Document 2jLrO4k5rG5z2kEGprxm46MN

Reproductin and Teratology Studies with Three Polychlorinated Biphenyls in Albino Rats .Otis E'. Fanchcr\ M. L. Keplinger, J. B. Plank, E. P. Wheeler^ and J. C. Calandra Industrial BIO-TEST Laboratories, Inc. Northbrook, Illinois Running Title: Reproductive Studies with 3 PCB's in Albino Rats. Present Address: 624 N. Abrego Drive, Green Valley, Arizona 85614, to whom proofs are to be sent. 2 Monsanto Company, St. Louis, Missouri. DSW 347218 STLCOPCB4085004 V ABSTRACT Reproduction and Teratology Studies with Three Polychlorinated Biphenyls in Albino Rats. Fancher, Otis E. , Keplinger, M. L. , Plank, J. B. , Wheeler, E. P. , and Calandra, J. C. (0000). Toxicol. Appl. Pharmacol, 00, 00 - 00. Three generation reproduction studies and teratology studies were conducted with 3 polychlorinated biphenyls (Aroclors 1242, 1254 and 1260) in albino rats. Aroclor 1242 at a ' 2< . dietary level of 100 ppm adversely affected mating and incidence of pregnancy during the second generation. Except for a. suggestion of some decrease in the inclination to mate among other groups fed 10 or 100 ppm of the test materials during the second and third genera tions, no other reproductive parameter was affected. None of the materials was judged to be teratogenic at dosage levels of 10 and 30 mg/kg. DSW 347219 STLCOPCB4085005 \ INDEX TERMS Aroclors Aroclor 1242 Aroclor 1254 Aroclor 1260 Polychlorinated Biphenyls Reproductive effects of, in albino rats Teratology study of, in albino rats / DSW 347220 STLCOPCB4085006 Reproduction and Teratology Studies with Three Polychlorinated Biphenyls* in Albino Rats Otis E. Fancher2 , M. L, Keplinger, J. B.' Plank, E. P. Wheeler^ and J. C. Calandra ' Industrial BIO-TEST Laboratories, Inc. Northbrook, Illinois Reproduction and Teratology Studies with Three Polychlorinated Biphenyls in Albino Rats. Fancher, Otis E. , Keplinger, M. L. , Plank, J. B. , Wheeler, E. P. , and Calandra,' J. C. (0000). Toxicol. Appl. Pharmacol. 00, 00 - 00. Three generation reproduction studies and teratology studies were conducted with 3 polychlorinated biphenyls (Arcolors 1242, 1254 and 1260) in albino rats. Aroclor 1242 at a ` dietary level of 100 ppm adversely affected mating and incidence of i' pregnancy during the second generation. Except for a suggestion of i some decrease in the inclination to mate among other groups fed 10 ' or 100 ppm of the test materials during the second and third genera tions, no other reproductive parameter was affected. None of the materials was judged to be teratogenic at dosage levels of 10 and 30 mg/kg. Background toxicological information has been summarized by Fancher et al. (0000) in the introductory paper of this series. Be cause of the demonstrated reproductive effects of polychlorinated DSW 347221 STLCOPCB4085007 biphenyls in various avian species and in view of the reported occurrence of jiveak deformities in chicks hatched from eggs treat ed with Aroclor 1242, the present study was conducted for assess ment of reproductive and teratogenic effects of 3 Aroclors in a standard mammalian species. . METHODS A. 3-Generation Reproduction Study The animals employed were weanling albino rats of the Charles River strain(Charles River Breeding Laboratories, North Wilming ton, Mass. ). A control group and 9 treatment groups, each consist ing of 8 male and 16 female animals, were employed, 3 treatment groups for each of the 3 Aroclors studied. All diets were prepared fresh each week by mixing the appropri ate Aroclor at levels of 1, 10 and 100 ppm with a standard pulver- .... ized stock ration(Special Mix Mouse Chow, Ralston Purina Company, St. Louis, Missouri). The control animals were fed the basic ration alone. Both food and water were available ad libitum. Except during mating trials, each animal was individually housed. All parental animals in all groups were maintained on the appropriate diets with out interruption until sacrifice which followed weaning of the Fb (second) litters. Initially the body weight of each parental animal was determined and all animals were wei ghed weekly until mating trials