Timed-mated CD® rats, pregnant based on gestational weight gain of greater than 50 g, were exposed to hexachloro-1,3-butadiene (HCBD, CAS No. 87-68-3) at 100, 200, 400, 750, 1100 or 1500 ppm in the diet from gestational day (GD) 17 to postnatal day (pnd) 10, eight or nine dams per dose. Dams were weighed on GD 0, 6, 11, 16, and 17 through pnd 10, and observed twice daily for clinical signs of toxicity. Food and water consumption was measured throughout the exposure period. Dams were allowed to deliver their litters. The day following the delivery of the entire litter was designated pnd 1. On pnd I through 3, litters were evaluated for number, sex and weight of each pup. On pnd 4, pups were weighed and counted, and litter size culled to 10 with equivalent sex ratio if possible. Pups were also weighed and counted on pnd 7 and 10. On pnd 10, one pup/sex/litter from each dose group was selected for collection of urine and serum. Urine samples were evaluated for osmolality. Urine and serum samples were subjected to clinical chemistry determinations. Also on pnd 10, 10 pups (one/sex from each of five litters per dose group) were selected for evaluation of renal competence by collection of urine prior to and after 4 and 6 hours of isolation from the dam. Urine samples were evaluated for osmolality. All pups were euthanized, sex confirmed, and liver and kidneys weighed and retained in fixative for subsequent histopathologic examination. Maternal tests on pnd 10 were as follows. Two to four dams per dose group were randomly selected for hydropenia evaluation by 20 hour (overnight from pnd 10 to pnd 11) fluid deprivation; urine sample pre- and post-deprivation were analyzed for osmolality. Three randomly selected dams per dose group were also selected for milk collection and milk samples were analyzed for HCBD content. All dams were euthanized and livers and kidneys weighed and fixed for subsequent histopathologic examination.
All dams at 1500 ppm and one dam at 200 ppm were sacrificed in extremis on GD 20 - pnd 1; dams and pups at 1100 ppm were also sacrificed in extremis on pnd 1-3. (Kidneys were collected from these animals.) Maternal body weights were reduced in a dose-related pattern at greater than or equal to 100 ppm, during both gestation and lactation. Maternal food consumption was reduced throughout the exposure period at greater than or equal to 200 ppm; maternal water consumption was reduced at greater than or equal to 400 ppm only during lactation. Even though maternal food consumption was reduced, HCBD intake increased with increasing levels in the diet. Dose-related clinical observations included weight loss, excessive urinations, hindlimb weakness, tremors, lethargy, and other non-specific indicators of stress, with only the first sign persisting into the lactation period at 200-1100 ppm and excessive urination persisting only at 750-1100 ppm. Maternal kidney weights were increased at 200-750 ppm (with the renal weights at 1100 and 1500 ppm confounded by maternal status and time of sacriice). Histopathologic findings in maternal kidneys included tubular regeneration at all dose levels with severity dose related, and tubular distention observed at higher doses. Maternal urine osmolality exhibited a dose-related decrease on pnd 10; fluid deprivation did not appear to affect urine-concentrating ability at any dose, based on a limited number of observations. Clinical chemistry data were also minimal with only urinary glucose appearing lower with increasing dose in replete dams.
The effects of HCBD on offspring were confounded by the extreme maternal toxicity observed, including mortality during gestation (at 1500 ppm) and very early in the postnatal period (1100 ppm). In addition, three dams at 750 ppm delivered all dead pups. Postnatal pup mortality appeared increased at 750 ppm through pnd 10. Postnatal pup body weights appeared reduced at all doses by pnd 10 with pups at 400 and 750 ppm emaciated and dehydrated. Comparison of maternal and offspring intake of HCBD, based on maternal dosed feed consumption and HCBD content in the diet versus HCBD content in milk and published estimates of milk intake by pups, indicated that pups consumed 3-7% as much HCBD as consumed by their dams at each dose. Histologic examination of pup kidneys revealed only reduced size and retention of the subcapsular metanephric blastemal zone, indicative of delay in postnatal development and not pathology per se. Osmolality of pup urine was slightly increased without deprivation only in pups selected for hydropenia tests. Clinical chemistry evaluation of pup urine indicated no treatment- related differences; clinical chemistry of pup serum indicated slight dose-related increases in serum glucose and BUN (blood urea nitrogen), with very few samples available. Fluid deprivation resulted in slightly increased urine osmolality in all HCBD treatment groups for both dams and offspring.
In conclusion, administration of HCBD in the diet during the perinatal period (GD 17-pnd 10) in rats resulted in maternal mortality at 1100-1500 ppm, and maternal toxicity at all lower doses tested, 100-750 ppm. The only indications of maternal toxicity observed at 100 ppm were slightly reduced food consumption and histologic renal lesions. Offspring toxicity was observed at all doses with profound effects at 400 ppm and above, confounded by concurrent maternal toxicity. The only indication of offspring toxicity at 100 ppm was slightly reduced body weights. Therefore, a No Observable Adverse Effect Level (NOAEL) was not established for either dams or offspring in this study.