Document 3Q8epLLK9D7N49BrOqxKOarmE

%` R & DREPORT DOW CHEMICAL U.S.A. RESTRICTED: for use within The Dow Chemical Company only. DCPARTMCNT Toxicology Research Laboratory CHI NUMIIR OUT COO* HET K-002661-(11) Auqust 3, 1978 ntl*7'WWff--BSo 4 NO, (E) 91 91 8 01 ll 21 II 11 01 9 CR1 NUMBER Kmhryotoxicitv of Inhaled Benzene in Mice and Rabbits uthor(si p Murray, j.a. John, L.W. Rampy, A.A. Crawford, K.D. Nitschke, R.P. Kalninss,, and B.A..//SSccffrayeyteztz____. tUTHS*(l) IIOHITUIIl{ / 1. AAjaAAAU t, flAO /i _______ft. AKUasiMi. !*- -V______P-fi JthJrunS /^CLu^ 7? HAS ftlVKWllt l SeIiuGnNAATi UwRnE> a \A- SPAOmiw^hr K. S. Rao This report is: if DINTERIM G9 FINAL and mainly: DATA REFERENCES (book and page): 47 PAGES IN FULL REPORT 38au wjrTTToiTL ?* NEW review (Refer also to earlier related reports and publications.) PATENT STATUS: I I disclosure submitted I DESCRIPTIVE SUMMARY WITH CONCLUSIONS: I case filed I X I no patent action required The effect of inhaled benzene on embryonal and fetal development was assessed in mice and rabbits. CF-1 mice and New Zealand white rabbits were exposed to 0 or 500 ppm of benzene for 7 hours per day from days 6 through 15 (mice) and 6 through \8 (rabbits) of gestation. Little evidence of maternal toxicity was discerned in either species. Exposure to benzene altered the incidence of some minor skeletal variants among the offspring of both species. In mice, a significant decrease in fetal body weight was noted. Although some evidence of embryotoxicity was seen in both species, a teratogenic effect was not observed in either mice or rabbits inhaling 500 ppm of benzene. DISTRIBUTION: See Back Pag* FORM C-4*4*0 36AI 3 C0NFl<^NTtAL DI-6560 EMBRYOTOXICITY OF INHALED BENZENE IN MICE AND RABBITS By F. J. Murray, J. A. John, L. W. Rampy, A. A. Crawford, K. D. Nitschke, R. P. Kalnins, and B. A. Schwetz Reviewed by K. S. Rao August 3, 1978 Toxicology Research Laboratory Health and Environmental Research Dow Chemical U.S.A. Midland, Michigan 48640 This work was sponsored jointly by Exxon Corporation and The Dow Chemical Company. ,41 A EMBRYOTOXICITY OF INHALED BENZENE IN MICE AND RABBITS By: F. J. Murray, J. A. John, L. W. Rampy, A. A. Crawford, K. D. Nitschke, R. P. Kalnins, and B. A. Schwetz Summary The effect of inhaled benzene on embryonal and fetal develop ment was assessed in mice and rabbits. CF-1 mice and New Zealand white rabbits were exposed to 0 or 500 ppm of benzene for 7 hours per day from days 6 through 15 (mice) and 6 through 18 (rabbits) of gestation. Little evidence of maternal toxicity was discerned in either species. Exposure to benzene altered the incidence of some minor skeletal variants among the offspring of both species. In mice, a significant decrease in fetal body weight was noted. Although some evidence of embryotoxicity was seen in both species, a teratogenic effect was not observed in either mice or rabbits inhaling 500 ppm of benzene. DO 136*15 t0pMTtAt CONF INTRODUCTION Benzene (benzol, CgHg) is used primarily as a starting material in the production of a wide variety of organic chemicals. Benzene is also used as an industrial solvent and as an anti-knock agent in gasoline. Acute exposure to benzene vapors produces central nervous system depression (Browning, 1965). Occupational exposure to high levels of benzene on a chronic basis has been associated with leukemia (Aksoy et al., 1972a; 1974; Berlin, 1974), aplastic anemia (Saita, 1973; Aksoy, 1972b), and chromosomal aberrations (Pollini and Colombi, 1964; Vigliani and Forni, 1969; Forni et al^., 1971). The OSHA-proposed standard for an occupa tional exposure to benzene for 8 hours per day, 5 days per week is 1 ppm (OSHA, 1977). Several studies have been conducted in laboratory animals to evaluate the potential teratogenicity of benzene. Watanabe and Yoshida (1970) reported cleft palate, agnathia, and micrognathia among the offspring of mice given a massive subcutaneous injection of 3 ml benzene/kg body weight on day 13 of gestation. In another study, pregnant rats were exposed to 0, 10, 50, or 500 ppm of benzene for 7 hours per day from days 6 through 15 of gestation (unpublished data. 00 1.3641.6 OONFTDFNTT AL. 2- - Hazleton Laboratories Corp., Vienna, Va.). A low incidence of malformations (exencephaly, angulated ribs, and non sequential ossification of forefeet) was observed among the litters of rats exposed to 500 ppm, suggesting a possible teratogenic effect at that concentration. In contrast, Green et al., (1978) found no evidence of a teratogenic effect in Sprague-Dawley rats inhaling 300 or 2200 ppm of benzene for 6 hours per day from days 6 through 15 of gestation. The purpose of the study presented herein was to assess the effect of inhaled benzene on embryonal and fetal development in two additional species. For these studies, pregnant CF-1 mice and New Zealand white rabbits were exposed to 500 ppm of benzene by inhalation for 7 hours per day from days 6 through 15 (mice) and 6 through 18 (rabbits) of gestation. Since the development of leucopenia has been demonstrated in rats, guinea pigs, and rabbits following repeated exposure to benzene vapors (Wolf et al_., 1956), blood samples from the female mice and rabbits and from rabbit fetuses were collected for hematologic evaluation. This work was sponsored jointly by Exxon Corporation and The Dow Chemical Company. 00 136417 CONFIOFNTTAL -3- MATERIALS AND METHODS Test Material. Benzene, identified as ID95-01000-0070, was supplied by Exxon Chemical Company U.S.A., Houston, Texas. The test sample was analyzed at the Analytical and Information Division, Exxon Research and Engineering Company, Linden, New Jersey (Table 1). On at least two days of exposure, however, the test animals were exposed inadvertently to a sample of benzene obtained from Mallinckrodt Chemical Company; the Mallinckrodt benzene was identified as nanograde quality. Lot No. 1043B3. Samples of both the Exxon and Mallinckrodt benzenes have been retained for possible compara tive analysis. Test Animals. Virgin CF-1 mice (Charles River, Portage, Michigan) and New Zealand white rabbits (Langshaws Rabbitry, Augusta, Michigan) were housed in wire mesh cages in a room controlled for temperature (703F), humidity (45+5%), and light cycle (12 hrs light and dark). The animals were maintained on commercial laboratory chow (Ralston Purina Company, St. Louis, Missouri) and tap water free choice except while in the exposure chambers. Mice and rabbits were allowed at least two and three weeks, respectively, for ' acclimatization to the laboratory before use. The day on oo 106A18 OONF -4- which a vaginal plug was observed in mice or. the day on which rabbits were bred was considered day zero of gesta tion. The animals were uniquely identified by means of an ear punch (mice) or a metal ear tag (rabbits). Experimental Design. Groups of 35 and 37 bred mice were exposed in chambers to filtered room air or 500 ppm of benzene, respectively, for 7 hours per day from days 6 through 15 of gestation. Concurrently, groups of 20 bred rabbits each were exposed in the same chambers to filtered room air or 500 ppm of benzene for 7 hours per day from days 6 through 18 of gestation. Inhalation studies were conducted under dynamic airflow conditions in stainless steel and glass Rochester-type chambers of 4.3 cubic meter volume. The