Burden of disease and costs of exposure to endocrine disrupting chemicals in the European Union: an updated analysis.

A previous report documented that endocrine disrupting chemicals contribute substantially to certain forms of disease and disability. In the present analysis, our main objective was to update a range of health and economic costs that can be reasonably attributed to endocrine disrupting chemical exposures in the European Union, leveraging new burden and disease cost estimates of female reproductive conditions from accompanying report. Expert panels evaluated the epidemiologic evidence, using adapted criteria from the WHO Grading of Recommendations Assessment, Development and Evaluation Working Group, and evaluated laboratory and animal evidence of endocrine disruption using definitions recently promulgated by the Danish Environmental Protection Agency. The Delphi method was used to make decisions on the strength of the data. Expert panels consensus was achieved for probable (>20%) endocrine disrupting chemical causation for IQ loss and associated intellectual disability; autism; attention deficit hyperactivity disorder; endometriosis; fibroids; childhood obesity; adult obesity; adult diabetes; cryptorchidism; male infertility, and mortality associated with reduced testosterone. Accounting for probability of causation, and using the midpoint of each range for probability of causation, Monte Carlo simulations produced a median annual cost of €163 billion (1.28% of EU Gross Domestic Product) across 1000 simulations. We conclude that endocrine disrupting chemical exposures in the EU are likely to contribute substantially to disease and dysfunction across the life course with costs in the hundreds of billions of Euros per year. These estimates represent only those endocrine disrupting chemicals with the highest probability of causation; a broader analysis would have produced greater estimates of burden of disease and costs.

Evolution of DOHaD: the impact of environmental health sciences.

Environmental exposures have a significant influence on the chronic health conditions plaguing children and adults. Although the Developmental Origins of Health and Disease (DOHaD) paradigm historically has focused on nutrition, an expanding body of research specifically communicates the effects of chemical exposures on early-life development and the propagation of non-communicable disease across the lifespan. This paper provides an overview of 20 years of research efforts aimed at identifying critical windows of susceptibility to environmental exposures and the signaling changes and epigenetic influences associated with disease progression. DOHaD grants funded by the National Institute of Environmental Health Sciences (NIEHS) in 1991, 2001 and 2011 are identified by grant-analysis software, and each portfolio is analyzed for exposures, disease endpoints, windows of exposure, study design and impact on the field based on publication data. Results show that the 1991 and 2001 portfolios comprised metals, PCBs and air pollutants; however, by 2011, the portfolio has evolved to include or expand the variety of endocrine disruptors, pesticides/persistent organic pollutants and metals. An assortment of brain-health endpoints is most targeted across the portfolios, whereas reproduction and cancer increase steadily over the same time period, and new endpoints like obesity are introduced by 2011. With mounting evidence connecting early-life exposures to later-life disease, we conclude that it is critical to expand the original DOHaD concept to include environmental chemical exposures, and to continue a research agenda that emphasizes defining sensitive windows of exposure and the mechanisms that cause disease.

Designing Endocrine Disruption Out of the Next Generation of Chemicals.

A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best achieved when information about a chemical's potential hazardous effects is obtained as early in the design process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science. To aid chemists in avoiding this hazard, we propose an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED) has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences. TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at "the drawing board." It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays. To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system. The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances. In this paper we present the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability. In a 'proof-of-principle' test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers. We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health.

Linking environmental agents and autoimmune disease: an agenda for future research.

Autoimmune diseases are influenced by multiple factors including genetics, age, gender, reproductive status, hormones, and potential environmental contaminants. A workshop, "Linking Environmental Agents and Autoimmune Diseases," was convened at the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 1-3 September 1998, to review current knowledge about links between environmental exposures and autoimmune disease, to identify and prioritize research needs, and to develop an integrated, multidisciplinary research agenda. Participants spent the last half-day of the workshop in small group discussions for the purpose of developing consensus on research needs. Research needs identified were a) develop research tools needed to explore links between environmental agents and autoimmune disease; b) establish a disease registry or surveillance system; c) develop and validate strategies for screening chemicals for the potential to induce or exacerbate autoimmune disease; d) develop an emergency response strategy to gain information from accidental exposures; and e) conduct hypothesis-driven research in occupationally exposed groups and/or in experimental animals. There was consensus that meetings like this workshop and projects that facilitate interactions between specialties should be encouraged. A multidisciplinary approach is needed to address this problem.

