Document X72wLor5GkdnaRebvZknDD8JG

ru\o vme\tal and applied toxicology i 4 A Three-Generation Rat Reproductive Toxicity Study of Vinvlidene Chloride in the Drinking Water12 K.D. NtTSCHKE, F.A. SMITH, /,F. QUASI, J.M. NOKKIS ond B.A. SCHiVETZ Toxicology Research Laboratory, Health and Environmental Sciences, U S.A., Don Chemical U.S.A., Midland, MI aS640 ABSTRACT A Three-Generation Rat Reproductive Toxicity Study of Vinylidene Chloride in the Drinking Water. Nitschke, K.D., Smith, F.A., Quast, J.F., Norris, J.M. and Schwetz, B.A. (1983). Fundam, Appl. Toxicol. 3:75-79. A reproduction study was conducted with Sprague-Dawley rats to evaluate the effects of parental exposure through three generations to drinking water containing 0, 50,100 or 200 ppm vinylidene chloride (V DC) on reproduction and the development of the resultant offspring. A low fertility rate was observed in three of the four groups including controls, for the fo generation rats. Remating the fo adults to produce the fib litters resulted in a lower fertility of dams ingesting 200 ppm VDC than of dams ingesting 0,50 or 100 ppm. However, the fertility index of the fo dams ingesting 200 ppm VDC in the drinking water was higher for the fib litters than for the fu litters. No overall decrease in fertility was observed in subsequent matings to produce the fj, f3, f3b or f^ litters. Although neonatal sur vival was decreased from concurrent control values in the h and fi. litters of dams ingesting VDC in the drinking water, the survival indices of the f2 litters were within the range of control values for this strain of rat in this laboratory. The apparent effect seen in the fo. litters was not repeated in subsequent matings of the same adults to produce either the fsb or the fac litters. Consequently, the decreased survival observed in the (3, was interpreted as being due to chance. Histopathologic examination of tissues of rats (f\ and f2 adults) exposed to vinylidene chloride in the drinking water in utero, during lactation, and post weaning revealed slight hepatocellular fatty change and an accentuated hepatic lob ular pattern of a reversible nature in the adult rats. In con clusion, ingestion of drinking water containing vinylidene chloride at concentrations which caused mild, dose-related changes in the liver, did not affect the reproductive capacity of rats through three generations which produced six sets of litters. INTRODUCTION Vinylidene chloride(1,1 -dichloroethylene, VDC) is an impor tant material in the manufacture of various resins and films. Reports on the acute toxicity of vinylidene chloride m rats indicates moderate toxicity when administered by gavage (Jenkins, 1972). Vinylidene chloride was not teratogenic when ingested by pregnant female rats at concentrations as high as 200 ppm in the drinking water (Murray, era/, 1979), However, no informa tion was found in the literature on the effects of ingested vinylidene chloride upon reproduction in rats Therefore, the purpose of the study presented herein was to evaluate the effects of ingested vinylidene chloride on the reproductive capacity of rats receiving the compound continuously through out three generations. Rats were given the test material in the drinking water rather than in the diet because VDC was found to be stable in water for 24 hours but was rapidly lost due to its volatility when incorporated in the diet, MTH9S Test materiaI Production grade vinylidene chloride3 was used. The test material purity was 99.5% minimum as determined by gas chromatography. Since the vinylidene chloride used in various copolymers for food packaging applications is distilled to remove the inhibitor MEHQ. the test chemical was also dis tilled prior to addition to the drinking water. Animats Male and female Sprague-Dawley rats* were housed sepa rately by sex except during mating periods in suspended wirebottom cages (two per cage). The rats were allowed laboratory chows and tap water ad libitum. Animals were housed m a room controlled for temperature (70F), humidity (50%) and light cycle (12 hours light and 12 hours dark). Experimental design Throughout the three generations, the test animals were continuously given tap water containing 0, 50,1Q0or200ppm VDC as the only drinking fluid. The