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The Toxicity of Vinyl Chloride as Determined bv Repeated Exposure of Laboratory Animals T. R. TORKELSON. M.S.. F. OYEN. *ad V. K. ROWE, Mi. Biochemical Research Laboratory, The Date Chemical Company. Midland. M chtgan Group? oi laboratory animals were exposed repeatedly ior up to ?ix month? to ?ither 500. 200. 100 or 50 ppni vinyl chloride tn air. Detectable change? occurred at aJ but the lowest concentration. The results are discussed and handling precautions suggested. Introduction n.WL chloride (CH.-*"CHCli is a chemical o: great industrial importance. It is used in tiie preparation oi polyvinyl chloride resin, as a copolymer in saran and other plastics, as a eitemicai intermediate uud as a solvent. Be* cause ot the flammability oi vinyl chlonde. it has been generally assumed that the greatest hazard associated with vinyl chloncie is that due to its flammability rather than us toxicity.1 The toxicity oi vinyl chloride lias been re viewed by von Oetungen5 and more recently by Moitrottucteo et al.` von Oettingen concluded that the gas was anesthetic in high concentra tions and tiut considerable interest had been shown m the use oi vinyl chloride as a surgical anesthetic. However, its effect on the circulatory system ha? discouraged exploitation oc this prop erty. No doubt the hazard trom flammability has also hindered this use. Only limited repeated exposures which were reported by Schaumann' were ui?cus?ed by von Uettmgen. These repeated exposures indicated little or no chronic effects even trom anesthetic concentrations. Mostronutteo er air also discussed the pub lished data and reported the results oi single exposures ot mice, rats and guinea pigs which confirmed the low acme toxicity of vinyl chlonde. Mastronmceo reported that only two human fatalities due to vinyl chlonde had been reported. It can be concluded from the published toxi cological data that anesthesia is rhe onlv sicnuicant effect of acme exposure. Sufficient re peated exposures have not been reported to draw conclusions about chronic toiacuy. The Threshold Limit Value of 500 ppm suggested by the American Conference of Governmental In dustrial Hygienist? iACGIHi1 is reported b\ Smyth* to be based on single animal exposures and human experience. The following report summarize? the results of repeated exposure? of Liberator-- animals to either 500. 200. 100 or 50 ppm ot v.nyl chloride. The significance of the results is ascussed and recommendations tor a threshold emir vaiue are nude. Experimental Procedures Material* Tested Vinyl chlonde, CH-"*CHCI. i? a colorless gas. It has a boiling point of -- 13.35'C i-r7.9o*Fi ami a freezing point of -- 1$4*C -- 2+4.52'F). The material po!ymertzes readily :ad hence is sometimes inhibited with phene I or tertiary butylcatechol. Vinyl chlonde i? very flammable, its flashpoint being --7>*C i-10vFi. The ex plosive limits are from 4G to 22' by volume. It has little odor although high . mcemratioii? may smell faintly sweet. A single, uninhibited sample was used in these studies. It was shown by mass ;?actroerapinc analysis to be essentially pure CE--CHCl. air beme the only impunry detecte-: :n eas phase samples. yoiirce and Feeding o' A/timaU The rats and rabbits used in th^ study were obtained irom the stock colony c; this labora tory, the guinea pigs were obtained from a commercial grower and the dogs "cere purebred beagles obtained from a local kennel. The rat? and dogs were fed Purina Laboratory Chow* or Famo Laboratorv Ration* The rabbits and euinea pigs received Famo Rabbit Breeder Ration. The guinea pig diet was supplemented with carrots. Experimental Protocol The experiment? were conduced m three phase?. In the tir-c phase, groups of 10 male $ Industna. and 10 tec day five month?. I clumber ; growto re' were obui scopic exa rats serve* The ?ec hour daih male rats, three mai male and or 100 ppi two group and mate:, weight. T no esposu The ?ecor hour exporo the oiii Tin? grou; rol group 7-liour ua: live male or 100 pp; The pro. ically the and morta The livers posure am the liver c Pre-exposi mutation? ietemunat L nne sum represent.!oi rhe exp. overntrhc and rabbit djv after killed by thiopental, oi blood u animal# to" i*e. semi anuc-pyru ntm-elutan The orxan hi?topathni were not stopped w. sacrificed i The rhir ot repeate. inrl 24 fem yie*:. d mai RSV 0016863 ned oh l ,e results mmais to v iiionue -*ea ami ' .mi? arc- riei* sas. -7.93:F> .44.S2*Jb. lieuce is term ry -mmable. Th? exvoiume. titration; i in tnese rograpiuc HCl. :ur -a; phase udv were ; iabora; from . purebre^. The r.'i i; rv Chow' obit? and Breeder demented in three 10 maie Industrial Hugiens Journal .iiia 10 female rat; were exposed evm hour; per and one femaie do? to 50 ppm vunl chlonde. uay rive uays ]>er k to 500 ppm for 4.5 Euuai matched group? served a; unexjrased and months. Exposure; were given in a 160-liter .ur-exposed controls. Three additional groups oi chamber previously described." Mortality and lu male rats each were exposed lor 4. 2 or 1 hour growth records were kept. Final organ weights ;>er day to 50 ppm. were obtained and tissue; were saved for micro The procedure* followed were e*-enTiailv the scopic examination. Five maie and live lemaie ?ame as u*ed for exposure? to 200 aim 1U0 pjmi rat* served a* unexposed controls. except that hematological examinations were The second phase consisted of rfpeated 7- made on the docs after three months and liver hour daily exposures of 20-24 male and 24 fe biopsies were made only pnor to exposure. Urine male rats, ten maie and eight lemaie guinea pig;, was collected from the rabbit* as well a? tne rat* three male and three lemaie rabbit; and one and dogs. The rat* which were not sacrificed at maie and one female do? to either 200 ppm the end of the exposures were pastured ior six or 100 ppm of vinyl chlonoe. These animals ana weeks and then sacrificed. two groups of controls were raretuily selected and matched on the basis of age. condition and Equipment L'sca weight. The first group oi controls received The exposure equipment used has been de- no exposure and served a; unexposed controls. .-cnbed previously.'1 However, m order to cam The second group received repeated daily 7- additional data about the results ot repeated liour exposures to room air in a chamber similar hort daily expo*ures. it was neees?.iry to devise o the one used for exposures to the chemical. a method of introducing additional croups of This group i; referred to a; the air-exposed con* rats. This was done without opening the door; roi group. In addition to the animal; receiving of the chamber by dropping the rat; through 7-hour daily exposures, eight separate groups of chutes made of 3.25-iuch stainless -teel tubing. five male rat; each were exposed to either 200 These chutes were closed with a rubber stopper or 100 ppm for 4. 