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A commentary in Environmental Health titled “Safety of Safety Evaluation of Pesticides: developmental neurotoxicity of chlorpyrifos and chlorpyrifos-methyl” by Mie et al. [15] erroneously suggests that developmental neurotoxicity studies conducted for chlorpyrifos and chlorpyrifos-methyl and then submitted to regulatory authorities were misleading and impaired the authorities’ ability to perform a valid evaluation. We will address the specific technical points, assertions and assumptions individually and demonstrate why the chlorpyrifos study - conducted more than 20 years ago - was reviewed and accepted by global regulatory authorities, including the U.S. Environmental Protection Agency, Australian Pesticides and Veterinary Medicines Authority, Health Canada Pest Management Regulatory Agency and the European Union. Labeled uses of chlorpyrifos rest on five decades of experience in use, health surveillance of manufacturing workers and applicators, and more than 4000 studies and reports examining the product in terms of health, safety and the environment. We will also respond to the allegations made about the chlorpyrifos-methyl study which was conducted more recently. Brain weight Mie et al. [15] challenge the chlorpyrifos DNT study by stating that “For high dose pups, the test laboratory reported a significant reduction of total brain weight and the dimensions of several brain regions on PND 11, but not on PND 65. The test lab argued that these observed effects do not indicate DNT…To support this interpretation, the test laboratory calculated that the average effect on all brain regions is similar to the effect on brain weight…Accordingly, the EPA has identified this analytical approach as an inappropriate and inconclusive manipulation of the data, but a correction was apparently not requested from the pesticide producer submitting the report.” The methodology used in the DNT studies was sound. The highest dose tested in the chlorpyrifos DNT study produced an appropriate level of maternal toxicity, verifying the use of adequate dose levels in this study. The toxicity observed (reduction in bodyweight gain and food consumption, adverse clinical observations, reduction in cholinesterase levels) did not interfere with the production of offspring but did result in a reduction in pup body weight and as noted by Mie et al. [15], a reduction in brain weight and the size of several brain regions in PND 11 pups but not in PND 65 adult rats. In addition to the reduction in weight and measurements of brain areas, the study design included a functional evaluation (i.e., behavioral assessments) in the offspring post weaning and a neurohistopathological assessment of the brain at two ages (i.e., PND 11 and 65) by a board certified neuropathologist. In the chlorpyrifos study, effects in the high dose pups were only observed on the weights and brain area measurements and these effects were not apparent when the pups grew up (at PND 65). The neurohistopathology of the CNS and peripheral nervous system and behavioral assessments were unaffected at the highest dose tested. When results such as observed in this study are produced, there could be several explanations. What the “testing laboratory was arguing” was supported by previously published articles [5, 6, 10] that demonstrated effects on brain weight and the size of various areas of the brain, without any other evidence of damage to the nervous system, is often due to undernutrition. This undernutrition would be expected in offspring of mothers who were growth retarded and food deprived during gestation. In addition to the literature references, a calculation of the % reduction in brain weight to the % reduction in the size of various brain regions was conducted to further support this theory of undernutrition causing the effects in the highest dose group. The real difference in opinion from the evaluation of the study results was in the interpretation of statistically significant reductions in the size of selected regions of the brain in the lower dose groups on PND 11. These differences were not apparent in the middle dose group of the adult rats (PND 65), the tissues were histologically normal at all doses, and there were no functional deficits at any dose, so it was argued that no developmental neurotoxicity was occurring at any dose level but clearly none was occurring at the middle dose level.

Bis-hexanoyl (R)-1,3-butanediol (BH-BD) is novel ketone ester undergoing development as a food ingredient to achieve nutritional ketosis in humans. Male and female Crl:CD(SD) rats were administered BH-BD twice daily at 9000, 12,000 or 15,000 mg/kg/day, by oral gavage in a 90-day toxicity study with 28-day recovery period; and an interim 28-day phase. Test substance-related early deaths occurred in four females at 15,000 mg/kg/day. A dose-dependent increase in acute transient postdose (1–3 h) observations of incoordination at ≥12,000 mg/kg/day and decreased activity at all dose levels were noted in both sexes. Postdose observations were likely associated with peak ketonemia and were considered adverse at 15,000 mg/kg/day. These daily observations decreased over the study without any persistent effects, as determined during weekly pre-dose observations. Adverse histopathological changes included ulceration/erosion in non-glandular stomach at ≥ 12,000 mg/k/day and in glandular stomach at 15,000 mg/kg/day. These histopathological findings were not noted after 28-days of recovery. Due to unlikely human relevance of the rat non-glandular stomach effects for BH-BD and test substance-related mortality at 15,000 mg/kg/day, the no-observed-adverse-effect level (NOAEL) for subchronic toxicity of BH-BD was determined to be 12,000 mg/kg/day.

Background To protect crewmember health, the U.S. Navy sets exposure limits for more than 200 components of submarine atmospheres. The addition of females to nuclear submarines required a reevaluation of these exposure limits, originally established for all-male crews. In the case of carbon dioxide (CO2 ), the only available data suitable for deriving an exposure limit were from a 2010 study sponsored by the British Royal Navy that reported a debatable interpretation casting doubt on whether current U.S. Navy exposure limits served to protect fetal developmental health. Methods About 120 time-mated female Sprague-Dawley rats (Crl: CD[SD]) were exposed to CO2 at levels of 1.5%, 2.0%, 2.5%, and 3.0% from gestation days 6 to 20. Dams were euthanized and fetuses were examined. Results Findings with implications for exposure limits for CO2 during pregnancy were an increased mean litter proportion of early resorptions and a lower mean litter proportion of viable fetuses in the 3.0% CO2 group. Conclusion The results yield a No Observed Adverse Effect Level (NOAEL) of 2.5% and a Lowest Observed Adverse Effect Level (LOAEL) of 3.0%. The results reasonably allow a point of departure of 2.5% CO2 for deriving an exposure recommendation. An interspecies uncertainty factor was applied to derive a recommended 90-day continuous exposure limit (CEL) of 0.8% for CO2 . As reproductive endpoints that are developmental in nature must be assumed to result from a single exposure at a critical point during gestation, it is further recommended that the 24-hr emergency exposure limit (EEL) also be 0.8%.

Histopathologic examination of the testis from juvenile rats is often necessary to characterize the safety of new drugs for pediatric use and is a required end point in male pubertal development and thyroid function assays. To aid in evaluation and interpretation of the immature testis, the characteristic histologic features of the developing rat testis throughout postnatal development are described and correlated with published neuroendocrine parameter changes. During the neonatal period (postnatal day [PND] 3–7), seminiferous tubules contained gonocytes and mitotically active immature Sertoli cells. Profound proliferation of spermatogonia and continued Sertoli cell proliferation occurred in the early infantile period (PND 8–14). The spermatogonia reached maximum density forming double-layered rosettes with Sertoli cells in the late infantile period (PND 15–20). Leptotene/zygotene spermatocytes appeared centrally as tubular lumina developed, and individual tubules segregated into stages. The juvenile period (PND 21–32) featured a dramatic increase in number and size of pachytene spermatocytes with the formation of round spermatids and loss of “infantile” rosette architecture. In the peri-pubertal period (PND 32–55), stage VII tubules containing step 19 spermatids were visible by PND 46. The presented baseline morphologic and endocrinologic information will help pathologists distinguish delayed development from xenobiotic effects, determine pathogenesis when confronted with nonspecific findings, and identify sensitive time points for targeted study design.

The male and female pubertal assays, which are included in the U.S. Environmental Protection Agency's (EPA) Endocrine Disruptor Screening Program (EDSP) Tier 1 battery, can detect endocrine-active compounds operating by various modes of action. This article uses the collective experience of three laboratories to provide information on pubertal assay conduct, interlaboratory reproducibility, endpoint redundancy, and data interpretation. The various criteria used to select the maximum tolerated dose are described. A comparison of historical control data across laboratories confirmed reasonably good interlaboratory reproducibility. With a reliance on apical endpoints, interpretation of pubertal assay effects as specifically endocrine-mediated or secondary to other systemic effects can be problematic and mode of action may be difficult to discern. Across 21–23 data sets, relative liver weight, a nonspecific endocrine endpoint, was the most commonly affected endpoint in male and female assays. For endocrine endpoints, patterns of effects were generally seen; rarely was an endocrine-sensitive endpoint affected in isolation. In males, most frequently missed EPA-established performance criteria included mean weights for kidney and thyroid, and the coefficient of variation for age and body weight at preputial separation, seminal vesicle weight, and final body weight. In females, the frequently missed EPA-established performance criteria included mean adrenal weight and mean age at vaginal opening. To ensure specificity for endocrine effects, the pubertal assays should be interpreted using a weight-of-evidence approach as part of the entire EDSP battery. Based on the frequency with which certain performance criteria were missed, an EPA review of these criteria is warranted.