maximum concentration of vinylidene chloride given in the drinking water, 200 ppm. was the upper limit of solubility of the compound in water. The fo rats were randomized into groups containing 15 male and 30 female (control group) or 10 male and 20 female rats (50,100 or 200 ppm groups) at 6-7 weeks of age. After 10O days of ingesting VDC in the water, the fo rats were mated to produce the f;, litters. Ten days after the last litter was weaned, the f0 rats were remated to produce the fib litters The rats serving as parents for the f2 and U generation were randomly selected with the aid of a random number table from 'This study ssas supported by the companies sponsoring research on stnshdene ohlonde and administered by The Manulacturing Chenmi > Association IHiesemed. in pari, at Ihe 17th Annual Meeung of the Society ot 1 oxicoloet Sun Francisco. CA, March 12-16, 1978. 'The Dow. Chemical Company, f-icepuii, TV ;Rats were obtained from Sparun Re*e.irch Animal', Inc . Hatlett, Ml Hunna Lboraior> Chgw, Ralston Purina Co . Si Louis. MO Copyright H&J, Society of Tqmcology Fundtmenttl and Applied Toxicology m 3-*/B3 75 TABLE 1 Fertility Index 'FI) of Rats Ingesting Vinylidene Chloride (VDC) Generation fo adults: fu litters fit litters ft adults* h litters f> ddLilts, ha litters f3b litters ?3c litters 0 FI" % 17/30 23130 57 77 27/30 90 29/30 32/36 20136 97 89 56 ppm VDC in the drinking water 50 100 FI FI % 200 Ft 19/201' 95 13 20 65 9/20 15120 73 lo 20 80 11/20 45 55 14/20 70 lo 20 80 20/20 100 19/20 22/26 16/20 95 16 20 80 19/20 85 1'23 S3 21/24 so 0'23 39 17/24 95 58 71 'Fertility Index (FI) = number of females delivering a litter number of females placed with a male. hSignificant!y different from the control value bv Fisher s exact probability test, p < 0.05, the f(a and f> litters, respectively. The fib andfj rats were mated at approximately 1 10 days of age to produce the h and f3 litters, respectively The fa rats were remated to produce the fab and fac litters, ten days after the last fj. and f3b litters were weaned, respectively. Preparation of test solutions Throughout the reproduction study, the water bottles were emptied and refilled daily from freshly prepared stock solu tions of vinylidene chloride The concentration of VDC present in the water decreased approximately 10% in a 24 hour period. Mating procedure For the initial mating of the fo rats, a male was placed with two females from the same group for a 15-day mating period (three estrus cycles). After weaning the f,, litters, the fo rats were remated to produce the f^ litters Each female was placed with a male for three 10-day mating periods. The pan beneath the cage was examined each morning for the pres ence of a vaginal plug. If a vaginal plug was observed, the female was placed in a separate cage. The remating and altered mating procedure was undertaken because of the low fertility rate observed in the initial mating of thefn rats, including the controls. In the subsequent matings of the fi and fj rats, a female was placed with a male for two 6-day mating periods (different male for each period) separated by a 6-day resting period. The mating period for the U and f3 generations was shortened to six days because we found that the fo rats did not mate after six days of cohabitation. After the mating period, the females were placed in individ ual cages containing ground corn cob litter for nesting. A screen floor was placed within each cage at the end of the first week of lactation. Cross fostered fitters Pups from eight dams ingesting 200 ppm VDC in the drink ing water from the fjv litters were randomly reduced to 8 per litter on the day of birth and raised by dams ingesting only water. Pups from eight dams ingesting water from the f3b litters were randomly reduced to eight per litter on the day of birth and raised by dams ingesting 200 ppm VDC. 76 Observations Since the animals from the fo generation were a subgroup from a larger group of animals used for concurrent 90-day and 2-year toxicity studies of vinylidene chloride in the drinking water, no body weights and no food or water consumption data were obtained from the fo animals in this reproduction study. Body weights and food and water consumption data were obtained from the subgroup