Black cohosh (BC; Actaea racemosa L.), a top‐selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose‐related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross‐sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC‐naïve women. Twenty‐three women were enrolled in the BC‐exposed group and 28 in the BC‐naïve group. Use of any brand of BC‐only supplement for at least 3 months was required for inclusion in the BC‐exposed group. Supplements were analyzed for chemical composition to allow cross‐product comparisons. All participants were healthy, with no known exposures (e.g., x‐rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.

Black cohosh (BC; Actaea racemosa L.), a top-selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose-related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross-sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC-naïve women. Twenty-three women were enrolled in the BC-exposed group and 28 in the BC-naïve group. Use of any brand of BC-only supplement for at least 3 months was required for inclusion in the BC-exposed group. Supplements were analyzed for chemical composition to allow cross-product comparisons. All participants were healthy, with no known exposures (e.g., x-rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.

Buprenorphine, an analgesic commonly used in rodent surgery, requires repeated dosing every 4 to 6 h in order to provide adequate analgesia. However, redosing requires repeated handling, which may itself cause stress. Buprenorphine SR-LAB, which reportedly maintains serum levels of buprenorphine greater than 1 ng/mL for 48 to 72 h, is commercially available. However, the viscosity of the product and small dosing volumes make accurate dosing a challenge. Simbadol is a concentrated formulation of buprenorphine hydrochloride labeled for use in cats with recommended dosing frequency of every 24 h. We measured serum concentrations over time after a single injection of this product in C57BL/6NCrl mice and compared it to standard buprenorphine (Buprenex) and Buprenorphine SR-LAB. Male and female mice were injected subcutaneously with one of the 3 buprenorphine formulations at a dose of 1 mg/kg at time 0. Groups of mice (n = 8) were euthanized at 1, 4, 8, 12, 16 h for all groups and 24 h for the Simbadol and the Buprenorphine SR-LAB. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to determine concentrations of buprenorphine in each serum sample. High concentrations were observed in both Simbadol and standard buprenorphine groups one hour after injection (>50 ng/mL). These groups had similar buprenorphine concentration curves, including rates of decline. The standard buprenorphine group had mean concentrations less than 1 ng/mL by 12 h and the Simbadol group by 16 h. In contrast, the Buprenorphine SR-LAB group remained above the 1 ng/mL therapeutic threshold throughout the 24 h. In addition, clinical signs, including increased activity, that lasted for up to an hour after the injection in the Simbadol and standard buprenorphine groups. We conclude that Simbadol does not offer dosing advantages over the standard buprenorphine formulation when given at 1 mg/kg. Buprenorphine SR-LAB maintained a steady concentration of buprenorphine above 1 ng/mL for at least 24 h, and as such is a superior choice for providing long-term analgesia.

Ulcerative dermatitis in laboratory mice remains an ongoing clinical problem and animal welfare issue. Many products have been used to treat dermatitis in mice, with varying success. Recently, the topical administration of healing clays, such as bentonite and green clays, has been explored as a viable, natural treatment. We found high concentrations of arsenic and lead in experimental samples of therapeutic clay. Given the known toxic effects of these environmental heavy metals, we sought to determine whether the topical administration of a clay product containing bioavailable arsenic and lead exerted a biologic effect in mice that potentially could introduce unwanted research variability. Two cohorts of 20 singly housed, shaved, dermatitis free, adult male CD1 mice were dosed daily for 2 wk by topical application of saline or green clay paste. Samples of liver, kidney and whole blood were collected and analyzed for total arsenic and lead concentrations. Hepatic and renal concentrations of arsenic were not different between treated and control mice in either cohort; however, hepatic and renal concentrations of lead were elevated in clay treated mice compared to controls in both cohorts. In addition, in both cohorts, the activity of δ-aminolevulinate acid dehydratase, an enzyme involved with heme biosynthesis and a marker of lead toxicity, did not differ significantly between the clay-treated mice and controls. We have demonstrated that these clay products contain high concentrations of arsenic and lead and that topical application can result in the accumulation of lead in the liver and kidneys; however, these concentrations did not result in measurable biologic effects. These products should be used with caution, especially in studies of lead toxicity, heme biosynthesis, and renal α2 microglobulin function.