began. No further weights were determined except for terminal weights just STLCOPCB4085008 prior to sacrifice. Parental animals were observed daily for mort ality and behavioral reactions. Mating trials were initiated with each generation when the animals were 100 days old. The 16 females in each group were caged in pairs and mated with a male randomly selected from within the same group. Daily observations for evidence of copulation (copulatory plug or vag inal blood) were made and the male was removed if, during the next estrous cycle of ftye days, no further signs of mating were observed. \f Any male failing to mate within days was replaced by another male from the same group. Females were permitted access to a maximum of 3 males. Mating Indices and Fertility Indicies were calculated. All pups at birth were examined for gross physical abnormal ities and the numbers of viable and stillborn pups of each litter were recorded. Records of survival at designated intervals during the lactation period were maintained and litters of more than 10 pups were reduced to that number by random sacrifice on the fifth day of the lactation period. Lactation, Live Birth and Survival Indices were calculated. A final examination for physical abnormalities was made at weaning. Tire Fa (first) litters were weaned 21 days postpartum and the parental females were again mated after 10 days to obtain Fb(sccond) litters. Eight males and 16 females from the Fb progeny of each group were selected at weaning for use as parental animals for the next generation. ' DSW 347223 STLCOPCB4085009 A gross autopsy was performed on any parental animal which died during the study and tissues were preserved for microscopic examination when feasible. After weaning of the Fb litters, parental animals were sacrificed after determination of final body weights. All males and 8 females from each group were subjected to gross patho logic examination. The weights of the liver, kidneys, spleen, gonads, heart and brain were recorded and organ to body weight and organ to brain weight ratios were calculated. Microscopic pathologic studies were conducted on tissues from fiye male and five female parental animals from the control group and from each high treatment level group except for the F2 parents treated with Aroclor 1242. In this case tissues from parental animals treated at the intermediate level were examined. B. Teratology Study - Albino Rats The animals employed were female Charles River albino rats bred at the Charles River Breeding Laboratories, North Wilmington, Mass. These were confirmed pregnant by sperm-positive results of vaginal examination and were shipped on Day One of gestation, Day Zero being the day of insemination. . A control group and 6 treatment groups (treatment levels of 1 0 and 30 mg/lcg/day for each of the Aroclors) were emjdoyed, each consisting of 26 animals. The test materials were administered orally as solutions in corn oil using a hypodermic syringe equipped with a ball-tipped intubation needle. Solutions were prepared daily DSW 347224 STLCOPCB4085010 in concentrations such that all animals received equivalent quantities of corn oil on a mg/kg basis. Control animals were treated with corn oil alone. All animals were treated once daily.from the sixth through the fifteenth day of gestation (lOdoses). Food and water were available ad libitum. All animals were weighed daily during the dosing period and just prior to sacrifice. Daily records of mortality and reactions were maintained. All females were sacrificed by carbon dioxide asphyxiation on the 20th day of gestation. An incision was made in the abdominal wall and the full extent of both uterine horns was exposed. Fetal swellings, implantation sites and corpora lutea were counted, special attention being paid to -resorption sites or any other uterine abnormalities. The number of viable fetuses present in the uterus was determined, spontaneous movement and a more ruddy color distinguishing live from dead animals. Each fetus was removed by placing a small hemostat on the umbilical cord, flush with the abdominal wall, and cutting