atmosphere was generated by metering liquid benzene at a known rate into a heated vaporization flask and then into the airstream being drawn into the chamber. Concentrations achieved in the chambers were analyzed daily throughout exposure by alter nately sampling the experimental and control chambers at 60 and 30 minute intervals, respectively, using a Miran I infrared spectrophotometer at a wavelength of 3.25y. DO C0^F -5- Standard samples were prepared daily by Injecting known quantities of benzene into 100 liter plastic bags. The time weighted analytical concentration of benzene in the exposure chamber was 5Q712 ppm (mean standard deviation). Maternal Observations. The animals were observed daily from days 6 of gestation until sacrifice for indications of toxicity from the test material. The mice were weighed on days 6, 8, 10, and 16 of gestation; the rabbits were weighed on days 6, 8, 10, 12, and 19 of gestation. In addition, maternal body and liver weights were recorded at the time of cesarean section, day 18 and 29 of gestation for mice and rabbits, respectively. Food and water consumptions were recorded during the experimental period at 3-day intervals for the mice and daily intervals for the rabbits. Fetal Examination. On day 18 and 29 of gestation, the mice and rabbits, respectively, were sacrificed by carbon dioxide inhalation. The uterine horns were exteriorized through a mid-line incision in the abdominal wall, and the number and position of live, dead, and resorbed fetuses were noted. The uterus of each apparently non-pregnant female was stained with a 10% solution of sodium sulfide and examined for evidence of early resorption sites (Kopf et al., 1964); this procedure HO 136*20 co nf-ioentiai- was conducted solely for the purpose of determining the incidence of pregnancy, not the incidence of resorptions. After being weighed, measured (crown-rump length), and sexed, all fetuses were examined for external alterations and cleft palate. One-third of the fetuses of each litter, selected at random, was examined immediately for evidence of soft-tissue alterations by dissection under a low power stereo microscope (Staples, 1974). The head of each mouse fetus which was examined for soft-tissue alterations was placed in Bouin's solution and subsequently examined by the razor section technique of Wilson (1965) . All of the fetuses in each litter were cleared and stained with alizarin red-S (Dawson, 1926) to permit examination for skeletal alterations. Hematologic Evaluation. At the time of cesarean section blood samples were obtained through an incision in the neck of individual rabbit fetuses from five different litters % of the control and benzene-exposed groups. The following parameters were analyzed: packed cell volume, hemoglobin content, red blood cell counts, and white blood cell counts. Hematologic determinations were not conducted on fetal mice since sufficient quantities of blood to perform the evaluations could not be obtained. 00 00^ -7- Additional groups of eight bred mice and four bred rabbits each were exposed to 0 or 500 ppm of benzene for 7 hours per day from days 6 through 15 (mice) and 6 through 18 (rabbits) of gestation. Blood samples were obtained from maternal mice by decapitation on day 16 of gestation and from maternal rabbits by cardiac puncture on day 19 of gestation. The following parameters were analyzed: packed cell volume, hemoglobin content, red blood cell counts, white blood cell counts, and differential white blood cell counts. Statistical Evaluation. The Wilcoxon test as modified by Haseman and Hoel (1974) was used to evaluate the incidence of fetal alterations and resorptions. Maternal and fetal body weights, maternal liver weights, food and water consump tion data, and hematologic data were analyzed statistically by a one-way analysis of variance (Steel and Torrie, 1960). In all cases, the level of significance chosen was p<0.05. DO 1 364-22 CONFIDENTIAL RESULTS Maternal Toxicity. Exposure of bred mice and rabbits to 500 ppm of benzene for 7 hours per day had no significant effect on the dams1 appearance# demeanor# body weight, body weight gain# or liver weight (Tables 2 and 3). Food and water consumptions of pregnant mice were not altered by inhalation of benzene (Table 4). In comparison, the amount of food and water consumed by the pregnant rabbits exposed to benzene was significantly greater than that of the con trols on a few days toward the end of the exposure (Tables 5 and 6). Embryo- or Fetotoxicity. Inhalation of 500 ppm of benzene did not significantly affect the incidence of pregnancy in either mice or rabbits (Tables 7 and 8). No significant effect on the average number of live fetuses per litter or on the incidence of resorptions was discerned in either species. Mean fetal body weight but not crown-rump length was decreased significantly among litters of mice exposed to benzene (Table 7). In rabbits# neither fetal body measurement was altered significantly by exposure to benzene (Table 8). DO CONF -9- Fetal Alterations. Among the offspring of mice exposed to benzene, no major malformation occurred at an incidence significantly different from that of the controls (Table 9). Cleft palate was observed in single fetuses in both control and benzene-exposed groups of mice; in the latter case, the fetus with cleft palate also exhibited asymmetric vertebrae as well as forked and fused ribs. Significant increases in the occurrence of several minor skeletal variants were seen in litters of benzene-exposed mice, including delayed ossifi cation of sternebrae, delayed ossification of skull bones, and unfused occipital bones of the skull. In rabbits, exposure to benzene did not significantly alter the occurrence of fetal malformations, when considered individually or collectively (Table 10). A single case of gastroschisis was noted in a litter exposed to benzene. Fused thoracic vertebrae accompanied by fused and forked ribs were observed in another benzene exposed litter. Another exposed fetus exhibited fused ribs only. No major malformations were observed among the control group. Two minor skeletal variants, i.e., 13 rib(s) and lumbar spurs occurred significantly less often among the litters of rabbits exposed to benzene. 