Recently, there has been a renewed interest in the use of the minipig as an alternative to dogs and non-human primates for conducting toxicological assessments in non-rodent species. Since the T-dependent antibody response (TDAR) is one of the most widely-accepted assays used in the assessment of immunocompetence, the present study was undertaken to characterize the primary and secondary TDAR to keyhole limpet hemocyanin (KLH) in the Göttingen Minipig(®). Following primary immunization with either 2 or 10 mg KLH, anti-swine IgM and IgG ELISAs were optimized and individual animal responses were evaluated over time. Immunization with 10 mg KLH on Day 0 promoted primary IgM responses that peaked 6-9 days after antigen administration, while primary IgG levels peaked on Day 13 or 14. Secondary IgG antibody levels (following secondary injection with 2 mg KLH on Day 14) plateaued on Days 20-22. Anti-KLH antibody levels were decreased in minipigs treated with cyclophosphamide (CPS), a known immunosuppressant, at doses ranging from 12.5-50 mg/kg/day, while antibody levels in animals treated with 2.5 mg CPS/kg/day were similar to levels in saline-treated swine. These results demonstrate that the Göttingen Minipig(®) can be a useful alternative non-rodent species to the dog and the non-human primate for evaluating the TDAR to KLH in regulatory assessments of immunotoxicity.

BACKGROUND: Decabromodiphenyl ether (DecaBDE; CASRN 1163-19-5) is a flame retardant used in a variety of manufactured products. A single oral dose of 20.1 mg/kg administered to mice on postnatal day 3 has been reported to alter motor activity at 2, 4, and 6 months of age. METHODS: To further evaluate these results, a developmental neurotoxicity study was conducted in the most commonly used species for studies of this type, the rat, according to international validated testing guidelines and Good Laboratory Practice Standards. DecaBDE was administered orally via gavage in corn oil to dams from gestation day 6 to weaning at doses of 0, 1, 10, 100, or 1,000 mg/kg/day. Standard measures of growth, development, and neurological endpoints were evaluated in the offspring. Motor activity was assessed at 2 months of age. Additional motor activity assessments were conducted at 4 and 6 months of age. Neuropathology and morphometry evaluations of the offspring were performed at weaning and adulthood. RESULTS: No treatment-related neurobehavioral changes were observed in detailed clinical observations, startle response, or learning and memory tests. No test substance-related changes were noted in motor activity assessments performed at 2, 4, or 6 months of age. Finally, no treatment-related neuropathological or morphometric alterations were found. CONCLUSIONS: Under the conditions of this study, the no-observed-adverse-effect level for developmental neurotoxicity of DecaBDE was 1,000 mg/kg/day, the highest dose tested. Birth Defects Res (Part B) 92:17–35, 2011.© 2011 Wiley-Liss, Inc.

The potential for neurotoxicological and immunotoxicological effects of ethylbenzene was studied in young adult Crl:CD(SD) rats following 90-day oral (neurotoxicity) or 28-day inhalation (immunotoxicity) exposures. In the neurotoxicity study, ethylbenzene was administered orally via gavage twice daily at 0, 25, 125, or 250 mg/kg per dose (total daily dosages of 0, 50, 250, or 500 mg/kg bwt/day [mg/kg bwt/day]) for 13 weeks and the functional observational battery (FOB), automated tests for motor activity and neuropathological examination were conducted. In the immunotoxicity study, animals were exposed by inhalation to 0, 25, 100, or 500 ppm ethylbenzene (approximately 26, 90, or 342 mg/kg bwt/day as calculated from physiologically based pharmacokinetic modeling). Immunotoxicity was evaluated in female rats using the splenic antibody-forming cell plaque-forming assay in sheep red blood cell sensitized animals. The no-observed-effect level for the oral gavage study was 50mg/kg bwt/day based on increased relative weights of the liver and kidneys in the male rats. The no-observed-adverse-effect level (NOAEL) for adult neurotoxicity was the highest dose tested 500 mg/kg bwt/day. The NOAEL for the immunotoxicity evaluation was the highest tested exposure concentration, 500 ppm (342 mg/kg bwt/day).

Perfluorooctanesulfonate (PFOS), a persistent and bioaccumulative compound, is widely distributed in humans and wildlife. Exposure of the human fetus and neonate to PFOS can occur in utero and via the mother's milk, respectively. Developmental studies have been conducted with PFOS in the past, including some developmental neurotoxicity endpoints. The objective of this study was to evaluate the functional and morphological changes to the nervous system in rats having gestational and lactational exposures to PFOS per current test guidelines (EPA OPPTS 870.6300 and OECD 426). Female SD rats (25/dosage group) were given daily oral doses of either 0.0, 0.1, 0.3, or 1.0mg/kg-d potassium PFOS (K(+)PFOS) from gestation day (GD) 0 through postnatal day (PND) 20. Offspring were observed through PND 72 for growth, maturation, motor activity, learning and memory, acoustic startle reflex, various behavioral manifestations, and brain weight. Specimens were taken from dams, fetuses, and pups for serum and tissue PFOS concentration, thyroid status endpoints, and liver mRNA transcript analysis, and those results are reported in a companion article. No significant effect was noted on maternal health or reproductive outcomes from dosing of maternal rats with K(+)PFOS throughout gestation. Maternal body weights were statistically significantly lower in the 1.0mg/kg-d dosage group from PND 4 through the end of lactation. Offspring from K(+)PFOS-treated maternal groups did not differ significantly from controls with respect to birth weight, growth, age and weight at attainment of sexual maturation, learning and memory, acoustic startle, various behavioral endpoints, and brain weight. Male offspring from the 1.0mg/kg-d maternal treatment group displayed increased motor activity and reduced habituation on PND 17 but not on PND 13, 21, and 61. The maternal no-observed-adverse-effect-level (NOAEL) was 0.3mg/kg-d based on decreased body weights observed in lactation. The maternal dose associated with the NOAEL for male offspring was 0.3mg/kg-d based on increased motor activity and reduced habituation in the 1.0mg/kg-d maternal dose-group male offspring on PND 17. The maternal dose associated with the NOAEL for female offspring was >1.0mg/kg-d. Mean serum concentrations of PFOS reported in a companion article for the 0.3mg/kg-d group maternal rats are several hundred times higher than those reported for females in the United States general population.

Diacylglycerol (DAG) oil is a novel edible oil with similar taste and usability characteristics as conventional edible oils. Recent studies suggest that DAG oil may be helpful in the prevention and management of obesity. The objective of the present two-generation study was to evaluate potential adverse effects of DAG oil on reproductive processes. DAG oil was administered via gavage to rats (30/sex/group) for at least 70 days prior to mating, at dose levels of 0, 1.25, 2.5 or 5.0 ml/kg/day (0, 1160, 2320 and 4630 mg/kg/day). An additional group received a triacylglycerol (TAG) oil with a similar fatty acid composition to DAG oil. The rats were treated throughout the mating, gestation and lactation periods. Administration of DAG or TAG oil did not reveal any toxicologically significant effects on reproductive performance (mating, fertility and copulation/conception indices). DAG oil did not affect mean gestation lengths, the process of parturition, spermatogenic parameters, organ weights, histopathologic findings, mean numbers of pups born, implantation sites and unaccounted sites. F1 and F2 pup viability, live litter sizes, body weights, mean age of attainment of balanopreputial separation and vaginal patency were similar to those in the control group. Based on the results of this study, a dose level of 5.0 ml/kg (4630 mg/kg/day) was considered as the no-observed-adverse-effect level for reproductive and systemic toxicity, and neonatal toxicity.

