Early Developmental Exposure to Triclosan Impacts Fecal Microbial Populations, IgA and Functional Activities of the Rat Microbiome.

Triclosan (TCS), a broad-spectrum antibacterial chemical, is detected in human urine, breast milk, amniotic fluid, and feces; however, little is known about its impact on the intestinal microbiome and host mucosal immunity during pregnancy and early development. Pregnant female rats were orally gavaged with TCS from gestation day (GD) 6 to postpartum (PP) day 28. Offspring were administered TCS from postnatal day (PND) 12 to 28. Studies were conducted to assess changes in the intestinal microbial population (16S-rRNA sequencing) and functional analysis of microbial genes in animals exposed to TCS during pregnancy (GD18), and at PP7, PP28 and PND28. Microbial abundance was compared with the amounts of TCS excreted in feces and IgA levels in feces. The results reveal that TCS decreases the abundance of Bacteroidetes and Firmicutes with a significant increase in Proteobacteria. At PND28, total Operational Taxonomic Units (OTUs) were higher in females and showed correlation with the levels of TCS and unbound IgA in feces. The significant increase in Proteobacteria in all TCS-treated rats along with the increased abundance in OTUs that belong to pathogenic bacterial communities could serve as a signature of TCS-induced dysbiosis. In conclusion, TCS can perturb the microbiome, the functional activities of the microbiome, and activate mucosal immunity during pregnancy and early development.

Analysis of incidence data in developmental toxicity studies: Statistical tests to account for litter effects in fetal defect data.

BACKGROUND: When analyzing fetal defect incidence in laboratory animal studies, correlation in responses within litters (i.e., litter effects) can lead to increased false-positive rates if litter effects are not incorporated into the analysis. Studies of fetal defects require analysis methods that are robust across a broad range of defect types, including those with zero or near-zero incidence rates in control groups. METHODS: A simulation study compared power and false-positive rates for six approaches across a range of background defect rates and litter size distributions. Statistical methods evaluated included ignoring the litter effect as well as parametric and nonparametric approaches based on litter proportions, generalized linear mixed models (GLMMs), the Rao-Scott Cochran-Armitage (RSCA) trend test, and a modification to the RSCA (mRSCA) introduced here to improve estimation at low background rates. These methods were also applied to a common and a rare defect from two prenatal developmental toxicology studies conducted by the National Toxicology Program (NTP). RESULTS: At background defect rates of 1%, the mRSCA and parametric litter proportion methods provided gains in power over the nonparametric litter proportion method, the GLMM method, and the RSCA method. Simulations involving litter loss in high-dose groups showed loss of power for both litter proportion methods. CONCLUSIONS: The mRSCA test developed here compares favorably with other litter-based approaches and is robust across a range of background defect rates and litter size distributions, making it a practical choice for prenatal developmental toxicology studies involving both common and rare fetal defects.

Simultaneous Quantitation of 2-Hydroxy-4-Methoxybenzophenone, a Sunscreen Ingredient, and its Metabolites in Harlan Sprague Dawley Rat Plasma Following Perinatal Dietary Exposure.

2-Hydroxy-4-methoxybenzophenone (HMB) is a common ingredient in sunscreens and other personal care products and thus significant potential exists for human exposure. HMB was nominated to the National Toxicology Program (NTP) for testing due to its high exposure through consumer products and inadequate toxicological data at the time, which also included increasing concern for the potential effects of HMB on reproduction and development. HMB is metabolized to numerous metabolites in vivo and in vitro including 2,4-dihydroxybenzophenone (DHB), 2,3,4-trihydroxybenzophenone (THB) and 2,5-dihydroxy-4-methoxybenzophenone (2,5-DHMB) as well as their corresponding glucuronide and/or sulfate conjugates. In this study, we have developed and validated a liquid chromatography-tandem mass spectrometry method to quantitate free (unconjugated) HMB and DHB, and total (combined conjugated and unconjugated) HMB, DHB, THB and 2,5-DHMB. The method was successfully applied to quantitate these analytes in plasma from postnatal day 28 and 56 male and female Harlan Sprague Dawley rat pups following perinatal dietary exposure to 0 (control), 3,000, 10,000 and 30,000 ppm HMB beginning on gestational Day 6. All determined analyte concentrations increased with increasing dose and were significantly higher than the controls at both timepoints. All the total analytes were quantified in all plasma samples and total concentrations were considerably higher than free, suggesting extensive conjugation. Mean concentrations of total HMB and DHB were higher (~100-300-fold) than the free HMB and DHB concentrations, and total concentrations in plasma were approximately HMB≈DHB > 2,5-DHMB¼THB. Free and total analyte plasma concentrations were not sex-dependent and in general, both free and total analytes were detected in the control samples. Comparison of our rat data, using the internal dose, with human data available in the literature suggests that the rat doses used in our studies were within 4-fold of the human dose.

Exposure to butyl paraben during gestation and lactation in Hsd:Sprague dawley SD rats via dosed feed.

Butyl paraben (BPB) is an antimicrobial used in a variety of consumer products. Due to widespread human exposure and reported estrogenic activity, the National Toxicology Program quantified internal exposure during critical periods of development. Time-mated female Hsd:Sprague Dawley SD rats were administered 0, 1500, 5000 or 15,000 ppm BPB via NIH-07 feed, ad libitum, from gestation day (GD) 6 to postnatal day (PND) 28. Dam plasma, amniotic fluid and fetuses were collected on GD18 and pup and dam plasma were collected on PNDs 4, 10, 14, 21 and 28 and analyzed for free (unconjugated) and total (unconjugated and conjugated) BPB using liquid chromatography-tandem mass spectrometry. Free BPB was below the limit of quantitation in fetuses (LOQ 1.91 ng BPB/g fetus) and amniotic fluid (LOQ 0.17 ng BPB/mL amniotic fluid) at 1500 ppm. Analyte levels in amniotic fluid were less than 1% of maternal plasma, suggesting limited placental transfer. Total BPB in PND4 pup plasma was less than 5% of dam plasma in all exposure groups, suggesting low lactational transfer. However, at nearly all time points and exposure groups, there were higher levels of free BPB in pup versus dam plasma, suggesting limited conjugation in pups. Pup conjugation of BPB was age-dependent, not reaching the percent-conjugation in dams (>99%) until PNDs 21 to 28. These data illustrate low placental and lactational transfer of dietary BPB and that poor conjugation in pups during early lactation results in higher exposure to free BPB in pups compared to dams.