2. 1 or 0.5 hour; per dav. except during the hnef time it took to insert the The procedure; and equpmient used were bas rats. The chutes, which were ;lo]ied at a 45* an ically the same as reported previously.11 Growth gle, ended m covered, screened cages inside the and mortality record; were kept on ali groups. chamber. The additional group* were started 3. The livers of the dog; were biopsied prior to ex posure and afier o'-a month; of exposure, hence the liver of each doe served a; it; own control. Pre-exposure and terminal hematological deter minations were made on all dogs and terminal determination; on representative group; of rat;. Urine samples were collected from the dogs and representative groups of rats. At the termination of the experiment, part of the rat; were starved overnight and then these and all the guinea pigs 5. 6 3nd 6.5 hour; after the 7-hour exposure* started. Since all animals were removed after the 7-hour exposure was completed, this routine re sulted in exposures lasting 4. 2.1 or 0 5 hours. The vinyl chlonde was metered irom a saran plastic bag winch ?erved as a reservoir for the gas. Metering of the gas wa; .lone bv Dual >vnn*e Pumps." Total air flow through the chambers was measured by means of calibrated flow meters. and rabbits were killed by decapitation on the dav after thetr last exposure. The dog; were Anafwu of the Chamber Atmosphere? killed by exsangumanon after anesthesia with Anahse; of the air were made bv direct com thiopental, sodium iPentotlial. Abbott i. Sample; bustion of a known volume of air in a heated of blood were taken of representative group; of quam tube. The resulting chlonde was trapped animals for determination of alkaline phospha in IG- sodium formate--IG sodium carbonate tase. serum-urea-nitrogen iSUNi. serum-glutamic-pynmc-iraasanunase iSGPTl. and se rum-glutamic-oxalacetic-transaminase i?GOT). The organs were weighed and tissues fixed for -olution and subsequently Titrated bv a nncroVolhard technique. The results of .the individual analyses were within 15r* of the theoretical con centrations during the exposures to 200, 100 and histopathologicai examination. The rats which 50 ppm. The average concentrations recovered f were not killed the day after exposures were were 107, 100.5 and 4S.7 ppm respectively. stopped were pastured for eight weeks and then sacrificed in the previously described manner. Results of Repeated Exposures The third phase of this experiment consisted of repeated daily 7-hour exposures of 24 male Erooiure to 600 ppm Vinyl Chloride and 24 female rats, 12 male and 12 female guinea The rats exposed repeatedly at 500 ppm for pigs, 3 male and 3 female rabbits and one male 4 5 months, grew normally and no changes were RSV 0016864 ; A * > * V4 X 336 October, 1961 T*u 1 g.nwBnn- 0{ Terminal Avenge Body and Organ Weight* of Male Rat* Receiving Repeated Exposure* to Vinyl Chloride 3 Dan per Week .. Cbkcbvauob is pm )ImUi OB gxpBBur* l Duly Expmm. boon Rauo o( Survival Aft Body ftnefct. .Liui Hurl Liver Jtidwy SpietB Twb CaaipuMU aoBirol a. ;0 0 41 204 0.14 : 0.33 2-32 o.n 11 0 10 M0 Vonpaatd naval Air apaaad caoiral 200 sou y MV JOU 100 100 100 l'jO faipae0* eoatrel Air Hpawd* control JOO* 100* 4.1 7 10 312 0.M Q 33 3 00* 0.75 o i: 0.33 20 0 is i: 343 0.13 0 32 43 0 09 0.14 0 90 ? : ,4 10 12 3M 0.12 0.32 2.12 o.ro on 0.74 Ml* .6 : 1! 341 0.12 0.32 0.11 0.17 0 94 a. .4 4 l 340 0 47 }.90* 0 34 0 14 0.SJ 2* 41 341 0.M 0 30 its* 0 44 010 l 0 99 4 a.M1 i i ua 0.41 2-44 0 11 0.13 ' 0 74 4 0.1 41 139 0.11 0 29 2.33 0.47 0.14 0 91 s 7 : i: 31' 0.31 0.29 j.4l* 0 13 0 13 o.si *) 4 31 36U 0 31 0 24 2.42 0.41 0 13 0 74 4 2 41 131 0.13 0.31 i) 47 0.14 < 0 93 i 1 3J 2.12 03i: 0.49 0.24 0 44 13 0 74 343t 0i 11 0.41 0.30 * 4J 0 44 0.14 0.79 0 0 10/12 Q.44334 0.49 0 31 : 0 44 Oil < : 1 12 2.11393 0.39 0.30 0.43 013 0.79 < r 89 331 0.47 0.29 2.10* 0 44 0 11 . 071 6: 376 0. IT 0.21 2.13' 0.41 a. i4 0 74 4 Future lot >ku lur aaoesutr* ermard. i. p - >>.en lb' P - oi.Oul |C; P - U.07 (di P - i) l {* P - 0.02 <( Pm o.li apparent grossiy at autopsy. Microscopically then livers showed increased central lobulaT granular degeneration and the kidneys showed interstitial ana tubular changes. The average weight increase oi the livers of the male rats was statisticallv stcmhcanc l P = 0.0011. Table I. The average weight ot the livers of the female rats was above that of the controls but not statis tically significant, Table II. SUN. SGPT. 5GOT. ami alkaline phosphatase determinations were within normal limits (Table IHi. Exposure to -V) ppm Vinyl Chloride All groups exposed seven hours per day I3S14-4 times m 204 days were normal in appearance, mortality and growth. Hematological, SUN. $GOT. SGPT and alkaline phosphatase values iTabie III i and the results of the urinalysis were wuhin normal limits. Gross pathology' was normal in all species. Micropachology was normal in the rats, guinea pigs and dogs, how ever. adverse effects were noted in the liver* of rabbits of both sexes. In the tnaie rabbits this was characterized by central lobular granular de generation and necrosis with some foamy vacuolation. In the female rabbits the changes noted were central lobular granular degeneration and necrosis with penportal cellular infiltration. All organ weights were normal except for the livers of the male and female rats which were in creased significantly alter repeated daily 7- hour exposures i Tables I. II. IV. V, VI, ana VII l. In the males this increase was still appar ent eight weeks after exposure ceased, although the liver weights appeared to be decreasing and returning to normal. The male rats exposed to 200 ppm of vinyl chloride for either four or two hours per day for six months had elevated average liver weights fTable I). However, the small size of the ex perimental groups made the findine statistically insignificant (P = 0.07 and 0.1 \. The groups ex posed for eiih*r 1 or 0.5 hours per day were enttrelv normal. Industrial nmimarv Female pomre-> i e x is l Mtimail wr iroi MO VHisuMln troJ Aif ripcv cvmroi eooTrrpl K\r ffpmr maim 100* l'mi pawl e tfOl Air npo*. mauat L'aetuuaad* i.r xyu i-OBIfOl** W * PuiUT ** Puiul* *< P - u h- P - M C p . IJ -l-p.