BACKGROUND: This study was conducted to evaluate the potential adverse effects of di-2-ethylhexyl terephthalate (DEHT) on reproductive capability from exposure of F0 and F1 parental animals. METHODS: Four groups of male and female Crl:CD (SD)IGS BR rats (30/gender/group) were exposed to 0, 0.3%, 0.6%, and 1.0% DEHT in the feed for at least 70 consecutive days before mating for the F0 and F1 generations. Exposure for the F0 and F1 males continued throughout the mating period until euthanasia. Exposure for the F0 and F1 females continued throughout mating, gestation, and lactation. The F1 and F2 pups were weaned on postnatal day (PND) 21. Assessments included gonadal function, estrous cyclicity, mating behavior, conception rate, gestation, parturition, lactation, and weaning in the F0 and F1 generations, and F1 generation offspring growth and development. RESULTS: DEHT exposure did not affect clinical observations. However, lethality was observed in F0 and F1 dams consuming the 1.0% diet during the post-weaning period. No treatment-related mortality occurred in any of the male groups exposed to DEHT or in the female groups exposed to 0.3% or 0.6% DEHT. Male rats consuming the 1.0% diet in both parental generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either the F0 or F1 generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, reproductive organ weights, lengths of estrous cycle and gestation, live litter size, developmental landmarks, and postnatal survival were similar in all exposure groups. Additionally, ovarian follicle counts for the F1 females in the high-exposure group were similar to the control values. No adverse exposure-related macroscopic pathology was noted at any exposure level in the F0 and F1 generations. CONCLUSIONS: Increases in liver weights were found in the male and female animals exposed to 0.6% or 1.0% DEHT in the diet. Because there were no accompanying histopathologic changes, this effect was not considered adverse. Significant decreases in feed consumption in the female animals from the groups consuming 1.0% DEHT in the diet during lactation accompanied reduced postnatal pup body weights and rate of weight gain. Reductions in pup body weights later in lactation may also have been due to direct consumption of the treated feed by the pups or taste aversion to the same. Reduced relative spleen weight was found in male weanling pups from the 1.0% group in both generations and reduced relative spleen and thymus weights were found in female pups from the 1.0% group in the F2 generation at necropsy on PND 21. Therefore, for parental and pup systemic toxicity, 0.3% DEHT in the diet (182 mg/kg/day) was considered no-observed-effect level (NOEL). The 1.0% DEHT (614 mg/kg/day) in the diet exposure concentration was considered a NOEL for F0 and F1 reproductive toxicity endpoints. Birth Defects Res (Part B), 2007. © 2007 Wiley-Liss, Inc.

This two-generation reproduction study assessed the reproductive hazard potential of decamethylcyclopentasiloxane (D(5)). Sprague-Dawley rats (30/sex/group) were exposed by whole-body vapor inhalation to a target concentration of 30, 70, or 160 ppm D(5) or filtered air for 6h/day. Exposures for the F(0) and F(1) generations started at least 70 days prior to mating and lasted through weaning of the respective pups on postnatal day (PND) 21. Female exposures were interrupted from gestation day (GD) 21 through PND 4 to allow for parturition and to permit continuous maternal care for the early neonates. F(2) pups were not directly exposed to D(5). There were no exposure-related mortalities, clinical signs of toxicity, or effects on body weight or food consumption. There were no treatment-related gross findings or organ weight effects at the F(0) and F(1) necropsies. Other than minimal alveolar histiocytosis in all exposed groups, there were no noteworthy microscopic findings. Reproductive parameters (number of days between pairing and mating, mating and fertility indices, gestation length, and parturition), spermatogenic parameters and ovarian primordial follicle counts and numbers of corpora lutea in the F(0) and F(1) parental animals were not significantly changed between treated and control groups. Mean live litter sizes, number of pups born, sex ratios, pup body weights, postnatal pup survival and general physical condition of offspring in each generation were not affected. The slight, but statistically significant, increase in the mean F(1) male pup AGD in the 160 ppm group was not considered to be related to treatment. Vaginal patency and balanopreputial separation were unchanged compared to controls. Thus, the No-Observed-Adverse-Effect-Level (NOAEL) for parental and reproductive toxicity was determined to be 160 ppm D(5).

This study evaluated the potential toxicity of whole-body vapor inhalation of octamethylcyclotetrasiloxane (D(4)) on reproductive capabilities in exposed F(0) and F(1) parental animals and the potential effects on neonatal survival, growth, and development of the F(1) and F(2) offspring. F(0) male and female Sprague-Dawley rats (30/sex/group) were exposed to D(4) vapor at concentrations of 0, 70, 300, 500 or 700 ppm 6h per day for at least 70 consecutive days prior to mating and lasted through weaning of the pups on postnatal day (PND) 21. Female exposures were suspended from gestation day (GD) 21 through PND 4 to allow for parturition and permit continuous maternal care for the early neonates. Starting on PND 22, F(1) weanlings were exposed to D(4) as described for the F(0) generation. The F(2) pups were not directly exposed to D(4). F(0) animals were mated once to produce the F(1) generation; F(1) parental animals were mated twice to produce two F(2) litters. In addition, the F(1) males were mated with unexposed females. Prolonged estrous cycles, decreased mating and fertility indices were observed in the F(1) generation exposed to D(4) for the first and second matings. Significant reductions in the mean number of pups born and mean live litter size were observed in the 500 and 700 ppm groups for both the F(0) and F(1) generations. Implantation sites were also reduced at 700 ppm for both F(0) and F(1) generations. No adverse effects were observed at any exposure level on anogenital distance, vaginal patency and preputial separation. No adverse effects were seen on male functional reproductive parameters, spermatogenic endpoints, microscopic evaluation of male reproductive tissue, or when the D(4)-exposed F(1) males were mated with the unexposed females, demonstrating that the reproductive toxicity observed was due to D(4) exposure to the females. Based on the lack of effect on reproduction when the D(4)-exposed males were mated to näive females, the NOAEL for male reproductive toxicity was considered to be 700 ppm. Based on the statistically significant effects on fertility and litter size, NOAEL for female reproductive toxicity was considered to be 300 ppm. The findings observed in this study are consistent with suppression or delaying of LH surge as well as acceleration of the onset of female reproductive senescence in the rat. While analogous pathways control ovulation in both rats and humans, there are significant differences in the mechanism for timing and release of LH and resulting changes in the control of ovulation and mating behavior between the two species. If D(4) delays rather than causes a prolonged suppression or ablation of the LH surge, the reproductive mode of action of D(4) would not likely be relevant for humans.

BACKGROUND: This study was conducted to evaluate the potential adverse effects of whole-body inhalation exposure of F0 and F1 parental animals from a 2-generation reproduction study of ethylbenzene on nervous system functional and/or morphologic end points in the F2 offspring from four groups of male and female Crl:CD® (SD)IGS BR rats. METHODS: Thirty rats/sex/group for F0 and 25/sex/group for F1 were exposed to 0, 25, 100, and 500 ppm ethylbenzene for six hours daily for at least 70 consecutive days prior to mating for the F0 and F1 generations. Inhalation exposure for the F0 and F1 females continued throughout mating and gestation through Gestation Day (GD) 20. On lactation days (LD) 1–4, the F0 and F1 females received no inhalation exposure, but instead were administered ethylbenzene in corn oil via oral gavage at dosages estimated to result in similar internal maternal exposure based upon PBPK modeling estimates (0, 26, 90, and 342 mg/kg/day, respectively, divided into three equal doses, approximately two hours apart). Inhalation exposure of the F0 and F1 females was reinitiated on LD 5 and continued through weaning on postnatal day (PND) 21. Survival, body weights, and physical landmarks were assessed in selected F2 offspring. Neurobehavioral development of one F2-generation treatment derived offspring/sex/litter was assessed in a functional observational battery (FOB; PND 4, 11, 22, 45, and 60), motor activity sessions (PND 13, 17, 21, and 61), acoustic startle testing (PND 20 and 60), a Biel water maze learning and memory task (initiated on PND 26 or 62), and in evaluations of whole-brain measurements and brain morphometric and histologic assessments (PND 21 and 72). RESULTS: There were no adverse effects on reproductive performance in either the F0 or F1 parental generations exposed to up to 500 ppm ethylbenzene [Faber et al. Birth Defects Res Part B 77:10–21, 2006]. In the current developmental neurotoxicity component, parental ethylbenzene exposure did not adversely affect offspring survival, clinical condition, body weight parameters, or acquisition of developmental landmarks of the F2-generation treatment derived offspring. There were no alterations in FOB parameters, motor activity counts, acoustic startle endpoints, or Biel water maze performance in offspring attributed to parental ethylbenzene exposure. A few isolated instances of statistically significant differences obtained in the treatment-derived groups occurred sporadically, and were attributed to unusual patterns of development and/or behavior in the concurrent control group. There were no exposure-related differences in any neuropathology parameters in the F2-generation treatment derived offspring. CONCLUSIONS: The no observed adverse effect level (NOAEL) for maternal reproductive toxicity, developmental toxicity, and developmental neurotoxicity in this study was considered to be 500 ppm/342 mg/kg/day ethylbenzene, the highest exposure level tested in the study. Birth Defects Res (Part B) 80:34–48, 2007. © 2007 Wiley-Liss, Inc.