Formamide and dimethylformamide: reproductive assessment by continuous breeding in mice.

Reproductive toxicity in Swiss mice, during chronic exposure to formamide (FORM) or dimethylformamide (DMF), was evaluated using the Reproductive Assessment by Continuous Breeding Protocols. FORM administered in drinking water at 0, 100, 350, and 750 ppm (approximately 20 to 200 mg/kg/d) reduced fertility and litter size in F0 animals without generalized toxicity at 750 ppm FORM. Crossover matings suggested that females were the affected sex. After F1 mating, FORM reduced F2 litter size, increased days to litter, reduced relative ovarian weight, and lengthened estrous cycles at 750 ppm. The No-Observed-Adverse-Effect-Level for generalized toxicity was 750 ppm for the F0 and 350 ppm for the F1 generation. Reproductive performance was normal at 350 ppm for both F0 and F1 mice. Chronic exposure to DMF in drinking water at 0, 1000, 4000, and 7000 ppm (approximately 200 to 1300 mg/kg/d) reduced fertility by the first litter at 4000 ppm, reduced body weight in F0 females at 7000 ppm, and increased liver weights at all doses in both sexes. A crossover mating at 7000 ppm identified F0 females as the affected sex. F1 postnatal survival was reduced at > or =4000 ppm DMF. F1 mating reduced F2 litter size and live pup weight at > or =1000 ppm. At necropsy, body weight of F1 males and females was reduced at > or =4000 ppm. DMF-treated pups (both F1 and F2) and F1 adults had cranial and sternebral skeletal malformations. Only DMF caused overt developmental toxicity. A No-Observed-Adverse-Effect-Level for DMF was not established.

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for organ toxicity.

In the past decade in vitro tests have been developed that represent a range of anatomic structure from perfused whole organs to subcellular fractions. To assess the use of in vitro tests for toxicity testing, we describe and evaluate the current status of organotypic cultures for the major target organs of toxic agents. This includes liver, kidney, neural tissue, the hematopoietic system, the immune system, reproductive organs, and the endocrine system. The second part of this report reviews the application of in vitro culture systems to organ specific toxicity and evaluates the application of these systems both in industry for safety assessment and in government for regulatory purposes. Members of the working group (WG) felt that access to high-quality human material is essential for better use of in vitro organ and tissue cultures in the risk assessment process. Therefore, research should focus on improving culture techniques that will allow better preservation of human material. The WG felt that it is also important to develop and make available relevant reference compounds for toxicity assessment in each organ system, to organize and make available via the Internet complete in vivo toxicity data, including human data, containing dose, end points, and toxicokinetics. The WG also recommended that research should be supported to identify and to validate biological end points for target organ toxicity to be used in alternative toxicity testing strategies.

General, reproductive, developmental, and endocrine toxicity of boronated compounds.

Boric acid and inorganic borates are abundant in nature. They are widely used in industrial, agricultural, cosmetic, and numerous smaller applications. These compounds are toxic to all species tested at high doses, but they are not carcinogenic or mutagenic. The major toxicities are reproductive and developmental. Testicular effects occurred at approximately 26 mg boron equivalents/kg body weight (bw)/d (26 mg boron equivalent (BE)/kg bw/d). New data on endocrine toxicity includes altered follicle stimulating hormone and testosterone within 14 d of treatment. Because these hormonal changes may be secondary effects of testicular toxicity, borates are not suspect as endocrine disrupters. The most sensitive of all the endpoints are prenatal growth and morphologic development in the rat; these changes occurred at a dose of 12.9 mg BE/kg bw/d. The no observed adverse effect level for rat fetal development was 9.6 mg/kg BE. Considering the estimated human exposure levels and a safety factor of 30, humans are not at significant risk of reproductive failure due to borates from environmental sources. The margin of exposure is estimated at 72 for males and 129 for females. Thus, the likelihood of human toxicity caused by boric acid and inorganic borates from exposure during normal activities is remote.

