Method to assess component contribution to toxicity of complex mixtures: Assessment of puberty acquisition in rats exposed to disinfection byproducts.

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.

Risks of obstructive genitourinary birth defects in relation to trihalomethane and haloacetic acid exposures: expanding disinfection byproduct mixtures analyses using relative potency factors.

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.

Comprehensive assessment of a chlorinated drinking water concentrate in a rat multigenerational reproductive toxicity study.

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.

Developmental toxicity evaluations of whole mixtures of disinfection by-products using concentrated drinking water in rats: gestational and lactational effects of sulfate and sodium.

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.

Identification of developmentally toxic drinking water disinfection byproducts and evaluation of data relevant to mode of action.

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.

Concentration, chlorination, and chemical analysis of drinking water for disinfection byproduct mixtures health effects research: U.S. EPA's Four Lab Study.

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.

Disinfection By-Product Exposures and the Risk of Musculoskeletal Birth Defects.

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.

Analysis of proportional data in reproductive and developmental toxicity studies: Comparison of sensitivities of logit transformation, arcsine square root transformation, and nonparametric analysis.

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.

The role of developmental toxicity studies in acute exposure assessments: analysis of single-day vs. multiple-day exposure regimens.

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.

Integrated disinfection by-products mixtures research: assessment of developmental toxicity in Sprague-Dawley rats exposed to concentrates of water disinfected by chlorination and ozonation/postchlorination.

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.

Integrated disinfection by-products mixtures research: comprehensive characterization of water concentrates prepared from chlorinated and ozonated/postchlorinated drinking water.

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.

Integrated disinfection by-products research: assessing reproductive and developmental risks posed by complex disinfection by-product mixtures.

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.

Associations Between Disinfection By-Product Exposures and Craniofacial Birth Defects.

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.

Disruption of testosterone homeostasis as a mode of action for the reproductive toxicity of triazole fungicides in the male rat.

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.

Disinfection By-Product Exposures and the Risk of Specific Cardiac Birth Defects.

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.

Effects of perfluorooctanoic acid exposure during pregnancy in the mouse.

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.

Effect of conazole fungicides on reproductive development in the female rat.

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.

A systematic evaluation of the potential effects of trichloroethylene exposure on cardiac development.

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.

Reproductive toxicity of a mixture of regulated drinking-water disinfection by-products in a multigenerational rat bioassay.

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.

Serum hormone characterization and exogeneous hormone rescue of bromodichloromethane-induced pregnancy loss in the F344 rat.

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.