-l Ezvoture All spec ride, sevet dai s, weri lowing cr. hematotoc GPT. alii nalisis an H- RSV 0016865 er. 1961 fffni rrs -- ;S Tnici 0 *9 0 14 *4 - )1 0.76 i: 0 94 16 0 ss < ie 0 w - ii 0 78 < ii 0 *5 ii 0 M 0 74 16 U.93 1 13 0 78 .1 16 0.75 <1 14 U.<4 <> 11 0 78 <J 11 11 It 0.71 0.78 rabbits this rranular tieuuv vacuo:Qces noted oration and .rration. All >r the livers h were in ti daily 7V. VI, and ?ull appard. although -reasing and ;im of vinyl - per day for iver weights e of the ex: statistically ie groups exlav were en- lu/iusinal Huoiene Journal Ta*LE II Mimman- of Average Bodv and Organ Weicinis of Female Rats Receivtnc Repeated 7-Hour Ex posure# to Vinvl Chloride 5 Davs per Week 9 , ; ;s O(m1a00 U* nSeMnvu, troi 0 S S 194 41 3 D 0 SO 0.19 M 4 j 10 193 IS 3.22* n 91 0.20 I'utMMnatroi u 10 12 JO* jj u 92 o.i: Alt MpotM roovtot 6 12 123 5* j. v si o.i* W 6 10 12 J'*5 U 3.39 0 *4 0.11 100 6 1! I' 221 39 3.W' 0 79 0.20 t'sMPOsre* rostral (1 ? 12 S3 37 2. ST ( 2 0-11 Air npaaed* rostral 6 SC IOC* 6 6 I'impn--desstto* 0 Ur ttposrd rostra! 6 6 9 12 240 12 13 253 12 12 23J 11 12 .*11 !!1J 202 9 12 204 37 2.97 0.U 0.11 U 3 W I'.Sl 10.30 40 3.17 0.S3 <0.21 39 3.02 0 *3 0.20 3 5 .3 u.M P . 37: 3 02 O N 0.20 V flrrcosrd** rostral 0 Air rtpnsrd coDimr* 8 n 12 19} * 12 221 40 2.SS 0.91 0.23 40 3.01 0.S7 0.19 a 12 233 30' 2.S3 0.92*0.20 * Pscturea lar 9 *N list npwim sed. " PuninS lar 0 11*11 liter timum auM. (|i P 0 17 (bt P - 003 (el P - 0 01 id) P O N Expoture to IOO ppi I'niuf Chloride .Ml species exposed to 100 ppm of vinyl chlo ride, seven hours per day, 13S-144 tunes in 204 days, were judged normal on the basis of the fol lowing criteria: appearance, mortality, growth, hematological examination. 5UX, 5GOT, S* GPT. alkaline phosphatase determination, uri nalysis and gross and microscopic examination of tissue. However, rlight increases were found in the averse* weight; ot ihe hveti oi mate and icmaie rats i Table? I and II i. Although not #12niheant statistically, an increase was mso seen in the average weight of the iivcrs 01 the maie rats exposed to 10Q ppm for either four or two hours per dav. The groups exposed for either 1.0 or 0.5 hour per day were entirely normal. Exposures to 50 ppm Vinyl Chloride The increase in weight oi the rat livers, the only significant finding in animals exposed to 100 ppm, did not occur m rats exposed repeatedly to 50 ppm of vinyl chlonde, 130 tunes in 1$9 davs. All groups of animals were judged normal in all other respects. See Tables II. V, VI. VII. VIII and IX. A srausticallv significant decrease in kidney weight which was seen m the female rats 1 Table II i was considered to be an artifact since it was not seen at higher concentrations. Discussion Repeated exposure to vinyl chloride at con centrations considerably below the level that has been considered safe for human exposure has been shown to have an effect on the liver and kidney of laboratory animals. Histopathoiogicai changes and increased liver weights id male and female rats were noted after repeated exposure at 500 ppm- Repeated exposure 3t 200 ppm for six months resulted in an increase in the average weight of the livers of male and female rats and micropatholoneal changes in the livers of the male and lemaie rabbits. The only effect noted after repeated daily 7.hour exposures to 100 ppm for six months was a slight increase in the aver age weight of the rat livers. Repeated 7-hour exposures to 50 ppm for six months had no effect on any species studied. Hence, the highest con centration without detectable effect on any spe cie? was 50 ppm. It is interesting to speculate on the apparent discrepancy between the effects observed at 100 ppm in this experiment and the lack of reported injury in humans using a threshold limit of 500 ppm. Several factors may be contributory. First, the effects noted in animals at 500 ppm were only slight to moderate: growth, mortality, and gen eral appearance were unaffected. Hence, it is possible that if humans were exposed repeatedly at 500 ppm slight injury might occur but due to the lack of subjective symptoms the injury would escape detection. Secondly, the injury was apparently reversible. The liver weights of both male and female rats which had shown definite increases after exposures at 200 and 100 ppm were normal or approaching normal after 6-3 weeks of recovery (Tables I, II and tTII). RSV 0016866 a Table III Nummary of Average Biochemical Value* Determined for Animals Receiving Repeated Exposures to Vinyl Chloride 5 Day* per Week specie* sad Doc No Coaceetrstiee ia ppm Months on Exposure Duration ot Daily Ezaosur*. hour* Number ABuiuh AlLsiter PtllM- phause kisr Arottrenc Latu Blood Lrcs VlUOCTB. me 100 mi SCRT 'GOT $icma-Fr**el Can Doc IU1 IU 41 M DO| DIM IU7 SIM *110 M I'snmnl control 0 Atr*ipo*J satral JuO iou F VacapoceU control 0 Atrexpnced eoatrol * mo t IDO u 1 U. * 24 I* 4 4.1 19 V 7 s4 n IS 4% i 4 1 14 :i 1? il u w> i 14 l : 14 1 24 1 i 10 U> u Rut Rut . Rabbit Rabbit .. .. M VBfipoattl control mo JUO 30U Jill IOU IOU mu F I'ocxpmcd eoatrol W mu too \! Control .<00 IOU F Control mo ll 4.4 4 t <> 4 * ,, 4 4 4 i 4 4 4 7 7 7 4 > 0 : 0 : fc.-J* 20.* V' 4 ]>V 11.i 31 17 7 14.3 It 4 4 i.: 33 I Si ir.: 43 4 V* 4 11 0 ? u r IS 3 ,o 111 4 IJ ; 1 ; 14 1 33 3 24 i 11 4 32.3 * * A "1 i 4 -l 11 14 1.1 31 31 - 1 i it 34 4 I.) 11 j 14 T. 11 1 4.I 14 U * Because of ion unusually hi(h individual vaiuaa ibis croup Uimc average t-aiueeare elevated aad do ao< correspond with the value* obtained uo tiimlar control croup* id our laboratory Normal eoeirol values are m iheaamt react* as those of tM epenmeBtal group* listed in tius table Table IV Mininury ot Average Hematological Values tor Animals Receiving Repeated 7-Hour Exposures ro Vinyl Chloride 0tein or Do* * Canctnif&Lieft in ppm Montnt Eepcrimtnt N* oi ArmiLii* Hemoioam i9 Hemtocm U'B\.C,i X ?ftu .Mufio> cym ir> P^lle Rat Rat Rat Rat Doc tm Doc slUl Doc * 1 Dot a I1J Doc sill Do< Doe 9 Doc *V4 Doc aius Doc IDS Doc eiu: Doc 107 Doc *1 Doc alM Doc SHU Doc a 1 III m Air noo*oo control M JW F Air t*on**d control F *U0 M L'arspioc'J esmeroi M l oaapuaad cuatrol M Air tiMtU ruauol M Air exposed eobirol M 2W M 300 -M iuu M mu r L otspna^J rootroi F L'ntipcitkl roturoi F Air ttpcwea control F Vir capote J control y .'no F *uu F urn F iou 4 4 4 0 4 U 6 >) 4 U 4 II 4 < 4 U 4 i) 6 * 14 1 ii 11 4 34 1 n.3 ;1 It 4 m 14 T 1' rr i " 3 U 0 4U 1 111 33 5 71.5 ij ;i hi 47 4 IM .`4 > J 10 i ] ---- ---- -- ---- ii ii 14 3 4n i. M l 14.1 so 111 41 :i 1 ii 14.1 13 IJ.3 it u 4 4 l U 4T 14.3 14 3< 1 14 1 it 11 4 >. 