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Background: Some disinfection byproducts (DBPs) are teratogens based on toxicological evidence. Conventional use of predominant DBPs as proxies for complex mixtures may result in decreased ability to detect associations in epidemiological studies., Objective: We assessed risks of obstructive genitourinary birth defects (OGDs) in relation to 12 DBP mixtures and 13 individual component DBPs., Methods: We designed a nested registry-based case-control study (210 OGD cases; 2100 controls) in Massachusetts towns with complete quarterly 1999-2004 data on four trihalomethanes (THMs) and five haloacetic acids (HAAs). We estimated temporally-weighted average DBP exposures for the first trimester of pregnancy. We estimated adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for OGD in relation to individual DBPs, unweighted mixtures, and weighted mixtures based on THM/HAA relative potency factors (RPF) from animal toxicology data for full-litter resorption, eye defects, and neural tube defects., Results: We detected elevated aORs for OGDs for the highest of bromodichloromethane (aOR = 1.75; 95% CI: 1.15-2.65), dibromochloromethane (aOR = 1.71; 95% CI: 1.15-2.54), bromodichloroacetic acid (aOR = 1.56; 95%CI: 0.97-2.51), chlorodibromoacetic acid (aOR = 1.97, 95% CI: 1.23-3.15), and tribromoacetic acid (aOR = 1.90; 95%CI: 1.20-3.03). Across unweighted mixture sums, the highest aORs were for the sum of three brominated THMs (aOR = 1.74; 95% CI: 1.15-2.64), the sum of six brominated HAAs (aOR = 1.43; 95% CI: 0.89-2.31), and the sum of nine brominated DBPs (aOR = 1.80; 95% CI: 1.05-3.10). Comparing eight RPF-weighted to unweighted mixtures, the largest aOR differences were for two HAA metrics, which both were higher with RPF weighting; other metrics had reduced or minimally changed ORs in RPF-weighted models., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: In developmental and reproductive toxicity studies, analysis of litter-based binary endpoints (e.g., incidence of malformed fetuses) is complex in that littermates often are not entirely independent of one another. It is well established that the litter, not the individual fetus, is the proper independent experimental unit in statistical analysis. Accordingly, analysis is often based on the proportion affected per litter and the litter proportions are analyzed as continuous data. Because these proportional data generally do not meet assumptions of symmetry or normality, data are typically analyzed by nonparametric methods, arcsine square root transformation, or logit transformation., Methods: We conducted power calculations to compare different approaches (nonparametric, arcsine square root-transformed, logit-transformed, untransformed) for analyzing litter-based proportional data. A reproductive toxicity study with a control and one treated group provided data for two endpoints: prenatal loss, and fertility by in utero insemination (IUI). Type 1 error and power were estimated by 10,000 simulations based on two-sample one-tailed t tests with varying numbers of litters per group. To further compare the different approaches, we conducted additional analyses with shifted mean proportions to produce illustrative scenarios., Results: Analyses based on logit-transformed proportions had greater power than those based on untransformed or arcsine square root-transformed proportions, or nonparametric procedures., Conclusion: The logit transformation is preferred to the other approaches considered when making inferences concerning litter-based proportional endpoints, particularly with skewed distributions. The improved performance of the logit transformation becomes increasingly pronounced as the response proportions are increasingly close to the boundaries of the parameter space., (© 2020 Wiley Periodicals LLC.)

Background: Epidemiological studies suggest that exposure to water disinfection by-products (DBPs) may increase the risk of certain birth defects. However, evidence for musculoskeletal defects (MSDs) is limited. Previous MSD studies have not examined DBPs beyond trihalomethanes (THMs) and have not separately examined limb or diaphragm defects which may have distinct developmental etiologies., Methods: We calculated adjusted odds ratios (aORs) in a registry-based case-control study of birth defects in Massachusetts with complete quarterly 1999-2004 data on four THMs and five haloacetic acids (HAAs). We matched 10 controls each to 187 MSD cases based on week of conception. Weight-averaged town-level first-trimester DBP exposures were individually assigned based on residence at birth. We adjusted THM models for exposure to the sum of five HAAs (HAA5), and HAA models for the sum of four THMs (THM4)., Results: We detected positive exposure-response associations for all grouped MSDs with THM4 quintiles (aOR range: 1.90-3.18) and chloroform quartiles (aOR range: 1.30-2.21), and for reduction of upper or lower limbs with chloroform quartiles (aOR range: 2.39-3.52). We detected elevated aORs for diaphragmatic hernia with DBP9 (sum of THM4 and HAA5), and chloroform and bromodichloromethane tertiles and an exposure-response relationship for THM4 tertiles (aOR range: 1.67-1.80)., Conclusions: This is the first epidemiological study to examine HAAs in relation to MSDs. Given the indirect nature of our exposure assessment data and small case numbers, the exposure-response relationships that we detected for THM4 and chloroform warrant further investigation., Competing Interests: CONFLICTS OF INTEREST: None declared

Objective: The aim of this study was to examine associations between craniofacial birth defects (CFDs) and disinfection by-product (DBP) exposures, including the sum of four trihalomethanes (THM4) and five haloacetic acids (HAA5) (ie, DBP9)., Methods: We calculated first trimester adjusted odds ratios (aORs) for different DBPs in a matched case-control study of 366 CFD cases in Massachusetts towns with complete 1999 to 2004 THM and HAA data., Results: We detected elevated aORs for cleft palate with DBP9 (highest quintile aOR = 3.52; 95% CI: 1.07, 11.60), HAA5, trichloroacetic acid (TCAA), and dichloroacetic acid. We detected elevated aORs for eye defects with TCAA and chloroform., Conclusion: This is the first epidemiological study of DBPs to examine eye and ear defects, as well as HAAs and CFDs. The associations for cleft palate and eye defects highlight the importance of examining specific defects and DBPs beyond THM4.

A method based on regression modeling was developed to discern the contribution of component chemicals to the toxicity of highly complex, environmentally realistic mixtures of disinfection byproducts (DBPs). Chemical disinfection of drinking water forms DBP mixtures. Because of concerns about possible reproductive and developmental toxicity, a whole mixture (WM) of DBPs produced by chlorination of a water concentrate was administered as drinking water to Sprague-Dawley (S-D) rats in a multigenerational study. Age of puberty acquisition, i.e., preputial separation (PPS) and vaginal opening (VO), was examined in male and female offspring, respectively. When compared to controls, a slight, but statistically significant delay in puberty acquisition was observed in females but not in males. WM-induced differences in the age at puberty acquisition were compared to those reported in S-D rats administered either a defined mixture (DM) of nine regulated DBPs or individual DBPs. Regression models were developed using individual animal data on age at PPS or VO from the DM study. Puberty acquisition data reported in the WM and individual DBP studies were then compared with the DM models. The delay in puberty acquisition observed in the WM-treated female rats could not be distinguished from delays predicted by the DM regression model, suggesting that the nine regulated DBPs in the DM might account for much of the delay observed in the WM. This method is applicable to mixtures of other types of chemicals and other endpoints., (Copyright © 2017. Published by Elsevier B.V.)

Background: Epidemiological studies suggest that women exposed to disinfection by-products (DBPs) have an increased risk of delivering babies with cardiovascular defects (CVDs)., Objective: We examined nine CVDs in relation to categorical DBP exposures including bromoform, chloroform, dibromochloromethane (DBCM), bromodichloromethane (BDCM), monobromoacetic acid (MBAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), and summary DBP measures (HAA5, THMBr, THM4, and DBP9)., Methods: We calculated adjusted odds ratios (aORs) in a case-control study of birth defects in Massachusetts with complete quarterly 1999-2004 trihalomethane (THM) and haloacetic acid (HAA) data. We randomly matched 10 controls each to 904 CVD cases based on week of conception. Weight-averaged aggregate first-trimester DBP exposures were assigned to individuals based on residence at birth., Results: We detected associations for tetralogy of Fallot and the upper exposure categories for TCAA, DCAA, and HAA5 (aOR range, 3.34-6.51) including positive exposure-response relationships for DCAA and HAA5. aORs consistent in magnitude were detected between atrial septal defects and bromoform (aOR = 1.56; 95% CI: 1.01, 2.43), as well as DBCM, chloroform, and THM4 (aOR range, 1.26-1.67). Ventricular septal defects (VSDs) were associated with the highest bromoform (aOR = 1.85; 95% CI: 1.20, 2.83), MBAA (aOR = 1.81; 95% CI: 0.85, 3.84), and DBCM (aOR = 1.54; 95% CI: 1.00, 2.37) exposure categories., Conclusions: To our knowledge, this is the first birth defect study to develop multi-DBP adjusted regression models as well as the first CVD study to evaluate HAA exposures and the second to evaluate bromoform exposures. Our findings, therefore, inform exposure specificity for the consistent associations previously reported between THM4 and CVDs including VSDs. Citation: Wright JM, Evans A, Kaufman JA, Rivera-Núñez Z, Narotsky MG. 2017. Disinfection by-product exposures and the risk of specific cardiac birth defects. Environ Health Perspect 125:269-277; http://dx.doi.org/10.1289/EHP103., Competing Interests: The views expressed in this manuscript are those of the authors and do not necessarily reflect the views or policies of the U.S. EPA or the ASPPH. The authors declare they have no actual or potential competing financial interests.