the cord distal to the clamp. Blotting paper was used to remove excess amniotic fluid and blood prior to weighing the animals. A\ External examinations were made for detection of abnormalities which might include hydrocephaly, exencephaly, meningoenceplralocele, simple meningocele, anophthalmia, microphthalmia, cleft lip, man dible or palate, oblique facial cleft, micrognathia, external ear abnormalities in size, shape or position, unusual size or position of DSW347225 STLCOPCB4085011 the limbs, number and disposition of the digits, umbilical hernia, gastroschisis, myelomeningocele, spina bifida and scoliosis. Two-thirds of the fetuses from each litter were examined for skeletal development using the alizirin red staining technique de scribed by Hurley (1965). Internal development of the remaining fetuses was evaluated using the free-hand razor blade sectioning technique of Wilson and Warkany (1965). ' RESULTS Statistical treatment of all data analyzed consisted of Analysis of Variance followed by application of "t"-tests to any significant dis closures. A. 3-Generation Reproduction Study No effect on body weights of either parental animals or weanlings was observed among any group of any generation. No behavioral reactions were observed and mortality was unaffected. Pathologic examination of the occasional animal which died during the study failed to reveal any relationship between ingestion of the Aroclors and the death of the animals. No gross pathologic findings among parental animals could be re lated to the ingestion of lest materials. Evaluation of organ weight and organ to body weight and organ to brain weight ratio data disclosed several randomly occurring intergroup differences. Only those effects related to the liver might be attributed to treatment with the Aroclors. There is an indication that the llil-ce materials at the level of 100 ppm DSW 347226 STLCOPCB4085012 may cause increased liver weights and increases in liver to body weights and/or liver to brain weight ratios. These effects, if they are real, were moderate in degree and inconsistent in pattern with respect to dose level, sex and generation. It has been shown in a chronic rat study (Fancher et al. , (0000) that each of the Aroclors caused liver effects at a dietary level of 100 ppm,, but the effects r, were delayed in development, being apparent at the 24-month, but_ not '* at the 12-month or earlier sacrifices. In the present study, the animals were treated for approximately 6 months only. No changes which could be correlated with treatment were observed among tissues from sacrificed parental animals. Findings for treated animals were essentially the same as those for control animals. Focal epithelial hyperplasia of the bladder was noted among some F2 animals; one control female, one male treated with Aioclor 1242 at 10 ppm, one male and 2 females (from the same litter) treated with Aroclor 1254 at 100 ppm, and one female treated with Aroclor 1260 at 100 ppm. This hyperplasia was associated with focal cystitis and, in some cases, with mineralized calculi. Mating indicies were somewhat decreased by all tliVee materials at levels of 10 and 100 ppm, particularly during the second and third generations, being most evident with Aroclor 1242 at the 100 ppm level. In this case also the incidence of pregnancy was low and since O only two females produced F2h litters, it was not possible to continue this level through the third generation. Data for reproduction perform- 0S'N3227 STLCOPCB4085013 ance are presented in Table 1. The number of live pups per litter born was not affected for any treatment group, and all pups delivered appeared normal. Erratic variations were observed in the number of pups weaned per litter but no consistent compound or dose correlation was apparent. Weanling body weights were normal for all groups and gross examination at weaning disclosed no external abnormalities. . Survival data are pre sented in Table 2. B. Teratology Study Maternal body weights and weight gains were slightly decreased by treatment with Aroclors 1254 and 1260. These data are given in Table 