4 oo 1 C0^F t 0^ T Al- -10- Hematologic Evaluation. Exposure to 500 ppm*-of benzene from days 6 through 18 of gestation did not produce any signifi cant hematologic changes among the fetal rabbits (Table 11). Hematologic evaluation of fetal mice was not performed since a sufficient quantity of blood could not be obtained. Hematologic values of adult bred mice and rabbits exposed to benzene were not significantly different from the control values. DISCUSSION Benzene was not teratogenic in either mice or rabbits inhal ing 500 ppm of the compound for 7 hours per day. The few malformations which were observed among the benzene-exposed litters were types that have been noted to occur spontaneously at a low incidence in previous control groups. In comparison, a possible teratogenic effect was reported in rats exposed to 500 ppm of benzene for 7 hours per day from days 6 through 15 of gestation (unpublished data, Hazleton Laboratories Corp. Vienna, Va.); however. Green et al. (1978) did not observe teratogenicity in rats inhaling up to 2200 ppm of benzene for 6 hours per day on the same days of gestation. -11- The lack of teratogenicity observed in the present study contrasts with the findings of Watanabe and Yoshida (1970); in that study, cleft palate was observed in three of fifteen litters of mice given a single subcutaneous injection of 3 ml benzene per kg body weight on day 13 of gestation. However, it is possible that the low incidence of cleft palate which was attributed to benzene by Watanabe and Yoshida was actually produced by the stress of such a massive subcutaneous injec tion; unfortunately, a negative control group was not included in that study. In the present study, one fetus with cleft palate was observed in each of the control and exposed groups. Although a teratogenic effect was not discerned, some evidence of fetotoxicity was noted in mice inhaling benzene. Increased incidences of delayed ossification of skull bones and sternebrae were observed among the offspring of mice exposed to benzene. This effect was associated with a decrease in the average fetal body weight. In rabbits, the only evidence of an effect on embryonal or fetal development was a decrease in the occurrence of two minor skeletal variants: 13 ribs (the normal number is 12 or 13) and lumbar spurs. DO 1364?7 CONFTDFNTTAl -12- Green et al. (1978) reported increased frequencies of t delayed ossification of sternebrae and missing sternebrae in female but not male offspring of rats exposed to benzene. A similar correlation between the occurrence of minor skeletal variants and the sex of the fetus was not observed in the present study. The concentration of benzene tested in this study, 500 ppm, was markedly higher than the OSHA-recommended standard of 1 ppm (OSHA, 1977). However, the margin of safety may be even greater than indicated by exposure level alone since the amount of air inhaled by mice and rabbits in proportion to their body weight is approximately 5 and 10 times, respec tively, that of humans (Handbook of Respiration, 1971). Thus, the findings of the present study, along with those of Green et al^. (1978) do not indicate that benzene is uniquely hazardous to the unborn at levels of exposure which might be encountered in the workplace. In summary, a teratogenic effect was not discerned in either mice or rabbits inhaling 500 ppm of benzene for 7 hours per day during the period of major organogenesis. Exposure to 00 136428 OONFTDFNTT At -13- 500 ppm of benzene resulted in some minor variations in the development of the fetal skeleton in both species, but serious embryotoxicity or fetotoxicity was not observed. WRITTEN BY: , ^ MAaau, (Aks) ^OutpS F. J. Murray , PH.D. 1803 Building August 3, J.978 0" HO, n jx a. ^JOhn, M.S. 1803 Building August 3, 1978 j /T ^ it L. W. Rampy, Plf.D.\ 1803 Building August 3, 1978 A. A. Crawford', B. S. 1803 Building August 3, 1978 [J (MQ. fV 7 K. D. Nitschke, 1803 Building August 3, 1978 B.S. 'P J. aJrurs /dtiu i 7f R. ip. Kalnins , B.S.,' 1803 Building August 3, 1978 August 3, 1978 REVIEWED BY: K. S. Rao, D.V.M., Pfl.D. i Toxicology Research Laboratory Health and Environmental Research 1803 Building Dow Chemical U.S.A. Midland, Michigan 48640 August 3, 1978 oo 1364?9 CONFIDENTIAL -14- REFERENCES 1. Aksoy, M., Dincol, K., Erdem, S.# Akgun, T., and Dincol, G. 1972a Details of blood changes in 32 patients with pancytopenia associated with long term exposure to benzene. Brit. J. Ind. Med. 29, 56-64. 2. Aksoy, M., Dincol, K., Erdem, S., and Dincol, G. 1972b Acute leukemia due to chronic exposure to benzene. Am. J. Med. 52, 160-166. 3. Aksoy, M., Erdem, S., Dincol, K., Hepyuksel, T., and Dincol, G. 1974 Leukemia in shoe-workers exposed chronically to benzene. Blood 44, 837-841. 4. Berlin, M., Cage, J., and Johnson, E. 1974 Increased aromatics in motor fuels: A review of the environmental and health effects. Work Environ. Health 11, 1-20. 5. Browning, E. 1965 Toxicity and Metabolism of Industrial Solvents. Elsevier Publishing Company, New York, N.Y. 6. Dawson, A. B. 1926 A note on the staining of the skeleton of cleared specimens with alizarin red-S. Stain Tech. 1, 123-124. 7. Forni, A. M., Cappellini, A., Pacifico, E., and Vigliani, E. C. 1971 Chromosome changes and their evolution in subjects with past exposure to benzene. Arch. Environ. Health 23, 385-391. 8. Green, J. D., Leong, B.K.J., and Laskin, S. 1978 Fetotoxicity of inhaled benzene in rats. Toxicol. Appl.'Pharmacol. In press. 9. Handbook of Respiration 1971 National Acadamy of Sciences, National Research Council, W. B. Saunders Company, Philadelphia, Pennsylvania. 00 136430 CONFIDENT! Al -15- 10. Haseman, J. K., and Hoel, D. G. 1974 Table of Gehan's generalized Wilcoxon test with fixed point sensoring. J. Statist. Comput. Simul. 3, 117-135. 11. Kopf, R., Lorenz, D., and Salewski, E. 1964 Procedure for staining implantation sites of fresh rat uteri. Naunyn-Schmiedebergs Arch. Exp. Path. Pharmacol. 247, 121-135. 12. OSHA 1977 Occupational Exposure to Benzene. Depart ment of Labor, Occupational Safety and Health Administration, Federal Register. 42 (103), 27453-27477. 13. Pollini, G., and Colombi, R. 1964 Lymphocyte chromo some damage in benzene blood dyscrasia. Med. Lavoro 55, 241-255. 14. Saita, G. 1973 Benzene-induced hypoplastic anaemias and leukaemias, in Girdwood, R. H., ed.. Blood Disorders Due to Drugs and Other Agents. Amsterdam, Excerpta Medica, p. 127-146. 15. Staples, R. E. 1974 Detection of visceral alterations in mammalian fetuses. Teratology 9, A-37. 16. Steel, R.G.D. and Torrie, H. H. 1960 Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York, N.Y. 17. Vigliani, E. C., and Forni, A. 1969 Benzene, chromo some changes, and leukemia. J. Occup. Med. 11, 14 8149. 18. Watanabe, G., and Yoshida, S. 1970 The teratogenic effect of benzene in pregnant mice. Acta. Medica et Biologica. 17, 285-291. 19. Wilson, J. G. 1965 Teratology: Principles and Techniques. The University of Chicago Press, Chicago Illinois, 262-277. 20. Wolf, M. A., Rowe, V. K., McCollister, D. D., Hollingsworth, R. L., and Oyen, F. 1956 Toxicological studies of certain alkylated benzenes and benzene. A.M.A. Arch. Industr. Hlth. 14, 387-398. DO 1.3643 1 CONFTDFNTT Al -16- TABLE 1 RESULTS OF ANALYTICAL TESTING BENZENE - ID 95-01000-Q070a TEST Color, Acid Washed (ASTMD848) RESULTS Sample Layer No. 0 Acid Layer No. 1 Color, Platinum-Cobalt (ASTM D 1209 Pt-Co 5 Distillation, ASTM D350 760 mm Hg @ 26C Sulfur Content, Total (AM-S 60.23) 5% 10% 20% 30% 40% 50% 60% 70% 80% 90% 95% IBP FBP Recovery Loss Bottoms <1 ddhi 80.0C 80.1 80.1 80.3 80.3 80.3 80.3 80.3 80.3 80.3 80.3 79.4 80.6 97.5% 2.4% 0.1% H,,S Ratinq Specific gravity @ 60/60F G.C. For Trace Impurities Total Non-Aromatics Toluene Bromine Index, ASTM 02710 Corrosion, Cu Strip, ASTM D 130 Acidity, ASTM D1613 >0.0, <0.5 0.88437 68 ppm 199 ppm 4.15 1A - 3 hrs. @ 212F Basic Sample aAs reported by J. M. Mulhall, Analytical and Information Division, Exxon Research and Engineering Company, Linden, New Jersey, 07036. 00 13643? CONFIDENTIAL -17- TABLE 2 BODY AND LIVER WEIGHTS OF PREGNANT MICE EXPOSED TO BENZENE BY INHALATION Number of dams** Maternal body weight (g) on gestation day 6 8 10 16 18 Control 26 34+2C 34+2 36+3 45+4 50+4 Benzene3 30 34+3 34+2 36+2 46+3 50+4 Maternal weight gain (g) on gestation days 6-7 8-9 10-15 16-17 Total 0.4+2 2+2 9+4 6+2 17+4 -0.3+2 2+2 9+2 5+1 16+3 Liver weight on day 18 of gestation absolute^ relative0 3.08+0.38 61.1+5.4 3.16+0.38 62.5+5.9 aMice were exposed to 500 ppm benzene for 7 hrs/day on days 6-15 of gestation. ^Data from non-pregnant mice were not included in these analyses. cMean +_ S.D. dg, mean + S.D. emg liver/g body weight, mean +_ S.D. 'No values were significantly different from control values by a one-way analysis of variance, p<0.05. See Table A-l for individual animal data. DO 136433 CONFIDENTIAL -18- TABLE 3 BODY AND LIVER WEIGHTS OF PREGNANT RABBITS EXPOSED TO BENZENE BY INHALATION Number of does*5 Maternal body weight (kg) on gestation day 6 8 10 12 19 29 Control 18 4.06+0.31c 3.96+0.20 4.02+0.28 4.06+0.32 4.10+0.34 4.17+0.35 Benzene 19 3.93+0.29 3.88+0.35 3.89+0.29 3.90+0.33 4.00+0.28 4.12+0.27 Maternal weight gain (kg) on gestation days 6-7 8-9 10-11 12-18 19-28 Total -0.05+0.06 0.02+0.06 0.20+0.03 0.08+0.11 0.07+0.16 0.11+0.25 -0.07+0.15 0.02+0.12 0.02+0.10 0.09+0.08 0.11+0.16 0.19+0.17 Liver weight on day 29 of gestation absolute0 relative6 98+16 23.7+4.0 105+10 25.5+2.4 aRabbits were exposed to 500 ppm benzene for 7 hrs/day on days 6-18 of gestation. bData from non-pregnant rabbits were not included in these analyses. cMean + S.D. dg, mean + S.D. eg liver/kg body weight, mean +_ S.D. No values were significantly different from control values by a one-way analysis of variance, p<0.05. See Table A-2 for individual animal data. 00 136434 CONFTDFNTTAt -19- TABLE 4 FOOD AND WATER CONSUMPTION OF PREGNANT MICE EXPOSED TO BENZENE BY INHALATION Number of dams^ Amount of food consumedc daily during gestation days 6-8 9-11 12-14 15-17 Amount of water consumedc daily during gestation days 6-8 9-11 12-14 15-17 Control 26 6+i 6+2 7+2 8+2 10+2 12+3 13+2 14+2 Benzene' 30 6+1 6+2 7+2 8+1 9+2 11+2 12+2 14+2 aMice were exposed to 500 ppm benzene for 7 hrs/day on days 6-15 of gestation. ^Data from non-pregnant mice were not included in these analyses. Expressed as grams/mouse/day. dMean + S. D. No values were significantly different from control values by a one-way analysis of variance, p<0.05. 00 136435 CONFIDENTIAL -20- TABLE 5 FOOD CONSUMPTION OF PREGNANT RABBITS EXPOSED TO BENZENE BY INHALATION L Number of does0 Control 18 Benzene* 19 Amount of food consumed0 on gestation 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 121+36d 144+34 146+35 149+42 148+60 132+29 140+35 134+29 142+33 139+50 134+31 124+43 136+44 140+52 159+40 143+42 125+42 118+48 111+60 98+64 86+66 85+52 100+53 142+53 143+46 161+36 160+46 160+24 156+43 139+27 143+23 146+23 159+41 162+37e 166+36e 164+39 188+41e 167+46 141+53 129+60 118+63 108+48 98+44 105+49 111+42 123+45 "Rabbits were exposed to 500 ppm benzene for 7 hrs/day on days 6-18 of gestation. Data from non-pregnant rabbits were not included in these analyses. A 'Expressed as grams/rabbit/day. DO 136436 *Mean + S.D. CONFTDENTTAl 'Significantly different from control value by a one-way analysis of variance, p<0.05. -21- TABLE 6 WATER CONSUMPTION OF PREGNANT RABBITS EXPOSED TO BENZENE BY INHALATION Number of does*3 Control 18 Benzene 19 Amount of water consumed0 on gestation day 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 187+110d 268+64 298+79 278+63 281+75 258+46 271+80 267+78 278+65 242+60 270+85 237+84 233+88 252+75 270+78 256+88 256+84 295+126 272+109 234+129 237+103 237+136 201+100 142+128 246+100 300+72 295+76 295+65 269+76 264+50 298+97 297+52 2 9 2+6 2e 290+38 287+84 327+64e 338+69e 311+93 274+101 265+98 249+112 239+98 256+86 245+73 231+73 277+77e aRabbits were exposed to 500 ppm benzene for 7 hrs/day on days 6-18 of gestation. bData from non-pregnant rabbits were not included in these analyses. Expressed as grams/rabbit/day. d Mean + S.D. no 1 CONFTDFNTTAl Significantly different from control value by a one-way analysis of variance, p<0.05. -22TABLE 7 OBSERVATIONS AT CESAREAN SECTION AMONG MICE EXPOSED TO BENZENE BY INHALATION Control Benzene3 No. of bred females No. maternal deaths Percent pregnant^ Percent pregnant, total0 Proportion of pregnant animals detected by sulfide stain No. litters examined Implantation sites/dame No. fetuses/1ittere Resorptions/1ittere,f Reporptions/implantations^ Litters with resorptions^ No. litters totally resorbed^ Resorptions/litters with resorptions^ Dead fetuses/total fetuses Sex ratio, M:F Fetal body weight, g^ Fetal crown-rump length, mm^ 35 (0/35) 74%(26/35) 8635(30/35) 1335(4/30) 26 12+2 11+2 2+1 1255(39/318) 69%(18/26) 0 2.2(39/18) (0/279) 50:50 1.01+0.09 24.1+1.3 37 (0/37) 8155(30/37) 8955(33/37) 955(3/33) 30 13+2 11+2 2+2 1535(56/381 ) 7355(22/30) 0 2.6(56/22) 0.3(1/325) 52:48 0.95+0.10 23.8+1.1 aMice were exposed to 500 ppm benzene for 7 hrs/day on days 6-15 of gestation. ^Number of females with visible implantation sites at the time of cesarean section/total number of bred females. cNumber of females with implantation sites as observed either visually at the time of cesarean section or after staining the uterus with sodium sulfide stain/total number of bred females. ^Number of females with implantation sites detected only after staining the uterus with sodium sulfide/total number of females with implantation sites. eMean + S.D. Resorptions detected by sodium sulfide staining were not included in these calculations. fylean of litter means + S.D. ^Significantly different from control value by a one-way analysis of variance, p<0.05. See Table A-3 for individual animal data. 00eVoF^U -23" TABLE 8 OBSERVATIONS AT CESAREAN SECTION AMONG RABBITS EXPOSED TO BENZENE BY INHALATION Control Benzene No. of bred females No. maternal deaths Apparent pregnancy rateb Total pregnancy rate0 Proportion of pregnant animals detected by sulfide staind No. litters examined Corpora lutea/dame Implantation sites/dame Pregnancy wastagee,f No. fetuses/1ittere Resorptions/1ittere Resorptions/implantations^ Litters with resorptions^ No. litters totally resorbed^ Resorptions/litters with resorptions* Dead fetuses/total fetuses Sex ratio, M:F Fetal body weight, g Fetal crown-rump length, mmb 20 (0/20) 90%(18/20) 90% (18/20) (0/18) 18 9+1 9+2 1+1 8+2 1+1 5%(9/l68) 44%(8/18) 0 1.1(9/8) (0/150) 59:41 39.43+5.67 97.32+7.54 20 (0/20) 95%(19/20) 95%(19/20) (0/19) 19 10+2 9+2 1+1 8+2 1+1 7%(13/182) 47%(9/19) 0 1.4(13/9) (0/152) 45:55 38.46+5.05 95.12+6.02 aRabbits were exposed to 500 ppm benzene for 7 hrs/day on days 6-18 of gestation. ^Number of females with visible implantation sites at the time of cesarean section/total number of bred females. cNumber of females with implantation sites as observed either visually at the time of cesarean section or after staining the uterus with sodium sulfide stain/total number of bred females. ^Number of females with implantation sites detected only after staining the uterus with sodium sulfide/total number of females with implantation sites. BMean + S.D. p Pregnancy wastage = number of corpora lutea minus number of implantations. ^Resorptions detected by sodium sulfide staining were not included in these calculations. "Mean of litter means + S.D. do 136439 CONFt^PNT TAl. No values were significantly different from control values by a one-way analysis of variance or by Fisher's exact probability test (incidence data), p<0.05 See Table A-4 for individual animal data. -24- TABLE 9 INCIDENCE OF ALTERATIONS AMONG LITTERS OF MICE EXPOSED TO BENZENE BY INHALATION Control Benzene8 No. Examined Fetuses (Litters) External Examination Soft Tissue Examination Skeletal Examination Bones of the Skull 279(26) 97(26), 253(24) 165(24) 325(30) 112(30), 286(28)' 189(28) % Affected (No. Affected) External Alterations Cleft palate* Subcutaneous hemorrhage. head region Fc 0.4(1) L 4(1) F0 L0 0.3(1) 3(1) 0.3(1) 3(1) Soft Tissue Alterations Slightly dilated 3rd ventricle of brain F0 L0 KD 3(1) Subcutaneous hemorrhage. head region F KD L 4(1) 0 0 Subcutaneous hemorrhage. around brain Skeletal Alterations F0 L0 1(1) 3(1) Sternebrae delayed ossification F 10(24) L 42(10) 18(51)d 64(18) fused F0 L0 1(3) 11(3) unfused F 6(15) L 38(9) 10(29) 43(12) extra site of ossification F L 4(9) 12(3) 1(2) 7(2) DO 136440 OONFIDENTTAl -25- TABLE 9 (Continued) INCIDENCE OF ALTERATIONS AMONG LITTERS OF MICE EXPOSED TO BENZENE BY INHALATION misshapen (#6) extra cartilage misplaced cartilage Ribs extra forked or fused* I irregular ossification Vertebrae asymmetric* lumbar spurs delayed ossification of centra delayed ossification of cervical arches ! forked atlas misshapen atlas delayed ossification of atlas Control F0 L0 F 1(2) L 4(1) F 1(2) L 8(2) F 12(30) L 58(14) F0 L0 F0 L0 F0 L0 F 6(14) L 42(10) F0 L0 F L F L F L F0 L0 Benzene3 0.4(1) 4(1) 0 0 0 0 12(35) 46(13) 0.3(1)e 4(1) 0.3(1) 4(1) 0.3(1 )e 4(1) 5(15) 43(12) 0.3(1)e 4(1) 0.3(1 )e 4(1) 0.5(1) 4(1) 0.5(1) 4(1) 0.5(1 )e 4(1) 00 136441 CONFIDENTIAL -26- TABLE 9 (Continued) INCIDENCE OF ALTERATIONS AMONG LITTERS OF MICE EXPOSED TO BENZENE BY INHALATION Skull delayed ossification foramen, occipital or parietal unfused occipital unossified, occipital misshapen, interparietal TOTAL MALFORMATIONS Control F 17(28) L 58(14) F0 L0 F 1(1) L 4(1) F0 L0 F0 L0 F 0.4(1) L 4(1) Benzene3 34(64)d 68(19) (2 7(2) 5(10)d 21(6) 1(2) 7(2) 0.5(1) 4(1) 0.5(1) 4(1) aMice were exposed to 500 ppm benzene for 7 hrs/day on days 6-15 of gestation. bTwo litters from the control group and two litters from the benzene group were not examined for skeletal alterations. The fetuses from these litters were inadvertently mixed together during the staining procedure. CF = fetuses; L = litters. j Significantly different from control value by a modified Wilcoxon test, p<0.05. eThis alteration occurred in the single fetus with cleft palate. Considered to be a major malformation. See Table A-5 for individual litter data. 00 1 3644-2 GONFIDFNT T AL -27- TABLE 10 INCIDENCE OF ALTERATIONS AMONG LITTERS OF RABBITS EXPOSED TO BENZENE BY INHALATION External Examination Soft Tissue Examination Skeletal Examination External Alterations Gastroschisis Fc L Soft Tissue Alterations Cystic calculus attached to gall bladder F L Fused innominate and left carotid arteries F L Satellite vessels off aorta, innominate or carotid arteries F L Skeletal Alterations Sternebrae - delayed ossification extra site of ossification fused unfused F L F L F L F L Ribs 13th rib or ribs lumbar spur or spurs F L F Control Benzene3 No. Examined Fetuses (Litters) 150(18) 56 18). 142(17)b 152(19) 59(19) 152(19) % Affected (No. Affected) 0 0.6(1) 0 50) 0 0 88(49) 100(18) 18(10) 33(6) 2(1) 5(1) 81(48) 100(19) 32(19) 47(9) 89(126) 100(17) Kl) 6(1) 0 0 0 0 83(126) 100(19) 0 0 0.6(1) 5(1) 1(2) 11(2) 63(89) 94(16) 5(7) 41(7) DO 136443 CONFIDENT! Al.. 43(65)d 89(17) 2(3)d 11(2) -28- TABLE 10 (Continued) INCIDENCE OF ALTERATIONS AMONG LITTERS OF RABBITS'EXPOSED TO BENZENE BY INHALATION fused or forked F L Vertebrae - fused vertebrae and hemi-centra, thoracic region F L misshapen or dumbell-shaped centra F L unfused centra F L extra site of ossification between vertebrae F L Skull - extra suture line within parietal bone F L foramen - parietal- or interparietal k F L TOTAL MALFORMATIONS F L Control 0 0 0 0 0 0 0 0 1(1) 6(1) 0.7(1) 6(1) 2(3) 18(3) 0 0 Benzene3 1(2) 11(2) KD 5(1) 3(4) 21(4) 1(2) 11(2) 0 0 1(2) 11(2) 4(6) 32(6) 2(3) 16(3) aRabbits were exposed to 500 ppm benzene for 7 hrs/day on days 6-18 of gestation. bDue to problems in fixation, one litter of 8 fetuses was not examined for skeletal alterations. CF = fetuses; L = litters. Significantly different from control value by a modified Wilcoxon test, p<0.05. Considered to be a major malformation. See Table A-6 for individual litter data. 1%44a TABLE 11 -2 9 - HEMATOLOGIC EVALUATION OF FETUSES AND DAMS EXPOSED TO BENZENE Fetuses - Rabbit Control Benzene-exposed - Mouse Not determined Bred females*1 - Rabbit r~) bo zo -n --t Control Benzene-exposed O"FI O' _ Mouse z Control r-4 IF Benzene-exposed PCV N Percent 5 43.6+5.5 5 46.7+3.5 4 39.9+6.4 4 38.9+3.5 8 49.7+1.5 8 48.1+1.6 RBC x 10/mrrr Hgb q/100 ml WBC x lO'Vimr NBC Differential Count {%) Neut Lymph Mono Eos in Baso 3.7+0.4 4.1+0.4 12.2+1.4 13.9+1.2 0.65+0.13 0.89+0.32 Not Determined 5.9+1.1 13.3+2.4 6.9+2.9 46 46 4 5.8+0.7 12.7+1.2 5.8+0.7 44 52 2 8.6+0.4 16.3+0.6 7.2+1.1 22 74 2 8.4+0.2 16.0+0.6 6.9+1.6 28 69 2 13 11 20 20 aBlood was collected on day 29 of gestation from the offspring'of rabbits exposed to 0 or 500 ppm of benzene for 7 hours per day from days 6 through 18 of gestation. bBred female mice and rabbits were exposed to 0 or 500 ppm of benzene for 7 hours per day from days 6 through 15 (mice) and 6 through 18 (rabbits) of gestation; blood was collected on days 16 (mice) and 19 (rabbits) of gestation. No value differed significantly from the control value by a one-way analysis of variance, p<0.05. 30- Animal Number *6-9000 46-9001 46-8013 46-8014 46-8015 46-8016 46-6018 46-8021 46-6062 46-8063 46-8064 46-8065 46-8066 46-8067 46-8074 46-6075 46-8077 46-6062 46-8063 46-8069 46-8090 46-8091 46-8097 46*8102 46-8103 46-6104 Mean 1 S,D, 4 36 31 34 37 35 35 36 34 35 35 34 33 33 35 35 33 33 35 30 27 30 31 33 35 36 34 34 1 2 TABLt A-l INDIVIDUAL 1O0Y AND LXY11 WIOTS Of PllCMAKT MCE UPOSED TO BENZENE BT INHALATION* Exposure Level: 0 ppe Maternal lody Weight (i) A 1 iff- ?<) oi <eatatloa 1& 16 Maternal Body Weight Cain (a) A-f no 10-16 16-18 33 33 39 42 31 32 46 50 36 37 47 51 37 39 47 51 37 40 49 51 37 40 49 54 35 37 46 51 33 37 49 54 35 35 42 50 36 36 46 56 30 34 39 47 33 35 46 55 33 35 41 47 34 35 43 51 36 35 47 54 35 37 47 55 34 40 46 52 37 36 42 48 31 29 52 57 30 29 34 37 32 33 38 42 36 36 46 51 32 35 44 50 33 36 46 51 36 36 46 51 32 35 46 52 306 3 0 1 14 4 2 1 10 4 028 4 2392 239 5 -1 2 9 5 1 2 12 5 007 8 1 2 8 10 4 4 5 8 0 2 11 9 026 -1 1 a 1 -1 12 6 8 7 2 2 10 8 1 68 4 2 -1 6 6 1 -2 23 5 3 -1 5 3 2154 5 0 10 5 -1 3 9 6 -2 3 10 5 028 3 -2 3 11 6 34 2 36 3 *3 * * 50 4 0..* * 2 22 94 62 Maternal Liver Weight (Per IS) Absolute Relative 8 (lAl Body W.liht) 2.67 3.00 3,10 3.34 2.91 3.77 2.57 3.37 2.83 3.60 3.23 3.27 2,86 3.10 3.73 3.32 2.54 3.00 3.62 2.33 2.88 2.83 KDb 2,71 3.19 3.12 63,6 60.0 60.8 65,5 57.1 69,6 50,4 62.4 56,6 64.3 68.7 59.4 60.8 60.8 69,1 60,4 46.6 62.5 63.5 63.0 68.6 55.5 ND^ 53,1 62.6 60,0 3.08 0.30 61.1 t 5.4 *Hle* Mere exposed to 0 or 500 pp* beoxeae for 7 Hre/daj oo days 6-15 of gestation, bThe liver weight mob inadvertently not determined for thie animal. on CONFIOFNTT Al. -31- Afiiul *6-8003 46-8004 46-8022 46-8023 46-8024 46-8023 46-8026 46*8027 46-8028 46-8029 46-8068 46-8069 46-8071 46-8072 46-8078 46-8080 46-8081 46-8084 46-8086 46-8087 46-8088 46-8092 46-8093 46-8094 46-8095 46-8106 46-8107 46-8108 46-8110 46-8112 M**Q S.D, i 40 36 35 36 31 32 31 34 34 36 32 37 36 39 36 34 36 30 31 35 34 31 29 34 33 36 37 37 35 35 34 * 3 TA1L1 A-1 (CofttlnuodJ Expo*wr Livili 500 ppa Katinvftl 0*. W'iltlt () ------- DirTIj Of GUt?lon A 10 16 18 Katarnal Body Usliht Colo (t) 6-8 *-10 npis ms 39 35 35 36 32 29 30 31 35 35 33 34 36 37 33 31 37 31 33 34 34 30 32 37 35 35 38 37 33 35 34 * 2 39 34 37 39 34 34 31 36 37 39 37 35 38 41 39 34 39 33 33 35 36 36 34 38 38 36 40 39 35 36 36 2 50 47 46 44 41 41 39 45 50 50 46 41 45 52 50 44 45 46 43 45 45 44 49 45 48 45 47 51 42 45 46 * 3 56 52 49 50 45 46 43 51 55 56 51 45 51 55 57 50 51 50 48 52 51 47 54 47 52 50 50 56 47 49 50 4 -1 -1 0 0 1 -3 -1 -3 1 -1 1 -3 0 -2 -3 -3 1 1 2 -1 0 *1 3 3 2 -1 1 0 -2 0 0.3 2 0 -1 2 3 2 5 1 5 2 4 4 1 2 4 6 3 2 2 0 1 2 6 2 1 3 1 2 2 2 1 2 12 11 13 9 5 7 7 a 9 13 11 9 6 7 11 11 10 6 13 10 10 9 8 15 7 10 9 7 12 7 9 92 6 5 3 6 4 5 4 6 5 6 5 4 6 3 7 6 6 4 5 7 6 3 5 2 4 5 3 5 5 4 5*1 Mjtcmil Llvtr WclahC (Day 18) AbtoluCft teiatlv* ____ 1 (g/kc Body tfoljtht) 3,76 2.91 3.02 3.13 3.01 2.76 2.48 3.15 3.00 3,16 2.81 2.51 3.23 3.09 3.62 2.62 3.52 3.11 2.82 3.03 3.30 3,28 3.82 3.29 3.83 3.08 3.67 3.82 2.80 3.15 67.1 56.0 61.6 62.6 66.9 60.0 57,7 61.8 54.6 56.4 55.1 55.8 63.3 56.2 63.5 52.4 69.0 62.2 58.8 58.3 64.7 69.8 70.7 70,0 73.6 61.6 73.4 68.2 59,6 64.3 3.16 * 0.38 62.5 i 5.9 nD 1 36447 CONFTOFNTTAV. Anlnal Nubtf 47-8003 47-0006 47-0007 47-0008 47-0031 47-8032 47-8033 47-8036 47-0039 47-8041 47-8046 47-8047 47-0048 47-0049 47-0054 47-8055 47-0056 47-8057 Mean * S.D. -32- TABU A-2 INDIVIDUAL BODY AND LIVXI WEl(ZTS Of PREGNANT 8A88IT3 EXPOSED TO BENZENE BY UKALATION* Bipoiurt Ltvtl; 0 pft Wtifml Body Weight (t)______________ Hatsraal Body Weight Cain (i) --r~----------- r---------- nr-----------IT" ......."U D.yU } oi Caseation 29 ST 8-10 nFn~"lTFIf"' 19-29 5,03 3.91 4,13 3.03 3.90 4.12 3.99 3.82 4.00 4.13 4.43 4.03 3.82 3.97 4.01 4,38 3.81 3.76 HD*1 ND ND ND 3.06 4,04 3,77 3.02 3.99 4.14 4.31 4.00 3.00 3.91 3.99 4.36 3,81 3.65 4.93 3.89 4.00 3,82 3.85 4.03 3.94 3.90 3,93 4.15 4.32 4.04 3.81 3.96 3.99 4.30 3.83 3.65 3.02 3.92 4.09 3.86 ND ND ND 3,91 3,96 4,21 4.35 4.01 3.83 4.00 3,98 ' 4.28 3.81 3,67 5.22 3.95 3.87 3.98 3.86 3.69 3.99 4.00 3.97 4.33 4.33 4.11 3.97 4,09 4.14 4,51 9.90 3.72 5.04 4.08 3.94 4.14 3.98 3,44 4.13 4.26 4.06 4.43 4,40 4.17 4.15 4.10 4.25 4.76 3.84 3.09 -- -- -- -.04 -.08 -.22 0 -.01 +.01 -.12 -.03 -.02 -.06 -.0* -.02 0 -.11 -- -- -- -,01 -.01 .17 .08 -.06 .01 .01 .04 .01 .05 0 -.06 .02 0 .09 .03 .01 .04 -- -- -- .01 .03 .06 .03 -.03 .02 .04 -.01 -.02 -.02 .02 .20 .03 -.22 .12 -- -- -- .09 .01 .12 .02 .10 .14 .09 .16 .23 .09 .05 -.18 .13 .07 .16 .12 -.45 .14 .26 .09 .10 .07 .06 .18 .01 .11 .25 -.06 .17 4.06 60.31 3,96 60.20 4.02 60.20 4.06 0.32 4.10 60.34 4.17 6 -0,05 60.06 0.02 0.06 0.02 0.03 0.08 60.11 0.07 60.16 Maternal Liver Weight (Day 29) Abaoluta Relative <*> (ft/kit Body Waijtht) 99 19.64 74 18,14 101 25.63 97 23.43 102 25.63 97 28.20 113 27.36 85 19.95 90 24,14 123 27.77 68 15.45 82 19.66 115 27.71 96 23.41 04 19.76 125 26.26 111 26.91 101 25.96 98,39 615.67 23.72 4.02 *labbit* w*r* eapotad to 0 or 500 ppa benzene for 7 hre/day on days 6-18 of geetatlon. b)lot determined for thla animal DO 136448 CONFTDFNTTAL. -33- Animal Nwbtr 47-8009 47-8010 47-8011 47-8012 47-8034 47-8035 47-8036 47-8037 47-8042 47-8043 47-8044 47-8045 47-8050 47-8051 47-8052 47-8053 47-8059 47-8060 47-8061 Main i S.D. 3.73 3.84 3.91 3.79 3.99 4.03 3.79 4.05 3.68 3.90 3.89 3.72 4.82 4.17 3.48 4.12 3.92 4.29 3.55 3.93 0.29 Matamal 8067 Wyltht (1) --ni l6 19 M0* NO NO NO 4.01 4.01 3.94 4.05 3.62 3.83 3.96 3.46 4.74 4.08 3.41 3.62 3.92 4,19 3.45 3.71 4.01 3.83 3.82 3.97 4.00 3.74 4.03 3.65 3,87 3,80 3.71 4.80 4.15 3.45 3.91 3.91 4.17 3.47 3.72 4.01 3.87 3.60 NO NO NO ND 3.63 3.90 3.96 3.70 4.67 4.16 3.45 4.10 3.92 4.30 3,49 3.83 4.04 4.10 3,78 3.94 4.12 4.03 4.12 3.76 3.92 3,89 3.77 4.75 4.29 3,60 4.23 4.09 4.29 3.53 3.88 0.33 3.89 0.29 3.90 0.31 4.00 0.28 TABU 4-2 (Caatlaitfd) Expoura Lt1; 500 pfa Matarnal Body ttaliht Cain (1) Day(i) of Captation 29 6-8 8-10 10-12 12-19 4.08 . .01 .11 4,16 -- -- 0 .03 4.36 -- -- .50 .23 3.81 -- -- -.22 .18 4.06 4.17 3.99 4.12 .02 -.02 .15 0 -.04 -.01 -.20 -.02 ---- --- -- ---- ---- 4.06 -.06 .03 -.02 .13 4.39 -.05 .02 .03 .02 4.15 3.93 .07 -.26 -.16 .25 +.16 -.01 -.07 .07 4.86 -.08 .06 -.13 .08 4.13 3.58 -.09 -.07 .07 .04 .01 .13 0 .15 4.03 -.50 .29 ,19 .13 4.31 4.32 3,75 0 -.10 -.10 -.01 -.02 .02 4.01 .13 .02 .17 -.01 *04 3115 .25 .12 .26 .03 .12 .05 .04 0 .30 .47 .26 .16 .11 -.16 -.02 -.20 .22 .03 .22 4.12 0.27 -0,07 0.15 *o0..0u2 0,02 0.10 0.09 0.08 0.11 0.16 Matarnal Llvar Vtight (Day 29) Abaoluta Ralactva (a) (a/** Body Valght) 219 118 90 95 100 101 94 105 104 122 106 104 101 98 99 97 126 111 101 29.17 28.37 20.64 24.93 24.63 24.22 23.56 25.49 25.62 27.79 25.54 26.46 20.78 23.73 27.65 24.07 29.23 25.69 26.93 104,79 10.00 25.50 2,42 Not dtc*ln*d for this animal 00 136449 CONFIDENTIAL -34 TABLE A-3 INDIVIDUAL LITTER SUMMARY OF OBSERVATIONS MADE AT THE TIME OF CESAREAN SECTION OF MICE EXPOSED TO BENZENE BY INHALATION Exposure Level: 0 ppm ANIMAL NUMBER LITTER SEX RATIO BODY CROWN-RUMP IMPLANTATIONS RESORPTIONS SIZE M:F WEIGHT (g) LENGTH (mm) 46-8000 46-8001 46-8013 46-8014 46-8015 46-8016 46-8017 46-8018 46-8019 46-8020 46-8021 46-8062 46-8063 46-8064 46-8065 46-8066 46-8067 46-8074 46-8075 46-8076 46-8082 46-8083 46-8089 46-8090 46-8091 46-8096 46-8097 46-8098 46-8099 46-8100 46-8101 46-8102 46-8103 46-8104 46-8077 11 2 9 6:3 0.84 23.4 13 1 12 6:6 1.02 25.1 13 2 11 8:3 1.00 24.3 10 0 10 3:7 1.05 24.4 13 0 13 5:8 0.97 23.9 13 5 8 1:7 1.14 25.2 Non-Pregnant 13 1 12 4:8 1.01 22.3 Non-Pregnant Non-Pregnant 13 1 12 7:5 1.11 24.8 12 0 12 7:5 1.03 25.1 13 0 13 7:6 1.11 25.8 9 2 7 3:4 0.90 24.4 14 1 13 7:6 1.13 26.1 9 0 9 5:4 1.16 26.1 12 1 11 6:5 1.07 25.3 14 3 11 4:7 0.97 24.3 12 0 12 7:5 1.09 25.0 Implantation sites detected only after sodium sulfide stain 13 2 11 6:5 0.96 23.9 13 2 11 4:7 1.07 24.9 6 2 4 3:1 1.00 24.2 8 2 6 4:2 0.82 21.4 14 4 10 6:4 0.93 22.9 Implantation sites detected only after sodium sulfide stain 15 4 11 5:6 1.03 23.1 Non-Pregnant Implantation sites detected only after sodium sulfide stain Non-Pregnant Implantation sites detected only after sodium sulfide stain 14 1 13 6:7 0.83 21.6 12 3 9 3:6 0.96 21.8 13 0 13 6:7 1.07 24.2 16 0 16 10:6 0.98 23.6 MEANiSD 122 21 11*2 50:50 1.01*0.09 24.1*1. aMice were exposed to 0 or 6-15 of a gestation. benzene for 7 hrs/day on days -35- TABLE A-3 (continued) Exposure Level: 500 ppn ANIMAL NUMBER LITTER SEX RATIO BODY CROWN-RUf IMPLANTATIONS RESORPTIONS SIZE M:F WEIGHT (g) LENGTH (n 46-8002 46-8003 46-8004 46-8022 46-8023 46-8024 46-8025 46-8026 46-8027 46-8028 46-8029 46-8068 46-8069 46-8070 46-8071 46-8072 46-8073 46-8078 46-8079 46-8080 46-8081 46-8084 46-8085 46-8086 46-8087 46-8088 46-8092 46-8093 46-8094 46-8095 46-8105 46-8106 46-8107 46-8108 46-8109 46-8110 46-8111 46-8112 Implantation sites detected only after sodium sulfide stain 13 1 12 5:7 1.03 24.5 16 0 16 10:6 0.82 22.8 12 1 11 5:6 1.02 24.8 13 2 11 6:5 0.92 23.0 8 1 7 6:1 1.14 25.0 11 0 11 7:4 0.96 24.3 13 4 9 5:4 0.93 24.3 13 0 13 5:8 0.93 24.3 14 2 12 5:7 1.07 24.7 14 0 14 9:5 1.04 24.9 14 1 13 8:5 0.96 24.4 10 3 7 3:4 0.80 22.2 Implantation sites detected only after sodium sulfide stain 11 1 10 4:6 0.98 24.7 15 2 13 7:6 0.96 24.0 Non-Pregnant 14 1 13 8:5 0.90 23.2 Removed from study due to leakage of water bottle 14 0 14 5:9 0.76 22.0 7 1 6 3:3 0.96 24.7 16 3a 12 6:6 1.04 24.9 Non-Pregnant 11 0 11 6:5 1.02 24.5 13 2 11 4:7 0.90 22.9 12 2 10 8:2 0.83 22.0 10 3 7 2:5 0.85 22.8 14 0 14 7:7 0.92 23.3 14 4 10 7:3 0.80 22.0 16 6 10 4:6 0.89 23.2 Implantation sites detected only after sodium sulfide stain 14 3 11 7:4 0.88 22.4 15 10 5 4:1 1.16 25.6 12 2 10 5:1 1.12 24.9 Non-Pregnant 9 0 9 6:3 0.86 22.6 Non-Pregnant 13 1 12 2:10 0.97 24.0 MEANiSD 13*2 2*2 11*2 52:48 0.950.10b 23.8*1.1 a0ne fetus was dead at the time of cesearean section ^Statistically significant by Dunnetts' Test p<0.05 HO 136451 CONFIDENTIAL TABLE A-4 INDIVIDUAL LITTER SUMHARY OF OBSERVATIONS MADE AT THE TIME OF CESAREAN SECTION OF RABBITS EXPOSED TO BENZENE BY INHALATION3 Exposure Level: 0 j Animal Number 47-8005 47-8006 47-8007 47-8008 47-8031 47-8032 47-8033 47-8038 47-8039 47-8041 47-8046 47-8047 47-8048 47-8049 47-8054 47-8055 47-8056 47-8057 Corpora Lutea 10 10 9 9 12 11 10 11 10 8 10 8 8 8 8 9 8 9 Implantations 12 9 8 9 9 11 9 11 10 7 9 8 8 8 5 9 8 9 Pregnancy Wastage 0 1 1 0 3 0 1 0 0 1 1 0 0 0 3 0 0 0 Resorptions 1 1 1 0 1 1 2 0 0 0 0 0 0 0 1 0 0 1 -Itter Size 11 8 7 9 8 10 7 11 10 7 9 8 8 8 4 9 8 8 Sex Ratio M:F 8:3 3:5 4:3 6:3 6:2 5:5 1:6 7:4 5:5 5:2 4:5 5:3 6:2 6:2 0:4 6:3 6:2 6:2 Fetal Body Weight (g) 39.9 42.8 36.4 44.0 36.3 24.9 39.3 38.5 34.2 46.4 41.6 38.7 39.1 36.8 51.2 45.2 39.7 34.7 Crown-Ruj Length (i 100.3 101.1 95.8 103.7 90.0 75.2 90.3 97.2 93.9 106.2 100.2 95.6 99.1 96.2 108.4 105.7 98.2 94.7 MEANSD 91 92 11 0.50.6 82 59:41 39.45.7 97.37.: -36 days 6--18 of gestation Oo j 3645? C'-ONF rDfrNTiAj TABLE A-4 (Continued) Exposure Level: 500 ppm Animal Number Corpora Lutea 47-8009 47-8010 47-8011 47-8012 47-8034 47-8035 47-8036 47-8037 47-8042 47-8043 47-8044 47-8045 47-8050 47-8051 47-8052 47-8053 47-8059 47-8060 47-8061 9 7 15 9 10 8 7 9 10 9 11 11 15 10 7 10 8 10 7 Implantations 6 8 14 9 6 8 7 5 10 8 11 11 12 10 8 9 7 10 7 Pregnancy Wastage 3 0 1 0 4 0 0 4 0 1 0 0 3 0 0 1 1 0 0 Resorptions 1 1 0 0 1 0 0 0 0 0 2 1 4 1 0 0 0 1 2 Litter Size 5 7 14 9 5 8 7 5 10 8 9 10 8 9 8 9 7 9 5 Sex Ratio M:F 0:5 1:6 7:7 7:2 2:3 2:6 2:5 3:2 7:3 2:6 3:6 5:5 6:2 7:2 5:3 3:6 2:5 4:5 1:4 Fetal Body Weight (g) 50.3 36.5 37.3 38.7 33.0 40.5 40.6 46.2 40.0 43.8 39.9 29.4 40.4 36.0 36.3 33.4 31.0 37.1 40.3 Crown-Rump Length (mm) 107.4 96.9 96.9 99.0 84.4 91.0 94.6 99.3 98.4 103.0 98.6 87.8 97.6 89.7 91.2 85.2 90.2 97.2 98.9 MEANiSD --t 6o zo -S'n--t ^ n O' z--t ^ --S vO a> 102 92 11 0.71.0 82 45:55 38.55.0 95.16.0 I u> vi I O oooooooo o -38- Aniul Number Control: 24 25 26 27 60 41 4Z 43 44 Mean S.O. TABLE A-7 HEMATOLOGIC EVALUATION OF PREGNANT MICE EXPOSED TO BENZENE BY INHALATION3 PCV Percent RBC * iq5/m3 Hgb x/100 al WBC * 1Q3/bb1 WBC Differential Count (X) Nent Lymph 47.0 50,0 49.0 51.5 50.0 46,5 50.0 49.0 52.0 49.7 1.5 8,0 9.0 8,1 8.9 9.0 8.1 9.0 8.7 8.7 8.6 0.4 15.4 16.9 16.3 17.3 15.9 16.1 16.2 15.9 16.7 16.3 0.6 7.6 8.9 8.5 7.2 5,9 6.9 6.6 7.6 5.7 7.2 1.1 39 56 3 2 22 76 2 0 20 75 4 1 7 89 2 2 15 82 1 2 20 79 1 0 24 72 4 o 30 66 0 4 26 68 3 3 22 74 2 2 Benzene-exposed: 77 49.5 78 47.0 79 47.0 80 48.0 93 49.0 94 50.0 95 45.0 96 49.0 Mean S.O. 48.1 1.6 8.3 8.4 8.2 8.4 8.5 8.7 8.0 8*7 8.4 0.2 16.8 15.8 14.9 16.1 15.9 16.4 15.4 16.3 16.0 0.6 9.6 7.0 6,9 4.5 7.3 5.2 6.9 7.9 6.9 * 1.6 18 81 1 31 66 2 26 74 0 20 77 2 36 57 1 23 74 2 29 69 1 37 56 3 28 69 2 0 1 0 1 5 1 1 3 2 aBred female nice were exposed to 0 or 500 ppn of benzene for 7 hours per day frofli days 6 through 15 of gestation; blood was collected on day 16 of gestation. No value differed significantly from the control value by a one-way analysis of variance , p <0.05. oooooooo 00 136454 CONFIDENTIAL -39 Animal Number Fetusea* Control: 47-8031 47-8032 47-8033 47-8046 47-8047 Mean S.D. TABLE A~8 HEMATOLOGIC EVALUATION OF RABBIT FETUSES AND OAHS EXPOSED TO BENZENE BY INHALATION PCV Percent RBC X lQ^/iml Hgb g/100 *1 NBC x lO^/imai WBC Differential Count (2) Neut Lymph Mono Eosin Bsso 43.0 35.0 48.0 43.0 49*0 43.6 5.5 3.9 3.1 4.0 3.6 3.8 3.7 0.4 12.7 9.9 13.6 12.1 12*6 12.2 1 1.4 0.52 0.53 0.75 0.66 0.81 0.65 0,13 Not Determined Benzene-exposed 47-8034 47-8035 47-8036 47-8037 47-8050 49.0 44,5 45.5 43.0 51.5 Mean S.D. 46.7 3.5 3,7 4.2 3.9 3.9 4.7 4.1 0.4 14.3 13.3 13.3 12.7 15.7 13.9 1.2 1.14 0.67 0,48 0.88 1.26 0.89 t 0.32 Not Date mined Bred Females** Control: 602 80 810 122 Mean S.D. 34.0 37.5 49.0 39.0 39.9 6.4 5.0 5.3 7.4 5.9 5.9 1.1 11.0 12.3 16.7 13.2 13.3 2.4 6.7 5.2 11.0 4.6 6.9 2,9 66 27 18 78 68 21 32 59 46 46 41 20 82 10 41 2 2 i 8 3 Benzene-exposed: 133 4006 187 698 35.0 5,0 11.4 4.9 54 42 2 2 43.5 6,7 14.2 6.0 46 51 2 1 39,0 6.2 12.9 5.6 30 64 2 1 38,0 5,5 12.3 6,6 47 49 3 0 Mean S.D. 38.9 3.5 5.8 0.7 12.7 1,2 5.8 0.7 44 52 2 1 0 0 3 1 1 *Blood was collected on day 29 of gestation fro the offspring of rabbits exposed to 0 or 500 pp of benzene for 7 hours per day fro days 6 through 18 of gestation* ^Bred female rabbits were exposed to 0 or 500 ppm of benzene for 7 hours per day fro days 6 through 18 of gestation; blood was collected on day 19 of gestation. No value differed significantly fro the control value by a one-way analysis of variance, p<0,05. DO 136455 CONFTDENTTAI -40 TABLE A-5 or orrnomOML unit sswun mu althatiom amosc uitus mici txposts to uicm n itouatioh* Expnnnra Lerel 0 pm man or rrrusis examined ANIMAL NUKBI1S 46-4000, 01, 13, U, 13, 16, 18, 11, 82, 83, 84, 85, 88, 87, 74, 73, 77, 82, 83, 89, 90, 91, 97, 102, 103, 104, LXTEMAL EXAMINATION S0TT-TI5SIZ EXAMINATION SKELETAL EXAMINATION ONES Of TUB SKULL 9 12 u 10 13 8 12 12 12 1) 7 13 9 11 11 12 16 11 11 4 6 10 11 13 344 34 34444 343 444 3443 3 3 4 4 0* 12 11 10 13 8 Ob ioe 12 13 7 13 9 8C 11 12 18 11 11 4 6 10 11 13 Oh 8 7 7 9 5 oh 8 8 9 4 9 6 4 7 8 11 7 7 1 3 7 7 9 9 13 44 9 13 39 Niton OP PETUSI8 AFFECTED Total Ma.flor Malformations 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 ' 0 0 0 0 0 0 0 0 0 0 EITIEWAL ALTTlATIONi Cl.ft P.l.C. Subcutaneous hemorrhage, hood raflon 00000001000000000000000 0 0 0 00000000000000000000000 0 0 0 SOFT TISSUE ALTERATIONS Slightly 4U*t(d 3rd ventricle of brain Subcutaneous hemorrhage, head teflon Subcutaneous hamocthaga, around brain 0000000000000a000000000 0 0 0 000000000000000000001 00 0 0 0 0000000Qa000a000Q000000 0 0 0 SKELETAL ALTERATIONS steraebree - delayed ossification fused uafu*ed extra ilte of oaelflcatlon alashapmn (#6) extra cartilage misplaced cartilage 00100 0 00 00 0 1030 00000 00000 00000 00100 000 300020 1 2 1 0300 6 2 1 0000000000000000 0 0 0 00020 1 0 1 00 1 0000 1 4 1 0 001 0 1 00000000000 0 0 7 0000000000000000 0 0 0 0000000000000000 0 0 2 0000000000000000 0 0 1 Elba * extra forked or fused Irregular oaalflcatlon 1 1 000 00000 00000 2 2 3 1 3020 , 20000 1 0 2 4 3 0000000000000000 0 0 0 0000000000000000 0 0 0 Vartebrao aaynnatrlc lumber spore delayed oaalflcatlon of centra delayed oaalflcatlon of cerylal arebaa forked atlaa misshapen atlas delayed oaalflcetlon of atlas 0 0 0 0 t> 1 I000 00 0 0 0 000 0 0 0100 1 00000 00 0 0 0 0000000000000000 0 0 0 1 ] 30000 0 1 000002 0 2 1 1 0000000000000000 0 0 0 0000000000000000 0 0 0 0000000000000000 0 0 0 0000000000000000 0 0 0 0000000000000000 0 0 0 Skull delayed oaalflcatlon foranan, occipital or parietal unfuaad occipital unoaelfled, occipital nlsahepen, interparietal 2102 1 00000 000 1 0 00000 00000 200 1 000 200 1 1 , 30 1 9 1 0 0000000000000000 0 0 0 0000000000 000000 0 0 0 0000000000000000 0 0 0 0000000000000000 0 0 0 TOTAL TOTAL musts litteis 279 28 97 28 253 24 165 24 11 11 00 00 11 00 24 10 00 IS 9 93 00 2l 22 30 14 00 00 00 14 10 00 00 22 00 00 28 14 00 L1 00 00 *Mlce were exposed to 0 or 300 ppm became for 7 hre/dsy on daye 8-13 of gestation. bfwe lletera fra the control group and two litters from tbe bnnxenn groap worn not examined for eketal alterations. The fetuses from them Uttera were iiadverteotly nixed together during tho staining procaduro, c$ona skeletons trm Httare 8021 and 8067 am nlaelng from the skeletal"mxamlnatlon because they where Inadvertently damaged during the staining procedure. `Considered to be n mnjor malformation. DO 13645ft CONFTDFNTTAl. -41- TA1LI A-) (Ceeti*od) tutTTWB! LXTT11 SUMUH CT VITAL ALTEAATHMi AMMG LlTTItS Of MICK HTCSID TO until IT INHALATION b^wuri Lml 300 wwm ov mwM iiahuto 48INAL roOU; 46-8003, 04, IS, M. 24, a. 26, 27, 28, 29, M, 69, 71, 72, 78, 0, 81, 84, >6, 7, M, 92, 93, 94, 9), 106, 107, 108. 110, 112, --re--sy EXAHXNAtlOH sott-tissui uamination SKELETAL UAMlNATIOg ONES (V TIE SKULL 11 14 11 11 4 54 4 12 flfr it 11 807 7 7n 34 7 11 47 9 13 IS 344 0* 12 12 08 14 1) 34 3 13 39 7 10 1) 1) 14 33443 N 10 13 1) 14 4 79 99 6 l) 11 11 10 3444 3 6 13 11 11 10 3 9777 7 14 10 10 3 3 33 7 14 10 10 49 77 U 4 11 7 5 10 33 5 10 27 9U 34 9 12` 68 KJKttl 091 flEUUS ametto Total Ha tor Ralfomatlons 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Q 0 0 0 0 0 EXTERNAL ALTERATIONS ci.it p.i.t." Subcutaneous hseorrhsge. head region 00000000 000 100000000000 00 0 0 0 0 0 0 0 000 0 0 0 1 000 0 000000 0 00 0 00 0 0 0 0 0 SOFT TISSUE ALTERATIONS Slightly dilated 3rd ventricle of brain Subcutaneous haeorrhage, head region Sobcoteneoue heeorrhsge. arot^ knit 0000000000100000000000000 0 0 0 0 0 0000000000000000000000000 0 0 0 0 0 00000Q00 1 0000000000000000 0 0 0 0 0 SKELETAL ALTERATIONS Sternabrae " diliTtd salification fuaed unfueed extra aita sf salification elaahapea (96) extra cartilage alspleced cartilage 2 0 2 0 0 0 0 14 0 0110 00 10 0 000 00 00 00 1 01 0 0 00 8 20 7 0 2) 2 0 0 10 1 0 0 13 2 1 30 030 0 o 1 0 0 0 0 0 0 0 00 0 10 0 00 0 0 0 0 0 10 0 6 0 10 0 0 0 0 0o0o0 Q 06320 02 0 0 0 00 0 0 0 0 0 0 0 1 0 0 o 0 0 10 01 0 0 0 0 0 o o o o o o 0 0 0 0 0 0 1 0 0 ooo 0 0 0 0 0 00 0 0 0 0 00000 00 0 0 0 0 0 0 0 0 0 o 00 0 0 0 0 0000 0 00 0 0 0 0 0 Riba extra forked or fused* Irregular oealflcetlon 1 0 0 000 0 0000 0000 7 0 0 3 1 1 0 10 0 400 1 2 30 0000l000000 0000 0 00 0000000000 1 0000 0 00 009 000 000 Vertebrae aiT^Btrlc liMber epure delayed salification sf centre delayed ossification of cerrlal arches forked atlae lifhapeo sties delayed oaslflcatioo of atlea ftull delayed ossification fnraean, occipital or perietal oafused occipital anoeaifled, occipital nlaehapen, ioterperietel 0 0 0 0 0 0 0 0 0 0 0 0 0000 0 0 10 0000 0000 0 10 0 0000 0000 0202 0000 000 0 0000 000 0 01 30 00 00 20 40 40 39 1 100 ) 8 30 10 0 0 000 000 000 000 001 000 002 000 0o0 o00 1 00000 00000 1 000100 2 0 0 1 0 0 30 00 00 04 00 01 00 00 0 0 0 0 0 TOTAL FETUSES 324 112 266 169 1 J 1 1 0 1 51* 3 29 2 1 0 0 35 1* 1 1* 15 1* 1* 1 1 1* 64* 2 10 2 1 TOTAL UTTER 30 30 26b 26* 1 1 1 1 0 1 16 3 12 2 1 0 0 1) 1 1 1 12 1 1 1 1 1 19* 2 6 2 1 idee were exposed to 500 ppe besets for 7 hre/dey at days 6-15 af gaatatloa. fcfto litters fr the control group atd tee Utter* fre* the hamate grmp mto net far eketal alterations. The fetuaee free these Utters mn lsedmrtently tilted cogs ther during the s telsieg procedure. c5<*e akeletsts froe Utters 6027 sad 6049 srs alsslsg free the skeletal meelnstioe beceuse they t*re inadvertently dsesgsd during the staining procedure. *SlgalflcMtly different trm control mien by t Modified Wlcmen tost, p<0.05. This alteration oecurr^ In tha alngla fstus with cleft palate. `Considered to be e eejor eelfometion. DO 1 36*57 DFNTT Al. C.ONF't -42- TA1LI 1-6 m or <FIHD dual UTTER SUMMARY FETAL ALTUAT10M5 AMONG LITTERS RABBITS INBALIHG BIHEEHE BT IMAUTIOP Expoauret 0 ppm hpheer or fetuses examined ANIMAL HUMBER External Examination Soft Tiaaue Examination Skalatal Examination 47-8005, 04, 07, 0. 31. 32, 33, 38, 39, 1. 46, 47* 48, 4*. 34, 33. 3*. 37, 8 a11 8 7 9 10 7 11 10 7 9 8 8 4 9 a a a a4 3 3 3 3 3 3 4 3 3 3 3 3 3 3 3 3 3 11 7 9 0* 10 7 11 10 7 9 8 49 FETUSES 150 56 142 TOTAL MAJOR MALFORMATIONS; DUMBER OF FETUSES AmCTID 0 Enttrul Uttptlm Gaatroacblala* 0 Soft Tiaauo Altaratlona Cygtie eoleului attached to goll bladdar Fused Inoonltuta and loft carotid artarlaa Satalllta veeeal* off aorta, Ina^lnata or carotid artarlaa 0 49 10 Skalatal Altaratlona Starnabraa - delayed oaalflcatloo astro alto of oaaifleatlon fuaod wafvaod 11 0 00 100 100 100 0 0 1126 0 0 Riba 13th rib or riba lunbar apur or apura fuaod or forbad* 8;9 0 Vartabraa fuaod vertebrae end henlcentra, thoracic raglon* laihapoa or duabell* ahopd centra uofuaad contra extra alto of oaalflcatlon between vartabraa Skull extra auturo llna wtthln perietal bona formo - parietal or interparietal 000 010 UTTERS 18 18 17* 0 0 0 18 6 171 0 0 16 07 *Rabblta wore exposed to 0 or 500 ppa banxona for 7 hrs/day on days 6-18 of |aatatlon* bDue to problme In fixation, one litter of 8 fotuaea one not exanloed for ahelatal altaratlona, ^Considered to be a major nalfomatloo, > i DO 136458 CONFTDFNTT At, 43- TARLI *-4 (OMtlttMi) INDIVIDUAL LITTER SUKMARI ON UTAL ALTERATIONS AHONC LITTER* ON RARIITS EBOSED TO lEieKRI IT INHALATION ANIMAL RtMttl Espooor*i 500 ppa rnwnman of eeahubp 47-0009, 10, 11, 12, 34, 33, 31, 37, 42, 43, 44, 43, SO, 31, 52. 53, 39, 40, 41 TOTAL FETUSES External Examination Soft tleeuft Examination Skeletal Examination TOTAL M4J01 MALFORMATIONS; External Alteration Cutroac^liliv 5 7 14 9 3 0 7 3 10 0 9 10 0 9 8 9 7 9 5 3333 333333333 33333 3 ; 14 9 3 0 7 5 10 9 10 0 9 9 7 9 5 nunisr or musts affected 0000 00 000 001 1 000 1 0 0 * 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 00 132 1S529 3 1 Soft Tiaaua Alterations Cyatic calculua attaebad to gall bladder Fused lnn<mlnate and left carotid arteries Satellite vessel* off aorta. innominate or carotid aftorlaa 000 0000000000 1 00000 224 33i 1 22 322233233 3 0 2 2 02 i 00 2 2 000 2 2 02 d 0 l 40 f 19 Skeletal Alteretlona Sternsbraa - delayed ossification extra site of notification fused unfused ft4 5 14 0 5 5 5 3 10 0 8 7358794 00 00 0 000 000000 0 0000 000 00000000 i0000000 00000000 0000000010 1 126 0 1 2 Ribs ' 13th rib or riba lumbar spur or spur* fuaad or forked 51 53 1 75030344733l15 000 0 00 0000 1 O2 000000 00000000000 1 0000 1 00 653c* 2 Vertebrae fuaad vertebrae and heml~ centra, thoracic region* misshapen or dunbell* hapad centre unfuaad centra extra site of ossification , between vertebrae ikull iextra future Hoe within perietal bone forsmen - pari*tel or Interparietal 000 0000000000000 1 00 000 0000000001 000 1 1 1 00a 000000000000010 1 0 00 00 0000 000 000 0 000 10 0 0 0 0 0 0 0 0 1 0 0 0 0 0 000 10 0 0 0 0 0 0 0 1 1 0 0 0 1 0101 1 4 2 0 2 6 TOTAL UTTER* 19 19 19 3 1 1 19 9 109 1 1 127 2 1 4 2 0 2 6 *|abblte were exposed to 0 or 500 ppm bonnene for 7 hre/dey on deys 6-18 of Reetation, to problems In fixation, on* llttor of 8 ftuoo a* not examined for skeletal alterations, c$ignificetly different fro* control value by * nodifled Wilcoxon teat, p<0.0J. ^Considered to be major nelfomatlon. DO , CONF10ENTtft