The 2011 EPA trichloroethylene (TCE) IRIS assessment, used developmental cardiac defects from a controversial drinking water study in rats (Johnson et al. [51]), along with several other studies/endpoints to derive reference values. An updated literature search of TCE-related developmental cardiac defects was conducted. Study quality, strengths, and limitations were assessed. A putative adverse outcome pathway (AOP) construct was developed to explore key events for the most commonly observed cardiac dysmorphologies, particularly those involved with epithelial-mesenchymal transition (EMT) of endothelial origin (EndMT); several candidate pathways were identified. A hypothesis-driven weight-of-evidence analysis of epidemiological, toxicological, in vitro, in ovo, and mechanistic/AOP data concluded that TCE has the potential to cause cardiac defects in humans when exposure occurs at sufficient doses during a sensitive window of fetal development. The study by Johnson et al. [51] was reaffirmed as suitable for hazard characterization and reference value derivation, though acknowledging study limitations and uncertainties., (Published by Elsevier Inc.)

Background: Trihalomethanes (THMs) and haloacetic acids (HAAs) are regulated disinfection by-products (DBPs); their joint reproductive toxicity in drinking water is unknown., Objective: We aimed to evaluate a drinking water mixture of the four regulated THMs and five regulated HAAs in a multigenerational reproductive toxicity bioassay., Methods: Sprague-Dawley rats were exposed (parental, F1, and F2 generations) from gestation day 0 of the parental generation to postnatal day (PND) 6 of the F2 generation to a realistically proportioned mixture of THMs and HAAs at 0, 500×, 1,000×, or 2,000× of the U.S. Environmental Protection Agency's maximum contaminant levels (MCLs)., Results: Maternal water consumption was reduced at ≥ 1,000×; body weights were reduced at 2,000×. Prenatal and postnatal survival were unaffected. F1 pup weights were unaffected at birth but reduced at 2,000× on PND6 and at ≥ 1,000× on PND21. Postweaning F1 body weights were reduced at 2,000×, and water consumption was reduced at ≥ 500×. Males at 2,000× had a small but significantly increased incidence of retained nipples and compromised sperm motility. Onset of puberty was delayed at 1,000× and 2,000×. F1 estrous cycles and fertility were unaffected, and F2 litters showed no effects on pup weight or survival. Histologically, P0 (parental) dams had nephropathy and adrenal cortical pathology at 2,000×., Conclusions: A mixture of regulated DBPs at up to 2,000× the MCLs had no adverse effects on fertility, pregnancy maintenance, prenatal survival, postnatal survival, or birth weights. Delayed puberty at ≥ 1,000× may have been secondary to reduced water consumption. Male nipple retention and compromised sperm motility at 2,000× may have been secondary to reduced body weights.

Some epidemiological studies report associations between drinking water disinfection byproducts (DBPs) and adverse reproductive/developmental effects, e.g., low birth weight, spontaneous abortion, stillbirth, and birth defects. Using a multigenerational rat bioassay, we evaluated an environmentally relevant "whole" mixture of DBPs representative of chlorinated drinking water, including unidentified DBPs as well as realistic proportions of known DBPs at low-toxicity concentrations. Source water from a water utility was concentrated 136-fold, chlorinated, and provided as drinking water to Sprague-Dawley rats. Timed-pregnant females (P0 generation) were exposed during gestation and lactation. Weanlings (F1 generation) continued exposures and were bred to produce an F2 generation. Large sample sizes enhanced statistical power, particularly for pup weight and prenatal loss. No adverse effects were observed for pup weight, prenatal loss, pregnancy rate, gestation length, puberty onset in males, growth, estrous cycles, hormone levels, immunological end points, and most neurobehavioral end points. Significant, albeit slight, effects included delayed puberty for F1 females, reduced caput epidydimal sperm counts in F1 adult males, and increased incidences of thyroid follicular cell hypertrophy in adult females. These results highlight areas for future research, while the largely negative findings, particularly for pup weight and prenatal loss, are notable.

A developmental toxicity bioassay was used in three experiments to evaluate water concentrates for suitability in multigenerational studies. First, chlorinated water was concentrated 135-fold by reverse osmosis; select lost disinfection by-products were spiked back. Concentrate was provided as drinking water to Sprague-Dawley and F344 rats from gestation day 6 to postnatal day 6. Maternal serum levels of luteinizing hormone on gestation day 10 were unaffected by treatment for both strains. Treated dams had increased water consumption, and increased incidences of polyuria, diarrhea, and (in Sprague-Dawley rats) red perinasal staining. Pup weights were reduced. An increased incidence of eye defects was seen in F344 litters. Chemical analysis of the concentrate revealed high sodium (6.6 g/l) and sulfate (10.4 g/l) levels. To confirm that these chemicals caused polyuria and osmotic diarrhea, respectively, Na₂SO₄ (5-20 g/l) or NaCl (16.5 g/l) was provided to rats in drinking water. Water consumption was increased at 5- and 10-g Na₂SO₄/l and with NaCl. Pup weights were reduced at 20-g Na₂SO₄/l. Dose-related incidences and severity of polyuria and diarrhea occurred in Na₂SO₄-treated rats; perinasal staining was seen at 20 g/l. NaCl caused polyuria and perinasal staining, but not diarrhea. Subsequently, water was concentrated ∼120-fold and sulfate levels were reduced by barium hydroxide before chlorination, yielding lower sodium (≤1.5 g/l) and sulfate (≤2.1 g/l) levels. Treatment resulted in increased water consumption, but pup weight and survival were unaffected. There were no treatment-related clinical findings, indicating that mixtures produced by the second method are suitable for multigenerational testing., (Published 2012 by Wiley Periodicals, Inc.)

In rodent bioassays where chemicals are administered in the drinking water, water consumption data for individual animals are needed to estimate chemical exposures accurately. If multiple animals share a common water source, as occurs in some studies, only the total amount of drinking water consumed by all animals utilizing the common source is directly measurable, and water consumption rates for individual animals are not available. In the Four Lab Study of the US Environmental Protection Agency, which included a multigenerational rodent bioassay, a complex mixture of drinking water disinfection by-products was delivered to multiple Sprague-Dawley rats from a common drinking water container. To estimate disinfection by-product mixture exposure for each animal, authors developed four log-linear regression models to allocate water consumption among rats sharing a common water container. The four models represented three animal lifestages: Gestation, Lactation, and Postweaning, with separate Postweaning models for male and female. Authors used data from six Sprague-Dawley rat bioassays to develop these models from available individual cage data for the Postweaning models, and available individual animal data for the Gestation and Lactation models. The r(2) values for the model fits were good, ranging from 0.67 to 0.92. The Gestation and Lactation models were generally quite accurate in predicting average daily water consumption whereas the Postweaning models were less robust. These models can be generalized for use in other reproductive and developmental bioassays where common water sources are used and data on the explanatory variables are available.

In complex mixture toxicology, there is growing emphasis on testing environmentally representative doses that improve the relevance of results for health risk assessment, but are typically much lower than those used in traditional toxicology studies. Traditional experimental designs with typical sample sizes may have insufficient statistical power to detect effects caused by environmentally relevant doses. Proper study design, with adequate statistical power, is critical to ensuring that experimental results are useful for environmental health risk assessment. Studies with environmentally realistic complex mixtures have practical constraints on sample concentration factor and sample volume as well as the number of animals that can be accommodated. This article describes methodology for calculation of statistical power for non-independent observations for a multigenerational rodent reproductive/developmental bioassay. The use of the methodology is illustrated using the U.S. EPA's Four Lab study in which rodents were exposed to chlorinated water concentrates containing complex mixtures of drinking water disinfection by-products. Possible experimental designs included two single-block designs and a two-block design. Considering the possible study designs and constraints, a design of two blocks of 100 females with a 40:60 ratio of control:treated animals and a significance level of 0.05 yielded maximum prospective power (~90%) to detect pup weight decreases, while providing the most power to detect increased prenatal loss.

Reactions between chemicals used to disinfect drinking water and compounds present in source waters produce chemical mixtures containing hundreds of disinfection byproducts (DBPs). Although the results have been somewhat inconsistent, some epidemiological studies suggest associations may exist between DBP exposures and adverse developmental outcomes. The potencies of individual DBPs in rodent and rabbit developmental bioassays suggest that no individual DBP can account for the relative risk estimates reported in the positive epidemiologic studies, leading to the hypothesis that these outcomes could result from the toxicity of DBP mixtures. As a first step in a mixtures risk assessment for DBP developmental effects, this paper identifies developmentally toxic DBPs and examines data relevant to the mode of action (MOA) for DBP developmental toxicity. We identified 24 developmentally toxic DBPs and four adverse developmental outcomes associated with human DBP exposures: spontaneous abortion, cardiovascular defects, neural tube defects, and low birth weight infancy. A plausible MOA, involving hormonal disruption of pregnancy, is delineated for spontaneous abortion, which some epidemiologic studies associate with total trihalomethane and bromodichloromethane exposures. The DBP data for the other three outcomes were inadequate to define key MOA steps., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Few studies have investigated the long-term effects of atrazine (ATR) following in utero exposure. We evaluated the effects of gestational exposure of Sprague Dawley dams to ATR (0, 1, 5, 20, or 100mg/kg-d) on the reproductive development of male offspring. We also quantified the distribution of ATR and its chlorinated metabolites in maternal, fetal, and neonatal fluid and tissue samples following gestational and/or lactational exposure. Dose-dependent levels of chlorotriazines, primarily diamino-s-chlorotriazine, were present in most samples analyzed, including fetal tissue. In utero exposure to 1-20mg/kg-d ATR did not alter testosterone production, the timing of puberty, play behavior, or other androgen-dependent endpoints of male offspring. Significant maternal toxicity and postnatal mortality were observed at 100mg/kg-d. We conclude that, although levels of chlorotriazines within the fetus were considerable, gestational exposures of 1-20mg/kg-d do not lead to alterations in the measures of male development examined in this study., (Published by Elsevier Inc.)

Atrazine (ATR) is an herbicide that exerts negative reproductive effects. We examined the effects of vehicle or ATR (1, 5, 20 and 100mg/kg-d), administered to Sprague-Dawley rats on gestational days 14-21, once daily or divided into two doses per day, on female offspring reproductive indices. Offspring body weights at birth were reduced and mortality increased in the 100mg/kg-d group shortly after birth; by PND 21 there were no significant effects. Vaginal opening was delayed in this group, indicating delayed puberty. No significant differences in mammary gland development were apparent at PND 45, or estrous cyclicity through PND 272. There were no differences between dosing regimens. Lower ATR doses (0-20mg/kg-d) showed few effects in females prenatally exposed to ATR, while the high dose (100mg/kg-d) reduced offspring body weight and delayed vaginal opening. Nonetheless, it is unlikely that environmental exposure comparable to the high dose would be encountered., (Published by Elsevier Inc.)

Chlorination of drinking water yields hundreds of disinfection by-products (DBPs). Among the DBPs, four trihalomethanes (THMs; chloroform, bromodichloromethane, chlorodibromomethane, bromoform) and five haloacetic acids (HAAs; chloroacetic, dichloroacetic, trichloroacetic, bromoacetic, and dibromoacetic acid) are U.S. EPA regulated. We assessed the combined toxicity of these DBPs. F344 rats were treated with mixtures of the four THMs (THM4), the five HAAs (HAA5), or nine DBPs (DBP9; THM4+HAA5). Mixtures were administered in 10% Alkamuls(®) EL-620 daily by gavage on gestation days 6-20. Litters were examined postnatally. All three mixtures caused pregnancy loss at ≥ 613 μmol/kg/day. In surviving litters, resorption rates were increased in groups receiving HAA5 at 615 μmol/kg/day and DBP9 at 307 μmol/kg/day. HAA5 caused eye malformations (anophthalmia, microphthalmia) at ≥ 308 μmol/kg/day. Thus, both HAAs and THMs contributed to DBP9-induced pregnancy loss. The presence of THMs in the full mixture, however, appeared to reduce the incidence of HAA-induced eye defects., (Published by Elsevier Inc.)

The U.S. Environmental Protection Agency's "Four Lab Study" involved participation of researchers from four national Laboratories and Centers of the Office of Research and Development along with collaborators from the water industry and academia. The study evaluated toxicological effects of complex disinfection byproduct (DBP) mixtures, with an emphasis on reproductive and developmental effects that have been associated with DBP exposures in some human epidemiologic studies. This paper describes a new procedure for producing chlorinated drinking water concentrate for animal toxicology experiments, comprehensive identification of >100 DBPs, and quantification of 75 priority and regulated DBPs. In the research reported herein, complex mixtures of DBPs were produced by concentrating a natural source water with reverse osmosis membranes, followed by addition of bromide and treatment with chlorine. By concentrating natural organic matter in the source water first and disinfecting with chlorine afterward, DBPs (including volatiles and semivolatiles) were formed and maintained in a water matrix suitable for animal studies. DBP levels in the chlorinated concentrate compared well to those from EPA's Information Collection Rule (ICR) and a nationwide study of priority unregulated DBPs when normalized by total organic carbon (TOC). DBPs were relatively stable over the course of the animal studies (125 days) with multiple chlorination events (every 5-14 days), and a significant portion of total organic halogen was accounted for through a comprehensive identification approach. DBPs quantified included regulated DBPs, priority unregulated DBPs, and additional DBPs targeted by the ICR. Many DBPs are reported for the first time, including previously undetected and unreported haloacids and haloamides. The new concentration procedure not only produced a concentrated drinking water suitable for animal experiments, but also provided a greater TOC concentration factor (136×), enhancing the detection of trace DBPs that are often below detection using conventional approaches.

Rodent toxicity studies typically use water bottles to administer test chemicals via drinking water. However, water bottles provide inconsistent exposure of volatile chemicals due to varying headspace, and lead to excessive waste of test material. To refine drinking water toxicity studies in rodents by enhancing sample quality and consistency, and minimizing waste, we designed and implemented a novel water delivery system that keeps the water chilled, headspace free and protected from light. Materials used were resistant to chemical interaction. In this gravity-fed system, a 6-L Teflon water bag, stored in a polystyrene cooler on the cage rack, was connected to a stainless steel manifold delivering water to five cages via specialized drinking valves. Due to the absence of headspace in the water bag, this system allows consistent exposure of volatile chemicals. In addition, small diameter tubing throughout the system reduces the amount of test material residing in the system and minimizes chemical waste.

In accordance with most toxicity guidelines, developmental studies typically utilize repeated exposures, usually throughout gestation or during organogenesis in particular. However, it is known that developmental toxicity may occur in response to single exposures, especially during specific windows of susceptibility. An overview of the available literature gave sufficient evidence that for many agents, the same endpoints observed in repeated dose, multiple-day studies were also observed in single-day exposures, thus indicating the relevance of developmental toxicity to health assessments of acute exposures. Further, results of benchmark dose modeling of developmental endpoints indicated that for embryo lethality, single-day exposures required a two- to fourfold higher dose than the multiple-day exposures to produce the same level of response. For fused sternebrae, exposures on specific days produced equivalent levels of response at doses that were more similar to those utilized in the repeated exposures. Appreciable differences in biological half-life (and corresponding dose metrics) as well as specific windows of susceptibility may partially explain the observed multiple- vs. single-day exposure dose-response relationships. Our results highlight the need of a more thorough evaluation of outcomes from repeated dose developmental toxicity studies in regards to their importance to chronic and acute risk assessments.

Epidemiological and animal toxicity studies have raised concerns regarding possible adverse health effects of disinfection by-products (DBPs) found in drinking water. The classes and concentrations of DBPs are influenced by the choice of disinfection process (e.g., chlorination, ozonation) as well as source water characteristics (e.g., pH, total organic carbon, bromide content). Disinfected waters were found to contain more than 500 compounds, many of which remain unidentified. Therefore, a "whole-mixture" approach was used to evaluate the toxic potential of alternative disinfection scenarios. An in vivo developmental toxicity screen was used to evaluate the adverse developmental effects of the complex mixtures produced by two different disinfection processes. Water was obtained from East Fork Lake, Ohio; spiked with iodide and bromide; and disinfected either by chlorination or by ozonation/postchlorination, producing finished drinking water suitable for human consumption. These waters were concentrated approximately 130-fold by reverse osmosis membrane techniques. To the extent possible, volatile DBPs lost in the concentration process were spiked back into the concentrates. These concentrates were then provided as drinking water to Sprague-Dawley rats on gestation days 6-16; controls received boiled, distilled, deionized water. The dams (19-20 per group) were allowed to deliver and their litters were examined on postnatal days (PD) 1 and 6. All dams delivered normally, with parturition occurring significantly earlier in the ozonation/postchlorination group. However, no effects on prenatal survival, postnatal survival, or pup weight were evident. Skeletal examination of the PD-6 pups also revealed no treatment effects. Thus, approximately 130-fold higher concentrates of both ozonated/postchlorinated and chlorinated water appeared to exert no adverse developmental effects in this study.

This article presents a toxicologically-based risk assessment strategy for identifying the individual components or fractions of a complex mixture that are associated with its toxicity. The strategy relies on conventional component-based mixtures risk approaches such as dose addition, response addition, and analyses of interactions. Developmental toxicity data from two drinking-water concentrates containing disinfection by-products (DBP) mixtures were used to illustrate the strategy. The results of this study showed that future studies of DBP concentrates using the Chernoff-Kavlock bioassay need to consider evaluating DBP that are concentrated more than 130-fold and using a rat strain that is more sensitive to chemically-induced pregnancy loss than Sprague-Dawley rats. The results support the planned experimental design of a multigeneration reproductive and developmental study of DBP concentrates. Finally, this article discusses the need for a systematic evaluation of DBP concentrates obtained from multiple source waters and treatment types. The development of such a database could be useful in evaluating whether a specific DBP concentrate is sufficiently similar to tested combinations of source waters and treatment alternatives so that health risks for the former may be estimated using data on the latter.

This article describes the disinfection by-product (DBP) characterization portion of a series of experiments designed for comprehensive chemical and toxicological evaluation of two drinking-water concentrates containing highly complex mixtures of DBPs. This project, called the Four Lab Study, involved the participation of scientists from four laboratories and centers of the U.S. Environmental Protection Agency (EPA) Office of Research and Development, along with collaborators from the water industry and academia, and addressed toxicologic effects of complex DBP mixtures, with an emphasis on reproductive and developmental effects that are associated with DBP exposures in epidemiologic studies. Complex mixtures of DBPs from two different disinfection schemes (chlorination and ozonation/postchlorination) were concentrated successfully, while maintaining a water matrix suitable for animal studies. An array of chlorinated/brominated/iodinated DBPs was created. The DBPs were relatively stable over the course of the animal experiments, and a significant portion of the halogenated DBPs formed in the drinking water was accounted for through a comprehensive qualitative and quantitative identification approach. DBPs quantified included priority DBPs that are not regulated but have been predicted to produce adverse health effects, as well as those currently regulated in the United States and those targeted during implementation of the Information Collection Rule. New by-products were also reported for the first time. These included previously undetected and unreported bromo- and chloroacids, iodinated compounds, bromo- and iodophenols, and bromoalkyltins.

Bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss, i.e. full-litter resorption, in F344 rats when treated during the luteinizing hormone (LH)-dependent period. This effect is associated with reduced maternal serum progesterone (P) and LH levels, suggesting that BDCM disrupts secretion of LH. To test the hypothesis that BDCM also affects luteal responsiveness to LH, we used ex vivo and in vitro approaches. For the ex vivo study (i.e., in vivo exposure followed by in vitro assessment), dams were dosed by gavage on gestation days (GD) 6-9 (plug day=GD 0) at 0 or 100 mg/kg/d. One hour after the GD-9 dose, rats were killed, blood was collected, and tissue concentrations of BDCM were assessed. Corpora lutea (CL) were incubated with or without hCG, an LH agonist, to stimulate P secretion. For the in vitro study, CL were pooled from untreated F344 rats on GD 9 and cultured with BDCM at 0, 0.01, 0.10 or 3.0 mM. BDCM was found at highest concentrations in adrenal, ovarian, adipose, and hypothalamic tissues. BDCM treatment decreased serum P and LH levels in vivo. Ex vivo, however, BDCM-exposed CL showed >2-fold increases in P secretion relative to controls. Both control and BDCM-exposed CL displayed a 2.4-fold increase in P secretion in response to hCG challenge. In contrast, in vitro exposures reduced CL responsiveness in a dose-related fashion while baseline levels were unaffected. It is unclear if the ex vivo 'rebound' reflects the removal of the CL from a possible direct inhibitory influence of BDCM, or a response to diminished LH stimulation in vivo. Thus, these data suggest that BDCM disrupts pregnancy in F344 rats via two modes: disruption of LH secretion, and disruption of the CL's ability to respond to LH.

Atrazine, a chlorotriazine herbicide, is used to control annual grasses and broadleaf weeds. In this review, we summarize our laboratory's work evaluating the neuroendocrine toxicity of atrazine (and related chlorotriazines) from an historic perspective. We provide the rationale for our work as we have endeavored to determine: 1) the underlying reproductive changes leading to the development of mammary gland tumors in the atrazine-exposed female rat; 2) the cascade of physiological events that are responsible for these changes (i.e., the mode of action for mammary tumors); 3) the potential cellular mechanisms involving adverse effects of atrazine; and 4) the range of reproductive alterations associated with this pesticide.

Triazole fungicides associated with a range of reported male reproductive effects in experimental animals were selected to assess potential toxic modes of action. Wistar Han rats were fed myclobutanil (M: 100, 500, or 2000 ppm), propiconazole (P: 100, 500, or 2500 ppm), or triadimefon (T: 100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 120. One male per litter was necropsied on PND1, 22, 50, or 92. Measurements included anogenital distance (AGD) at PND0, body and organ weights, serum hormone levels, age at preputial separation (PPS), sperm morphology and motility, and fertility and fecundity. AGD was increased by the high dose of all three triazoles, indicating hypervirilization. Triadimefon delayed PPS, consistent with delayed puberty, at 1800 ppm. Relative liver weights were increased at PND1, 50, and 92 by all three triazoles. Hepatocellular hypertrophy was present at PND50 from propiconazole and triadimefon and at PND92 from all three high-dose triazole treatments. Relative pituitary weights were decreased at PND92 by middle- and high-dose myclobutanil treatment. Absolute testis weights were increased at PND1 by myclobutanil, at PND22 by myclobutanil and triadimefon, and at PND50 by propiconazole and triadimefon treatment. Relative ventral prostate weights were increased at PND92 by myclobutanil and triadimefon treatment. Serum testosterone was increased at PND50 by triadimefon and at PND92/99 by all three triazole treatments. Insemination and fertility were impaired by myclobutanil and triadimefon treatment. In addition to the reproductive system effects, total serum thyroxine levels were decreased at PND92 by high-dose triadimefon. These reproductive effects are consistent with the disruption of testosterone homeostasis as a key event in the mode of action for triazole-induced reproductive toxicity.

Three triazole fungicides were evaluated for effects on female rat reproductive development. Rats were exposed via feed to propiconazole (P) (100, 500, or 2500 ppm), myclobutanil (M) (100, 500, or 2000 ppm), or triadimefon (T) (100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 98. Body weight (BW) and anogenital distance (AGD) at PND 0, age and BW at vaginal opening (VO), estrous cyclicity, and body and organ weight at necropsy were measured. BW at PND 0 was unaffected by treatment. AGD was increased by M2000. VO was delayed by M2000 and T1800. Estrous cyclicity was initially disrupted by P500, P2500 and T1800, but later normalized. At PND 99 there was a decrease in BW by T1800, an increase in liver weight by P2500 and T1800, and an increase in ovarian weight by M2000 and T1800. It is concluded that exposure to P, M and T adversely impacted female rodent reproductive development.

