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THE JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY Volume 20 FEBRUARY, 1938 Number 2 MORPHOLOGICAL CHANGES IN THE LIVERS OF RATS RESULTING FROM EXPOSURE TO CERTAIN CHLORINATED HYDROCARBONS * Granville A. Bennett, Cecil K. Drinker, and Madeleine Field AVauren Department of Pathology, Harvard Medical School, and the Department of Physiology, Harvard School of Public Health, Boston, Massachusclts HLORINATED hydrocarbons, C particular^' chlorinated naph thalenes and chlorinated di to determine what, systemic effects, if any, would result from the adminis tration of a number of these com phenyl, have been used extensivpeolyunds to experimental animals. in certain industries. Their use in the The known findings in the three manufacture and preparation of many fatal cases of jaundice, together with types of electrical equipment is con a review of the pertinent literature stantly increasing. Although it is have already been reported (1). known that, some of these compounds The present paper describes the cause acne, only recently has the pathological changes observed in rats possibility of more serious systemic that bad been exposed to various effects been recognized. chlorinated naphthalene compounds During the spring of 1936 we were and to chlorinated diphenyl. informed of the occurrence of three fatal cases of jaundice in workmen using chlorinated naphthalenes and chlorinated diphenyl. At the request of the manufacturers of these com pounds, we undertook an invest igation * Received for publication September 5, 1037. _ Tibs is tlie second of three papers read at n symposium on chlorinated hydrocarbens piven at the Harvard .Sc hool of I'ublio, llcaHb, Poston, Jane 30, 1037. Tho third J'Apor will appear shortly. Materials and Methods A detailed description of the ap paratus and technic employed by the authors in this investigation has been published (1), White rats, main tained on a diet of Purina Dog Chow, supplemented by lettuce, eggs, milk, and cod liver oil, were used throughout the experiments. DSW 025553 'r,*>vT7T^vrr. NrT;' STLCOPCB4009508 98 JOURNAL OF INDUSTRIAL IIYGIFNF AND TOXICOLOGY M. SO, no. s The chlorinated hydrocarbons tested were (be following: Compound A. A mixture of Iri and tetracb1ornaphtb alenes.* Chlorine content 49.4 per cent. Compound B. A mixture of tetra- and pentuohlomaphtha- lencs. Chlorine content 66.4 per cent. Compound C. A mixture of tetra- and pentachlornapblba- lencs plus chlorinated diphenyl. Chlorine content 43.5 per cent.| Compound D. A mixture of penta- and bexachlornaphtha- lenes. Chlorine content 62.6 per cent. Compound E. A mixture of penta- and hexaehlornnphtba- ' lenes. Chlorine content 62.6 per cent. Compound F. A mixture of 90 per cent penta- and hexa- ehlornaphtkalenes, plus 10 per cent chlorinated diphenyl. Chlorine content 63 per cent. Compound G. Chlorinated di phenyl. Chlorine content 65.0 per cent. , These preparations were selected because of their relative, importance in industry. It should be noted that they also represent a. wide range of chlorination. All of the above materials were administered orally in varying doses. Compounds A, D, F, and G were selected for the inhalation experiments. * Tin's compound contained only small amounts of tetiacldormiplitiialencs. In this paper it will therefore he referred to ns trichiornaplitlinlenos. t This compound consisted of a mixture of compounds R and C! but in addition con tained two plasticizers which have been considered to he. inert.. Without these materials the chlorine content of this com pound would be between that of compounds R and D. Jnhalalinn. Experiments We were most interested in the results of the inhalation experiments because they more nearly simulate the type of exposure to which the workmen tiro subjected (1). The in halation experiments were carried out in air-tight; wooden compartments through which air, containing the volatilized compound being tested, was driven by electrical blowers. With this apparatus we were able to tost simultaneously the efleet of four compounds. Eighty rats were ex posed to each compound. The meth ods for determining the daily air concentration (mgms. per cu. m.) of the chlorinated hydrocarbon being used, os well as the rate of airflow have been described (1). Compound A (trichlornaphtlmlenes) was administered by inhalation to two groups of animals. In the first of these experiments, the rats were ex posed to low air concentrations (aver age 1.31 mgms. per cu. m.) 16 hours daily for 134 days. In the Bccond experiment the concentration was increased to an average of 10.97 mgms. per cu. m. This 16 hour daily exposure was continued for 102 days. Compound D (hexa- and pentachlornaphthalenes) was administered to three groups of rats. In the first experiment the exposure consisted of 16 hours daily for 134 days, with an average air concentration of J.Hi mgms. per cu. m. In a second experi ment the average air concentration was 1,44 mgms. per cu. m. This was maintained 8 hours daily for 143 days. In the final experiment, the average air concentration was in creased to 8.88 mgms. per cu. m. ami exposure, 16 hours daily, was con tinued over a period of 52 days. r\*7 nr' y>-*y ."T DSW 025554 STLCOPCB4009509 Y.i no. .r'.rlt h'. Ri tlio r_ 'iTTCThnentS riinullltc ^;.xh the The icarried out ::ir-' -i.-tmerits r.r.'i:.:rig the tested, -hi; hi over,5. e wire able to ; ifeet of four tv..-: were cx~ i. The meth;he daily air rer cu. in.) rxirl<.n being r-R.e o: airflow 'Tjm'mi.alencs) Tuition to two z. the first of : rvi-> were ex-`Tiiirris (averii. ;r..) lo hours lr. the second miration was rage of 10.97 " is 16 hour daily -I for 102 "days, arid pcntachlordmimsiered to . In the first itv consisted of 1 days, with an ation of 1.16 n second experi- conec-ntrfttion r cn, m. 'This mrs daily for 143 experiment, the trntion was in , ]xw cu. m. and daily, was conf 52 days. j * t i i [f t i \I ( f*r+ - n\Nr*v*-;i(rr'y /-,h, LW1 CHLORINATED HYDROCARBONS 99 Compound F (hexa- and penlafhloninphthalcnes SO per cent plus rid urinated diphenyl 10 per cent) was administered' in low concentrations lo two groups of rats. In the first experiment an average air concentra tion of 1.37 mgms. per cu. m. was maintained 1G hours daily for 134 (lavs. In the second experiment, an average air concentration of 1.06 logins, per cu. m. was inhaled 8 hours daily for 143 days. Compound G (chlorinated diphenyl) was aihniiii.sl.ered to two groups of journals in low concentrations. An average concentration of 0.57 mgms. per cu. nr. was employed 16 hours daily for 134 days in the first experi ment. In the second experiment (em ploying an average air concentration of 0.93 mgms. per cu. m.) the animals were exposed 8 hours daily for 143 days. Feeding experiments All compounds tested were employed in the feeding experiments. A weighed amount of finely ground material was mixed with rat food. This mixture was placed daily in one food container to supply the rats in ft given cage, Thus each animal had mi equal opportunity to ingest the compound supplied. As the number of rats per cage was reduced, the dosage supplied was proportionately decreased. Most compounds were '-applied in large and small amounts. Supplementary experiments were conducted on a smaller number of fats by feeding several of these com pounds in small known amounts by Momaeh tube or by injecting them subcutaneously as suspensions in gum in-aria. Carbon Ichachloridc and alcohol administration to experi mental animals Seemingly the incidence of acute yellow atrophy in workers exposed to chlorinated naphthalenes and chlo rinated diphenyl is very low. This single fnct is of some importance in Hint it suggests that certain indi viduals may be more susceptible to the compounds or that in these in stances the liver damage may have been intensified by some, other agent. With this in mind, groups of animals that had been exposed for varying periods of time to certain chlorinated naphthalene compounds and chlorin ated diphenyl were subsequently given a sublot.bal dose* of carbon tetrachloride mid ethyl alcohol by stomach tube (0.75 cc. of each per kgin.). This dosage did not result in a single death among the 1G control animals and the degree of liver damage produced was quite constant and never very great. Administration of carbon tetrachloride nd alcohol t.o rats exposed to the more highly chlorin ated naphthalene compounds and chlorinated diphenyl caused extensive liver damage and proved widely fatal. Therefore, the use of carbon tetra chloride and alcohol lias been employed regularly on representative groups of rats from each inhalation experiment. Examination of tissues Animals were sacrificed for patho logical examination after varying peri ods of exposure. Complete autopsies were performed. Liver weights were recorded. In representative animals * One cithic centimeter of carbon tetra chloride plus 1.0 co. ethyl alcohol per kilo gram when administered orally to normnl white rale finises a M per cent mortality (2). DSW 025555 STLCOPCB 100 JOURNAL OK INDUSTRIAL HYGIENE AND TOXICOLOGY [vol. SO, no. t from each experiment, all organs excepting the central nervous system were examined microscopically. The livers of all animals were studied microscopically. Tissues were fixed in Kenker's fluid, 10 per cent formalde hyde solution, and absolute alcohol. The routine paraffin sections were stained with cosin mctliyle.no blue. Other stains employed were hematoxy lin and eosin, phosphotungslic acid hematoxylin (Mallory), Foot's modifi cation of Bielschowsky's stain (reticu lum), Best's carmine stain, and Iron reaction with ferrocyanide of potas sium (Mallory). In addition, frozen sections from certain liver specimens were stained with scarlet, rod, Lugol's solution, and methyl violet. Results Compound A. Tnchlornaphthahncs (a) Exposure by inhalation to low concentrations (average 1.31 mgms. per cu. ni.) 16 hours daily.--The 80 rats subjected to these conditions showed no ill effects. Autopsies were per formed on groups of 3 to 8 animals after 37, 72, 105 and 131 day:' exposure. There were no significant abnormalities in liver weights.* Mticroscopically the majority' of the livers appeared normal although an occa sional one was paler than norma'. Rarely slight mottling was observed. Microscopically, tire liver cells often appeared slightly enlarged, more gran ular than normal, and occasionally they were vacuolated. These slight abnormalities and the presence of a rare mitotic figure suggested that very slight injury to tire liver cells had occurred. There was no demon strable increase in the above changes after the first exposure period (37 days). Occasional livers of rats ex posed for the total period showed * Calculations pertaining In the weight: of livers in these and .subsequent animal.; arc based on data in H. II. DonaldsonV book "The ltat," 2nd ed. The Wisti.i Institute, Philadelphia, 1921 (p. 211). PLATE I Fins. I ani> 2. Camera lucida drawings showing the portal areas of the livcia of two rats that were fed largo doses of a mixture of tetra- and pcnlnchlnrnaphIhnlcnos and chlorinated diphenyl. Tbo changes illustrated in figure 1 occurred within IS days aftrr exposure was begun. One should note the marked accumulations of hyaline globules in the coll cytoplasm, and the marked bm oiling of liver cells. In creased numbers of mitotic figures were also present. iSimilar changes are apparent in figure 2 which was made from the liver of a rat exposed 12a days. Degeneration in the centra) portions of the liver lobules is also present. EjCis. 3 ani> 4. These drawings illustrate the marked swelling ami tatty vacuolization of liver cells observed in all rats that were fed small doses of tetra- and pentnehlormiphtlmlencK. The changes shown in fig ure 3 were present after 20 days' exposure, those in figure. 4 after -IS (lays' exposure. Note (he mitotic figures in figure. 3. The compound responsible for the. changes con tained no chlorinated diphenyl. Otbenviso it was similar to the compound rcsponsibl' for the lesions illustrated in figures 1 and 2 I'Tcs. >r> and C. Camera lucida drawing' of the portal areas of the livers of two raf fed moderntc sized daily doses of a mixliin of penta- and hexaehlornaphthalenes for li and 29 days respectively. In the first 'I these figures one notes swelling of liver eel!' with narrowing and distortion of the sine soids. The liver cells show nn increas'd granularity of the cytoplasm wilh n massinr of basophilic granules near the nuclei There, is also an excess of fat in small ana medium sized vacuoles. This was num prominent in the central portions of th liver lobules. An increased spacing he tween fiver cells nod between liver reliaml sinusoidal endothelium is present After 29 days' exposure (fig. 0) the nbnv changes are greatly increased- Numcnne large intercellular spaces containing soreunrecipitalc, strands of fibrin and loucoryfi had developed between liver cells. Tinspaces were bounded by distorted fi',; cells. .See also plate HI, fig. 1. Camera lucida drawings X 395. . DSW 025556 STLCOPCB4009511 -'Y |rd. i'j, no. t .iilitiMio f! Viced :ikh-.-'. xvorc- j:er3 (o 3 aui.T.als -lid I3i days' p no gigniicant .t`if;!il.s.* Mac- :iiy of the iivrrs louitL ail f-cea- ILan iy. r:'.al. : \vi..= ob,-.>. m'ed. :ivcr eeKs efteu ted, more gran- od ooctisic :.a!!v i. TJjffP `light jijv.-f-ncc- of a .rested thai verv liver eclif bad s no de :;:on- above cl: aligCS .ire pcriix (37 era of rr.\ 5 ex- period si. fi'.vcd ..'i; to the w li'iit.. ser(UMit a: i'.nls 11. J)e>'..'d 'i: n 'h cd. Tlii- (star HIM (J). :in. yuiul rcsperdl'k' !i figure.' 1 aud 2. lucida dri'.vi:'.;:.-; livers of t'.vo rats '.ise* n,r :i Hiixuro ;)litlis!vi)f> for 14 : In tlir f.r>t iif .liiip o! livet cell? l-ioil Of 111.'.' Fit'lliw iiii iiicn'SFOiI n wiih r, run-.dm: car (lie i:,'i i*i. fat- i:. clan'.! and This was more jioi-i inns of tlit.0(1 Fjoioinc Inween Ijvci colls i'M is present. jr. (\) tlic nS>ovc sod. Numerous iiiilniiiinir frr.ms lnl lelirocvt.-s or cells. TIicfc distoj-u-d liver B- 1. X oM. -- -Viiij `.1/fc i ii. ,, , m I'b.vn: i v > As'?< v.f' '' - V \r ' ..." It AV"'''#' o'-to- '''"V!* '.r / t, . ' O <: '' i:- .;S ;<?H V'l'A,.-ivW-fec.1 yt/A 'ft ' jV.V M 9'M A' r- ' '. %:9..".5.,4. . : : V . . . -i.,-v/ (r '>f-r,. i-X. C ya . V? ,-^J - f> (fr'...ro-* S^>- '... t, y .(<> /S\ o of/'f"',!.-/i),: 1 ;A \ r \ , . i V5'-vA?-v ' >V' , -o' fHSo.lv y-*tM fy.. . *- X;'. : - -.>-vj,hy-..; -;y ,w -^vqH y. p ' y/-.. ^vj y.i- f.c Aim -t 9fe '* j[ `/. I . .V. v.t- --X,- PutffksW. WtP (LA .a;- v <-o.-'b) ./V ^r-v''v'iV?' vr '. > s * x. s t < h // VV e. .ilyy -1 ->.-' -. -i^'c: \y?^pJ 4-hfefy \r v>. -.' - 'P? '- 3 y .<?> ' 4".. /<>/. .. -.HHriHH--'- *'i-;y c'Vv-V-.y .-' <^ij b 'i v . *x > * i '' ' * ` *' i f--A ^ * !-'LvV`1HA,^1AA;-' t \.v' - .i,v; : -A . .Va ' .' ! . t (, ' ,, '..ir i.V------ l....... . iff ' . .*....L ..;> x ; -'h .'LL- -. .... , v: ,, ''y-: AV' );' :' . >-. I r . ... tJU* iJ ; .>v "vLi L- - vl;; ^'V^>v.v'^. K * *i ,' fy ' , / V Ir.-JA-Jinr ^ s'r4,.-'<4.i (wl DSW 025557  <0* I-'4`Wim w/<. V I' 102 JOURNAL or INDUSTRIAL JlYGUvNE AND TOXICOLOGY \vl. SO, m, t no definite microscopic change. (Sec contrast, to the. persistent hepatic fig. 3, plate II.) All other organs were lesions resulting from exposure to the normal.* compounds of higher chlorination. Further evidence that the liver l tissue injury had been very slight (b) Exposure by inhahlion to high was obt,(lined from the carbon tetra conamU'olion {10.1)7 mgms. per cm. m.) chloride and alcohol test. Of 10 rats 16 hours daily.--In this experiment, exposed for 144 days in the. above t-he concentration of trichloniaphlha- manner and then fed 0.75 ec. per lenes was approximately eight times kgm. each of carbon tetrachloride that previously employed. Exposure and ethyl alcohol by stomach tube, was continued for 102 days. The 50 none died. At. autopsy and on mi animals subjected to this concentra croscopic examination there was little tion appeared normal. Groups of 3 or no evidence of Jiver injury greater to 17 rats were sacrificed after expo than that produced by the same dose sure periods of 31, 48, 59, 72 and 102 of carbon tetrachloride and alcohol days. Most of the. livers were pale in normal control rats. (Sec plate IV, yellow in color and slightly mottled. figs. 1 and 2, and plate V, fig. 1.) There tvas, however, no significant The livers of 8 animals exposed to variation in liver weight. Micro trichlomnphtlmlencfi for 105 days and scopic examination revealed swollen autopsied after a 2 month recovery' liver cells with slightly increased period showed no definite pathological granularity and vacuolization of the change. This is further evidence that cytoplasm. Occasional degenerating this compound is only slightly toxic. and regenerating cells were observed. These findings represent a marked Those changes, which were slightly * In none of the experiments to follow were significant changes found in any organ other than the liver. more marked in the central portion of the liver lobules, were present after the first month of exposure. They PLATU II Fias. 1, 2, anti 3. Livpr changes result ing from lire administration of frichloinaphthnlencs arc illustrated in these camera lucidn drawings. In figures 1 and 2 me shown the most marked changes observed after prolonged (100 and J30 days respec tively) feeding in daily doses of 3 gm. per 10 rats. The changes observed consisted of slight, to moderate swelling of liver rolls, increased granularity of the cytoplasm and finally, after prolonged exposure, fatty vacuolization of the majority of cells and complete fatty degeneration' of occasional cells. Figure 3 is a drawing from a section of the liver of n rat exposed by inhalation to low concent rations of tricldoninphthnlenes for 100 days. No striking or constant changes were observed in rata so treated. Occasional celts are slightly swollen and show increased granularity of the cyto plasm, A rare mitotic figure was observed m occasional sections. Fins, -f, 6, ani> 0. Microscopic changes observed following the administration of n mixture of penta- and hcxaehlurnnphthnjenes are illustrated in these, three draw ings. Figure 4 shows the elianges ohserved after feeding this preparation in 3 gm. daily doses per 10 rats, for a period of 30 days. The liver cells are markedly swollen and vacuolated. There is also necrosis and degeneration of scattered cells. Large intercellular spaces are observed, (.See plate VI, figs. 1 and 2). Less marked hut similar changes are observed in (lie livers of vats fed smaller (loses of this preparation (see fig. 51. (2li days' exposure.) Figure ti illustrate* the liver cell changes that oc curred after inhalation of low concentra tions. of penta- and hexachlorimphthnlcnes for 75 days. Note the swelling of the liver cells, the inereared vacuolization and pres ence of large numbers of hyaline globules within cells. .Mitotic figures were fre quently observed. Camera lucidn drawings X 305. V' ft> * r V * \ ,i i ft l. t It I *i 6- Vk,1 - - ` 'r. * t ( "V u OSM 025558 STLCOPCB4009513 -*r -v* ^s^.* v/v$ P, ;&&<';<>..: (fife#,* 4 'Y^-' :;. * .