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K^0D/H/I- 0+1 Huntingdon u'e Sciences STUDY NO. 96-4080 VINYL CHLORIDE COMBINED INHALATION TWO-GENERATION REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDY IN CD RATS Developmental Toxicity Study Final Report Submitted to: Chemical Manufacturers Association Chemstar Department 1300 Wilson Boulevard Arlington, Virginia 22209 Attn: Hasmukh C. Shah, Ph.D. Date: 30 January 1998 Page l DO 136288 CONFIDENTIAL Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 3 Final Report SCIENTIST SIGNATURE PAGE The following Scientists were responsible for the overall conduct of this study. Departmental supervisory personnel are listed on the personnel page ofthis report (Appendix Q). Raymond E. Schroeder, M.S., DABT Study Director So <r Date 'o-m/ Date SCIENTTFIC REVIEW The following Scientist has reviewed and approved this report. Dean E. Rodwell, M.S. Vice President, Toxicology Operations CoOoNFTDFNTTAl Huntingdon Life Sciences Developmental Toxicity Study 96-4080 QUALITY ASSURANCE STATEMENT Page 5 Final Report Day 21 Lactation Pup Selection and Sacrifice Developmental Toxicology Report Developmental Toxicology Analytical Report 10 Sep 97 26 Jan 98 to 29 Jan 98 29 Jan 98 10 Sep 97 29 Jan 98 29 Jan 98 Date DO CONF 1 TDFN'Vf AV Huntingdon Life Sciences Developmental Toxicitv Studv 96-4080 TABLE OF CONTENTS Page 7 Final Report STATEMENT OF COMPLIANCE.............................................................. 2 SIGNATURE PAGE...................................................................................... ,,.3 QUALITY ASSURANCE STATEMENT................................................................................-......... 4 ABSTRACT..TM...................................................................................................................................... 6 TABLE OF CONTENTS............................................................................... 7 1. INTRODUCTION. ___10 2. MATERIAL AND METHODS 2.1. Regulatory References.....................................................................-------- ....-----10 2.2. Study Management................................................................... --.........................11 2.3. Experimental Outline................................................ .......................................-..... 12 2.4. Study Dates.....................................................................................-............... ~......12 2.5. Test Material.......................................................................................... -................ 13 2.6. Test Animals.......................................................................... 14 2.7. Selection/Group Assignment................................................. ................................. 15 2.8. Mating................................................................................................................ .......15 2.9. Animal Identification...............................................................................................16 2.10. Veterinary Care...................................................................................... 16 2.11. Husbandry During Non-Exposure Periods.................................................... ......... 16 2.12. Test Material Administration.................................................................................... 18 2.13. Experimental Evaluations..................................................... .................................. 19 2.14. Postmortem .................................................20 2.15. Statistical Analysis..................................................................... 23 2.16. Data Storage............................................................................. 