41 ii 11 1 13 IS 1 4o 40 4 4 I 1* 14 I* 6 SI U si 'i x u 1 51 > i 4J 17 It j 111 14 14. & 34 41 1 4 i Id v; N 4 Vi 13 4 i 14 i: u r Vo -'1 j U m i: i 44 4' 5 ; 1*) 14 1* 3 4*> ii IJ l 13 1 u 14 s <i 17 '1 l 11 1 14 14 4 HV io i SS8 i Industrial M L a#iou W Air tt pootr M S* M It*1 M Inftpi fMtr M A if ftp n>Bt? M> r vos> Ar ets nir L a*%p> mntr Air rtp ii P . <b. P - ' ici P* umman* Weight* 7-Hour 1 Week to ? m M A.r M M \| M \l F Lart F rr r F r RSV 0016867 89891-00 ASH suomuiuaoijoa q2ii{ .vjauiima pac luaoidmba aqi ut i^uaj jjava* aicotput pfno.u aoiiciuiaouoa jrja uas e qdiq stqi muu iuc[d aouanpojd at aidd 00? Jc Afsnonuuao) pasodsa sq oi susmnq ioj d|q -usod aq p(noAv 11 jaqiaqai (tipqnop st it 'Ajpitti^ (MW ninmlit 4*i|* nM 9 M| ptinuij , :* 0 ti ot# 0 it: n ost o OK St S! 09 t: o ti 0 h o tt Sos o cf 0 o:s St SI : tt 0 ti u ;; o;:c: ts osf o KS j m 1 :t n is o :t 0 its Ot $ 0. 0tt 0 oi 0:$ t :t oat 0 szt 01 9 ta 09f 0 6 0 6fl cro If 0 its 01 1 :s 0 trot# ott t it oof o #t St St t t* 0 ti n oi o it : :t o if o its SI St s*0si nu a:t vcc oof 0 ait 5 6 0 ,pinu ji\- fCAaw tpBdn8.] Of Of Of CR l&jiaoj 9 pwodn ji^|0J10'V> P4tMXM 1 ^ x T- " Z " n --z WJUft <909 iunt x *mai.ft u*iiq f* * rf 'm if A Ii i TB ^i s| l r Jfw,V\ -t<l #-icQ e jpuojqj |.vui.\ ot ajn#od -\3 pamaday Suuiaoay u#H *(B!M jo siqjti^ utJjq put Vpog sjIciaay (cutuuaj, jo .unmuny Ill.\ r>'x cs 0 ;t oit : t; o:a o t'J 1 t fs'ooro it s ti'oo: 0 Ell 1 t tt 0 tt'OOt t ti 0 64 11 9 i t t IS U Ct O M 'S g: n ns 0 i II t 1 *; 0 ft o*i i t: o>. 0 t Ot 1 1 st 0 ct on toi o - u s t I i; n ct o :9': it ii 1: 11 9 1 I 1 rt 11 c: oft oot:on* 0 9i I ; Of foiiaa* PWrtQu ity fOJtqoA pH09lW.^ 001 dec (Mia pMDdu ity fOJIBOd D9 191 ffl Ifl ntt tot ;ot 4m C31 J J J J i 1 J tv tt ivk otvowtri 0:: 0 9 01 1 1 11 0 s; a wo aft ?: awo ftt t t ft nit'o st oor: m 09* 0 0 St 1 1 St OfCOIfOOf'S 98 0 toi : 01 t 1 M ost 0 :t 0 n : ^ u*9 0 f 1 1 t ft n;sms 0 9t : a om H 0 Cl 1 [ pwm jty 1011004 puiliw.i 001 0l) lOJIQM pwoflii jry |OJU pmuu^ i ;si v> 111 stt tot IV IV IV IV IV IV Mini K fi ii S'm m*ns\ AQog 3 mi I *14313 ft UC31Q is S1 5 7 iz sqmoK 0 joj ijaa.w JJd 'Artl ? apuom^ j.vut \ o sajci^od \3 jooh- Piwdag JutAtaoag ssorj 10 siqaia.w uca^ij pur .ipug jruij >0 .ucumnu II.\ 3W1 1 tft t t ot fajiaoj 1 s Si pHn jv [0X1009 1 IS t f-f wo n o at iroin1 SI t c. t pqnfliie^ 001 1 01 t s; oos (OAQA) 1 18 E s c paaodi9 jry i Cl t t s paod3*0.1 1 0: t 11 Of raAack> tt 0 wn wo ircito :t 01 ii'C S [ pBDdsa jty 1 Oi s 1 ft c 1- It c c; ss ct 1 one 2 : 1 19 S t s - 3C /J * ini 11s431**11\4\31.140Suuji^ > ?2 s. *R ptMtis,1] 001 oos fOJlVOJ pMOflta iry pmttflltti ^ S' s*1 i 1 d 1 j j d a IV K JV IV IV IV IV ^qiuojv 9 Joj qaa.u jad *ACQ o apuojtQ |.Vin \ ot saansodsy jnoy-* A[tc<j patvaday aut.uajay mqqsy jo siqiia^v ucSji) pue -Vpog adcaaAy {Btmujaj. 50 .ut'uuun. I.\ VX t:o - d i' tsn - <1 'qj u - 4 < U 0 89 0 90* st it w |OJIOOJ 91 u n 0 ptMn ic> 104130-1 H O 69 0 its reo j: 11 M000390.1 :i on 0 IK S #s-n Sf 11 tfOB 0 *09 S :S 0 it: 0 001 04V JOJUIOJ tt nu n set wo m u ;m M*od>4 J'V fOJldnJ 'D ore :: o ;9 0 vrost 11 tJ 0 MS 9* II II IQJltlOd Of 11 ST '0 W0 It s :S 11 It II Sf 0 iTOrt'i :r it ntfi tt 0 60 0 0# ' WO IK'O sen ss 11 :: <1 ifo pWfW ** J(V JOUBUJ pMOdSMI.X OOE OOC |OJlfl(W p*fnflw ity 10X1904 paiMSM 1 <pog t tvt\ 9 tliftio ^ Mjq njtuojv 9 JOJ ^aa_\Y Jd sacq c anuotqj |.vut \ 01 i-ajnmodv^ ^ A(icfl patraday JuiAia-tay >2it{ camnr> 10 iqjta\\ itraoQ pur -vpog aarjj.xy |cutuijaj_ jo .unuui; ' A 3`3>'X wiunor )'i3iD>lf{ ]DU7Snp'<J 360 October, 1961 Tablc IX summary of Average Hematological Values for Dogs Receiving Repeated 7-Hour Exposures to Vinyl Chloride Do* * Sn Montn* ..___ CtunmuM in pete OB No. of Ecpcn- AotmnU ! m. Hmu* VVBN x Ncutrotoeni 10* phili Ln*Spn> Eewi cym pnile rnent : 11 * Do* ms:... Do* *143 Da* *141 Do* HM Do* a 144 Do* I44 Dm ni2 Do* *IU . Do* |43 Dm * i4i Do* nlil Dm isi Do* m Do* *144 Dm *144 Dm mi Dm *141 Do* !! >1 CneipMctl control >1 Vonpaned control >1 L'nMpnacd aooiTTU >1 Air npenad control M Air opened control M Air Mooted control .11 40 11 40 M 30 F Uacipcctd coctrol F L'aezpoacd control r V ncioaacd control F Air tienwl control f Air czpoaed eootrol F Air tipoacd eootrol F 40 F 40 F SO 0 ] i\ 3 6 0 3 4 0 3 * 3 6 0 3 4 11 u i: s 44 4: i 4 14 43 19 4 43 > 4 4 14.4 44 IS 4 S3 33 ------ -- ------ It.} so 13.4 44 3 14 40 11.1 4S 12 10.4 39 13 6 4* .* 1 14.4 44 tt.r 4* 4' 1 14.4 4.1 u.j 41 j' 3 11 41 13. s 44 ;o V 14.4 S3 33 6 6: 34 S 14.4 14 5 4*> 13 'J 11 46 14 41 :? : 16 Sir 11.3 *> * 14 4 46 IT 0 S3 10.4 34 li.J Si ... n 0 14.4 44 13.4 So 0 14.4 46 13.3 43 ~ n adjacent to the ieaki. The flammability of vinyl chloride precludes such severe leaks if this haz ard oi vinyl chlonde is to be controlled. Industrial Hygiene standard The data presented indicate that little like lihood ot injury would be expected if repeated daily 7- to >-hour exposures are iinuted to 100 ppm or less. Although the level or 100 ppm may not ap pear to oner an\ margin oi saiety since rats ex posed repeatedly to this level were very slightly affected, the vast amount oi human experience that is available while operating under an MAC of 500 ppm indicates that imury is not likely Likewise the effects ot even rather severe over exposure are not rerious. hence this level seems reasonable. A time-weighted average for all exposure should probably not exceed 50 ppm Summary Vinyl chloride iCH-CHCl) is a monomer used in very large quantities id the production of plastics. Repeated exposures of laboratory ani mals to several concentrations of vinyl chlonde in air were conducted to determine the chronic toxicity of this material towards animals in or der to assess the hazard to humans. Vinvl chlo nde was found to have a flight capacity to cause liver and kidney injurv on repeated exposures Male and female rats showed micropathologicai changes after repeated daily 7-hour exposures at 500 ppm for -4.5 months. Repealed 7-hour ex posures at 200 ppm for six mocas resulted in micropathologicai changes in the livers of rab bits and statistically sigmncanr njreases in the average weight oi rhe livers or and female rats but no detectable change; in dogs and guinea pigs. Repeated 7-hour exrosures at 100 ppm resulted in slight increase? a; the average weight of rat livers, the other st--:ies were not affected. All species *tudied toi-rated repeated daily 7-hour exposure- ro 50 ppm : >r six months with no detectable iniurv. Repeated dailv 1-hour exno4ures ai 200 and 1U0 ppm oi vinyl chloride were without effect, ionger exposures c:iii?eu a slight :i Tease in lit er weight. The standard for evaluating regular dailv 7to 6-hour exposures may be deAr-rU as the con centration below which practical!" all analytical results must fall. The value of h'O ppm is sug gested as this standard for vin\. 