Rodents form an early inverted yolk sac placenta (invYSP) by apposing the yolk sac to the uterine wall. The invYSP supplies nutrients via histiotrophic nutrition involving pinocytosis of materials from uterine gland secretions, lysosomal degradation, and transfer of the products to the embryo. Interference with histiotrophic trafficking through the invYSP by high-molecular-weight molecules (such as trypan blue) causes malformations and resorptions. Later in gestation, rodents form a definitive chorioallantoic placenta (CAP). By contrast, humans and dogs never develop an invYSP, relying exclusively on the CAP. Given their large size (approximately 250 kD), hemoglobin-based oxygen carriers (HBOC), being developed as blood substitutes, could be expected to interfere with histiotrophic trafficking through the invYSP. During initial toxicity testing, intravenous infusions of HBOC caused pronounced developmental toxicity in rats exposed during the pre-CAP period. Assuming that HBOC interfered with invYSP function, we hypothesized that these findings would not apply to humans or dogs, which lack an invYSP. Subsequent extensive developmental toxicity studies in dogs produced no developmental toxicity after intravenous infusion at the maximum tolerated dose. In view of the existing species-specific placental differences and HBOC's demonstrated, exclusive interference with invYSP histiotrophic nutrition, HBOC is not expected to cause abnormal development in humans or other mammals that do not develop an invYSP.

This study was conducted to assess potential adverse functional and/or morphological effects of styrene on the neurological system in the F2 offspring following F0 and F1 generation whole-body inhalation exposures. Four groups of male and female Crl:CD (SD)IGS BR rats (25/sex/group) were exposed to 0, 50, 150, and 500 ppm styrene for 6 hr daily for at least 70 consecutive days prior to mating for the F0 and F1 generations. Inhalation exposure continued for the F0 and F1 females throughout mating and through gestation day 20. On lactation days 1 through 4, the F0 and F1 females received styrene in virgin olive oil via oral gavage at dose levels of 66, 117, and 300 mg/kg/day (divided into three equal doses, approximately 2 hr apart). Inhalation exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through weaning of the F1 or F2 pups on postnatal day (PND) 21. Developmental landmarks were assessed in F1 and F2 offspring. The neurological development of randomly selected pups from the F2 generation was assessed by functional observational battery, locomotor activity, acoustic startle response, learning and memory evaluations, brain weights and dimension measurements, and brain morphometric and histologic evaluation. Styrene exposure did not affect survival or the clinical condition of the animals. As expected from previous studies, slight body weight and histopathologic effects on the nasal olfactory epithelium were found in F0 and F1 rats exposed to 500 ppm and, to a lesser extent, 150 ppm. There were no indications of adverse effects on reproductive performance in either the F0 or F1 generation. There were exposure-related reductions in mean body weights of the F1 and F2 offspring from the mid and high-exposure groups and an overall pattern of slightly delayed development evident in the F2 offspring only from the 500-ppm group. This developmental delay included reduced body weight (which continued through day 70) and slightly delayed acquisition of some physical landmarks of development. Styrene exposure of the F0 and F1 animals had no effect on survival, the clinical condition or necropsy findings of the F2 animals. Functional observational battery evaluations conducted for all F1 dams during the gestation and lactation periods and for the F2 offspring were unaffected by styrene exposure. Swimming ability as determined by straight channel escape times measured on PND 24 were increased, and reduced grip strength values were evident for both sexes on PND 45 and 60 in the 500-ppm group compared to controls. There were no other parental exposure-related findings in the F2 pre-weaning and post-weaning functional observational battery assessments, the PND 20 and PND 60 auditory startle habituation parameters, in endpoints of learning and memory performance (escape times and errors) in the Biel water maze task at either testing age, or in activity levels measured on PND 61 in the 500-ppm group. Taken together, the exposure-related developmental and neuromotor changes identified in F2 pups from dams exposed to 500 ppm occurred in endpoints known to be both age- and weight-sensitive parameters, and were observed in the absence of any other remarkable indicators of neurobehavioral toxicity. Based on the results of this study, an exposure level of 50 ppm was considered to be the NOAEL for growth of F2 offspring; an exposure level of 500 ppm was considered to be the NOAEL for F2 developmental neurotoxicity.

Histopathologic examination of the immature ovary is a required end point on juvenile toxicity studies and female pubertal and thyroid function assays. To aid in this evaluation and interpretation of the immature ovary, the characteristic histologic features of rat ovary through the developmental periods are described. These histologic features are correlated with published changes in neuroendocrine profiles as the hypothalamic–pituitary–gonadal axis matures. During the neonatal stage (postnatal day [PND] 0–7), ovarian follicle development is independent of pituitary gonadotropins (luteinizing hormone [LH] or follicle-stimulating hormone [FSH]), and follicles remain preantral. Antral development of “atypical” follicles occurs in the early infantile period (PND 8–14) when the ovary becomes responsive to pituitary gonadotropins. In the late infantile period (PND 15–20), the zona pellucida appears, the hilus forms, and antral follicles mature by losing their “atypical” appearance. The juvenile stage (PND 21–32) is the stage when atresia of medullary follicles occurs corresponding to a nadir in FSH levels. In the peripubertal period (PND 33–37), atresia subsides as FSH levels rebound, and LH begins its bimodal surge pattern leading to ovulation. This report will provide pathologists with baseline morphologic and endocrinologic information to aid in identification and interpretation of xenobiotic effects in the ovary of the prepubertal rat.

Bovine-derived hemoglobin-based oxygen carriers (HBOCs) have been investigated for use in humans (HBOC-201) and approved for veterinary medicine (HBOC-301). We infused pregnant beagles with HBOC-201 to test whether HBOC-induced developmental toxicity previously observed in rats would occur in a species devoid of an inverted visceral yolk sac (invVYS). Phase 1 assessed developmental toxicity of 6 g/kg HBOC-201 on gestational day (GD) 21. Phase 2 investigated single infusions of 6 g/kg HBOC-201 on one of GDs 21, 25, 29 or 33. Phase 3 studied multiple sequential infusions on GDs 21, 23,25,27,29, 31, and 33 at 0.52 g/kg/day (3.6 g/kg total dose). Mild to moderate maternal toxicity occurred in all phases. There was an unequivocal absence of developmental toxicity in all phases. Overall, our hypothesis that HBOC, which interferes with the function of the invVYS, would not affect the offspring in dogs was supported. The implications relative to human risk are discussed.

Highlights • Infusion HBOC into pregnant rats causes developmental toxicity. • Sensitive from GD 7 to 11 when inverted yolk sac placenta (invYSP) supplies nutrition. • Controls for protein content, oncotic properties and Hb show effects are due to Hb. • Whole embryo culture verified HBOC interference with invYSP function. • Humans lack invYSP and are unlikely to be affected by HBOC., HBOC-201 is a bovine-derived, cross-linked, and stabilized hemoglobin (250 kDa) in physiological saline. Daily intravenous infusions of HBOC (1.95, 3.90, or 5.85 g/kg/day) during gestational days (GDs) 6–18 in Sprague-Dawley rats caused fetal mortality, reduced birth weight, and malformations. Subsequent single-day infusions (5.85 g/kg/day) showed that developmental toxicity was limited to GDs 7–9 when histiotrophic nutrition via the inverted visceral yolk sac (invVYS) is essential. Histiotrophic nutrition is receptor-mediated endocytosis of bulk maternal proteins and subsequent lysosomal degradation providing amino acids and other nutrients for embryonic growth. Controls for protein content, oncotic properties, and hemoglobin content indicated that toxicity was due to hemoglobin. Rat whole embryo cultures verified HBOC interference with invVYS transport capacity and resultant deficient embryonic nutrition. These mechanisms of action are not expected to impact human development based on differences in VYS morphology and function, although a complete understanding of early human embryonic nutrition is lacking.

Numerous studies have suggested that single-day intraperitoneal (IP) injection of inorganic arsenic results in failure of neural tube closure and other malformations in rats, hamsters, and mice. Most of these studies involved treatment of limited numbers of animals with maternally toxic doses of arsenic (generally AsV), without defining a dose-response relationship. In the present Good Laboratory Practice-compliant study, sodium arsenate (AsV) was administered IP and arsenic trioxide (AsIII) was administered either IP or orally (by gavage) on gestational day 9 to groups of 25 mated Crl:CD®(SD)BR rats. Only at dose levels that caused severe maternal toxicity, including lethality, did IP injection of arsenic trioxide produce neural tube and ocular defects; oral administration of higher doses of arsenic trioxide caused some maternal deaths but no treatment-related fetal malformations. In contrast, IP injection of similar amounts of sodium arsenate (based on the molar amount of arsenic) caused mild maternal toxicity but a large increase in malformations, including neural tube, eye, and jaw defects. In summary, neural tube and craniofacial defects were observed after IP injection of both AsV and AsIII; however, no increase in malformations was seen following oral administration of AsIII, even at maternally lethal doses. These results demonstrate that the frequently cited association between prenatal exposure to inorganic arsenic and malformations in laboratory animals is dependent on a route of administration that is not appropriate for human risk assessment. Teratology 60:283–291, 1999. © 1999 Wiley-Liss, Inc.

