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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.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).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.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.

Deoxynivalenol (DON) is the most widely distributed trichothecene mycotoxin in grain-based foods and animal feed. Exposure to DON is widespread as it has been detected in food sources from around the world. The objective of this work was to develop a method to quantitate DON in biological matrices and apply it in a preliminary assessment of gestational and lactational transfer of DON following exposure of pregnant rats. The method used protein precipitation followed by ultra-performance liquid chromatography–tandem mass spectrometry. The method was evaluated in male Sprague Dawley rat plasma over the concentration range ∼2–1,000 ng/mL. The method was linear (r ≥ 0.99), accurate (mean relative error ≤ ±4.9%) and precise (relative standard deviation ≤ 5.5%). The mean absolute recovery was 85.9%. The limit of detection was 0.35 ng/mL. The method was also evaluated in gestational day (GD) 18 Hsd:Sprague Dawley®SD® dam plasma and fetal homogenate (mean % relative error ≤ ±16.9; % relative standard deviation ≤ 9.5). Concentrations of DON in dam plasma stored at −80°C for at least 29 days and in fetal homogenate for at least 43 days were within 97.9 to 120% of Day 0 concentrations, demonstrating that DON is stable in these matrices. The method was used to quantitate DON in rat maternal plasma, amniotic fluid, GD 18 fetuses and postnatal day (PND) 4 pups following exposure of dams to 0 (control) and 1 mg/kg DON beginning on GD 6 and continuing through gestation and lactation for a preliminary assessment of maternal transfer. In animals exposed to 1 mg/kg/day, similar concentration of DON was found in GD 18 dam plasma and fetuses, demonstrating significant gestational transfer. The concentration of DON in PND 4 dam plasma was similar to that in GD 18 dam plasma. However, DON was not detected in PND 4 pup plasma above the limit of detection of the assay, demonstrating absence of transfer of DON to pups via lactation.

2,2ʹ-Dimorpholinodiethyl ether (DMDEE) is a specialty amine catalyst used in the production of flexible foams, adhesives and coatings. The potential for occupational exposure to DMDEE is high, but toxicity data are very limited. The objective of this work was to develop a method to quantitate DMDEE in biological matrices to assess gestational and lactational transfer of DMDEE in rats following exposure of dams The method used protein precipitation, followed by removal of phospholipids and analysis of supernatant by ultra-performance liquid chromatography-tandem mass spectrometry. Rat fetuses were homogenized in water prior to protein precipitation and delipidation procedures. The method was evaluated in male Sprague Dawley rat plasma over the concentration range 5 to 1000 ng/mL. The method was linear (r ≥ 0.99), accurate (mean relative error (RE) ≤ ±11.9%) and precise (relative standard deviation (RSD) ≤ 2.7%). The mean absolute recovery was 106%. The limit of detection was 0.262 ng/mL. Standards as high as ∼100,000 ng/mL could be successfully diluted into the calibration range (mean %RE = −14.9; %RSD = 0.5). The method was evaluated in Sprague Dawley rat dam plasma, post-natal day 4 pup plasma, gestational day (GD) 18 amniotic fluid and fetal homogenate (mean %RE ≤ ±11.9; %RSD ≤ 2.3). Concentrations of DMDEE in rat dam plasma, amniotic fluid and fetal homogenate stored for at least 29 days and in pup plasma for at least 18 days at −80°C were within 87.7 to 99.5% of Day 0 concentrations, demonstrating that DMDEE is stable in these matrices. The method was used to quantitate DMDEE in rat plasma, amniotic fluid and fetus samples from a dose range finding toxicology study in which dams were dosed via gavage with DMDEE from GD 6 at doses of 0 (control), 62.5 and 250 mg/kg/day. DMDEE concentration increased with the dose in all matrices examined. The concentration in GD 18 fetuses was almost 2-fold higher than GD 18 dams demonstrating gestational transfer of DMDEE. However, the concentration in post-natal day 4 pup plasma was more than an order of magnitude lower than corresponding dam plasma suggesting less potential for transfer of DMDEE from dams to pups via lactation. There was no significant difference in concentration for male and female pup plasma.