- .' % \ ';J-?r\ iJ?. --^u <v'y>'v-- ' fic' v/-iUy,{|-fac.fa, \^,.>w? T.:/4 .? VVSv PVW/A$S*};>\\*4A *"?`>:J- f ;#'"' t* ..$,$ $ 7^V^4^#b j?yf.-^. .. . ^ .-. _< -i t- fc;-%'.<-3* 6rVV jW Y%?r ; ^ tvr;'^?./>0^/t'>.: '<<;?.>`-'f 5 -v.':-.T^/r>yi->V:j' {,*'&$*& -Z'fOtf$ OSH 025559 >,; 'yyy-_r7-''tr'fm>ii; . ; <fpf 11 ^ -**~. STLCOPCB4009514 -:<*UW**4MVMUEW'.nuii i^Mk*JW 104 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY (rol. SO, no. i were somewhat inercascd will) longer most marked of these (fig. 2, plate. Y), exposure. After 102 days the liver the central one-half or two-thirds of . t cells were more markedly swollen and each liver lobule was necrotic and in contained large and small fat vacuoles. occasional areas two or more adjacent t Small hyaline droplets in the. cell lobules had undergone degeneration. cytoplasm were occasionally observed. Hemorrhage and extensive leucocytic Mitotic figures, although never numer infiltration had occurred in those ous, were increased in number. No necrotic areas. The. leas damaged structural changes had occurred in liver cells at- the periphery of the the liver lobules. lobules showed large numbers of Carbon tetrachloride and ethyl al mitotic figures, indicating accelerated cohol were administered by stomach regenerative activity. tube, to 9 rats kept under these condi 1 tions. At autopsy their livers were (e) Exposure by feeding.--Nine of 1 slightly to moderately enlarged. They the 10 rats fed trichlornaphtbalenes in were yellow in color and the majority doses of 3 gm. per day per 10 rats of them were mottled. The. micro were sacrificed at varying periods of scopic findings were variable. In one time from 9 to 136 days. The tenth t animal the observed necrosis was no rat died of a respiratory infection after greater than that seen after the 182 days' exposure. There was no 1 administration of a similar dose, of significant abnormality in the. weights i 1 i11 carbon tetrachloride and alcohol to normal control rats. The livers of 4 animals showed increased fatty de of the livers of these animals. After 2 months' exposure, microscopic exam- . ination of the livers showed slight generation in the central portions of swelling of liver cells. Tins was the lobules with small necrotic foci. accompanied by increased vacuoliza In the 4 remaining animals extensive tion of the cytoplasm due to the 1 central necrosis had occurred. In the accumulation of abnormally largo PLATE III Fjor. 1, 2, and 3. Liver changes resulting and hyalinization were apparent (fig. from the administration, by feeding and 3). inhalation, of a mixture of pnntn- and Fios. 4, 6, and 6. The jnierosenpir hoxachloninphtlmleues (00%) and chlor changes resulting from exposure to chlor . i inated diphenyl (10%) arc illustrated in inated diphenyl are illustrated in these 1 these drawings. _ Figure 1 is u drawing figures. Figure 4 was made from the liver made from a section stained with P.T.A.H. of 11 ml fed large daily doses for only <> In addition to other changes it demon days. Marked swelling of cells and rapid strates the presence of large and small regenerative activity arc apparent. Figure intercellular spaces, interpreted ns dilated 5 illustrates the changes that were uni bile capillaries. These spaces contained formly produced by the feeding of small serous precipitate, fibrin, and occasional doses of this preparation. Note the ex loucocyles. Note the sharply outlined tremely numerous hyaline inclusions in boundaries of the. spaces urn! the maimer liver cells. This rat- was exposed for 29 in which those spaces ramify between cells. days. Very similar changes were produced This rat was exposed by the feeding of large by the administration of this compound by daily doses for 12 days. Similar but less inluilatioii methods (fig. 0). The rat from marked changes resulted from the feeding which Ibis drawing was made had hern of small doges over a period of 28 days exposed to Joiv concentrations for a period (see fig. 2). After 75 days' exposure by of 107 days. inhalation to low concentrations of tins Fio. 1. Camera lueida drawing Xl''* preparation, marked liver changes, con Fins. 2 to 0. Camera lueida drawing'' sisting of cellular swelling, vacuolization, X 395. OSW 025560 STLCOPCB4009515 hr. ****.'. K OMCOLOCV Ur,!. SO, no. i d of iLese- (Sc. 2, plate V), one-half or two-thirds of obule was necrotic and in m` :wo or more adjacent . urid-'rzorie degeneration. and extensive leucocytic had occurred in these eft.*. Tlio les* damaged at the periphery of the iwed large numbers of ires, indicating accelerated e acti'.ity. sure hr fo:dirtg.--Nine of fed iricblomaphthalenes in gm. pr day per 10 rats :ced at varying periods of II to 13d days. The tenth a respiratory infection after exposure. Thrre was no abnormality in the weights 's of these- animals. After xjKtS'.ire, microscopic examthe livers showed slight if liver cells. This was 'd by increased vacuolizaie cytoplasm due to the *on of abnormally large i ' : j ' ; , ' ' j j i j ; izftttca were apparent (Hr- 5. a.si> 6. The microscopic ilting from cxj'.'suro to clilornyl are illustrated in these ure t ey made from the liver i large ciaily doses for only 0 .:! ar.-.cjinjr of colls and rapid activity arc apparent. Figure s the ciiat'it.'s that were uniiured by the feeding of small is preparation. Note the exineroir* hyaline inclusions in This rat was exposed for 2!t similar chance* were produced mistration of thi compound l>y net hois tfig. 0). The rat from drawing. was made had been low foni'entratioris for a period .'amera lueida drawing X 95. TO 0. Camera lueida drawings i i ! ' , ; i i ! ' ; : 1 J i ;\Twynp-msycm i.1*- iw*,eiyT^ PLATE III DSW 025561 ifcw-iwy.CTw- rrrrfvr STLCOPCB4009516 100 JOURNAL or INDUSTRIAL ItYGIKNE AND TOXICOLOGY M. HO, n. 2 amounts of fat. These; changes in creased slightly with longer exposure. (Sec plate II, figs. J and 2.) There were no architectural changes in the liver. Because the effects resulting from large doses of frichlornuphthalcnes were slight, this compound was not fed in small amounts. Compound B. Tetra- and pentachlornaphthalenes wore present in increased numbers, indicating accelerated regenerative ac tivity. The microscopic changes re sulting from the feeding of tetra- and pcntaelilornaphthalcne.s are illustrated in plate I, figures 3 and 4. They arc those of diffuse fatly infiltration and fatty degeneration. Compound C. Tetra* and Pe.ntachhrnaphthalcncs plus Chlorinated Diphenyl Exposure by feeding.--This prepara tion was employed only in feeding experiments. The daily food ration for 10 rats contained 0.5 gm. of this compound. All animals fell ill and cither died or were sacrificed by the sixty-third day. The livers were pale yellow in color, friable, and occasionally showed mot tling. There were no significant varia tions in weights. On microscopic examination the same changes were observed in all rats. These consisted of moderate to marked swelling and rounding of the liver cells. The sinusoids were markedly narrowed. The. majority of the liver cells con tained large numbers of small fat vacuoles. This finding was most, marked and first observed in the central portions of the lobules. In tbc least altered cells there was promi nent massing of basophilic granules near the nuclei. Occasional necrotic cells were observed. Mitotic figures Exposure by feeding.--Feeding (3 gm. doses daily per 10 ruts) was continued with but one 4 day inter ruption for 130 days. Ten rats were so treated. Seven rats were sacrificed at varying periods of 47 to 124 days. Three rats died after exposure periods of 122 to 130 days. In all animals the livers were enlarged (33 to 00 per cent). The average weight increase was 71 per cent. They were also friable, pale yellow in color, and somewhat mottled. On microscopic, examination, const aid changes were observed. Practically every liver cell was swollen and rounded. Their cytoplasm contained large numbers of hyaline bodies. These were circular or oval in shape and varied in size from about half the size of a rod blood corpuscle to twice the size of the nucleus of a liver cell. (See figs. I and 2, plate 1.) In numer ous instances, many small hyaline bodies lmd fused, forming large circular masses as large or larger than a normal PLATI2 IV Fio. 1. Photomicrograph X -15 illustrat ing the extent- of the liver damage produced in IS hours hy 0.75 er. per kgtn. each of earhon tetruehloride and ethyl nleohol when administered hy stomach tube to a normal control rat. The. degenerative changes were limited to the central portion of the lobules. Pin. 2. Photomicrograph X 220 of n section of liver tissue front a rat exposed hy inhalation methods to trichlnrnaphthuh'iics in low concentration for 150 days. Follow ing this exposure 0.75 ee. per kgm. each "f carbon tetrachloride and ethyl nleohol was administered hy stomach tube. Tin' animal survived and was sacrificed 17 days later. No significant liver changes arc demonstrable. DSW 025562 STLCOPCB4009517 la&mi*, lioT. tO, no. *(l number#, [iterative nrchanges ro of lclrn- and iir illustrated 4. They are filtration and 1 Pentachlorlorinoted --Feeding (3 , 10 rot#) was 4 day inter Ton rat# were were sacrificed 7 to 124 days, tposure periods all animals the to 90 per cent), nerense was 71 riso friable, pale aewbat mottled, nation, constant ed, Practically tp swollen and iplasm contained hyaline bodies, or oval in shapean about half the orpusch* to twiee .ms of a liver cell, tte I.) In nttmerny small hyaline . wing large eirntlar ,1'ger than a normal PLATE IV ;\ l. - ' B i X*i^\ ,'V''-''f- `f.'J tv.v-*".v. >. f, < ! ' .. it ;.,A*f 'J--i r %'' .-* ,.'-s *;** /-j.v--i: ' j * *7,# . ' '!{. . ri ,. . . ./ ;.vv' .\ . : > Fin. 1 from n rat I'xpnscct l>y ,t Irii'lilonmplitli.'ileni'S for I.W days, l-'otlmv5 cr. per Icgm. each of and ethyl alcohol was stomach tuho. The was anerifired 17 days nt liver chanties are l I OSM 025563 STLCOPCB4009518  .. "**--V.. -n frfi-, -y, f y i nrfif T,yi,` w | r > t Ml>r I ir*tl If * 108 JOFKXAL OF INDUSTRIAL HV01KNK AND T0X1C01.0GV |t{. 20, f. J , liver coll. These bodies slid tied bril tion of these bodies was iiieotHni,: liantly with rosin dye. They were The larger vacuolated hyaline often laminated and occasionally con contained Rinall droplets of fat w|,,! tained small clear fat vacuoles in the majority of the smullcr tlmplii. their central portions. Mitotic figures did not stain red. Although *inii!,, in liver cells were sufficiently numerous hyaline droplets were observed j. to indicate an increased rate of re livers of rats exposed to various ehlm,. generation (fig. 1, plate I). In the nated naphthalenes, this lype livers of 2 ruts exposed for 12-1 and 125 degeneration occurred much eailii. days respectively, there were, in and to a much more marked dew., addition to the above changes, large in those rats that were exposed j. areas of complete liver cell degenera preparations containing chloririsic.j tion (fig. 2, plate I). For the most, diphenyl (figs. 1 and 2, plate 1) or i.. part this was limited to the central chlorinated diphenyl alone (tigs, 1, half or third of the liver lobules. and 6, plate III). Occasionally, however, there was com plete degeneration of all liver cells except for a narrow zone of colls around Compound D. Penta- and hcxarhlw . naphthalenes t the portal areas. In such livers there . (a) Exposure by inhalation to was a heavy polymorphonuclear and concentrations (average 1.16 mgnis. y mononuclear inflammatory cell infil cu. m.) 16 hours daily.--Eighty ran tration in the necrotic areas where living under these conditions appenm! liver cells were being removed. normal throughout the exposure peri-: The most conspicuous feature of the (134 days). Representative animal microscopic changes in the livers of were sacrificed in groups of 3 to I', these rats was the presence of large animals after varying exjrosure period : numbers of circular hyaline droplets as described in the experiments cn in the cytoplasm of the liver cells. ploying trichloriinplithalencs. Then- This material did not stain in a manner were no significant alterations in liv i characteristic of amyloid and its weights. Macroseopieally the major staining properties were not. like those ity of the livers were light, yellow i: of the hyaline observed in alcoholic color and slightly mottled. .Wi cirrhosis. In frozen sections stained the initial exposure period of 37 day with Scharlaeh It, the staining reac there was evidence of slight injury ' PLATE V Flc,. 1. A photomicrograph X 120 show ing small areas of necrosis in central areas of the liver lobules. This rat bad been exposed by inhalation to low eoneen(rations of triclilornaplitlndenes for ldl days follow ing which carbon tetrachloride and ethyl nleohol were administered by stomach tube in doses of 0.75 cc. each per kiriu. The rat was sacrificed 5 days later, The extent of necrosis is similar to that resulting from carbon tetrachloride and alcohol atone. Firs. 2. In this photomicrograph X -15 are shown the most marked liver changes that were observed following the sdniifi:- trntion of carbon tetrachloride and ah "I (0.75 cc. of each per kgm.) to rats which t been exposed try inhalation methods to hh concentrations of trichlornnpliibnlvic' This rat bad been exposed t.o tricldnaphthalenes for 1 month. Very cxlemi' necrosis was present in the central ureacurb liver Inhute. lu occasional areas t major portion of several adjacent Mm1' were necrotic. This rat was nutopsh-d *' hours offer the administration of i-.id' tetrachloride. DSU 025564 STLCOPCB4009519 MMMWtttwfcu-. JOK&&IMiX* **':'* v- C50J.00Y t>, no. tidies was inconstant, uolatod hyaline bodies 1 droplets of fat while if the smaller droplets ed. Although similar Ip were observed in posed to various chloriilenos, this type of leenrred murh earlier more marked degree that were exposed to ontnining chlorinated ] and 2, plate I) or to 'henyl alone (figs. 4, 5, 1).- Penta- and kcxachloriphthalcnes by inhalation to low iaverage 1.J6 mgins. pa irs (faiVy.--Kighty ruts ese ronditions appeared iut the exposure period 'lepresentative animals i in groups of 3 to J5 arying exposure periods u the experiments emornaphthaleues. There cant alterations in liver 'roseopicnily the major 's wore light yellow in .ightly mottled. After iKstti'e period of 37 days .fence of slight injury to ved following the ml minis 'll tetrachloride amt alcohol jterkem.) to rats which had ill tin In t ion met hods to high of ttirhlorimphthalenes. sieen exposed to trichlor<r ] month. Very extensive sent in the central areas of c. In occasional arena the if several adjacent- lobules Tin* rat ivii nutopsied -18 administration of carbon *-.v. 'fp~- t^OT]Mn PLATE V DSU 025565 mwer'r^rfi;rr.,`ii>u>t r fmiiwart*-1 7w.; STLCOPCB4009520 ia,v. ,`A^ t v4^> ' '}*,*A^v*+.`yliiyf-tsOjl^i^L /1.1 U..<JA*.'ijlVn)`.'<i4.,t'iifc 4^i M 1 * - r iff' *T i ^irT*-' I- 110 .10UJ5NAI, or IXIH'KTlilAI- HYGIliXK AND TOXJCXILOGY [col. to, no. S live." cells whioli appeared swollen and figs. 3 and 2, pinto VII). The centra) more granular than normal. The one-half or two-thirds of each liver cytoplasm of occasional cells con lobule showed complete liver cell < tained small acidophilic hyaline drop necrosis and degeneration, Diffuse lets and there was a moderate excess extravasation of erythrocytes had of fat in the form of tiny vacuoles occurred and numerous polymorpho (fig. 6, plate II). All microscopic nuclear and mononuclear leucocytes abnormalities increased slightly during had invaded the necrotic areas. In the second period (37 to 72 days) but the narrow periportal zones where the no significant advance was detected liver cells were less markedly damaged, between the. 72nd and 134th day of there were large numbers of mitotic exposure. figures (fig, 2, pinto VII). The livers of rats exposed for 105 dajrs and then removed from exposure (b) Exposure by inhalation to low for a period of 2 months still showed concentrations (average. mgms per cellular changes similar to those ob cu. m.) S hours daily.--Animals were served at the beginning of the recov subjected to these conditions for 143 ery period. In a few of the specimens days. Groups of animals sacrificed they appeared slightly less marked, after 42, 77, 98, 119, and 143 days' indicating that some repair had taken exposure showed liver changes that place. In no instance was there were essentially like those seen in evidence of increased damage. rats exposed 16 hours daily. The Carbon tetrachloride and ethyl alco carbon tetrachloride, and alcohol test- hol administered to rats exposed in was uniformly fatal and produced I the above manner for 144 days proved liver changes that were identical to highly fatal, 9 out of 10 rats dying those, already described and illustrated within the first- 6 days---6 within 72 (figs. 1 and 2, plate VII). hours. At autopsy the livers of these rats were yellow and mottled and (c) Exposure by inhalation to high they were increased in weight (16 to concentrations (average S.S8 mgms. per 44 per cent). Microscopic examina cu, m.) ]G hours daily.--Pcnta- and tion showed widespread central necro hexnohlornnphtlmlcnos when adminis sis similar to that observed in early tered in this concentration proved acute yellow atrophy of man (see highly toxic. Eighty animals were PLATK VI I'm. I. Photomicrograph X 220 of tiic live r of 11 i-nt exposed to penta- and hexnchtonmphthalcnoz by the feeding of large dnilv doses for 10 days. The liver cells me swollen und markedly vacuolated, There is distortion of (he liver eetl columns. In frozen M et ion preparat ions stained with Seharlaeh U. there was evidence of marked fatty degeneration. In addition to these changes, numerous oval, round and irregu lar intercellular spaces are present through out the liver lobules. >Sueli spaces contain n granular serous precipitate, strands of fibrin and occasional leucocytes. Those spaces, insofar ns could he. determined, were due to Hilntation of the bile capillaries and eannlieuli. Fin. 2. This photomicrograph X 215 when compared with the above figure dem onstrates the similarity of the liver changes produced by the same compound admin istered in u different manner. In this instance the rut iuhulcd high concentrations of pentn- and hcxaehlornaphtlmlciics for U1* days. DSW 025566 STLCOPCB4009521 OG'i ,c->^. /'), no, VIlj. The central ri<Is of ^tf.li liver :pl'-V' liver eel! wruvi'.u. DifTu'e erylLr<'/-ytes had rous {tf/lyrnorpholUeK-.r kueomes erroli'- areas. In :1 v.'heie the ifivhf-iy damaged, mU-rs of mitotic II;. inhcbdion to low 'C I..Vt per .'.--Animals were omlitions for 143 nimaU sacrificed and 143 day.-' or changes that (hose seen iii urs daily. The and alcohol test fuid produced |-civ identical to d find illustrated ill). halnU-.n to high 8.SS mjms. per iy.--Penta- and s when adminisitrntion proved animals were tteocyres, Then* I be determined, lie bile capillaries mtpraph X 215 bnvr figure dnntho liver chances (impound adminDfliitier. In this :li r.ineetitratiotis pht hateties for 3$ PLATE VI r- .V " -v ' ' ' .*v'.; ' ''rv V; . ,, ? > ,f * s .* ..,, . r' ~vV-'-*` ' * . V-`"-s' . V.< i & *: '' . .V' .,1.: I r' . f* 4*VNi' * .. . ef ' - v * ;/,* ' V * .<; ^ ' ; JI?, -w. ' : - T. . -c * . t >.. vi-i( -4 ; . ' I . -'it. .. -*r . .-.-J " ,4 '' ' J ' \ - ..o' ^ . 'V'-vvV' . i-\d"v."-*t% .' '; ,xi/;v(>'''j';**iy. ,1 ( . 'I3 Fia. 1 y,, f ':\..vV:v rr.'*h. ',! t- -* 'j'v f V : tV.: -A >.r *r*7ww- . v-T/ -4v'- :V/^ v; w "i vVjS#^,0;i T V.V- ' |r . ** ... tTe . y' r^.'-'i- f\v>; `V^K. .*>.; V, . t' ^1-r-.,vr ... ' r%* L- ^ V*'- n . . ` *'.' .- A -'InAi'.. -v ' \ / 'A .<' r '^(r ' ;;/ - ^,i`` Hk W. l,^fV ,*f<11 syuiV.,,,. DSW 025567 STLCOPCB4009522 ; '1 112 JOURNAL OF INDUSTRIAL JTYC.1KKK AND TOXICOLOGY \ol. go, ? used, all of which lost, weight and lints removed from exposure hr. appetite. Four died by the end of the tween the third and fifth week con first month, 55 within 52 days. Most tinued to die, the longest survival time .4 of these were markedly jaundiced. .being 35 days. Microscopic examina The remainder were sacrificed for tion of the livers of these animal- pathological examination or suc revealed no evidence of recovery. cumbed to the effects of carbon Administration of carbon t.etr. < tetrachloride and alcohol. Only 8 chloride and alcohol again proved < animals survived the 52 day exposure. fatal to animals exposed in the above { The majority of livers woe moder manner. The livers of such animal- i< ately or markedly enlarged. They showed the characteristic massive were all yellow and in addition the ma necrosis previously described and i!lu>- jority were markedly mottled. Occa truted. i sionally they' were granular and their cut surfaces showed minute spaces (d) Exposure by feeding.--When fed < which became more prominent after in doses of 3 gm. daily to a group of 10 fixation. After an 8 day exposure, rats, penta- and hexachlornaphtha- t the liver cells were markedly swollen lencs produced marked cellular and l and moderate fatty degeneration was structural changes in the liver in a i present. This was most marked in short period of time. All rats lost the. central areas nlthough all portions weight and appeared ill from the of the liver lobules were affected. beginning. The longest survival time The extent of liver injury rapidly was 33 days. Although the liver iIi increased so that after 30 days' expo edges were blunt and rounded, then* sure, marked structural as well as were no significant weight varia i cellular changes were apparent. The tions. The livers were friable, yellow < l microscopic appearances were identi and mottled. Microscopically they i cal to those to be described in rats sliowed marked swelling and vacuoliza I j ! fed large doses of penta- and hexa- tion of the cells. There was also chlornnphthnlenrs (figs. 1 and 2, plate complete degeneration of scattered i VI) and in rats fed similar doses of 90 cells. Occasional mitotic figures were per cent penta- and hexachlornaphthn- observed (sec fig. 4, plate II). Suit lenes plus 10 per cent chlorinated able stains revealed very marked fatty i diphenyl (see fig. 1, plate III and degeneration. Occasional cells con figs. I and 2, plate VIII). tained oval shaped or circular acid- PLATK VII Fio. 1. A low power photomicrograph X 45 illustrating the marked degree of degeneration of liver tissue resulting from the administration of n email dose of curhon tetrachloride mid ethyl alcohol (0.75 ee. of each per kpm.) to nn animal that- had been exposed previously by inhalation methods to low eoneentrutions of penta- and liexaoldonmpluhnlenes for 131 days. This animal died within 24 hours after the admin istration of earbon tetrachloride. Similar degrees of necrosis resulted front the administration of carbon tetrachloride and alcohol to rats which had been ex|>s>'il to a mixture of <K>% penta- and hexiichh'i- naplitlmlcnes and 10% chlorinated diphenyl or to refined chlorinated diphenyl. Fig. 2. A higher power photomicrograph X 225 of the liver illustrated in the above figure. \ DSW 025568 j STLCOPCB4009523 u `GY frol. MOt w>, $ n exposure beI fifth week con gest .survival time 'roscupic cxtuniimof these animals - of recovery, of carbon tetratol again proved "*osed in the above rs of Mich animals . victerisiie massive (described and iilus- /m/i'iip.---When fed aily to a group of JO hexnohlornaphtha- iflrked cellulnr and Is in the liver in a lime. All rats lost eared ill from the liongost survival time Although the liver and rounded, there cant- weight varin|s were friable, yellow ;Aficroseoj)ienlly they welling nnd vncuoliaa))s, There was also lerniion of scattered 'a! mitotic figures wore g. 4, plate II). Suitaied very marked fatty Occasional cells consped or rireulur ncid- I i l l r i i errosis resulted from ciirbim tetriu'lilorido job Imd been expired n-ntn- nnd hexncblordilorliinted diphenyl i?d diphenyl. iver pluilomicro^rnp1 uitrnled in the l,l,v,! i j i K-,, PLATE VII Fig. 2 ... DSW 025569 STLCOPCB4009524 it***"*" < t. i i i 1 \ t t 1 i I . <JI1 .) I i( I< '*' *-~-': O'-* <*-- *1/-`'rhrVe, 114 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY \vol. tO, no. i ophilic hyaline inelusion.s. In addition to these cyfological changes there were numerous varying sized intercellular spaces throughout the livers (sec figs. 1 and 2, plate. VI and fig. 1, plate III). These spaces were usually circular or oval in shape, although many of them ramified between cells in the liver columns or between liver cells and the sinusoidal endothelium in such a way as to suggest that they represented greatly diluted bile capil laries or eanaliculi (fig. 1, plate III). They contained serous precipitate, a few strands of fibrin, and occasionally a few leucocytes. Rarely one ob served one or two degenerating liver cells within these spaces. In some of the more markedly damaged livers, red blood corpuscles were also present. The above microscopic changes ap peared identical to those observed in the livers of rats subjected to high concentrations of this compound by inhalation methods (figs. 1 and 2, plate VI). They were also similar to the changes that resulted from the feeding of another penta- and hoxachlornaphthalcne compound (figs. 5 and 6, plate I) and from the feeding . of a mixture of penta- and hexachlornaphthnlene (90 per cent) and chlori nated diphenyl (10 per cent). When this preparation of penta- and hcxftohlornaphthalones was fed in smaller amounts (0.5 gm. every second day to a group of 4 vats) the above described liver changes occurred less rapidly and to a lesser degree (see fig. 5, plate II). Compound 15. Penta- awl Hcxachhrnaphthalenes Exposure by feeding.--Feeding in doses of 1 gm. daily for 10 rats was continued with but one 4 day inter ruption. AH 10 rats had died or had been sacrificed by the fifty-fifth day, These livers were friable and yellowish, and exhibited moderate to marked mottling. One animal was jaundiced. Of the 5 animals that were sacrificed, three showed enlargement of the liver (30 to 40 per cent); the remaining livers were normal in size. On micro scopic examination the livers of rats exposed for 2 necks showed marked cellular changes that appeared to he degenerative in nature. Although the liver cell injury was somewhat greater in the central portions of the liver lobules, no portion of the liver was spared. The liver cells showed vary ing degrees of swelling, increased granularity and vacuolization of the cytoplasm. Early injury was indi cated by a massing of basophilic granules and small rod-shaped struc tures near the. cell nuclei. The pe ripheral portions of such celts were acidophilic and vacuolated. Small irregular spaces wore present between PLATE VIII Flo. 1. A photomicrograph X 05 of the liver of n ml fed a mixture of 00% pen laand hcxneldoninphthalones nnd 10% chlori nated diphenyl, in 3 gm. doses |>cr 10 nits for 12 days. Tills rut survived for 23 days after feeding was stopped. A eompariBon of the liver of this rat with those of rats sacrificed earlier indicated that the liver changes had progressed considerably after administra tion of the.compound had been terminated. One should note the marked change in the liver architecture. The spares like those previously illustrated (tigs. 1 and 2, plate VI) are very large nnd the,remaining livet tissue is compressed. The liver cells show marked fatty degeneration. Fin. 2. A higher power photomicrograph X 235 from another area in the liver illus trated in the above figure. DSW 025570 STLCOPCB4009525 'X ICOhOGV M. to, M. g mp of 4 rats) the above, or change.* occurred less to a le.-sor drgroo (see I). \ Pntla- and Heracldornajddhaktm by feeding.--Feeding in ni. daily for 10 rats was tli but one 4 dny inter- 10 rats had died or had d by the fifty-fifth day. *vere friable and yellowish, d moderate to marked |no animal was jaundiced, .nals that were sacrificed, d enlargement of the t per cent); the remaining rirnial in size. On micro nation the livers of rats 2 week? showed marked ges that appeared to be in nature. Although the !:ry was somewhat greater al portions of the liver Portion of the liver was liver cells showed vnrvof swelling, increased aid vacuolization of the Early injury was indimassing of basophilic small rod-shaped strueue cell mielci. Tlie peions of such cells wore and vacuolated. Small 'es were present between w the marked change in the are. Thr spaces like those United (figs. 1 and 2, plate <rge mat the remaining liver eiiged. The liv`r cells show cRciicrution. dier power photomicrograph ithcr area in the liver illusnve figure. > V-'rt i ttf'iitLft.i jb* vji>'-y PLATE VIII - M**- ,Mi> Fig. 1 ? S ..J r ' Tr'rv- '/''t.v.c:- :> *..* . Me. \> W, . *. 'ix.s . v-- > W Jf V* "`V'-' ' . ;3 w . n.i < 2^:.^, f W .** ** S'- , -- '.-h- 'iWl.t 0 f ' V SVt.SV J 4. '.v * . * V* ` / . - Flo. 2 DSW 025571 STLCOPCB4009526 %-`Jjjriit-'j+*A.r<ii a'lltff X'StihU*lfKbOMtfi*T-' -- ',-' t IK) JOUHXAL OF INDFSTItlAL HYGIEXK AXK TOXICOLOGY M. go, ,,,,. ^ liver cells and between the columns of liver cells and the sinusoidal endo thelium (see fig. 5, plate I), These were interpreted to be the early stages in the formation of large intercellular spaces (fig. 6, plate I) which subse quently developed and which were identical to the spaces described in the foregoing section. After longer exposure tlx: liver eells allowed very marked swelling and fatty infiltration. Occasional degenerating and regener ating cells were observed. Compound F, 90 per cent Pcnta- and Hcxachlornaphthalenes and 10 per cent Chlorinated Diphenyl (a) Exposure by inhalation.--Inha lation experiments were carried out on two groups of rats. The first of these groups of 80 animals were exposed to low concentrations (average 1.37 mgms. per cu. m.) 16 hours daily for 134. days. The second group, also comprised of 80 rats, was exposed to low concentrations (average 1.06 mgms. per cu. m.) 8 hours daily for 143 days. No evidence of illness was noted in the living animals in cither group. In each experiment represent ative groups of nnimals were sacrificed after exposure periods corresponding to those used in low concentration inhalation experiments with trichlornaphthnlenes and pcnta- and hexaclilornnplithalenos. Since there were no recognizable differences in the amount or type of liver change observed after similar exposure periods in these two groups of rats, the second group (exposed 8 hours daily) will not be discussed. Maeroscopicnlly the livers wore usually light yellow. After pro longed exposure, this abnormality was more marked and in many instances mottling was present. There, were no very significant alterations in i),,. weights of livers although the major ity were slightly swollen (average of 20 per cent increase in weight). Microscopically the livers showed constant changes after 37 days {if exposure. These increased somewhat during the second period of 35 days. Longer exposure produced little if anv increase in liver damage. The ob served pathological changes consisted of swelling and rounding of liver cells accompanied by a definite, increase in the prominence of the cytoplasmic granules. Hyaline droplets in the altered cytoplasm were a conspicuous feature (see fig. 3, plate III). Mitotic figures were present in abnormally large numbers. In an occasional liver, as was the case with similar exposure to both pcnta- and hoxaehlomaphthalenes alone and refined chlorinated diphenyl, there was a marked deposit of finely divided granular and re fract ilc yellow or yellow brown pig ment. This was present within liver and Kupffer cells. Larger amounts were found in the central portions of the liver lobules. It failed to stain in a manner characteristic of either hemosiderin or hemofusdn. The livers of rats removed from exposure for 2 months after an expo sure period of 105 days were similar to those of animals sacrificed at the end of 105 flays' exposure. There was, however, no evidence of any increase in the pathological changes. The administration of carbon tetra chloride and alcohol was almost uni formly fatal. Macroscopic examina tion of livers of these rats revealed marked enlargement (approximately osw 025572 STLCOPCB4009527 moav ivoi. so, * od in many instance* 'Kent. There were no : alterations in t he although the majory swollen (average of increase in weight). the livers showed os after 37 days of <e increased somewhat d period of 35 days. ` produced little if any ir damage. The obiwd changes consisted rounding of liver cells a definite increase in of the cytoplasmic ,ine droplets in the m were a conspicuous 3, plate III). Mitotic ivseut in abnormally In an occasional liver, with similar exposure md hexachiornaphthnd refined chlorinated was a marked deposit ed granular and reor yellow brown pig-' j present within liver `Us. Larger amounts he central portions of *s. It failed to stain iiaraeteristic of cither ucmofuscin. f rats removed from months after an expo105 days were similar inals sacrificed at the jys' exposure. There no evidence of any pnthological changes, ration of carbon tetracoliol was almost uniMneroseopie examinnif these rats revealed ament (approximately f ft . IPW) CHLORINATED HYDROCARBONS 117 j.-! r< r ecnl). Such livers were yellow ^4 mottled. Again there was extenB-,"f Hml widespread liver necrosis on tr.i-: loscopic examination (see figs. 1 tr .l 2, plate VII). (b) Fxjmurc by feeding.--When fed H hi>re doses (3 gm. daily for 10 rats) Icompound proved to be very p.nir. All animals appeared ill and fading of the compound was stopped idifi Vi days. Despite this the rats continued to die, the last dying on the ,tMh day. Subsequent microscopic examination revealed that the liver lunges had continued to progress idler feeding was stopped. MacroKopicitlly, the majority of the livers were enlarged, the largest showing an increase in weight of 118 per cent; the average increase was approxi mately *10 per cent. All livers were yellow, friable, and many of them were markedly mottled. Subsequent micro scopic examination indicated that much of the mottling was duo to hemorrhage into spaces within the liver tissue. In addition, the external and cut surfaces of the livers appeared lightly pit ted or granular. The micro scopic changes observed in the livers (I the animals autopned after 10 to M days' exposure consisted of moder ate to marked swelling of liver cells accompanied by marked fatty vacuvliruiion. Occasional liver cells Kbnxvcd additional signs of injury such w nuclear degeneration and poly morphonuclear leucocytic invasion, b uttered circular or oval shaped interrcllular spaces (fig. 1, plate III) like tlnwe previously described in experi ments where penla- and hexachlornr.phthulcncs were employed (figs. 1 *nd 2, plate VI) were observed. In the less markedly damaged livers, such spaces contained serous precipi tate, strands of fibrin, and small numbers of leucocytes. In rats sur viving 18 to 35 days, these spaces were much larger and often had attained the size of a normal liver lobule (see figs. 1 and 2, plate VIII). In these more severely damaged specimens, extensive hemorrhage had occurred (fig. 2, plate VIII). In sections stained with P.T.A.H. these spaces were seen to be bounded by flattened liver cells which showed sharp cell borders. Liver cells between such spaces were distorted, swollen, and showed marked fatty degeneration. In many of these livers there was a slight increase in connective tissue between the remaining cords of liver cells and around rccognizablo portal areas. Proliferative changes were occasionally observed in the bile ducts. This was indicated by increased num bers of ducts in certain areas and by mitotic figures in the bile duct epi thelial cells. No recognizable dilata tion of medium sized or large bile ducts was present. In many sections the architecture of the liver was so completely altered that it was difficult to recognize the portal areas, In all sections there was evidence of fatty and hyaline degeneration of the re maining liver cells. Slight polymor phonuclear leucocytic infiltration was present. This was somewhat more marked in the periportal areas. Feeding of this mixture of pentaand hexachlornaphtlialcnes and chlo rinated diphenyl in smaller doses (0.5 gm. every second day for 4 rats) resulted in liver damage that devel oped loss rapidly. Tho rats were sacrificed after exposure periods of trp itjmw *jmm r DSW 025573 STLCOPCB4009528 A'.wto-Mii'iA.'ifc .ivifcHt'n i >';,,.i'.iili.if.'ii^-.,>.io,.-.1 . y y,-iy , , u. 118 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY \vol. HO, no. g 21, 34, 58, and 81 days. Their livers were yellow, increased in size and showed slight mottling. Microscopi cally, the chief abnormality observed was marked fatty vacuolization of liver cells. No intercellular spaces like those described above were present. (. Coinj/ound G. Chlorinated Diphenyl (a) Exposure by inhalation.--Expo sure by inhalation methods to low concentrations of chlorinated diphenyl was carried out on two groups of animals, each comprised of 80 rats, in a manner identical to that de scribed in the foregoing sections. In the first group a 16 hour daily expo sure, with an average concentration of 0.57 mgms. per cu. m., was employed. The second group of animals was exposed 8 hours daily to an average concentration of 0.93 mgms. per cu. m. Neither group of rats appeared ill. Representative animals were sacrificed in groups of 3 to 21 rats, after varying periods of exposure. In the first experiment, the animals were sacri ficed after 37, 72, 105, and 134 days of exposure, in the second experiment after 42, 77, 98, 119, and 143 days. The livers were pale or slightly yellow and somewhat mottled. There were no constant variations in the weights of the livers. The microscopic find ings in the two groups indicated that the different exposures had resulted in the. same degree of liver injury. The pigmentation of the liver and Kupfler cells was somewhat greater in animals exposed 8 hours daily. In both experimental groups the carbon tetrachloride and alcohol test was highly fatal and led to the same exten sive central necrosis of liver tissue that was observed in rats exposed to the toxic' chlorinated naphthalene compounds (figs. 1 and 2, plate VII). The microscopic changes resulting from exposure to low concentration of chlorinated diphenyl alone were simi lar to those resulting from inhalation of ponta- and hexachlomaphthalcnes, plus 10 per cent chlorinated diphenyl. The most conspicuous change was hyaline degeneration (fig. 6, plate III) although swelling of liver cells, in creased prominence of cytoplasmic granules and increased vacuolization were present. Mitotic figures were present in increased numbers. No microscopic evidence of recovery was found in the livers of rats exposed for 105 days and then removed from exposure for a period of 2 months. (b) Exposure by feeding.