Prospective Power Calculations for the Four Lab Study of A Multigenerational Reproductive/Developmental Toxicity Rodent Bioassay Using A Complex Mixture of Disinfection By-Products in the Low-Response Region

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

Defects in cervical vertebrae in boric acid-exposed rat embryos are associated with anterior shifts of hox gene expression domains.

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

The skeleton : biochemical, genetic and molecular interactions in development and homeostasis. : Effects of boric acid on Hox gene expression and the axial skeleton in the developing rat.

Gestational exposure to boric acid (BA) causes reduced incidences of supernumerary ribs and shortening/absence of the 13th rib in the progeny of multiple laboratory species. To further explore this, Sprague-Dawley rats received 500 mg/kg b.i.d. on gestation days (GD) 6, 7, 8, 9, 10, or 11 (plug day = GD 0). GD-21 fetuses were stained for skeletal examination. BA's most noteworthy effects were apparent homeotic shifts in the axial skeleton; i.e., a given vertebra anatomically resembled an adjacent vertebra, thus leading to altered numbers of cervical, thoracic, or lumbar vertebrae. Whereas most groups generally had no such effect, about 90% of the GD-9 exposed fetuses had only six cervical vertebrae. Deficiencies in the C3-C5 region, C6, or C7 were observed in 67%, 1%, and 23% of the exposed fetuses, respectively. In contrast, GD-10 treatment caused agenesis of a thoracic/lumbar vertebra in over 60% of the fetuses. In these fetuses, the deficient region was usually T11. In view of the 90% incidence of six-cervical vertebrae in GD-9 exposed fetuses, we used this exposure regimen (500 mg/kg b.i.d. on GD 9) as an experimental model for the study of homeotic shifts. We sought to determine if these skeletal alterations could be explained by modifications of the hox code, involved in the establishment of positional information along the cranio-caudal axis of the embryo. Embryos were collected on 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. A cranial shift in the cranial limit of expression of hoxc6 and hoxa6 was evident in the prevertebrae, whereas no difference was observed between control and treated embryos in the expression of hoxd4, hoxa4, hoxc5, and hoxa5. Anteriorization of the expression domain of hoxc6 and hoxa6 is consistent with the posterior transformation of cervical vertebrae, and may partially account for the phenotype observed on GD 21 in BA-exposed fetuses

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

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