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. Keywords: Butyl paraben, Maternal transfer, Placental transfer, Lactational transfer, Conjugation, Metabolism ontogeny

2,4-Dichlorophenoxybutyric acid (2,4-DB) is principally used in the United States on peanuts, soybeans, and alfalfa. In Europe, it is used on cereals, undersown cereals, lucerne (alfalfa), clover, and clover mixtures. Doses in the 2-year chronic/oncogenicity rat study were 0, 60, 600, and 1800 ppm. No evidence of an oncogenic potential for 2,4-DB was evident and the study clearly established a NOEL of 2.48 mg/kg/day (60 ppm, males) and 3.23 mg/kg/day (60 ppm, females), as well as an MTD of 78.0 (1800 ppm, males) and 110.6 mg/kg/day (1800 ppm, females), for chronic effects of 2,4-DB in the rat. Doses in the 18-month mouse oncogenicity study were 0, 25, 250, and 750 ppm. No oncogenic effect was noted in the study. In summary, the findings of these studies indicate low chronic toxicity of 2,4-DB and the lack of oncogenic response to 2,4-DB following chronic dietary exposure of 2,4-DB in the rat and mouse.

Forms of 2,4-dichlorophenoxyacetic acid (2,4-D) are herbicides used in the control of a wide variety of broadleaf and woody plants. Subchronic toxicity studies in dogs were conducted on three forms of 2,4-D: the parent form, 2,4-D acid (ACID); 2,4-D dimethylamine salt (DMA); and 2,4-D 2-ethylhexyl ester (2-EHE). The three studies were designed to allow for comparison of the toxicity of the three forms. Doses in the subchronic studies, on an acid equivalent basis, were 0, 0.5 (ACID only), 1.0, 3.75, and 7.5 mg/kg/day. Treatment related findings in the three studies included reductions in body weight gain, and food consumption, and minor increases in blood urea nitrogen, creatinine, and alanine aminotransferase. The data from the three subchronic studies demonstrated the comparable toxicity of ACID, DMA, and 2-EHE and support a subchronic no observed adverse effect level (NOAEL) of 1.0 mg/kg/day for all three forms. Due to the similarity in toxicity of the three forms of 2,4-D, a 1-year chronic toxicity study was performed on the parent ACID to fully characterize the potential toxicity of 2,4-D in the dog. ACID was well tolerated at doses of 0, 1.0, 5.0, and 7.5 mg/kg/day. The clinical pathology alterations were similar to those seen in the subchronic studies and were not progressive. The histopathology alterations observed were not severe in nature and the no observed effect level in the chronic study was determined to be 1.0 mg/kg/day. There was no indication of any immunotoxic or oncogenic response in the studies. In conclusion, the findings of these studies indicate comparable toxicity among representative forms of 2,4-D and their generally low toxicity following subchronic and chronic dietary exposure in the dog.

Forms of 2,4-dichlorophenoxyacetic acid (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. Subchronic toxicity studies in rats were conducted on three forms of 2,4-D: the parent form, 2,4-D acid; 2,4-D dimethylamine salt (DMA); and 2,4-D 2-ethylhexyl ester (2-EHE). Doses in the subchronic studies (on an acid equivalent basis) were 0, 1, 15, 100, and 300 mg/kg/day. Major treatment related findings in the three studies included decreases in red cell mass, decreases in T3 and T4 levels, decreases in ovary and testes weights, increases in liver, kidney, and thyroid weights, and cataracts and retinal degeneration (high-dose females). These data demonstrated the comparable toxicities of 2,4-D acid, DMA, and 2-EHE and support a subchronic no-observed-effect level of 15 mg/kg/day for all three forms. In summary, the findings of these studies indicate comparable low subchronic toxicity potentials among representative forms of 2,4-D.

Inhibition of the enzyme acetylcholinesterase (AChE) using a carbamate compound was measured in 30 Crl: CD®BR Sprague Dawley rats. Erythrocyte, plasma, and brain tissues were analyzed using modifications of the Ellman technique 1 on two different clinical chemistry analyzers. Both EDTA and heparin anticoagulated whole blood were used for the erythrocyte and plasma tests. Results demonstrated similar inhibition of the enzyme in all three tissues between the control and dosed groups using the two technique modifications and instruments. Final inhibition of plasma and erythrocyte AChE for the control vs . treated groups (males and females combined) was 89.5% vs . 82% and 39% vs . 38% for the Technicon AutoAnalyzer™ vs . the Boehringer Mannheim Hitachi™ 704, respectively. Inhibition of the left and right brain segments for the control vs . treated groups (males and females combined) was 35% vs . 39% and 33.2% vs . 29% for the Technicon and the Hitachi, respectively. All inhibitions were significant at the 5% level using two tailed Dunnett's t -Test. Hemolysates prepared from EDTA whole blood packed cells gave more consistent results on the Hitachi 704.

