in liver, kidneys, and thyroid. In the kidneys the collecting tubules were primarily affected. Doses of 0.1 pg/kg/day for 31 days, a single dose of 5 pg/kg. and multiple weekly doses of 1.0 and 0.2 pg/kg did not produce changes in rats discernible with the light microscope. Toxicity studies were conducted in our labora tory with dichlorophene.`1 * The oral LD50 for adult female rats when the material was given m 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 amJ 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 sonic ulceration resulted but there was no systemic toxicity. The acute intravenous LDjD in adult male rats was 16.8 mg/kg when it was given in a saline lecithin suspension. In a two generation rat 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> the offspring did not reveal any morphological changes that could be related to dichlorophene exposure. These findings indicated that dichlorophene was not very toxic to rats. The great difference between the oral and the intravenous LL)*,, suggested either poor absorption or rapid break down of the material in the gastrointestinal tract. Martins61 lists the acute LDS0 for guinea pigs as 1,250 mg/kg and for dog?, 2,000 mg/kg. Rais fed for 90 days on a diet containing 2,000 ppm showed no evidence of toxicity. The acute toxicity of hexachlorophcne is much greater and varies in different animal species and is 4K CRC Critical Reviews In Toxicology DSW 031854 STLCOPCB4015816 also dependent on the route of administration and the solvent in which it is given. Gump1 15 lists the acute oral LDsn in mice as 168 mg/kg and cites LDs(i values reported by other investigators rang ing from 80 mg/kg to 215 mg/kg. Dogs and sheep were more susceptible to the toxic effects of hcxachlorophcne. The interest in the effect of hexacldorophcne 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 cliildren with orally administered hexachlorophcne in China.1,1 Intravenously administered hexa- chlorophcnc in rats resulted in an LDS0 value of 9.1 mg/kg and in rabbits of 8.5 mg/kg. More recently wc found that the oral LDS0 (single dose) for adult male rats was 66 mg/kg and for adult female rats 56 mg/kg: for weanlings it was 120 mg/kg when (he material was given in peanut oil by stomach tube to Sherman strain rats. The acute intravenous LD50 was 7.5 mg/kg in adult male rats.111' Nakauc c( ul.119 reported similar oral LDso values in Wislar rats. The single oral LD5o for sheep and cattle lies between 30 and 60 mg/kg.120 Single dermal LDS0 values have not been reported in the literature. We found in rats11" that single applications of 600 mg/kg hexachlorophenc in 95% ethyl alcohol solution caused death in I 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 alcoiiol was not established. The signs of acute hexachlorophene poisoning following a single oral dose varied with the age of the rats.118 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 (hey 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- plicnc poisoning has also been recorded in rats.119 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 al.119 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 the rats that were fed 200 ppm (about 13 mg/kg/day) for 16 weeks. In a two generation rat reproduction study which we conducted in our laboratory, we found a reduced survival in the offspring of the F, generation fed 100 ppm (11.8 to 5.5 mg/kg/day) in their diet 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.11* A dietary level of 20 ppm (2.3 to 1.1 mg/kg) did not influence reproduction. Thorpe'7' reported (hat the oral administration of hexachlorophcne to male rats as well as single doses of 25 and 50 mg/kg hexachlorophene to sheep produced de generation of spermatogenic cells; multinucleated cells were observed within the tubules of the testes. The doses given by Thorpe were higher titan 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 rats.12 2 ''13 It should be determined whether malformations can also be produced in other species and with lower doses 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 was mistaken for milk of magnesia, or by the applica tion of hexachlorophene to large areas of burned or otherwise damaged skin.,,,,174 Larson1 was January 1974 457 DSW 031855 the first to recognize that hunted patients developed convulsions and other toxic symptoms alia repeated exposure to hexachlorophene which was applied to burned areas of the skin. The incidence of convulsions in burned patients decreased when I he use of hexachlorophene was discontinued.1 2 5 Eleven cases have been reported156,127 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 hexachlorophene, became comatose and died. One additional dealh occurred in this group, however, this was most likely due to other causes. A 17-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 (he lips, and lateral shaking movements of the head. It was also noted in the case report that the child refused to lake the medicine, there was drooling and frequent vomiting, and it is not quite clear whether the child received 37 mg/kg/day. The cliild survived. A week after the hexachlorophenc 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 the child was healthy looking.128 Pilapil mentioned in his 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 an infant who by mistake got a spoonful of PhisoHex in each feeding bottle for approximately two weeks, when it suddenly died. Chung et al.117 treated a total of 105 patients 5 to 15 years old with hexachlorophene for chlonorchiasis sinensis infestation. Sixty of these patients received 1 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 to the central nervous system and to the Bastiointestinal 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 after 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 and Winsten12' in Scandinavia and two fairly recent cases with fatal outcome occurred in the U S.110 Herler131 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 bath without rinsing the baby. After four days, excoriations appeared on the child's face and buttocks, the infant developed convulsions, lus face twitched and his extremities jerked. A roving nystagmus was 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, Mullick130 also reported four cases children who died following the treatment for bums in two instances and the treatment for severe ichthiosis 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 Wlnsten129 reported two cases of poisoning following the dermal application of hexacbloio phene. Both patients were fairly extensively burned, and developed central and peripheral nerve damage as well as some kidney impairment. Lockhart132 mentioned six deaths that were reported to the FDA caused by the topical application of hexachlorophene on burned patients. In all of these 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 lend to forget at times. Damaged or abnormal skin may be 458 CRC Critical Review* In Toxicology HP.......................... QSW 031856 STLCOPCB4015818 I 10 lios of One I lie live ryes ever rred the flcr ;sivc also ifler and cent I JO >rl a rmal if a fant r the four and , his iving vitlthad nned oral cs l for for rath, who toms loroand wing iloroively heral nent. were ipical jmed was hexaent a nd to ay be more permeable, and the skin of prematures, newborns, anil children in general may have absorption characteristics other than those of adult human beings, another factor that is often neglected. Recently133,134 41 deaths due to hexachlorophenc poisoning have been reported in France. The deaths occurred in infants who were treated with a powder to which 6% hexachlorophene had been added by mistake. This powder was 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 will not be made public until litigation problems have been settled. Little published information is available on the toxicity of the germicides in Figure 2 aside from hexachlorophenc and dichlorophene. The acute oral LDno for triclosan (TCC) in mice and rats is about 4,000 mg/kg.135 The acute subcutaneous toxicity for rats was 14,700 mg/kg and the acute dermal loxicity was about 9.3 g in rabbits. Concentrations of t to 5% solutions caused eye * irritation in rabbits. Daily oral doses oTTCC up to 1,000 mg/kg/day for 4 weeks did not produce an effect in rats. When 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, 13R mg/kg/day. In baboons a daily oral dose of 100 mg/kg/day did not produce a toxic reaction. The minutes also 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. Chloroaniline as a result of heating during manu facture could be present as such an impurity. Of the anilines, according to Hamblin,1 36 only p- and m-chloroaniline may cause methemoglobinemia if absorbed through the skin. The o- and m-chloro aniline may cause liver and kidney damage. Tri closan itself is also absorbed through the skin and absorption is greater when applied to damaged ikin. SPECIAL TOXIC EFFECTS Skin Reactions Chloracnc This occupational skin disease can be produced by a number of chemical compounds.13 7 Poly chlorinated biphenyls as well as the chlorinated naphthalenes, a few isomers of the chlorinated dibenzodioxins, 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 is true for smooth, tender skin and it is possible that young children might be less susceptible because of the nature of their skin. Chloracne has also occurred in workers engaged in the production of 2,4,5-T1. During the indus trial production of Irichlorophenol a sudden rise in temperature within the reactor has occasionally resulted in accidents. In one instance this exo thermic reaction caused an explosion.10* When the rise in temperature occurred, 2,3,7,8-telrachJorodibenzodioxin was formed and severe out breaks of chloracne were observed among the workers.'378 The term chioracne was first used by Herxheimer in 1899,'38 who thought it was caused by free chlorine generated in certain factories. Wauer in 1918139 and Tcleky in 1927'40 suggested the term "Pcrnakrankheit." Another term sometimes used is halogcnwaxacnc. 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 (he 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 that make con densers.141'143 According to Braun,141 a few of January 1974 459 DSW 031857 i* 11 STLCOPCB4015819 the cases with manifest chJoracne started with changes in the skin of the face resembling photo sensitivity and the bearers of these lesions suffered severe puritns 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 chloracnc. In addition to the pustules and comedones of typical chloracnc, brownish keratinization of the skin was observed in some patients. Braun'41 pointed out that (he 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.13144 Jones and Alden'4s 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 1930s and 1940s 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 what 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 Kligman*46 and Plewig'47 showed (hat the skin lesions could also be produced by applying the chloracnegenic compound to the skin. After occupational chloracne manifested itself, the patients also complained of systemic effects such as loss of appetite, nausea, edema of the face and hands, abdominal pain, vomiting, and burning and soreness of the eyes. Particularly with the chlorinated naphthalenes, hepatotoxic effects have also been observed. Application of chemicals to the skin can lead to their absorption and cause systemic toxicity. Whether a systemic toxic effect results will depend on the skin surface area covered, the amount of the toxic substance applied, and the ability of the skin to absorb it. II dermal absorption is poor, a single application will usually not be very toxic. However, if the com pound is not easily metabolized and exacted, repeated dermal application may result in suffi cient accumulation in the body to cause systemic toxicity. Crowe144 has pointed out that chloracne due to clilorinated naphthalenes has decreased because these compounds have been used less since the Second World War. However, Weber in I'W;'4" reported an outbreak of chloracne in a company engaged in the production of electric coils that were coated with a mixture of chlorinated naph thalenes with the tradename "Nibrenwax " Detailed examination by several German invest igators indicated that the capacity of 2,4.5trichlorophenol as well as 2,4,5-T (2,4.5-tnchlomphenoxyacetic acid)149,1 50 to produce chloiacne was due to contamination with 2,3,7,8-tctrachlorodibenzofuran or 2,3,7,8-tetrachlorodibenzodioxin. Schwartz et al.'51 indicated (hut the most potent chloracne producing agents were the chlor inated naphthalenes, chlorodiphcnyls. and chlurodiphenyloxides. Persons who worked with chlorinated naphthalenes usually developed acne after a month or