24 2.17. Protocol Deviations............................................................. .................. ...................25 cnoonfidfnttw Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 9 final Report TABLE OF CONTENTS APPENDICES A. Individual Clinical Observations During Gestation. _ A-I B. Individual Gestation Body Weights (grams)............ ...B-I C. Individual Gestation Body Weight Gain (grams).... ,,.C-1 D. Individual Gestation Food Consumption (grams/animal/day)_______________________ -D-l E. Individual Gestation Food Consumption (grams/kg/day)............................. .................... ...E-l F. Individual Female Reproduction Data and Mean Fetal Weight Data_________________ ...F-I G. Individual Gravid Uterine Weight and Net Body Weight Change (grams)G-I H. Individual Fetal Body Weights (grams)................................................................................... H-l I. Individual Maternal Necropsy Observations.............................................. ,,............................. I-I J. Individual Organ and Final Body Weights................................................. J-I K. Individual Fetal External Observations.......................................................................... K-l L. Individual Fetal Visceral Observations............................................................................... L-l M. Individual Fetal Skeletal Observations...................................................................................... M-I N. Inhalation Report........................................................................................................... N-I O. Analytical Report - Gas Chromatography Methodology and Validation_______ _____ 0-1 P. Huntingdon Life Sciences Historical Control Data............................................... P-1 Q. Testing Facility's Personnel...................................................................................... Q-l R. Protocol............................................................................................................ ...................______R-l CnOoNFTOHNTTAl Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 11 Final Report Rule, Federal Register, Volume 56, No. 32, February 15, 1991, pp. 6426*6505 effective March 18,1991. 2.1.4. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY Currently acceptable practices of good animal husbandry were followed e g-, Guide for the Care and Use of Laboratory Animals. National Academy Press, 1996. Huntingdon Life Sciences, East Millstone, New Jersey is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). 2.2. STUDY MANAGEMENT 2.2.1. SPONSOR Chemical Manufacturers Association Chemstar Department 1300 Wilson Boulevard Arlington, Virginia 22209 I i 2.2.2. SPONSOR REPRESENTATIVE Hasmukh C. Shah, Ph.D. 2.23. TESTING FACILITY Huntingdon Life Sciences P.O. Box 2360 Mettlers Road East Millstone, New Jersey 08875*2360 23.4. STUDY DIRECTOR Raymond E. Schroeder, M.S., DABT DO 136793 CONFIDFNTTAl Huntingdon Life Sciences Developmental Toxicity Study 96*4080 2.4.5. TERMINATION OF EXPOSURES 10 April 1997 2.4.6. TERMINAL SACRIFICE 2-11 April 1997 2.4.7. STUDY TERMINATION Date Final Report is signed by the Study Director. 2.5. TEST MATERIAL Vinyl Chloride 2.5.1. SUPPLIER The GEON Company Pedricktown, NJ 08067 2.52. LOT NUMBER 1 2.55. DATE RECEIVED 28 August 1996 2.5.4. DESCRIPTION Colorless gas 2.55. PURITY >99.9% 25.6. STORAGE Room temperature Page !3 Final Report Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 15 Final Report 2.6.4. AGE AT RECEIPT Females were 57 days in age at receipt 2.6.5. WEIGHT AT GESTATION DAY 0 Mean: 213 grams Range: 172-252 grams 2.6.6. JUSTIFICATION FOR ANIMAL SELECTION The rat is a rodent animal model commonly utilized m reproduction/developmental toxicity studies as recommended in the referenced guidelines. In addition, a historical data base is available at Huntingdon Life Sciences, East Millstone, New Jersey with this strain of rat for comparative evaluations. 2.6.7. ACCLIMATION PERIOD Approximately two weeks; all animals were observed for viability twice daily. Prior to mating, all animals were examined by the technical staff to ascertain suitability for study. 