--When fed in large daily doses (3 gm. for groups of 10 rats), chlorinated diphenyl proved highly toxic. Feeding was discontinued after 6 days. Seven rats died within the first 8 days. The 3 remaining rats gained in weight after the feeding was stopped and were finally sacrificed. The last animal was sacrificed 03 days after feeding was begun. The majority of livers showed a moderate increase in weight. Micro scopically there was evidence that liver injury had occurred within the first few days. The cells were swollen, at times sufficiently to obscure the sinu soids. The cytoplasm of the liver cells was acidophilic in staining quality and contained small vacuoles. Small eosin-stniiied hyaline granules and globules were present. Mitotic figures were numerous (sec fig. 4, plate III). There was little inflammatory cell infiltration and no structural changes were observed. DSW 025574 7 ' <4 t III iw r ;=> 1 I I- l . v t .. < V * *. It I. V. ; ! I ',.1 I. r 1 . I. | f.. * L- :* t!.< ! I. h - ,1 . 'i STLCOPCB4009529 A-ft/' . l'(h., IMI CHLORINATED HYDROCARBONS 119 When smaller doses (0.5 gm. every M'cond day) of this compound were administered to 10 rats, the first death occurred after 9 days. Two addi tional deaths occurred after 12 days and two more by the end of the fifth wi'ok. The remaining animals were sacrificed between the 40th and the 190th day. The livers were enlarged, the average increase in liver weight bring 33 per cent. They were pale and slightly mottled. The most out standing microscopic abnormality was the presence of large numbers of varying sized acidophilic hyaline glob ules in the cytoplasm (see fig. 5, plate 111). These hyaline globules, although larger and more numerous, appeared identical to those encountered in the livers of rats exposed to other chlori nated diphenyl preparations. The liver cells were markedly swollen and showed fatty vacuolization. There was also increased regenerative activ ity, No structural changes were ob served in any of the livers. Discussion Published reports concerned with the possible systemic effects of chlori nated naphthalenes have been few in numbers. Lehmann (3) in 1919 re ported that animals exposed to chlori nated naphthalenes by inhalation or feeding refused to eat and at death showed "peculiar'' lesions in the liver. In 1936, Klinn and Jarvik (4) reported the results observed following the daily subcutaneous injections into 30 rabbits of enormous doses (30 mgra.) <d certain chlorinated naphthalene roinpounds dissolved in paraffin oil. 1 lie rabbits receiving a mixture of trifcnd letruchlornapbthalcncs lived and when sacrificed, after a 2 months' exposure, no pathological changes attributable to the compound injected were demonstrable. The mixtures of tetra- and pentachlornaphthalenes and of penta- and hexachlornaphthalencs proved highly fatal and degeneration of liver tissue was observed. Prom tlveir findings Flinn and Jarvik con cluded that certain chlorinated naph thalenes or impurities contained in them arc capable of producing yellow atrophy of the liver in the rabbit. It is noteworthy, in the light of the present experiments, that the com pound of lowest chlorination (a mix ture of tri- and tetrachlomaphthalenes) had no apparent effect on the well being of the animals and did not produce demonstrable changes in the liver although injected daily for a period of 2 months. The present experiments demon strate that chlorinated naphthalene compounds and chlorinated diphenyl arc capable of producing marked liver damage in the white rat without demonstrable microscopic changes ap pearing in the other organs. Further more, the characteristics of the liver lesions resulting from comparable amounts of any given compound are the same, regardless of the method of administration (inhalation, feeding, or subcutaneous injection). An attempt has been made through out this study to grade the toxicity of each of the several compounds tested in accordance with the severity of the liver damage produced. Although some of these compounds appeared to produce similar degrees of liver injury, the toxicity of each compound seemed directly related to its degree of chlorin ation. Thus, a mixture of trichiornaphthalenes (chlorine content 49.4 DSW 025575 STLCOPCB4009530 u*. * J'S. t*CUt\tl-M*.ti*~A. 120 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY |vol. to, no. t per cent) proved to be the least toxic of any of the compounds tested, whereas chlorinated diphenyl (chlorine content 65.0 per cent) was highly toxic even in very low concentrations. Trichloruaphthalencs, when com pared to other naphthalene compounds of higher degrees of chlorination, were relatively innocuous. Even after pro longed feeding of large amounts of this material, the rats showed no evidence of ill health. After 2 months' exposure, microscopic examination of the livers revealed only slight to moderate degrees of fatty infiltration and degeneration of liver cells (plate II, figs, 1 and 2). These changes were Blight when compared to those result ing from short exposures to compounds of higher chlorination. Exposure by inhalation to low concentrations of trichloruaphthalencs produced liver cell alterations that were never more than minimal (see fig. 3, plate II). Such animals withstood additional liver Injury from carbon tetrachloride and alcohol in a manner indistinguish able from that of the normal animal. Somewhat greater liver injury re sulted from inhalation when the air concentration of this compound was increased more than eight times that used in the previous experiment but again the liver changes were slight compared to those produced by com parable exposure to the more highly chlorinated compounds. The mixture of tetra- and pentachlornnphthalencB employed in these experiments appeared to be consider ably more toxic than was the mixture of trichlornaphthalenes, Animals fed email doses of the former of these preparations fell ill and died or were sacrificed by tho 63rd day. Their livers showed extensive fatty infiltra tion and fatty degeneration (figs. 3 and 4, plate I). Feeding of tetra- and pcntachlor- naphthalenes in combination with chlorinated diphenyl resulted in pro nounced liver changes. These livers had increased in weight (average 71 per cent). Microscopic examination revealed a peculiar type of hyaline degeneration involving practically every liver cell (see figs. 1 and 2, plate I). This type of cell degeneration was more marked and occurred earlier after exposure to preparations con taining chlorinated diphenyl than to any other compounds tested. Fur thermore, it was most marked in the livers of animals exposed to refined chlorinated diphenyl (figs. 4, 6, and 6, plate III). Comparable exposure of rats to penta- and hexachlornaphthalenes (Compound D), penta- and hexaehlor- naphthalenes (Compound E), and penta- and hexachlornaphthalenes (90 per cent) plus chlorinated diphenyl (10 per cent) (Compound F), resulted in liver changes having few detectable differences. The morphological changes were slightly greater in the livers of rats exposed to tho lost named compound. In these instances tho hyalin degeneration of the cell cytoplasm was more marked. Al though rats inhaling low concentra tions of compounds D and F showed no demonstrable signs of ill health, microscopic examination of their livers revealed marked liver cell injury (fig. 6, plate II, and fig. 3, plate III). These lesions were still demonstrable after a 2 months' recovery period. Further evidence that, liver damage had resulted from inhalation in low concentrations of these compounds was obtained from the carbon tetro- DSW 025576 dilorid were resulle, (figs, i three c toxic n 0 pe of in Inigi 1, plnte 1 and chungrs lntion ii pound such li feature filtered! routine specially ented tl from di The reas. apparent big dilat bile duct lobules i ever obs< bined cli Juts was) in the no ous liver i to high c pounds, most um (hose of mieroscop Of the carbons I gave cvidr When ndi very low < to 0,93 m changes v the first c striking d of (he cell Hi). Sue STLCOPCB4009531 ffb.. /WI CHLORINATED HYDROCARBONS 121 chloride and alcohol test. Such tests essentially unchanged after a 2 month were almost uniformly fatal and recovery period. In these animals resulted in widespread liver necrosis email sublethal doses of carbon tetra (figs. 1 and 2, plate VII). These chloride and alcohol uniformly pro three compounds proved to be very duced extensive liver necrosis and was toxic and resulted in a most unusual highly fatal to them (figs. 1 and 2, type of liver change when administered plate VII). Chlorinated diphenyl fed in large amounts by feeding (see fig. in small doses produced similar but 1, plate III, fig. 1, plate VI, and figs. more marked liver injury (see fig. 5, 1 nnd 2, plate VIII). Identical plate III). In large doses this com changes were observed following inha pound was highly fatal. The liver lation in high concentrations of com changes in animals dying after short pound D (see fig. 2, plate VI). In exposures were inconspicuous and such livers the most conspicuous consisted mainly of swelling of cells feature was the presence of large and active regeneration (see fig. 4, intercellular spaces. Examination of plate III). However, animals re routine sections, supplemented by moved from exposure before being specially stained preparations indi fatally poisoned, subsequently devel cated that these spaces had resulted oped hyaline degeneration of liver from dilatation of bile canaliculi. cells similar to that produced by pro The reason for such dilatation was not longed administration of small doses apparent. There was no correspond of this compound. ing dilatation of the large and small Thus tho results of tho present bite ducts at tho periphery of the liver study, as well as certain field studies lobules and no inspissated bile was that have been made (1) suggest that ever observed. The organically com the solution of the industrial hazard bined chloride in the livers of these involved is dependent largely on a rats was no greater than that observed reduction of the air concentration of in the normal animal. Although seri- ' these compounds to a level that will oils liver damage results from exposure not produce liver damage. The pres to high concentrations of these com ent experiments indicate that lower pounds, tho resulting changes are air concentrations must be obtained most unusual and do not resemble in the case of the more highly chlori thoso of acute yellow atrophy on nated naphthalene compounds and microscopic examination. chlorinated diphenyl than for trichlor- Of the various chlorinated hydro nnphthalenes if a safe environment for carbons tested, chlorinated diphenyl workmen is to be assured. Because gave evidence of being the most toxic. of the pronounced toxic effect of small When administered by inhalation in doses of carbon tetrachloride on the very low concentrations (average 0.57 livers of nnimals already injured by to 0.93 mgms. per cu. in.) liver cell exposure to chlorinated naphthalenes changes were veiy pronounced after and chlorinated diphenyl, its use as a the first exposure period. The most solvent for these compounds would striking change was the hynlinlzation appear to be very hazardous. of the cell cytoplasm (see fig. 0, plate Although this investigation was Hi), Such cellular alterations were designed to determine what systemic / DSW 025577 STLCOPCB4009532 w*V--^VVJwi^<Uh.Wn*V^*.^ iMMW-tkVv^ 'i>*4i|)^ iejHtO*(V<OJ^i *-..' ,,V *-J** > 122 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY |w>. 0, no. effects, if any, would be produced in experimental animals by a group of chlorinated hydrocarbons, the patho logical changes produced have been of such a nature as to justify further comment. Exposure of rats to highly chlorinated naphtbaleno compoimds and chlorinated diphenyl results in a type of liver injury differing consider ably from the liver lesions resulting from carbon tetrachloride (5, 6) and chloroform poisoning (7). The changes resulting from exposure to the compounds employed in this study involve all portions of the liver lobule. This is true despite the fact that the initial morphological changes are more pronounced in the liver cells nearest the hepatic veins. The changes ob served are degenerative in nature. The successive degenerative changes were cloudy swelling, fatty infiltration and fatty degeneration and finally, the complete disintegration of the cell. Degeneration characterized by the accumulations of large amounts of acidophilic hyaline material was also observed. This feature was much more marked in the livers of animals exposed to the compounds containing chlorinated diphenyl. It is of interest that widespread degenerative changes of all liver cells can exist for several months without producing any evi dence of ill health hr the animals or without causing important structural changes in their livers. Such findings suggest that the alterations take place slowly and that the scattered necrotic cells ore efficiently removed and re placed by new cells which also become injured. There was no observable morphological evidence to indicate that the regenerating liver colls had acquired an increased resistance to the injurious effects of tlio compound being used (8). The fact that liver cell changes were still present after a 2 month period during which the ani mals were removed from exposure to the more highly chlorinated com pounds is further ovidence that this type of injury is persistent and only slowly recovered from. The administration in high concen trations of two compounds of inter mediate degrees of chlorination re sulted in marked structural changes in addition to marked degenerative changes in liver cells. The most conspicuous change was the occurrence of progressively enlarging intercellular spaces. These were interpreted as markedly dilated bile canaliculi. We are unaware of such changes having been previously described. From the above it is apparent that exposure to highly chlorinated naph thalenes and chlorinated diphenyl not only results in liver changes having marked differences from those caused by other well known toxic agents, but also that the characteristics of the liver changes produced can be mark edly altered quantitatively and quali tatively by varying the degree of exposure. For theso reasons one would seem justified in suggesting that detailed studies on animals in which the types of liver injury observed in these experiments had been produced might further our understanding of liver cell function. Summary 1. Studies concerning the effect upon white rats of a group of chlorin ated naphthalene compounds and chlorinated diphenyl are reported. This investigation was undertaken because of the recent occurrence of 3 fatal cases of jaundice in men work Fch., ms) ing in im; compound compound. fhlonmiii,, l"htli\c i,( Four were arliini air coiiv !t! were vjiritested }iy nnunmts. were tivt,., m/ds l,y xu! effects of not appear method of marked <J( di/Tcroncfs worn) con ing the niv in feeding 3. Aim- e.vmiiiudii exposed to Unit thc festr'd oxmuimifif rigniliennt 1. Dhinm: pililr clilori 1!. I.IV-MIK 3. I.UIIMASN Altiril T.i-I, D'U . 1'l.ISN, of III Ile li\ M.-A, A. Gaiiuni POK, 1 M. J r. i) DSW 025578 STLCOPCB4009533 . | ^^[.!1-t.>.M>jT:i.-.>Ww'^.<W*Ufc>J^r <^1***>/:I ^,v*/>aMT *v .>.i AJ-1 . V-A2l+..^t-&****--*miJ X'<> M., MJ CHLORINATED HYDROCARBONS 323 ing In industrial plants where these compounds were being used. The compounds tested were selected as representative of a certain range of chlorination and because of their relative industrial importance. 2. Four of the. compounds tested were administered by inhalation. The nir concentration and exposure periods were varied. All compounds were tested by feeding in large and small amounts. In addition three of them were tested on small numbers of ani mals by subcutaneous injections. Tho effects of any given compound did not appear to be influenced by the method of administration. However, marked quantitative and qualitative differences in the effects of each of several compounds resulted from vary ing the air concentrations in inhalation experiments and the dosage employed in feedbig experiments. 3. Macroscopic and microscopic examination of the tissues of rats exposed to these compounds indicated that tho injurious effects are mani fested solely in the liver. Blood examination during life revealed no significant abnormalities. 4. The liver changes result,ing from exposure to the chlorinated hydro carbons employed have been described and illustrated. As indicated by their ability to injure liver tissue the toxicity of these compounds increases with increasing degrees of chlorination. Tims a mixture of trichlornaphthalenes is relatively innocuous in low concentrations and when compared with compounds of higher chlorina tion, its toxic properties even in higher concentrations are relatively slight. Chlorinated diphenyl appears to be the most injurious compound of all those tested. 5. Administration of small Bublcthal doses of carbon tetrachloride and ethyl alcohol to rats whose livers have already been injured by the com pounds under consideration is highly fatal and produces massive necrosis of the liver. 6. The significance of this study in relation to the industrial problem involved is discussed. 7. It is suggested that the types of liver injury observed in this study might be employed in experiments designed to study liver cell function. BIBLIOGRAPHY J. Drinker, C. if., Warren, M. F., and Bennett, G. A.: The problem of pos sible systomio effects from certain chlorinated hydrocarbons. Tms J.r 10, 283 (1037). 2. Lamson, P. D.: Personal communication. 3. Lei?mann, K. B.: IvurzcB Lohrbuoh dcr Arbeit und Gowerbchygiene, S. Hirzol, Leipzig, 1010 (p. 261). 4. Flinn, I1'. B., and Jarvik, N. E.: Action of cortain chlorinated naphthalenes on the liver. Proc. Soc. Exp. Biol, and Med., 85,118 (1030). 6. Gardner, G. n., Grove, It. 0., Gustaf son, U, IC., Maine, E. D., Thompson, M. J., Wells, H. S., and Lamson, P. D.: Studios on the pathological histology of experimental carbon tetra chloride poisoning. Bull. Johns Hop kins 1-Iosp., S8, 107 (1025). 0. Cameron, G. It., and Kauonaratne, W. A. J3,: Carbon totrachloride oirrho sis in relation to liver regeneration. .T. Path, and Bact., 43,1 (1030). 7. Whittle, G. H., and Sperry, J. A.: Chloroform poisoning. Livor necrosis and repair. Bull. Johns Hopkins Hosp., SO, 278 (1000). 8. MacNider, W. A.: A study of the Re quired resistance of the fixed tissue cells morphologically altered through proocflsea of repair. J. PJmrm. and Exp. Therap., 66, 350 (1030). DSW 025579 STLCOP