more of exposure. Light microscopic observations made of the 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 acanthotic widening of the external root sheath ol 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 cell tows of granular ceils, in later skin biopsies, folding of the basal 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 raihei than sebum. The sebaceous glands also underwent changes. Within the alveoli of the glands the basophil cells increased in number and basal cel] hyperplasia was noted. The ducts of the sebaceous glands were Tilled with a solid mass of basophil epithelial cells. The sebaceous glands gradually 60 CRC Critical Rca/ewt In Toxicology m DSW 03185b STLCOPCB4015820 disappeared and in lalcr secretions were only be investigated in chloracne patients who manifest d recognized as solid protrusions of tlie comedo hypcrpigmentalion. Oil wall. Aside from alropliy of the sebaceous glands, In none of the human case reports on chloracne cc If ill hypoplasia lias ulso been described and many large sebaceous glands forming huge cysts filled pri have the mucous membranes been studied to any great extent. Vagina! smears in women and sputa, marily with keratinous material have been corneal, and nasal scrapings may give helpful li d, observed. Hyperkeratosis and acanthosis of the information in this respect. Since in animals fi- surrounding epidermis has usually accompanied these lesions. Foreign body granulomala which are exposed lo polychlorinated polycyclic compounds low vitamin A levels have been observed in the lie thought to result from rupture of the follicular liver, it is possible that the compounds may affect je cysts have been found in the skin.'47,151 In the vitamin A absorption and storage, and adequate sc Japanese poisoning incident (Yuslio), the predom utilization in the liver and elsewhere in the body. lie inant skin lesion was marked hyperkeratosis of the If the lesion is produced by inadequate utilization I epidermis, cystic dilatation of the hair follicles, of vitamin A, then administration of vitamin A per and an increase in llic melanin pigment in the basal se may not necessarily alter the course of the ly at cells of the epidermis. Foreign body granulomala disease. Protein deficiency may also mimic hypo- >h- were also observed. The contents of the cysts vitaminosis 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 st- the hyperkeratosis was even more pronounced, and these stores may be affected in liver disease. .5- accompanied by atrophy of the epidermis; cystic The microscopic appearance of the skin lesions o dilation of hair follicles was observed, especially in resembles in some respects observations made in ne the skin of the head. According to earlier skin of patients suffering from vitamin A >r- reports151 some differences exist in the micro deficiency. In vitamin A deficiency, hyperkeratosis Ji- 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 )Sl pounds that can produce the lesion. It is, of plugs in the sebaceous glands. Dryness and .ir- course, possible that these histopathologic vari- scaliness as well as furunculosis of the skin may ro- alioiis arc in reality only differences that exist also be present. In contrast, night blindness has ih bciwccn 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 lo explain acne outbreaks are often not well investigated and, he the development of chloracnc. None is satis because Of problems with litigation, accounts are al, factory. The concept of mechanical plugging of often only published many years after the out I he the hair follicles on skin exposure to polychlor break has occurred if they are published at all. |)I1. inated polycyclic compounds is not supported by Chloracne is a very persistent skin disease and ! of the fact that chloracne develops following in the Japanese outbreak of poisoning with chloro- or ingestion of these compounds. It is possible that diphenyls, the skin changes were still present in a lo the skin lesions arc caused by a toxic effect on the number of patients three years after exposure to cd, epithelium of the skin and its appendages which is the chlorodiphenyls had been discontinued. What lar manifested in proliferation and loss of normal needs to be investigated is whether this persistence sal function of the cell. Since the chemicals are lipid of the skin disease is caused only by the fact that vas soluble they could occur in a more concentrated once the lesion has developed it regresses very nd form in sebum and therefore affect sebaceous slowly, or whether it may be sustained by the glands more than sweat glands or salivary glands. chloracne-producing chemicals which have been cd The reports on the Yuslio outbreak suggest that absorbed and stored in the body, particularly the lan 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 (he presence of the ell indicate a functional disturbance resulting in offending polycyclic polychlorinated compounds. >us increased melanin production. Hypcrpigmentalion If adipose tissue is unobtainable, the sebum of the ihil is also observed when the adrenals are damaged, as ear canal or the content of the skin lesions may lly in Addison's disease, and adienal function should represent a good substitute. January 1974 461 DSto 031859 Apparently (here is no known effective (realntcnl for chlorncnc and the best control measure is prevention. The manufacture of chlorinated hydrocarbons and the coating of wires and con densers will) insulating materials containing them should be done in totally enclosed areas so that the fumes of the insulating substances and of the solvents if any arc used do not come in contact with the 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 arc not unduly delayed after work. Special synthetic wetting agents' *' 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 (Jig 2,3,7,8-letrachlorodibenzo-pdioxin107 and with Halowax 1014, pentachloronaphlhalene, and hexachloronaphlhalene.147 Experimental work in animals has shown that most species do not develop the same type of dermatitis. The rabbit's skin,'" 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 a hepatotoxic effect.'50 Microscopic examination of the changes in the rabbit ear were described by Jones and Krizek.'*3 Inagami and Koga1 SA 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 hyperkeratosis 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-disease in Animals Chloracne-lype lesions do not usually occur in other species except for the rabbit and the hairless mouse, already mentioned. However, in 1947 Olafson1 ss described a disease in cattle which was designated as X-disease or hyperkeratosis. Symptomatology included excessive lacrimation, diarrhea, polyuria, marked salivation, and dis charge from the nostrils. The animals developed a chronic cough, poor appetite, numerous iol maculae in the 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 greatly inflamed, swollen, and edematous with many superficial ulcers.15'' 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-discase could be produced in cattle by the ingestion of highly chlorinated naphthalenes, and also by petroleum products such as crankcase oil. Bell'57 tested the ability of the various compounds of the chlorinated naphthalene group to produce X-diseasc. He demonstrated in his studies that dichlorinatcd and trichlorinaled naphthalenes did not result in X-disease, while tetrachloronaphthalene had an effect and the higher chlorinated naphthalenes such as pentachloronaphthalene, hexachloro naphthalene, hept achloronaphthalene, and octachloronaphthalene caused severe disease Oclachioronaphthalene was less toxic than hexaand heptachloronaphthalene. Wagencrl5B repott ed the occurrence of hyperkeratosis in cattle m Germany between 1946 and 1948. The cause of this outbreak was traced to a wood preservative which was used in postwar Germany. The author proved experimentally that animals housed m cabins painted with the wood preservative develop ed typical X-disease. The poisonous ingredient in the wood preservative appeared only in certain lots of (his product and later lots did not contain it The poisonous ingredient was never identified by chemical analysis. Animal experiments were conducted with the toxic ingredients of rice bran oil that produced Yusho in Japan. These toxic substances consisted of a mixture of chlorinated biphenyls containing 48% chlorine, with a (race 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 skin around the neck, foreleg}, and sides of the chest. The author 462 CRC Critical Rrrimt in Toxkolofiy 1 031860 STLCOPCB4015822 \poihaul. > and ns on liscrvollcn, 66 In tin A indiit I lie ( j | ! cut a spccl- Inally :cd in mated ulucis lily of inalcd 2. He :d and jll in ad an lalcncs :h!on>- and iseasc. hexareportItic in use of rvalivc author sed in evelopicnl in tin lots lain it. Tied by j j ; itli (he nduced nsisted taming iphlhan for 3 nges of eration rir, and ind the author! that, at the end of (he experiment, some .lunges in tire skin similar to those in the experimental mice were seen in some of the control mite also, which makes it difficult to interpret their findings. in I'>57 a disease that will be discussed in pealci detail later in this article occurred in a large number of chickens. It was termed chick edema disease and was found to be caused by ceituin mxic fat. Allen and Carstens' 60 fed this substance lo monkeys and found that, among other changes which will be discussed later, the monkeys developed a generalized alopecia and subcutaneous edema one to two months before death. School tie el al.'6' produced hyperkeratosis of the skin in rats by feeding them hcxachloronaphthalenc. Young swine'65 and sheep'6:1 did not develop ikin lesions as a result of exposure lo chlorinated naphthalenes. riiutoconiaci Dermatitis and Irritation of the Skin As was pointed out earlier, (lie hyperpigmenta- liiiii observed in patients with clrioracne as well as the reaction described as cable rash could also be the result of a photosensitive reaction. This photo sensitive reaction is the predominant lesion in the skm produced by a number of germicides, namely, biiliionol (Figure 2), tetrachlorosalicylanilide (TCSA), and a number of other halogenated phenolic compounds including fenticlor. Patients whu develop photodermatitis complain initially of a burning sensation, and this is followed by Hilling. A rasli develops which in (he beginning is ciyihcmatous and associated with varying degrees of edema. As it subsides, pigmentation is observ ed. 164 Wilkinson'66 was the first one to describe eases where patients developed a photo allergy which was due to the incorporation of TCSA into popular soaps. He observed (hat the outbreak of dcmiulitis was confined to (he areas that usually wcic exposed to light. Most of the cases reported fiom England were transient and as soon as exposure to the compound was discontinued the icaction subsided. Jillson and Baughman'66 and iillson,'67 on the other hand, reported that their eases were more persistent. Willis and Kligman16* demonstrated (hat the phoiocontact allergy of (he skin with these various compounds was simply a contact sensitization. The reason that allergic reactions of the skin developed only after exposure lo the sun was due to the photodecomposilion of the various products to which the skin had been exposed. If various brominaled and chlorinated salicylanilidcs were irradiated in vitro in an appropriate solvent, the resulting breakdown products caused an aller gic reaction on contact, without exposure to UV light. These studies were performed because of information provided in earlier studies by Coxon et al.,'69 who had irradiated brominaled and chlorinated salicylanilides and found that these compounds gave off halogens. With 3,5 substituted salicylanilides, the halogen substituted on the 3 position was lost. Tetrachlorosalicylanilide photo sensitized subjects reacted to 3\ 4f, 5-lrichlorosalicylanilide and to 3\ 4'-dichlorosalicylanilide. A reaction to nonhalogenated salicylanilidc 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 to tetrachlorosalicylanilide reported by Wilkinson,'65 a group of patients in a shop of a factory was also included. This same outbreak was reported by Jones,' 70 who found that among 106 employees who used the same soap very frequent ly, 31% were affected by a dermatitis due to contact with tetrachlorosalicylanilide present in the soap. Additional cases of photodermatitis due to the exposure to tetrachlorosalicylanilide were reported by Calnan el al.,'7' 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'75 reported an additional nine cases of acute contact dermatitis of the hands due to tetrachlorosalicylanilide. Epstein173 described a photoallergic reaction in two patients