2.7. SELECTION/GROUP ASSIGNMENT More females than required for the study were purchased and acclimated. Animals considered suitable for study on the basis of pretest physical examinations were included into the mating phase of the study. Females which mated were randomly assigned to groups daily in such a way as to most nearly equalize both the Day 0 gestation mean body weights between groups and the distribution of animals into groups. This was done by ranking the animals by body weight from lightest to heaviest and randomly assigning them to groups in blocks using a random numbers table. 2.8. MATING Females selected for mating were co-housed with male rats from an in-house breeding colony nightly in a 1:1 ratio. Vaginal smears were taken early in the morning following nightly intervals of co-housing. The females were considered to have mated if sperm was observed microscopically in the vaginal smear and/or a vaginal plug was observed. The day on which evidence of mating was observed was defined as Day 0 of gestation. 00 136295 OONFTDFNTTAL Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 17 Final Report 2.11.5. MONTHLY WATER ANALYSES Monthly water analyses, provided by the supplier, are maintained on fil at Huntingdon Life Sciences. 2.11.6. BIANNUAL WATER ANALYSES Biannual chemical and microbiological analyses of water samples collected from representative rooms in this facility were conducted to assure that water met standards specified under EPA National Primary Drinking Water Regulations (40 CFR Part 141). The results of these analyses are being maintained on file at Huntingdon Life Sciences. 2.11.7. CONTAMINANTS There were no known contaminants in the feed or water which were expected to interfere with the results of this study. 2.11.8. ENVIRONMENTAL CONDITIONS Light/Dark Cycle Twelve hour light/dark cycle controlled via an automatic timer. Temperature Temperature was monitored and recorded twice daily. Desired: Actual: 20to24C 20 to 23C Relative Humidity Relative humidity was monitored and recorded once daily. Desired: Actual: 40 to 70% 32 to 76% Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 19 Final Report equivalent of the limit dose of 1000 mg/kg body weight/day. The high exposure level of 1100 ppm was also expected to produce effects on the liver and other organ systems (ATSDR, 1993). The middle and low exposure levels were selected to provide a dose response for the observed effects and a no-observed effect level, respectively. 2.13. EXPERIMENTAL EVALUATIONS 2.13.1. VIABILITY CHECKS Animals were observed, at least twice daily in their cages, for mortality, general appearance and signs of severe toxic or pharmacologic effects. Animals in extremely poor health or in a possible moribund condition were identified for further monitoring and possible euthanasia. 2.13.2. PHYSICAL EXAMINATIONS Animals were removed from their cages and examined on Days 0 and 620 of gestation. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration. Unusual behavior and physical signs were recorded. During the test period, observations were performed postexposure when animals were being transferred to their home cages. 2.13.3. BODY WEIGHT Animals were removed from their cages and weighed using a Mettler Balance, Model PE4000 (Mettler Instrument Corporation, Hightstown, New Jersey). Body weights were recorded on Days 0, 6,9, 12, 15 and 20 of gestation. 2.13.4. FOOD CONSUMPTION Food was available, without restriction, 7 days/week, except during exposure periods. Animals were presented with weighed feeders on Days 0, 6, 9, 12 and 15 of gestation. Feeders were removed on Days 6, 9, 12, 15 and 20 of gestation and a "feeder-out" weight recorded using a Mettler Balance, Model PE4000 (Mettler Instrument Corporation, Hightstown, New Jersey). The resulting weight was subtracted from the Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 21 Final Report Reproductive System The intact uterus (ovaries attached) was removed from the abdominal cavity and weighed intact (with fetuses and placenta) using a Mettler AK.-160 balance (Mettler Instrument Corporation, Hightstown, New Jersey). The uterine weight data was then used to calculate a corrected Day 20 gestation body weight for each animal. The corrected Day 20 gestation weight was determined by subtracting the gravid uterine weight from the terminal Day 20 gestation weight. The uterus was then opened along the antimesometrial border and the number and location of the following were recorded for each uterine hom: live fetuses dead fetuses - movement in response to touch. lack of movement in response to touch with no visible degeneration. late resorptions - recognizable dead fetus undergoing degeneration regardless of size. early resorptions - evidence of implantation but no recognizable fetus. implantation sites total of resorptions and fetuses. Uteri without grossly visible implantations were stained according to the procedure of Salewski (1964) to identify the presence of sites of early resorption. If no stained sites were present, the female was considered not pregnant Ovanes The ovaries were dissected free from the uterus and evaluated for the presence and number of corpora lutea. 2.143. FETAL EVALUATIONS External Evaluations Each fetus was individually weighed using a Mettler Balance, Model PE4000, sexed externally (ano-genital distance) and examined for external malformations/variations to include observation for palatal defects. 136398 CONFTDFNTTA! Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 23 Final Report 2.15. STATISTICAL ANALYSIS The following items were analyzed statistically in the final report: 2.15.1. CONTINUOUS DATA: j Body weights (all recorded intervals) Body weight change (between all recorded intervals to include Days 6-20 of gestation using the actual and corrected Day 20 gestation weights) Food consumption values (all recorded intervals) Organ weight data absolute and relative to the Day 20 gestation weight Number of corpora lutea Uterine implantation data (fetuses, resorptions^, implantations) Pre-implantation loss^ Post-implantation loss*5 Mean percent male and female fetuses per litter Fetal weight distinguished by sex and as a composite for both sexes (l1itter as experimental unit) 2.15.2. CONTINUOUS DATA - MULTIPLE GROUP ANALYSIS: Employed when data for more than one treated group are compared to control. All statistical tests were conducted at the 5% and 1%, two-sided risk levels. StatisticaI evaluation of equality of means was made by die appropriate one way analysis of variance (ANOVA) technique, followed by a multiple comparison procedure, if needed. If ANOVA showed no difference, no additional comparisons were made. If ANOVA was significant, Dunnett's test was used to determine which-data, if any, differed from the control. References for these techniques are as follows: Dunlap, W.P, and J.A. Duffy (1975) "Fortran IV functions for calculating exact probabilities associated with Z, Chi-square, T and F values." Behav. Res. Methods and Instrumentations, 7:59-60. Dunlap, W.P., M.S. Marx and G.J. Agamy (1981) "Fortran IV functions for calculating probabilities associated with Dunnett's Test", Behav. Res. Methods and Instrumentations, 13:363-366. ^The Kruskal-Wallis test (2 X n) was used to test equality of means. Ifno D0 136799 cDoNFtr*NTTAL Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 25 Final Report Archives of Huntingdon Life Sciences for a. period of ten years after submission of the signed final report The Sponsor will be contacted in order to determine the final disposition ofthese materials. 2.17. PROTOCOL DEVIATIONS The following protocol deviations occurred during the study but were not considered to have compromised the validity or integrity ofthe study: Relative humidity in the animal quarters was out of the protocol specified range on several occasions. The protocol states that detailed physical examinations were to be performed on Days 0 and 6-20 (daily) during gestation; however, one animal (Group IV female No. 4625) was not observed on Day 0 due to technician error; the initial observations for this animal were recorded on Day 3 of gestation. no 136300 CONFTDFNTT At Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 27 Final Report 3.2.2. PREGNANCY RATES (Table 1; Appendix F) Pregnancy rates were comparable between the control and vinyl chloride-treated groups. Pregnancy rates for the control, 10, 100 and 1100 ppm treated groups were 92%, 96%, 88% and 96%, respectively. 3.23. PHYSICAL OBSERVATION DATA (Table 2; Appendix A) No adverse effect of treatment with vinyl chloride was indicated from clinical findings noted during the detailed physical examinations. There was a slight increase in the incidence of females with alopecia of the extremities and/or snout in the 100 ppm group but in the absence of a similar increase in the 1100 ppm group, this was not considered treatment-related. Other clinical findings seen in the treated groups occurred at low incidence or with similar frequency as control and were not considered treatment-related. 33.4. BODY WEIGHT DATA-GESTATION PERIOD (Tables 3,4 and 8; Appendices B, C and G) Mean body weights were comparable for the control and treated groups throughout the treatment period. Mean body weight gains for the treated groups were comparable to control data during the pretreatment period (Days 0-6) and early in the treatment period (Days 6-9, 9-12, 12-15). However, for Days 15-20 of gestation, mean body weight gains for all three treated groups were significantly lower than control. Since these differences were slight (less than 10% in comparison to control data in the 100 and 1100 ppm groups and 12% in the 10 ppm group) and were not dose-response related, and in the absence of an effect of treatment on food consumption, the reduction in weight gains in these groups was considered related to the slight reduction in litter size seen in the treated groups (about one fetus/litter) and the slightly lower gravid uterine weights (Table 8). The mean body weight gains for all treated groups over the Day 6 to 20 gestation interval using the corrected Day 20 gestation weights (actual Day 20 gestation weight minus the weight of the gravid uterus) were comparable to control data. Thus, these slight changes in body weight gain are not likely to be treatment-related. Huntingdon Life Sciences Developmental Toxicity Studv 96-4080 Page 29 Final Report Kidney weights, absolute and relative to the Day 20 gestation weights, were comparable to control in the 10 ppm group. In the 100 and 1100 ppm groups, absolute kidney weights were comparable to control data but relative weights were slightly higher. These latter differences were statistically significant and considered indicative of a treatment-related response. 3-3. FETAL DATA 3.3.1. FETAL BODY WEIGHT DATA (Table 7; Appendices F and H ) No adverse effect of treatment with vinyl chloride at a concentration level up to and inclusive of 1100 ppm was seen from fetal body weight data. Mean fetal body weights distinguished by sex and for both sexes combined were comparable between the control and treated groups. 3.3.2. FETAL SEX DISTRIBUTION DATA (Table 7; Appendix F) No adverse effect of treatment with vinyl chloride at a concentration level up to and inclusive of 1100 ppm was seen from fetal sex distribution data. The mean percentage of male and female fetuses per litter was comparable between the control and treated groups. 3-33. FETAL EXTERNAL EXAMINATION DATA (Tables 11 and 12; Appendix K) No malformations were seen in the external examination of the 318 control fetuses from 23 litters, 297 Group II (10 ppm) fetuses from 24 litters or 283 Group III (100 ppm) fetuses from 22 litters. In the 1100 ppm group, craniorachischisis, a severe malformation involving the cranium and spinal cord, was seen in one fetus from the litter of female No. 4622. No other external malformations were seen in the remaining 304 fetuses examined in the 1100 ppm group from 24 litters. Though not seen in recent historical control data for this laboratory (Appendix P, page P-7), a low incidence of craniorachischisis has been noted in the recently published MARTA/MTA compilation of historical control data for the CrI:CD BR rat (average fetal and litter occurrences were DO 13630? CONFIDENTIAL Huntingdon Life Sciences Developmental Toxicity Study 96-4080 Page 31 Final Report No adverse effect of treatment was evident from fetal ossification variation data. The litter incidences for the various ossification variations seen during the study were generally comparable between th control and treated groups. In the 1100 ppm group, the incidence of litters containing at least one fetus with reduced ossification of th cervical vertebral arches was significantly lower than control. This was not considered to represent an adverse effect of treatment 4. CONCLUSION In this study, vinyl chloride was administered via whole body inhalation (6 hours/day) to pregnant rats at targeted concentration levels of 0,10,100 and 1100 ppm during Days 6~ 19 of gestation. No maternal or developmental toxicity was seen at the 10 ppm exposure level. At the 100 ppm exposure level, the only maternal toxicity seen was an increase in the kidney weight relative to the Day 20 gestation weight At the 1100 ppm exposure level, the only maternal toxicity observed was an increase in kidney and liver weights relative to the Day 20 gestation weights. No developmental toxicity was seen at either the 100 or 1100 ppm exposure levels. Thus, the no-observed-effect level (NOEL) for maternal toxicity with vinyl chloride in the pregnant rat was 10 ppm and the NOEL for developmental toxicity was 1100 ppm. DO 136303 CONFTDFNTTA1 Huntingdon Life Sciences 96-4080 Developmental Toxicity Study_______________________________________ CALCULATIONS REFERENCES AND CALCULATIONS Preimplantation Loss: no. of corpora lutea - no. of implantation sites Percent Preimplantation Loss: no. of corpora lutea - no. of implantation sites/no. of corpora lutea Postimplantation Loss: total no. of early and late resorptions and dead fetuses Percent Postimplantation Loss: no. of early and late resorptions and dead fetuses/no. of implantation sites Page 33 Final Report DO 136304 conftdfnttai 29-JAN-19-. 12:05 Huntingdon Life Sciences 96-40000 Developmental Toxicity Study TABLE 1 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF SURVIVAL AND PREGNANCY Group Concentration (ppm) No. of females mated N Pregnant - Died/sacrificed moribund - El ctive sacrifice N N N Nonpregnant - Died/eacrlficed moribund - Elective sacrifice N N N Total no. of females died/ sacrificed moribund N t Examined at scheduled c-section N - Nonpregnant N - With total implant loss N % - With viable fetuses N % No statistically significant differences I 0 25 23 0 0 2 0 0 0 0.0 25 2 0 0.0 23 92.0 II 10 25 24 0 0 1 0 0 0 0.0 25 1 0 0.0 24 96.0 III 100 25 22 0 0 3 0 0 0 0.0 25 3 0 0.0 22 88.0 N = number of animals r> oo Z-n O r-t -1 aTt dZ z--A O -t ui i> PAGE 1 IV 1100 25 24 0 0 1 0 0 0 0.0 25 1 0 0.0 24 96.0 Huntingdon Life Sciences 96-4060D Developmental Toxicity Study Group Concentration (ppm) DAY 0 to 20 TERMINAL SACRIFICE General Appearance RED EXUDATE FROM ANO-GENITAL AREA Dermal-General ALOPECIA - EXTREMITIES/SNOUT ALOPECIA - GENERAL Ocular CHROMODACRYORRHEA - UNILATERAL LACRIMATION - UNILATERAL Oral/Buccal INCISORS BROKEN/MISSING INCISORS MALOCCLUDED TABLE 2 'ride Combined Inhalation Two-Generation and Developmental Toxicity Study in CD Rate OBSERVATIONS DURING GESTATION (frequency/animals) I II III 0 10 100 25/25 0/ 0 10/ 2 4/ 1 6/ 1 2/ 1 0/ 0 9/ 1 25/25 1/ 1 11/ 1 0/ 0 4/ 2 0/ 0 11/ 1 8/ 2 25/25 0/ 0 45/ 6 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 PAGE 1 IV 1100 25/25 0/ 0 32/ 3 0/ 0 0/ 