Evaluation of the developmental toxicity of methacrylamide and N,N'-methylenebisacrylamide in Swiss mice.

Timed-pregnant CD-1 outbred albino Swiss mice received either methacrylamide (MAC; 0, 60, 120, or 180 mg/kg/day) or N,N'-methylenebisacrylamide (BAC; 0, 3, 10, or 30 mg/kg/day) p.o. in distilled water on gestational days (GD) 6 through 17. Maternal clinical status was monitored daily. At termination (GD 17), confirmed-pregnant females (27-30 per group, MAC; 24-25 per group, BAC) were evaluated for clinical status and gestational outcome; live fetuses were examined for external, visceral, and skeletal malformations. For MAC, no treatment-related maternal mortality was observed. Maternal body weight on GD 17, maternal weight gain during treatment and gestation, and corrected maternal weight gain were reduced at the high dose. Relative maternal food and water intake was not adversely affected; neurotoxicity was not observed. Relative maternal liver weight was increased at > or = 120 mg/kg/day; gravid uterine weight was decreased at 180 mg/kg/day. The maternal no-observed adverse effect level (NOAEL) was 60 mg/kg/day. The NOAEL for developmental toxicity was also 60 mg/kg/day. At > or = 120 mg/kg/day, mean fetal body weight was reduced. At 180 mg/kg/day, increased postimplantation death per litter was observed. Morphological development was not affected. The maternal NOAEL for BAC was 10 mg/kg/day. At 30 mg/kg/day, decreased maternal body weight on GD 17, maternal body weight change during treatment and gestation, corrected maternal body weight, and gravid uterine weight were observed. Relative maternal liver weight increased at 30 mg/kg/day. The developmental NOAEL was 3 mg/kg/day BAC. Mean fetal body weight was reduced at 30 mg/kg/day. At > or = 10 mg/kg/day, an increased incidence of fetal variations (extra rib) was observed, although fetal malformation rate was unaffected. MAC and BAC were not teratogenic to Swiss mice at the doses tested. BAC was more potent than MAC in causing adverse maternal and developmental effects.

Differential follicle counts as a screen for chemically induced ovarian toxicity in mice: results from continuous breeding bioassays.

Ovaries from National Toxicology Program Reproductive Assessment by Continuous Breeding (RACB) bioassays were used to directly compare differential ovarian follicle counts and reproductive performance for 15 chemicals. Ovaries of 10 animals per group from 16 studies in CD-1 mice and 1 study each in C3H and C57BL/6 mice were sectioned serially at 6 microm. Counts of small, growing, and antral follicles were obtained in every 10th section. For all follicle types, younger mice had more follicles than older mice, and CD-1 mice had more follicles than age-matched animals from either inbred strain. The in-life portion of the RACB protocols demonstrated that 9 of 15 chemicals altered reproductive outcome in one or both sexes of mice, with six agents affecting females (R. E. Morrissey et al., 1989, Fundam. Appl. Toxicol. 13, 747-777). Three of six female toxicants [2,2-bis(bromoethyl)-1,3-propanediol, BPD; ethylene glycol monomethyl ether, EGME; methoxyacetic acid, MAA] significantly decreased counts of small and/or growing follicles by 33 to 92% in CD-1 mice; EGME also reduced follicle counts in the other strains. Follicle counts were decreased in progeny of animals treated with EGME or its active metabolite, MAA. For BPD, reductions in follicle numbers were proportional to dose. In CD-1 mice, female toxicants di-N-hexyl phthalate, propantheline bromide, and tricresyl phosphate reduced reproductive performance but not follicle numbers. Counts were not affected by toxicants for which the susceptible sex could not be determined (bisphenol A, ethylene glycol, oxalic acid). Altered follicle counts without apparent reproductive impairment occurred in CD-1 mice at lower doses of BPD but were not observed for nontoxic chemicals. These data suggest that differential follicle counts (1) are a quantifiable endpoint of ovarian injury in conventional bioassays, and (2) in some instances, may provide a more sensitive indicator of female reproductive toxicity than fertility.

Influence of sampling on the reproducibility of ovarian follicle counts in mouse toxicity studies.

Different ovarian follicle counting procedures were investigated to reduce labor while retaining statistical power. Intact ovaries of untreated CD-1 mice (20/group) from National Toxicology Program Reproductive Assessment by Continuous Breeding (RACB) studies were serially sectioned at 6 microm. Mean numbers of small and growing follicles were used to assess sampling efficiency. In 10 mice per group, comparisons were made between 10% nonrandom samples from every 10th section starting at either the first or sixth section having follicles (approximately 40 sections per ovary). These 10% counts were compared with 5% (20 sections) and 20% (80 sections) nonrandom samples and with 1% (4 sections), 5%, or 10% random samples from the same 10 animals. For two studies, a 10% nonrandom sample was analyzed from 20 mice per group. Follicle counts for each group were comparable regardless of the sampling paradigm. Four to 10 animals provided 90% confidence that a 20% difference in mean counts would be detected. The 1% sample had a larger error term and, thus, slightly reduced statistical power. These data suggest that follicle counts from 1% or 5% random samples may provide a suitable screen for ovarian toxicity.

Evaluation of the developmental toxicity of methacrylonitrile in Sprague-Dawley rats and New Zealand white rabbits.

Timed-pregnant Sprague-Dawley (CD) outbred rats and New Zealand White rabbits were dosed by gavage with methacrylonitrile (MACR) in distilled water during major organogenesis. Rats were dosed on Gestational Days (GD) 6 through 15 (0, 5, 25, or 50 mg MACR/kg/day) and rabbits on GD 6 through 19 (0, 1, 3, or 5 mg MACR/kg/day). Maternal clinical status was monitored daily during treatment. At termination (GD 20, rats; GD 30, rabbits), confirmed-pregnant females (25-26 per group, rats; 17-22 per group, rabbits) were evaluated for clinical status and gestational outcome; each live fetus was examined for external, visceral, and skeletal malformations. In rats, no treatment-related maternal clinical signs or mortality were observed, nor was there any adverse effect on maternal body weight or food or water consumption. At necropsy, absolute, relative, and adjusted maternal liver weight was increased at the mid- and high-dose groups, an effect that may be indicative of induction of hepatic enzymes rather than toxicity. In the absence of any indication of maternal toxicity, the no-observed-adverse-effect level (NOAEL) for maternal toxicity in this study was >/=50 mg MACR/kg/day. The NOAEL for developmental toxicity in rats was also >/=50 mg MACR/kg/day. There was no effect of treatment on postimplantation loss, mean fetal body weight per litter, or morphological development. In rabbits, maternal mortality and clinical signs were not dose related. Maternal food consumption, body weight, and liver weight were not adversely affected by treatment. Thus, the maternal NOAEL was >/=5 mg MACR/kg/day. Maternal toxicity, including death, was observed >/=7.5 mg/kg/day in preliminary studies. The developmental NOAEL was also >/=5 mg MACR/kg/day. There was no adverse effect of treatment on postimplantation loss or fetal body weight. A significant decrease in the percentage male fetuses per litter was observed, although there was no effect on total live litter size, suggesting that the reduction in the ratio of live male fetuses in the high-dose group was not biologically significant. MACR had no adverse effect on morphological development. In summary, oral administration of MACR to rats and rabbits during organogenesis, at doses that did not cause persistent maternal toxicity (50 mg MACR/kg/day, rats; 5 mg MACR/kg/day, rabbits), also did not cause any adverse developmental effects.

The developmental toxicity of boric acid in rabbits.

Boric acid (BA), an ingredient of many pharmaceutical, cosmetic, and pesticide products, was previously shown to induce reproductive and developmental toxicity in laboratory rodents. In this study, BA (0, 62.5, 125, or 250 mg/kg/day, po) was administered on Gestational Days (GD) 6-19 to New Zealand White rabbits (18-23 pregnant/group). Maternal body weight, food consumption, and clinical condition were monitored at regular intervals throughout gestation. At termination (GD 30), the numbers of uterine implantations, resorptions, dead fetuses, and live fetuses were determined. Fetuses were weighed, and live fetuses examined for external, visceral, and skeletal defects. Maternal food intake decreased during treatment at 250 mg/kg/day and increased at >/=125 mg/kg/day after treatment. Maternal body weight (GD 9-30), weight gain during treatment, gravid uterine weight, and number of ovarian corpora lutea decreased at 250 mg/kg/day. In contrast, maternal corrected gestational weight gain increased at >/=125 mg/kg/day. Maternal liver weight was not affected. Relative (but not absolute) maternal kidney weight increased at 250 mg/kg/day, and microscopic evaluation revealed no treatment-related renal pathology. At 250 mg/kg/day, prenatal mortality was increased (90% resorptions/litter vs 6% for controls), the proportion of pregnant females with no live fetuses was increased (73% vs 0%), and live litter size was reduced (2.3 fetuses/litter vs 8.8). As a result, there were only 14 live fetuses (6 live litters) available for evaluation in the high-dose group, compared to 153-175 live fetuses (18-23 live litters) in the other groups. The percentage malformed fetuses/litter was increased at 250 mg/kg/day, primarily due to cardiovascular defects in 72% of high-dose fetuses vs 3% of controls. The most prevalent cardiovascular malformation (interventricular septal defect) was observed in 57% of high-dose fetuses compared to 0.6% among controls. At 250 mg/kg/day, average fetal body weight/litter was 92% of the average control weight (not statistically significant). In summary, no definitive maternal or developmental toxicity was observed at 62.5 or 125 mg/kg/day BA. Mild maternal effects and severe developmental toxicity were observed at 250 mg/kg/day.

Nitrofurazone: reproductive assessment by continuous breeding in Swiss mice.

Nitrofurazone (NTFZ), a nitrofuran antibiotic, was evaluated for reproductive toxicity in Swiss CD-1 mice using the Reproductive Assessment by Continuous Breeding protocol. Male and female mice were cohabited for 15 weeks and exposed to NTFZ in feed at concentrations of 0, 100, 375, and 750 ppm (14-102 mg/kg/day). Fzero 750-ppm breeding pairs had significantly reduced fertility after 7 days of exposure to NTFZ (17% fertile compared to 98% for control pairs) and were infertile after the second litter. Fzero mid-dose pairs had progressively decreasing fertility (47% by the fifth litter), reduced litter size, and reduced proportion of pups born alive. Crossover breeding of control and high-dose Fzero animals confirmed infertility in high-dose males and reduced litter size and pup weight in high-dose females when compared to the control x control group. At necropsy, there were no effects on body weight, but Fzero males had reduced testis weight at the high dose and reduced epididymal sperm concentration and abnormal sperm morphology at all doses of NTFZ. Increased liver as well as kidney and adrenal weights (combined) were observed at 375 and 750 ppm; hepatic hypertrophy was noted microscopically at 750 ppm. Fzero females had reduced body weight, hepatic hypertrophy, and altered estrous cycles at 750 ppm and reduced ovarian weight at all doses. In the second generation, F1 mice at 375 ppm had reduced postnatal survival and body weight and produced smaller F2 litters compared to control mice. At necropsy, F1 males had reduced testes weight and epididymal sperm concentration, abnormal sperm morphology, hepatic hypertrophy at 375 ppm, and borderline nephropathy at 100 and 375 ppm. F1 females had decreased body, liver, and ovarian weight at 375 ppm and altered estrous cycles at 100 and 375 ppm. Thus, NTFZ at > or = 100 ppm (> or = 14 mg/kg/day) caused adverse reproductive effects in Fzero male and female and F1 female mice in the presence of relatively mild systemic toxicity.

Developmental toxicity evaluation of sodium fluoride administered to rats and rabbits in drinking water.

Sodium fluoride (NaF; Cas No. 7681-49-4) is used in fluoridating municipal water supplies, resulting in chronic exposure of millions of people worldwide. Because of a lack of pertinent developmental toxicity studies in the literature, sodium fluoride was administered ad libitum in deionized/filtered drinking water (to mimic human exposure) to Sprague-Dawley-derived rats (26/group) on Gestation Days (GD) 6 through 15 at levels of 0, 50, 150, or 300 ppm and New Zealand White rabbits (26/group) on GD 6 through 19 at levels of 0, 100, 200, or 400 ppm. Higher concentrations via drinking water were not practicable due to the poor palatability of sodium fluoride. Drinking water (vehicle) contained less than 0.6 ppm sodium fluoride (limit of detection) and sodium fluoride content of the feed was 12.4 ppm fluoride (rats) and 15.6 ppm fluoride (rabbits). Maternal food, water, body weights, and clinical signs were recorded at regular intervals throughout these studies. Animals were killed on GD 20 (rats) or 30 (rabbits) and examined for implant status, fetal weight, sex, and morphological development. In the high-dose group of both studies there was an initial decreased maternal body weight gain which recovered over time and a decreased water consumption--attributed to decreased palatability. No clear clinical signs of toxicity were observed. Maternal exposure to sodium fluoride during organogenesis did not significantly affect the frequency of postimplantation loss, mean fetal body weight/litter, or external, visceral or skeletal malformations in either the rat or the rabbit. The NOAEL for maternal toxicity was 150 ppm sodium fluoride in drinking water (approximately 18 mg/kg/day) for rats, and 200 ppm (approximately 18/mg/kg/day rabbits. The NOAEL for developmental toxicity was > or = 300 ppm sodium fluoride (approximately 27 mg/kg/day) for rats and > or = 400 ppm (approximately 29 mg/kg/day) for rabbits administered during organogenesis in drinking water. The total exposure to fluoride (mg F/kg body weight/day from food and drinking water combined) in the mid- and high-dose groups for both species was > 100-fold higher than the range at 0.014-0.08 mg F/kg/day estimated for a 70-kg person from food and fluoridated (1 ppm) drinking water.

A novel hypothalamic peptide, pituitary adenylate cyclase-activating peptide, regulates the function of rat granulosa cells in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel peptide that was isolated from ovine hypothalamic tissue on the basis of its ability to stimulate cAMP accumulation in cultured rat pituitary cells. Recently we demonstrated that PACAP can stimulate cAMP accumulation and secretory function in cultured rat Sertoli cells. Since ovarian granulosa cells share many properties with Sertoli cells, we have examined the effect of PACAP (consisting of 38 or 27 amino acid residues) on cultured granulosa cell function. Granulosa cells were obtained from the ovaries of 25-day-old rats implanted with a silastic capsule containing diethylstilbestrol 5 days prior to culture. PACAP 38 (0.1 microM-0.01 pM), both alone and in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine, stimulated cAMP accumulation 4-8-fold with an ED50 of approximately 100 pM. Maximal PACAP 38 or PACAP 27 stimulation of granulosa cell cAMP was significantly greater than that produced by a maximally effective concentration of FSH. Because PACAP 38 and 27 have 68% sequence homology with vasoactive intestinal peptide (VIP), and since VIP stimulates granulosa cell cAMP accumulation and estradiol and progesterone secretion, we examined the possibility that PACAP could be acting via the VIP receptor. VIP stimulated cAMP only at concentrations of 10 nM or greater, whereas the PACAP stimulation was evident at 10 pM. Moreover, only one of three potent VIP antagonists inhibited VIP stimulation of cAMP accumulation, and only at 1 microM or greater. This VIP antagonist did not inhibit PACAP 38 action at 2000-fold excess concentration. Interestingly PACAP 38 was more effective than PACAP 27 with regard to steroid secretion and the ability to induce LH responsiveness. PACAP and VIP stimulation of granulosa cell cAMP accumulation or estradiol or progesterone secretion was not additive. Thus, these data support the hypothesis that granulosa cells have specific PACAP 38 receptors and that VIP acts via these receptors. In addition, PACAPs 38 and 27 are more potent stimulators of cAMP accumulation in luteinized granulosa cells than LH. These results both pre- and postovulation, along with previous data indicating that the PACAPs are found in the ovaries, suggest a role for PACAP in the regulation of ovarian function.