designated for the 90-day study (Quast, 1983). For the remaining generations, body weight and water consumption of male and female rats were recorded at least weekly until mating. After mating, the females were observed daily for signs of normal or abnormal parturition. The following records were maintained: date of parturition (Day 0), number of live and dead newborn, number of live pups on days, 1, 7, 14 and 21, litter weights on days 1, 7 and 14, and individual weanling weights on day 21. On days 1,7,14 and 21 each pup was examined for external anomalies, signs of toxic ity and sex. Necropsy, organ weights and histopathoiogy - weanlings All weanlings which were not selected as future parents were necropsied. Body weights and liver and kidney weights TABLE 2 Average Number of Days Between First Cohabitation and Parturition Among Rats Ingesting Vinylidene Chloride (VDC) ppm VDC in the drinking water Generation 0 50 100 200 fo adults: fi. litters fib litters 28 4* 26 2 27 4 26 4 27 3 33 9* 28 5 26 2 ft adults; fj litters 27 3 26 3 26 2 26 3 h adultsf3. litters fib Inters fi litters 25 2 25 2 26 3 23 2 24 2 25 * 2 25 2 25 t 3 27 4 26 3 23 X 3 25 X 3 "Mean t S D. ''Significantly different from the control value by Dunnetts test, p < 0 05. Fundam. Appl Toxicol P) March/April I9SJ SX, 70781 TABLE 3 Average Number of Pups per Litter at Birth Among Rats Ingesting Vinylidene Chloride (VDC) Generation fi. fits h fu fk ppm VDC in the drinking Hater 0 50 100 200 10 2* s4 94 10 4 12 J 10 3 94 93 11 3 11 4 11 4 11-3 10 = 3 9=4 12 r 2 12 = 3 11 = 4 10-5 ii -- i 0"3 ie = 3 n=4 10 = 5 =4 Mean numbers of pups (live and dead) S.D. No values were significantly different from control value by Dunnett's test, p < 0.05. from two to seven 21-day old pups/sex/concentration level from thefis, U and fab litters were recorded. Paraffin-embedded sections of the liver and kidneys from these animals were stained with hematoxylin and eosin and examined histologically. Necropsy, organ weights and histopathofogy -- adults Rats found dead or moribund during the course of the study were necropsied. The h and f; adults were also necropsied after the fj and fac litters, respectively, were weaned. Blood urea nitrogen (BUN) levels and serum glutamic pyruvic trans aminase (SGPT) and alkaline phosphatase (AP) activities were determined from 10 fa adult rats/sex/dose level Weights of brain, heart, liver, kidneys, and testes were recorded from at least 10 male and 20 female fj and f3 generation rats at each dose level. Paraffin-embedded sections of the liver and kid neys from at least 10 fi and f3 adult rats/sex/dose level were stained with hematoxylin and eosin and examined by light microscopy. In addition, due to the advanced age of the f. rats, all grossly visible masses were examined histologically after similar preparation. Statistical analysis Statistical evaluation of the survival indices was made by the Wtlcoxon Test as modified by Haseman and Hoel (1974). Fertility index, sex ratio and certain gross and histopathologic data were statistically analyzed by the Fisher Exact Probability Test (Siegel. 1956). Analysis of the neonatal and maternal body weights, number of days between observing a vaginal pi ug and parturition, and number of days between first cohabi tation and parturition, food consumption and water consump tion data was made by an analysis of variance and the means were compared with control values by Dunnett's test (Steel and Torne. 1960) The level of significance reported m all cases was p <1 0.05. RESULTS Concentration of VDC in tha water Analyses of the water solutions for VDC content were con ducted 35 times during the test period. The mean ( S 0.) concentration of VDC in the drinking water, as determined by gas chromatographic analyses, was 68 21 ppm, 99 22 ppm and 206 33 ppm for the respective nominal concentra tions of 50. 100 and 200 ppm. Clinical observations Ingestion of water containing as high as 200 ppm VOC did not significantly alter the physical appearance of the f0 ani mals. The body weight, water consumption and food consump tion of the rats from the concurrent 90-day toxicity study were not significantly affected by the addition of vinylidene chloride to the drinking water (Quast, 1983). Likewise, the physical appearance, body weight and water consumption of the fi, fj and f3 rats was not affected by drinking water containing as high as 200 ppm VDC. Fertility The fertility of the T rats was unusually low in thecontrol rats and 2 of the~3Tr~dubsexposed to VDC for the fi. litters (Table 1). When the fo adults were remated to produce fib litters, the fertility of dams ingesting 200 ppm VDC was lower than the fertility in dams ingesting 0, 50 or 100 ppm but higher than it was when these dams ingesting 200 ppm VDC were mated the first time to produce the ft. litters. When thef? rats were mated to produce the f* offspring, a decreased fertility was observed in thecontrol and 100 ppm groups; the age range for the h rats at the time of mating for the litters was 276-298 days and the advanced age probably contributed to the decreased fertility. No significant increase from the control values was ob served in the mean number of days from copulation to delivery i n any generation of rats ingesting as high as 200 ppm VDC. A significant increase in the average number of days from cohab itation to delivery was observed in the second mating of the fo rats ingesting 100 ppm VDC in the drinking water (Table 2);** however, there was no effect on the mean number of days > from cohabitation to delivery at the higher concentration of VOC in any generation. TABLE 4 Postnatal Survival Index (PSII Among Litters of Rats Ingesting Vinylidene Chloride (VDC) ppm VDC in the Drinking Water 0 50 100 200 Generation PSP ao PSI PSI PSI % fu 153/178 So 145 lo$ 53 115,126 91 81/102 79 fih 155il0 52 n: mo 50 101.140 72 65/87 75 h 234/252 3 121 14 51" 145,142 1691206 82b . f,. 215/296 73 211 45= 1 74 192 33s i 60/215 2Sb ' f3b 214/306 70 153 2cC o 167.207 81 114/148 77 fv 193/205 143 171 55 86'91 94 117/152 77 ^Postnatal Suf\ ivj! Index (PS{i is the r/jmber of iueborn jpups surviving to 21 days of age/number of (iveborn pup> Significant!) different from the cort ro! *. alue b\ t he Kilcoxon test as modified by Haseman a nd Hoel, p< 0 05, Fundamental and Applied Toxicology (3j3-t/S3 77 table 5 Postnatal Body Weight Among the Offspring of Rats _______Ingesting Vinyiidene Chloride *VDC)______ ppm VDC in the Drinking Water Generation Day 0 50 100 200 fl. 1 7 X 1* 71 71 7 I 7 15 X 2 13 15 - 7 13 z 3 14 28 Z 4 17 - 7 2S - 3 25 z 4 21-M 42 z 7 44 42 z 5 41 Z 21-F 41 = 7 40 X A 42 z 6 3o z 6 fib 1 7z1 7- 1 7zi 7X1 7 14 z 3 14 - 3 16 z 1 14 z 5 14 29 r 7 28 - 5 30 z 5 27 Z 7 21-M 45 z 5 45 = 7 46 z 9 42 r 11 21-F 43 z 7 41 - 7 43 - 9 39 3 h 1 8z 1 7- 1 7X1 7X 1 7 16 z 3 14 z 14 - 14 - 3 14 28 z 5 25 z 4 23 5b 2o - 5 21-M 42 r 5 35 = o5 36 z 6 38 X 8 21-F 40 - 7 35 5 34 z 7 39 3 fa. 1 7z 1 7X 1 / X1 61 7 14 21-M 14 r 2 25 r 4 36 r 7 12 - 5 24 - 5 36 - 9 12 z 3 22 z 6 35 z 3 11 z` 20 -4 4k 35 -k- 4 21-F 34 = 0 32 = 9 33 - 7 30 X 6 hb 1 7xI 7X 1 8-1 7X 1 7 15 z 4 14 z 3 14 z 3 13 z 4 14 26 z O 26 z 3 25 - 4 24 z 6 21-M 43 z 10 37 z s 39 - 6 38 > 8 21-F 42 - 5 57 - 5 33 X 9 36 -L. 8 fib 1 3X I 7X 1 8X2 7 3: 2 7 16 3 16 - 3 17 X 4 16 X 4 14 30 = 5 29 ~ 6 30 * 6 30 X 6 21-M 49 z 9 46 - 10 47 - n 51 X 9 21-F 45 - 9 45 r S 48 r 14 45 X 8 "Mean of litter means x S D. (gl Significantly different from the control value by Dunnett's test, p < 0.05. Litter sue and survival The average number of pups per litter (live anddead) for any group ingesting VDC in the drinking water was nni-.-significantly different from the respective control value in any gener ation (Table 3) No significant decrease in the percentage of pups alive at birth was observed in any group ingesting VDC. The postnatal survival index among the groups ingesting VDC (the percentage of liveborn pups surviving until 21 days of age) was not significantly different from control values in the fu and fib litters (Table 4). The postnatal survival index for the f3 litters ingesting 50, 100 or 20(3 ppm VDC in the drinking water wa significantly decreased from the concurrent control group. The decreasedpostnataj survival in the U generation (Table 4) was most likely due to the larger.litter size at.birth (Table 3). The postnatal survival index for the f3, litters was significantly decreased in a dose related mannerTn'dams given VDC SubT sequent breeding of the same f2 dams to produce the fjb and f litters did not show a significant decrease in postnatal survival from the concurrent control groups Similarly, the postnatal survival index for the two sets of cross fostered litters (0 and 200 ppm VDC in the drinking water) were comparable to each other arid the fib control litters (data not shown). Postnatal body weight The average body weight of the T, or fib pups from dams ingesting vinyiidene chloride in the water was not significantly different from that of the control pups (Table 5). Significant decreases in postnatal body weight were observed in the ft litters from dams ingesting 50 or 100ppm VOC in the drinking water. However, the decreased body weight in these litters relative to controls was most likely due to the increased number of pups/litter at birth (Table 3) from the dams ingest ing 50 or 100 ppm VDC. The postnatal body weights of pups in the U litters from dams ingesting 200 ppm VOC in the drinking water were not significantly different from control litters. The average body weight of the fu pups from dams ingesting 200 ppm vinyiidene chloride was occasionally significantly de creased from that of the control pups. No effect on postnatal body weight was detected in the fa. pups from dams ingesting 50 or 100 ppm VDC or from the f3k or fu litters ingesting 50, 100 or 200 ppm VOC in the water. The body weights of the dams ingesting VDC were not signif icantly affected during the period of lactation. Gross pathology, organ weights and histopathoiogy -- weanlings A necropsy was performed on the 21 -day old pups that were not randomly selected as parents for the next generation. No gross lesions due to the ingestion of VDC in the drinking water were observed in the pups. Nor was any effect observed m the absolute or relative liver or kidney weights of 21 -day old pups from dams ingesting VDC. Light microscopic examination of 78 Fundam. Appl. Toxicol (3) March/April J9S3 SL 100789 the liver and kidneys of the weanlings revealed no histopatho logic alterations considered to be related to the ingestion of VDC in the drinking water. Gross pathology, organ weights and histopathology -- adults Gross examination at necropsy of the f, and f5 adults revealed no pathologic alterations considered to be related to the ingestion of vinylidene chloride in the drinking water Upon gross examination, the h male rats ingesting 200 ppm VDC in the drinking water had an increased incidence and severity of chronic renal disease which was accompanied by a loss of body fat (mtraabdominal deposits). Chronic renal disease occurs spontaneously at a high incidence among the male rats of this strain hence its relation to VDC is uncertain Gross examination of the liver of the fj female rats ingesting 100 or 200 ppm VDC in the drinking water revealed a pale appear ance, small pale foci and an accentuated lobular pattern. No organ weight changes considered to be related to the ingestion of VDC in the drinking water were observed m the fj adults; elevated relative liver weights of the female rats ingest ing 200 ppm VDC were observed in the U generation. An increase in SGPT activity (25% above control mean) was observed in female f* rats ingesting 200 ppm VDC in the water and may have been due to ingestion of VDC No changes occurred in values for BUN or AP inf,> rats ingesting as much as 200 ppm VDC in the water. Chronic renal disease, which occurs spontaneously at a high incidence in males of this strain, was observed histologi cally at a higher incidence among the male fi rats ingesting 200 ppm VDC in the drinking water. Light microscopic examin ation of the liver and kidneys revealed an accentuated hepatic lobular pattern and a minimal degree of hepatocellular fatty change in the ft rats ingesting 100 or ZOO ppm VDC in the water. Similar hepatocellular fatty changes were observed m each group of fj ratsingesting vinylidene chloride in the drink ing water. Due to the advanced age of the f2 adults, 384-406 days old, all grossly visible masses were also examined by light microscopy but no effect due to ingestion of water containing VDC was observed. DISCUSSION This study evaluated the potential of vinylidene chloride to interfere with the reproductive capacity of rats ingesting the compound in drinking water during three successive genera tions which produced six sets of litters. Fluctuations in reoroductive performance were observed but were not relatable to ingestion of VDC in the drinking water. For example, at the two highest concentrations (200 and 100 ppm), and in the controls, fertility was slightly decreased in the fo generation relative to historic controls. An improvement in reproductive perfor mance was observed in subsequent generations. The survival indices of neonates during lactation were decreased in a non-dose response manner in the f> generation and in a dose related manner in the f?. generation when com pared to concurrent controls. Survival in the fn, litters of dams ingesting 100 or 200 ppm VDC was higher than in concurrent controls. The mean and standard deviation of the neonatal survival from all control litters in this Study and from historical background data is 83 10% and 83 3%, respectively. Except for the f n set of litters, mean values for neonatal survi val for the 50. 100, and 200 ppm groups was 80. 83, 78%, respectively; these values fall within the limits of historical control data. In an effort to determine whether or not the decreased neonatal survival in thef i, litters was due to causes other than chance, ihe ft adults were remated to produce the f-.i, generation and several of the litters were cross-fostered, i.e., the litters from dams ingesting 200 ppm vinylidene chlo ride were raised by dams ingesting only water and vice versa No decrease in litter size was observed in either of the crossfostered sets of Inters or in the litters raised by their own mother.- In fact, survival in the litters of dams ingesting 100 or 200 ppm VDC was higher than concurrent controls This would indicate that there was no treatment-related effect on the neonates, the dams, or the neonates and dams combined In addition, a third mating of the f2 adults to produce the f* offspring did not-reveal any significant effect oh neonatal sur vival which was attributable to treatment. Consequently, the decreased survival observed in the f3, generation was inter-' preted as being due to chance. At necropsy and in the subsequent histopathologic exami nation, the adult fi rats ingesting 100 or 200 ppm VDC and the adult fj rats ingesting 50, 100 or 200 ppm VDC exhibited a significant increase in the incidence of slight hepatocellular fatty change and an accentuated hepatic lobular pattern. Similar hepatocellular fatty changes and accentuated lobular patterns were observed in rats inhaling 25 or 75 ppm VDC for 18 months (personal communication, J.F. Quest); these changes were found to be no longer discernible within 6 months after the exposure to VDC had terminated. Therefore, this alteration was considered to be readily reversible. No fatty changes were observed in the livers from fj, fj and fa generation weanlings examined histopathologically at 21 days of age. In conclusion, ingestion of vinylidene chloride in the drink ing water by rats at concentrations which cause mild, doserelated changes in the liver did not affect the reproductive capacity through three successive generations which pro duced six sets of litters. REFERENCES Haseman, J.K. and Hoel, D.G. (1974). Table of Gehan's generalized Wilcoxon test with fixed point sensoring. J. Statist. Comput. Simut. 3:117-135. Jenkins, L.J., Jr,, Trabutus, M.J. and Murphy, S.D. (1972). Bio chemical effects of 1,1-dichIoroethylene in rat*. Comparison with carbon tetrachloride and 1,2-dichloroethylene. Toxicol. Appl. Pharmacol 13:501-510. Murray, F.J., Nitschke, K.D., Rampy, L.W. and Sehwetz, B.A. (1979), Embryotoxieity and fetotoxicity of inhaled or ingest ed vinylidene chloride in rat* and rabbits. Toxicol. Appl. Pharmacol. 49:189-202. Quast, J.F., Humiston, C.G., Wade, C.E., Ballard, Beyer, J.E., Sehwetz, R.W. and Norri*. J.M. (1983). A chronic toxicity and oncogenicity study in rats and subchronic toxicity study in dogs on ingested vinylidene chloride. Fundam. Appl. Toxicol. 3:55-62. Siege), S. (1956). Sonparametric Statisticsfor the BehavioralSciences p 96, McGraw-Hill Book Co., Inc., New York, NY. Steel, R.G.D. and Torrie, H.H. (I960). Principles and Procedures of Statistics, pp. 101, 194, McGraw-Hill Book Co., Inc,, New York, NY, Fundamental and Applied Toxicology (3)3-4/83 SXj 100790 79