'hlonde. with a time-weighted average for all er?o.-ures not to exceed 50 ppm. References 1. Manuiacturtnc Chcrmna Auoc Cn?;_cal Saletv DU SPicct SD-S6 MW> 1 vov Otmscts. IV. T - T/faioornc?-4 Toxicity and Potr*ttal Danorri. . r` Public Snltb Same*. Publication No lit.I Goi'iniufi! Pnbiiai Oftrt WMhiaron. D C H944i. 3. M*To>tTTto E. A. M. riSRCt 2. CnmicTt*. v* D. DtMiciH Amt* [abtltnon Touew oi Vinvl Chlo ride to Laboratory .Uumali .(m. /no. Pm- Astoc- 1. -'I 3M UMOi. l. ckcmaxn. O. eit*d bv K. S Lomas txo F. FtcT. a Indust Tone sonar kin* S A.C C o' E* 4. Smtti swio it i: * Chcrr * Ralaii 9. Ham- 1 r I O h i* eu n t: n t; P ti si g' R RSV 0016869 r. 1961 Eon Industrial Hygiene Journal S6J Toxieoi. u. Hy*. atr t-eoBaeno LeatacaaittaJ. J. ipnaor D*ru&. mt p. 128. iFttm tfet uwUm Taaco,aey oa Hggm> c Jnaminat Solvmuti by L. Kinc uid H. F. Smvui. Jr.. IMIi. 5 l.CG.I.H. TliraanoM Limn Valuai tor lMO. A.WA Aren. |V n eir. Htalrh I 02 I IM0>. 6. Smith. H. F.. J*.: Inprai'M CoamuaiBtMB--Hvvmt Sumapo: tor rhulv InliaiaiMio. Am. Ittd. Hgg. Aime. J. 17* .lttti. r. ChifTT Hill Fubi, Camdan New Jantjr. Hainan Puna* Company, St. Louu. Miaaoun. 9. Ham* Millmf Company. .Mount Pwaaaat. 10. Srcxtta. H. C. V. K. Ron'S. C M. Adaui. D D. McCoujiTca. and D. t>. laian: Vapor Tosioty oi Etbvton* Dirntonor ai Dricnuaail by Espanmaau on Labonion* Atuaau A.'/.l Aren. ina. Hits, nan Otnp. ,V0. i: *82 UMli. 11. TonscLaos. T. R.. M. X. Molt. F. OrtN. aab V. iL Rove: Vapor Touaiy oi Allvl Ctiiono* aa Oatarmioao oo Laboratory Animal*- Am. Jmd. H w. aims. J. K: 217 iltHi. 12. Dual Synatt Fratlrr Pump*. Moira Matalenil, Mid land. Micbiaas. t- :iour es-ulted in - of rab? in tlie J femaie ogs and s at ]00 average ere not repeated : month- 200 and n effect. 1 m liver daily 7he connolytical n is nitide. with e* nor to L. dTocm*be*ii'll* Baalth -iun* iimi, *n# Viavi Chlo* iwc. J. II: Industrial Hygiene Certification THE AMERICAN BOARD OF INDUSTRIAL HYGIENE invites inquiries from those who are interested is applying for voluntary certification in the profession of industrial hygiene. Certificates nil] be issued on the basis of an examination to those who have eight yean of practice, following a suitable baccalaureate degree. No examination will be required of those who have fifteen years of practice with recognized contributions to the field, following acceptable education. Certain specialists within the field will be certified when one-fourth of their time has consistently been devoted to application of their specialty to industrial hygiene. A brochure describing the certification program, and giving more details of eligibility will be sent to those who direct inquiries to Henry F. Smyth, Jr., Secretary-Treasurer. American Board of Industrial Hygiene. 4400 Fifth Avenue. Pittsburgh 13, Pennsylvania. The Board was chartered as a non-profit corporation under the laws of the Common wealth of Pennsylvania in September 1960. The incorporators were selected by the American Conference of Governmental Industrial Hygienists and the American In dustrial Hygiene Association from among their respective memberships. They are John C. Soet. Chairman; Lester V. Craliey, PhD.. Vice-Chairman; Henri' F. Smyth, Jr.. Ph.D., Secretary-Treasurer: and Edgar C. Barnes. William R. Bradley, .Ulan L. Coleman. Hervry B Elkins. Ph.D.. Louis F. Garber. Thomas F. Mancuso. MB., Kenneth M. Morse. James H. Sterner, MB., and Charles D. Ynffe. Industrial Hygiene Engineering and Chemistry he DIVISION OF OCCUPATIONAL HEALTH. Public Health Service, will Tconduct two simultaneous courses January s--19.19G2. at the Occupational Health Research and Training Facility, Cincinnati. Ohio. "Industrial Hygiene Engineering'' is designed for industrial hygienists and engineers in the field of occupational health who have a degree m engineering or in the physical sciences. "Industrial Hygiene Chem istry'1 is designed for chemists in this field who have a degree m chemistry or chemical engineering, or in the physical sciences with a minor in chemistry. Dunng the first week, trainees for both courses meet together for instruction in industrial hygiene and medicine, toxicology, and principles pertaining to evaluation of the environment. For the hygienists, the second week's work coven temperature and humidity measure ments, illumination, noise measurement and control, and industrial ventilation: for the chemists, laboratory analyses for lead, free silica, and solvents, and spectroscopy, poiarography, x-rav diffraction, electron microscopy, and gas chromatography. Much tune m both courses is spent is the laboratory. It should be noted that applications for the course or requests for information should be addressed to the Chief. Training Program. Robert A. Taft Sanitary En gineering Center. 4676 Columbia Parkway, Cincinnati 26. Ohio, or to a DHEW Regional Office Director. RSV 0016870 Toxicology. 11 (1978) 45--54 Elaener/North-HoU&nd Scientific Publishers Ltd. EFFECTS OF VINYL CHLORIDE EXPOSURE ALONE AND IN COMBINATION WITH TRYPAN BLUE -- APPLIED SYSTEMATICALLY DURING ALL THIRDS OF PREGNANCY ON THE FETUSES OF CFY RATS GY. UNGVARY, ARANKA HUDAK, ERZSEBET TATRAI, M. L6RINC2 and G.FOLLY Deportment* of Experimental Pathology and Chemistry, State Institute of Occupational Health, Budapest, H-1450 Budapest P.O.B. 22 and Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, H-1450 Budapest P.OJ}. 67 (Hungary) (Received December 29th, 1977) (Revision received May 22nd, 1978) (Accepted May 23rd, 1978) SUMMARY Vinyl chloride (VC) has been shown to be present in the fetal and maternal blood as well as in the amniotic fluid after the exposition of pregnant CFY rats to VC at an atmospheric concoitration of 5500, 18 000 or 33 000 mg/ms (--2000, 7000 or 12 000 ppm) for 2.5 h on the 18th day of preg nancy, indicating the permeability of the placenta to the agent. Teratological investigation of the offspring of pregnant rats exposed continuously to VC at an atmospheric concentration of 4000 mg/m3 air (1500 ppm) during the first, second or last third of pregnancy has shown that VC has no teratological effect in the rat and has no embryotoxic effects either, when applied during the second or last third of pregnancy in the above concentration. Exposition to VC during the first third of pregnancy resulted in an increased fetal mortality and in the manifestation of embryotoxic effects. Fetal losses and induction of central nervous system malforma tion due to trypan blue administration were not potentiated by a combined exposure of pregnant rats to VC and the dye. INTRODUCTION VC is ranking 23rd from among the 50 most widely used industrial chemi- Supported, in part, by the Scientific Research Council, Ministry of Health, Hungary. 6-11'0401-03-1/MU. Abbreviation: VC, vinyl chloride. 45 RSV 0016871 cals. Carcinogenicity of the compound has been reported in rats, mice and hamsters [1--3). In man the occurrence of hemangiosarcoma -- a rare malignant neoplasm -- of the liver has been brought into possible causal relationship with VC exposure [4--8). VC or its metabolite, chloroethylene oxide, have been shown to induce mutagenic effects [9--14]. Chromosome aberrations have also been described in plant workers after VC exposure of various lengths (15--17). However there are only a few data on the terato genicity of VC. The results of the epidemiological studies are controversial. Infante [18] reported an increase in the incidence of congenital malforma tions of the central nervous system among people living near chemical plants working with VC. Further epidemiological study (19) did not confirm this finding. Infante et ah [20] found higher mortality in families where the father was subject to occupational exposure to VC. Paddle (21), however, questioned methodological aspects of this approach. The only study in experimental teratology with VC was conducted by John et al. [22]. They found no teratogenic effect after inhalation of VC at an atmospheric con centration of 50, 500 or 2500 ppm for 7 h daily, during organogenesis in S-D rats, CF-1 mice or N-Z rabbits. The large scale' production of VC and the wide usage of the polymer PVC, the great number of people exposed, the reported hazardous effects, muta genicity and carcinogenicity of the compound as well as controversial data of epidemiologic studies of teratogenicity call for a detailed study of experi mental teratology of the compound. The present work was aimed to answer the following questions: (1) Does VC, inhaled by the pregnant animal, cross the placenta and result thus in a direct intrauterine exposure of the fetus? (2) Does VC in itself possess teratogenic or embryotoxic effects? (3) When given in combination with a known teratogenic agent does VC potentiate the teratogenic effect of the former compound? MATERIALS AND METHODS Female CFY rats of 240--280 g body wt. were mated in a harem system. The day of finding sperm in the vaginal smear was considered as the first day of gestation. The animals were kept on a standard rat pellet [LATI*, Godollft] and tap water ad lib. Body weights were recorded once a week. Groups of 3 rats were exposed to VC for 2.5 h on the 18th day of gesta tion at 5500,18 000, 33 000 mg/m3 (~2000,7000,12 000 ppm) atmospheric concentrations. At the end of the exposure the animals were sacrificed and maternal and fetal blood and amniotic fluid samples were collected for VC determination performed by the method of LArincz [23]. Allocation of other pregnant rats to experimental groups can be seen in Table I. Groups LA, IC, HA and QIA inhaled air in an inhalation chamber for 24 h/day on the days of pregnancy 1--9, 8--14 and 14--21, respectively. Groups IB, ID, HB and IHB were exposed to VC in an atmospheric concen- * Institute of Laboratory Animala, GtfdtflkS. 46 RSV 0016872 TABLK I SUMMAHI'/.KD DATA OK KXPEH IMKNTAL GHOUPS OK PHEGNANT CI'Y RATS EXPOSED TO VINVL CHLORIDE. TRYPAN BLUE OR BOTH Tmimunt *4 hfday Daya ol prc| nancy No. of littera Maternal weicltl gain** <%> No. of fetuaea Live Dead Rcaoibed <%> Mean litter al/e Mean fetal weight (1) Mean placental weight <1 Weight rctardede fetuaea (%> Liver Livtr wt./ wt./budy reduced wl. ratio body wl.* <%l ratio {%) (1) A Ale Inhalation Phya. aal. a.e.* B VC Inhalation7 Phya. Ml. .e,c C Air Inhalation Trypan blue a.e,* t) VC Inhalation7 Trypan blue a.c.* (ID A Air Inhalation 1 - 7-8 1-9 1-1 1-8 7-6 1-9 7-8 B--14 B VC inhalation7 8-14 (III) A Air Inhalation 14-21 B VC Inhalation7 14-21 (IV) Untreated control -- 13 64.89 t 171 1 2 3 18 19 65.18 t 223 1 12 2 09 13 66.68 l 131 3 32 2 86 18 61 12 i IB8 3 34 2.63 14 49.36 l 167 -- 6 1.86 28 61.70 t 374 1 18 1.12 18 62.72 t 212 1 12 2 91 22 40.93 J 244 -- 14 1.67 28 62.46 t 316 1 10 123 1.7 6.5t 21. m 16.7*7 13.16 t 0.64 11.69 t 0.39 10.16 * 0.64 11.00 t 0.69 3.61 t 0.02 3.74 * 0.03 3.71 l 0.03 3.76 t 0.03 3.19 4.64 11.29 t 0.81 13.36 1 0.37 3.96 1 0.04 3.96 I 0.02 6.9 11.79 t 3.66 t 0.94 0.03 6.4 11.18 l 3.64 t 0.74 0.03 3.37 11.26 i 3.83 t 0.64 0.02 0.63 t 0.006 0.66 t 0.006 0.67 i( 0.006 0.64 t 0.008 0.66 i 0.007 0.62 t 0.004 0.62 i 0.007 0.67 1 0.007 0.61 1 0.006 2.3 6.7 6.3 10.6 6.3 3.21 11.8 12.3 2.9 3.71 t 0.09 4.28 !* 0.09 4.16 0.06 4.61 t * 0.14 4 41 i 0.13 4.92 l** 0.09 4.70 * 0.08 6.31 0.11 4.06 i 0.08 4.26 t* 0 06 4.67 ! 0.06 6.03 t* 0.07 3.76 1 0.06 3.60 t 0.06 4.29 Oil 4.12 t 0.07 3.89 t 0.01 4 -60 t 0.00 * In per cent of Martina body weight. b In per rent of total Implantation alter. { Per cent of living feluaea welching leu than 3.3 c. Maternal weight (total weight of leluaea placentae) O.S ml/lOO gbody wl./dey * 4000 tng/m* (1600 ppm) 0.5 ml/lDOg body wt./dav of 1% (w/v) aoluilon. * P < O.OS; * P < 0.01; ((teat) t P < 0.06; tt= P< 0.01; (Mann Whitney U teat): t S.E.M. RSV 0016873 tration of 4000 mg/m3 (`*-1500 ppm) for the same length during the same periods of gestation as their respective controls. The volumes of the inhala tion chambers were 0.13 ma, the vertical flow rate of the air 2 m3/h at a regulated temperature of 24--25C and 50--55% relative humidity. VC concentration in the inhalation chamber was determined by means of a type 5840 A Hewlett Packard digital gaschromatograpb [24]. The rats in groups IC and ID were given subcutaneously 2 injections of 50 mg/kg body wt. trypan blue (1% solution) on the 7th and 8th day of gestation. Group IV was kept in the animal quarters during the whole period of gestation. On the 21st day of gestation the animals were anesthetised with ether. Abdominal wall and uterine horns were cut open and the number, position of fetuses living, dead or resorbed were noted. Fetuses and placentae were excised, weighed and macroscopic investigation was carried out. Half of the fetuses of each mother were put into Bouin's fixative and dissected after fixation under the stereomicroscope [25]. Organs with macroscopic abnorm alities were embedded and hematoxylin-eosin stained sections were studied further. Histological investigation of representative other organs was also carried out. In order to investigate the skeletal system the other half of the fetuses were fixed in alcohol and stained with alizarin-red-5 [26]. The mothers were dissected and their livers were processed in routine histology. Arithmetic means and standard errors were calculated. Student's f-test was used for statistical comparison. The litter was regarded as the experi mental teratological unit [27]; affected over total fetus ratios were calculated. Mann-Whitney U-test was used for the statistical comparison of the ratios obtained. RESULTS Considerably high VC concentrations were found in the blood of pregnant rats as well as their fetuses, when the mothers were exposed to VC at an atmospheric concentration of 5500,18 000 or 33 000 mg/m3 (~2000, 7000, 12 000 ppm) for 2.5 h on the 18th day of pregnancy. The presence of VC in the amniotic Quid was also detectable (Table ID- Maternal loss was not encountered in the experiments- No difference in the weight gam of pregnant rats expressed as percentage of the starting body weight was found with the exception of group ttto exposed to VC during the third week of pregnancy. The weight gain in this group was lower, than in the other groups (Table I)- The maternal liver weight and liver weight/body weight ratio increased in response to trypan blue as well as to VC applied in the first or second week of pregnancy (P < 0.01 and P < 0.05, respectively) while no difference was seen in these parameters after VC exposure during the third week of pregnancy (Table D- No pathological change was observed in the liver of VC treated mothers at the light microscopic level. There was a marked periportal histiocytic reaction in the liver of trypan blue injected animals. The number of resorbed fetuses as well as the fetal loss taken as percent- l > . 48 RSV 0016874 TABLE II VINYL CHLORIDE LEVELS IN MATERNAL AND FETAL BLOOD AND AMNIOTIC FLUID OF CFY RATS ON THE 18TH DAY OF PREGNANCY AFTER 2.5 H OF EXPOSURE Inhalation chamber mg/m*) Maternal blood (ui/ml) Fetal blood (ug/ml) Amniotte fluid <Mf/al) 0 5500 (-*2000 ppm) 18000 (-*7000 ppm) 33000 -12000 ppm) 0x0 19.02 < 1.70 32.40 s 2.12 48.43 x 1.95 0x0 12.80 x 2.92 22.67 t 2.75 30.52 x 3.77 0x0 4.27 t 0.42 4.93 x 0.18 13.50 x 2.99 z,SM. ige of the total number of implants was significantly increased in the group exposed to VC during the first 9 days of pregnancy (P a 0.05); there was a endency of increased resorption and fetal loss, though not significant in the *roup exposed to VC during fire second week of pregnancy (P < 0.1) and no iifference in the parameters was seen after an exposure to VC during the bird week of pregnancy. There was an increase in the number of resorbed 'etuses as well as fetal loss in the group injected with trypan blue (P < 0.01). combined trypan blue administration and VC exposure was not more effective than the dye injection alone. The number of deal fetuses was ncreased by the trypan blue treatment (Table I). A slight tendency of increase in the ratio of retarded fetuses was seen in he groups exposed to VC during the first 9 days of gestation or injected rith trypan blue, but no significant change in the mean weight of fetuses or placentae and in the ratio of fetuses with weight retardation (less than 3,3 g) vas seen no matter during which time of gestation the VC exposure occurred, although the ratio of retarded fetuses was higher in the groups inhaling air, >r VC in the inhalation chambers during the third week of gestation, this ifference was not significant and probably may be due to the stressor effect f novel environment. The findings of the dissection and skeletal investigation of fetuses are hown on Table III. None of the malformations or anomalies could be ttributed to VC. >lSCUSS!ON Continuous exposure of rats to VC results in a permanent elevation of its lood level [28]. A permanent increase in blood level and the low molecular /eight of VC facilitate a rapid extnvascular distribution of the chemical 28]. One can assume, that during a continuous exposure of pregnant rats 49 U o TABLE III FINDINGS OF DISSECTION AND SKELETAL INVESTIGATION OF FETUSES Treatment Inhalation 24 h/day of pregnancy Air 1-9 VCb Air 1--9 1-9 VCb 1-9 S.c. injection days of pregnancy Phy*. sal* 7-8 Phya. aal Trypan b* 7-8 7-8 Trypan be 7-8 No. of littera examined 13 19 13 18 No. of live fetuses 171 223 131 196 Externa) malformations Exencephaly Umbilical hernia -- -- --2 ---- -- 1 No. of fetuses dissected 83 106 63 95 Internal malformations Internal hydrocephalus Anophthalmia Microphthalmia Polycystic lungs Thymus with processus Pyelectasia Dilatation of urinary bladder -- -- -- -- 4 2 - -1 12 1-- --2 69 46 3-- 1 2 2 -- 9 2 6 Air VCb 8-14 8-14 __ ---- 14 28 157 374 ---- ---- 76 185 1 -- -- -- 6 6 1 -- -- -- -- 9 20 8 Air VCh 14-21 14-21 _-- ---- 18 22 212 244 -- -- 102 1 -- -- 3 9 6 7 -- -- 117 _ 1 9 -- - -- _ -- 28 316 -- 166 __ _ _ ___ 3 RSV 0016876 Skeletal retardation aigns Poorly oaalfied aternebrae Bipart, vertebra centra Shortneaa of 13th rib. 2 8 Skeletal anomatiee Fueed aternebrae Supernumerary riba. 2 Skeletal matformattone Miaalng orbita Multiplex * 0.6 ml/100 g body art./day. b 4000 mg/m* (1600 ppm). 4 0.6 ml/100 g body art./day of 1% (ar/v) eotution cn J3 CD to VC the fetuses like the mothers are also permanently exposed. Our detection of VC in the amniotic fluid and in the blood of fetuses of pregnant rats exposed on the 18th day of gestation (Table II) justify this assumption. In order to study the possible teratogenic and embryotoxic effects of the compound, rats were exposed to VC at 4000 mg/m3 (1500 ppm) atmos pheric concentration continuously during the fust, second or third week, of pregnancy. In spite of the exposure at this very high level of concentra tion no obvious alteration in the behaviour of experimental animals was observed; their food and water consumption and weight gain did not differ from that of the controls and activation of self protective mechanisms [29] was not seen either. Although our study of the great number of fetuses gave essentially negative results, some tendencies found -- not reaching the level of signifi cance -- might deserve further attention. There was an increase in the number of resorbed fetuses in the groups subjected to VC during the first and second week of pregnancy. Though the increase in the number of implantations and higher birth rate might explain this difference in the group exposed during the second week; no increase in the number of implants was seen in the rats exposed during the first week. Thus it is most probable that the toxic effects of VC might explain the increased fetal loss close to the level of significance (P a 0.05). This is all the more probable, because toxic agents independent of their chemical nature have been shown to result in similar embryotoxic effects, when applied during the first third of pregnancy [30]. Among the offspring of mothers exposed to VC during the first week of pregnancy one case of microphthalmia and an other case of anophthalmia occurred. In spite of the fact that this was not consistent with a significant increase in the incidence of congenital malformations, these cases deserve further attention for the following reasons. None of these malformations was observed in the group exposed to air in the chamber, or in the untreated controls. Both malformations are related to the central nervous system, and an increased incidence of congenital malformations of the central nervous system have been brought into causal relationship with VC exposure by Infante [18]. On the basis of our results VC exposure in itself has no teratogenic effect in CFY rats, but an embryotoxic effect of VC exposure during the early stages of pregnancy at high atmospheric concentrations should be taken into consideration. A similar lack of teratogenic effect of VC has been reported by John et al. [22]. Their conclusion is based on studies of the effect of VC applied during arbitrarily chosen short periods of organogenesis. One could emphasize here that it is a minimal requirement of experimental studies aimed to reveal the teratogenic effect of any particular chemical that the pregnant mothers are exposed to the chemical in such a way as to provide continuous exposure of the fetuses during the whole period of organogenesis. The fulfilment of this RSV 0016878 requirement is particularly important with VC and other toxic agents taken up by inhalation and exhaled rapidly. In our view the teratogenic effect of a chemical cannot be excluded in studies using shorter exposure times than the whole period of organogenesis. We are not aware of any data on the effect of VC in early pregnancy. Even if the results of our animal experiments cannot be applied directly to the human and considering that an exposure to VC at 250 ppm atmospheric concentration would result in the saturation of the metabolizing capacity of VC [31], the hazards of an occupational exposure of women in the fertile age requires thorough consideration. Infante [18] claims that the high incidence of congenital malformations of the central nervous system in the neighbourhood of PVC producing plants is due to VC. Edmonds et al. [19] were not able to confirm this view. Ln this context we studied the possibility that even if VC has no teratogenic effect to induce malformations of the central nervous system it might potentiate a teratogenic effect of other agents. Trypan blue has been reported to be embryotoxic and bring about malformations of the nervous system [32--34]. Negative results have been obtained in this respect; a concomittant exposure :o VC did not affect either the teratogenic or the embryotoxic effects of rypan blue, as no higher incidence of congenital neural malformations exencephaly, anophthalmia, microphthalmia, aplasia of the orbit) or higher ?tal losses were encountered in the group with combined VC and trypan lue treatment. rKNOW^EDGEMENT The inical assistance of Mr Gy. Krasznai, Miss A. C&onka, Mrs Gy. 3moUu>yi, Mrs J. Nyilas is gratefully acknowledged, v TERENCES PX. Viol*. A. Bigotti and A. Caput, Cancer Rea., 31 (1971) SX6. C. Maltoni and G. Lefemine, Environ. Rea., 7 (1974) 387. Z. Maltoni and G. Lefemine, Ann. N.Y. Acad. Sci., 246 (1975) 185. i.X Creech and M.N. Johnaaon, J. Occup. Med., 16 (1974) 150. .X. Thomas and H. Popper, Ann. N.Y. Acad. Sci., 246 (1975) 268, .. Makk, P. Debitor*, XX Creech, Jr., L.X Ogden, B., E.H. Fadeil, C.X Songster, . Clanton, M.N. Johnaaon and W.M. Cristopherson, Cancer, 37 (1976) 149. ; M. Smith, D.MJ. William* and D.M.D. Evan*, Bull. N.Y. Acad. Med., 52 (1976) 17. Zimmermans and H. Edc, Virchows Arch. A. PathoL Anal. HiatoL, 368 (1975) Banach, C. Malaveiile end R. Monteaano, lot. J. Cancer, 15 (1975) 429. Malaveille, H. Baitach, A. Barbin, H.M. Camua, R. Monteaano, A. Croiay end P. quinan, Biochem. Biophys. Rea. Cenmun., 63 (1975) 363. -tubennan, H. Bartach and X Sacha, lot. J. Cancer, 16 (1975) 639. Loprieno, R. Barsle, S. Baroncelli, C. Bauer, G. Brocuetti, A. CammeUini, G. agnani, C. Com, G. Gervasi, C. Leporini, R. Nteri, AM. Road, G. Stretti and urehi, Mutat. Rea., 40 (1976) 65. 13 V. Rannog, R. GOtha sad CA. Wachtmekter, Chem.-Biol. Internet., 12 (1976) 251. 14 AJ. Cairo, J.B. Gottenplan and P. MUry, Mutat. Rat., 38 (1976) 61. 15 V. Dueatman, K. Huscbom and Li. SeUkoff, Mutat. Ret^ 31 (1975) 163. 16 F. Funai-Cmvioto, B. Lambert, i. Linaten, L. Ehrcaberg, A-T. Nataiajan and S. Oaterman-Golkar, Lancet, 1(1975) 459. 17 J. Ssentasi, E. Hornyak, Gy. Ungraty, A. Czeiaal, Z. Bognir and M. Timdr, Mutat. Raa. 37 (1976) 313. 16 P.F. Infanta, Lancet, ii( 1975) 1098. 19 L-D. Edmonidi, H. Falk and J.E. Ntscim. Lancet, ii (1975) 1098. 20 P.F. Infanta, J.K. Wagoner, AJ. MeMlehael and RJ. Waxwailer, Lancet, i (1876) 734. 21 GJ4. Paddla, Lancet, i( 1976) 1079. 22 JA. John, PA Smith, BJLi. Leong and BA. Scbwetz, Toxicol Appl Pharmacol., 39(1977) 497. 23 M. L&rincs, Munkarddalam, 24 (1976) 47. 24 NIOSH Manual of Analytical Methods, Method No.P and CAM-127 (1974). 25 J.G. WU*on and F. Warkany, Method* for admini*taring agent* and detecting mal formation* in experimental animal*, in Teratology: Principle* and Technique*. The Unitersty of Chicago Praaa, Chicago, 1965. 26 R.E. Staple* and V.L. 8chnaU, Stain. Technol, 39(1964)62. 27 JJK. Haaeman and Mi). Hogan, Teratology, 12 (1976) 166. 28 JJt. Witbey, J. Toxicol Environ. Health, 1 (1976) 381. 29 E.S. Reynold*, M.T. Molaas, S. Ssabd and RJ. Jaeger, Re*. Common. Cbem. Pathol. PhaxmacoL, 12 (1975) 685. 30 J. Elk, Proc. Ear. Sac. Toxicol., 16 (1975) 133. 31 Hil. Bolt, RJ. Laib, H. Kappu* and A. Bucbter, Toxicology, 7 (1977) 179. 32 L-Denkar, Teratology, 15(1977)179. 33 J, GOlmann, C. Gilbert, L Spence and T. GOlmann, S. Afr. J. Scl., 13 (1948) 47. 34 J. Warkany, J.G. Wilaoa and J.F. Geiger, J. Comp. Neurol, 109 (1958) 35. I \ ToxicoL O ELaev EMBR METH ARAN? Dtpertrr H-14S0 (Receive (Reviaio (Accept* SUMMA CFY 1500 rr h/day f toluene (266 p: mice * 24 h/d; inhaling None tions d; anomaL Benzen ment. The pregnar. hygiene INTROE The r and ter occupat in reape Most Support*. 6-11-0401 54 c RSV 0016880