The reproductive and neural toxicities of acrylamide and three analogues in Swiss mice, evaluated using the continuous breeding protocol.

Acrylamide is a known genetic, reproductive, and neural toxicant, although it is not known if one effect is predominant. The toxicities of several structural analogues of acrylamide have been incompletely characterized, and the relative sensitivity of the second generation is not known. The present studies were designed to explore the relationship between neurotoxicity and reproductive toxicity, to further characterize the toxicities of three acrylamide analogues, and to evaluate the relative sensitivity of a second generation to these compounds. For the F0 generation, male and female Swiss CD-1 mice were provided drinking water containing acrylamide (ACR; 3, 10, 30 ppm), N,N'-methylenebisacrylamide (MBA; 10, 30, 60 ppm), N-(hydroxymethyl)acrylamide (HMA; 60, 180, 360 ppm), or methacrylamide (MACR; 24, 80, 240 ppm) during and after a 14-week cohabitation. The last litter was reared and dosed after weaning until mating at 74 +/- 10 days of age with the same level of compound given to the parents Neurotoxicity was assessed at several times in both generations by measuring forelimb and hindlimb grip strength. In the F0 generation, ACR caused an 11% decrease in pup number without measurable neurotoxicity; female fertility was not affected. Although both generations consumed the same amount of ACR, there were larger changes in the fertility-related endpoints in the F1 mice than in the F0's, with no concomitant change in organ weights or sperm parameters. In F0 mice, MBA reduced the number of live pups and their adjusted weight, with no neurotoxicity and no change in F0 female reproduction. MBA caused greater adverse effects in the second generation, concomitant with increased consumption. In the F0 generation, HMA caused the largest decrease in pup number during cohabitation (26%) together with a small effect on grip strength. Female reproduction was not affected. The second generation consumed more HMA and showed slightly greater toxic effects. In both generations, MACR was negative for both neurotoxicity and reproductive toxicity. Dominant-lethal studies showed that the fertility effects for ACR, MBA, and HMA could be explained by a male-mediated increase in postimplantation loss. These studies found that dominant lethality occurred without structural effects on the reproductive system in the presence of only minor effects on grip strength and without detectable neural histopathology. Female reproduction was not significantly affected by these compounds at the doses used. Thus, these data confirm the male as the affected gender and that the reproductive toxicity was greater than motoneuron toxicity when measured as grip strength.

Assessment of the reproductive toxicity of a complex mixture of 25 groundwater contaminants in mice and rats.

The potential reproductive toxicity of a mixture of 25 chemicals (MIX) formulated to simulate contaminated groundwater supplies near hazardous waste dumps was evaluated in CD-1 Swiss mice and Sprague-Dawley rats using the reproductive assessment by continuous breeding protocol. Male and female mice and rats were exposed to MIX in the drinking water at concentrations of 1, 5, and 10% of a technically achievable stock solution. For mice, body weight and feed consumption were not affected by MIX but water consumption was decreased for both the 5 and 10% MIX groups in both F0 and F1 animals. For F0 mice, the number of live pups/litter was decreased at 10% MIX and the number of females/litter was decreased 10 and 17% at the mid and high MIX dose, respectively. Vaginal cytology was normal, as were testis weight and testicular spermatid head count. For F1 mice, fertility was unaffected, but there was a decreased number of female pups/litter (19%) and a decreased adjusted live pup weight at 10% MIX. At necropsy, cauda epididymal sperm concentration and spermatid head count were reduced (20%) in the presence of normal testis, epididymis, prostate, seminal vesicle, liver, and kidney/adrenal weight. Female estrous cyclicity was altered at 5 and 10% MIX with normal kidney/adrenal, uterus, and ovary/oviduct weight. For rats, F0 body weight and feed consumption were not affected by MIX but water consumption was decreased 10, 30, and 40% in the low-, medium-, and high-dose MIX groups, respectively, and 39% in the high-dose MIX F1 animals. Rat fertility was normal but there was a decreased number of male pups/litter (11%) and a decreased live pup weight (6%) at 10% MIX. Male and female (F1) pup weights were decreased on Postnatal Days 0, 4, 7, 14, and 21 (10% MIX) and remained lower through necropsy on Day 120 +/- 10. F1 fertility was normal but F2 pup weights were decreased (10% MIX). At necropsy, F1 (10% MIX) male body weight was decreased 16% and relative kidney, testis, epididymis, and prostate weights were increased in the presence of normal sperm concentration percentage motile sperm and percentage abnormal sperm. Estrous cyclicity was normal as were kidney/adrenal and ovary weight while female liver weight was reduced 14%. In summary, a "cocktail" of 25 chemicals commonly found in contaminated groundwater at or near hazardous waste sites was administered in drinking water at doses which resulted in severely decreased water consumption in both mice and rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Carisoprodol: reproductive assessment by continuous breeding in Swiss mice.