3. No dose-effect correlation was apparent. No deaths occurred among animals of any group and no abnormal reactions were noted. ' The results of the sacrifice on Gestation Day 20 are presented in Table 4. Animals in all groups were free of gross uterine ab normalities. There is a suggestion that the Aroclors may exert some effect on the incidence of resorptions but the slight magnitude of any effect and the absence of any clear-cut dose correlation make this of doubtful significance. No other reproductive parameter was affected. None of the materials at either level affected fetal body weights or sex ratios. A-fl fetuses appeared normal and were free of gross abnormal- OSVM 347228 STLCOPCB4085014 ities. Data regarding fetal skeletal development are summarized in Table 5. None of the materials had an effect on skeletal development at either treatment level. Examination of fetuses for internal development revealed atria of abnormal size among the progeny from all groups. The incidence of this abnormality did not differ significantly among the control and ; \. ' test groups. Twelve percent of the fetuses from dams treated with Aroclor 1254 at the dose level of 30 mg/kg had caudal renal ectopia. ^ \ ' % . ' \ `N _ - A .^ This abnormality was not seen among the progeny of any other group. / ' j (_.t / ` .u Data with respect to fetal internal development are summarized in lj.'- c ` Table 6. . ' ' DISCUSSION Y Aroclor 1242 at a dietary level of 100 ppm adversely affected mating and the incidence of pregnancy among albino rats during the ' second generation of a scheduled 3-generation study. Except for a- suggestion of decreased inclination to mate among other groups fed 10 or 100 ppm of the test materials during the second and third gen erations and some erratic variations in pup survival to weaning age, no other reproductive parameter was affected. None of the materials are judged to be teratogenic in albino rats at levels of 10 and 30 mg/kg. The non-occurrence of remil ectopia with 10 mg/kg of Aroclor 1254 or among the progeny of rats treated at either level with the closely related Aroclors makes it doubtful that this defect is a specific teratogenic response. 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'ndJ1 (Si in G3 H o * r-4 aJ > H > Jh d CO o Va id P O (SI N in H o O p-* V-O t'*" o V-Q n: o co co m co m o co <n ^ ro m h o m co co O lAvD cn ^ o O' v) O' CO ^ ^0s co co co oo f* h- co co id x o . T3 CCD PH cd > in u> 2 cn oo qs so O' co (\J *--< O O m <\J co <n h h m o vD pj m i- ^ o v^o t""" o r- r-- r-n h c> h co o m h- co 00 ^ ^ t4- o O' co xo r- m vO o co co os h X C3 2 o d ffi id > H1 (SI r--l tdo m -d1 mo ^ in h- in O m (S3 (\3 '--1 O O O r-~ o co o vo co co o --i o --i in i--i *d* O CO 00 O o o o o o o o o o o co o O O O CO o o P in cq X 43 1) P >d P --i o cj oo o in r~i cs3 1--i '--i in CO lO 00 N d oo >--i o o o co c> cc (mmh n co in r- o so o CS O O O O O' O CS O' O O' c- O' CO O' O' CO (C a 4+-->> pd ri P id P rt do id -O id P d P i P d ^ id P --i p-i oo na m m i--i i--i (M nj m tn --i --i (s] N m cn P P P P P P p p p p p p P PP P P P P O sD co P >-i 3(l Ho 'c o>^ r-* r> p p *a l-l O i--( u o 1-4 < o o o r-H r--< o o -X~ p Id P du 4- d o cq CQ rD a, d H P > <D in ex d r--i fd id p> m oo oo pp I1 X 03 x) c U d> Iu 2 in o o X M in P id P <d PQ id 4-> aJ in P, 0 d ,0 Pd * i--^ 4) > 3 in id ft >H d P o U-l O Oo P ii c o -M 4d-J 0 V d r0 1--c1 O o o o c ud o f-l fQ o P P, in P& Ph d 0P rH no d *H >o V-l p o -iud oo p II p4-1 IH PQ x o 0) T3 >d P 4ao->> p X in P id P fd4 o4-> id P -D id > in Pi ,n d id H P> 1+4 o oo Pp II X 03 T3 P^ dj i--H Q tf > m> 14 d co PSW 347235 STLCOPCB4085021 .+U> 'd -ruf O o O CO n H nj C n3 (,A5 m co o co t--i< rQ co 1--1 w <1 CO iP rH <fP > w rH H o rH O u o Mu MO-t < Q txo d o CQ T e s t M a te ria N one W e ig h t (g ra m s ) "a tio n D a y 12 15 D tal W e i G a in (gram s A M rH CO m m O CO rH c- CO CO co --H 1--< rH H o CM cm 0s- m CO co o rH O c- 0s 00 oo CO CO CO co CO CO CM C O CO co CO CO in t- c- c- co IT) CO ro CO CO CO co CM CO *>, O 'g O fP in in rH CO co Is- rH in H rH CO CO CO . CO co CO co CO CO CO CO v> CO rH o v> o CO CO o rH o o o CO CO CO CM CO CO CO vD CO ` o o rH o CO in o O o o CC CO CO rH CM CO rH iH o Ti to ./ ri O ^ <2 > t a> c D ^ - 1 o oo o oo 1 rH CO rH co rH CO r--H CO o nF m vO CO co CO rH rH rH uuu ooo rH rH rH Ouu Ooo uuu <<< DSW 347236 STLCOPCB4085022 TABL i 23. 54. o vO cm r--( d in cm cm CM w u o I--I n) Pi <ivi u o u o fcH rO <Vi i--I o u <y '>+hi o > O P *o0 Vi Pi 0) Pi >1 in o o ri iJ +J c rH u o P. o p kt d o I-- Pi Vi to P o vT "E d J w d Pi ft) 00 O' in o in o -H m r1 sD 00 00 o o 00 t--4 r--i nJ r- cm r- co o o o. o M1 O' o' t~ CM IT> 00 m O O' -H rQ m CM o --i O ft) r-- nJ tn o O^ CO *-H o LO CM CM r- o rM i--< i--H r--< i--i o r--r o 1--< -H J-< O r3 0< 0 U tJ o2 uA 0 i--4 ni 0 d 0 ro CO 0 O r--{ d*--* d 0 xtd to W to oo to > O ft) Q^ 0D r- CM O sD Sf r- r--< CM (M o t~4 CM i--i i"-( i--( t-H r--( r--1 fH i--( MO CM O CO 00 CM CM CM *-t --t O o oo T--l CO --< oo CO -H o CO oJ f--4 4- O 10 o rt H CO (M o <0 c o 2! f--I u o u LO CO u o f--( u o u < o vO CM u o u o u < CO 0 4-J CO 0 c^ to 4i -> CO 0 o d o Pi Vi Pi o to 0) Vi o to Vi a) Vi CM T) nj .n) rd ,d 0 a) r-i >--i n) nJ 0-H V0H 00 dd oo rO 3*1^ QS^ STLCOPCB4085023 Pk Im -H r- vO ^ m - ^ vD O t'- *-h O \D rH rH CHO or--t CO --* o^ r- \D O vO 00 vO iH vO O' (M h- r~ CO CO oi--H 00 <y 4-> ou H 4C->O do sO o CHO 0H d VH o in co r w Q FQ o bo o f-1 co d* CO Pk to H o JJ 1--oH nu oo fi p aa I--t d 40-*) r*H a CO ;/kg I 4. 27 co ^ O' (M O' o co in in o Pi co (M (NJ co CO W HH CO in --r-Hi tC-O co co cn f--i CO CO vO Pk o 11 11 11 1 1--t t-H ! 1 l l I 1l vD CO Tf in in o CO Pk ic--oH CO LO r--i O--t' vrOH CO rH w, O C h-H Q --< co CO m oo CO CcoO r--i m o rH 2t--i fa CO co in vO in vO Pk rr---( CO o oo T--i c0 in CO --t fQ HH O m m ii-nA (rN-(J (<\J-l o "H CO CO co r- co O oo o Pk o o t-H iH o o <: CO H tH d co --* f--( t--( oo in O > O CU QJ Ih n-> o U1 *-> 43 H^ l1 oo t-H CO oo *-H CO oo rH CO r--o1 O CO o^ M CO <1 ^ u rHo U O in u ro <- Ih r--O( u o u o vO CO < (0 CO CO H +d4 0 u 4<->u CO WH o 0 o 4o- o ~ u d TJ 0 d d o CO r--< d d c~l 4-> rU Q fo-c "M UH * t--1 (0 f-< to ?dh O a) a o 0 *0 0^ VM CO .o ^o (0 0-< +j oo i--I '-iH o > -r<t o *0 g r* CO <D CO 4d-> a> d O. 0 <y CO rQ 'a 0 a) o *O o o o 0 *0 u -j d u -- m d &0 10 0U -d D .n o ^ o <! & Pk Wr*> 11 t I <<* n O Q ^ h H P< ri DSW 347238 STLCOPCB4085024 d H(\]^H(\]Ot^HHH . SO H 0s ^ IT) o rH g t 'g g g g g g o r'OON'^fOMNin^lMlMt'HMMOrtvOh i-H fH rH (\J H o & o o d <D 'Ot'N^NNNHtOHOlflHCOHH I--< .--I Id H u 2 d a> P R to +j do v) o2 d m c 3d 'tf in no H dco * -1 o p Qh ^10--44 m O (0 u o C <J a) O b o <D > d to d --\ d frd P< o u o Pi a to <D QJ <D P. CO to d r-* XJ dH " J Eh 4- f--4 CO a 4-> UH +-> to 'TJ a) .3 .2 " ! d ^ -* --i p rj d r| d d ^ <J a> to f--I O W d to a u rs *-? d tyo, o; 1--4 u 4O-> r-4 O CO U d d r"^ ^ . d t) d d c t_i d 4M-> d o I--I dit .Hd d d U >h d t--4 to u <d r-H O rH tuO ^d O tUO d 'd d . d a p o .3 - rt d o to- i; d i-^ ?H +J d tr----<t d o to M sd d dd dd d d d d ^2 d WhlPWhlOwJOioJwJUio J 2 CO h4. o > o to a10 o Q | I o o oo oO fOrH CO rH ro i--H 4(-D> d 4J a to d ao H fsj to r--4 u o rH u o u < ID C4 t--4 u o . r-H u o u < o vO C4 1-H u o 1--4 V o u < DSW 347239 STLCOPCB4085025 FOOTNOTES ^ Aroclors 1242, 1254 and 1260. Monsanto Company, St. Louis, Missouri ^ Present address: 624 N. Abrego Drive, Green Valley, Arizona 85614 2 Monsanto Company, St. Louis, Missouri . DSW 347240 STLCOPCB4085026 REFERENCES Fancher, Otis E. , Keplinger, M.L., Wheeler, E. P. and Calandra, J. C. (0000). Toxicolog.icaLgtudies of three polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 00. 00 - 00. Fancher, Otis E. , Keplinger, M. L. , Plank, J. B. , Wheeler, E. P. , and Calandra, J. C. (0000). Chronic toxicity of three polychlorinated biphenyls in albino rats. .Toxicol. Appl. Pharmacol. 00, 00 - 00. Hurley, L. S. (1965). Demonstration A-alizirin staining of bone. Supplement to Teratology Princi pies and Techniques pp. 121 - 122. Berkeley, California. Wilson, J. G. and Warkany, J. (1965). Teratology Principles and Techniques pp,271 - 277. Univ. of Chicago Press, Chicago, Illinois. DSW 347241 STLCOPCB4085027