The potential for 2,4-D and seven of its salts and esters to induce cytogenetic abnormalities in mammalian cells in vivo was investigated in the mouse bone marrow micronucleus test. All the test materials were administered to male and female mice by oral gavage and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow were determined at intervals of 24, 48 and 72 h following dosing. There were no significant increases in the incidence of MN-PCE in the treated mice at any of the bone marrow sampling times. These results are consistent with the reported lack of in vitro genetic toxicity for these materials in various in vitro genotoxicity assays as well as the absence of carcinogenic potential for 2,4-D in both mice and rats.

2,4-dichlorophenoxyacetic acid and several of its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity in vitro of 2,4-D and seven of its salts and esters were examined by employing gene mutation in bacteria (Ames test) and induction of DNA damage and repair in rat hepatocytes. In addition, an in vivo unscheduled DNA synthesis (UDS) assay was performed on 2,4-D. There were no indications of genotoxic potential for 2,4-D acid, or any of its derivatives, in these assays. These results are consistent with the reported lack of carcinogenic potential for 2,4-D in both mice and rats.

Forms of 2,4-dichlorophenoxyacetic acid (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. Single-dose acute and 1-year chronic neurotoxicity screening studies in male and female Fischer 344 rats (10/sex/dose) were conducted on 2,4-D according to the U.S. EPA 1991 guidelines. The studies emphasized a Functional Observational Battery (which included grip performance and hindlimb splay tests), automated motor activity testing, and comprehensive neurohistopathology of perfused tissues. Dosages were up to 250 mg/kg by gavage for the single-dose study, and up to 150 mg/kg/day in the diet for 52 weeks in the repeated-dose study. In the acute study, gavage with 250 mg/kg test material caused slight transient gait and coordination changes and clearly decreased motor activity at the time of maximal effect on the day of treatment (day 1). Mild locomotor effects occurred in one mid-dose rat (75 mg/kg), on Day 1 only. No gait, coordination, or motor activity effects were noted by day 8. In the chronic study, the only finding of neurotoxicologic significance was retinal degeneration in females in the high-dose group (150 mg/kg/day). Body weights of both sexes were slightly less than controls in the mid-dose group, and 10% less than controls in the high-dose group. In summary, the findings of these studies indicated a mild, transient locomotor effect from high-level acute exposure, and retinal degeneration in female rats from high-level chronic exposure. Based on the results from these two studies, the no-observed-adverse-effect level for acute neurotoxicity was 15 mg/kg/day and for chronic neurotoxicity was 75 mg/kg/day.

The effects of long-term caloric restriction on the hepatic phase II drug metabolizing enzymes were investigated in the male Fischer 344 rat. Rats that had been restricted to 60% of their pair-fed control consumption from 14 weeks post-partum exhibited altered conjugating enzyme activities at 22 months. Caloric restriction significantly reduced the age-related decrease in glutathione-S-transferase activity towards 1,2-dichloro-4-nitrobenzene, but did not significantly alter the age-related changes in UDP-glucuronyltransferase or sulfotransferase activities towards hydroxysteroids. Caloric restriction appeared to increase hepatic microsomal UDP-glucuronyltransferase activity toward bilirubin and gamma-glutamyltranspeptidase activities. These observations suggest that caloric restriction has multiple effects on the hepatic phase II drug metabolizing enzymes in the rat. Such effects may alter hepatic metabolism and activation or detoxification of drugs and carcinogens.

The effects of long-term caloric restriction on the hepatic cytochrome P-450 dependent monooxygenase system were investigated in the 22-month-old Fischer 344 rat. Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver. Caloric restriction also increased hepatic microsomal testosterone 6β-hydroxylase, lauric acid 12-hydroxylase and 4-nitrophenol hydroxylase activities over corresponding values in both ad libitum fed 22-month and 60-day-old control male rats. This suggests that cytochrome P-450 isozymes, P-450 pcn 1&2 , P-452 and P450 j may be induced by caloric restriction. Such changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat.