Document LKVQ2mMjjqbKrK4GvRV7rn3vq

Dim DOW CHEMICAL U.S.A. November 28, 197 7 MIDLAND, MICHIGAN 48640 Ms. Lucille Henschel Manufac. luring Chemists Association 1825 Connecticut Avenue, N.W. Washington, D.C. 20009 cc: W. V. Andresen, American Cyanamid Company Z. G. Bell, Jr., PPG Industries, Inc. H. A. Cantor, A. E. Staley Manufacturing Co. R. B. Jaeder, BASF Wyandotte Corporation M. N. Johnson, B. F. Goodrich Chemical Co. A. Konishi, Kureha Chemical Industry Co., Ltd. J. R. McDonough, Tennessee Eastman Company W. A. Peterson, W. R. Grace & Co. L. Rapoport, Olin Corporation J. M. Smith, Rohm and Haas Company R. J. Turner, Morton Chemical Company P. J. Vanderhorst, E.I. duPont de Nemours & Co. P. L. Wright, Monsanto Company J. Yamada, Asahi-Dow Limited Dear Lucille: A draft copy of the document entitled "The Effect of Maternally In haled or Ingested Vinylidene Chloride on Rat and Rabbit Embryonal and Fetal Development" is enclosed for your review. This document will be discussed at the December 7-8, 1977 MCA meeting. Sincerely, /?/ Li* > ' M. Norris Associate Scientist Government Registration Health and Environmental Research _____J |V,,. 3 u 1977 Enclosure dpt' r' m'pHi A.fH.rs J*n AIM OPERATING UNIT OF THE DOW CHEMICAL COMPANY SL 083282 THE EFFECT OF MATERNALLY INHALED OR INGESTED VINYLIDENE CHLORIDE ON RAT AND RABBIT EMBRYONAL AND FETAL DEVELOPMENT BY F. J. Murray, K. D. Nitschke, J. A. John, J. G. McBride, M. F. Balmer, A. A. Crawford, J. S. Murray, J. F. Quast, L. W. Rampy, and B. A. Schwetz DRAFT Reviewed by B. A. Schwetz November 10, 1977 Toxicology Research Laboratory Health and Environmental Research Dow Chemical U.S.A. Midland, Michigan 48640 ThiA Atudy wola Aupponted by the. companteA AponAOAtng xeAeanch on vlnylidene chloK4.de and admlntAtened by The Manu^actuntng ChemtAtA AAAoctatton. SL 083283 THE EFFECT OF MATERNALLY INHALED OR INGESTED VINYLIDENE CHLORIDE ON RAT AND RABBIT EMBRYONAL AND FETAL DEVELOPMENT By: F. J. Murray, K. D. Nitschke, J. A. John, J. G. McBride, M. F. Balmer, A. A. Crawford, '5'r^-S.^Mur ray, J. F. Quast, L. W. Rampy, and B. A. Schwetz Reviewed by: B. A. Schwetz SUMMARY The teratogenic potential of inhaled or ingested vinylidene chloride was evaluated in Sprague-Dawley rats and New Zealand rabbits. Both species were exposed to the test material by inhalation for 7 hrs/day at concentrations of 20 (rats only), 80, or 160 ppm. For the ingestion study, rats were maintained on drinking water containing 200 ppm vinylidene chloride. The animals received the test material on days 615 (rats) and 6-18 (rabbits) of gestation. A teratogenic effect was not evidenced in rats or rabbits inhaling up to 160 ppm vinylidene chloride for 7 hrs/day or in rats given drinking water containing 200 ppm vinylidene chloride. Toxicity to both the dam and her developing embryo was observed among the rats inhaling 80 or 160 ppm and among the rabbits inhaling 160 ppm. At exposure levels which caused little or no maternal toxicity, 20 ppm in rats and 80 ppm in rabbits, no effect on embryonal or fetal development was discerned. Among the rats given drinking water containing 200 ppm VDC, there was no evidence of toxicity to either the dam or to the developing embryo and fetus. SL 083284 2- - INTRODUCTION Vinylidene chloride (1,1-dichloroethylene, VDC, CJ^CClj) is used in the manufacture of various plastics, including films and coatings for food packaging. Reports on the acute toxicity of vinylidene chloride in laboratory animals have indicated moderate toxicity when given by inhalation or by gavage (Carpenter et aJ., 1949; Rylova, 1953; Siegel et al., 1971; Jenkins et al^. , 1972; Jaeger et al. , 1973). Repeated inhalation exposure to 100 ppm vinylidene chloride for 6 weeks resulted in some weight loss in rabbits and monkeys, but not in rats, guinea pigs, or dogs (Prendergast, 1967). Rats exposed for up to 4 weeks to 500 ppm vinylidene chloride exhibited nasal irritation, decreased weight gain, and liver cell degeneration; at 200 ppm, only slight nasal irritation was discerned (Gage, 1970). Balmer et al. (1976), recently reported minimal reversible changes in the liver of rats inhaling 25 or 75 ppm vinylidene chloride for 90 days. Similar hepatic alterations were seen in rats maintained for 90 days on drinking water containing 200 ppm vinylidene chloride, but not among those given water containing 100 ppm or less of vinylidene chloride (Humiston et al. , 1975) . SL 083285 3- - Vinylidene chloride has been shown to be a weak mutagen in certain strains of bacteria (McCann et^ al. , 1975; Greim at al. , 1975; Bartsch et al., 1975). This effect was seen in bacterial systems only under conditions of activation with microsomal enzymes, implicating a metabolite of vinylidene chloride rather than vinylidene chloride itself as the causative agent. A review of the scientific literature on vinylidene chloride revealed a lack of information regarding its teratogenic potential. Thus, the studies presented herein were undertaken to evaluate the potential of inhaled and ingested vinylidene chloride to adversely affect embryonal and fetal development in rats and rabbits. For the inhalation studies, both rats and rabbits were exposed to vinylidene chloride at concentrations of up to 160 ppm. The Threshold Limit Value of vinylidene chloride is 10 ppm (ACGIH, 1977). In order to simulate exposure by ingestion, rats were given the test material in the drinking water. The concentration of vinylidene chloride given in the drinking water, 200 ppm, was the upper limit of solubility of the compound in water. These studies were conducted under the auspices of the Manufacturing Chemists Association in the Toxicology Research Laboratory, Health and Environmental Research, The Dow Chemical Company, Midland, Michigan. SL 083286 4- - MATERIALS AND METHODS Test Material. The test material used for this study was production grade vinylidene chloride obtained from The Dow Chemical Company, Midland, Michigan. Each sample of the test material had a minimum purity of 99.5% and contained 200-400 ppm monomethylether of hydroquinone (MEHQ) to inhibit poly merization. Since the vinylidene chloride used in various copolymers for food packaging applications is distilled to remove the inhibitor MEHQ, the ingestion portion of the study was conducted on distilled vinylidene chloride having a residual concentration of 1-5 ppm MEHQ. Each sample used for the study was analyzed by gas-liquid chromatography for determination of impurities (Table 1). Animals. Adult New Zealand white rabbits (Langshaws Rabbitry, Augusta, Michigan), weighing 3.4-4.7 kg, and Sprague-Dawley rats (Spartan Research Animals, Haslett, Michigan), weighing approximately 250 gm, were used. The day on which sperm were found in the vaginal smear of rats was considered day zero of pregnancy; in rabbits, the day of natural mating was designated day zero of pregnancy. The animals were uniquely identified by means of an ear punch (rats) or a metal ear tag (rabbits). Animals were housed individually in wirebottom cages and maintained on commercially available SL 083287 5- - laboratory animal chow (Ralston Purina Company, St. Louis, Missouri) and tap water free choice. Animals did not have access to food or water while in the exposure chambers. The food and water consumption of rats was measured at intervals of three days or less during the experimental period. Experimental Design. In the initial inhalation experiment, groups of 30 bred rats and 18 bred rabbits were exposed to 160 ppm vinylidene chloride for 7 hrs/day on days 6-15 and 6-18 of gestation, respectively. Subsequently, additional groups of 30 bred rats and 22 bred rabbits were exposed to 80 ppm. Because some embryotoxicity was observed in rats exposed to 80 ppm, an additional group of 44 bred rats was exposed to 20 ppm. Groups of 20-47 bred rats and 16 bred rabbits were exposed in chambers to filtered room air to serve as negative controls; there was a separate control group for each level of exposure. Pregnant animals were exposed in stainless steel and glass Rochester-type chambers of 4.3 cubic meter volume under dynamic airflow conditions. The chamber atmosphere was generated by metering vinylidene chloride at a known rate into a heated vaporization flask and then into the airstream being drawn into the exposure chamber. The concentration of vinylidene chloride in the chamber was analyzed continuously during SL 083288 6- - exposure using an infrared spectrophotometer. The timeweighted mean analytical concentrations of vinylidene chloride in the chamber for the 20, 80, and 160 ppm exposures were 201, 80+5, and 158+6 ppm (mean + standard deviation), respectively. For the ingestion study, 26 bred rats were given tap water containing 200 ppm vinylidene chloride on days 6-15 of gestation. A group of 24 bred control rats was given tap water without vinylidene chloride. The water was supplied from 16 ounce wide-mouth glass bottles with plastic screw caps fitted with stainless steel sipper tubes containing stainless steel balls. The bottles were emptied and re filled daily from freshly prepared stock solutions. In an earlier study, vinylidene chloride was determined to be relatively stable in water for 24 hours. Maternal and Fetal Observations. Animals were observed daily throughout the experimental period for indications of toxicity from the test material. The maternal body weights of rabbits were recorded on days 6, 12, and 19 of gestation. The body weights of rats were recorded on days 6, 10, and 16 of gestation. In addition, maternal body weight and the weight of the maternal liver were recorded at the time of cesarean section, day 21 in rats and day 29 in rabbits. SL 083289 7- - On the day of cesarean section, the females were sacrificed by carbon dioxide inhalation; the uterine horns were ex teriorized through a mid-line incision in the abdominal wall, and the number and position of live, dead, and resorbed fetuses were noted. The uteri of apparently non-pregnant animals were stained with a 10% solution of sodium sulfide and examined for evidence of early resorption sites (Kopf et al., 1964); this procedure was conducted for the sole purpose of determining the percentage 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 rat 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 eviscerated and placed in 95% ethanol and were cleared and stained with alizarin red-S (Dawson, 1926) to facilitate examination for skeletal alterations. Statistical Evaluation. The incidences of pregnancy and resorptions were analyzed statistically by the Fisher exact SL 083290 8- - probability test (Siegel, 1956). The Wilcoxon test as modified by Haseman and Hoel (1974) was used to evaluate the incidence of fetal alterations. Fetal body weights and body measurements, maternal body weights, weights of the maternal livers, and food and water consumption data were analyzed statistically by a one-way analysis of variance (Steel and Torrie, 1960). The level of significance chosen for all cases was p<0.05. RESULTS RATS Maternal Toxicity. No maternal deaths were seen among the rats receiving vinylidene chloride either by inhalation or by ingestion of the compound in the drinking water. Single dams exposed to 80 or 160 ppm vinylidene chloride by inhala tion delivered a litter prematurely on days 18.and 21 of gestation, respectively; early deliveries were not seen in any of the other groups of rats. Sialodacryoadenitis, as diagnosed by the presence of swollen salivary glands, was observed in one of the dams inhaling 20 ppm vinylidene chloride, as well as in one concurrent control dam; this disease, presumably of viral origin, was not evident among the other rats. SL 083291 9- - The mean body weight of the pregnant rats exposed to 20 or 160 ppm vinylidene chloride by inhalation was significantly less than that of the concurrent control rats on day 10 of gestation, (Table 2); at 80 ppm, a significant decrease was observed in maternal body weight on day 16 of gestation. Maternal weight gain on days 6-9 of gestation was decreased in a dose-related manner at 80 and 160 ppm. A significant increase in maternal weight gain was noted during the post exposure period among the dams exposed to 160 ppm. No significant effect on maternal body weight gain was discerned among the dams inhaling 20 ppm of vinylidene chloride or among those receiving the compound in the drinking water. Inhalation exposure of the dams to 160 ppm vinylidene chloride resulted in a significant increase in maternal liver weight relative to body weight, but the absolute liver weight was unaffected. The maternal liver weight was not altered by exposure to the lower levels of inhaled vinylidene chloride or by ingestion of drinking water containing vinylidene chloride (Table 2). The amount of food consumed by the pregnant rats exposed to 160 ppm vinylidene chloride by inhalation was significantly decreased on days 6-8 of gestation and significantly increased on days 12-14 and 18-20 of gestation (Table 3). At 80 ppm, SL 083292 -10- significant decreases in food consumption were seen between days 6-17 of gestation. Exposure to 20 ppm vinylidene chloride by inhalation or ingestion of drinking water con taining 200 ppm vinylidene chloride had no significant effect on the amount of food ingested (Tables 3 and 4). The amount of water consumed by the pregnant rats exposed to 160 ppm vinylidene chloride was significantly greater than that of its control group throughout days 6-20 of gestation (Table 3). Significant increases in water consumption were also seen in the dams inhaling 80 ppm (days 15-17 of gestation) and 20 ppm (days 12-17 of gestation). Inclusion of vinylidene chloride in the drinking water did not significantly affect the amount of liquid consumed by the dams (Table 4). Based on the amount of water consumed, it was calculated that the dams given vinylidene chloride in their drinking water received an approximate daily dose of 40 mg vinylidene chloride/kg body weight/day. Embryo- and Fetotoxicity: Observations made at the time of cesarean section of rats are recorded in Table 5. Exposure to vinylidene chloride by inhalation or by ingestion did not significantly affect the incidence of pregnancy. A statistically significant increase in the average number of corpora lutea per dam was discerned among the rats receiving vinylidene SL 083293 -11- chloride in the drinking water but not among those exposed by inhalation; since the number of corpora lutea is estab lished before day 6 of gestation, this increase was not a compound-related effect. No significant effect on the number of implantations or on the number of live or dead fetuses was discerned among litters of rats given vinylidene chloride by either route of administration. The incidence of resorptions among the litters of rats exposed to 80 ppm vinylidene chloride was significantly greater than among the controls; however, this increase was attributable to a single 80 ppm-exposed litter in which all of its 13 implan tations were resorbed. The incidence of resorptions was unaffected among the litters of rats exposed to 20 or 160 ppm vinylidene chloride by inhalation or among those re ceiving vinylidene chloride in the drinking water. Exposure to vinylidene chloride by either route of administration did not alter the fetal sex ratio or body weight. The mean fetal crown-rump length among the litters of rats receiving drinking water containing vinylidene chloride was significantly greater than that of the controls; this parameter was un affected among the litters of rats exposed to the compound by inhalation. The incidence of major malformations, considered individually or collectively, among the litters of rats given vinylidene chloride by inhalation or ingestion was not significantly SL 083294 -12- different from that of the concurrent control litters (Table 6). No external or soft-tissue alteration occurred signifi cantly more often among the litters of rats receiving vinylidene chloride than among the control litters. Several minor skeletal alterations occurred significantly more often among the litters of rats exposed to 80 or 160 ppm vinylidene chloride by inhalation (Table 6). At 160 ppm, these included delayed ossification of the interparietal bone of the skull, unfused parietal and interparietal bones of the skull, delayed ossification of centra of cervical vertebrae, and wavy ribs. At 80 ppm, statistically signi ficant increases were observed in the following skeletal alterations: delayed ossification of the parietal and interparietal bones of the skull, lumbar spurs (bilateral), and wavy ribs; in addition, the incidence of unfused parietal and interparietal bones of the skull was significantly decreased compared to the control value. Fetuses with wavy ribs were classified as either type I, those in which only a gentle curve in one or more ribs was observed, or type II, those in which one or more ribs had either a callous or a "U"-shaped bend (Figures 1-3). Among the litters of rats exposed to 160 ppm of vinylidene chloride, the incidence of .type II but not type I wavy ribs was significantly increased; in contrast, type I but not type II wavy ribs occurred significantly more often at 80 ppm. No skeletal alteration SL 083295 -13- occurred among the litters of rats inhaling 20 ppm vinylidene chloride or ingesting water containing 200 ppm vinylidene chloride at an incidence significantly different from that of their respective control groups. RABBITS Maternal Toxicity. Single maternal deaths were observed in each of the two control groups and in the 80 ppm-exposed group; two deaths were seen among the rabbits exposed to 160 ppm vinylidene chloride. All of these deaths were associated with pneumonia, enteritis, or perimetritis, and thus did not appear to be compound-related. One dam each from the 80 ppm-exposed group and its concurrent control group delivered a litter on day 28 of gestation, i.e., one day prior to the scheduled day of cesarean section. The body weight of the dams exposed to 160 ppm vinylidene chloride was not significantly different from that of the controls (Table 7); however, the body weight gain of these dams was significantly decreased on days 6-11 and 12-18 of gestation, (during the period of exposure) and significantly increased on days 19-28 (post-exposure). At the beginning of the experiment (day 6 of gestation), the 80 ppm-exposed dams weighed significantly less than the controls. The maternal weight gain of the 80 ppm-exposed dams was not significantly different from the control value during the SL 083296 -14- period of exposure, but a significant increase was observed during the post-exposure period. The liver weight of the dams exposed to 80 ppm was signifi cantly increased on both an absolute and relative basis. No effect on maternal liver weight was discerned among the dams exposed to 160 ppm. Embryo- and Fetotoxicity. Observations made at the time of cesarean section of rabbits exposed to vinylidene chloride by inhalation are presented in Table 8. The percentage of pregnancy was not significantly altered by exposure to either concentration of vinylidene chloride. No statisti cally significant effect on the number of corpora lutea, implantations, or live or dead fetuses was noted among the rabbits inhaling vinylidene chloride. The incidence of resorptions among the litters of rabbits exposed to 160 ppm vinylidene chloride was significantly increased compared to that of its control group. Among the dams exposed to 160 ppm vinylidene chloride, four exhibited litters in which all or most of the implantations were resorbed; each of these four dams also showed a greater weight loss than the other dams exposed to 160 ppm, losing an average of approximately 450 gm of body weight during days 6-17 of gestation. Among the litters of dams exposed to 80 ppm vinylidene chloride, SL 083297 -15- the incidence of resorptions was slightly lower than that of its control group. Exposure to either concentration of vinylidene chloride had no significant effect on fetal body weight, crown-rump length, or sex ratio. The incidence of major malformations was not significantly affected by exposure to vinylidene chloride (Table 9). No single external alteration occurred at an incidence signifi cantly different from the control incidence among the litters of rabbits inhaling vinylidene chloride. The soft-tissue examination revealed a statistically significant decrease in the occurrence of minor satellite vessels branching off the innominate artery among the litters of rabbits exposed to 160 ppm but not among those exposed to 80 ppm. No other soft-tissue alterations were observed at an incidence that differed significantly from the control incidence. The skeletal examination revealed a significant increase and decrease in the incidence of 13 pairs of ribs and of delayed ossification of the 5th sternebrae, respectively, among the litters of rabbits inhaling 160 ppm vinylidene chloride. At 80 ppm, no skeletal alteration was observed at an incidence significantly different from that of the controls. SL 083298 -16- DISCUSSION The results of these studies, summarized in Table 10, in dicate that vinylidene chloride was not teratogenic in rats or rabbits inhaling the compound for 7 hrs/day at concen trations sufficiently high to produce maternal toxicity. In rats, inhalation of 80 or 160 ppm vinylidene chloride pro duced significant maternal effects including decreased weight gain, decreased food consumption, increased water consumption, and increased liver weight (160 ppm only). At 20 ppm, the only effects noted among the dams were minor changes in body weight and in the amount of water consumed. In comparison, pregnant rabbits inhaling vinylidene chloride exhibited decreased maternal weight gain at 160 ppm but not at 80 ppm. A statistically significant increase in liver weight was noted among the rabbits exposed to 80 ppm vinyli dene chloride; because a similar effect was not discerned at 160 ppm, this increase was not interpreted to be related to vinylidene chloride exposure. No single major malformation occurred among the litters of vinylidene chloride-exposed rats or rabbits at an incidence significantly different from that seen in the control litters. In addition, the incidence of total major malformations, when considered collectively, was not significantly altered by exposure to vinylidene chloride in either species. The SL 083299 -17- few major malformations which were observed among the vinylidene chloride-exposed litters have been noted to occur among control litters in past studies. While a teratogenic effect was not observed, some evidence of embryotoxicity and fetotoxicity was noted in both rats and rabbits. In rats, exposure to inhaled vinylidene chloride resulted in a significantly increased incidence of several skeletal alterations at 80 and 160 ppm, which were maternally toxic concentrations. These alterations included wavy ribs and delayed ossification of various bones. In addition, a statistically significant increase in the inci dence of resorptions was observed among the rats exposed to 80 ppm vinylidene chloride; however, this increase was not considered to be compound-related because it was attributable to a single litter which was entirely resorbed and because a similar effect was not seen at 160 ppm. At 20 ppm, a con centration which caused little evidence of maternal toxicity, no adverse effect on embryonal or fetal development was discerned. Rabbits exposed to 160 ppm vinylidene chloride exhibited a marked increase in the incidence of resorptions. The oc currence of resorptions in each litter correlated well with the degree of toxicity observed in the individual dams. SL 083300 -18- Thus, it is not clear whether the increased incidence of resorptions was due directly to the compound or indirectly to maternal toxicity. Exposure of rabbits to 160 ppm vinylidene chloride also resulted in a significant change in the incidence of several minor fetal alterations, including an increased incidence of 13 pairs of ribs (the normal number is 12 or 13) and decreased incidence of minor satel lite vessels and of delayed ossification of the 5th sternebra. At 80 ppm, a level which was not maternally toxic to rabbits, no significant evidence of embryotoxicity or fetotoxicity was observed. Most of the fetal alterations which occurred at an increased incidence in either species were minor variants, commonly seen in teratologic studies when maternally toxic dose levels of test agents are employed. However, increased occurrences of wavy ribs, as seen among the litters of rats inhaling 80 or 160 ppm vinylidene chloride, are seen less frequently and appear to be more compound-specific. The induction of wavy ribs has been associated with several compounds including insulin (Lichtenstein et al., 1951), clomiphene and other basic phenolic ethers (Diener and Hsu, 1967) and hexachlorophene (Kimmel, 1972). In the present study, the incidence of wavy ribs among the fetuses and SL 083301 -19- litters of rats exposed to vinylidene chloride was 4 and 33%, respectively, at 80 ppm and 7 and 38%, respectively, at 160 ppm. The frequency of this alteration among the control groups from eight other recent studies involving 2,708 fetuses and 229 litters ranged from 0-2% among fetuses and 0-17% among litters. These figures are consistent with the range of control values in the present study, 0-2% among fetuses and 0-18% among litters. The severity of the wavy ribs appeared to increase in a dose-related manner. At 160 ppm most of the fetuses with wavy ribs exhibited a callous or even a "U"-shaped bend in the rib, whereas at 80 ppm a gentle wave in the rib was seen more often. The increased occurrence of wavy ribs in rats is regarded to be a manifestation of embryotoxicity but is not generally considered to constitute a teratogenic effect (personal communication, J. G. Wilson1). In fact, there is some evidence indicating that wavy ribs are a reversible phenomenon (Diener, 1973). To evaluate the embryotoxic potential of ingested vinylidene chloride, rats were given the compound in their drinking water at a concentration of 200 ppm, the solubility limit of the compound in water. No evidence of maternal toxicity or teratogenicity was discerned among the rats maintained on Childrens Hospital Research Foundation, Elland and Bethesda Avenues, Cincinnati, Ohio. SL 083302 -20- the vinylidene chloride containing water. The only statis tically significant response observed among the litters of rats given the compound in the drinking water was an in crease in the average fetal crown-rump length; although this response was statistically significant, it was not considered to be toxicologically significant. Maintenance of pregnant rats on drinking water containing 200 ppm of vinylidene chloride had less effect on the dam or her offspring than exposure to either 80 or 160 ppm of the test material by inhalation. The average daily dose of vinylidene chloride received by the rats given the compound in their drinking water was calculated to be approximately 40 mg/kg/day. Estimates of the total amount of vinylidene chloride metabolized in rats indicate that a single gavage administration of 40 mg/kg vinylidene chloride is approximately equivalent to a single 7-hr inhalation exposure to 120 ppm of the compound (personal communication, M. J. McKenna2). Thus, the sensitivity of the rat to vinylidene chloride was apparently greater when vinylidene chloride was inhaled than when it was ingested. Diurnal variation in the metabolism of vinylidene chloride may have been responsible for the apparent route-dependent 2Toxicology Research Laboratory, The Dow Chemical Company, Midland, Michigan. SL 083303 -21- difference in the sensitivity to vinylidene chloride. A major pathway for the detoxification of vinylidene chloride is conjugation of a reactive metabolite with glutathione (McKenna et al., 1977). Levels of glutathione are known to undergo diurnal variation (Jaeger et al., 1973; Watanabe et al., 1976), rising markedly during the late evening and early morning hours (the period during which the rat normally eats and drinks). Therefore, it is hypothesized that, in the drinking water study, the available levels of glutathione may have been sufficient to prevent toxicity; whereas in the inhalation study, toxicity may have resulted due to the lowered avail ability of glutathione during the exposure period (approximately 8:30 AM - 3:30 PM). In these studies, adult rats appeared to be slightly more sensitive than adult rabbits to the toxicity of inhaled vinylidene chloride. However, the susceptibility of the developing embryo to vinylidene chloride appeared to be comparable in the two species since embryotoxicity was observed only at levels of exposure which caused maternal toxicity. In conclusion, vinylidene chloride was not teratogenic in rats or rabbits inhaling up to 160 ppm of the compound for 7 hrs/day or in rats given water containing 200 ppm of the compound. Some evidence of embryotoxicity or fetotoxicity SL 083304 -22- was observed in both rats and rabbits exposed to vinylidene chloride by inhalation; these effects were associated with maternally toxic levels of exposure. At concentrations of vinylidene chloride which had little or no effect on the dam, no adverse effect on embryonal or fetal development was discerned in either species. SL 083305 REFERENCES ACGIH (American Conference of Governmental Industrial Hygienists) Threshold Limit Values for Chemical Sub stances and Physical Agents in the Workroom Environ ment with Intended Changes for 1977. Balmer, M. F., Rampy, L. W., and Quast, J. F. (1976). 90-Day repeated inhalation toxicity study of vinylidene chloride in rats. Report to the Manufacturing Chemists Association. Bartsch, H., Malaveille, C., Montesano, R., and Tomatis, L. (1975) . Tissue-mediated mutagenicity of vinylidene chloride and 2-chlorobutadiene in Salmonella typhimurium. Nature 255, 641-643. Carpenter, C. P., Smyth, H. F., Jr., and Pozzani, U. C. (1949). The assay of acute vapor toxicity, and the grading and interpretation of results on ninety-six chemical compounds. J. Ind. Hyg. Toxicol. 31, 343-346. Dawson, A. B. (1926). A note on the staining of the skeleton of cleared specimens with alizarin red-S. Stain Tech. 1, 123-124. Diener, R. M. (1973) . The wavy rib syndrome. The Bulletin of the Society of Pharmacological and Environ mental Pathologists. No. 3, 8-9. Diener, R. M. and Hsu, B.Y.D. (1967) . Effects of certain basic phenolic ethers on the rat fetus. Toxicol. Appl. Pharmacol. 10^, 565-576. Gage, J. C. (1970). Subacute inhalation toxicity of 109 industrial chemicals. Brit. J. Ind. Med. 27 (1), 1-18. Greim, H., Bonse, G., Radwan, Z., Reichert, D., and Henschler, D. (1975). Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmacol. 24, 2013-2017. SL 083306 -24- 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. Humiston, C. G., McCollister, S. B., and Quast, J. F. (1975) . Results of a 90-day study incorporating vinylidene chloride in the drinking water of rats. Report to the Manufacturing Chemists Association. 12. Jaeger. R. J., Conolly, R. B., and Murphy, S. D. (1973). Diurnal variation of hepatic glutathione concentration and its correlation with 1,1-dichloroethylene inhalation toxicity in rats. Res. Commun. Chem. Pathol. Pharmarcol. 6, 465-471. 13. Jenkins, L. J., Jr., Trabulus, M. J., and Murphy, S. D. (1972). Biochemical effects of 1,1-dichloroethylene in rats. Comparison with carbon tetrachloride and 1,2-dichloroethylene. Toxicol. Appl. Pharmacol. 23, 501-510. 14. Kimmel, C. A., Moore, W., and Stara, J. F. (1972). Hexachlorophene teratogenicity in rats. Lancet 2_, 765. 15. 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. 16. Lichtenstein, H., Guest, G. M., and Warkany, J. (1951). Abnormalities in offspring of white rats given protamin zinc insulin during pregnancy. Proc. Soc. Exp. Biol. Med. 78, 398-402. 17. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. (1975) . Detection of carcinogens in the salmonella microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A. 72_, 5135-5139. 18. McKenna, M. J., Watanabe, P. G., and Gehring, P. J. pharmacokinetics of vinylidene chloride in the rat. Env. Health Perspectives, in press. The SL 083307 -25- 19. Prendergast, J. A., Jones, R. A., Jenkins, L. J., Jr., and Siegel, J. (1967) . Effects on experimental animals of long-term inhalation of trichloroethylene, carbon tetrachloride, 1,1,1-trichloroethane, dichlorodifluromethane, and 1,1-dichloroethylene. Toxicol. Appl. Pharmacol. 10, 270-289. 20. Rylova, M. L. (1953). Toxicity of 1,1-ethylene dichloride. Farmakol. i. Toksikol. 16, 47-50. 21. Siegel, J., Jones, R. A., Coon, R. A., and Lyon, J. P. (1971) . Effects on experimental animals of acute, repeated, and continuous inhalation exposures to dichloroacetylene mixtures. Toxicol. Appl. Pharmacol. 18^, 168-174 . 22. Siegel, S. (1956). Non-Parametric Statistics for the Behavioral Sciences. McGraw-Hill Book Company, Inc New York, New York. 23. Staples, R. E. (1974). Detection of visceral alterations in mammalian fetuses. Teratology 9, A-37. 24. steel, R.G.D. and Torrie, H. H. (1960). Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York, New York. 25. Watanabe, P. G. Hefner, R. E. Jr., and Gehring, P. J. (1976). Vinyl chloride-induced depression of hepatic non-protein sulfhydryl content and effects on bromosulphalein (BSP) clearance in rats. Toxicology 6., 1-8. 26. Wilson, J. G. (1965). Teratology: Principles and Techniques. The University of Chicago Press, Chicago, Illinois,-262-277. SL 083308 TABLE 1 CONCENTRATION OF VARIOUS IMPURITIES IN THE SAMPLES OF VINYLIDENE CHLORIDE USED FOR THE TERATOLOGY STUDIES Concentration in ppm (wt/wt)^ Vinyl chloride Trans 1,2-dichloroethylene Cis 1,2-dichloroethylene 1,1,1-trichloroethane 1,1,2-trichloroethane Monomethylether of hydroquinone (MEHQ) Sample Ia Sample II Sample III Sample IV 43 109 32 2 <0.2 209 66 340 47 8 <0.2 400 N.D.C N.D. 210 20 100 220 25 9 0.6 N.D. N.D. N.D. Sample I was used for the 160 ppm inhalation exposure; Sample II, the 80 ppm inhalation exposure Sample III, the 20 ppm inhalation exposure; Sample IV, the drinking water study. ^Determined by gas-liquid chromotography. Q N.D, = not detected. SL 083309 TABLE 2 BODY AND LIVER WEIGHTS OF PREGNANT RATS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION OR BY INGESTION Route of Exposure ppm Vinylidene Chloride Number of dams Maternal body weight (g) on gestation day 6 10 16 21 Maternal weight gain (g) on gestation days 6-9 10-15 16-20 Liver weight on day 21 of gestation c absolute relative** 0 20 Inhalation Drinking Water 0 80 0 160 0 200 40 40 20 28 17 26 24 26 255+26 27228 310+29 390+39 24627 258123* 301124 382+30 244+15 264115 307+19 382+24 242+18 253123 293124* 366+34 278+21 292124 329118 400148 268+21 269120* 314+24 394129 290113 309116 348120 421128 284+30 301+22 342126 414135 1512b 3717 8018 12112 44+14 80+12 2018 43112 75+15 11114* 41118 72116 14+13 3617 64110 2110* 44114 80111* 1917 3916 72116 16118 40+14 72+20 15.21.9 15.1+1.6 39.03.8 39.5+2.9 15.911.0 15.711.7 41.7+2.5 43.113.7 15.9+1.4 39.812.2 16.711.9 42.513.9* 15.911.7 37.913.9 15.2H.4 37.013.4 SL 083310 aRats were exposed to 20, 80, or 160 ppm vinylidene chloride for 7 hrs/day or were given drinking water containing 200 ppm of the compound on days 6-15 of gestation. bMean S.D. c g, mean S.D. **g liver/kg body weight, mean S.D. *Significantly different from the concurrent control value by a one-way analysis of variance, p<0.05. Individual animal data are presented in Table A-l. TABLE 3 AMOUNT OF FOOD AND WATER CONSUMED BY PREGNANT RATS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION ppm Vinylidene Chloride3 0 20 0 80 0 160 Food consumption (g) on gestation days 6-8 9-11 12-14 15-17 18-20 204b 224 244 274 293 203 224 25+4 273 294 223 265 25+2 29+4 28+2 184* 20+3* 22+3* 273* 27+4 223 22+4 236 27 5 26+4 186* 21+6 26+3* 294 36+8* Water consumption (g) on gestation days 6-8 9-11 12-14 15-17 18-20 42+10b 48+8 5210 64+11 62+9 42+7 519 589* 69+10* 64+10 34+12 50+10 536 5910 586 34+10 4512 49+13 667* 60+8 4011 38+16 4014 56+13 55+13 53+13* 6013* 65+10* 7616* 7412* Rats were exposed to vinylidene chloride for 7 hrs/day on days 6-15 of gestation. ^Expressed as grams/rat/day, mean S.D. *Significantly different from the concurrent control value by an analysis of variance, p<0.05. SL 083311 TABLE 4 LIQUID AND FOOD CONSUMPTION BY PREGNANT RATS MAINTAINED ON DRINKING WATER CONTAINING VINYLIDENE CHLORIDE Days of Gestation 6 7 8 9 10 11 12 13 14 15 16 1 J17 18 ^ } J19 20 LIQUID__________ Control Vinylideng Chloride11 4 7 + 8b 49 + 8 50 + 9 49 + 6 ^l J1 508 54 + 12 51+8 51 + 9 ^\ J1 ____________ FOOD Vinylidene Control Chloride 24 + 2b 23 + 3 24 + 3 23 + 3 53+13 54 + 14 54 + 12 59 + 11 l / 24 + 2 24 + 3 56 + 14 58 + 10 59 + 9 1 25 + 2 59 + 7 J 25 + 2 59 + 9 67 + 9 64 + 10 5912 66 + 12 62 + 19 l / * } *J 27+2 31 + 8 26+4 31 + 9 6216 6410 \ > *) 25 + 7 24 8 54 + 11 54 + 9 24 + 3 24 4 a Vinylidene chloride was given in the drinking water at a concentration of 200 ppm on days 6--15 of gestation. g/rat/day, mean + S.D. No values differed significantly from control by an analysi of variance , p<0.05. SL 083312 TABLE 5 OBSERVATIONS MADE AT THE TIME OF CESAREAN SECTION OF RATS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION OR BY INGESTION Route of Exposure ppm Vinylidene Chloride Ho. of bred females Ho. deaths Percent Pregnant^ Pregnancies detected by stain** 0 47 0 85(40/47) 0 20 44 0 91(40/44) 1 Inhalation3 0 80 21 30 00 95(20/21) 1 97 (29/30)c 1 0 18 0 94(17/18) 0 160 30 0 90(27/30)C 0 Percent pregnant, totale Humber of litters Corpora lutea/dam^ Implantation sites/dam^ Pregnancy wastage^ Live fetuses/litter^ Resorptions/litter^ X Implantations resorbed^ X Litters with resorptions^ X Litters totally resorbed^ Resorptions/litters with resorptions^ Z Dead fetuses Fetal sex ratio, M:F Fetal body weight, g*1 Fetal crown-rump length, mm** 85(40/47) 40 132 12+3 1+2 12+3 0.2+0. 5 2(9/479) 18(7/40) 0(0/40) 1.3(9/7) (0/479) 50:50 5.60+0.40 43.9+1.1 93(41/44) 40 14+3 12+2 2+2 12+2 0.4+0.7 3(16/478) 28(11/40) 0(0/40) 1.5(16/11) (0/478) 53:47 5.61+0.28 44.0+0.9 100(21/21) 20 12+2 , 12+2 1+2 12 + 2 0.40.9 3(8/238) 25(5/20) 0(0/20) 1.6(8/5) (0/230) 43:57 5.540.26 44.4+1.0 100(30/30) 94(17/18) 27 17 12+2 142 11+3 13 + 2 1+2 1+1 10+4 12 + 2 0.8 + 2.4 0.40.7 8(24/316)* 3(7/216) 39(11/28) 29(5/17) 4(1/28) 0(0/17) 2.2(24/11) 1.4(7/5) (0/292) (0/209) 50:50 48:52 5.720.45 5.52+0.26 45.1+1.3 43.8+0.8 90(27/30) 26 13+2 12+2 12 11+2 0.50 .7 4(14/305) 42(11/26) 0(0/26) 1.3(14/11) (0/291) 51:49 5.47+0.82 43.6+2.6 Drinking Water 0 200 27 26 00 89(24/27) 0 100(26/26) 0 89(24/27) 24 12 + 2 11 + 3 22 10t3 0.310.5 3(8/258) 33(8/24) 0(0/24) 1.0(8/8) (0/250) 49:51 5.79+0.44 46.6+2.4 100(26/26) 26 14 + 3 12+4 3+3 llc4 0.4iO.8 4(11/301) 27(7/26) 4(1/26) 1.6(11/7) (0/290) 50: 50. 5.70+0.30 47.8 tl.3** aBred rats were exposed to 20, 80, or 160 ppm vinylidene chloride for 7 hrs/day or were given drinking water containing 200 ppm of the compound on days 6-15 of gestation. DNo. of females with visible implantation sites at the time of cesarean section/total no. of bred females. C0ne dam at the intermediate and high dose levels delivered a litter on days 18 and 21 of gestation, respectively; these dams jwere included in the calculation of the pregnancy rates. Their litters were not examined for fetal alterations. CO No. of females with implantation sites detected only after staining the uterus with sodium sulfide stain. F O CNo. of females with implantation sites as observed either visually at the time of cesarean section or necropsy or co after staining the uterus with sodium sulfide stain/total no. of bred females. LJ Mean S.D. OJ ^Resorptions detected only by sodium sulfide staining were not Included in these calculations. I-1 LJ Mean of the litter means + S.D. *Significantly different from the concurrent control value by Fishers exact probability test, p<0.05. **Significantly different from the concurrent control value by a one-way analysis of variance, p<0.05. Individual litter data are presented in Table A-2. TABLE 6 INCIDENCE OF FETAL ALTERATIONS AMONG LITTERS OF RATS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION OR BY INGESTION Route of Exposure4 ppm Vinylidene Chloride EXTERNAL AND SKELETAL EXAMINATION SOFT TISSUE EXAMINATION SKULL BONE EXAMINATION 0 470/40 162/40 300/39 Inhalation 20 0 80 0 160 No. Fetuses/No. Littersi Examined 462/40 158/40 304/39 230/20 77/20 153/20 292/27 103/27 189/26 209/17 70/17 139/17 291/26 100/26 191/26 Drinking Water 0 200 250/24 86/24 164/23 290/25 100/25 190/24 Total Malor Malformations^ FC L External Examination Short trunk and multiple defects* Subcutaneous edema Omphalocele* Narrow head, pointed snout* Subcutaneous hemorrhage head, neck, or back F L F L F L F L F L Soft Tissue Examination Diaphragmatic hernia* Yellow-colored focus on liver Hemorrhage, liver Hydroureters Dilated renal pelvis Ectopic ovaries* Head - subcutaneous hemorrhage^ Head - thickened dermal and subcutaneous tissue In ocular region F L F L F L F L F L F L F L F L % Affected (No. Affected) 0.4(2) 0 0.4(1) 0.7(2) 2(4) 5(14) 5(2) 0 5(1) 7(2) 12(2) 15(4) 0.2(l)d 2(1) 0 0 0 0 0 0 0.6(3) 8(3) 0 0 0.2(1) 2(1) 0 0 0 0 0.4(2) 2(1) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.3(1) 4(1) 0.3(1) 4(1) 0.3(1) 4(1) 0.4(l)e 6(1) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.2(1) 5(2) 0.6(1) 2(1) 0 0 0 0 0.6(1) 2(1) 0 0 0 0 O 0 0 0 1(1) 5(1) 0 0 0 0 0 0 0 0 0 0 2(2) 10(2) 0 2(3) 0 0 8(3) 0 0 0 0 0 1(1) 4(1) 0 0 0 0 0 0 2(2) 4(1) 0 0 0 0 1(1) 6(1) 0 0 0 0 1(1) 6(1) 1(1) 6(1) 1(1) 6(1) 0 0 0 0 0 0 0 0 4(4) 11(3) 0 0 0 0 0 _0 0 0 00 00 0 0 0 0 0 0 0 0 0.4(1) 4(1) 0 0 0 0 0 0 0 0 1(4) 8(2) 0 0 0 0 1(1) 4(1) 1(1) 4(1) 0 0 0 0 3(3) 12(3) 0 0 0 0 0 0 0 0 3(3) 12(3) 0 0 0 0 3(3) 12(3) 0 0 SL 083314 TABLE 6 (Continued) Route of Exposure1 ppm Vinylidene Chloride Skeletal Examination Skull - delayed ossification of parietal bone -delayed ossification of interparietal bone -delayed ossification of any skull bone -unfused parietal and interparietal -bone island, interparietal -misshapen parietal and interparietal -extra site of ossification between skull bones -delayed ossification of zygomatic arch Vertebrae - delayed ossification of atlas -delayed ossification of cervical centra -delayed ossification of thoracic centrum -unfused cervical centrum -unfused thoracic centrum -missing some thoracic, all lumbar and all sacral vertebrae*' -missing one thoracic vertebra and one lumbar vertebra* -any missing vertebrae -fused vertebrae, thoracic region* -extra thoracic vertebra and pair of ribs* F L F L F L F L F L F L F L F L F L F L F L F L F L F L F L F L F L F L __________________________Inhalation 0 20 0 80 0 160 Drinking Water 0 200 3(8) 13(5) 14(42) 44(17) 14(42) 44(17) 0.3(1) 3(1) 0 0 0 0 0 0 0 0 0 0 7(34) 42(17) 0 0 0 0 0.2(1) 2(1) 1(3) 8(3) 1(2) 10(2) 6(11)** 4(6) 27(7) 12(2) 12(36) 8(13) 20(38)** 17(24) 46(18) 35(7) 50(13) 47(8) 12(36) 8(13) 20(38)** 17(24) 46(18) 35(7) 50(13) 47(8) 0.3(1) 3(1) 11(17) 4(8)** 45(9) 19(5) 4(6) 24(4) 0.3(1) 2(1) 1(1) 5(1) 0 0 0 0 0 Kl) 0 0 5(1) 0 0 0 0 0 0.5(1) 0 ,0 0 4(1) 0 0 0 1(2) 0 0 0 8(2) 0 000 000 0 0 13(58) 10(24) 16(47) 48(19) 70(14) 63(17) 14(30) 77(13) 0 0 0.3(1) 0 0 0 4(1) 0 0 0 0.3(1) 0 0 0 4(1) 0 0.2(1) 0.4(1) 2(1) 5(1) 1(2) 7(2) 0 0 7(13) 27(7) 30(57)** 77(20) 31(59)** 77(20) 17(33)** 54(14) 2(3) 8(3) 0 0 0 0 0 0 0.3(1) 4(1) 37(107)** 77(20) 0 0 0.3(1) 4(1) 0.3(1) 4(1) 0.2(1) 0 0 0 0.5(1) 0 2(1) 0 0 0 6(1) 0 0 000 0 000 1(2) 6(1) 5(14) 15(4) 0.2(1) 0 0 0 2(1) 0 0 0 1(3) 6(1) 5(14) 15(4) 0 0 0 0 0.5(1) 0 0 00 0 6(1) 0 0 0 0 0 0.5(1) 0 0 000 6(1) 0 2(3) 9(2) 3(5) 12(3) 12(19) 43(10) 17(32) 46(11) 12(19) 43(10) 17(32) 46(11) 57(93) 87(20) 55(104) 83(20) 0(0) 0(0) 1(2) 8(2) 00 00 1(1) 4(1) 0 0 00 00 0 0 18(46) 67(16) 0 0 14(42) 72(18) 00 00 00 00 1(2) 8(2) 0.3(1) 4(1) 00 00 00 00 00 00 00 00 00 00 SL 083315 TABLE 6 (Continued) Route of Exposure*InhalationDrinking Water ppm Vinylidene Chloride 0 20 0 80 0 160 0 200 Ribs - unilateral spur on 1st lumbar F L -bilateral spurs on 1st lumbar F L -any spurs on 1st lumbar F L -unilateral wavy ribs F L -bilateral wavy ribs F L -any wavy ribs -Class I wavy ribs*1 -Class II wavy ribs1* -any shortened 12th rib1 -missing 12th rib - unilateral1 -missing 13th pair of ribs only*'^ -missing ribs - multiple ribs*' F L F L F L F L F L F L F L -any missing ribs F L -extra ribs F L Sternebrae - delayed ossification of sternebrae F L -unossified sternebrae F L -unfused sternebrae F L -fused sternebrae F L Limbs and Digits - delayed ossification of metacarpels or metatarsels F L 13(59) 12(56) 65(26) 65(26) 8(40) 5(25) 50(20) 35(14) 21(99) 18(81) 78(31) 75(30) 1(5) 12(5) 1(4) 10(4) 1(4) 10(4) 2(9) 18(7) 2(9) 12(5) 3(13) 18(7) 1(5) 12(5) 2(10) 18(7) 1(4) 10(4) 1(3) 5(2) 00 00 o 0 00 00 00 0.2(1) 2(1) 0 0 0.2(1) 2(1) 0 0 1(6) 0.2(1) 8(3) 2(1) 4(21) 4(20) 32(13) 30(12) 00 00 1(5) 12(5) 0.2(1) 2(1) 1(3) 8(3) 0 0 12(28) 13(38) 65(13) 67(18) 13(28) 71(12) 5(11) 10(30)** 8(16) 35(7) 63(17) 59(10) 17(39) ' 23(68) 75(15) 85(23) 21(44) 82(14) 0 1(4) 1(2) 0 15(4) 6(1) 0 2(7)** 0.5(1) 0 26(7) 6(1) 0 4(11)** 1(3) 0 33(9) 6(1) 0 3(9)** 1(2) 0 30(8) 6(1) 0 1(2) 0.5(1) 0 7(2) 6(1) 00 00 1(2) 6(1) 0 0 0.5(1) 00 6(1) 00 00 1(2) 6(1) 0 0 0.5(1) 00 6(1) 00 00 1(3) 6(1) 0.4(1) 5(1) 1(2) 7(2) 2(4) 12(2) 5(12) 3(10) 30(6) 30(8) 4(9) 41(7) 00 00 0 0 0 1(2) 0.5(1) 0 4(1) 6(1) 00 00 0 0 16(45) 73(19) 10(30) 62(16) 26(75) 85(22) 1(3) 8(2) 7(19)** 35(9) 8(22)** 38(10) 3(9) 19(5) 4(13)** 35(9) 9(11) 15(4) 1(9) 8(2) 5(14) 15(4) 0 0 5(15) 15(4) 2(6) 19(5) 5(15) 31(8) l(4)c 4(1) 0.3(1) 4(1) 0 0 00 00 00 00 0 4(13) 0 12(3) 13(32) 16(46) 79(19) 84(21) 6(14) 7(19) 33(8) 40(10) 18(46) 22(65) 88(21) 84(21) 0.4(1) 0.3(1) 4(1) 4(1) 0 1(2) 0 4(1) 0.4(1) 4(1) 1(3) 8(2) 0 1(3) 0 8(2) 0.4(1) 4(1) 0 0 00 00 00 00 00 00 00 00 00 00 4(1) 4(1) 0 0 5(13) 3(9) 42(10) 24(6) 0.4(1) 4(1) 0 0 1(2) 8(2) 0 0 00 00 00 00 aPregnant rats were exposed to 20, 80 . r 160 ppm vinylide ne chloride for 7 hrs/day or were given drinking water ^containing 200 ppm of the compound on days 6-15 of gestation. ^Total major malformations detected by external, soft tissue, or skeletal examination. jF = fetuses; L = litters. In addition to short trunk, this fetus exhibited hypoplastic tail, inward rotation of the hindlimbs, ectopic ovaries, missing 4-13th thoracic vertebrae, missing all lumbar and all sacral vertebrae, gand missing 4-13th pairs of ribs. This fetus demonstrated short trunk, acaudia, anteriorally displaced ovaries and uterine horns, missing f3-13th thoracic vertebrae, missing all lumbar and sacral vertebrae, and missing 2-13th ribs. This alteration was observed during the examination of the heads fixed in Bouin's solution, ^This alteration was observed only among fetuses exhibiting a short trunk in the external examination, I Type I gentle wave; Type II - callous or "U"-shaped bend in rib. This alteration occurred only in fetuses missing one thoracic and one lumbar vertebrae. *This alteration was considered to be a major malformation. **Significantly different from control value by a modified Wilcoxon test, p<0.05. Individual litter data are presented in Table A-3. SL 083316 TABLE 7 BODY AND LIVER WEIGHTS OF PREGNANT RABBITS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION a ppm Vinylidene Chloride 80 160" Number of dams 14 18 13 15 Maternal body weight on gestation day 6 4.33+0.32b 4.04+0.34* 12 4.28+0.34 4.02+0.30* 19 4.3410.33 4.00+0.44* 29 4.42+0.29 4.2210.35 4.0310.27 4.01+0.27 4.12+0.29 4.1910.32 4.07+0.38 3.90+0.37 3.86+0.40 4.0710.40 Maternal weight gain (kg) on gestation days 6-11 0.05+0.06 -0.03+0.11 12-18 0.06+0.07 -0.02 + 0.22 19-28 0.0710.10 0.23+0.15* -0.0210.09 -0.17+0.14* 0.12+0.08 -0.04+0.14* 0.07+0.22 0.21+0.13* Liver weight on day 29 of gestation absolute0 re.la.t.ive d 99 + 14 22.312.9 107+12* 25.3+3.0* 104+16 24.8+4.0 109+19 26.513.6 a Pregnant rabbits were exposed to 80 or 160 ppm vinylidene chloride for 7 hrs/day on days 6-18 of gestation. bMean 1 S.D. c g, mean 1 S.D. dg liver/kg body weight, mean 1 S.D. *Significantly different from the concurrent control value by a one-way analysis of variance, p<0.05. Individual animal data are presented in Table A-4. SL 083317 TABLE 8 OBSERVATIONS MADE AT THE TIME OF CESAREAN SECTION OF RABBITS INHALING VINYLIDENE CHLORIDE No. of bred females No. deaths Percent pregnant'1 Pregnancies detected by stain Percent pregnant, total1 0 16 1 100(16/16)c ppm Vinylidene Chloride 80 0 22 16 1 'l 91(20/22)c 88(14/16) 0 100(16/16) 1 96(21/22) 0 88(14/16) 160 18 2 94 (17/18)^ 0 94(17/18) Number of litters 14 Corpora lutea/dams 9+2 Implantation sites/dam a Pregnancy wastage 9+2 0.1+0.4 Live fetuses/litter ah Resorptions/litter* 8i2 0.8+1.2 Z Implantations resorbed 9(12/128) Z Litters with resorptions11 43(6/14) Z Litters totally resorbed (0/14) Resorptlons/litters with resorptions 1.5(9/6) Z Dead fetuses (0/116) Fetal sex ratio, M:F Fetal body weight, g* Fetal crown-rump length, ram* 52:48 36.8*3.7 94.6*2.8 18 9+2 9*1 0.30.8 9+1 0.7+0.9 7(13/168) 50(9/18) (0/18) 0.8(7/9) (0/155) 56:44 36.9+5.0 93.44.1 13 9*2 9*3 1.0*1.8 8+3 0.3*0.6 3(4/115) 23(3/13) (0/13) 15 8*3 9*2 0.6+0.8 6*4 2.7*3.9* 31(41/132)** 53(8/15) 20(3/15) 1.3(4/3) (0/111). 52:48 37.4*7.6 93.4+6.2 5.1(41/8) 1(1/91) 53:47 36.0+4.2 92.9*4.7 *Bred rabbits were exposed to 80 or 160 ppm vinylidene chloride for 7 hrs/day on days 6-18 of gestation. ^No. of females with visible implantation sites at the time of cesarean section or necropsy/total no. of bred females. g One BO ppm-exposed dam and one dam from its control group which both delivered litters on day 28 of gestation were included In the calculation of the pregnancy rates. Their litters were not examined for fetal alterations. ^The single non-pregnant rabbit exhibited a purulent inflammation of the ovaries. *No. of females with implantation sites detected onlv after staining the uterus with sodium sulfide stain, f No. of females with implantation sites as observed either visually at the time of cesarean section or necropsy or after staining the uterus with sodium sulfide stain/total no. of bred females. Mean S.D. ^Resorptions detected only by sodium sulfide staining of the uterus were not included in these calculations. *Mean of litter means S.D. Significantly different from the control mean by a one-way analysis of variance, p<0.05. **SignifIcantly different from the control value by Fishers exact probability test, p<0.05. Individual litter data are presented in Table A-5. SL 083318 TABLE 9 INCIDENCE OF FETAL ALTERATIONS AMONG LITTERS OF RABBITS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION ppm Vinylldene Chloride* _0__________80 0________ 160 No, Fetuses/No. Litters Examined EXTERNAL AND SKELETAL EXAMINATION 116/14 SOFT TISSUE EXAMINATION 44/14 155/18 56/18 111/13 42/13 91/12 37/12 b Total Major Malformations X Affected (No. Affected) c F 0 L0 2(3) 17(3) 2(2) 15(2) 2(2) 16(2) External Examination Edema - neck and abdominal region F0 L0 0 0 0 1(1) 0 8(1) Soft Tissue Examination Dilated cerebral ventricles* F L Heart - thinning of right ventricular wall* F L Satellite vessel off aorta F L Satellite vessels off innominate artery F L Fused left carotid and innominate arteries F L Kidney - cortical Indentation, F unilateral L 0 0 0 0 0 0 9(4) 21(3) 71(31) 93(13) 0 0 2(1) 5(1) 0 0 0 0 20(11) 44(8) 55(31) 72(13) 2(1) 5(1) 0 0 0 0 10(4) 15(2) 31(13) 62(8) 60(25) 77(10) 0 0 0 0 3(1) 8(1) 0 0 8(3)** 17(2) 78(29) 100(12) 0 0 Skeletal Examination Skull - delayed ossification F L -frontal ridge* F L -foramen, frontal bone F L -foramen, parietal bone F L -extra suture linos, parietal bone F L -bone island, parietal bone F L -extra site of ossification between occipital condyles F and atlas L Ribs - 13th rib - bilateral F L -13th rib - unilateral F L -lumbar spurs F L -shortened first thoracic F L -11 ribs on left and 12 ribs on right* F L 0 0 0 0 0 0 0 0 0 0 0 0 0 0 48(56) 93(13) 16(19) 71(10) 7(8) 29(4) 1(1) 7(1) 0 0 0 0 1(1) 5(1) 0 0 1(2) 11(2) 1(1) 5(1) 1(1) 5(1) 0 0 45(70) 89(16) 8(12) 56(10) 2(3) 17(3) 0 0 0 0 2(2) 8(1) 0 0 2(2) 8(1) 2(2) 15(2) 0 0 0 0 2(2) 17(2) 0 0 0 0 0 0 1(1) 8(1) 1(1) 8(1) 0 0 31(34) 69(9) 10(11) 54(7) 6(7) 31(4) 0 0 Id) 8(1) 1(1) 8(1) 50(45)** 100(12) 11(10) 58(7) 3(3) 25(3) 0 0 0 0 SL 083: TABLE 9 (Continued) ppm Vinylidene Chloride________ 0 80 0_________ 160 Vertebrae - extra cervical vertebrae* F L -extra and misshapen lumbar vertebrae* F L -hemivertebrae in thoracic region fused or forked rib & misshapen centra* F L Sternebrae - delayed ossifica tion of 2nd sternebra F L -delayed ossification of 5th sternebra F L -delayed ossification of 6th sternebra F L -unfused F L -fused F L -extra site of ossification between 5th and 6th sternebrae F L Limbs and Digits - delayed ossification of phalanges in first digit, bilateral F L 0 0 0 0 0 0 3(3) 21(3) 41(48) 86(12) 33(38) 79(11) 1(1) 7(1) 1(1) 7(1) 0 0 1(1) 7(1) 1(1) 5(1) 0 0 1(1) 5(1) 1(1) 5(1) 27(42) 67(12) 20(31) 67(12) 0 0 2(3) 11(2) 0 0 0 0 0 0 1(1) 8(1) 0 0 0 0 0 0 0 0 40(44) 85(11) 21(23) 69(9) 0 0 0 0 1(1) 8(1) 2(2) 17(2) 21(19)** 58(7) 23(21) 58(7) 1(1) 8(1) 2(2) 17(2) 0 1(1) 0 8(1) 00 00 Rabbits were exposed to 80 or 160 ppm vinylidene chloride for 7 hrs/day on days 6-18 of gestation. bTotal major malformations detected by external, soft tissue, or skeletal examination. CF = fetuses; L = litters. *This alteration was considered to be a major malformation, **Significantly different from the concurrent control value by a modified Wilcoxon test, p<0.05. Individual litter data are presented in Table A-6. SL 083320 TABLE 10 SUMMARY - VINYLIDENE CHLORIDE TERATOLOGIC STUDIES ppm Vinylidene Chloride Maternal Toxicity Rats Inhalation ~T5 ISTT Drinking Water ---------Z?R5---------- Rabbits Inhalation "SO ----------HTTT demeanor Body weight gain Food consumption Water consumption Liver weight - absolute relative no -- inc no dec dec inc -- ----- no dec dec inc -- inc no -- -- -- no -- ND ND inc inc no dec ND ND -- Embryo- and Fetotoxicity Resorptions Fetal body weight Fetal crown-rump length External alterations Soft tissue alterations Skeletal alterations Major malformations -- inc -- -- -- -- ---- ---- -- inc ---- -- -- -- -- -- inc -- --- inc -- -- -- -- inc ---- --" -- dec -- yes "* a(--) = not significantly different from the concurrent control value; dec = decreased; inc = increased; ND = no data. ^Decreased incidence of minor satellite vessels off innominate artery. CDecreased incidence of delayed ossification of the 5 th sternebra incidence of 13 pairs of ribs. and increased SL 083321 TABLE A-l INDIVIDUAL BODY AND LIVER WEIGHTS OF PREGNANT RATS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION OR INGESTION Route; Inhalation - Exposure Level: 0 ppm (Concurrent Controls for 20 ppm) Animal Number Maternal Body Weight (g) Maternal Body Weight Cain fii Da v (s) of Gestation 6 10 16 21 6-9 10-15 16-20 44-7746 7747 7748 7749 7751 7752 7753 7754 7755 . 7756 7758 7759 7760 7761 7763B 7764B 7765B 7766B 7767B 7768B 7769B 7770B 7779 7780 7781 7782 7783 7784 7795 7796 7797 7798 7799 7800 7801 7802 7810 7813 7815 7816 225 242 226 229 254 267 244 236 240 287 227 217 280 284 252 ND 224 270 255 212 ND 227 275 252 297 277 267 314 291 231 280 207 270 271 274 227 276 271 252 253 249 262 243 242 270 284 265 242 252 296 223 217 268 279 279 311 248 285 281 233 295 257 295 277 307 310 285 325 294* 321 261 284 295 242 306 266* 314 283 270 244 273 300 289 292 317 294 309 302 288 345 232 273 273 330 315 351 305 315 320 277 331 266 325 316 309 358 321 334 340 307 328 247 318 342 310 308 365 343 312 308 328 372 377 367 390 367 382 360 351 440 309 332 364 393 410 435 385 385 405 351 443 355 406 400 435 451 426 380 380 394 421 286 415 431 399 384 465 4 34 390 387 24 20 17 13 16 17 21 6 12 9 -4 0 -12 -5 27 ND 24 15 26 21 ND 30 20 25 10 33 18 11 3* 90 -19* 77 25 -29 32 39 38 12 18 -9 24 38 46 50 47 10 44 60 36 49 9 56 5 51 36 40 57 30 39 44 36 9 30 39 2 48 36 9 46 -14* 67 -37* 23 100* 4 42* 51 60 42 64 55 72 B8 75 73 73 73 58 63 95 77 59 91 63 95 84 80 70 85 74 112 89 81 84 126 - 93 105 46 40 87 93 39 97 89 89 76 100 91 78 79 Maternal Liver Weight (Day 21) Absolute Relative (g) (g/kg Body Weight) 13.61 13.17 15.47 13.86 14.88 14.75 16.18 14.41 16.03 17.61 11.80 11.67 15.60 13.76 15,69 19.67 17.23 16.43 17.00 14.48 19.28 14.77 14.22 13.67 13.89 18.30 16.45 16.77 16.25 15.22 15.88 12.46 16.01 15.46 13.52 13.27 16,80 14.37 13.81 12.80 41.49 35.40 41.03 37.76 38.15 40.19 42.36 40.02 45.67 40.02 38.19 35.15 42.86 35.01 38.27 45.22 44.75 42.68 41.96 41.25 43.52 41.60 35.02 34.18 31.93 40.58 38.62 44.13 42.76 38.63 37.72 43.57 38.58 35.87 33.88 34.56 36.13 33.11 35.41 33.07 MEAN+SD 255126 272128 310129 390139 15+12 37H7 80+18 15.16*1.86 39.01+3.79 SL 083322 TABLE A-l (Continued) Route: Inhalation - Exposure Level: 20 ppm Animal Humber 44-7762A 7763A 7 764A 7765A 7766A 7767A 7768A 7769A 7771 7774 7775 7776 7777 7778 7788 7 789 * 7790 7 791 7 79 2 7793 7 794 7803 7804 7805 7807 7808 7817A 7818A 7821A 7822A 782 3A 7824A 7818A 7819B 7821B 782 2B 7823B 7824B 782 5B 7826B 6 251 240 235 208 225 285 231 230 248 222 224 254 229 262 280 295 249 233 2 79 272 257 263 281 245 239 223 290 265 310 214 250 262 207 221 210 236 261 240 200 220 Maternal Body Weight fol 10 16 258 293 252 268 227 285 222 277 230 282 277 333 240 285 237 281 253 302 237 289 247 298 290 331 250 300 287 330 286 325 296 316 259 301 257 297 287 323 283 337 270 315 290 290 270 336 260 294 226 295 241 278 295 326 255 297 302 359 243 306 266 303 274 315 231 264 232 277 229 267 245 304 287 337 255 306 224 250 244 285 Maternal Body Weight Gain Day(s) of Gestation 21 6-9 10-15 16-20 340 364 351 355 355 429 364 365 382 369 385 421 390 407 411 399 386 364 415 419 388 354 424 376 382 360 408 341 466 396 375 390 347 358 329 384 426 396 327 366 7 12 -8 14 5 -8 9 7 5 15 23 36 21 25 6 1 10 24 8 11 13 22 -11 15 -13 18 5 -10 -8 29 16 12 24 11 19 9 26 15 24 22 35 47 16 96 58 66 55 78 52 73 56 96 45 79 44 84 49 80 52 80 51 87 41 90 50 90 43 77 39 77 20 83 42 85 40 67 36 92 54 82 45 73 0 64 66 88 34 82 69 87 37 82 31 82 42 44 57 107 63 90 37 72 41 75 33 83 45 81 38 62 59 80 50 89 51 90 26 77 41 61 MEAN+SD 246+27 258123* 301+24 382+30 12+12 44+14 80+12 Maternal Liver Weight (Day 21) Absolute Relative <R) (g/kg Body Weight) 15.00 14.22 14.95 11.50 14.95 16.79 13.78 14.15 13.77 15.37 16.07 16.60 13.95 17.74 16.48 15.59 15.34 13.98 15.50 17.97 15.21 13.24 17.54 12.34 15.68 12.78 14.84 15.16 18.29 16.54 15.20 14.04 13.10 14.82 14.40 15.94 16.41 14.89 12.64 15.50 44.12 39.06 4 2.59 32.39 42.11 39.14 37.86 38.7 7 36.05 41.65 41.74 39.43 35.77 43.59 40.10 39.07 39.74 38.41 37.35 42.89 39.20 3 7.40 41.37 32.82 41.05 35.50 36.37 44.46 39.25 41.77 40.53 36.00 37.75 41.40 43.77 41.51 38.52 37.60 38.65 42.35 15.06+1.55 39.482.90 $L 083323 TABLE A-l (Continued) Rouge: Inhalation - Exposure Level: 0 pp* (Concurrent Controls for 80 ppm) Animal Number 33-6439 6440 6441 6442 6443 6444 6445 6446 6453 6454 6455 6456 6457 6458 6459 6459A 6461 , 6463 6464 6465 6 262 254 223 244 260 240 262 257 258 215 265 220 24 5 235 248 245 237 256 230 232 Maternal Body Weight (g) Maternal Body Weight Gain (g) Dav(s) of Gestation 10 16 21 6-9 ikT5 16^20 27 2 272 232 257 273 268 267 273 284 243 287 241 266 251 268 267 257 275 258 249 307 317 285 314 323 306 318 299 336 299 332 277 274 294 311 349 303 312 296 291 355 386 354 391 400 383 402 352 402 367 401 337 383 351 405 430 382 409 382 374 10 18 9 13 13 28 5 16 26 28 22 21 21 16 20 42 20 19 28 17 35 48 45 69 53 69 57 77 50 77 38 79 51 84 26 53 52 66 56 68 45 69 36 60 8 109 43 57 43 94 62 81 46 79 37 97 38 86 42 63 MEANlSD 244+15 264+15 307+19 382+24 20+8 43+12 7515 Maternal Liver Weight (Day 21) Absolute Relative (g) (g/kg Body Weight) 15.85 14.91 13.57 15.28 16.27 16.76 16.87 14.65 16.14 16.23 16.39 15.16 17.90 14.40 15.92 17.20 17.26 15.72 16.21 15.95 44.65 38.63 38.33 39.08 40.68 43.76 41.96 41.62 40.15 44.22 40.87 44.98 46.74 41.02 39.31 40.00 45.18 38.44 42.43 42.65 15.93+1.05 41.74+2.52 SL 083324 TABLE A-l (Continued) Route: Inhalation - Exposure Level: 80 ppm Animal Number 33-6371 6372 6373 6374 6375 6376 6377 6378 6379 6380 6381 6382 6391 6392 6393 6394 6395 6396 6397 6398 6400 6401 6402 6404 6405 6406 6407 6408 , 6 234 227 240 270 263 227 229 288 260 2 57 244 259 251 228 230 207 260 260 236 237 220 220 219 244 237 248 239 246 Maternal Body Height Col Maternal Body Height Gain (r) Day(s) of Gestatlon 10 16 21 6-9 10-15 16-2(5 256 290 378 22 34 68 245 254 282 18 9 28 254 296 363 14 42 67 288 315 398 18 27 83 278 322 415 15 44 93 242 27 5 361 15 33 86 244 270 315 15 26 45 298 335 406 10 37 71 275 317 399 15 42 82 267 309 378 10 42 69 240 267 299 -4 27 32 268 318 399 9 50 81 249 279 353 -2 30 74 187 251 336 -41 64 85 229 279 338 -1 50 59 217 303 378 10 86 75 261 313 389 1 52 76 270 303 370 10 33 67 2 39 287 353 3 46 66 240 277 361 3 37 84 250 296 378 30 46 82 228 265 354 8 37 89 233 265 334 14 32 69 271 267 329 27 -4 62 275 306 379 38 31 73 262 321 415 14 59 94 253 314 398 14 61 64 257 319 382 11 62 63 HEAN1SD 242+18 253+23 293+24* 366+34 11114* 41 18 72116 Maternal Liver Weight {Day 21) Absolute Relative (8) (R/kg Body Weight) 16.12 12.97 16.20 17.59 18.52 13.22 13.90 17.13 15.39 15.68 13.02 14.36 16.93 16.39 15.25 16.64 18.78 16.35 16.72 16.98 16.99 15.71 14.86 11.58 14.59 18.10 15.33 15.32 42.65 45.99 44.63 44.20 44.63 36.62 44.13 42.19 38.57 41.48 43.55 35.99 47.96 48.78 45.12 44.02 48.26 44.19 47.37 47.04 44.95 44.38 44.49 35.20 38.50 43.61 38.52 40.10 15.741.74 43.113.73 SL 083325 TABLE A-l (Continued) Rouge: Inhalation - Exposure Level: 0 ppm (Concurrent Controls for 160 ppm) Animal Humber 33-6268 6269 6270 6272 6273 6276 6276 6277 6282 6283 6284 6286 6287 6288 6289 6290 6291 KEAH+SD Maternal Body Weight (8) Maternal Body Weight Gain _(*i Day(s) of Gestation 6 10 16 21 6-9 10-15 16-20 278 292 332 382 14 40 50 293 311 352 431 18 41 79 303 312 343 402 9 31 59 262 285 313 377 23 28 64 262 272 302 354 10 30 52 258 285 312 373 27 27 61 294 303 343 418 9 40 75 274 286 333 403 12 47 70 274 285 KD 423 11 ND ND 272 284 KB 374 12 ND ND 254 262 KD 372 8 ND HD 324 348 KD 496 24 ND ND 303 324 ND 412 21 HD ND 289 277 KD 422 -12 ND ND 260 247 KD 329 -13 HD ND 247 286 KD 401 39 ND ND 282 310 KD 429 28 ND ND 27821 292+24 329118 400138 14113 3617 64+10 Maternal Liver Weight (Day 21) Absolute Relative _i> (g/kg Body Weight) 15.39 17.51 16.20 15.26 15.62 13.53 16.98 15.63 16.80 14.94 14.83 19.17 14.41 17.00 14.27 15.07 17.48 40.29 40.63 40.30 40.48 44.12 36.27 40.62 38.78 39.72 39.95 39.86 38.65 34.98 40.28 43.37 37.58 40.74 15.89+1.44 39.80+2.20 SL 083326 TABLE A-l (Continued) Route: Inhalation - Exposure Level: 160 ppa Animal Number 33-6200 6201 6202 6203 6204 6205 6206 6207 6208 6209 6210 6212 6213 6214 6215 6216 6217 6218 6219 6221 6222 6223 6224 6225 6227 6229 MEANiSD 6 280 281 240 274 256 272 286 261 303 259 251 228 292 24 7 263 250 266 285 291 250 273 300 295 225 2 59 276 Maternal Body Weight (k) Maternal Body Weight Gain (ft) Day(s) of Gestation 10 16 21 6-9 10-15 16-20 285 323 401 283 326 426 234 279 357 283 321 404 256 307 393 274 316 392 297 356 434 253 298 362 308 348 440 263 287 342 262 336 423 255 256 341 300 343 452 245 299 377 272 322 397 245 285 368 272 305 366 287 319 398 274 314 395 253 297 384 275 324 406 301 350 432 265 334 410 235 295 368 255 300 376 270 318 388 5 2 -6 9 0 2 11 -8 5 4 11 27 8 -2 9 -5 6 2 -17 3 2 1 -30 10 -4 -6 38 78 43 100 45 78 38 83 51 86 42 76 59 78 45 64 40 92 25 55 74 87 1 85 43 109 54 78 50 75 40 83 33 61 32 79 40 81 44 87 49 82 49 82 69 76 60 73 45 76 48 70 26621 26920* 31424 39429 2+10* 44+14 8011* Maternal Liver Weight (Day 21) Absolute Relative (k) (g/kg Body Weight) 18.89 18.09 17.05 17.69 16.35 15.57 18.84 13.88 17.33 15.45 17.27 13.41 19.43 12.66 15.31 16.40 17.83 18.52 14.85 16.70 19.00 18.31 19.75 16.45 16.12 13.91 47.11 42.46 47.76 43.79 41.60 39.72 43.41 38.34 39.39 45.18 40.83 39.32 42.99 33.58 38.56 44.56 48.72 46.53 37.59 43.49 46.80 42.38 48.17 44.70 42.87 35.85 16.73+1.94 42.533.93* CO r1 083327 TABLE A-l (Continued) Animal Number 33-59 50 5951 5952 5953 5954 5955 5963 5964 5965 5966 5967 5968 5977 5978 59 79 5981 5983 5994 5995 5996 5997 5998 5999 6000 MEANtSD Route: Drinking Water - Concentration: 0 ppm Maternal Body Weight (a) Maternal Body Weight Gain . (8) Day(s) of Gestation 6 10 16 21 6-9 10-15 16-20 288 312 351 418 305 340 386 476 290 315 358 445 300 321 360 432 237 3U9 346 38u 283 309 349 422 309 327 381 461 283 313 354 430 260 283 319 391 293 306 342 405 275 295 331 408 302 323 352 390 280 298 341 409 302 320 364 443 294 304 331 365 308 336 383 478 292 304 348 407 , 285 299 337 424 269 289 330 409 294 312 34 7 443 266 266 303 386 287 303 335 412 307 325 366 434 296 312 349 422 24 39 35 46 25 43 21 39 22 37 26 40 16 54 30 41 23 36 13 36 20 36 21 29 18 43 18 44 10 27 28 47 12 44 14 38 20 . 41 18 35 0 37 16 32 18 41 16 37 67 90 B7 72 40 73 80 76 72 63 77 38 68 79 34 95 59 87 79 96 77 77 68 73 290+13 309+16 348+20 421+28 19+7 396 72+16 Maternal Liver Weight (Day 21) Absolute Relative -(B)-____ (g/kg Body Weight) 13.37 17.93 14.15 17.84 .U.99 14.03 16.36 16.19 14.15 15.64 15. 78 14.43 17.14 17.55 18.64 19.64 14.88 16.41 14.80 17.10 13.24 15.15 15.73 17.17 31.98 37.67 31.80 41.30 38.83 33.25 35.49 37.65 36.19 38.62 38.68 37.00 41.91 39.62 51.07 41.09 36.56 38.70 36.23 38.60 34.30 36.77 36.24 40.69 15.93+1.70 37.93+3.90 SL 083328 TABLE A-l (Continued) Animal Number 33-5956 5957 5958 5959 5960 5961 5962 5970 5971 5972 597 3 5974 5975 5976 5984 5985 5966 5987 5988 5989 6001 6002 6003 6004 6005 6006 MEAN+SD Route: Drinking Hater - Concentration: 200 ppm Maternal Body Height (g) Maternal Body Height Gain (g)______ Day(s) of Gestation 6 10 16 21 6-9 10-15 16-20 315 335 371 454 273 291 326 393 270 293 297 390 235 303 345 437 J 2t>8 313 393 307 324 361 407 235 267 300 362 32 5 305 386 471 289 307 347 415 289 303 337 392 318 334 375 448 276 296 340 421 323 331 351 361 314 326 382 461 310 320 ND 441 310 317 ND 457 250 27 2 ND 334 ,3C4 304 ND 433 295 284 ND 431 258 267 ND 381 274 295 330 415 275 275 323 385 301 320 355 410 300 317 365 458 210 275 319 392 274 292 326 422 20 36 16 35 23 4 68 42 6 17 37 32 33 -20 81 18 40 14 34 16 41 20 44 8 20 12 56 10 ND 7 ND 22 ND 0 ND -11 ND 9 ND 21 35 0 46 19 35 17 48 65 4h 18 34 83 67 93 92 80 46 62 85 68 55 73 81 10 79 ND ND ND ND ND ND 85 62 55 93 73 96 28430 301i22 34226 414+35 16+18 40+14 72+20 Maternal Liver Height (Day 21) Absolute Relative (6) (g/kg Body Height) 16.59 15.06 14.02 17.41 15.14 14.17 14.43 15.04 14.26 12.88 14.52 13.64 14.60 17.29 17.43 17.97 14.74 14.74 14.18 13.09 16.97 14.79 17.40 15.84 13.18 15.24 36.54 38.32 35.95 39.84 38.52 34.82 39.86 31.93 34.36 32.86 32.41 32.40 40.44 37.50 39.52 39.32 44.13 34.04 32.90 34.36 40.89 38.42 42.44 34.58 36.7 2 36.11 15.25+1.43 36.97+3.36 aExcluded from the calculations due to a likely scale error on day 10 of gestation. ND _ No data available. Significantly different from the control value by a one-way analysis of variance, p<0.OS. SL 083329 TABLE A-2 INDIVIDUAL LITTER SUMMARY OF OBSERVATIONS MADE AT THE TIME OF CESAREAN SECTION OF RATS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION OR BY INGESTION Rout*: Inhalation - Exposure Level: 0 ppm (Concurrent Controls for 20 ppm) Animal Corpora Number Lutea Implantations 44-7746 7747 7748 7749 7750 7751 7752 7753 7754 7755 7756 7757 7758 7759 7760 7761 7763B 7764B 7765B 7766B 7767B 7768B 7769B 7770B 7779 7780 7781 7782 7783 7784 7785 7786 7795 7796 7797 7798 7799 7800 7801 7802 7810 7811 7812 7813 7814 7815 7816 MEAN+SD 10 10 15 13 14 14 15 12 Non- pregnant 13 12 11 10 14 12 10 10 11 12 15 15 Non-pregnant 12 13 88 14 14 12 11 12 12 13 14 11 14 15 11 13 12 11 12 16 15 13 13 15 12 13 13 15 16 13 15 14 14 12 1 Non-pregnant Non-pregnant 14 6 13 11 14 12 10 6 13 12 18 14 13 11 16 14 14 14 Non-pregnant Non-pregnant 16 15 Non-pregnant 12 12 13 12 13+2 123 Pregnancy Wastage 0 2 0 3 1 1 2 0 0 0 0 0 0 1 0 0 0 4 1 0 1 0 3 0 0 0 0 11 8 2 2 4 1 4 2 2 0 1 0 1 U2 Litter Resorptions' Size 0 10 0 13 0 14 0 12 1 11 0 10 0 12 0 10 0 12 1 14 0 13 08 1 13 1 10 0 12 0 14 0 14 0 11 0 12 0 12 0 15 0 13 0 12 0 13 0 16 1 14 0 14 01 Sex Ratio M:F 5:5 8:5 6:8 7:5 Fetal Body Weight <8> 5.48 5.40 5.68 5.78 4:7 5.36 5:5 5.45 6:6 5.32 1:9 5.99 5:7 5.17 8:6 5.36 4:9 2:6 7:6 3:7 5:7 8:6 4:10 5:6 7:5 4:8 10:5 7:6 9:3 8:5 8:8 5:9 11:3 1:0 5.06 6.69 5.17 5.96 5.67 5.22 4.99 5.22 5.38 5.17 5.30 5.16 4.96 5.93 5.61 5.52 5,66 7.01 Crown-Rump Length (na) 43.2 42.7 44.0 44.2 42.6 43.6 43.6 43.5 43.1 43.4 41.8 46.3 42.5 44.6 44.7 42.9 42.2 43.2 43.5 42.0 45.8 42.9 44.2 43.1 43.4 45.5 43.3 45.8 2 4 2:2 5.88 45.0 0 11 7:4 5.56 43.6 0 12 8:4 5.68 44.4 2 4 2:2 5.87 44.7 0 12 6:6 5.62 44.4 0 14 8:6 5.42 43.2 0 11 7:4 5.69 44.2 0 14 5:9 5.70 44.6 0 14 5:8 5.66 45.5 0 0 0 0,2+0.5 15 12 12 12+3 6:9 6:6 7:5 233:237 6.07 5.61 5.71 5.60i0.40 45.4 44.7 44.6 43.9+1.1 SL 083330 TABLE A-2 (Continued) Route: Inhalation - Exposure Level; 20 ppm Animal Humber Corpora Lutea Implantations Pregnancy Wastage Resorptions Litter Size Sex Ratio M:F Fetal Body Weight (g) Crown-Rump Length (mm) 44-7762A 7763A 7764A 7765A 7766A 7767A 7768A 7769A 7771 7772 7773 7774 7775 7776 7777 7778 7787 7788 7789 7790 7791 7792 7793 7794 7803 7804 7805 7806 7807 7808 7809 7817A 7818A 7819A 7820A 7821A 7822A 7823A 7824A 7818B 7819B 7820B 7821B 7822B 7823B 7824B 7825B 7826b 14 9 5 0 9 5; 4 74 43.7 12 12 0 0 12 10:2 38 42.5 12 11 1 0 11 5:6 28 43.1 14 12 2 1 11 4:7 74 44.0 10 10 0 0 10 5:5 83 45.3 14 14 0 0 14 9:5 5.81 44.5 12 12 0 0 12 6:6 5.42 43.4 15 11 4 0 11 6:5 5.07 42.5 22 13 9 1 12 6:6 6.22 45.8 Removed from study due to dehydration resulting from mechanical failure of watering system. Removed from study due to dehydration resulting from mechanical failure of watering system 12 12 0 1 ii 6:5 5.70 43.8 15 11 4 0 ii 6:5 5.44 43.6 14 14 0 0 14 6:8 5.71 44.3 14 14 0 1 13 5:8 6.04 45.2 14 12 2 0 12 7:5 5.66 43.9 Removed from study due to dehydration resulting from mechanical failure of watering system 13 13 0 2 11 6:5 5.57 43.3 15 14 1 0 14 7:7 5.16 42.5 15 13 2 0 13 9:4 5.67 44.0 17 12 5 1 11 7:4 5.60 43.6 16 16 0 1 15 10:5 5.26 43.2 19 13 6 1 12 4:8 5.58 43.7 15 12 3 0 12 4:8 5.68 44.0 11 11 0 3 8 4:4 5.65 44.3 14 9 5 0 9 5:4 5.73 44.9 17 12 5 0 12 7:5 5.78 44.8 Implantation sites detected only after sodium sulfide s talning. 13 11 11 11 2 0 11 5:6 5.58 44.3 0 0 11 7:4 5.66 44.1 Non-pregnant 14 14 12 3 0 1 13 10:3 5.41 43.7 9 0 3 1:2 6.26 46.1 Non-pregnant Non-pregnant 21 16 17 14 12 11 15 13 15 13 14 13 5 1 15 5:10 5.51 43.9 3 0 14 5:9 5.32 42.2 1 2 9 3:6 5.70 44.7 2 0 13 4:9 5.61 44.0 2 0 13 11:2 5.35 43.9 1 0 13 8:5 5.53 44.2 Removed from study due to dehydration resulting from mechanical failure of watering system 10 9 1 0 9 7:2 6.07 45.7 14 12 2 0 12 5:7 5.12 43.2 14 13 1 0 13 7:6 5.78 45.2 14 11 3 0 11 9:2 5.94 45.4 15 11 4- 0 11 5:6 5.49 43.9 10 11 0 0 11 6:5 5.21 43.6 MEAN+SD 143 L2+2 2+2 0.4+0.7 12+2 247:215 5.610.28 44.0+0.9 SL 083331 TABLE A-2 (Continued) Route: Inhalation - Exposure Level: 0 ppm (Concurrent Controls for 80 ppm) Animal Number Corpora Lutea Implantations Pregnancy Wastage Resorptions Litter Sire Sex Ratio M:F Fetal Body Weight (g) Crown-Rump Length (nm.) 33-6439 6440 6441 6442 6443 6444 6445 6446 6453 6454 6455 6456 6457 6458 6459 6459A 6460 6461 6462 6463 6464 6465 10 11 0 4 7 3:4 5.30 43.8 12 12 0 0 12 4:8 5.29 43.7 11 11 0 0 11 2:9 5.30 44.0 11 11 0 0 11 2:9 5.95 45.9 11 12 0 0 12 6:6 5.69 45.5 13 13 0 0 13 7:6 4.18 43.2 10 11 0 0 11 6:5 5.57 45.0 99 0 1 8 3:5 5.63 45.0 14 14 0 1 13 6:7 5.08 42.9 15 13 2 0 13 6:7 5.62 43.5 10 8 2 0 8 2:6 5.70 44.0 12 10 2 1 9 4:5 5.74 45.7 15 14 1 0 14 4:10 5.14 42.7 10 10 0 0 10 5:5 5.72 44.3 15 15 0 0 15 8:7 5.56 43.8 20 13 7 0 13 4:9 5.51 44.5 Implantation sites detected only after sodium sulfide staining. 14 13 1 1 12 5:7 5.68 45.2 Removed from study due to dehydration :resulting from mechanical failure of watering system 13 13 0 0 13 7:6 5.96 45.4 13 13 0 0 13 7:6 5.37 44.1 13 1? 1 0 12 7:5 5.79 45.5 MEAN+SD 122 12*2 112 0 .410.9 122 98:132 5.54+0.26 44.411.0 SL 083332 TABLE A-2 (Continued) Route: Inhalation - Exposure Level: 80 ppm Animal Corpora Number Lutea Implantations Pregnancy Wastage Resorptions Litter Size Sex Ratio M:F 33-6371 6372 6373 6374 6375 6376 6377 6378 6379 6380 6381 6382 6391 6392 6393 6394 6395 6396 6397 6398 6399 6400 6401 6402 6403 6404 6405 6406 6407 6408 14 14 0 1 13 7:6 51 11 11 13 13 14 15 4 0 1 0:1 0 0 11 7:4 0 1 12 9:3 0 0 15 10:5 15 14 1 0 14 5:9 11 5 6 0 5 3:2 14 14 0 2 12 5:7 14 14 0 0 14 5:9 12 12 0 0 12 4:8 15 13 2 13 0 0:0 16 15 1 1 14 7:7 12 9 3 0 9 6:3 14 12 2 1 11 4:7 11 8 3 0 8 4:4 11 11 0 0 11 2:9 13 12 1 1 11 5:6 10 7 3 0 7 4:3 10 9 1 1 8 4:4 13 9 4 0 9 4:5 Dellvcri'il on tiny 18 of gestation 12 U 1 1 10 3:7 11 11 0 0 11 6:5 99 0 0 9 5:4 Implantation sites detected only after sodium sulfide staining. 9 10 13 13 0 0 10 8:2 0 1 .12 8:4 15 15 0 0 15 8:7 15 14 1 0 14 8:6 15 15 0 1 14 4:10 Fetal Body Weight (8) 5.44 7.42 5.61 5.42 5.49 5.50 5.75 5.72 5.52 5.46 " 5.74 5.88 5.30 5.59 5.58 5.39 6.06 5.94 5.89 5.74 5.54 5.52 6.77 5.84 5.35 5.59 5.36 MEAN+SD 12 i2 11+3 1+2 0.82,4 10+4 145:147 5.72+0.45 Crown-Rump Length (mm) 44.3 49.6 44.5 44.3 44.7 44.7 46.0 45.4 45.0 44.2 " 45.8 45.5 43.5 44.7 44.5 44.4 46.5 46.2 46.2 46.0 44.8 44.3 47.1 44.8 43.1 43.6 43.6 45.1+1.3 SL 083333 TABLE A-2 (Continued) Route: Inhalation - Exposure Level: 0 ppm (Concurrent Controls for 160 ppm) Animal Number 33-6268 6269 6270 6271 6272 6273 6274 6275 6276 6277 6282 6283 6284 6285 6286 6287 6288 6289 6290 6291 Corpora Lutea Implantations Pregnancy Wastage Resorptions Litter Size Sex Ratio M;F Fetal Body Weight Cg> Crown-Rump Length (mm) 18 15 13 12 13 13 3 0 15 8:7 4.96 42.8 1 0 12 7:5 5.51 43.7 0 0 13 4:9 5.50 43.8 Non-pregnant 15 11 4 0 11 5:6 5.62 44.2 12 9 3 0 9 3:6 5.38 43.6 15 13 2 1 12 5:7 5.56 44.0 Remov ed from study due to dehydration resulting from mechanical failure of watering system 15 12 3 0 12 7:5 6.18 45.9 16 14 2 0 14 7:7 5.43 44.5 15 15 0 0 15 9:6 5.45 44.0 11 11 0 2 9 5:4 5.37 43.6 16 13 3 1 12 2:10 5.41 43.8 Remov ed from study due to dehydration resulting from mechanical failure of watering system 14 14 0 0 14 6:8 5.65 44.1 14 14 0 0 14 8:6 5.56 42.5 14 14 0 2 12 4:8 5.63 44.1 12 11 1 1 10 5:5 5.30 43.0 12 12 0 0 12 8:4 5.90 43.8 14 13 1 0 13 7:6 5.36 43.1 MEANtSD 142 13+2 111 0.4+0.7 1212 100:109 5.520.26 43.8+0.8 SL 083334 TABLE A-2 (Continued) Route: Inhalation - Exposure Level: 160 ppm Animal Number Corpora Lutea Implantations Pregnancy Wastage Resorptions 33-6200 6201 6202 6203 6204 6205 6206 6207 6208 6209 6210 6211 6212 6213 6214 6215 6216 6217 6218 6219 6220 6221 6222 6223 6224 6225 6226 6227 6228 6229 MEANtSD 13 10 3 2 14 14 0 0 14 12 2 1 14 11 3 1 16 14 2 0 14 11 3 0 15 15 0 1 11 10 1 0 14 14 0 0 8620 13 12 1 1 Non-pregnant 11 11 0 1 16 14 2 2 13 13 0 0 13 13 0 0 12 13 0 0 15 6 9 0 11 10 1 0 12 11 1 0 Deliv ered on morning of day 21 of gestation 12 14 0 1 14 14 0 2 14 12 2 0 12 9 3 1 12 12 0 1 Non-pregnant 13 13 0 0 Non-pregnant 10 11 0 0 132 122 12 0.510.7 Litter Site 8 14 11 10 14 11 14 10 14 6 11 Sex Ratio M:F 1:7 9:5 9:2 7:3 8:6 5:6 7:10 4:6 6:8 2:4 6:5 Fetal !Body Weight (g> 5.91 5.37 3.93 5.74 5.66 6.04 5.64 5.92 5.69 2.81 3.92 Crown-Rump Length (ms) 45.1 44.5 39.5 44.2 43.5 45.9 44.2 44.9 44.5 35.2 39.1 10 5:5 12 7:5 13 9:4 13 7:6 13 5:8 6 4:2 10 6:4 11 7:4 5.79 5.66 5.92 5.67 5.26 5.17' 5.92 6.64 44.0 43.8 44,7 44.3 42.1 42.9 44.5 46.9 13 7:6 12 4:8 12 4:8 8 2:6 11 10:1 5.68 5.49 5.67 4.95 5.72 44.2 44.0 44,4 41.6 45.2 . 13 7:6 11 2:9 5.42 6.72 43.9 48.0 112 147:144 5.47i0.82 43.62.6 SL 083335 TABLE A-2 (Continued) Route: Drinking Water - Concentration: 0 ppm I Animal Corpora Number Lutea Implantations 33-5950 5951 5952 5953 5954 5955 5963 5964 5965 5966 5967 5968 5969 5977 5978 5979 5980 5981 5982 5983 5994 5995 5996 5997 5998 5999 6000 MEAN+SD 12 11 13 13 15 14 13 12 12 5 11 11 11 11 12 12 10 10 11 9 13 11 13 4 Non- pregnant 12 12 13 12 81 Non- pregnant 16 14 Non-pregnant 12 11 14 13 16 14 13 13 13 13 10 10 99 15 13 12i2 11+3 Pregnancy Wastage 1 0 1 1 7 0 0 0 0 2 2 9 0 1 7 2 1 1 2 0 0 0 0 2 2i2 Resorptions 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 1 1 1. 1 0 0 0 1 0.3+0.5 Sex Litter Ratio Size M: F 10 7:3 13 6:7 14 8:6 12 6:6 5 3:2 10 2:8 11 3:8 12 6:6 10 3:7 8 5:3 11 5:6 4 2:2 Fetal Body Weight (6) 6.38 5.98 5.63 5.60 6.18 5.64 6.02 5.81 5.67 5.68 5.67 6.28 12 6:6 12 5:7 1 1:0 5.15 5.63 7.29 14 11:3 5.44 10 6:4 12 6:6 13 6:7 12 3:9 13 8:5 10 3:7 9 6:3 12 5:7 5.84 5.62 5.41 5.55 5.36 5.80 6.01 5.41 10+3 122:128 5.79+0.44 Crown-Rump Length (mm) 45.9 44.6 47.1 42.6 43.9 43.0 47.5 45.7 46.6 46.8 45.5 48.4 42.6 43.4 53.2 46.9 49.2 47.2 47.3 47.7 47.4 48.3 49.5 47.5 46,62.4 SL 083336 TABLE A-2 (Continued) Route: Drinking Water - Concentration: 200 ppm Animal Corpora Number Lutea Implantations 33-5956 5957 5958 5959 5960 5961 5962 5970 5971 5972 5973 5974 5975 5976 5984 5985 5986 5987 5988 5989 6001 6002 6003 6004 6005 6006 MEANiSD 15 19 12 12 13 10 14 15 13 13 13 14 7 16 17 19 13 14 16 17 15 12 12 16 21 13 14i3* 14 9 11 14 12 8 12 13 13 9 13 14 1 16 11 15 6 13 14 15 15 10 5 14 12 12 12*4 Pregnancy Wastage Resorptions 10 10 0 10 00 10 20 22 20 00 40 00 00 61 00 60 41 73 10 20 20 02 21 7 020 91 10 33 0.4+0.8 Litter Size 14 9 11 14 12 8 10 13 13 9 13 14 0 16 11 14 3 13 14 15 13 9 5 14 11 12 Sex Ratio M;F 8:6 5:4 9:2 4:10 5:7 3:5 6:4 8:5 5:8 5:4 5:8 5:9 0:0 3:13 4:7 12:2 3:0 8:5 10:4 6:9 5:8 5:4 2:3 7:7 7:4 6:6 Fetal Body Weight (g) 5.57 5.90 5.56 5.30 5.47 6.27 5.88 5.79 5.16 5.94 5.66 5.64 5.42 5.70 5.69 6.51 5.46 5.76 5.21 5.60 5.89 5.92 5.76 5.57 5.84 Crown-Rump Length (mm) 46.7 47.9 46.8 45.9 46.7 50.6 48.2 47.4 45.7 48.7 48.1 48.5 47.8 48.0 48.0 49.7 47.2 47.3 44.8 47.8 49.0 48.6 48.6 47.5 49.5 11+4 146:144 5.70+0.30 47.811.3* *Significantly different from concurrent control values by a one-way analysis of variance, p<0.05. SL 083337 TAIK 40 IMMVIuuaj. UTtU iiatuMT Of rrrxj. ALtEfAtlMl R^ta: Inhalation R*peai,re Levali 0 ppn (Cuicvrrant Central* fee 20 Pf") bales! hAMm CinkMU ElAHtHATION lorr Ttbsur fjia,hinat(QI IKKItLUETAXJ,. 4ElAIW^IUlKtTAlOTMtO* Total iLilof ttjtfcrfunlan* 4*0244, 47, 41, 41, 11, 12, 11, 14, 11, M, If, JO, 16, II, IJ, *4, U, H, *7, If. *. >0. 71. *0. !, *, >. ** **' ** *7 * **' 100, Q1, 02, 10, 1), 11, ll hmaar af Fetusca daalnid 10 13 14 12 u 10 12 10 12 14 1) 1) 10 12 14 U n 12 lJ 1) I) 12 1) 14 14 1 4 U U 4 12 14 It 14 14 IS It It 122 4 10 13 14 12 11 10 12 10 11 U 13 13 10 It 14 14 11 12 It 15 U 12 ll H 14 14 1 4 U 12 14 11 14 14 13 12 12 I 7 I I 10 I Tetal fetal 2te4 tltteri Mcr >f Values* affected hlttl Wwlnet low than trunk end (Uf deficta twkbtanaiiu* *4*W tephalecala harrow Hr.->4 painted aaaut >WbCuC4fteut hWJffh#** * hud, Mtk and back faft flatiia laaaliutl-- DlsphraiMttc hanld TallOw-calarad fecua a* liver bswrihata, liver ydrovra car* Dilate renal palvta Re caste *na faaf - put outanaouS ha*orrlW|e Haaf - thUkened daniUl and H^utanaoua tlasee la otculu raflan tkalatal tualiatlaa tlhll * delayed aaiirieatlM af ptMtlil bona Halay*d aaeiflcettae *f intnrparletel bane delayed unification of uy Skull ben wnfuand parietal and interparietal bee* lal*^ . lntarparletal -OlaaHaper parietal and lnterp.irtetal ^itra altf of tMlfltatld betweaa skull baoa delayed oealfUatlea af Ijiaaitlc arch Vertebras - delayed eaalflcatlan af atlas Halayad etalficatlsn af thnrac l( centre* ^bfuird rarvlcal cantrip* -dfc/eied thoracic eantnut Hellyad etetficallaa of cervical centrun -atattnj a.ie thoracic vartabra and one limber vertebra -alaa(n| all lunbar aod acral vertebra -any nlailnf vertebra -tubed vertebrae thoracic rrJn eitra tlwiwK vertebra I pair af riba Riba - unilateral pur m 1i 11. hr bilateral apuf* *n lit lwbar any spun an lit {unbar vMlatml v,irr rlk bilateral wavy riba any wavy tlbe Claaa 1 anly, wavy riba -Claaa 11 only, vivy rib* any iharirncd 12th rib -niacins lilh rib - unilateral elaalnp |J(h piir ef rlbe only ^laalnj ilbc mitllple riba any tUilKi (lb# -aitra tiba Itcrncblae dela>cd eaalflcatlan af arntnebraa unfyaid *<rrnbfea litiid atrrfKbre* Llohe 4 Dlyit* ~ 4vWcd e*el1irntlon e( oerc riiwli delayed aaalflretlen of netilftraola 2)11 SL 083338 IhUi InlrtUtlM - Uf*#<** U**l ** PI* U-?7*It IS, l, IS, II, I7, M. II. M, 71, IS. 71, 77, I*. M. *. *0, 11. I.', II, **. *01, to, 01. 07. 00, 171, 10*. 2U. 131, IJA, 1*0, in, iti, no. no, no, n, iso, ; Ml*i ! 7<laH> baalaaO * DTCBWa toMlKATtO* ton tissue nutlwttoa HrLfTM. U.V4! bATIOM UtAL Hit tM-'UKAriM 9 11 11 ll 10 14 II U II 11 11 I* is II U U IS II IS l: 11 244*1444444*4 *4)4l}4l I 11 II It 10 14 12 u 12 II U 1 12 11 II IJ 124 a4 U4 11 11 11 11 11 Tul HiIt m)la(9ll4l till r af fataito *ft*<t#4 fatal**! tmimiw Unit tr4 i4 kif <*>( MHatiiwM 4*M OWMlMtU Hum tail, palatto atow( WbcuCtMfra Wwrrhi|* Wl, baM, *1*4 #eb toll ttim* trt*lmli IllfUipiUc baraU t*ll#i^*0lai*4 facua as llfir hMnU|i, ll[ *r4fwi*ri |l|ai4 nul palvla hu|l( a*4(lai to*4 aubcuiaiwt* 9M4 - thlrkrM* laraal aid tiKallHta* dtM la hi,|.i **!"* Qlalatal Hall ' 4alay*4 *iti(l<*((M ut ftrUial buaa '4*i*T*4 aa*!U<*|laa *1 Ml*rw4 *1 a#7 kail lava aklMM *r|aial an* lataif-tr 1*4*1 4M lalM ` lltl*<r||iil alaahtfn pariflal all Iniarparlatll Min alia a! aaaiflcaliM b*c***a #1*11 hm 4iltj44 Hllt;Kl*a a< iriMtii wi **nlca* - 4alar*4 aaalllutlM *f *(laa aaalflcatlaa *f Ikprat it ciatna anluaal <r*tcal aaatrak -aafuaaO tharatlc iitIM lalipH aiallltillH *f nr. ku 1 tiniM -Mlaalna an* thatatlt **na|a an* aat Idt.r vattalft -alaalaf all l#tf aM attfll .h (abra .IMwr4* tutrlMae#ria*briaharacW t*l>* -^ita ttoraclr nlltlu 4*4 *lf *1 riba lib* aallatatal apur * 1*4 UkMf -ltl*fll apura aa lal laMir aa? aaut# a* lit likMr kkllataral #j>r tlba bilateral wav? ilba i>r *i*r alba -CUai l H**r riba Cl*M II riba a.7 IlNt'Ml ll(h 111 al**lM tf(b fU - *ll*taral *|aala* ISth pair t| rtb* t*l| MMlH 4*4* * 4*l4l*l4 ilba aa* #<*(*( riba aaira III* It.rwlrat *rlrM aaa7l**tl*k oi atriarnraa *nf**4 ,...i_bi4a f**M Bt"arbrat t taka I Dla 11 a *,ljya* MaXtcattaa al ,. *nml* 4,'|a*r* ##*tfl,atlva < SL 083339 TAlit.l-. A-l ((Out*! Inhalation - Exposure Level; 00 pj>4 Aflinsl NiMbetaj ETTEftXAL EXAMINATION Soft T1SSUL tXAWlNATJON SKELETAL EXAMINATION SKULL BONE EXAMINATION Total Major Halforaatlon* External Expiration fhort trunk and other deface* Subcutaneous edtAS Omphalocele Her rev head, pointed snout Subcutaneous huaorrhag* - neck, head, and back '0371, 72, 73, 74, 75, 74, 77, 78. 79, BO, >2, 01, 92, 93, 94, 9S, 96, 97, 90. 400, 01, 02, 04, 03, 00, 07, 00 N4*b*r nf Putusc* Eunlntd 13 1 11 12 IS 14 s 12 14 12 14 9 11 B 11 11 7 0 9 10 11 * 10 12 13 14 14 A l 4 4 5 3 3 A s A 5 3 4 3 .4 4 3 3 3 3 43 34 3 5S 13 1 11 12 15 14 5 12 14 12 14 9 11 8 11 11 7 0 4 10 11 9 10 12 15 14 14 9 0 7 10 9 2 8 9 9 0 7 S 7 7 4 S 6 7 76 7 0 10 * 9 Nunber ef fetuses Affected 0 0 0 0 0 0 0 1 l 0 0 0 0 0 0 0 0 0 0 0 00 00000 Total Total Lilt.ra Ft-tuwys 27 292 103 292 189 27 27 26 22 0 00 0 0000000000000000 0 00 00000 0 0 000 0000 0000000000 00 0 0 0 0 00 0 1 0 0 000 0 0 00 00 0 00 0 00 00000 00000 0 1 1 1 00000000 1 0 000000000 0 00 00000 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 1 Soft tissue Examination Diaphragmatic hetoU Tellov^ceiorvd focus on liver Noorrt^|*i Itvaf Bydrourctef* Dilated renal pelvl* Ectopic ovariae Mead - subcutaneous h^orrhag# ooooo ooooo 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 ooooo 00000 ooooo ooooo ooooo 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 0 0 0 0 0 0 0 00 0 0 00 oooo 0 0 00 oooo 0 0 0 0 0 0 00 00 ooooo 0 0 20 0 0 0 0 00 oooo Bead - chickened dorsal and subcutaneous ciasue In eccular region 0 0 0 0 oooo Skeletal Examination SWU - delayed ossification of parietal boo* 0 0 2 2 2 1 1 2 3 10 0 0 0 0 oooo -delayed oaaiflcJtlon of interparietal bone 20 5 3 5 0 1 3 4 4 0 0 0 0 0 3 00 2 -delayed oaaiftc-itlon of any akull bonr 2 0 5 3 5 0 1 3 4 4 0 0 0 0 0 10 0 2 -unfuaod parietal and Interparietal 0 0 0 2 2 0 0 10 0 0 0 o 0 0 oooo -bone la Land - lntecpai total 0 0 0 0 0 0 0 oooo 0 0 Q 0 oooo -misshapen parle'al and Interparietal 0 0 0 0 0 0 0 oooo 0 o 0 0 oooo -extra ait* of oi filtration between akull bone 0 0 ooo 0 0 10 0 0 0 0 0 0 oooo -delayed ossification of tygooatlc arch 0 0 0 0 1 0 0 10 0 0 0 0 0 0 oooo Vertebra* - delayed ossification of atlas ^-delayed ossification of tteracle centres unfused cervical cant run -unfuaed thoracic centrum -delayed ossification of cervical eentrun -missing one thor.iclc vertebra and on# lumbar vertebra visaing all luni ar and sacral vertebrae -any Biasing vertebra -fused vertebra* - thoracic region -#Xtro thoracic vertebtt 4 pair of riba Bib* - unilateral 'pur on 1st lumbar -bilateral spurt on lat lumbar -any spurs on lat lumbar -unilateral wavy ribs -bilateral wavy riba any wavy riba Oast I wavy rib* -Class II wavy ribs -any tUortvucd 12th tib -mtsaiug 12th rih - unilateral -BtSHfii)-, nth pair of ribs Only -missing rlbi - i>.jltl|'1e rib* -any mitinKig rib* -extra riba 0 0 ooo 0 0 oooo 0 o 0 0 oooo 0 0 ooo 0 0 oooo 0 o 0 1 oooo 0 0 ooo 0 0 0 0 ' 0 1 0 0 0 0 oooo 0 0 ooo 0 0 0000 0 1 0 1 oooo 3 0 10k 3 1 1 1 o B 2 1 0 4 3 2 10 0 0 ooo 0 0 oooo 0 o 0 0 oooo 0 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 000 0 0 oooo 0 0 0 0 oooo 01 1 1 22 1 2 0 3 2 0 2 2 0 3 0 10 0 0 2 0 0 1 0 2 112 0 6 1 3 0 0 11 0 l 3 1 2 3 X 4 14 4 0 B 3 3 3 0 27 0 0 1 0 1 0 0 oooo 0 0 1 0 0 0 0 1 1 0 1 0 0 0 1 110 0 0 0 0 0 0 0 0 1 1 0 2 0 1 0 X 110 0 0 0 1 0 0 0 0 2 1 0 2 0 1 0 1 10 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 1 Q 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 ooo 0 0 0 0 0 0 0 0 0 0 oooo 0 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 0 0 0 0 0 oooo 0 1 0 0 0 0 10 SiiTu.huit' ' oanlfUation of i i nt br<tf *un'Hi< I ( l >1 hti'Tiuhta* - uni u-i ,| >i ( rn< l>i it -fu-i'd nU-r n, t'fitt' 0 0 0 0 0 I 0 0 t 0 0 0 0 0 1 oooo 0 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 ooo 0 0 oooo 0 0 0 0 oooo 0 0 ooo 0 0 oooo 0 o 0 0 oooo t.lmhtt 4 hlgl i 4 - J< lav. J pan l f It at Ion ol t n nr(>i'la -J. I iv<`d o>i* 11 li nt ton of wlih ni a.1 In 0 0 ooo 0 o oooo 0 0 0 0 oooo O 0 ooo 0 o oooo 0 0 0 0 oooo 0 00 0 00 ooo 0 00 0 00 ooo ooo ooo 0 oooo 0 oooo 0 oooo 0 oooo 0 oooo 0 oooo o oooo 0 oooo ooo 0 10 0 10 0 00 ooo ooo 0 00 ooo o 0000 0 4 10 0 0 2 4 1 10 00 0 o o oooo 0 oooo 0 oooo o. oooo 0 0 0 0 oooo 00 0 OOO ooo 0 oooo 0 oooo o oooo 00 3 000 o 4 0 10 0 oooo 0 0 0 0 oooo 0 0 0 0 oooo 0 0 0 0 oooo 00 0 20 0 1 01 30 1 00 0 0 10 01 0 0 10 00 0 00 0 OOO 00 0 ooo 00 0 00 0 0 2 0 2 0 0 0 0 0 0 0 0 0 0 0 oooo 3 0 11 4 10 2 7 113 oooo oooo oooo oooo oooo oooo oooo oooo oooo oooo oooo 0 2 0 1 2 0 11 0 0 0 0 oooo 0 7 0 0 oooo 0 0 0 0 oooo 0 0 0 0 oooo OOO 0 oooo 0 00 0 1 1 0 00 0 0 2 0 1 0 0 11 7 30 30 0 0 13 5 0 0 11 22 00 1 1 2 1 1 2 47 17 00 0 0 0 0 0 0 00 30 30 10 17 40 4 23 A 77 11 9 98 22 00 00 00 00 0 2 0 2 10 0 0 2 0 0 1 0 00 00 SL 083340 RuuU: Inli.il.it, lun - U|..... ItVi-l: 0 |i|iin (lIiiiKurr, nL Lout roll foe SO ppm) Anlm.il NumbtM: EXTERNAL EXAMINATION SOI'r TISSUE examination SKELETAL EXAMINATION SKULL SONE EXAMINATION Total Major Malformations 35-64 39, 40, 41, 42, 43. 44, 45, 46, 53, 54, 55, 56. 57, 58, 59, 59A, 61, 63, 64, 65 Number of Fetuses Examined 7 12 11 11 12 13 11 3444444 7 12 12 11 22 13 n 487 7 89 7 0 000000 s n 13 8 9 14 10 15 n 12 13 13 12 344 3 35 35 4 4 44 4 8 ] 3 13 8 9 H 10 15 13 12 13 13 12 5 9 9 5 6 9 7 10 98998 Number of Fetuses Affected Q000 1 00 0 00000 Total Ft tUSl '3 230 77 230 153 1 External Examination Short trunk and other defect* Subcutaneous edema Omphalocele Narrow head, pointed snout Subcutaneous hemorrhage - neck, head, and back 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00000000 0000000 0 0 00 0 00000 00000 0 0 00 0 00000 0 0 0 0 0 Soft Tissue Examination Diaphragmatic hernia Yellow-colored focus on liver Hemorrhage, liver Hydroureter Dilated renal pelvis Ectopic ovaries Head - subcutaneous hemorrhage 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 Q 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 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 0 0 0 0 0 0 0 0 0 Head - chickened dermal and subcutaneous tissue in occular region 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00000 00000 00000 00000 00000 00000 00001 00000 1 0 0 0 0 0 2 0 Skeletal Examination Skull - delayed ossification of parietal bone 000001 000000000 -delayed ossification of interparietal bone 000 l 0 300 1 0000 1 0 -delayed ossification of any skull bone 0 0 0 1 0 3 0 0 1 0 0 0 0 1 0 -unfused parietal and interparietal 00001 01030001 1 1 -bone island - Interparietal 00 000000 000 000 0 -misshapen parietal and Interparietal 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 -extra site of ossification between skull bone 0000000000 00 000 -delayed ossification of zygomatic arch 0000000 00 00 0 000 Vertebrae - delayed ossification of atlas -delayed ossification of thoracic centrum -unfused cervical centrum -unfused thoracic centrum -delayed ossification of cervical centrum -missing one thoracic vertebra and one lumbar vertebra -missing all lumbar and sacral vertebrae -any missing vertebra -fused vertebrae - thoracicregion -extra thoracic vertebra 4 pair of riba Ribs - unilateral spur on 1stlumbar -bilateral *pur* on 1st lumbar -any spurs on 1st lumbar -unilateral wavy ribs -bilateral wavy ribs -any wavy ribs -Cl.ms I wavy ribs -Class IJ wavy ribs -.my shortened 12th rib -nlssing 12th rib - unilateral -missing 13th pair of ribs only -missing ribs - multiple ribs - utv missing ribs -extra ribs 0 000 0 000 00 00000 00000 000 0 0 0 00 00 00000 000 0 0 0 0 0 0 0 00 100000 0Q00000 01 1 1 1 3 23 2 2 1 0 401 00000000 00000 00 00000000 0000000 00a 00 000 0 00 00 00 00000000 0 000000 0000000000 00000 4 22 300002 2 1 0 1 3 2 Q4 0i 0 1 00 1 2 00000 4 6 2 /* 0 1 0 0 3 4 1 0 1 3 2 000 0 00000 0 00 000 00000000 0000000 00 000000 00 0 00 00 0 0000000 0 00 0 0 0 0 0 00 000 00 0 0 0 000 0 0 0u n0 00 00 0 0 0 0 00 0 0000000 00 0 0 000 00000 0000000000 0 000 00 00 0 0 0 0000 0 0 0 0 0 0 0, 0 0 0 0 0 0 0 0 000000000 1 00000 SUnwbrae delayed ossification ot sternebrae -unoKHiflcd rttefnebrue -unfusod KtiTnobrae -luscd Hterncbtae 010002 200001 402 00000000 0 000000 0000000000 00000 0 0 0 000 00 00 0 0000 Limbs k Digits - delayed ossification ol metacarpels -delayed ohhIflent ton of M'tatarHcla 0 00 000 00 0 0 000 0 0 0 0 00 0000 0 000 00 0 1 0000 3 1 300 3 1 300 2 0 304 00100 00 Q00 0 0 00 0 00 00 0 00 00 0 00000 0 000 0 0 0 000 1 001 0 0 000 0 00000 00000 0 0 00 0 00000 2022 0 1 1 000 3 1 2 20 00000 0 0 00 0 00000 0Q 0QQ 0 00 00 0 0 00 0 0 000 0 0 000 0 00000 00000 00000 0 0000 0000 0 00 000 0000 0 00000 0 000 0 2 13 13 17 1 1 0 0 0 0 0 1 24 0 0 0 0 0 28 U 39 0 0 0 0 0 0 0 0 0 0 1 12 0 0 0 0 0 Total \ tters 20 20 20 20 1 0 0 0 0 0 1 0 0 0 0 0 2 0 2 7 7 9 1 1 0 0 0 0 0 1 14 0 0 0 0 0 13 7 15 0 0 0 0 0 0 0 0 0 0 1 6 0 0 0 0 0 SL 083341 9 lAhH'^O Uiu.t gout*: Initiation Exposure Lewis 160 p^ Antan 1 Numbers J 33-6200, 01, 02, 0). 04, OS, 06, 07, 08. 0*, 10. 12, 13, 1*. 15 16, 17, 11, 19, 21, 22, 23. 24, 25, 27, 2* Number of fetwaee Examined Total Total frtki-vJi Litters EXTERNAL EXAMINATION soft tissue examination SKELETAL EXAMINATION SKULL BONt EXAMINATION Totil Major Malformations g 14 U 10 14 11 14 10 14 354354535 8 14 a io u a u to 14 5*779 7 9 7 9 13 13 44 13 13 99 6 10 11 13 12 12 3 34 44 4 6 10 a 13 12 12 3 7 7 9 ft ft 11 13 11 3444 a 13 11 579 7 etusea Affected 0 0 0 00 00 0 0 000 291 lOo 291 191 14 2ft 2ft 2ft 26 4 External Examination Short trunk end other defects Subcutaneous edema Omphalocele Narrow head, pointed enout Subcutaneous hemorrhage - bead, neck, and hack 0 0 00 0 0 0 0 000 0 0 00000 000 000 000000000000 00000 00 00000 00000000 0000 00 00 00 00 00 Soft Tissue Examination Diaphragmatic hernia Yellow-colored focus on liver hemorrhage, liver Hydroureters Dilated renal pelvis Ectopic ovaries Head - subcutaneous hemorrhage Bead - thickened dermal nd subcutaneous tissue in occular region 000000000000 00000000 0000 00 00 00 00 0000 2 00000000000 00000 0 00 0000 00000 0 00 00 00 000000 000000 000 0 00000Q00 00 00 00 43 00 00 00 00 Skeletal Examination Skull - delayed ossification of parietal hone delayed ossification of interparietal bone delayed ossification of any hull hone -unfused perietal and interparietal bone island- Interparietal misshapen par.lvt.il and interparietal extra alts of ossification between skull bene -deleyed ossification of tygomaclc itch 00 22 22 23 00 0o 00 00 21 32 34 21 00 00 oo 00 Vertebrae - delayed ossification of atlas -delayed ossification of thoracic centrum unfused cervical centrum unfused thoracic centrum -delayed ossification of cervical centrum -miaslng one thoracic vertebra and one lumbar vertebra -visaing all lumbar and sacral vertebrae any missing vertebra -fused vertebrae - thoracic region extra thoracic vertebra ft pair of riba 000 00 00 oo 00 00 00 0 10 11 7 12 2 11 000 00 00 00 0 0 0 ooo 01 0 0 1 0 ooo ooo Elba - unilateral spur on 1st lumbar bilateral spurs on l*c lumbar -any Spurs on 1st lumber -unilateral wavy ribs -bilateral wavy ribs -any wavy ribs -Class 1 wavy ribs Class 11 wavy rlbs -any shortened 12th rib -missing l'th rih - unilateral missing 11th p.i)r of ribs only -hissing ribs - multiple ribs -any missing riha extra ribs Sternehr.iv - drlayid Qn* 1 f test Ion of itrrnrbrae -unosnlfltd sUriK'hfrtc unfu'i'J n|i'rn hrne -fuHd atk riii'hfnc 0 0 4 0 0 02 0 0 0 0 0 0 00 0 0 0 0 0 1 ooo 0 0 0 0 0 ooo 02 0o 00 00 Limbs g Mm i s - tiilrtycil ossification o( at t K iirpt'l* -dtlnyi'd i<m<(ffi ut lint of O0 O0 1 0 0 0 0 0 ooo ooo 2a 0 0 0o 00 000000 000010 4 4 03 3 0 200451 4 4 0 33 0 200 4 3 1 6 1 01 0 04 0000 1 1 1 0001 000000 000000000000 000000 000000 0 0000 0 00 0 00 0 13 7 37 20 59 20 33 14 33 00 00 00 0 000 0 00 0 0 00 0 11 000000000000 0 0000 0000000 00 1 0 0 0 000000 162101 22 3310 0 1 1 107 0 1 1 20 0 0000000 0000 14 4 000000 00 0000 00 00 0000000000 14 4 000000 000000 00 00 00 0 000 0 000 00 45 19 30 16 75 22 32 19 9 22 10 95 13 9 11 4 42 14 4 00 15 4 43 0000 00 1 0 000o 0o00 00 0 0 0 0 00 00 00 0000 8 1 ooo 0 0 0 o 0 0 ooo 0 0 0 o 0 0 13 0 SL 083342 Table a-3 (ConttmmJ) tout*: Drinking Water - Expoeure levelt 0 ppm A"4^al Mbrti KXTCRMAL examination OFT TISSUE L\AM!UT10N SKELETAL FXAv!lSATlON SKULL WNE EXAMINATION Total Ha 1t Malformations Total Total 33-9990, 11, 32, 93, 34. 39. 83, 44, 63. 66, 67, 68, 77, 78, 79, 81. 83, 94, 93. 96. 97. 98. *. oo Fetueau Litter* Huaiber of fetuses Examined 10 13 14 13 9434 10 13 14 12 7 998 3 10 11 12 10 3 3 44 3 3 10 11 12 10 2 1787 8 11 34 8 11 37 4 12 12 34 4 4 12 12 188 1 14 10 12 13 12 13 10 1 334444 3 1 14 10 12 13 12 13 10 09 7$9i9 7 9 12 34 9 12 1 230 86 230 164 26 26 26 23 Humber of Fctutea Affected 004400 40 00000 0 0 0 0 00 04 000 0 0 External Examination Subcutaneous hemorrhage * bead, neck, and back 0000 0 000 00 00000000001 0 40 1 1 loft Tiaeua Examination iydroureters Benorrha|*. llvtr Mead - subcutaneous hemorrhage 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 4 0 0 0 00 00 0 0 0 0 0 0 1 4 0 0 0 4 0 9 0 0 0 0 0 4 4 0 0 0100010 0000004001 Q00 000 1 1 3 1 1 3 Sk*l*t*l Examination Skull * d*la>*d ossification of parietal bun* delayed islfleer ion of Interparietal b*tv delayed edification of any eku 11 bomitnfwa*d p.irletel and Interparietal -bon* lalaut] - Interparietal misshapen patlat.il and intTpvTietal . -extra alfc of oetlficatlofl between akuii bone# -delayed '.IfRation of ygoaatic arch Vertebra# - delayed oaelfieadon of atlas -dalayad ossification of eervlcai centra delayed ossification of thoracic centrum -unfuSCd cervical centrum ^infused thoracic cancrua mlsalnft one thoracic vertebra and one Lumbar vertebra -niaalng ton* thoracic, all lumber and all sacral vertebrae any missing vertt-bra -fueed vertebrae, thoracic ration extra thoracic vertabra 4 pair of ribs Elba - unilateral spur on 1st lumber bilateral spun on 1st lusher any spurs on 1st lumbar unilateral wavy ribs -bilateral wavy riba eoy ve*y ribs -Claaa 1 wavy ribs Cleae 11 wavy ribs any ahorn'iii'd 12th rib -missing 1/ilt rli. - unilateral -ptaalng lltli piir of rib* only -Olssinr. ril'S - mult 1 p11' ribs any 1*h1<\a rib* (Kra rib* IteritL'bra*' - dnlnycd Ossification of aternwbr.ie -unoaslfhJ i.-rnibrae unfuHcd su.rn<br>c fu*vd *ti t nt bra*1 llmba 4 01rIi*< - delayed osaifRation of su taunt p*l* -delay**! On*l fRat Ion of out aunt? 1* 0 00 0 0 0 00 0 0 000 1 40 0 0 0 0 2 000 0 03 0 1 l 1 L 0 L 4006 0 1 0 1 0 04 000 00301 1 1 1 Q 1 000 6 41 0 1 4 04 000 7 7 4 1 2 2 4 7 6 3 7 06 80 7 7 2 0 9 0 4 1 3 00004 0000000000000000440 0 0400000 00004400000 00 0 4Q 0 0 00 0 0 00 0 0 0 00 0 0 1 0 0 4 0400 0 40000 0 00 00 00000000Q 00 00 0 000000 0000 004 000 00Q0 0 00 0 0 00000 00 00 0 040 0400000 0 0 0 00000 0 00 000 04 040 0 04 04 0 0 0 02 7 1 0 1 01 00 1 01 203 06 7 1 1 2 1 2 000000 0000 0 00 0 0 404 0 4 1 1 00 0 000Q 0 a0 0 0 Q Q00 0 4 0 4 0 44 0 0 0 00000 0 000 0 00400400 0 00 000 00000 000 40 0 400 0004 0 0 0 000 0 000 0 0 00 0 0 0000 0 0 0 40 0 0 0 00 00000000000 0000000000 000 4 3010132 1 1 01131 1 201 101 2 1 1 0 2 0 0 0 3 2 00 0 00 2 0 0 0 1 4 2 0 1 40 3 221 01 84 1 1 01 1 3 1 1 2 1 1 302 2 1 000000 00 00 000 1000 00 0 0000 000000 00 00 00000 0004 00 000 00444 0 00 0 0 004 1 0 00 0 0 00 0 00 00000000 00 00000000000 000 000000 00 00 0Q0100000 00 000 00000 0000Q 000 0000 04 0 0 00 0 0 0000 000 00000 000 000 00 000 000000 00004000000 4040900 0 0 0 0 0 0. 0 00 000400040400 0 0 0000 0 Q 00 00 0 0Q0Q0040 4 004 0 1 0000 0 00000000 04 00040 009 000000 1 1 2 0 001 00 24 1 1 2 1 4J 0 0 0004004400 0040 1 000 00000 000000000000400 1 000 1 0000 00000 0000000000000000400 0 00000 00000 00000 40000000 004400 00 D 0 004000 0 4009 000 9 19 19 *3 0 0 1 0 0 0 0 46 2 0 0 0 0 0 32 16 66 1 0 1 0 1 0 0 0 0 0 1 13 1 2 0 0 0 2 14 10 20 0 0 1 0 9 0 0 16 2 0 0 9 0 0 19 8 21 1 0 1 0 1 0 0 0 0 0 1 10 1 2 0 0 0 Slj 083343 tABI.F A-3 (Continued) touce; Drinking Water - Exposure Leval; 200 ppm Animal Number*! CmRNAL EXAMINATION SOIT TISSUE EXAMINATION SKELETAL EXAMINATION SKULL BONE EXAMINATION Total Major Malformations 33-3956, 37, 58. 5*. 40, 41, 42, 7Q, 71, 72, 73,, 74, 74, 84, 5. 86, 7. 88, 89, 01, 02, 03. 04, 03, 04 Dumber of rctusta Exemlned 114 3 U 9 11 14 12 34 3 4 9 11 14 12 6 99 8 10 13 13 3344 8 10 13 H 3799 9 13 14 14 11 14 34 3445 9 13 14 16 11 14 * 9 9 12 7 9 3 13 14 13 13 34 334 3 13 14 13 13 0 9 9 10 9 9 3 9 6 3 14 11 12 3344 3 14 u 12 Z 9 78 Number of Fetuses Affected 000000Q000 00 0000000000000 Total Total litters 290 ,, 100 23 290 23 190 24 00 Exttrnsl Examination Subcutaneous hesurthage * bead, oeck, and Lack 000003 0000 0002 0 00000000 00 4 2 Soft Tissue Examination Mydrouretcts Hemorrhage, Uvit Mead - subcutaneous hemorrhage 0 0 0 0 0 0 0 0 0 i 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 9 0 0 1 0 0 0 0 a 0 0 .0 00 00 0 0 0 * 0 3 3 0 3 Skeletal Examlftsclom Skull - delayed ossification et parietal Lone 000000 2000 00 200001 0000Q0Q 5 -delayed ossification of interparietal bone 1 0 1 0 1 0 4 0 0 0 0 0 1 1 0 0 `4 3 0 3 0 0 3 1 0 31 -delayed ossification of any skull ken* l 010 1 040 00 -unfused parietal and interparietal 333350i6 76 -bone island. Interparietal 000001 0000 misshapen parietal and interparietal 0 0 0 0 0 0 0 0 0 0 0 9 08 8 10 1 4 0 7 0 0 4 8 5 6 0 0 3 3 0 1 0 0 3 4 1 1 0 0 000 1 0 00 000 c 0 000 0 000000000c 0000 32 104 2 0 -extra alt* of osiificatioo between skull bones 0000000000 0000000000c 0000 0 -delayed ossification of ygometic arch 0000000000 0000000000c 0000 0 Vertebrae - delayed ossification of atlaa -delayed ossification of cervical centre -delayed ossification of thoracic centrum -unfused cervical centrum -unfused thoracic centrum -isaing one thoracic vertebra and one limber vertebra missing some thoracic, all lumbar, and all sacral vertebra* any missing vertebra -fused vertebrae - thoracic region -extra thoracic vertebra & pair of ribs Kibe - unilateral spurs on 1st lumbar -bilateral spurs on 1st lumbar any spun on 1st lumber -unilateral vevy ribs -bilateral wavy ribs -any wavy ribs Class X vavy ribs -Class It wavy ribs -any shortened 12th rib lasing l2th rib - unilateral -missing 13th pair of ribs only -missing ribs - multiple ribs -any missing ribs extra ribs 0 00000 000 0 0 0 0 00 00000 c 0000 0 200 3001 1 1 1 1 2 2 33 041 3 1 0023 0 0 0 0 o 0 0 o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 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 0 0 0 0 0 0 0 0 0 1, ') '' 0 0 0 0 0 0 0 0 Q 0 0 0 0 oo 00000 00 0000 0 0000 0 0 000 *1 0 0 1 0 0 00 o0 0 0 o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 '> 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0000000 0 0 00 0 00 0000 0 0 0000 0 Z 0 00 2 0 0 0 0 0 0 0 0 00 0 0 40 40 00 00 00 oo 00 00 00 00 00 00 00 2 1 3 0 0 0 0 0 0 0 0 0 0 0 2 0 2 0 0 0 0 0 0 0 0 0 0 0 2 2 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 z l 0 0 0 0 0 0 0 4 3 9 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 1 1 2 0 0 0 0 0 0 0 0 0 0 0 2 0 2 0 0 0 0 0 0 0 0 0 0 0 3 0 3 0 0 0 0 0 0 0 0 0 0 0 3 0 3 0 2 2 2 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 Q 0 0 1 3 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q 0 0 0 0 0 0 0 0 0 204 000 204 000 000 00 0 000 000 000 000 000 000 0 0* 0 000 1 1 2 0 0 Q 0 0 0 0 0 0 0 0 ) ) 3 J 1 5 3 J 0 0 0 J 13 13 28 00 00 00 00 00 00 00 00 00 00 00 i 0 1 0 0 0 0 0 0 0 0 0 0 0 2 1 3 0 0 0 0 0 0 0 0 0 Q 0 4 J* 43 1 2 3 3 0 0 Q 0 0 0 Strrncbrae - delayed ossification Of scrrm'l'Me -unonntf l,d ntcrn.'hris -unfueed stvrnvhraa -f gild i> nuUt.li 0 0 0 0 00 00 00 00 2 0 9 0 0 0 0 0 01 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 3 a 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 9 0 0 0 Limbs 4 Dibits - d< Uyrd ossification of su'IOi m |-* l* -dutayod oi*niri n Ion of mi-tatarsc) a 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 U 11 20 2 0 0 0 0 19 0 0 1 0 0 o 0 0 21 10 21 1 1 2 2 o 0 0 00 0 o 0 o 0 0 0 SL 083344 TABLE A-4 INDIVIDUAL BODY AND LIVER WEIGHTS OF PREGNANT RABBITS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION Exposure Level: 0 ppm (Concurrent Controls for 80 ppm) Animal Number Maternal Body Weight (kg) paternal Body Weight Gain (kg) Day(s) of Gestation 6 12 19 29 6-11 12-18 19-28 33-6431 -6432 -6433 -6434 -6435 -6436 -6437 -6447 -6448 -6449 -6451 -6452 -6466 -6467 4.17 4.51 4.75 4.51 4.39 3.91 4.52 3.58 4.48 4.64 4.19 4.51 4.46 3.98 4.07 4.60 4.74 4.47 4.26 3.84 4.47 3.52 4.40 4.53 4.16 4.51 4.45 3.93 4.13 4.65 4.73 4.62 4.34 3.79 4.55 3.60 4.49 4.48 4.30 4.59 4.40 4.05 4.35 4.75 4.81 4.68 4.39 4.00 4.56 3.82 4.46 4.47 4.51 4.65 4.35 4.05 -0.10 0.09 -0.01 -0.04 -0.13 -0.07 -0.05 -0.06 -0.08 -0.11 -0.03 0.00 -0.01 -0.05 0.06 0.05 -0.01 0.15 0.08 -0.05 0.08 0.08 0.09 -0.05 0.14 0.08 -0.05 0.12 0.22 0.10 0.08 0.06 0.05 0.21 0.01 0.12 -0.03 -0.01 0.21 0.06 -0.05 0.00 MEAN+SD 4.33+0..32 4.28+0.34 4.34+0.33 4.42+0.29 -0.05+0.06 0.06+0.07 0.07+0.10 Maternal Liver Weight (Day 29) Absolute Relative (8) (g/kg Body Weight) 112 100 122 102 113 79 90 95 95 93 119 92 99 70 25.75 21.05 25.36 21.79 25.76 19.75 19.74 24.87 21.30 20.81 26.39 19.78 22.76 17.28 99+14 22.31+2.86 SL 083345 TABLE A-4 (Continued) Exposure Level: 80 ppm Animal Number Maternal Body Weight (kg) Maternal Body Weight Gain (kg) Day(s) of Gestation 6 12 19 29 6^n-- 12-IB 19-28 33-6409 6410 6411 6412 6413 6414 6415 6417 6419 6420 6421 6422 6423 6424 6425 6427 6428 6430 3.98 3.94 4.10 3.43 3.89 4.10 4.15 4.19 4.30 3.93 4.38 3.50 4.14 4.03 3.97 4.55 4.73 3.51 4.12 4.12 4.08 3.41 3.89 4.17 4.11 4.11 4.32 3.91 4.29 3.56 4.10 3.94 3.85 4.46 4.41 3.43 4.25 4.24 4.18 3.14 3.89 4.02 3.84 3.94 4.97 3.76 3.94 3.58 4.17 3.64 3.84 4.53 4.60 3.40 4.45 4.37 4.38 3.38 4.19 4.25 4.04 4.22 4.72 4.18 4.41 3.84 4.54 3.95 3.93 4.69 4.62 3.77 0.14 0.18 -0.02 -0.02 0.00 0.07 -0.04 -0.08 0.02 -0.02 -0.09 0.06 -0.04 -0.09 -0.12 -0.09 -0.32 -0.08 0.13 0.12 0.10 -0.27 0.00 -0.15 -0.27 -0.17 0.55 -0.15 -0.35 0.02 0.07 -0.30 -0.01 0.07 0.19 -0.03 0.20 0.13 0.20 0.24 0.30 0.23 0.20 0.28 -0.15 0.42 0.47 0.26 0.37 0.31 0.09 0.16 0.02 0.37 MEAN1SD 4.04+0.,34* 4.020.30* 4.000.44* 4 .220.35 -0.03+0.11 -0.02+0.22 0.230.15* Maternal Liver Weight (Day 29) Absolute Relative (g) (g/kg Body Weight) 125 28.09 105 24.03 106 24.20 94 27.81 93 22.20 116 27.29 115 28.47 127 30.09 97 20.55 112 26.79 112 25.40 112 29.17 105 23.13 117 29.62 98 24.94 112 23.88 98 21.21 83 22.02 10712* 25.33+2.97* SL 0 8 3 3 4 7 TABLE A-4 (Continued) Exposure Level: 160 ppm Animal Humber Maternal Body Weight (kg) Maternal Body Weight Gain (kg) Day(s) of Gestation 6 12 19 29 6-11 12-18 19-28 33-6241 6242 6244 6245 6246 6248 6249 6250 6251 6252 6253 6254 6255 6256 6257 4.61 4.06 3.73 3.97 4.47 3.92 3.36 4.12 4.11 4.82 3.85 4.30 3.86 3.70 4.21 4.19 3.98 3.71 3.85 4.47 3.85 3.18 4.03 3.63 4.68 3.58 4.00 3.74 3.62 4.06 4.00 3.87 3.74 3.72 4.61 3.85 3.16 4.07 3.32 4.59 3.52 3.80 4.00 3.63 4.05 4.40 4.20 4.05 3.99 4.69 4.05 3.19 4.24 3.78 4.80 3.63 4.10 4.01 3.73 4.23 -0.42 -0.08 -0.02 -0.12 0.00 -0.07 -0.18 -0.09 -0.48 -0.14 -0.27 -0.30 -0.12 -0.08 -0.15 -0.19 -0.11 0.03 -0.13 0.14 0.00 -0.02 0.04 -0.31 -0.09 -0.06 -0.20 0.36 0.01 -0.01 0.40 0.33 0.31 0.27 0.08 0.20 0.03 0.17 0.46 0.21 0.11 0.30 0.01 0.10 0.18 MEAN+SD 4.07i0.38 3.900.37 3.86+0.40 4.07+0.40 -0.17+0.14* -0.04+0.14* 0.21+0.13* Maternal Liver Weight (Day 29] Absolute Relative () (g/kg Body Weight) 116 87 111 101 116 110 73 142 113 133 HO 133 91 90 112 26.36 20.71 27 .41 25.31 24.73 27.16 22.88 33.49 29.88 27.71 ND 32.44 22.69 24.13 26.48 109+19 26.53+3.61 ND no data available. See Table for summary data. *Significantly different from the concurrent control value by a one-way analysis of variance, p<0.05. 4 TABLE A-5 INDIVIDUAL LITTER SUMMARY OF OBSERVATIONS MADE AT THE TIME OF CESAREAN SECTION OF RABBITS INHALING VINYLIDENE CHLORIDE Exposure Level: 0 ppm (Concurrent Controls for 80 ppm) Animal Numb er Corpora Lutea Implanta tions Pregnancy Wastage Resorptions 33-6431 6432 6433 6434 6435 6436 6437 6438 6447 6448 6449 6450 6451 6452 6466 6467 MEAN+SD 99 00 10 10 00 10 11 03 12 12 01 54 12 98 00 99 00 Delivered on day 28 of gestation. 88 00 99 00 88 02 Dead on day 16 of gestation; pneumonia-related. 11 11 00 10 10 01 11 10 13 99 00 92 9+2 0.110.4 0.811.2 Litter Size 9 10 8 11 2 8 9 8 9 6 11 9 7 9 8+2 Sex Ratio M:F 1:8 4:6 3:5 5:6 2:0 4:4 2:7 Fetal Body Weight <g> 42.5 34.4 37.0 33.4 44.4 36.2 34.0 6:2 38.1 7:2 38.6 3:3 32.4 6:5 36.7 7:2 40.9 5:2 32.7 5:4 34.4 60:56 36.813.7 Crown-Rump Length (mm) 98.3 94.1 95.9 91.2 99.2 94.0 89.0 96.4 96.4 91.8 94.5 97.5 93.2 93.5 94.612.8 TABLE A-5 (Continued) Exposure Level: 80 ppm Animal Number Corpora Lutea Implantations Pregnancy Wastage Resorptions Litter Size Sex Ratio M:F 33-6409 6410 6411 6412 6413 6414 6415 6416 6417 6418 6419 6420 6421 6422 6423 6424 6425 6426 6427 6428 6429 6430 MEAN1SD 10 10 0 1 9 5:4 12 11 1 1 10 6:4 99 77 99 14 11 99 0 1 8 7:1 0 0 7 3:4 0 0 9 3:6 3 1 10 5:5 0 2 7 5:2 Delivered on day 28 of gestation. 12 12 0 1 11 6:5 Dead on day 23 of gestation; enteritis-related; pregnant. 79 99 12 11 0 2 7 4:3 0 0 9 5:4 1 3 8 6:2 88 99 78 10 11 0 0 8 6:2 0 1 8 4:4 0 0 8 6:2 0 0 11 2:0 Non-pregnant. 78 0 0 8 4:4 99 0 0 9 4:5 Implantation sites detected only after sodium sulfide staining. 88 0 0 8 6:2 92 911 0.310.8 0.710.9 911 87:68 Fetal Body Weight (g) 40.2 36.7 41.4 34.5 39.54 33.6 30.6 33.7 47.0 40.3 34.2 32.1 42.6 29.6 29.4 38.4 38.1 42.6 36.915.0 Crown-Rump Length (mm) 93.2 91.8 94.4 91.3 94.2 90.2 87.4 93.8 98.2 99.8 91.6 91.3 97.7 85.9 87.9 97.3 96.3 99.3 93.414.1 TABLE A-5 (Continued) Exposure Level: 0 ppm (Concurrent Controls for 160 ppm) Animal Number Corpora Lutea Implantations Pregnancy Wastage Resorptions Litter Size Sex Ratio M:F 33-6298 6299 6300 6301 6302 6303 6304 6305 6318 6319 6320 6321 6322 6323 6324 6325 MEANSD 95 85 88 4 05 3 05 0 08 Non-pregnant. Non-pregnant. 99 12 12 0 27 0 1 11 99 0 09 12 11 1 0 11 Dead on day 25 of gestation; pneumonia-related; pregnant. 10 14 0 0 14 68 11 11 89 12 12 72 0 08 0 0 11 0 09 0 0 12 5 11 3:2 2:3 4:4 4:3 8:3 4:5 7:4 6:8 7:1 5:6 2:7 5:7 1:0 92 93 1.0+1.8 0.30.6 83 58:53 Fetal Body Weight (g) 36.6 43.3 36.6 45.1 28.3 40.1 30.7 25.6 32.2 33.3 39.4 42.3 53.3 37.47.6 Crown-Rump Length (mm) 91.9 93.8 95.6 101.0 88.2 99.8 87.4 80.8 89.3 89.8 98.8 95.9 101.7..x 93.46.2 SL 083350 SL 083351 TABLE A-5 (Continued) Exposure Level: 160 ppm Animal Number Corpora Lutea Imp lantations Pregnancy Wastage Resorptions Litter Size Sex Ratio M: F 33-6241 6242 6243 6244 6245 6246 6247 6248 6249 6250 6251 6252 6253 6254 6255 6256 6257 6258 MEAN+SD 12 12 0 9 3 1:2 10 8 2 0 8 6:2 Dead on day 22 of gestation; perimetritis-related; preeggnnaanntt. 12 11 1 10a 8:2 10 10 0 10 5:5 99 0 8 3:5 Non-pregnant; purulent inflammation of the ovaries 11 11 76 00 10 11 5:6 6 4:2 9 9 0 2 7 3:4 8 6 2 60 10 8 2 2 6 5:1 0 9 0 90 7 11 88 87 0 11 0 0 0 8 3:5 1 0 7 2:5 7 7 0 0 7 3:4 Dead on day 28 of gestation; pneumonia-related; preeggnnaanntt. 8 3 92 0.60.8 2.713.9 * 6+4 48:43 Fetal Body Weight (g) 32.6 40.3 35.4 36.4 34.2 25.2 38.24 34.5 40.6 38.4 38,9 37.1 36.0+4.2 Crown-Rump Length (mm) 85.8 96.5 95.4 93.6 90.6 82.6 91.6 93.2 96.0 97.6 97.7 94.6 92.9+4.7 at the time of cesarean section. *Significantly different from the concurrent control value by a one-way analysis of variance, p<0.05. * -< * 1 TABLE A-6 INDIVIDUAL LITTER SUMMARY OF FETAL ALTERATIONS AMONG LITTERS OF RABBITS EXPOSED TO VINYLIDENE CHLORIDE BY INHALATION Exposure Level; 0 ppm (Concurrent Control for 80 ppm) Animal Numbers; EXTERNAL EXAMINATION SOU- TISSUE EXAMINATION SKELETAL EXAMINATION Total Maior Malformations 33-6431. 32, 33, 39, 35, 36, 37, 47, 98, 49. 51, 52, 66, 67 Number of Fctuei Examined Total Fetuses Total Litters 9 10 8 11 2 a 9 8 9 6 11 9 7 9 3334 23 33334334 9 10 8 ii 2 a 9 8 9 6 u 9 7 9 116 44 116 14 14 Number of Fetuse* Aff acted 000 00 000000000 0 0 External Examination Edema - neck and abdominal region 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Soft Tissue Examination Dilated cerebral ventricles Heart - thinning of right ventricular wall Satellite vessel off iorta Satellite vessel off Innominate artery FUsed left carotid ami innominate artery Kidney - cortical indentation unilateral 00 0000 00000000 00000 000000000 0 0 0 0 0 0 0 0 0 0 0 0 .0 0 2 0 001 i 00000000 31 341 3 1 1 3 24104 0 00 0 0 0 0 0 0 000 0 0 0 0 0 4 31 0 0 0 3 13 0 Skeletal Examination Skull - delayed ossification -frontal ridge -foramen, frontal -foramen, parietal -extra suture lines, parietal -bone island, parietal -extra site of ossification -between occipital condyles and atlas Vertebrae * hemivertebrae thoracic region with corresponding fused or forked rib & misshapen centra -extra and misshapen left Vertebrae -extra cervical vertebrae Ribs - ll ribs on Left 6 12 ribs on right -shortened 1st thoracic -13th ribs - bilateral -13th rib - unilateral -lumbar spurs rib Sternvbrae - delayed ossification of 2nd sternebra -delayed ossification of 5th -delayed ossification of 6th -unfu^ed -lust'd -extra site of ossification between Sth 6 6th Linbs - delayed ossification of phalanges in first digit, bilateral 0 0 0 0 00 00 00 11 56 13 19 10 84 33 46 12 36 11 11 11 SL 083352 TABLE A*6 (Contlwed) Exposure Lsvsl: 60 ppm Animal Numbers; EXTERNAL EXAMINATION SOFT TISSUE EXAMINATION SKELETAL EXAMINATION Total Malor Malformations Total Total 33-6409, 10, 11. 12. 13, 14, 15. 17, 19, 20, 21, 22. 23, 24, 25, 27, 28, 30 fetuses Litters Number of Fetuaea Examined 9 10 8 7 9 10 7 11 7 9 8 8 8 8 11 8 9 8 333 33 33 4 33 3 3 3 343 3 3 9 10 8 7 9 10 7 11 7 9 8 8 8 8 11 8 9 8 155 56 155 16 18 18 Number of Fetuaea Affected 001 000000000i001 00 3 3 External Examination Edema * neck and abdominal region 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Soft Tissue Examination Dilated cerebral ventricles Heart - thinning of right ventricular wall Satellite vessel off aorta Satellite vessel off Innominate artery Fused left carotid and Innominate arteries Kidney - carotid Indentation unilateral 00000000 00000001 00 00000 00 000000000 00 0000000 000000000 0 0 01 0001003 2 011001 01 3 20 03 2 04 2 3 021 2 0 2 23 00 000000 0000000 l 00 i 0 0 11 31 1 1 0 0 8 13 1 Skeletal E>.wiin.it ion Skull - delayed ossification -frontal ridge -foramen, frontal -foramen, parietal -extra suture lines, parietal -bone Island, parietal -extra site of ossification between occipital condyles and atlas 000 0 000 0 000 000 0 000 000000000000000 l 00 00000000 0000000000 000001 1 00000000000 0000 000 0 00 00 000 1 0 0 00000100000000 0000 00000000000 0000000 Vertebrae - hemivertebra# thoracic region with corresponding fused or forked rib and misshapen centra 001000000000000 1 00 -extra and misshapen lumbar vertebrae -extra cervical vertebrae 0000 000 0 0000000 000 0000 0000 0000100 000 Elba ~ 11 ribs on loft 6 12 riba on right -shortened 1st thoracic rib -13th rib - bilateral -13th rib - unilateral -lumbar t=i>urs 0Q0000000 0000000 00 00 00 0 0 0 00 0 0000000 0 2 4 0 3 2 5 1 6 3 4 6 6 6 0 10 5 5 2 1 1 1 2i 00002 101001 10 001 0000 001 0001 0000 Sternebrae - delayed ossification of 2nd sicrni'bra -delayed ossification of 5th 0000000 00000 01 0000 7 2 8 04 5 2 0 00 30 23 2 2 02 -delayed ossification of 6th 0 1 7 6 0 2 4 2 00 1 1 3 01 2 0 1 -unfused 0000000 00000000000 -fused 0000000 1 000002 0000 -extra site of ossification between 5th & 0th 0000 000 00000000000 Limbs - delayed ossification of phalanges in fiebt digit, bilateral 000 000000000000000 0 1 0 2 1 1 0 1 0 1 0 0 70 12 3 1 42 31 0 3 0 0 0 1 0 2 1 1 0 1 0 1 0 0 16 10 3 1 12 12 0 2 0 0 SL 083353 TAnu: A-6 (Continued) Exposure Level: 160 ppn Animal Numbers Total Total 33-6241, 42, 44, 45, 46, 48, 49, 50, 52, 55, 56, 57, 58 Fetuses Litter* EXTERNAL EXAMINATION SOFT TISSUE EXAMINATION SKELETAL' EXAMINATION Total Major Malformaclons Number of Fetuses Examined 3 8 10 10 8 33 3 3 3 3 8 10 10 8 11 6 7 6 8 7 7 0 43 3 3 3 3 3 0 11 6 7 6 8 7 7 0 Number of Fetuses Affected 1 0 0 1 0 00 0 0 0 0 0 0 91 37 91 2 12 12 12 2 External Examination Edema - neck and abdominal region 11 Soft Tissue Examination Dilated cerebral ventricles Heart - thinning of rlgh ventricular wall Satellite vessel off aorta Satellite vessel off Innominate artery Fused left carotid and Innominate arteries Kidney - cortical indentation unilateral 00 0 0 0 0 0 0 0 0 00 0 10 0 0 00 0 0 0 0 00 0 0 00 00 00 00 00 0 0 0 0 0 0 0 0 10 2 0 0 0 0 3 13 2 3 4 3 13 3 1 2 0 00 0 0 0 0 00 0 0 0 0 0 0 1 0 3 29 0 0 1 0 2 12 0 Skeletal Examination Skull - delayed ossifies:ion -frontal ridge -foramen, frontal -foramen, parietal -extra suture lines, p. rletal -bone island, parietal -extra site of ossification betvecn occipital condyles and atlas Vertebrae - hemivertebra: thoracic region with corresponding fused or forked rib and misshapen centra -lumbar vertebrae -extra cervical vertebrae Ribs - 11 ribs on left 4 12 riba on right -shortened 1st thoracic rib -13th ribs - bilateral -13th ribs - unilateral -lumbar spurn Sternebrae - delayed oaslflcation of 2nd aternebra -delayed ossification of 5th sternebrae -delayed ossification or 6th sternebrae -unfused -fused -extra sice of ossification between 5th 4 nth Limbs - delayed oS'.ifiistlon of phalange:: in fitat digit, hi Isle:n1 0 10 0 0 10 0 0 0 0 0 0 0000 000000 000 0000 000000 000 000 0 000 000 000 01 00 000000000 0 10 0 0 0 0 0 0 0 0 0 0 0000 l 00 000000 2 0 0 0 1 1 1 0001 000000000 0000 000 000 000 0000 0000 00 000 0 0 000 0 0 0 0 0 0 0 0 0 0 000 0 0 0 0 0 0 0 0 3 8 232 1 5 5 1 6 5 4 0 0 0 2 10 2 0 1 0 1 1 2 0 0 0 001 1 0 0 0 1 0 0 0 111 0 5 2 7 oi o 0 3 1 0 2 0 0 10 0 ooo 0 0 0 0 0 0 iooo L 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ooo 00 1 0 0 0 0 45 10 3 2 19 21 1 2 0 0 0 () I) oooooo 0 0 li 2 0 0 0 1 1 1 1 0 0 0 0 12 7 3 2 7 7 1 2 1 0 *Signlflcently different trum the coni nrreni eonirol value by u uoc-wny analysis of variance, p<0.05. SL 083354 TABLE A-6 (Continued) Exposure Level: 0 ppm (Concurrent Control for 160 ppm) Animal Numbers; EXTERNAL EXAMINATION SOFT TISSUE EXAMINATION SKELETAL EXAMINATION Totsl Major Malformations External Examination Edema - neck and abdominal region Soft Tiasue Examination Dilated cerebral ventricles Heart - thinning of right ventricular vail Satellite vessel off aorta Satellite vessel off innominate artery Fused loft carotid and Innominate entries Kidney - cortical Indentation unilateral Skeletal Examination Skull - delayed ossification -frontal ridge -foramen, frontal -foreman, parietal -extra suture lines, parietal "bone island, parietal -extra site of ossification between occipital condyles and atlas Vertebrae - hemivertebras thoracic region with corresponding fuaed or forked rib & misshapen centra -extra and misshapen lumbar vertebrae -extra cervical vertebrae Riba - 11 ribs on left 1 12 ribs on right -shortened 1st thoracic rib -13th ribs - bilateral -13th ribs - unilateral -lumbar spurs Stcrnebroc - delayed ossification Of 2nd sLornebra -delayed ossification of 5th sternebrac -delayed Omh 1 fLeal loo of 6tli Bturncbnic -unfurl'd -fused -extra site of ossification between 5th A 6th Limbs - delayed ossification of phalanges in first digit, bilateral 6296, Total Total 300, 03, 06, 05,, 18,, 20. 21. 22, 23, 24, 25 Fatuses Litters Numbtr of Fatuses Examined 55 33 5s 8 7 11 9 ii 14 8 11 9 12 1 334345 34 341 8 7 11 9 11 14 8 ii 9 12 1 in 43 111 13 13 13 Nimbr of rotUMO Affsctsd 00 00000110000 2 2 00 00000000000 0 0 00 Q0 00 11 12 00 00000 0000 00 0 0000000000 00103 000000 0201 3 1 3001 0 03 1 24 234 300 00000000000 0 0 4 13 25 0 0 0 2 8 10 0 00 00 00 00 00 00 000002 00000 0 0000 0 0 000 0 00000 200000 001 000001 00 00000 000000 000 00 000 0 00 00 0 0000 000 00 0 2 0 2 2 0 0 0 1 0 1 2 0 0 0 00 00 00 00 00 04 00 00 00000000000 0 000 0 01 0000 0000 0000000 00000 1 00000 0000 0000000 01 252603560 10320201 1 10 003 1 000201 0 0 1 0 1 0 34 11 7 0 1 0 1 0 9 7 4 54 12 00 00 00 A 5904 14 2 330 0 3 1 05 3 31400 0 00 00 0 0000 0 00 000 000 0 00 00000000000 44 23 0 0 0 00 000 00 00 000 0 0 11 9 0 0 0 0 SL 083355 -7- On the day of cesarean section, the females were sacrificed by carbon dioxide inhalation; the uterine horns were ex teriorized through a mid-line incision in the abdominal wall, and the number and position of live, dead, and resorbed fetuses were noted. The uteri of apparently non-pregnant animals were stained with a 10% solution of sodium sulfide and examined for evidence of early resorption sites (Kopf et al. , 1964); this procedure was conducted for the sole purpose of determining the percentage 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 rat 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 eviscerated and placed in 95% ethanol and were cleared and stained with alizarin red-S (Dawson, 1926) to facilitate examination for skeletal alterations. Statistical Evaluation. The incidences of pregnancy and resorptions were analyzed statistically by the Fisher exact SL 083356