A review of the literature revealed no published inhalational developmental toxicity studies of arsenic performed according to modern regulatory guidelines and with exposure throughout gestation. In the present study, inorganic arsenic, as arsenic trioxide (As 13 ,A s 2O3), was administered via whole-body inhalational exposure to groups of twenty-five Crl:CD t (SD)BR female rats for six h per day every day, beginning fourteen days prior to mating and continuing throughout mating and gestation. Exposures were begun prior to mating in order to achieve a biological steady state of As 13 in the dams prior to embryonal-fetal development. In a preliminary exposure range-finding study, half of the females that had been exposed to arsenic trioxide at 25 mg/m 3 died or were euthanized in extremis. In the definitive study, target exposure levels were 0.3, 3.0, and 10.0 mg/m 3 . Maternal toxicity, which was determined by the occurrence of rales, a decrease in net body weight gain, and a decrease in food intake during pre-mating and gestational exposure, was observed only at the 10 mg/m 3 exposure level. Intrauterine parameters (mean numbers of corpora lutea, implantation sites, resorptions and viable fetuses, and mean fetal weights) were unaffected by treatment. No treatment-related malformations or developmental variations were noted at any exposure level. The no-observed-adverse-effect level (NOAEL) for maternal toxicity was 3.0 mg/m 3 ; the NOAEL for developmental toxicity was greater than or equal to 10 mg/m 3 , 760 times both the time-weighted average threshold limit value (TLV) and the permissible exposure limit (PEL) for humans. Based on the results of this study, we conclude that arsenic trioxide, when administered via whole-body inhalation to pregnant rats, is not a developmental toxicant.

Assessment of risks to human health from chemical agents is a complex process that requires the assembly, careful analysis, and integration of human and animal data collected from studies performed at different times, for disparate purposes, and under varying conditions. The application of risk assessment methods to data without consideration of the relevance of critical experimental parameters such as route of exposure or magnitude of dose can lead to specious determinations of the risk posed by exposure to environmental agents. A case study of the purported risk of developmental toxicity from inorganic arsenic is presented to illustrate (1) the nature of the problem, (2) how extant data from all studies are useful, (3) how appropriately designed modern studies can clarify the situation, and (4) how conflicted data should be evaluated in terms of appropriateness for use in risk assessment.

Anogenital distance (AGD) is an endpoint that was recently added to the U.S. EPA testing guidelines for reproductive toxicity studies. This endpoint is sensitive to hormonal effects of test chemicals. It is possible that apparent alterations in AGD might occur after treatment with agents that affect overall pup body size. In such cases, hormonal activity might be associated incorrectly with the test treatment. The analyses in this report evaluated statistical correlations between pup body weight and AGD in control litters. AGDs were measured on postnatal day 1 in 1501 pups derived from 113 untreated female Sprague-Dawley rats in two independent two-generation reproductive toxicity studies. Significant correlations were detected between AGD and body weight and between AGD and the cube root of body weight. In males, AGD increased 0.26 mm for each 1 g increase in body weight. In females, AGD increased 0.13 mm per 1 g increase in body weight. Although there were essentially no differences between the regression models developed to predict AGD in either males or females using body weight as a covariate and those based on the cube root of body weight, such similarities in predictivity might not occur in larger animals with broader weight ranges than those encountered in this analysis. Normalization of AGD by dividing by body weight significantly overcompensated for differences in body size. Normalizing with the cube root of body weight resulted in an AGD/cube root of body weight ratio that was constant across the range of body weights observed in this study. In conclusion, as a preferred method to account for body size effects on AGD, analysis of covariance is recommended. If a normalization is done directly, the ratio of AGD to the cube root of body weight is the more appropriate metric.

We have isolated a cDNA encoding Xenopus laevis (Xl) heat-shock factor 1 (XHSF1). XHSF1, translated from the mRNA synthesized in vitro, will bind specifically to the X1 hsp70 promoter (hsp70). Microinjection of XHSF1 mRNA into Xl oocytes leads to synthesis of XHSF1 which accumulates in the nucleus and selectively activates Xl phsp70p activity at 18°C.

The effects of route and vehicle on blood and milk levels of decabromodiphenyl ether (DecaBDE; CASRN 1163-19-5) were investigated in the rat to assist in the design and conduct of a developmental neurotoxicity study. Blood plasma and/or milk concentrations were determined in dams, fetuses, and/or nursing pups after repeated DecaBDE administration by gavage throughout gestation or gestation and lactation using corn oil (CO) or soyaphospholipon/Lutrol F 127-water (SPL) as the vehicle. The impact of vehicle on plasma levels was also investigated in pups derived from naive dams after a single postnatal dose. This study reports for the first time fetal and neonatal plasma concentrations concurrent with those of maternal plasma and/or milk. Higher concentrations of DecaBDE were achieved in plasma and in milk with CO than with SPL. Furthermore, pups derived from dams treated with only SPL were lower in body weight, compared with those from dams treated with either CO, CO and DecaBDE, or SPL and DecaBDE. The study further shows that exposure to DecaBDE is relatively consistent across the dose range of 100 to 1000 mg/(kg · day) when administered in CO.

This study was conducted to determine the potential of bisphenol A (BPA) to induce functional and/or morphological effects to the nervous system of F(1) offspring from dietary exposure during gestation and lactation according to the Organization for Economic Cooperation and Development and U.S. Environmental Protection Agency guidelines for the study of developmental neurotoxicity. BPA was offered to female Sprague-Dawley Crl:CD (SD) rats (24 per dose group) and their litters at dietary concentrations of 0 (control), 0.15, 1.5, 75, 750, and 2250 ppm daily from gestation day 0 through lactation day 21. F(1) offspring were evaluated using the following tests: detailed clinical observations (postnatal days [PNDs] 4, 11, 21, 35, 45, and 60), auditory startle (PNDs 20 and 60), motor activity (PNDs 13, 17, 21, and 61), learning and memory using the Biel water maze (PNDs 22 and 62), and brain and nervous system neuropathology and brain morphometry (PNDs 21 and 72). For F(1) offspring, there were no treatment-related neurobehavioral effects, nor was there evidence of neuropathology or effects on brain morphometry. Based on maternal and offspring body weight reductions, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 75 ppm (5.85 and 13.1 mg/kg/day during gestation and lactation, respectively), with no treatment-related effects at lower doses or nonmonotonic dose responses observed for any parameter. There was no evidence that BPA is a developmental neurotoxicant in rats, and the NOAEL for developmental neurotoxicity was 2250 ppm, the highest dose tested (164 and 410 mg/kg/day during gestation and lactation, respectively).

The expression of the rat cellular retinol binding protein I (rCRBPI) can be upregulated in vivo by retinoic acid (RA). Here we have analyzed the rCRBPI promoter region and compared it to the corresponding mouse sequence. We find that the CRBPI 5′ flanking region has been highly conserved between rat and mouse, including a RA response element (RARE) ∼ 1 kb upstream of the start of transcription. The RARE is of the direct repeat type with a two nucleotide spacer. Like other direct repeat RAREs, this response element is activated by RARα and β but not by RARγ1. Furthermore, the rCRBPI-RARE is most effectively activated when both RAR and RXR are present. In addition RAR RXR heterodimers are required for efficient binding to the rCRBPI-RARE, while RARs or RXR alone do not interact effectively with this response element. The rCRBPI gene is therefore most likely activated in vitro by a rmRAR RXR heterodimer.

The objective of juvenile animal toxicity studies of pharmaceuticals is to obtain safety data, including information on the potential for adverse effects on postnatal growth and development. Studies in juvenile animals may assist in identifying postnatal developmental toxicities or other adverse effects that are not adequately assessed in the routine toxicity evaluations and that cannot be safely or adequately measured in pediatric clinical trials. Unlike the traditional reproductive and developmental toxicology studies that have been discussed in the accompanying reports, the design requirements for toxicity studies in juvenile animals are not explicitly defined in regulatory guidance. However, studies in juvenile animals can be useful in providing safety information necessary to enable pediatric clinical trials in pediatric patients or when there are special concerns for toxicities that cannot be safely or adequately measured in clinical trials. These juvenile animal toxicity studies are designed on a case-by-case basis. General design considerations and examples of study designs for assessment of juvenile animal toxicity are discussed. Birth Defects Res (Part B) 86:463–469, 2009. © 2009 Wiley-Liss, Inc.

Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is currently guided by the International Conference on Harmonization (ICH) S5(R2) document (available at http://www.ich.org). The studies that assess developmental hazard are generally conducted in rodents and rabbits. Based on the authors' collective experience, adequate designs (including range-finding studies) and the presentation of data for these studies are described in detail. In addition, the suggested initiation and then total duration of these studies in relation to clinical studies that enroll women of childbearing potential are described. Optional parameters that may be included in the studies are discussed, as are study designs that combine assessments of fertility and developmental toxicity. New methods that may replace or enhance current procedures are outlined. The details described herein will assist all laboratories performing these studies, individuals who need to plan for the studies, and regulatory agencies that ultimately review these studies.

Assessment of potential developmental and reproductive toxicity of human pharmaceuticals is currently guided by the ICH S5(R2) document, "Detection of Toxicity to Reproduction for Medicinal Products and Toxicity to Male Fertility." Studies that assess a candidate drug's effect on fertility are generally conducted in rats. The evolution of, and ultimate harmonization of, fertility study designs are reviewed, and specific elements of an acceptable design, as well as the recommendations for presentation of data, are described in detail. Additionally, the timing of nonclinical fertility studies in relation to clinical studies that enroll men and women of reproductive potential is reviewed. Possible strategies for combining fertility assessment with other study designs are also presented. This article provides testing laboratories, sponsors, and regulatory agencies with a comparison of current methods and designs, with the aim of providing a common understanding of the critical design features.

Cellular retinol-binding protein (CRBP) is a retinol-specific binding protein. A rat cDNA clone of CRBP was expressed in Escherichia coli. In order to determine amino acid residues in CRBP which may be important for the binding of all-trans-retinol, comparative model-building studies were performed in which strong sequence similarities were identified between CRBP and several other binding proteins. Based on this analysis, specific amino acids were predicted to be important in retinol binding, and these predictions were tested using the technique of site-directed mutagenesis to subtly alter the protein's structure and function. Specifically, site-directed mutagenesis was performed to alter the Gln-108 to Arg-108 (Q108R). Making use of fluorescence, Q108R was found to have a 3-fold lower affinity for all-trans-retinol, and the fine structure of the excitation spectrum of the Q108R.all-trans-retinol complex was also different than for the wild type.all-trans-retinol complex. The mutant bound 13-cis-retinol with an excitation spectrum identical to wild type bound to 13-cis-retinol, but with only one-half of the fluorescence intensity. In competition binding experiments, the Q108R mutant was found to have similar binding affinities for all-trans-retinol, all-trans-retinoic acid, 13-cis-retinoic acid, and retinal, while wild type CRBP was only able to bind to all-trans-retinol. Thus, altering a single amino acid in CRBP (Gln-108 to Arg-108) caused a significant change in the ligand binding specificity of the protein.

Spontaneous ejaculation has been reported in a variety of mammalian species and may occur either as a result of pharmacological treatment or as a component of the daily behavior of normal, untreated animals. Infrequently, increased numbers of spontaneous ejaculatory plugs have been reported among the clinical signs in rat toxicology studies. This mini-review presents an overview on the presence of ejaculatory plugs in rodents and provides recommendations to consider when attempting to understand the toxicological significance of increased numbers of ejaculatory plugs in rat toxicology studies.

Evidence suggests that the health benefits associated with green tea consumption are related to tea catechins. The objective of this study was to evaluate potential maternal and fetal effects of standardized heat-sterilized green tea catechins (GTC-H). GTC-H was gavage administered to mated female rats from gestation day 6 through 17, at doses of 0, 200, 600, and 2000 mg/kg/day. There were no GTC-H-related deaths or macroscopic findings. During the entire gestation period in the high-dose (2000 mg/kg/day)-treated group and during days 6-9 and 6-18 in the 600 mg/kg/day group, mean body weight gain was lower. Mean feed consumption was lower during gestation days 6-9 in the 600 mg/kg/day group and during gestation days 6-9 and 9-12 in the 2000 mg/kg/day group. Compared to the control group, mean body weights in the 600 and 2000 mg/kg/day groups were up to 5.1% and 7.7% lower during gestation days 9-20. GTC-H administration did not affect mean gravid uterine weights or intrauterine growth and survival. There were no GTC-H-related fetal malformations or developmental variations. Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for GTC-H was 200mg/kg/day for maternal toxicity, and 2000 mg/kg/day for embryo/fetal development.

Perfluorooctanesulfonate (PFOS), a persistent and accumulative compound, is widely distributed in humans and wildlife. Human exposure can occur early in development, as evidenced by the detection of PFOS in umbilical cord blood and breast milk. As part of a developmental neurotoxicology study for which developmental endpoints, including those related to the developing nervous system, have been reported separately, groups of 25 pregnant Sprague Dawley rats were given daily oral doses of either vehicle control or potassium PFOS (K(+)PFOS) at 0.1, 0.3, and 1.0mg/kg-d from gestation day (GD) 0 (day positive for mating) through postnatal day (PND) 20. An additional 10 pregnant females per treatment group were treated through GD 19 and sacrificed on GD 20 in order to obtain maternal and fetal serum and tissue samples at the end of gestation. The present paper reports the results of samples of serum, liver, brain, and thyroid glands taken at various times to evaluate: (1) serum, liver, and brain PFOS concentrations by LC-MS/MS to establish the relationship between PFOS concentrations and study outcomes; (2) serum thyrotropin (TSH) concentrations by RIA; (3) thyroid follicular cell proliferation index by Ki-67 immunohistochemical staining; (4) thyroid follicle epithelial cell height and colloidal area by histomorphometric analysis; (5) selected liver mRNA transcripts by quantitative RT-PCR. PFOS concentrations in dam and pup serum, liver, and brain increased across treatment groups in approximate proportion to the proportional increases in maternal K(+)PFOS dose, and sex differences in PFOS concentrations were not apparent in pups on PND 21. In pups from K(+)PFOS maternal dose groups on PND 72, serum PFOS had decreased to about 3 and 11% of PND 21 concentrations in males and females, respectively, and liver PFOS had decreased to about 17% of PND 21 concentrations in both sexes. Liver PFOS concentrations were approximately 0.6-0.8 times serum PFOS in GD 20 fetuses, and increased to about 2-4 times serum concentrations on PND 4 and 21. GD 20 fetal and PND 4 pup brain PFOS concentrations were approximately 33% of the corresponding serum concentrations, dropping to approximately 10% by PND 21, in contrast to dam brain PFOS concentrations, which were approximately 4-9% of serum PFOS concentrations. Compared to controls, Cyp2b2 mRNA was increased (2.8-fold) in the 1.0mg/kg-d treatment-group dams on GD 20. In male pups on PND 21, Cyp4A1, ACoA, and Cyp2b2 were increased 2.1-, 1.5-, and 1.8-fold, respectively, and Cyp7A1 was decreased 3.5-fold. Serum TSH and thyroid follicular morphology were not altered by K(+)PFOS treatment. The mean number of proliferating thyroid follicular cells was increased 2.1-fold over control in GD 20 female fetuses from 1.0mg/kg-d-treated dams, yet the highest individual count was similar to that of controls (116 versus 113 in controls).

SLE66 a synthetic pseudo-ceramide, has been shown to reduce dryness/scaling/itching of human skin. Naturally occurring ceramides have been claimed to play a crucial role in cell proliferation, differentiation, and apoptosis including processes important for embryogenesis. The objective of this study was to evaluate the potential maternal and fetal effects of SLE66. SLE66 was administered orally (gavage) to mated female Crl:CD®(SD)IGS BR rats (25/group) once daily from gestation day 6 through 19, at dose levels of 0 (control), 150, 400 or 1000 mg/kg/day. No treatment-related clinical or internal (macroscopic) findings were noted and all animals survived to the scheduled necropsy on gestation day 20. SLE66 administration did not affect mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, or feed consumption. Similarly, SLE66 administration did not affect intrauterine growth and survival related parameters such as viable fetuses, pre-implantation loss, early and late resorptions, fetal weight and fetal sex. No SLE66-related fetal malformations or developmental variations were noted. Based on the results of this study, a dose level of 1000 mg/kg/day (highest dose used) was considered as the no-observed-adverse-effect level (NOAEL) for both maternal and developmental toxicity.

BACKGROUND: Endogenous opioids seem to regulate hypothalamic gonadotropin release in both males and females, as evidenced by the effects of opioid agonists and antagonists on LHRH release and reproductive hormone levels. The effects of long-term oral administration of opioid analgesics on reproductive function have not been well characterized. METHODS: The reproductive effects of oxymorphone, a potent opioid agonist, were investigated in male and female Crl:CD(SD) IGS BR rats at oral doses of 0, 5, 10, and 25 mg/kg/day (25 animals/sex/group). Males were treated for approximately 9 weeks (mated after 4 weeks of dosing). Females were treated for 14 days before mating, and through Gestation Day (GD) 7. Estrous cycling was evaluated during the premating period. On GD15, pregnancy status and the numbers of corpora lutea, implantation sites, live and dead embryos were determined. Epididymal and testicular sperm counts and epididymal sperm motility and morphology were evaluated in males. RESULTS: Two males given 25 mg/kg/day died. Behavioral changes and deficits in body weight gain occurred at all doses. There were no effects of oxymorphone on reproductive function or sperm parameters in males. The estrous cycle was prolonged in females given 25 mg/kg/day (mean of 5.3 vs. 4.3 days in controls). A small, but consistent decrease in the numbers of corpora lutea (with associated decreases in implantation sites and embryos) occurred in females given ≥10 mg/kg/day. There were no effects on mating or fertility in females. CONCLUSIONS: Oxymorphone seems to partially inhibit ovulation in female rats, with no significant effects on male reproductive outcome. Birth Defects Res (Part B) © 2007 Wiley-Liss, Inc.

BACKGROUND: These studies were conducted to evaluate the potential adverse effects of di-2-ethylhexyl terephthalate (DEHT) exposure on in utero development in mice and rats. In addition, a uterotrophic assay for estrogenic activity was conducted in sexually immature rats. METHODS: In the developmental toxicity studies, diet containing DEHT was fed to four groups of mated female Crl:CD(SD)IGS BR rats (25/group) from gestation day (GD) 0–20 or Crl:CD1(ICR) mice (25/group) from GD 0–18. Concentrations within the feed were 0, 0.3, 0.6, and 1.0% for the rats and 0, 0.1, 0.3, and 0.7% for the mice. Laparohysterectomies were carried out on the last day of exposure and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. The fetuses were weighed, sexed, and examined for external, visceral and skeletal malformations, and developmental variations. The dose rate from dietary DEHT exposure was 0, 226, 458, and 747 mg/kg/day in the rats and 197, 592, and 1382 mg/kg/day in the mice for the control, low, mid, and high-exposure groups, respectively. RESULTS: DEHT exposure did not affect clinical observations. A slight reduction in body weight gain was noted in the high-dose level rat group; the remaining groups were unaffected. At necropsy, increased liver weights were noted in the high-dose rat group and the mid- and high-dose mouse groups. Mean numbers of implantation sites and viable fetuses, mean fetal weights, and mean litter proportions of preimplantation loss, early resorptions, late resorptions, and fetal sex ratios were unaffected by DEHT exposures. No test article-related malformations or variations were observed at any concentration level in the rat and mouse developmental toxicity studies. In the uterotrophic assay for estrogenic activity, sexually immature female rats received oral gavage doses 20, 200, or 2000 mg DEHT/kg bw/day from postnatal day (PND) 19–21. A slight reduction in rate of body weight gain was noted on the first day of dosing in the high dose group, but no other indications of toxicity were evident. DEHT exposure did not affect wet or blotted uterine weight parameters in any of these dose groups. The NOEL for developmental toxicity in rats was 747 mg/kg/day and 1382 mg/kg/day in mice. The NOEL for estrogenic activity was 2000 mg/kg/day. The NOEL for maternal toxicity was 458 mg/kg/day in rats and 197 mg/kg/day in mice. CONCLUSIONS: The lack of adverse developmental effects with DEHT exposure are in contrast to the adverse developmental effects noted after di-2-ethylhexyl phthalate (DEHP) exposure. The difference between the effects noted with the ortho-constituent (DEHP) and the lack of effects reported with the para-constituent (DEHT) is due most likely to differences in metabolism and the formation of the stable monoester, mono-2-ethylhexyl phthalate (MEHP) from the DEHP moiety. Birth Defects Res (Part B). © 2007 Wiley-Liss, Inc.

Diacylglycerol (DAG) oil is an edible oil with similar taste and usability characteristics as conventional edible oil. Recent studies suggest that use of DAG oil may be helpful in the prevention and management of obesity. This study evaluated the potential maternal and fetal effects of DAG oil, following exposure to pregnant rats, during the critical period of major organogenesis. DAG oil was administered via gavage to four groups of mated female Crl:CD®(SD)IGS BR rats (25/group) once daily from gestation day 6 through 17, at dose levels of 0, 1.25, 2.5 or 5.0 ml/kg/day (0, 1160, 2320 and 4630 mg/kg/day) with total volume made to 5 ml/kg/day with triacylglycerol (corn) oil. No mortality or treatment-related clinical or internal findings were noted in any of the groups. Compared to animals in control group, mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption were not affected by DAG oil administration. Similarly, intrauterine growth and survival were not affected by DAG oil administration. No DAG oil-related fetal malformations or developmental variations were noted. A maternal maximum tolerated dose for DAG oil was not achieved in this study. Based on the results of this study, a dose level of 5.0 ml/kg (4630 mg/kg/day) was considered as no-observed-adverse-effect level (NOAEL) for both maternal and developmental toxicity.

S-111-S-WB (CAS No. 72968-38-8), a mixture of perfluoro fatty acid ammonium salts, was administered daily via oral gavage to 30 Crl:CD(SD) rats/sex/group at 0.025, 0.125 and 0.6 mg/(kg day) over two generations to assess potential reproductive toxicity. Reproductive performance, mean litter size, pup survival and pup weights were unaffected. Lower mean body weights were observed in 0.6 mg/(kg day) group F0 and F1 males. Higher liver weights, correlating to hepatocellular hypertrophy in the 0.6 mg/kg group, were noted for parental males in the 0.125 and 0.6 mg/(kg day) groups, parental females in the 0.6 mg/(kg day) group and F1 pups in the 0.125 and 0.6 mg/(kg day) groups. Higher kidney weights, correlating to renal tubule hypertrophy in the 0.6 mg/kg group, were observed for parental males and females in the 0.125 and 0.6 mg/(kg day) groups. Systemic exposure (measured only in females) to total S-111-S-WB was proportional to dose following 9 weeks of daily administration on the gestation day 19. Total S-111-S-WB concentration in the serum of male and female pups was 1.2–1.4-fold higher than in the dams 2 h following administration to the dams on lactation day 13. A dosage level of 0.6 mg/(kg day) was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive function. A dosage level of less than 0.025 mg/(kg day) was considered to be the NOAEL for F0 and F1 parental systemic toxicity based on microscopic hepatic findings in the males of all test article groups, and a dosage level of 0.025 mg/(kg day) was considered to be the NOAEL for neonatal toxicity based on higher liver weights in the F1 and F2 pups at 0.125 mg/(kg day) and higher.

Octamethylcyclotetrasiloxane (D(4)) has been shown to have effects on the female rat reproductive cycle. This study evaluated the phase of the female rat reproductive cycle affected by D(4) using a study design that allowed the complete female reproductive cycle, as well as phases of the cycle, from pre-mating through gestation, to be evaluated. Rats were exposed via whole body vapor inhalation up to 700 ppm D(4) during the overall phase (28 days prior to mating through gestation day (GD) 19), the ovarian phase (31-3 days prior to mating), the fertilization phase (3 days prior to the start of mating through gestation day 3), and the implantation phase (GD 2-GD 5) of the reproductive cycle. D(4) was associated with decreases in implantation sites and litter size in the overall and fertilization phases, but not in the ovarian or implantation phases. In order to further define the sensitive period for D(4) exposure, additional groups of rats were exposed on single days. A single 6h exposure to D(4) on the day prior to mating resulted in a significant reduction in fertility. These data indicate that there is a very narrow window, around the time of ovulation and fertilization, for D(4) to exert effects on the reproductive cycle of the female rat. Subsequent research, reported elsewhere, has elucidated the mode of action and assessed its potential relevance to humans.