Carisoprodol (CARI), a commonly prescribed neuromuscular relaxant, was evaluated for reproductive toxicity in Swiss CD-1 mice using the Reproductive Assessment by Continuous Breeding (RACB) protocol. Male and female mice were given CARI in corn oil suspension by daily gavage at doses of 0, 300, 750, and 1200 mg/kg body wt/day. Clinical signs of general toxicity in F0 animals included sedation, primarily in the high-dose group during the first week of exposure, and reduced body weight in high-dose females. CARI administration for 14 weeks did not affect the ability of the F0 animals to produce litters. However, decreases in proportion of pups born alive (4%) and absolute (5%) and adjusted live pup weight (7%) were observed at 1200 mg/kg CARI when compared to controls. In a crossover mating trial to determine the affected sex, there were no significant differences in the measured reproductive parameters. CARI at the high dose increased the proportion of time spent in proestrus and estrus, but cycle length was unaffected. At F0 necropsy (Week 27 of treatment), all sperm parameters were normal. Right epididymis and liver weights, relative to body weight, were increased (12 and 23%, respectively) over the control group for high-dose males. A mating trial to determine the fertility and reproductive competence of the F1 generation showed no effect of CARI on indices of mating, pregnancy, or fertility, the proportion of F2 pups born alive, the sex ratio of live F2 pups, live F2 pup weight, or gestation length.(ABSTRACT TRUNCATED AT 250 WORDS)

The developmental toxicity of boric acid in mice, rats, and rabbits.

Boric acid (BA) is a naturally occurring agent used in manufacturing processes and numerous consumer products. Because of the potential for both industrial and consumer exposure to boron-containing compounds, and the lack of developmental toxicity data, the National Toxicology Program evaluated the potential for boric acid to cause developmental toxicity in pregnant Swiss (CD-1) mice, Sprague-Dawley rats (n = 26-28/group), and New Zealand rabbits (n = 18-23/group). BA was provided in the feed to mice and rats at 0, 0.1, 0.2, or 0.4% throughout gestation to attain steady-state exposure as early as possible during development. Average doses (mg/kg/day) were 248, 452, or 1003 for mice, and 78, 163, or 330 in rats. A separate group of rats received 0.8% BA in the feed, or 539 mg/kg/day only on gestation days (gd) 6 to 15. Rabbits were given BA (0, 62.5, 125, or 250 mg/kg) by gavage administration on gd 6 to 19. Maternal body weight, food and/or water consumption and signs of toxicity were monitored at regular intervals. At termination, gd 17 (mice), 20 (rats), or 30 (rabbits), the uterus was examined to determine the number of resorptions, dead, or live fetuses. Fetuses were weighed and live fetuses were examined for external, visceral, and skeletal defects. Mouse dams exhibited mild renal lesions (> or = 248 mg/kg/day BA), increased water intake and relative kidney weight (1003 mg/kg/day BA), and decreased weight gain during treatment.(ABSTRACT TRUNCATED AT 250 WORDS)