to tribromosalicylanilide (TBS). With patch tests these patients showed a cross reaction to TCSA. A similar cross reaction pattern to a number of halogenated salicylanilides was also observed by Osmundsen17* in patients who had developed photodermatitis following exposure to tribromosalicylanilide. Because of the photosensitivity reactions and the cross reactions that were also found with bithionol, which is similar in structure to tetrachlorosalicylanilide (Figure 2), the U.S. Food and Drug Administration, on October 24, 1967, with drew ail new drug applications of all drugs that January 1974 463 DSW 031861 STLCOPCB4015823 contained bithionol. In the notice of withdrawal of approval in (lie Federal Register, the Commissioner of the Food and Drug Administra tion pointed out that bithionol may in some instances cause a very persistent photosensitization and sevete dermatitis may occur with exposure to sunlight without further contact with the sensitiz ing articles, and that bithionol may produce cross photosensitization with other commonly used chemicals such as certain luilogcnatcd salicylanilidcs and hcxachiorophenc. Hcxachlorophcnc preparations may also cause irritation of the skin if these preparations are used frequently. A true allcrgjc reaction to hexachloroplicnc is apparently rare.175 The more severe reactions were usually confined to the skin of the scrotum and consisted of a primary irritant contact dermatitis.176'*17 The cases reported by Baker ct al.17* also included a 33-month-old Caucasian female who developed a rash around the bullocks, vulva, and upper thighs following the use of a hcxaclilorophene preparation in her bath water. Wc observed severe skin irritation and ulcera tion in rats that were exposed dermally to a 3% hexachloropltene solution in propylene glycol ora detergent. The skin reaction was not as severe when the hexachlorophene concentration was reduced to I.5%.118 Boutwell et al.178 reported considerable skin irritation in mice that had 5 mg/mouse applied twice weekiy for 21 weeks. Harber et al.l7!> 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 pigs with primary photosensitivity to TCSA and TBS. Photosensitivity to trichlorocarbanilide (TCC) was not produced in guinea pigs. The guinea pig may represent a suitable animal 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. Dichlorophene used as an antimicrobial preserv ative may also cause contact dermatitis. Cross sensitivity to hexachlorophene was not demonstrated.180 Chick Edema Simpson ct al.* 8 1 and Sanger ct al.1 82 describ ed a new highly fatal disease in chickens. The first outbreak occurred in 1957 in Georgia. Large 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 a waddling, unsteady gait. Gross inspection of the birds revealed a pale heart, fluid was noticed in the pericardial sac, and the livers were pale, mottled, and had an irregular granular surface. In advanced stages of the disease the chickens had large distended abdomens that were filled with fluid Ecchymotic hemorrhages were present in the skin beneath the wings, on the legs, and over the 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 1957, when extensive losses of chickens and turkeys occurred throughout the southeastern pari of the U.S. Suspicion soon developed that losses were caused by the consumption of rations containing certain lots of animal fat.181 The animal fats contained tallows and greases obtained as 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.181 came to the conclusion that the edema in the chickens was the result of vascular changes that seemed to be the primary lesion. The vascular lesion, or endotheliosis, 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 tubular 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 to globulin ratio with a decrease in the albumin fraction. This could, of course, lead to edema formation, particularly when combined with matfunctioning glomerular capillaries. It has also been suggested that increased permeability of the eardiovascular bed contributed to the edema forma ,, , t u c ,, c, |\ t ri c a l> |\ d n it c p s< t c n p t! c li : a c h b si b> fa 2. su e< c< It n I. n <-' 11 b c 464 CRC Critical Reviews In Toxicology m DSW 031862 W STLCOPCB4015824 Tibfirst 31 pc ' .cks ` tical | ighl , and the a.Mi.'<,n'1"' 1" oliici studies the scrum protein .tmecniratioii did not differ from (/tat of the controls1"* 1 " 5 and increased permeability of the .jrdiovascular bed was advanced as the sole cause afilic fluid accumulation. In addition, pulmonary edema was observed in diseased chickens and perivascular lymphocytic infiltration as well as fjouia of the cardiac muscle with interstitial ly a lunpliocytic infiltration has been noted. 1 88 the Electron microscopic examination of the cardiac the led. iced arge uid. skin ie Q and ion:arly and part rsses conimal I i as loro- muscle revealed degeneration and loss of mito chondria.1 84 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 (lie 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 witlt a semipurified diet than with a natural grain rationi iodium chloride in the diet was a prerequisite for the development of the syndrome.181 If low rations of the toxic fat were fed to chickens they did not develop chick edema but reproduction and hnlchubility were drastically reduced. Toxic ful was nol the only product that caused the chick edema syndrome. Chlorinated biphenyls re ial caused the disease in chickens1 88 and in Bengalese irred finches.76 A mixture of penlachJoronaplilhalene n ct and hcxachloronaphtlialcnc fed to chickens also la in resulted in chick edema disease.189 Cantrell et inpes a).190 announced in 1967 that one of the toxic :ular compounds capable of producing chick edema nion found in toxic fal was 1,2,3,7,8,9-hexacliIorodi- s of benzo-p-dioxin. Then Higgenbotham et al.SJ Pro showed thut the chick edema factor was produced m of by two principal compounds isolated from toxic it no fats: 2,3,7-trichlorodibenzo-p-dioxin, and bular 2,3,7,8-lclrachlurudibcnzo-p-dioxin. They also aren- suggested fats and fatty acids (hat contain commercial chlorophenols as possible sources of that contamination. When crude fats and laliows are if the healed to produce fatly acids, chloroplicnol in to residues may be converted to a chick edema umin factor. Whether these last three compounds dema mentioned arc the only ones (hat produce chick malbeen : carornia- edema disease is questionable unless (lie chlori nated naphthalenes as well as chlorinated biphenyls dial produced chick edema were also conlammalcd with Ihcm. Another outbreak of chick edema disease occurred in 1969.41 In (his incident millions of birds in North Carolina were involved. The outbreak was traced (o 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 traps used to collect by-product fatty acids held as feed fats. Flick et al.1 88 have recently shown that pyro lytic products of 2,3,4,6-telraclilorophciiol were toxic to chicks and produced chick edema if 50 ppb were fed for 21 days. The product fed consisted of 53% liexa-, 45% heptu-, and 25% oclacliloro-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 1 ppm (200 fig total ingestion) of the material for 21 days. When a mixture of tri- and letraciilorodibenzo-p-dioxin was fed to the animals at a dietary concentration of 0.01 ppm (a calcula ted intake level of 1,9 fig), 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 fish meal that was processed for animal feed in a plant in Wilmington, North Carolina.1 9 1 From about April 1971 until July 1971, PCBs leaked into the fish meal and 16 thousand tons of contaminated fish meal were distributed to more than 60 companies in 10 states. One of the purchasers of the fish meal, The Holly Farms, the 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 hatchability of their eggs was drastically reduced to 18% of norma). 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 Kuncchlor 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 465 - - - |f S 0SW 031863 STLCOPCB4015825 rice bran oil, also contaminated with Kanechlor 400 and produced by the same company.192 Metcalfcl,J suggested that chick edema factor could be minimized if fleshings grease from hides preserved with technical pentachlorophenols con taining chlorinated dibenzodioxins were no longer used for food products. It was also pointed out that the use of glue emulsions containing chlorophcnols 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 (o be seen, since polychlorinated biphenyls that may be con taminated with chlorinated dibenzofurans have also produced chick edema disease. Yusho During the summer of 1968, 13 cases of cliloracne 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.'92 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 a total number of 89 males and 100 females that suffered from Yusho were examined, a variety of symptoms were found in these patients. Hie incidence of the different symptoms that varied a great deal is given in Table 2. The epidemic outbreak was finally, after very extensive investigation, pinpointed to Kanemi oil, manufac tured on February S, 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 adipose 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.19 4 The amount of oil necessary to produce disease was found to be at least 2.5 1. The younger the patient, the higher the morbidity rate with a smaller total amount of ingested oil. The average amount of chlorobiphenyl consumed by individual patients was calculated to be a total of 2 g.''M (Various reports written on this subject give different accounts of what actually was consumed and the amount can at best only be estimated.) When the rice bran oil was heated under reduced pressure, Kanechlor leaked from (lie old pipes in which holes were subsequently discovered The Yusho outbreak spread not only ova Fukuoka, Japan, but also over 20 other prefectures in the western part of Japan. A total of 1,057 poisoning cases resulted according lo the latest tabulation (August 1971, Ministry of Welfare).1,6 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 eruption, 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 in the limbs, and hearing difficulties. Most patients showed acnelike skin eruptions that resembled the chloracne observed after occupational exposure to 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 a lower risk for both males and females in the age groups over 60 years was noted. The proportion of severe cases among those age 13 to 29 was significantly largei 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 tire disease, delivered 10 liveborn and 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 and cystic dilation of hair follicles, especially ol those of the head. A marked increase of melanin pigment in the basal cells of the epidermis was also noted. Most fetuses that were bom were smaller 466 CKC Critic*1 Review? in Toxicology mmrr------------------------------ OSW 031864 STLCOPCB4015826 ,i s give , imed ilcd.) : ander c old | crcd. over oilier . lul of i the v of piom i was upper it ion. a t ion and palms lUICUS ten is; idicc, s and , and ogica) nsiem n I lie J lients ] :J Ihe urc lo cnyls. > were It sex sk for ver 60 eases larger i with (uslio, iscasc, . Nine brown ;ingiva nfunts ;xamilarked lis and ' those iclanin as also mailer tabu; 2 Symptoms (complaints) of Patient* (89 males, 100 female*, as of October 31, 1968)* Symptoms Mates % Females % Blackening of nails Black spots in all pores Fsccssive sweating in palms Acnelike skin rrtiplions Red spots on limbs Itching Change ill skin color . Swelling or Bands and led Hardening of backs and hands Pigmentation of mucous membranes Sebum (gum secretion in eyes) Hyperemia of mucous membranes in eyes Temporary failing of eyesight Jaundice Swelling of upper eyelids Sense of weakness Numbness of hands and feet fever Hearing difficulty Spasms of hands and feet Headaches Vomiting Diarrhea 83.1 64.0 50.6 . 87.6 20.2 42.7 75.3 20.2 24.7 56.2 88.8 70.8 56.2 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 5S.0 11.0 74.0 52.0 39.0 19.0 19.0 8.0 39.0 28.0 17.0 Reprinted with permission from kuralsune et al.1 ' ' than Ihe 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. Okumura and Katsuki1 97 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, Ihe initial symptoms were eye discharge associated with edema of the eyelids, defects in vision, generalized fatigue, anorexia followed by comedo and acneform eruptions of the face, and pigmentation and flutlcningof ihe 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 allowed a low grade fever. Most laboratory testa conducted on theae 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 a, 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 serum 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 hyperglyceridemia, an apparent con- i i mm January 1974 467 J GSW 031865 STLCOPCB4015827 genital anomaly. lisscutiul hypcrplyccridcmiu has been observed in siblings of a number of lantiltcs, bu( (lie mode of transmission is not known. The course of this disease is generally benign and is consistent with a normal life span.'" Observation of the familial eases shows that the mere elevation of triglyceride docs not in itself increase the risk of arteriosclerosis. Another interesting fact in this poisoning epidemic was that out of 23 eases with obvious Yusho. 10 showed symptoms or signs of a sensory neuropathy that included numbness, pain und hyperesthesia, and. in addition. I ease also showed aiellcxia.200,20 1 This effect on the sensory nerves would mu be immediately obvious in animal experiments and probably should be investigated further. Samples of sputa and adipose tissue collected from patients who suffered from Yusho contained chlorinated biphenyls on chemical analysis.194 The concentration in adipose tissue was much higher than in sputa. Recently Kuratsune et a!.*96 reported on the present status of (lie patients who suffered from Yusho. Many of tire patients were, still suffering from the illness. When the findings made in 159 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. Immunosuppression Several reports have indicated that PCBs and possibly some of (he cldorinated dibenzodioxins and furans may alter the immune response in animals. Atropiiy of the thymus and reduced total white count were observed in rabbits following tire dermal exposure to PCB fractions containing tetraand pentachlorodibenzofuran.96 In fish, exposure to Arocior 1254 for 14 to 45 days led to emaciation and funguslike skin lesions." However, no attempt was made to isolate organisms from these lesions. When 10-day-old mallard ducklings were fed diets containing PCBs (Arocior 1254) and were subsequently injected inlerpcritoneaily with a duck hepatitis virus, significantly more ducklings died than when they received the virus alone.202 In guinea pigs, subiethal dosage levels of 2,3,7,8-tetrachiorodibenzodioxin produced severe atrophy of the thymus with destruction of lymphocytes, lymphoid depletion in spleen and lymph nodes, as well as extensive hemorrhages in(o various organs including the adrenals. Reduced thymus and an spleen weights were also observed in rats that received a single dose of 25 #rg/kg TCDD and in mice the cell mediated immunity was found to be im: rep suppressed.203 The various findings listed here do not conclusively prove that the immune response is specificaily altered by these compounds as wc Bio P<>r encounter it following radiation exposure or therapy witli 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 is also accompanied by a reduction in lymphatic tissue. In this respect it is also of interest that when monkeys were fed "toxic fat" containing 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.160 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 10- acq cl i a ity frag (Po the sitiv nun slim hem (AL acul acui both subg li wori. acid tacii- day-old ducklings than the virus alone suggests a reduced immune response but could also merely represent an additive damaging effect on the liver, pliyri made more susceptible to infection by tire toxic mere, effects of PCBs. So far no convincing evidence of noted "wasting disease" as a result of exposure to reinvt chlorinated polycyclic compounds has been ST nd ic reported in rodents or any other species in the 73 in; literature. of tin. Atrophy of the thymus in newborn rodents ! first n reduces the capacity of these animals to produce and oi serum antibodies, resulting in wasting disease, also rinuria referred to as runt disease or homologous disease. (Yusln Sucli animals are then incapable of rejecting not re| foreign skin grafts,204 which probably represents the most convincing evidence for homologous Ap| as ret disease. A shift in the immune response could i porphv influence the development and growth of cancer porphi and alter the defense mechanism in infections. In j when l order to prove that PCBs as well as chlorinated j diet. T dibenzodioxins and furans do indeed influence the ; poisoni immune response, the effect of these substances | When f on newborn animals of several species should be j in the tested with adequate parameters such as skin < became 466 CRC Critical Rtvlewt In Toxicology W" DSW 031866 STLCOPCB4015828 pans and (lull ij in o be o do onse ! we or idies imds icily tsclf nans alive lioid d to cy is aalic (hat ning The tn as ;aone nore hick duck 10gcsts : rely iver, oxic :e of : to been the I | I I j | lents ducc also ease, cling sents goos mild inccr s. In lated e the mces d be skin grafting and the measuring of the development of antibodies. Whether the other chlorinated polycylic compounds in this article also affect 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 urnporphorinuria, photosensitiv ity as 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 sitivity can be produced experimentally by a number of drugs; all of these have the ability to stimulate the activity of the initial enzyme in the heme synthesis 6-aminolcvulinic acid synthetase (ALA). In addition to this acquired porphyria, an acute intermittent porphyria erthropoietica and an acute intermittent porphyria hepatica, which are both hereditary, exist together with a number of subgroups which will not be considered here. In 1964, Bleiberg et al.2 reported studies of 29 workers in a 2,4-D (2,4-dicldorophenoxyacetic acid) and a 2,4,5-T (tricldorophenoxyacetic acid) factory. Many of the workers had chloracne and II had abnormal excretion of urinary uroporpliyrins. Many of the workers with uroporphyrinuria had hirsutism, hyperpigmentation, and increased skin fragility. Liver dysfunction was noted in two hospitalized patients. Poland et al.1 reinvestigated tire workers of the same plant studied by Bleiberg et al.2 They studied a total of 73 male employees; chloracne was found in 80% of the workers. In this study six years after the first report, clinical porphyria was not observed and only one worker had persistent uroporphyrinuria. In the Japanese poisoning epidemic (Yuslio), increased uroporphyrin excretion was not reported. Apparently polychlorinated biphenyls, as well as tetrachlorodibcnzodioxins, 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 hcxacldorobenzene poisoning and human porphyria cutanea tarda.2* When female rats were fed 100 ppm Aroclor 1254 in the diet (approximately 7.9 mg/kg/day) 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 o! 550-fold, prophobilinogen 27-fold, and 6-aminolevulinic acid 18-fold. The major porphyrins found in urine from Aroclor treated rats were 8-carboxyporphyrin (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 porphyrin 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-fold 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 6-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 acid 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 Koeman95 reported that chemical porphyria was produced by the two European PCB products, Plienoclor DP6 and Clophen A60, as well as the American product, Aroclor 1260. The porphyrogenic action was observed in chickens, quail, and rats. It has not been established so far whether only certain isomers of the polychlori nated biphenyls or contamination with a chlorinated dibenzofuran is responsible for the production of hepatic porphyria cutanea tarda. Poland and Glover206 used fertile chicken eggs at a developmental stage of 15 to 20 days and injected them with 25 pi of the halogenated dibemo-p-dioxins. They found that 2,3,7,8-tetrachlorodibenzo-p-dioxin produced a dote-related January 1974 469 DSW Wi" 031867 I increase in ALA synthetase activity. The lowest dose tested, 4.66 xlO'15 mol/egg (1.5 ng) doubled the enzyme activity. These results were statistically significant. Studies witli other halogenated dibenzo-pdioxins gave the following results: the 2,3,7-trichIoro and 2,3,6-tribromo isomers are potent inducers of ALA synthetase while 2.3-dichloro and 2,7-dichloro, 2,8-dichloro, 1,3,6,8-te(rachloro, and 1,2,3,4-telrachloro isomers all failed to Induce ALA synthetase at doses up to 2.5 ing/egg. Of the limited number of halogenated dibenzo-p-dioxins that have been tested for fetal toxicity and the ability to produce chloracnc, 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 the chick embryo may also induce it in the mammalian system. Woods701 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-tetrachIorodibenzodioxin. 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 demonstale an increase in ALA synthetase and uroporphyrins in the liver in CS1 black male mice given 25 |ig/kg/week for 4 weeks.509 It is 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.50* Female animals and the embryo may be more susceptible than adult male animals. Effect on Other Enzymes and on Vitamin A A number of investigators have shown that the polychlorinated biphenyls induce liver microsomal enzymes.510'511 Norback and Allen511 correlated an increase of hepatic microsomal enzyme activity with smooth endoplasmic reticu lum proliferation. Litter*! et al.515 studied the effect of four PCB mixtures, namely, Arnchir 1242, 1248, 1254, and 1260. The animals were fed the material for 4 weeks and diciary levels employed were 500, 50, 5 and 0.5 ppm At higher dietary levels an increase in liver weights was observed. Elevation of triglycerides in the liver w as seen at 500 ppm with a maximum effect occurring at approximately 50% chlorine content. An increase in cytochrome P-450 was observed at the 50 and 500 ppm dietary levels of all ITIl mixtures. An Induction of tire pentobarbital hydroxylation and an increase in mellrylase activity were observed at the higher dietary levels. Induction of pentobarbital hydroxylation increased with Increasing chlorine content of the PCBs. Nitro reductase activity was markedly stimulated with increasing doses of PCB and the induction 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-metaboli/ing enzymes of the liver have been reported; one group to which phenobarbila! belongs resulted in increased P-450 content of the liver and induction of a variety of pathways including benzpyrene hydroxylase and demethylalion of ethylmorphine. The polycylic hydrocarbons such as 3,4-benzpyrene belong to the second group, which resulted in the formation of an abnormal cyto chrome. P448, and increased benzpyrene hydroxylation, but not ethylmorphine dcmeihylation. Alvares et al.513 reported that PCB treat ment produced an increase in cytochrome P-44H. an increase in benzpyrene hydroxylation, 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 prior administration ofaclinomycin D. When the effect of a number of PCBs on the beef heart mitochrondria! enzyme system was studied, it was found that they inhibited NADIloxidase, succinoxidase, and cytochrome oxidase activity. It has not been established whether these same effects will occur in vivo. Bentlic et al.*14 induced liver microsomal enzymes by tniraperitoneal injection of a single dose of Aroclor 1248 or 1232. Most of the microsomal enzymes have been studied in the rat, but they are affected in other species as well. Allen et ai.51 * reported liver 470 CKC Critical Reviews in Toxicology i i m 1 "im DSW 031868 STLCOPCB4015830 hypertrophy aftci feeding polychlorinated cantly in male rats for a period of up to 28 days. biphenyls (Aroclor 1248) or a polychlorinated Cytochrome b-5 content was also increased. k-ls diphenyl (Aroclor 5460) for three months to Hydroxylation of aniline was induced and amino- piiinntes. The increase in liver size was attributed pyrine demethylation rates were decreased. Micro to the increase in smooth endoplasmic reticulum somal proline was increased by approximately as and induction of microsomal enzymes was 15%. The induction of UDP glucuronyltransferase "6 observed. Proof of the induction of microsomal was most striking. Oxidative phosphorylation rates \ enzymes in birds is the finding made by Lincer and in rat liver mitochondria using succinate as a lie Pcakall21 6 that livers obtained from American substrate were unchanged. The enzyme induction cs. kestrels exposed to either Aroclor 1254 or Aroclor was observed as early as one day - the peak <m 1262 showed an increase in the in vitro breakdown induction was reached between three and ten days re of ocstradiol to a more polar metabolite. This after treatment. The enzyme induction was accom or effect on sex hormones is also emphasized by panied by an increase in smooth endoplasmic ith Platonow et al.: 17 Aroclor 1254 given to reticulum and a mild increase in rough endo ro- Yorkshire boars reduced the urinary excretion of plasmic reticulum.228 These changes were first 111 gonadal steroids. observed three days after the injection of the /as Both the polychlorinated biphenyls as well as tetrachlorodibenzodioxin. Thus, enlargement of It the chlorinated naphthalenes affect the vitamin A the liver during exposure to various chlorinated at content of the liver. This was particularly studied compounds is accompanied by an increase in ml with the chlorinated naphthalenes since hyperker smooth endoplasmic reticulum. atosis and other abnormal keratin formation of the Furthermore, if "inclusions" or "hyaline bodies" g skin is often accompanied by vitamin A are seen within the cytoplasm with the light -iic deficiency. Low vitamin A plasma levels were microscope, concentrically arranged membranes in observed in cattle with X-disease and young that often surround lipid vacuoles can be seen with on swine.'55,162 A decrease in vitamin A storage in the electron microscope (Figure 6). DC the liver of Japanese quail and rats that were fed Hexachlorophene, on the other hand, does not ic. Aroclor 1242 was also demonstrated.218 Since increase the size of the liver.'18 The livers show as vitamin A deficiency affects reproduction, this no ultrastructural changes and probably eh aspect should be further investigated to determine microsomal enzymes are not induced. Whether to whether the effect of PCBs on reproduction is other compounds listed in Figure 2 affect liver ne secondary (o vitamin A deficiency.219 microsomal enzymes is not known. Hexachlor hi Ecobiclion and Johnstone2 2 0 studied the effect ophene has an effect on a variety of other enzyme nt- of various PCB isomers on the hepatic enzyme systems than those mentioned thus far. Cammer 18, system. Biphenyl itself did not cause induction of and Moore2 2 5 and Caldwell et al.226 have shown nd hepatic drug metabolizing enzymes and neither did that hexachlorophene uncoupled oxidative lit monochlorobiplienyl (4-CI). Substitution of chloro phorphorylation, increased adenosine triphos on groups at the three and four positions of the di- phatase activity, but did not affect the activities of >ns and (elraclilorobiphcnyls caused induction of electron transport enzymes when compared to an microsomal monooxygenases. Induction of micro controls. Gould et al.22 7 demonstrated that hexa- lo somal monooxygenases occurred also when hexa- chtorophene inhibited succinoxidase activity of rat on and octachlorobiphenyls were given to the rats. heart, liver, and kidney preparations. According to fas Nitroreductase and carboxylesterase activities were unpublished material cited by Gluck,2 28 hexa >H- not affected while all compounds produced a chlorophene is conjugated to the glucuronide asc marked induction of the sulfobromophthalein conjugate which is excreted in bile. The esc conjugating system. Terphenyls as well as chlori proportion of conjugated hexachlorophene to I 4 nated terphcnyls increased the pentobarbital meta unconjugated hexachlorophene in blood may be ra- bolism in the rat with corresponding increase in more significant in determining the toxicity of lor smooth endoplasmic reticulum which suggested hexachlorophene than the hexachlorophene blood microsomal enzyme induction.2 2 1 ,2 2 2 levels as they are presently determined. ben Lucier et al.2 2 2 reported that a single oral dose her of 5 or 25 Mg/kg body weight of tetnchlomdi- Methemoglobinemia ver bemodioxin induced cytochrome P-450 signifi In the early I960* outbreaks of methemoglob- January 1974 471 DSW 031869 incmias were reported from hospital nur series.2"'1'10 One oulbreak involved a total of 18 infants with a level of methcmoglobin from 4.9 to 30% of the total hemoglobin. An epidemiological investigation traced the outbreak to 3,4,4'trichlorocarbanilide (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 slay is usually longer. It is also possible that chemicals penetrate more easily through the skin of prema tures. In another outbreak, methemoglobinemia occurred among a group of custodial 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-dichloroaniline have been suggested as 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 as the more studied persistent pesticides such as DDT and dieldrin. 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 the 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 tissues of rats given a single dose of the PCB Aroclor 12S4131 showed the highest concentration of PCBs in adipose tissue and the least in plasma. Brain, liver, and kidney had levels of the same order of magnitude but were lower. If, for instance, 1,600 mg/kg body weight of Aroclor 1254 were given by stomach tube, 24 hr later the mean concentration in 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 rats. The levels of Aroclor 1254 found in the brain showed a mean of 138 ppm and the values observed for Aroclor I 260 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 1254 was fed to male weanling rats for 58 days the mean concen tration of Aroclor derived materials found in (he 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 1 ppm. While PCBs were fed to the rats, the gas chromatogram obtained from the feces was superimposable on the one obtained from the standard Aroclor 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 the 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 adipose tissue had not decreased appreciably and 472 CRC Critical Reviews In Toxicology DSW 031870 STLCOPCB4015832 (fill showed values in (he neighborhood of about 400 ppm and 4.7 ppm was found in the 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 was fed for 240 days at the dietary level of 100 ppm, the concentration of the PCB derived material 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 rats for 2 years, the concentration in adipose tissue is about 95 ppm.1*5 In a separate study where 500 ppm of Aroclor 1254 was fed to male rats for 6 months and the exposure to PCB containing diets was subsequently discontinued for an additional 10 months, the concentration of the PCBs ranged from 924 to 1,688 ppm in adipose 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 ppm."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 al.135 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 gastrointestinal 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 gas 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.136 In a reproduction study, Heath et al.99 analyzed 2 second-year eggs of mallards fed 25 ppm Aroclor 12S4 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 samples. Lower organisms such as mussels and fish contained a higher concentration of PCBs with lower chlorination than birds.15 In mammals, excretion of PCBs in milk and transplacental passage is also observed. Feeding cows 200 mg/day of Aroclor 1254 for 60 days1* 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 was 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.137,13* 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.138 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.139 analyzed two human adipose tissue samples which contained 200 and 600 ppm PCBs ranging from pentachlorobiphenyl to decachlorobiphenyl and Price and Welch140 found 100 ppm PCBs in another human adipose tissue sample. According Jinuary 1974 473 r* DSW 031871 it* 1'iitY and Welch, 111 -II to lY,;. of the petieial populalion in ilio U.S. contain I |>|>m or more PCISs in their adipose tissue. Among I'CB sources they discussed (Ire food chain and house dust which, from residences in southwestern Michigan, contained up to I HO ppm I'CUs. Polychlorinated biphenyls were found in 31% of 637 samples of human adipose tissue that were collected from the general population of the IJ.S.. as part of the Human Monitoring Survey. Of the samples ana lyzed. 5% contained more than 2 ppm.24' IXTBs have also been found in human adipose tissues of the general population of Scandinavia2 4 2 and Japan,2 44 '24 5 and in human milk and human adipose tissue of tire general population of Germany.243 In general, low concentrations of RGBs were stored in adipose tissue in a certain proportion of the general population. 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 whether 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, dichlorophcne, bilhionols, fenticlor, triclosan, and tetrachlorosalicylanilide are almost 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, also called Jrgasan DP300 and CH 3565 (2,4.4'-lrichloro-2'hydioxydiphcnyl 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 (he glucuronide or sulfate m mine and fcccs over a 5-day period. Il lias been shown that TCC is absorbed through the skin. Tissue levels of the chlorinated terphenv! Aroclor 5460 were determined in codfish.24'* In this study it was found that Aroclor 5460 was apparently poorly absorbed from the gastrointest i nal trad; il was stored in all tissues that were analyzed. The highest concentration was found in the liver and following a single dose of I g some of it was sliil present in the 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 (he chromatograms of the standard with those obtained from chlorin ated terphenyls extracted from tissues revealed more of the 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 the 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 highly chlorinated naphthalenes were excrclcd in milk.156 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 lo them, but again not much information is presently available. Firestone et al.241 fed the unsaponifiable fraction isolated from "toxic fat" or 3% "toxic fat" itself to young cockerels. The "toxic fat" contained chlorodibenzodioxins with 2 to 8 chlorine atoms. When (he excreta were analyzed il was found that a certain percentage of the hexachlorodiben/odioxins had been absorbed from the gastrointest inal tract, while all of the octachlorodiben/odioxins remained in (he gastrointestinal tract, and only very little of the heptachlorodiben/.odioxm 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 CHC Critical Reviews in Toxicology DSW 031872 STLCOPCB4015834 v:is also found in 1 he bone and skill, while llic ,uhoi hip;ms contained vciy few dioxins. Some evidence for a possible metabolism of hexa- and hepiachlorodibcnzodioxin by the chick was also observed. Zilko and Wildish1** fed di-, tri-, letra-, and oclachlorodibcnzofuran to fish and delected only octachlorodiben/.ofuran in muscle and gut of dead fish. One problem with metabolism, storage, and distribution studies of chlorinated dibenzodioxins and furans is that not all of the com pounds arc readily available and, if they are available, any long-term feeding studies in large animals are expensive. The very toxic dioxin compounds can be given only at such low levels that they escape detection easily. In our labora tory we detected a chlorinated dibenzofuran in the urine of rats fed Aroclor 1254. We found this same dilorinaled dibenzofuran in the Aroclor itself*0 as a contaminant. Williams ct al.1*9 fed rats a total of 22.7 and 120.7 fig of oclachloiodibcnzodioxin. 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 (be absorption, excretion, and storage of these compounds, and their persistence. Studies with labeled material may eventually give us some information on their metabolism. Piper ct al.150 gave a single oral dose of C1* 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 tire feces and only 13% In urine and 3% in expired air. Most of the remaining radioactive material was found in the Jiver. In another study151 100 Ug of Cl30oclachlorodibenzodioxin 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 Cornish151 studied the urinary excretion of biphenyl and 4chlorobiphenyl in rabbits. Biphenylglucosiduronic acid and 4-hydroxybiphenyl were isolated from urine of rabbits fed biphenyls. The rabbits fed the 4-chlorobiphenyl excreted 4 - ( p ch!orophenyl)-phcnol and 4-chlorobiphenyl glu- cosiduronidc. Twice as much 4-chlorobiphcnyl as biphenyl was excreted as the glucosiduronic acid derivative. It is possible that other low chlorinated biphenyls and naphthalenes are excreted in urine in a similar manner; whether they would be delected by present gas chromatographic methods would have to be checked by combining the identification of radio labeled material with gas chromatography. Hulzinger el a).151 found evidence that some chlorinated biphenyls are hydroxylated by some species. Apparently trout is not able to hydroxylate 4-chlorobiphenyl, 4,4'-dichlorobiphenyl, and 2,2\5,5,-le(rachlorobiphenyI, while the rat and the pigeon both formed monohydroxychlorobiphenyl 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'tetrachlorobiphcnyl 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-chlorobiphenyl. No hydroxy metab olites could be identified in the excreta of the three species when they were given 2,2\4,4'5,5'hexachlorobiphenyl. No evidence of reductive dechlorination was observed in any species. Only very few studies have been conducted with radiolabeled hcxachlorophcne to study its absorption, distribution, and metabolism. Wit and Van Genderen15* gave rats and rabbits hexaclilorophcne-C1* (2,2'-melhylene-Cl * 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 (he feces as unidentified metab olites. The metabolites could not be extracted front the feces with ethanol. The biological halflife calculated front the experimental results was found to be approximately 2 7.8 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 Metabolites were not identified and tissue levels were not determined in litis study. The authors cited a study by Kok,555 who did determine Ik'Vu'hlt'H'i'hcuo in various organs of rats. i'.moil el al.JSh studied the absorption ofC-14 labeled licxachlorophene on the tail skin of the rat. Hcxachloropltcnc in a detergent solution was absorbed by the intact skin of the tail at the average rale of 1.7 jug/cm5 of exposed surface aica/hr. Immediately after the infliction of second degree burns, (Ire absorption rate increased 2.5 times that of the normal skin surface. After 24 hr tire absorption rate dropped to normal or sub normal levels. Immediately after traumatizing the tail, the absorption rale of hexachlorophene was even greater than following the infliction of a burn wound. Manowitz and Johnston5 5 7 found that hexa chlorophene was deposited on the skin when used in various formulations and could be extracted front the skin by soaking the'exposed areas (hands and forearms) irr alcohol. Tire 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 (be 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 blond of a few random samples of the general population. We found that the arithmetic mean concentration of hexachlorophene in adults was 0.028 ppm (/jg/g) of whole blood of a total of 14 samples. In infants who were washed with hexachlorophene in the nursery (he amount of hcxachloropltcnc found in whole blood after the infants bad anywhere from 2 to 14 washes averaged 0.10*1 ppm (gtg/g whole blood). Blood obtained from the cord at the time of birth contained an arithmetic mean of 0.022 ppm hexachlorophene.5 5 * ,55 * 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.5 59,560 At the time we reported these blood levels, our recovery rate was only about 75%. All 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 requesl of Sterling Laboratories'35 illustrated that newborn rhesus monkeys following total body bathing with a 3% detergent solution for 1 week had blood levels of about 1.5pg/ml (ppm). In subsequent months a plateau effect was observed and at 90 days the blood level was equivalent to 1.1 pg/ml These monkeys showed status spongiosus of the white matter of the brain, this indicates that at least in the monkey, blood levels of about 1.5 jug/ml whole blood were consistent with fluid accumulation in the myelin sheath of the central nervous system. Lockhart135 cited unpublished studies by Gluck, who found that hexachlorophene blood levels in term infants after washing with a 3% hexachlorophene detergent solution were some what higher than those reported by us with a mean concentration of 0.345 ppm (pg/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 15 premature infants that were washed with a 3% hexachlorophene detergent solution, the average blood concen tration amounted to 0.475 ppm (wg/g).5 61 Abbott et al.56J using a different washing regimen found a mean hexachlorophene blood concentration 12 to 18 hr after the initial wash of 0.154 ppm (pglg > i j j Cf-'C Cri:tio'Krririv: m DSW 031874 STLCOPCB4015836 of ill) of [lie lien Kill rlh pin Ion in rod ; of ;iys led esc out 'ere the wlf ma- of orn ith iod en( 90 ml. (lie al 1.5 uid Irul by ood 3% meican ad). ffer/try , or exaanis lene cenbott und i 12 Ug/ft whole blood) amt on (lie 7 lo 9ih day of life, 24 lo 4S In ai'iei the second or lbird application of ht-M.'hlo: .-phene. the nice * M.w- level e. --..nn-.ied .'.2'" rp:v. i_c c Vhc .inv.;::; found in con! blood of lo infants "as 0.013 ppm Urg/g). When hexachlorophene was applied only on the day of delivery, the hexachlorophene blood levels gradu ally decreased from a mean of 0.145 ppm on the first day to a mean of 0.057 ppm on the ninth day, following an increase to 0.194 ppm on tiie fourth day. The authors suggested that premature infants may absorb more hexachlorophene through their skin since in one infant 29 weeks of gestation and weighing 1.42 kg the blood level was 1.17 ppm 24 lu after the initial wash and 0.655 ppm at 48 hours. Alder et al.263 reported mean hexachlorophene blood levels of 0.18 ppm in infants who were dusted with a powder containing 0.33% hexachlorophene. Adults using hexachlorophene preparations also absorbed it via the skin.132 The blood levels obtained in adults that exposed their total body surface to a 3% hexachlorophene preparation twice daily for 60 days, 5 min each time, showed blood levels of approximately 0.68 ppm (jag/ml whole blood) with a range of 0.25 to 1.08 ppm (/ig/ml 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 /rg/ml. blood levels obtained in a patient with burns who died between 24 and 48 hr after the last hexachlorophene application were 2.2 ppm (/rg/g whole blood). The blood levels that have been reported all indicate that following dermal hexachlorophcne exposure the chemical can be found in blood, and from information through animal studies as well as some human experience, levels of approximately 1.5 to 2 ppm (ng/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 belter way of assessing a toxic dose would be to measure the amount of hexachlorophene in the brain, but for obvious reasons that is not possible. Female rats fed hexachlorophene at the rate of 500 ppm (25 mg/kg body weight/day) for 55 days with very definite lesions had about 4 ppm hexachlorophene in their brain and 3.91 ppm in the liver. Squirrel monkeys given S mg/kg body weigh (/day of hexachlorophene for 3N days showed a mean hexachlorophene couccnnation in *'".e or 0 35 ppm av.d -d'.-ov,- uvme h.> hexaebloiophnie coucoiiliatiou was 0.01 ppm/*' * The recovery rate in this study was only 75%. These squirrel monkeys had no overt clinical signs of toxicity, but showed mild status spongiosus of the white matter of the brain. Electroencephalographic changes had also been observed in these primates. Ulsamer et al.165 found hexachloro phene brain levels of about I ppm in newborn rats with signs of central nervous system toxicity. In 1 suspected case of human hexachlorophene poisoning in a child, the hexachlorophene brain level was about 2.2 ppm.261 Adult rats dying after the administration of a single dose of hexachloro phene showed concentrations of 6 to 9 ppm of hexachlorophene in the brain.26* Some variation of the concentration of hexachlorophene in the brain which can be related to neurotoxic 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 hexachloro phene would alter hexachlorophene brain levels has thus far not been reported. With present methods, we are probably only determining free hexachlorophene while the conju gated portion remains undetected. The toxicity of hexachlorophene will vary with tire ability of the organism to conjugate, metabolize, and excrete this material. We found in rats that hexachlorophene is excreted in milk of dams fed hexachlorophene 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 hexachlorophene did not seem 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 hexachlorophene, the possible storage of the material as well as its metabolism needs further investigation. Since hexachlorophene was found in cord blood of infants, it can be assumed that transplacental passage also occurred. Morphologic Effect on the Liver Some of the chlorinated naphthalenes, chlori- January 1974 477 mm DSW 031675 STLCOPCB4015837 nnlcil dihcnzodinxins, chlorinated dihciiiiorurans, (lie chlorinated biphenyls, as well as I lie cJilorinalcd lerplienyls have an effect on the liver in various species, including humans. Bennett et a!.3*1 compared (he toxicity of differently chlori nated naphthalenes and a biphenyl that contained 65% chlorine. Tri-chloronaphlhalene apparently was less hcpaloloxic than the higher chlorinated compounds and (he 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 described in the liver consisted of liver necrosis, fat accumulation, and the presence of hyaline bodies in the cytoplasm of the liver cells. A marked deposit of finely divided granular yellow or yellow-brown pigment was observed in Kupffer 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 course, all livers 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/nr1, 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 105 days and were removed from exposure for 2 months. Similar findings were made by Miller5* who studied the 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 pigs, rats, 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 guinea pig, less in the rabbit, and least in the rat. Nishizumi,5*9 who studied the 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 Kupffer 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 Koeman95 observed liver necrosis in chickens that were dosed with European PCBs, whijc a Monsanto product with 60% chlorine did not produce this effect. Increased amounts of iron were demonstrated with Perl's iron stain in the livers of all chickens exposed to the European as well as the American PCB sample. Vos and Beems96 studied the PCB-induced lesions in ilic 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, centrolobular liver cell atrophy, ceroid pigment in Kupffer cells, periportal fibrosis, and cytoplasmic hyaline degen eration. The feeding of Aroclor 5460, a chlorinated terphenyl, Aroclor 1254, ora chlorinated dibenzop-dioxin, not further specified in the article, led to liver hypertrophy of Sprague-Dawley rats within eight days to three weeks. Ultrastructurai 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.110 Rabbits given 300 mg of Aroclor 1242 and 1254 once a week for 14 weeks developed enlarged livers and the ones given Aroclor 1254 showed midzonal necrosis of the hepatic lobules. The livers of rabbits fed Aroclor 1221 in a similar fashion did not show any histologic changes.5 71 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 478 CRC Critical Rttiewt to Toxicology DSW 031876 STLCOPCB4015838 cr as > risl a )% ;e* of 'cr l in :rs an ri* an In he v a J&+- 1 7^.v, . y v v:. tr". ,*',rr' V <5 -v ' ^ v^irJyCrt - */ A .< . ' C:- r.. >'AW 4 in is, lid * IT * M on ry 4 he as nd FIGURF 3. Section of livet of u ral fed Aroclor 1260 for 90 days. This figure illustrates a mitotic he figure and a liypcuhromalic nucleus (arrows) H & E X 500. to ro ly single dose of 10.000 tng/kg ill peanut oil by di me I hanonaphthalene, and piperonyl 'CI stomach lube, and were saci'iliccd the next day, but oxide3 74 (a-(2(-butoxyethoxy)ethoxy)-4,5- ist the liver was essentially normal microscopically. melhylenedioxy-2-propyltoluene) They are identi ru Doses of 1.000 ppm (72 mg/kg) of Aroclor 1260 cal to the so-called hyaline bodies that were for 3 months produced only an increase in mitotic described in the earlier literature by Bennett et ed figures (Figure 3) and enlarged hepatocytes. When al.3<7 and Miller.28 to- groups of rats were fed dietary levels of 20, 100, Aside from these general changes, extensive, to 500, and 1,000 ppm of Aroclor 1260 and 20, 100, grayish-white, firm, glistening areas were noticed lin and 500 ppm Aroclor 1254 for 8 months, (he liver in the livers of a number of the experimental er- changes were pronounced.275 The hepatocyles animals, particularly at the high dietary levels. nd were enlarged in many of the rats of the experi Microscopic examination of these grayish-white ric mental groups and lipid accumulation, foamy areas showed that the hepatic parenchyma had io- cytoplasm, and a brown pigment which stained been replaced by glandular pale staining epithilial m- partially positive for hemosiderin were observed in cells that formed ducts and were surrounded by Us the livers. The pigment was primarily observed in proliferating fibrous tissue (Figure 5). Larger e a macrophages and Kupffer cells. Inclusions (Figure lesions often had extensive fibrosis and also nd 4) that stained slightly more eosinophilic than the contained collagen and the ducts that were formed nal surrounding cytoplasm were present in the cells. by epithilial pale staining cells were markedly of These inclusions within the cytoplasm have been dilated and contained necrotic debris or mucus. did described for many compounds such as mirex373 The lesion has been classified as adenofibrosis for (dodecachloro-octahydro-1,3,4-metheno-2H- the time being. It has been described in detail by ity cyclobuta|cd) pentalene), DDT7 74 , 1 ,1 ,1 -(tri- Edwards and White374 who observed it in rats lan chloro-2,2-bis(p-chlorophenyl) ethane dieldrin375, that were fed butter yellow (p-dimethylamino-azores 1,2,3,4,1 0,1 0-hexachioro-6,7-epoxy-1,4,4a- benzene). These lesions enlarge peripherally by the a a 5,6,7.7,8a-octahydro-l,4-endo-exo-5,8- formation of new glandular structures. Hepatic January 1974 OSW 03187? STLCOPCB4015839 T-'IGURL 4. Section of liver from ret fed Aroclor 1254. A cluster of inclusions is present in lire cytoplasm of one hcpatocyte (arrow). Usually individual cells contain only one of these formations at a time. Totuidine blue stain of miraglass embedded material X 300. tissue may be (rapped between the glands of the peripheral area of adenofibrosis. The older central parts of the 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 (he same liver small foci 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 conclusion271 that a precursor, other than bile ducts, existed from which adeno fibrosis developed. However some investigators of the lesions have assumed that it represented an atypical bile duct proliferation, also referred to as cholangiofibrosis.27* In addition to this lesion we also observed small clusters of glandular cells within areas of adenofibrosis as well as adjacent to blood vessels that were surrounded by rchiivelv normal appearing hepatic parenchyma with sum ing characteristics of salivary gland tissue. These clusters of cells very closely resembled panelcatie tissue.2 79 The distinction between adcnolibiosis and adenocarcinoma is difficult. The glands are 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 consum 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 the periphery and old and hyalinized towards the center of the lesion.277 Of course a liver showing adenofibrosis may also contain carcinomatous lesions. So far conclusive evidence is lacking in the literature (hat adenofibrosis gives rise to carci nomas. However, it has been found to coexist with carcinoma of the liver in rats and some investi gators feel (hat the lesion is in fact a precursor of cancer.2*0 Choline deficient rats apparently, in addition to other changes in the liver, also develop 480 CRC Crillctl Rrviewt in Toxicology DSW 031878 STLCOPCB4015840 I i vi'K IICURI. 5. Scclinn illuMriilcs an area of adenofibrosis of the liver of a rat fed Aroclor 1254. Note the epithelial cells forminp small ducts. The ducts contain cellular debris and are surrounded by fibrosis which can become quite pronounced, H 4 F. X 125. rely uintese itlic osis arc be , in ,rity tieris is 's in is is the the wing Ions i the arciwilh 'csti>r of in elop adenofibrosis.21 However, these mis were fed pcaiiui meal and soymeal and therefore the possi bility exists dial die experiment] diet was conlaminnied willi a mycoloxiu. Dciinet cl al.267 also described die lesion (adenofibrosis) in rals when a mixluic of chlorinated naphthalenes and chlorodiphenyls was fed to them. A critical review of Ihe hislopalhogcncsis of this lesion was made by Stewai t and Sncil.2 7 7 Ulirasiruclutal changes of the livers of animals exposed to ihe Aroclor consisted of an increase in smooth endoplasmic reticulum and atypical mitochondria. Many lipid vacuoles were observed, pailiculaily al Ibe liiglter dietary levels. They were it limes surrounded by concentrically arranged membranes. The "inclusions" or "hyaline" bodies observed in the cytoplasm with the light micro scope correspond to these formations when they ire examined under the electron microscope. Their ullrastructural appearance has led to (heir being referred to as "linger prints" (Figure 6). The epithelial component of the adenofibrosis outlined consisted of cubordal or columnar cells with the free surface lined by microvilli, granular cytoplasm with many ribosomes, and a few endoplasmic membranes. Some of the cells contained a great deal of mucus and resembled goblet cells. Tonofilamcnts 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.267 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 trichJorophenol, 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 DSW 031879 STLCOPCB4015841 *V 1` ' . 7 */* * " .* , A, ', ` * * :.-v' - *. .* '` ' :4 ' 4. ' ` ''; ,Vv--A ' " : *: ' i' * ' \t, .'.-.`, \t*f-ii"tvVv-'*.VV. f*V''.* s, . ' Vj. >y* V w,.-Jf .XJ**"*.- T ..-- .fr . ' # - ' v"-* * . . . ,, <i jf't? ' . ` v> \y*.... .' " V '/^.V s'.v * v' rVN ; . - ' -^.V r- ..; . . .. v'ZMX- mry-, FIGURE 6. Electron micrograph of a portion of the cytoplasm of a hepatocyte. The section shows concentrically arranged arrays of membranes which surround vacuoles containing lipid. Lead citrate, uranyl acetate X 34,200. particularly bad following exposure to halo-waxes, which are chlorinated naphthalenes.181 Cotter583 reported seven cases of pentachlorinated naphthalene poisoning in workers who were engaged in manufacturing wire cable during World War II for the navy. Four of the workers develop ed jaundice and two died. Microscopic examina tion of the liver of the two workers that died showed complete loss of liver cells 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 I to 10 /ig/kg 2,3,7,8tetrachlorodibenzodioxin given to rabbits produc ed a hepatotoxic effect. At the higher dosage level the animals died and the lower doses caused serious liver damage.10* Schulz107 had reported earlier that single oral doses of 20-50 pg/kg body weight resulted in fatal liver necrosis in rabbits. Weanling rabbits were treated wilh 2,3,7,8tetrachlorodibenzo-p-dioxin by applying a dose of 1.67 Fg daily to the inner aspect of both ears for three days. The total dose given was 7.05 Fg/kg body weight. When these rabbits were sacrificed 18 days following the last application of the dioxin, their livers were significantly larger than those of the controls. Microscopic examination of the livers showed enlarged hepatocytes. Some of the hepatocytes were multinucleated. The cytoplasm was foamy or vacuolated. A light brown pigment in some liver cells as well as Kupffer cells was also observed. Inclusions were seen in the cytoplasm in some liver cells, slight interstitial fibrosis was present, and two of three rabbit livers examined showed foci of necrosis that were surrounded by fibrosis.184 ; j JI I ! ) j 1 Studies with "toxic fat," the cause of chick j edema disease, showed that it had a necrotizing > 4S2 CJtC Critic/ Rertcwi 01 Toxicolofy ppm DSW 031880 STLCOPCB4015842 ciYcvi on ilk' 11vci in chickens. As we now know, die toxic agents responsible for chick edema disease are chlorinated diben/o-p-dioxins. Toxic fats also altered liver morphology in Macacca ntulatta monkeys.5*5 Hcpatocyies were enlarged, ntullinuclealcd and focal necrosis in the cenlroJobular /.one w'as observed. Many liver cells were vacuolated and stained positive for neutral fat. In our studies of rats given hcxachforwphcnc, 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* Thorpe151 on the other hand, observed periportal fatly changes in livers of sheep given three or four doses of 50 mg/kg hexachloropltenc and hislochcmica! changes were also observ ed in the hcpatocyies. Pugh and Crowley5* also observed hcpatotoxicity in sheep after giving them hexachiofophene. Of the other germicides discussed in this article, only Iriclosan (Irgasan DP-300) (2,4,4-trich!oro2'-hydroxydiphcnyl ether) has been reported to have a toxic effect on the liver at a dose of 125 mg/kg and enlarged the liver of rats at a dose of 25 mg/kg. according to live summary minutes of tire 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 furl her. / The accumulation of fat in the liver indicates thai either 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 "inclusions" will also disappear after a certain amount of time if exposure to the material is discontinued.5*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 rate, the stimulated liver also metabolizes some medica tions more rapidly,58* 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 cell is not known. The faci that smooth endoplasmic reticulum may become hypoaclive was first postulated by Hutterer et al.5*9 Fibrosis of the liver as well as necrosis and the adenofibrosis observed after PCB exposure in rats should definitely be considered a very serious lesion. Usually adenofibrosis occurs concomitantly with hepatomas or hepatocarcinomas in rodent livers. A recent publication by Kimura and Baba1*9* showed that hepatomas can be induced in rats 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',J90 as part of the symptomatology of poisoning, but in most reports not much emphasis was put on it. In the Yusho epidemic Murai and Kuroiwa590 per formed more detailed examinations in 21 cases admitted consecutively to Kuyushu University Hospital in the northern part or 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 a Finnish company where paper was impregnated with biphenyl.95 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 weighl/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.29' 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 41 FIGURE 7. Electron micrograph from the white matter of the brain of a rat, illustrating status spongiosus. Note the l.ngc empty appearing vacuoles lined by myelin (MY = myelin, A axon), ljead citrate, urany! 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.191 These vacuoles are smaller, usually very uniform, and at times contain a homogeneous pink staining materia). They are usually separated by a wider area of norma! appearing white matter than the vacuoles in status spongiosus. Brains that show this artefact do not weigh significantly more than those that do not show it. Electron microscopic examination of brains of rats given hexachlorophene showed that the vacuolation in the white matter of the brain revealed large vacuoles lined by myelin. The axons that were present appeared usually to be quite normal, occasionally strands of myelin had separated and , traversed the vacuoles. Tire vacuoles were other wise empty except for some electron dense granu lar material (Figure 7). No other alterations wetc observed in the white matter. This lesion is not unique for hexachlorophene; other chemicals that have produced status spongiosus as we have called the lesion include Iriethyllin,293 isoniazid,194 the halogenated benzaniiide, 2-acetoxy4-chlom-5.5diiodobenzanilide,195 and rafoxanide.,9<' which is the 3,5-diiodo-3'-chloro4,-(p-chloro-phcnoxy) saiicylanilide. Triethyltin seemed to produce the lesion at a much lower dietary level and moic rapidly than the other chemicals mentioned, par ticularly hexachlorophene. In an additional study, after feeding rats 500 ppm hexachlorophene in the diet for 10 weeks and then discontinuing the exposure to hexachloiophene, function began to return in the hind quarters of paralyzed rats after 2 weeks and wiiliiu 44 CRC Crlticel Review! in Toxicology DSW 031882 STLCOPCB4015844 6 weeks the rats had almost completely recovered clinically. However, microscopic examination of the biains of these rats showed that a few cyslic spaces were still present 12 weeks after the exposure to hexachlorophcnc had been discon tinued. In weanling rats, but not In adult rats, the bruin lesion and symptoms of paralysis could be produced with a single oral dose of 100 mg/kg of body weight given by stomach tube in peanut oil.11" Part of the reason for being able lo produce the brain lesion in the weanling rats with a single dose may be related to the fact that the weanling was able to survive a higher single dose than the adult rat. Other species that have also shown paralysis and other neurological deficits after exposure to hexachlorophcne are rabbits,397 pigs,39* and cats.399 -Sheep become blind according to Udall and Malone.'30 Hanig et al.399 recorded the neurological deficits produced in cats in more detail. Early symptoms consisted of lassitude, weakness and ataxia of the hindlegs, impaired righting reflex, patellar hyperreflexia which later developed into hyporcficxiu, urinary retention, and eventually complete flaccid paralysis. The cranial nerve functions remained intact except prctcrminally. In these cals an increase in cysternal cercbulspinul fluid pressure was observed. In our first studies we continued exposure to hexachlorophcnc for a period of M weeks because we had designed the exjrcrimcnl to conduct an approximate 90-day feeding study. It is not necessary to feed rats for this length of time. As we determined later, microscopic changes can be observed in rats after a feeding period of two weeks when the animals were exposed to levels high enough to produce the lesion. Some vacuolation can already be observed 3 to 4 days after onset of exposure to hexachlorophene in some rats. When lower dietary levels were fed in a reproduction study,1'* the dietary level of 20 ppm (2.4-0.9 mg/kg body weight/day) had no effect on the brain. At the dietary level of 100 ppm (11.8-4.6 mg/kg body weight/day) focal areas of vacuolation of the white matter of the brain were observed in some of the rats, but they were never extensive. The rats in these lower dietary levels did not show any clinical neurologi cal symptoms. The brain lesion observed in rats was also described in monkeys following the exposure to hexachlorophene. Newborn monkeys washed with 3% hexachlorophene detergent solution developed a diffuse status spongiosus of the white matter of the brain.3"0 Sanfolucito364 301 was able lo produce the vacuolation of the white matter, the so-called status spongiosus, by injecting squirrel monkeys daily subcutaneously with 5 mg hexachiorophenc/kg of body weight for 37 days. When monkeys were fed hexachlorophene they did not develop the brain lesion but showed eiectroencephalographic changes which need further investigation. The monkeys that showed the vacuolation also demonstrated electroencephalographic changes. Sheep have apparently also shown status spongiosus of the white matter according to Hale and Reid.303 Lampert et al.303 were able to 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 intraperiloneally did not stain the brain, an indica tion that (he 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 cases that died from hexachlorophene poisoning. The two adults in this group did not show vacuolation of the white matter of the brain. They had died less than 48 hr after exposure to hexachlorophene. The four children (two with congenital ichthyosis, two with burns) had at least three or more days of exposure to hexachloro phene baths in which a 3% hexacldorophene preparation was used. The final concentration of hexachlorophene in the bath water was not known. All four children showed status spongiosus of the white matter. The brain weight given in one of the cases, a 12-day-old child, was 425 g. The normal brain weight for a child that age is 382 g. Shuman et a).304 in a retrospective blind study were able to demonstrate that the incidence of status spongiosus in the brain stem reticular formation in infants who had died from a variety of causes could be correlated to hexachlorophene exposure. The authors found 21 cases in a series of 250 autopsies of children. Of these cases, 18 were premature infants weighing less than 1,400 g and were of less than 30 weeks' gestation. All but 2 of the cases had 3 or more total washes with 3% hexachlorophene. In the group of cases that were studied were number of stillborns who did not January 1974 485 DSW 031883 STLCOPCB4015845 vh,,w il,o k-MMU and ,<r comsc had not had dermal 1io\.k)i|i,ii,|>Iioik' exposure. Shuman el al.' iii.'imnnoil e.olirr cases of Lcllcrer Siwe's disease m uI.kI. they had observed status spongiosus of i|IC W|,,ie mutter. Each of tire children with I.CIICICI Siwe's disease had had pronounced exposuic to hcxaehlorophene because of the skin lesions wliich had been vigorously scrubbed with a detergent containing the chemical. The various ohscivations sltow that, in patients with abnormal skin as well as the skin of the premature, hexachlorophenc may be absorbed in sufficient amounts to cause status spongiosus. The fact that biood levels in premature infants washed with hexachlorophcne lend to be higher than in mature infants also substantiates these findings. Almost identical observations were made by Powell el al.305 In premature infants weighing less that, 1.400 g, 7 of 13 that had 4 or more exposures to hcxaehlorophene washes showed status spongiosus of the 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 more exposures to hexacldorophcnc washes showed the lesion, and I oF4 that was washed 4 to 8 times. Four stillborns and 13 infants washed 0 to 3 times with hexachlorophene and weighing less than 1,400 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 (hat 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 children306 (New York News, Dec. 13, 1972). Detailed accounts have 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 the skin, the more likely it is (hat 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 spongiosus of the white matter.306 These various accounts show that status spon giosus can also be produced in humans and probably represents a nonspecific reaction which can be elicited by a number of chemicals. The lesion status spongiosus of the white matter as far as we now know seems to represent a specific or restricted type of brain edema where the 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 spon giosus 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 diseases and the reader is referred to KJatzo,30 7 Ardonato and Lamperl,30* 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 et al.,303 microscopic changes in rodents did not completely regress and the fact that sheep do not recover their eyesight afier hexachlorophene poisoning'20 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 central nervous system. Gastrointestinal Lesions Only two types of compounds under discussion have produced lesions in the gastrointestinal tract. We observed in our study with Sherman strain rats that high single oral doses of 3,000 mg/kg Aroclor 1254 and 1260 or more caused ulceration of the gastric and duodena) 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.,105 in their reproduction study with mink, observed that Aroclor 1254 produced hem orrhage In the gastrointestinal tract of the off spring. Allen and Norback310 observed hyperplasia and dysplasia of the gastric mucosa in rhesus monkey*. Six of these rhesus monkeys were fed 4S6 CKC Critic*! Rtvicwt in Toxicology <*** DSW 031884 i 1 STLCOPCB4015846 l-'IGURE 8. Section of the glandular portion of the stomach of a rat given a single dose of 3,000 nig/kg of Aroclor 1254. This figure illustrates the focal loss of mucosa and the infiltration by inflammatory round cells. H & E X 50. 300 ppm Aroclor 1248 and six were given 5,000 ppm Aroclor 5460, a polychlorinated triphenyl. The glandular formations in this lesion were highly atypical; penetration of the muscularis mucosae and invasion of the subntucosa by the mucosal epithelium were observed. Allen and Carslens3*5 reported an effect of toxic fat on the gastric mucosa in 18 of 27 monkeys, Macaco 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 dibenzodioxins. It has also been observed that hemorrhaging occurred into the gastrointestinal tract of rat fetuses who were exposed to 2,3,7,8-tetrachlorodibenzo-o-dioxin in utero. It was not stated in the reports whether the gastrointestinal hemorrhages were caused by ulceration of the mucosa.110,111 Ulceration of the glandular portion of the adult rat stomach was also observed.113 Patients with chloracne frequently complained of loss of appetite, nausea, vomiting, and abdom inal pain, and Goldmann311 described a worker with a follicular dermatitis who died six months after exposure to 2,3,7,8-tetrachlorodibenzodioxin. 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 lower dietary levels and whether the contamination of the chemicals with various chlor inated dibenzodioxins and furans is responsible for them. Valuable information might be obtained from retrospective studies of workers following occupational exposure. Whether the ulcers are caused by a 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 naphtha lenes, and the chlorinated terphenyls is very low. January 1974 487 I B OSW 031885 STLCOPCB4015847 On lIk- other hand, lliey :irc poorly broken down in (he environment and (he metabolism of the biphenyls with more than four chlorine atoms seems to be negligible. Because of these character istics, the use, wherever possible, of chlorinated biphenyls that consist of mixtures of compounds with one to four chlorines has been proposed. Neither the 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 dibenzodioxins and chlor inated dibenzofurans. The possibility remains that the compounds illustrated In Figure 1 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 dibenzodioxins and the chlorinated dibenzofurans are highly toxic. The most toxic is 2,3,7,8-telrachlorodibenzodioxins, 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 very 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 hexachlorobenzene, chlorinated biphenyls, chlorinated dibenzodioxin, 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 dibenzo furans also produce porphyria. It has also 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 cuUnea tarda can apparently occur.3 It is also not known if the combined effect oT 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 effect 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 has not been satisfactorily demonstrated. Chick edema disease has been thought to be caused by proliferation of the vascular endothelium. This might explain the 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 better 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 doubt whether this is caused by a general debilitating effect on the animal of which atrophy of the lymphoendothelial system may be one manifes tation, or whether this is a specific alteration, as we know it from the effects of radiation and exposure 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 the information obtained from these studies, a tolerance level of 5 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. - i | i j , j I \ i 4M CftC CHlktl Rtiitwt In Toxicokfy DSrt 031866 STLCOPCB4015848 In infant nurseries in hospitals, one of the greatest problems is the constant threat of a staphylococcus infection of epidemic proportions. In order to prevent outbreaks of slapltyloccocus infections, a routine was developed in many hospitals in the U.S. and elsewhere in the early lUbO's wltcrc infants were washed once daily with a 3% hcxaclilorophcnc detergent solution, and in addition tire diaper area was washed several limes a day. Tlte detergent was subsequently rinsed off more or less vigorously, depending upon the philosophy of the different nurses. In some hos pitals the detergent was directly applied to the infant, in others it was first diluted in water. Since Die ncurotoxic effect of hcxachlorophcne has become known, many hospitals have changed their policies on the use of hexachlorophene. Another drawback to this routine, in addition to a possible neurotoxic effect, is that hexachlorophene keeps not only the staphylococcus of certain phage types from growing on the skin, but reduces all Gram-positive bacteria,315 which is highly unde sirable and may lead to disease. Recently Evans et al.313 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 lire fall: the prevalence of Escherichia coii 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 that good general housekeeping practices are ultimately more effective in preventing bacterial disease in newborns than various germicides. Unfortunately, breaks in technic do occur which in many hospitals makes the use of germicides on infants desirable. The information reviewed in this article shows that the toxic effects of the polycyclic polychlor inated compounds are complex. The mere know ledge of an LDso value in an animal species docs not give us a great deal of information about the toxicity of a compound. Toxicity or toxic effects are a relative concept and such household ildms as table salt, vitamins, pepper, and allyI 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 these 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 not. 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 the most vulnerable. ACKNOWLEDGMENT My sincere thanks go to my colleagues and former co-workers who have made this review possible, particularly to Drs. R. E, Zehr, J. E. Suggs, V. W. Burse, R. E. Jennings, R. E. Linder, V. E. Sedlak, J. A. Goldstein, E. C. Villaneueva, and T. B. Gaines for letting me incorporate some of the unpublished results into this manuscript. REFERENCES t. Poland, A. P., Smith, D., Metier, G., and Pusaick, P., A health survey of workers in a 2,4-D and 2,4,S-T plant, Arch. Environ. Health, 22, 316, 1971. 2. Bleiberg, J., Wallen, M., Brodkin, R., and Applebium, T. L., Industrially acquired porphyria. Arch. Dermatol., 89, 793, 1964. 3. Larson, D. L., Studies show hexachlorophene causes burn syndrome,/ Am. Hosp. Attoc., 42,63, 1968. 4. Schmidt, II. and Schulz, G., liber DiplienyIbasen, Annalcit, 207, 338, I SB 1. 5. Penning, C. H., Physical characteristics and commercial possibilities of chlorinated diphenyl, Ind. Eng. Chem., .22, 1180, 1930. 6. Jenkins, R. 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