Four triazole fungicides used in agricultural or pharmaceutical applications were examined for hepatotoxic effects in mouse liver. Besides organ weight, histopathology, and cytochrome P450 (CYP) enzyme induction, DNA microarrays were used to generate gene expression profiles and hypotheses on potential mechanisms of action for this class of chemicals. Adult male CD-1 mice were exposed daily for 14 days to fluconazole, myclobutanil, propiconazole, or triadimefon at three dose levels by oral gavage. Doses were based on previous studies that resulted in liver hypertrophy or hepatotoxicity. All four triazoles caused hepatocyte hypertrophy, and all except triadimefon increased relative liver/body weight ratios at the middle and high dose levels. CYP enzyme activities were also induced by all four triazoles at the middle and high doses as measured by the dealkylations of four alkoxyresorufins, although some differences in substrate specificity were observed. Consistent with this common histopathology and biochemistry, several CYP and xenobiotic metabolizing enzyme (XME) genes were differentially expressed in response to all four (Cyp2d26 and Cyp3a11), or three of the four (Cyp2c40, Cyp2c55, Ces2, Slco1a4) triazoles. Differential expression of numerous other CYP and XME genes discriminated between the various triazoles, consistent with differences in CYP enzyme activities, and indicative of possible differences in mechanisms of hepatotoxicity or dose response. Multiple isoforms of Cyp1a, 2b, 2c, 3a, and other CYP and XME genes regulated by the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) were differentially expressed following triazole exposure. Based on these results, we expanded on our original hypothesis that triazole hepatotoxicity was mediated by CYP induction, to include additional XME genes, many of which are modulated by CAR and PXR.

Four triazole fungicides were studied using toxicogenomic techniques to identify potential mechanisms of action. Adult male Sprague-Dawley rats were dosed for 14 days by gavage with fluconazole, myclobutanil, propiconazole, or triadimefon. Following exposure, serum was collected for hormone measurements, and liver and testes were collected for histology, enzyme biochemistry, or gene expression profiling. Body and testis weights were unaffected, but liver weights were significantly increased by all four triazoles, and hepatocytes exhibited centrilobular hypertrophy. Myclobutanil exposure increased serum testosterone and decreased sperm motility, but no treatment-related testis histopathology was observed. We hypothesized that gene expression profiles would identify potential mechanisms of toxicity and used DNA microarrays and quantitative real-time PCR (qPCR) to generate profiles. Triazole fungicides are designed to inhibit fungal cytochrome P450 (CYP) 51 enzyme but can also modulate the expression and function of mammalian CYP genes and enzymes. Triazoles affected the expression of numerous CYP genes in rat liver and testis, including multiple Cyp2c and Cyp3a isoforms as well as other xenobiotic metabolizing enzyme (XME) and transporter genes. For some genes, such as Ces2 and Udpgtr2, all four triazoles had similar effects on expression, suggesting possible common mechanisms of action. Many of these CYP, XME and transporter genes are regulated by xeno-sensing nuclear receptors, and hierarchical clustering of CAR/PXR-regulated genes demonstrated the similarities of toxicogenomic responses in liver between all four triazoles and in testis between myclobutanil and triadimefon. Triazoles also affected expression of multiple genes involved in steroid hormone metabolism in the two tissues. Thus, gene expression profiles helped identify possible toxicological mechanisms of the triazole fungicides.

Perfluorooctanoic acid (PFOA), a member of the perfluoroalkyl acids that have wide commercial applications, has recently been detected in humans and wildlife. The current study characterizes the developmental toxicity of PFOA in the mouse. Timed-pregnant CD-1 mice were given 1, 3, 5, 10, 20, or 40 mg/kg PFOA by oral gavage daily from gestational day (GD) 1 to 17; controls received an equivalent volume (10 ml/kg) of water. PFOA treatment produced dose-dependent full-litter resorptions; all dams in the 40-mg/kg group resorbed their litters. Weight gain in dams that carried pregnancy to term was significantly lower in the 20-mg/kg group. At GD 18, some dams were sacrificed for maternal and fetal examinations (group A), and the rest were treated once more with PFOA and allowed to give birth (group B). Postnatal survival, growth, and development of the offspring were monitored. PFOA induced enlarged liver in group A dams at all dosages, but did not alter the number of implantations. The percent of live fetuses was lower only in the 20-mg/kg group (74 vs. 94% in controls), and fetal weight was also significantly lower in this group. However, no significant increase in malformations was noted in any treatment group. The incidence of live birth in group B mice was significantly lowered by PFOA: ca. 70% for the 10- and 20-mg/kg groups compared to 96% for controls. Postnatal survival was severely compromised at 10 or 20 mg/kg, and moderately so at 5 mg/kg. Dose-dependent growth deficits were detected in all PFOA-treated litters except the 1-mg/kg group. Significant delays in eye-opening (up to 2-3 days) were noted at 5 mg/kg and higher dosages. Accelerated sexual maturation was observed in male offspring, but not in females. These data indicate maternal and developmental toxicity of PFOA in the mouse, leading to early pregnancy loss, compromised postnatal survival, delays in general growth and development, and sex-specific alterations in pubertal maturation.

Background: Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) regulate cell proliferation and differentiation in the embryo. The induction of cleft palate (CP) by all trans-retinoic acid (RA) was associated with altered expression of TGFalpha, EGF receptor, and binding of EGF. This study uses knockout (KO) mice to examine the roles of EGF and TGFalpha in teratogenic responses of embryos exposed to RA., Methods: Pregnant wild-type (WT) mice of mixed genetic background, EGF KO, C57BL/6J, and TGFalpha KO mice were given a single oral dose of RA (100 mg/kg, 10 ml/kg) or corn oil on GD 10 at 12 PM, GD 11 at 12 PM or 4 PM, or GD 12 at 8 AM or 12 PM (plug day = GD 0). GD 18 fetuses were examined for external, visceral, and skeletal effects., Results: After exposure to RA on GD 12, the incidence of CP in EGF KO was significantly reduced relative to WT. In TGFalpha KO fetuses, RA exposure on GD 10 increased the incidence of CP versus C57BL/6J. The incidence of skeletal defects in the limbs, vertebrae, sternebrae, and ribs were also affected by lack of expression of EGF or TGFalpha with region-specific amelioration or exacerbation of the effects of RA. In TGFalpha KO fetuses, incidences of forelimb long bone and digit defects increased relative to C57BL/6J. In EGF KO fetuses, relative to WT, the incidence of hindlimb oligodactyly was increased. In EGF KO, but not WT, RA produced short, bent radius, humerus, and ulna. Both TGFalpha and EGF KO mice had increased incidences of dilation of the renal pelvis and this was reduced by RA., Conclusions: RA exposure produced skeletal and visceral defects in all genotypes; however, EGF or TGFalpha KO influenced the incidence and severity of defects. This study supports a role for EGF and TGFalpha in the response to RA., (Published 2005 Wiley-Liss, Inc.)

Chemical disinfection of water is of direct public health benefit as it results in decreased water-borne illness. The chemicals used to disinfect water react with naturally occurring organic matter, bromide, and iodide in the source water, resulting in the formation of disinfection by-products (DBPs). Despite the identification of several hundred DBPs, more than 50% of the mass of total organic halide formed during chlorination remains unidentified. The toxic contribution of the DBPs that are formed and present but not yet chemically identified, the unidentified fraction, has been largely unexplored. A better understanding of the potential for adverse human health consequences associated with exposure to the DBPs present in drinking water will be gained by integration of knowledge on the toxicity of individual DBPs; simple, defined DBP mixtures; complex, environmentally realistic DBP mixtures with partial chemical characterization; and the unidentified fraction.

Epidemiological data suggest an association between exposures to bromodichloromethane (BDCM), a trihalomethane found in drinking water as a result of drinking water disinfection, and an increased risk of spontaneous abortion. We previously hypothesized that BDCM targets the placenta and showed that the secretion of chorionic gonadotrophin (CG) was reduced in primary cultures of human term syncytiotrophoblasts exposed to BDCM. In the present study we extend this observation by evaluating the effects of BDCM on the morphological differentiation of mononucleated cytotrophoblast cells to multinucleated syncytiotrophoblast-like colonies. Addition of BDCM to cytotrophoblast cultures inhibited the subsequent formation of multinucleated colonies in a dose-dependent manner, as determined by immunocytochemical staining for desmosomes and nuclei. The effect was seen at BDCM concentrations between 0.02 and 2 mM and was confirmed by quantitative image analysis. Secretion of bioactive and immunoreactive chorionic gonadotropin was also significantly inhibited in a dose-dependent manner under these culture conditions, and cellular levels of CG were also reduced. Trophoblast viability was not compromised by exposure to BDCM. We conclude that BDCM disrupts syncytiotrophoblast formation and inhibits CG secretion in vitro. Although other tissue targets are not ruled out, these data substantiate the idea that BDCM targets the placenta and could have implications for understanding the adverse pregnancy outcomes associated with BDCM exposure in humans.

Previously, we demonstrated that bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss in F344 rats when given on gestational days (GD) 6-10, encompassing the luteinizing hormone (LH)-dependent period of pregnancy (GD 7-10). Pregnancy loss, i.e., full-litter resorption, was associated with reduced serum progesterone levels; however, we were unable to identify an effect on serum LH. Here, we reevaluated serum LH levels using the more sensitive technique, DELFIA(R). We further sought to better define the temporal pattern of endocrine disruption caused by BDCM during pregnancy with more frequent sampling. Lastly, we attempted to prevent BDCM-induced pregnancy loss using exogenous progesterone or human chorionic gonadotropin (hCG), an LH-agonist. BDCM, in 10% Alkamuls(R), was dosed at 75 mg/kg/day by gavage to F344 rats on GD 6-10 (plug day = GD 0). BDCM-induced pregnancy loss was associated with marked reductions in serum progesterone and LH on GD 10. The decrease in serum LH consistently preceded the decrease in progesterone. In the hormone replacement studies, BDCM and progesterone were administered on GD 6-10, hCG on GD 8-10. BDCM was delivered at 100 mg/kg/day, progesterone at 10 mg/kg twice daily, and hCG at 0.5 IU/0.2 ml/rat. Both progesterone and hCG prevented BDCM-induced pregnancy loss. Thus, BDCM-induced pregnancy loss was associated with marked GD-10 reductions in serum LH and corresponding decreases in progesterone. Furthermore, coadministration of an LH agonist prevented pregnancy loss, supporting the hypothesis that BDCM-induced pregnancy loss in the rat occurs via an LH-mediated mode of action.

Bromodichloromethane (BDCM) is a trihalomethane found in drinking water as a by-product of disinfection processes. BDCM is hepatotoxic and nephrotoxic in rodents and has been reported to cause strain-specific full-litter resorption in F344 rats during the luteinizing hormone-dependent phase of pregnancy. In humans, epidemiological studies suggest an association between exposure to BDCM in drinking water and increased risk of spontaneous abortion. To begin to address the mechanism(s) of BDCM-induced spontaneous abortion, we hypothesized that BDCM targets the placenta. Primary cultures of human term trophoblast cells were used as an in vitro model to test this hypothesis. Trophoblasts were allowed to differentiate into multinucleated syncytiotrophoblast-like colonies, after which they were incubated for 24 h with different concentrations of BDCM (20 nM to 2 mM). Culture media were collected and assayed for immunoreactive and bioactive chorionic gonadotropin (CG). Cultures exposed to BDCM showed a dose-dependent decrease in the secretion of immunoreactive CG as well as bioactive CG. The lowest effective BDCM concentration was 20 nM, approximately 35-times higher than the maximum concentration reported in human blood (0.57 nM). Trophoblast morphology and viability were similar in controls and cultures exposed to BDCM. We conclude that BDCM perturbs CG secretion by differentiated trophoblasts in vitro. This suggests that the placenta is a likely target of BDCM toxicity in the human and that this could be related to the adverse pregnancy outcomes associated with BDCM.

This unit presents two basic protocols for testing prenatal developmental toxicity in rodents. The standard protocol for assessing prenatal developmental toxicity, often referred to as a Segment II study, describes the methods used for performing cesarean sections and examining cesarean-delivered fetuses for external, skeletal, and visceral alterations. The protocol for the Chernoff/Kavlock assay, a developmental toxicity screen, describes methods for examining parturition, and postnatal growth and viability of prenatally exposed litters.

Background: Previously, we showed that prenatal exposure to boric acid (BA), an industrial agent with large production, causes alterations of the axial skeleton in rat embryos, reminiscent of homeotic transformations. Indeed, Sprague-Dawley rats exposed in utero to BA on gestation day 9 (GD 9) had only six, rather than the normal seven, cervical vertebrae. This finding, observed in 91% of GD 21 fetuses, suggests posterior transformations of vertebrae. The present study attempts to determine if these skeletal alterations could be explained by modifications of the hox code, involved in the establishment of positional information along the craniocaudal axis of the embryo., Methods: Pregnant rats were treated by gavage with BA (500 mg/kg, twice) on GD 9. Embryos were collected on GD 11 or GD 13.5 and processed for in situ hybridization. Several hox genes were selected according to the position of their cranial limit of expression in the cervical and thoracic region., Results: At GD 13.5, we detected a cranial shift of the anterior limit of expression of hoxc6 and hoxa6. We observed no difference between control and treated embryos in the location of the cranial limit of expression of the other genes: hoxd4, hoxa4, hoxc5, and hoxa5., Conclusions: Our results demonstrate that following in utero exposure to BA on GD 9, a disturbance of the expression of hox genes involved inthe specification of most anterior vertebrae is observed at GD 13.5. Based on their expression domain and on their implication in the definition of the cervicothoracic vertebral boundary, it is likely that the anteriorization of hoxc6 and hoxa6 reported here is correlated to the morphological phenotype observed in BA-exposed fetuses at GD 21.

Background: The purine analog 2-chloro-2'-deoxyadenosine (2-CdA) caused ocular and limb defects in the mouse and rabbit. The current study examined the teratogenic potential of this drug in the rat and compared the adverse developmental outcomes with the other species., Methods: Timed-pregnant Sprague-Dawley rats were given a single intraperitoneal injection of various doses of 2-CdA ranging from 5-60 mg/kg, at gestational day (GD) 9.5 and GD 14. 2-CdA concentrations in maternal serum and embryos were measured by HPLC and termed fetuses were prepared for teratological examination., Results: Full-litter resorption was seen in dams receiving 50 mg/kg of 2-CdA at GD 9.5, whereas post-implantation loss was significantly increased and fetal weights significantly reduced at 40 mg/kg. Gross examination of the surviving fetuses revealed microphthalmia, a shortened body trunk and lumbar hernia, manifested by a soft mass protrusion at the lumbar region on one or both sides of the spine. Incidence of these defects increased in a dose-dependent fashion. Histological examination indicated that the hernia was associated with hypoplasia of the body wall, poorly developed skeletal muscle bundles surrounding the vertebral column in the lumbar region, and an absence of the lateral muscle groups that allowed protrusion of the abdominal viscera. The lumbar hernia was generally accompanied by spina bifida, deformed ribs and a wide spectrum of soft tissue-abnormalities that included kidney, genitourinary and heart defects. At GD 14, exposure to 2-CdA at 60 mg/kg produced oligodactyly in one of six litters., Conclusions: 2-CdA produced similar ocular defects in the rat and mouse, although the incidence was much lower in the former species. In contrast, the drug-induced lumbar hernia was only seen in the rat. These apparent disparities were not readily explained by species differences in pharmacokinetic parameters. the similarities between the teratological features of 2-CdA-induced lumbar hernia in the rat and the clinical description of lumbocostovertebral syndrome, however, may provide a key to unlock the etiology of this rare birth defect in humans., (Published 2002 Wiley-Liss, Inc.)

Bromodichloromethane (BDCM), a trihalomethane, is a by-product of the chlorination of drinking water. In a recent epidemiological study, consumption of BDCM was associated with an increased risk of spontaneous abortion in pregnant women. We have previously shown that BDCM causes pregnancy loss, i.e., full-litter resorption (FLR), in the F344 rat. The mode of action was investigated, with three main findings. First, there was a dramatic difference in sensitivity between F344 and Sprague-Dawley (SD) rat strains. Following aqueous gavage treatment on gestational days (GD) 6-10, F344 rats had a 62% incidence of FLR at 75 mg/kg/day, whereas all SD rats maintained their litters. Second, the critical period encompassed the luteinizing hormone (LH)-dependent period of pregnancy. Rats treated on GD 6-10 at 75 mg/kg/day had a 75% incidence of FLR, but rats treated on GD 11-15 at 75 or 100 mg/kg/day were unaffected. Third, 24 h after a single dose, all dams with FLR had markedly reduced serum progesterone levels; however, LH levels were unaffected. The high FLR rate during the LH-dependent period, the lack of response thereafter, and the reduced progesterone levels without an associated reduction in LH levels suggests that BDCM disrupts luteal responsiveness to LH.

Atrazine was administered by gavage, in 1% methylcellulose, to F344 Sprague-Dawley (SD), and Long Evans (LE) rats at 0, 25, 50, 100, or 200 mg/kg/day on gestation days 6 through 10. The dams were allowed to deliver and litters were examined postnatally. The F344 strain was the most sensitive to atrazine's effects on pregnancy, showing full-litter resorption (FLR) at >/=50 mg/kg. In surviving F344 litters, prenatal loss was increased at 200 mg/kg. In SD and LE rats, FLR occurred only at 200 mg/kg. Delayed parturition was seen at >/=100 mg/kg in F344 and SD rats. Regarding maternal toxicity, the SD dams were the most sensitive, with weight loss at >/=25 mg/kg. When 200 mg/kg was administered to F344 rats on days 11 through 15 (after the LH-dependent period of pregnancy), no FLR was seen. These findings suggest that atrazine-induced FLR is maternally mediated, and consistent with loss of LH support of the corpora lutea.

The aryl hydrocarbon receptor (AhR) mediates many of the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and transcriptional activation of genes encoding a number of xenobiotic metabolizing enzymes. Prenatal exposure of mice to TCDD causes severe alterations in embryo and fetal development, including hydronephrosis and cleft palate. However, the mechanisms underlying these effects are unclear. In this work, the teratogenicity of TCDD in AhR-null mice was evaluated to determine if this effect is mediated by the AhR. Homozygous wild-type (+/+) or AhR-null (-/-) female mice were mated with males of the same genotype overnight. On gestation day (GD)-10, mice were intubated orally with either corn oil (vehicle control) or 25 micrograms/kg TCDD. Fetuses were examined on GD18 for visceral and skeletal alterations. For non-TCDD-exposed litters, all developmental endpoints were comparable between genotypes, with the exception of a lower incidence of large interfrontal bones in (-/-) mice. For TCDD-exposed litters, (+/+) fetuses had a significantly greater incidence of cleft palate, hydronephrosis, small kidneys, tortuous ureters and greater dilation of the renal pelves and ureters compared to (-/-) fetuses. Interestingly, an increased resorption rate was observed in (-/-) fetuses exposed to TCDD. Results from this work demonstrate that fetal development per se is generally unaffected by the absence of the AhR or that other genes may have compensated for the loss of the AhR. More importantly, these data indicate that the AhR mediates TCDD-induced teratogenicity. Further, since a higher percentage of resorptions was observed in (-/-) litters from TCDD-treated dams, it is possible that AhR-independent mechanisms contribute to TCDD-induced developmental toxicity.

Prenatal exposure to elevated levels of boric acid (BA) causes reduced incidences of supernumerary ribs and shortening/absence of the 13th rib in multiple laboratory species. To explore this further, Sprague-Dawley rats received 500 mg/kg b.i.d. on gestation days (gd) 5-9, 6-9, 6-10, or on single days between gd 6 and 11 (plug day = gd 0); gd-21 fetuses were stained for skeletal examination. Following multiday exposures, malformations of the axial skeleton involved the head, sternum, ribs, and vertebrae. Shortening/absence of the 13th rib was seen particularly in the gd 5-9 and 6-10 exposure groups. Although most groups exposed on single days were generally unaffected, about 90% of the gd-9 exposed fetuses had only six cervical vertebrae; the deficient region was usually C3-C5. In contrast, gd-10 treatment caused agenesis of a thoracic/lumbar vertebra in over 60% of the fetuses; the deficient region was usually T11. For 13-ribbed fetuses, the length of rib 13 was shortened compared to controls. Postnatal assessment suggested increased mortality for gd-10 exposed pups. Embryos in culture showed reduced development when exposed to BA for 48 h. These findings demonstrate the critical periods for axial development in the rat and provide an experimental model for the study of homeotic shifts.

Guanethidine, a chemical that selectively blocks sympathetic noradrenergic neurons, was used to investigate the role of sympathetic innervation in the fertility of rat epididymal sperm, using both natural mating and in utero insemination protocols. This animal model correlates, at least in part, with spinal cord injury (SCI) in men. Adult male rats were treated daily by i.p. injections, for 21 or 42 days, with 0 or 6.25 mg/kg guanethidine. To compare the effects of guanethidine-induced sympathectomy with those following surgically induced sympathectomy, the inferior mesenteric ganglion and the proximal hypogastric nerves were removed in another group of rats. Both chemically and surgically induced sympathectomy increased the weight of the epididymis and seminal vesicles/coagulating glands as well as the number and the transit time of cauda epididymal sperm. Neither serum testosterone levels nor LH was affected by treatment with guanethidine. Using natural mating, no litters were produced by guanethidine-treated rats. Chemically denervated rats failed to produce copulatory plugs or ejaculate into the uterus. However, distal cauda epididymal sperm from chemically or surgically denervated rats displayed normal fertilization ability (80%) using in utero inseminations. In addition, the sperm of denervated rats did not show abnormal sperm chromatin structure using an assay that detects DNA damage. We conclude that sympathectomy delays the transit of sperm through the cauda epididymidis and produces ejaculatory dysfunction but does not compromise sperm quality in the distal cauda epididymidis. Moreover, these data provide compelling evidence that there is no association between the prolonged transit time of sperm within the epididymis, i.e., pre-ejaculatory sperm aging, and the fertility of those sperm, which has important implications for artificial insemination using sperm from men with SCI.

Several halocarbons have been shown to cause full-litter resorption (FLR) in Fischer-344 rats when administered orally in corn oil. Since halocarbons often occur as contaminants of drinking water, we sought to determine the influence of the vehicle, aqueous versus lipid, on the developmental toxicity of two of these agents. In separate assays, bromodichloromethane (BDCM) and carbon tetrachloride (CCl4) were administered by gavage to Fischer-344 rats on gestation days (GD) 6-15 at 0, 25, 50, or 75 mg/kg/day in either corn oil or an aqueous vehicle containing 10% Emulphor EL-620. Dams were allowed to deliver and the litters were examined postnatally. Uteri of females that did not deliver were stained with 10% ammonium sulfide to detect FLR. Effects of both agents on maternal weight gain were slightly more pronounced in the aqueous vehicle at lower doses, but at the highest dose, CCl4 was more maternally toxic in corn oil. Developmentally, both agents caused FLR at 50 and 75 mg/kg in both vehicles. At 75 mg/kg, dams receiving corn oil had significantly higher rates of FLR (83% for BDCM, 67% for CCl4) compared to their aqueous-vehicle counterparts (21% for BDCM, 8% for CCl4). Blood concentrations of BDCM following GD-6 gavage revealed a shorter elimination half-life in the aqueous dosing vehicle (2.7 h) compared to the oil vehicle (3.6 h). Benchmark doses of CCl4 were similar for the oil (18.9 mg/kg) and aqueous (14.0 mg/kg) vehicles. For BDCM, the corn oil vehicle yielded a less conservative (i.e., higher) value (39.3 mg/kg) than the aqueous vehicle (11.3 mg/kg), reflecting different confidence intervals around the estimated 5%-effect dose levels.

Several low-molecular weight halocarbons have been shown to cause full-litter resorption (FLR), i.e., pregnancy loss, in Fischer-344 rats treated during organogenesis. To determine periods of gestation sensitive to acute exposure, a single dose of 150 mg carbon tetrachloride (CCl4)/kg was administered on gestation day (GD) 6, 7, 8, 10, or 12. Fetuses were delivered by cesarean section on GD 20. Non-gravid uteri were examined for resorption sites, placed in 10% ammonium sulfide, and re-examined for stained resorption sites approximately 1 and 4.5 hr later. FLR was seen in 4% (1/27) of control dams and 36% (4/11), 54% (7/13), 72% (18/25), 54% (7/13), and 0% (0/12) of dams treated on GD 6, 7, 8, 10, and 12, respectively. Ammonium sulfide staining clearly yielded a more accurate account of the incidence of FLR. The technique was most effective when the staining period was extended to 4.5 hr, as two cases of FLR were revealed that had been undetected after 1 hr of staining. For dams with FLR, staining was required to detect resorption sites in all dams treated on GD 6 or 7, most dams treated on GD 8, and one dam treated on GD 10. Fewer implantation sites were detected in the dams treated on GD 6, and the size of the stained resorption sites increased as the day of treatment was delayed. These findings demonstrate a relationship between the time of toxicant exposure and the size and detectability of resorption sites near term, suggesting that the size of the resorption site may reliably reflect the time of embryonic death. Treatment on GD 8 caused the highest incidence of FLR and will be used in subsequent mechanistic research.

Recently, we demonstrated with short-duration tests that dibromoacetic acid (DBAA), a commonly occurring by-product of water disinfection, alters sperm morphology and motility in the male rat. These results suggested that the effects of DBAA on sperm quality were likely to compromise reproductive competence of the male rat early in subchronic exposure. The present studies were undertaken to investigate the dose response and time course of alterations in fertility and sperm quality. Proven breeder male rats were gavaged daily with 0, 2, 10, 50, or 250 mg DBAA/kg for up to 79 days; interim and terminal measurements of sperm quality and reproductive outcome were made. Because of the known neurotoxicity of the analogue, dichloroacetic acid, both natural breeding and artificial inseminations were evaluated in untreated females to distinguish between possible behavioral and spermatogenic effects. DBAA compromised male fertility during the second treatment week in naturally bred rats dosed with 250 mg/kg. The early antifertility effect appeared to be the result of behavioral changes since females artificially inseminated with sperm collected on Day 9 successfully produced offspring. However, sperm morphology and motility also were rapidly affected by DBAA treatment so that no offspring via natural insemination and only one litter via artificial insemination were produced subsequent to Day 15. Through 31 days, substantial effects on sperm motility, sperm morphology, and epididymal sperm numbers were observed, but there was no demonstrable effect on serum testosterone or sperm production. Because severe toxicity developed in the group given 250 mg/kg, exposure of these animals was prematurely terminated after 42 doses and their recovery was monitored through a 6-month posttreatment period; decreased testis weights and only limited recovery of reproductive performance were observed. Exposure to 50 mg/kg resulted in moderate changes in sperm morphology and motility and moderate decreases in epididymal sperm counts in rats dosed for 31 or 79 days. However, these males remained fertile, litter size was unaffected, and no paternally mediated developmental defects were noted in their offspring. No effects on sperm quality were detected at dosages of 2 or 10 mg/kg. However, compared to controls, naturally bred DBAA-treated rats tended to have fewer inseminations, fewer copulatory plugs, and fewer multiple litters, suggesting that DBAA may have altered mating behavior at dosages as low as 10 mg/kg.

A biologically-based dose-response (BBDR) model is a mathematical description of the biological events leading to expression of a toxic response. As an alternative to current approaches in non-cancer risk assessment, such models will reduce uncertainty in that they will provide a more comprehensive description of toxicity. We are involved in construction of a BBDR model for the developmental toxicity of 5-fluorouracil (5-FU) in the rat using multiple approaches. First, to identify critical events in the pathogenesis of 5-FU developmental toxicity, thymidylate synthetase (TS) inhibition and alterations in cell kinetics and growth were examined in embryos following maternal administration of 5-FU on day 14 of gestation. A dose-related decline in TS activity was observed within 1 h; however, maximal inhibition and recovery were similar at 10, 20 and 40 mg/kg. Dose-dependent cell cycle alterations were observed within 4 h after exposure and were maximal at 8 h. Hindlimb growth reduction was observed 24 h after exposure to 40 mg/kg, but not at lower doses. At term hindlimb defects were observed at doses above 30 mg/kg. An integrated dose-response model for hindlimb defects was derived from empirical relationships among these events. The resultant dose-response somewhat over-predicted the developmental toxicity of 5-FU, although results of a Monte Carlo simulation indicated that these data were not incompatible with model predictions. Overall, the results suggest that TS inhibition is a key component of the mechanism of 5-FU developmental toxicology, but the model does not capture all of the critical events in the induction of hindlimb defects. A preliminary mechanistic model for the inhibition of embryonic TS, DNA synthesis and cell cycle following maternal exposure to 5-FU, independently derived from literature data to further examine the potential role of this pathway in its developmental toxicity, predicted a dose-response for TS inhibition and DNA synthesis that closely reflected the observed patterns. These results further suggest that TS inhibition, resultant deficits in DNA synthesis and cell cycle perturbations represent a critical mechanistic pathway in the developmental toxicity of 5-FU.

In order to identify nonadditive effects on development, three compounds were combined using five dosages of each agent (a 5 x 5 x 5 full-bacterial design). Trichloroethylene (TCE), di(2-ethylhexyl) phthalate (DEHP), and heptachlor (HEPT), in corn oil, were administered by gavage to Fischer-344 rats on Gestation Days 6-15. Dose levels were 0, 10.1, 32, 101, and 320 mg/kg/day for TCE; 0, 24.7, 78, 247, and 780 mg/kg/day for DEHP; and 0, 0.25, 0.8, 2.5, and 8 mg/kg/day for HEPT. The dams were allowed to deliver and their pups were weighed and examined postnatally. Maternal death showed no main effects but DEHP and HEPT were synergistic. For maternal weight gain on Gestational Days 6-8, main effects for all three agents were observed, as well as 6-8 main effects for all three agents were observed, as well as TCE-DEHP synergism, and DEHP-HEPT antagonism. Maternal weight gain on Gestational Days 6-20 adjusted for litter weight showed main effects for TCE and HEPT, but no interactions. Main effects for all three agents were evident for full-litter resorptions and prenatal loss. The HEPT main effects were unexpected and were interpreted as reflecting potentiation by HEPT of the other agents. For full-litter loss, the TCE-HEPT and DEHP-HEPT interactions were antagonistic, perhaps due to a "ceiling" effect. For prenatal loss, the TCE-DEHP interaction was synergistic. Postnatal loss showed DEHP and HEPT main effects but no interaction. Analysis of pup weights on Day 1 revealed TCE and DEHP main effects and DEHP-HEPT antagonism; on Day 6, DEHP and HEPT main effects, DEHP-HEPT antagonism, and TCE-DEHP synergism were evident. Microphthalmia and anophthalmia incidences revealed TCE and DEHP main effects but no interactions. This extensive examination of a full-factorial design elucidates the complexities of studying and interpreting mixture toxicity. The data are available for further analysis.

Toxicity data collected under standardized test conditions may be of the utmost importance in health risk assessment, in which human exposure limits are often derived from laboratory experiments. A standardized approach to data collection is also important for evaluating the sensitivity and specificity of test methods used to determine toxic potential. Several experiments were undertaken to determine the effects of chemical exposures using a multidisciplinary screening battery, which included tests for systemic, neurological and developmental toxicity. The effects of 1- and 14-d exposures to 10 chemicals on systemic and neurological indices of toxicity were determined in female F344 rats using standardized test batteries. Parallel experiments determined chemical effects on prenatal and postnatal development following exposure of the dams for 14 d. The chemicals included four pesticides (carbaryl, triadimefon, chlordane, and heptachlor), four solvents (trichloroethylene, tetrachloroethylene, carbon tetrachloride, and dichloromethane), and two industrial compounds (phenol and diethylhexyl phthalate). The results showed that the chemicals produced markedly different qualitative patterns of effect on systemic, neurological, and developmental indices of toxicity. Differences in the pattern of systemic and neurological effects were also obtained that depended on dosing duration. Quantitative analyses indicated that the highest ineffective dose as well as the lowest effective dose could vary by as much as two orders of magnitude across the different indices of toxicity. These results clearly show that a test battery focused on a single endpoint of toxicity cannot be used to accurately predict either qualitatively or quantitatively a chemical's systemic, neurological, and developmental toxicity profile.

As part of the validation of an integrated bioassay for systemic toxicity, neurotoxicity, and developmental toxicity, we evaluated the effects of four pesticides, four chlorinated solvents, and two other industrial chemicals in Fischer 344 rats. The pesticides included carbaryl, triadimefon, chlordane, and heptachlor; the solvents included dichloromethane (DCM), carbon tetrachloride, trichloroethylene (TCE), and tetrachloroethylene (perchloroethylene, PER); and the industrial chemicals were di(2-ethylhexyl)phthalate (DEHP) and phenol. In the developmental toxicity studies, timed-pregnant rats were treated by gavage with vehicle or 1 of 2 dose levels of each compound on gestation d 6-19. The dams were allowed to deliver and their litters were examined on postnatal d 1, 3, and 6. Litter weights were determined on postnatal d 1 and 6. Implants were also counted to determine prenatal loss. Maternal toxicity was evidenced by dose-related alterations in weight gain for all 10 compounds. Clinical signs of maternal toxicity were present for all chemicals except chlordane and heptachlor. DEHP exposure resulted in the most pronounced developmental toxicity (high levels of pre- and postnatal mortality), whereas chlordane induced extensive postnatal loss. Of the solvents, only DCM did not cause a high incidence of full-litter resorption. Phenol, heptachlor, triadimefon, and carbaryl showed only slight potential for developmental toxicity. Malformations suggestive of teratogenicity included kinked tail (phenol), microphthalmia (TCE, PER, DEHP), and cleft palate with renal agenesis (DEHP). Although several findings (eye defects caused by TCE and PER, full-litter resorption and delayed parturition caused by PER, and delayed parturition/dystocia associated with triadimefon) have not been previously reported, the results are generally consistent with previous reports and highlight the importance and relative ease of incorporation of developmental evaluations into a multidisciplinary screening battery.