0 0/ 0 0/ 0 0/ 0 29-JAN-19> o 12:10 Huntingdon Life Sciences 96-4080D Developmental Toxicity Study TABLE 3 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats Group Concentration (ppm) DAY 0 1 MEAN S.D. N DAY 6 MEAN S.D. N DAY 9 MEAN S.D. N DAY 12 MEAN S.D. N DAY 15 MEAN S.D. N DAY 20 MEAN S.D. N No statistically significant differences SUMMARY OF GESTATION BODY HEIGHTS (GRAMS) I II 0 10 III 100 214 13.7 23 243 14.8 23 254 16.0 23 270 15.8 23 287 19.0 23 353 25.3 23 214 12.3 24 240 13.3 24 250 13.8 24 266 16.7 24 282 15.8 24 340 19.2 24 210 12.9 22 240 13.7 22 251 13.2 22 266 13.2 22 202 14.5 22 341 23.6 22 n oo 2n O --t - a"h OCo' 2H Oa) M \/ y> IV 1100 214 10.5 24 242 10.8 24 250 10.2 24 266 11.4 24 202 11.6 24 342 15.6 24 29-JAN-19>u 12:10 Huntingdon Life Sciences 96-4080D Dev lopmental Toxicity Study Group Concentration (ppm) TABLE 4 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF GESTATION BODY WEIGHT GAIN (GRAMS) I II III 0 10 100 DAYS 0 TO 6 MEAN S.D. N DAYS 6 TO 9 MEAN S.D. N DAYS 9 TO 12 MEAN S.D. N DAYS 12 TO 15 MEAN S.D. N DAYS 15 TO 20 MEAN S.D. N DAYS 6 TO 20 MEAN S.D. N Statistical key: * p<0.05 29 5.6 23 10 5.8 23 16 3.5 23 17 4.3 23 66 8.7 23 110 13.5 23 26 5.8 24 10 5.7 24 16 5.7 24 16 4.0 24 58* 8.2 24 100* 13.8 24 30 8.9 22 10 4.6 22 16 3.9 22 15 4.8 22 60* 12.7 22 101* 14 .0 22 o oz oo T* M O {*> ZW -i O w 3> IV 1100 28 10.3 24 8 3.6 24 15 4.8 24 16 3.9 24 60* 7.8 24 99* 10.4 24 29-JAN-1998 12:13 Huntingdon Life Sciences 96-4080D Developmental Toxicity Study Group Concent ration (ppm) TABLE 5 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF GESTATION FOOD CONSUMPTION (GRAMS/ANIMAL/DAY) I II III 0 10 100 DAYS 0 TO 6 MEAN 19 19 19 S.D. 2.5 1.7 1.7 N 23 24 22 DAYS 6 TO 9 MEAN 18 19 19 S.D. 1.6 1.9 2.0 N 23 24 22 DAYS 9 TO 12 MEAN 20 20 20 S.D. 1.7 2.6 1.7 N 23 24 22 DAYS 12 TO 15 MEAN 21 22 22 S.D. 2.0 1.7 2.2 N 22 24 22 DAYS 15 TO 20 MEAN 24 23 23 S.D. 1.9 1.9 2.2 N 23 24 22 No statistically significant differences O oo zo oT1 jOi' z oo ---t"t ov0 3> PAGE 1 IV 1100 19 1.7 22 18 1.4 22 20 1.3 22 22 1.6 22 24 2.3 22 Huntingdon Life Sciences 96-40B0D Developmental Toxicity Study Group Concentration (ppm) TABLE 6 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF GESTATION FOOD CONSUMPTION (GRAMS/KG/DAY) I II III 0 10 100 DAYS 0 TO 6 MEAN S.D. N DAYS 6 TO 9 MEAN S.D. N DAYS 9 TO 12 MEAN S.D. N DAYS 12 TO 15 MEAN S.D. N DAYS 15 TO 20 MEAN S.D. N No statistically significant differences 90 10.2 23 76 5.2 23 79 6.3 23 79 4 .0 22 64 4.2 23 89 8.5 24 77 6.7 24 81 8.0 24 81 5.3 24 83 5.9 24 93 10.7 22 77 8.0 22 81 5.6 22 81 7.3 22 82 4.7 22 PAGE 1 IV 1100 89 8.4 22 73 5.3 22 79 3.9 22 82 4.9 22 84 8.2 22 29-JAN-19so 12:15 Huntingdon Life Sciences 96-4080D Developmental Toxicity Study Group Concentration (ppm) TABLE 7 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF CESAREAN SECTION DATA I II III 0 10 100 Pregnant N Dams with no Viable Fetuses N Dams with viable Fetuses N Corpora Lut a No. per animal TOTAL MEAN S.D. Implantation Sites No. per animal TOTAL MEAN S.D. Preimplantation Loss No. p r animal TOTAL MEAN S.D. 1 per animal Live Fetuses No. per animal Males Females r> O3 zo n aw Tt O' ZD --l -1 MEAN% S.D. TOTAL MEAN S.D. TOTAL MEAN% S.D. TOTAL MEAN% S.D. No statistically significant differences N number of animals 23 0 23 351 15.3 1.45 327 14.2 1.38 24 1.0 1.19 6.6 7.22 318 13.8 1.64 149 46.7 14.39 169 53.3 14.39 24 0 24 355 14.8 3.41 309 12.9 1.80 46 1.9 2.75 11.1 11.21 297 12.4 1.84 149 50.7 16.50 148 49.3 16.50 22 0 22 337 15.3 4.31 289 13.1 2.55 48 2.2 3.02 11.8 12.68 283 12.9 2.57 133 46.5 12.41 150 53.5 12.41 PAGE 1 _ ^_ IV 1100 24 0 24 363 15.1 2.68 320 13.3 2.30 43 1.8 1.61 11.6 9.99 305 12.7 2.27 151 50.1 10.36 154 49.9 10.36 Huntingdon Life Sciences 96-4080D Developmental Toxicity Study Group Concentration (ppm) TABLE 7 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rata SUMMARY OF CESAREAN SECTION DATA I II III 0 10 100 Fetal Body Weight (g) MEAN S.D. N Male Fetuses MEAN S.D. Female Fetus s MEAN S.D. No statistically significant differences 3.4 0.17 23 3.5 0.22 3.3 0.18 3.4 0.17 24 3.5 0.19 3.3 0.18 3.4 0.22 22 3.5 0.24 3.3 0.22 PAGE 3 IV 1100 3.3 0.22 24 3 .4 0.21 3.2 0.23 oo zO n r-i -1 OT| aC)'' z -A -1 -I '0 29-JAN-l. V. .3:05 Huntingdon Life Sciences 96-40B0D Developmental Toxicity Study TABLE 6 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF GRAVID UTERINE WEIGHT AND NET BODY WEIGHT CHANGE (GRAMS) Group Concentration (ppm) I 0 II III 10 100 NET BODY WT. CHANGE GRAVID UTERINE WT. NET WEIGHT CHANGE MINUS UTERINE WT. MEAN S.D. N MEAN S.D. N MEAN S.D. N 110 13.5 23 75 9.7 23 35 8.5 23 100* 13.0 24 69 10.0 24 31 9.1 24 101* 14.0 22 70 12.0 22 31 9.0 22 Statistical k y: * pcO.05 NET BODY WT. CHANGE - TERMINAL BODY WT. MINUS DAY 6 BODY WEIGHT NET WEIGHT CHANGE - NET BODY WT. CHANGE MINUS UTERINE WEIGHT r> oo zO ~n --i -* a'll to z to -H to 3> PAGE IV 1100 99* 10.4 24 69 10.4 24 30 7.6 24 29*JAN-1 J :45 Huntingdon Life Sciences 96-4080D Developmental Toxicity Study DAMS Group Concentration (ppm) N TABLE 9 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF MATERNAL NECROPSY OBSERVATIONS I II III 0 10 100 25 25 25 GROSS EXAM N1 0 0 ALOPECIA N1 0 0 % 4.0 0.0 0.0 KIDNEY N0 I 0 KIDNEY DISTENDED N 0 1 0 % 0.0 4.0 0.0 BLADDER N0 0 0 BLADDER FLUID FILLED: MODERATE N 0 0 o {THICK WHITE) % 0.0 0.0 0.0 UTERUS N0 0 0 UTERUS DISTENDED N 0 % 0.0 0 0.0 0 0.0 H " Humber of animals PAGE 1 IV 1100 25 1 1 4.0 0 0 0.0 1 1 4.0 1 1 4.0 Huntingdon Life Sciences 96-4080D Dev lopmental Toxicity Study Group Concentration (ppm) TABLE 10 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats ABSOLUTE ORGAN WEIGHTS I II III 0 10 100 FINAL BODY WEIGHT g MEAN S.D. N 353 25.3 23 LIVER g MEAN S.D. N 14.418 0.959B 23 KIDNEYS g MEAN S.D. N 1.871 0.1256 23 MEAN INCLUDES ONLY DAMS WITH VIABLE FETUSES AT TERMINATION No statistically significant differences 340 19.2 24 14.022 1.0B70 24 1.84 9 0.1388 24 341 23.6 22 14.267 1.4462 22 1.944 0.1322 22 PAGE 1 IV 1100 342 15.6 24 14.610 1.0688 24 1.924 0.1496 24 TABLE 11 TABLE 12 I 00 136317 CONFlOFNTTAl Huntingdon Life Sciences 96-4080D Developmental Toxicity Study Group Concentration (ppm) TABLE 12 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF FETAL OBSERVATIONS - VARIATIONS I II III 0 10 100 SQUAMOSAL(S} REDUCED OSSIFICATION Fetal Incid nee 6 18 15 Litter Incidence 3 10 10 MAXILLA(E) REDUCED OSSIFICATION Petal Incidence 482 Litter Incidence 262 MALAR(S) REDUCED OSSIFICATION Fetal Incidence Litter Incidence 4 13 15 3 7 10 PARIETAL(S) REDUCED OSSIFICATION . P tal Incidence Litter Incidence 1 7 4 11 6 9 6 FRONTAL(S) REDUCED OSSIFICATION Fetal Incidence 310 Litter Incidence 110 O3 PRESPHENOID REDUCED OSSIFICATION Zo T| Fetal Incidence --( 1 0 0 Litter Incidence o 05 Tt O' 1 0 0 MALAR UNOSSIFIED Fetal Incidence Z -A --* rA 05 0 0 1 Litter Incidence P0 0 1 SUPRAOCCIPITAL UNOSSIFIED Fetal Incidence Litter Incidence 001 001 No statistically significant differences PAGE 2 IV 1100 6 5 3 3 3 3 5 4 0 0 0 0 0 0 0 0 Huntingdon Life Sciences 96-4080D Developmental Toxicity Study Group Concentration (ppm) TABLE 12 Vinyl Chloride Combined Inhalation Two-Generation Reproduction and Developmental Toxicity Study in CD Rats SUMMARY OF FETAL OBSERVATIONS - VARIATIONS II III 10 100 CAUDAL ARCH(ESJ UNOSSIFIED Fetal Incidenc Litter Incidence 32 37 35 12 14 14 CAUDAL CENTRUM(A) UNOSSIFIED Fetal Incidenc Litter Incidence 0 0 STERNEBRA(E) S AND/OR 6 UNOSSIFIED Fetal Incid nee 110 89 94 Litter Incid nee 23 23 21 STBRNEBRA(E) 1,2,3 AND/OR 4 UNOSSIPIED Fetal Incidence Litter Incidence 2 2 STERNEBRA(E) 1,3 AND/OR 4 REDUCED OSSIFICATION Fetal Incidence Litter Incidence 02 023 RIB(S) 13TH RUDIMENTARY Fetal Incidence Litter Incidence 0 2 2 RIB(S) BENT Z-n o t -1 Fetal Incidence Litter Incidence %'i 0 0 1 1 0 0 32 RIB (S) REDUCED OSSIFICATION Fetal Incidence -i >0 T> 3 Litter Incidence 1 No statistically significant differences PAGE 4 IV 1100 27 12 111 24 0 0 0 0