This study was conducted to evaluate the potential adverse effects of ethylbenzene (EB) on reproductive capability from whole-body inhalation exposure of F0 and F1 parental animals.Four groups of Crl:CD(SD)IGS BR rats (30/sex/group for F0 and 25/sex/group for F1) were exposed to 0, 25, 100, and 500 ppm EB for 6 hr/day for at least 70 consecutive days before mating. Inhalation exposure for the F0 and F1 females continued throughout mating, gestation through gestation day (GD) 20, and lactation days (LD) 5-21. On LD 1-4, females received EB in corn oil via oral gavage at dose levels of 26, 90, and 342 mg/kg/day (divided into three equal doses, approximately 2 hr apart), as calculated from a physiologically-based pharmacokinetic (PBPK) model to provide similar maternal blood area-under-concentration (AUC) as provided by inhalation. Pups were weaned on postnatal day (PND) 21 and exposure of the F1 generation started on PND 22. Estimates of internal exposure were determined by measuring EB concentrations in blood collected from F1 dams (4/group) and their culled pups 1 hr after the last gavage dose on PND 4. On PND 22, blood was collected from these same F1 dams and their weanlings for EB analysis 1 hr after a 6-hr inhalation exposure. The remainder of the F2 generation was not directly exposed.EB exposure did not affect survival or clinical observations. Male rats in the 500 ppm group in both generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, ovarian follicle counts, reproductive organ weights, lengths of estrous cycle and gestation, live litter size, pup weights, developmental landmarks, and postnatal survival were unaffected. No adverse exposure-related macroscopic pathology was noted at any level.Increased liver weights were found in the animals exposed to 500 ppm. F1 maternal whole blood EB concentrations of 0.49, 3.51, or 18.28 mg/L were found 1 hr after administration of a composite oral dose of 26, 90, or 342 mg/kg/day, respectively, but no detectable EB was found in blood samples of their F2 PND 4 culled pups. F1 maternal mean whole blood EB levels 1 hr after a 6-hr inhalation exposure on postpartum day (PPD) 22 was 0.11 mg/L (25 ppm), 0.56 mg/L (100 ppm), and 11 mg/L (500 ppm). For the offspring exposed with their dams on PND 22, F2 pup blood EB concentrations ranged from 0.017-0.039 mg/L (25 ppm), 0.165-0.465 mg/L (100 ppm), and 8.82-15.74 mg/L (500 ppm). Because decreased weight gain in the 500 ppm males was transient and no histopathological changes were associated with the increased liver weights in the 500 ppm male and female groups, these changes were not considered adverse. Therefore, for parental systemic toxicity, 100 ppm was considered a NOEL and 500 ppm a NOAEL in this study. The 500 ppm exposure concentration was considered a NOAEL for F0 and F1 reproductive toxicity and offspring developmental endpoints.

Di-isoheptyl phthalate (DIHP) is a branched, phthalate ester with seven carbon alkyl side chains. Since structurally similar phthalates have been shown to produce developmental and/or reproductive effects in rodents, the potential for DIHP to produce developmental and reproductive toxicity was assessed. In a developmental toxicity study, female rats were given DIHP by oral gavage on gestational days 6–20. There were significant reductions in uterine weight, increased resorptions and reduced fetal weight in the high dose (750 mg/kg) group. Fetal examination revealed malformations and variations of both the skeletal system and the viscera including ectopic testes. The intermediate dose, 300 mg/(kg/day), was a no effect level in this study. In a two-generation reproductive toxicity study, DIHP was given in the diet at 1000, 4500 and 8000 ppm. In the 8000 ppm group of the first (F1) generation, anogenital distance was reduced, time to balanopreputial separation was increased, there was a significant increase in thoracic nipples and testicular abnormalities, and weights of testes and accessory reproductive organs were significantly reduced. Testicular sperm counts and daily sperm production were significantly reduced. Fertility was also significantly reduced in the 8000 ppm group. In the second (F2) generation offspring, anogenital distance was significantly reduced and there was evidence of reduced weight gain during lactation in both the 4500 and 8000 ppm groups. The overall no effect level (NOEL) in the reproductive toxicity study was in the range of 64–168 mg/(kg/day) (gestation–lactation periods). By comparison, estimated average human exposures in the general population are

The potential for nelfinavir mesylate (VIRACEPT) to produce reproductive toxicity was evaluated in rats administered oral doses of 200, 500, or 1000mg/kg/day. In the fertility and early embryonic development to implantation study, male rats were treated beginning 28 days prior to mating until necropsy and females for 2 weeks prior to mating and through gestation day (GD) 7. In the pre- and postnatal development study, pregnant rats were treated from GD 6 through lactation day (LD) 20. Selected F(1) pups from this study were evaluated in sensory and behavioral tests and were subsequently mated. Pregnant F(1) females were euthanized on GD 20 and their F(2) fetuses were examined. F(1) animals were not directly dosed with the drug. No treatment-related effects were observed on any male reproductive parameters. Administration of nelfinavir did not produce adverse effects on fertility, pregnancy, embryo-fetal development, parturition, or lactation in the F(0) generation. Similarly, no adverse effects of nelfinavir treatment were observed on pre- and postnatal growth, development, reproductive performance and embryo-fetal development in the F(1) offspring. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for developmental and reproductive toxicity in rats was considered to be 1000mg/kg/day, the highest dose tested.

The potential for nelfinavir mesylate (VIRACEPT) to induce maternal and embryo-fetal toxicity was evaluated in rats and rabbits following oral administration. The drug was administered by gavage to rats at doses of 200, 500, or 1000 mg/kg/day on days 6–17 of gestation and to rabbits at doses of 200, 400, or 1000 mg/kg/day on days 7–20 of gestation. Dams and does were euthanized on GD20 and 29, respectively, and the offspring were weighed and examined for external, visceral, and skeletal alterations. Maximum plasma nelfinavir concentrations (Cmax) in rats were comparable to Cmax values in humans and were 3- to 6-fold higher than the reported human trough levels, while plasma nelfinavir levels in rabbits were approximately 0.13–0.17× the human Cmax and 0.25–0.5× the human trough. In rats, no treatment-related maternal or embryo-fetal toxicity was observed at any dose level and the NOAEL for both maternal and fetal toxicity was considered to be 1000 mg/kg/day. Two rabbits in the 400 mg/kg/day group died prior to scheduled termination. Because no deaths occurred in the high dose group and there were no other treatment-related signs of clinical toxicity in any dose group, these deaths were considered unrelated to nelfinavir. Group mean body weight loss in rabbits was observed at 1000 mg/kg/day on gestation days 7–10. Food consumption was also reduced in this treatment group throughout the dosing period. There were no treatment-related findings in other maternal or fetal parameters. Thus, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity in the rabbit was considered to be 400 mg/kg/day (based on maternal body weight loss in the high dose group), while the NOAEL for embryo-fetal toxicity in the rabbit was considered to be 1000 mg/kg/day. Thus, under the conditions of this study, nelfinavir was not considered to be toxic to the rat or rabbit conceptus.

A thorough review of the literature revealed no published repeated-dose oral developmental toxicity studies of inorganic arsenic in rats. In the present study, which was conducted according to modern regulatory guidelines, arsenic trioxide was administered orally beginning 14 days prior to mating and continuing through mating and gestation until gestational day 19. Exposures began prior to mating in an attempt to achieve a steady state of arsenic in the bloodstream of dams prior to embryo-foetal development. Groups of 25 Crl:CD(SD)BR female rats received doses of 0, 1, 2.5, 5 or 10mg/kg/day by gavage. The selection of these dose levels was based on a preliminary range-finding study, in which excessive post-implantation loss and markedly decreased foetal weight occurred at doses of 15 mg/kg/day and maternal deaths occurred at higher doses. Maternal toxicity in the 10mg/kg/day group was evidenced by decreased food consumption and decreased net body weight gain during gestation, increased liver and kidney weights, and stomach abnormalities (adhesions and eroded areas). Transient decreases in food consumption in the 5mg/kg/day group caused the maternal no-observed-adverse-effect level (NOAEL) to be determined as 2. 5mg/kg/day. Intrauterine parameters were unaffected by arsenic trioxide. No treatment-related foetal malformations were noted in any dose group. Increased skeletal variations at 10mg/kg/day were attributed to reduced foetal weight at that dose level. The developmental NOAEL was thus 5mg/kg/day. Based on this study, orally administered arsenic trioxide cannot be considered to be a selective developmental toxicant (i.e. it is not more toxic to the conceptus than to the maternal organism), nor does it exhibit any propensity to cause neural tube defects, even at maternally toxic dose levels.