The National Toxicology Program tested two common radiofrequency radiation (RFR) modulations emitted by cellular telephones in a 2-year rodent cancer bioassay that included interim assessments of additional animals for genotoxicity endpoints. Male and female Hsd:Sprague Dawley SD rats and B6C3F1/N mice were exposed from Gestation day 5 or Postnatal day 35, respectively, to code division multiple access (CDMA) or global system for mobile modulations over 18 hr/day, at 10-min intervals, in reverberation chambers at specific absorption rates of 1.5, 3, or 6 W/kg (rats, 900 MHz) or 2.5, 5, or 10 W/kg (mice, 1,900 MHz). After 19 (rats) or 14 (mice) weeks of exposure, animals were examined for evidence of RFR-associated genotoxicity using two different measures. Using the alkaline (pH > 13) comet assay, DNA damage was assessed in cells from three brain regions, liver cells, and peripheral blood leukocytes; using the micronucleus assay, chromosomal damage was assessed in immature and mature peripheral blood erythrocytes. Results of the comet assay showed significant increases in DNA damage in the frontal cortex of male mice (both modulations), leukocytes of female mice (CDMA only), and hippocampus of male rats (CDMA only). Increases in DNA damage judged to be equivocal were observed in several other tissues of rats and mice. No significant increases in micronucleated red blood cells were observed in rats or mice. In conclusion, these results suggest that exposure to RFR is associated with an increase in DNA damage. Environ. Mol. Mutagen. 61:276–290, 2020. © 2019 Wiley Periodicals, Inc.

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10–12 %), compared to control, occurred at 5,000–10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9–13 %) were increased at 2,500−5,000 ppm and 1250–10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000–10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0–10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5–50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10–50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10–50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250−1000 μg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

The role of ERα36 in regulating BPA's effects and its potential as a risk factor for human uterine fibroids were evaluated. BPA at low concentrations (10-6 μM - 10 μM) increased proliferation by facilitating progression of hormonally regulated, immortalized human uterine leiomyoma (ht-UtLM; fibroid) cells from G0-G1 into S phase of the cell cycle; whereas, higher concentrations (100 μM-200 μM) decreased growth. BPA upregulated ERα36 gene and protein expression, and induced increased SOS1 and Grb2 protein expression, both of which are mediators of the MAPKp44/42/ERK1/2 pathway. EGFR (pEGFR), Ras, and MAPKp44/42 were phosphorylated with concurrent Src activation in ht-UtLM cells within 10 min of BPA exposure. BPA enhanced colocalization of phosphorylated Src (pSrc) to ERα36 and coimmunoprecipitation of pSrc with pEGFR. Silencing ERα36 with siERα36 abolished the above effects. BPA induced proliferation in ht-UtLM cells through membrane-associated ERα36 with activation of Src, EGFR, Ras, and MAPK nongenomic signaling pathways.

Activation and/or upregulated expression of p38δ are demonstrated in human skin malignancies including cutaneous squamous cell carcinoma, suggesting a role for p38δ in skin carcinogenesis. We previously reported that mice with germline deletion of the p38δ gene are significantly protected from chemical skin carcinogenesis. Here, we investigated the effects of cell-selective targeted ablation of p38δ in keratinocytes and in immune (myeloid) cells on skin tumor development in a two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical mouse skin carcinogenesis model. Conditional keratinocyte-specific p38δ ablation (p38δ-cKO∆K) did not influence the latency, incidence, or multiplicity of chemically-induced skin tumors, but led to increased tumor volume in females during the TPA promotion stage, and reduced malignant progression in males and females relative to their wild-type counterparts. In contrast, conditional myeloid cell-specific p38δ deletion (p38δ-cKO∆M) inhibited DMBA/TPA-induced skin tumorigenesis in male but not female mice. Thus, tumor onset was delayed, and tumor incidence, multiplicity, and volume were reduced in p38δ-cKO∆M males compared with control wild-type males. Moreover, the percentage of male mice with malignant tumors was decreased in the p38δ-cKO∆M group relative to their wild-type counterparts. Collectively, these results reveal that cell-specific p38δ targeting modifies susceptibility to chemical skin carcinogenesis in a context-, stage-, and sex-specific manner.

Genetic variability in humans is a key characteristic underlying susceptibility potential for responses to environmental chemicals, yet animal testing paradigms typically do not capture the breadth of human diversity.Diversity Outbred (J:DO) mice are a population of outbred animals with well-randomized genetic sequence polymorphisms and a genetic architecture similar to that of human populations.This study was designed to evaluate baseline characteristics of J:DO mice that could serve as a foundation for future toxicological studies using this population-based model.Adult male DO mice (n = 75) were fed a control diet and monitored for 13 weeks.Clinical chemistry values and calculated reference ranges, organ and body weights, and background histopathology for the liver and kidney are provided.Measurements of sperm parameters and levels of leptin and insulin are included to assess variability in reproductive and endocrine parameters.Power calculations are provided for endpoints having comparator data from prior recent NTP studies, allowing researchers to determine the number of DO mice needed-per endpoint-to observe a comparable statistical difference to that using conventional rodent stocks (80% power, p = 0.05).The data suggest sample sizes for guideline toxicological studies when using DO mice would need to be increased to at least 16-34 mice for most conventional endpoints.In contrast, hormonal and reproductive endpoints would require >100 mice per endpoint to achieve the same power for determining a difference in experimental groups comparable to that of conventional studies, which may impact study design.The results of this initial characterization of DO mice suggest an inherent variability in the underlying biology that might provide utility for evaluating population dynamics in responses relevant to genetically diverse human populations.

Black cohosh extract (BCE) is a widely used dietary supplement marketed to women to alleviate symptoms of gynecological ailments, yet its toxicity has not been well characterized. The National Toxicology Program (NTP) previously reported significant increases in micronucleated erythrocytes in peripheral blood of female Wistar Han rats and B6C3F1/N mice administered 15–1,000 mg BCE/kg/day by gavage for 90 days. These animals also developed a dose-dependent nonregenerative macrocytic anemia characterized by clinical changes consistent with megaloblastic anemia. Both micronuclei (MN) and megaloblastic anemia can arise from disruption of the folate metabolism pathway. The NTP used in vitro approaches to investigate whether the NTP's test lot of BCE, BCEs from various suppliers, and root powders from BC and other cohosh species, were genotoxic in general, and to gain insight into the mechanism of action of BCE genotoxicity. Samples were tested in human TK6 lymphoblastoid cells using the In Vitro MicroFlow® MN assay. The NTP BCE and a BC extract reference material (XRM) were tested in the MultiFlow ® DNA Damage assay, which assesses biomarkers of DNA damage, cell division, and cytotoxicity. The NTP BCE and several additional BCEs were tested in bacterial mutagenicity assays. All samples induced MN when cells were grown in physiological levels of folic acid. The NTP BCE and BC XRM produced activity patterns consistent with an aneugenic mode of action. The NTP BCE and five additional BCEs were negative in bacterial mutagenicity tests. These findings show that black cohosh preparations induce chromosomal damage and may pose a safety concern. Environ. Mol. Mutagen. 59:416–426, 2018. © 2018 Published 2018. This article is a US Government work and is in the public domain in the USA.

Poly- and perfluoroalkyl substances (PFAS) are chemically and thermally stable, hydrophobic, lipophobic compounds used in stain repellants and water and oil surfactants, and associated with immunosuppression and peroxisome proliferator activity. Perfluoro-n-decanoic acid (PFDA, (CF3(CF2)8COOH), a fluorinated straight chain fatty acid compound, is reported to induce thymic atrophy and reversible bone marrow hypocellularity in rodent models. The objective of this study was to assess potential immunotoxicity of PFDA, due to its structural similarity to other immunosuppressive PFASs. Female Harlan Sprague–Dawley rats were exposed to 0–2.0 mg PFDA/kg by oral gavage daily for 28 d. Female B6C3F1/N mice were exposed once/week to 0–5.0 mg PFDA/kg by gavage for 4 weeks. Animals were evaluated for effects on immune cell populations in spleen and bone marrow, and innate, humoral-, and cell-mediated immunity. Mice were also evaluated for resistance to Influenza virus. Treatment-related hepatocyte necrosis and hepatomegaly were observed in rats treated with 0.5 mg PFDA/kg/d. In mice, hepatomegaly (26–89%) was observed following exposure to ≥0.625 mg PFDA/kg/week, while splenic atrophy (20%) was observed at 5.0 mg PFDA/kg/week. At 5.0 mg PFDA/kg/week, total spleen cells, and Ig + and NK + cells were decreased (17.6–27%). At ≥ 1.25 mg PFDA/kg/week the numbers of splenic CD3+, CD4+, CD8+, and Mac3+ cells were decreased (10.5–39%). No changes were observed in leukocyte subpopulations in PFDA-exposed rats. Phagocytosis by fixed-tissue macrophages was decreased in liver (specific activity, 24–39%) at ≥0.25 mg PFDA/kg/d in rats. PFDA-induced effects on humoral- and cell-mediated immunity, host resistance, and bone marrow progenitor cells were limited. These data suggest that exposure to PFDA may induce adverse effects in rat liver in a manner consistent with the PFAS class, and may also alter the balance of immune cell populations in lymphoid tissues in mice.

Sterilization of rodent feed by steam autoclaving is a common practice in many research institutions. Often we only consider the beneficial effects of this process-the reduction of microbial contamination-and forget that the high temperatures and pressures can have negative effects on diet quality. The purpose of our study was to assess both the physical and chemical changes to a standard rodent feed autoclaved at multiple sterilization temperatures and the effects of the treated diets on mice. Pelleted NIH31 rodent feed was autoclaved at 4 sterilization temperatures (230, 250, 260, and 270 °F). Feed pellet hardness and the acrylamide concentrations of the diets were tested and compared with irradiated NIH31 feed. Study diets were fed to mice for 28 d, after which tissue samples were collected for analysis of acrylamide, glycidamide (the active metabolite of acrylamide), and genotoxicity. Both feed pellet hardness and acrylamide concentration increased with increasing sterilization temperatures; however, neither affected feed intake or body weight gain. Plasma acrylamide and glycidamide were significantly elevated only in mice fed NIH31 diet autoclaved at 270 °F compared with the irradiated feed, whereas urine acrylamide and glycidamide metabolites were significantly elevated in most autoclaved diets. Liver DNA adducts, which correlate with genotoxicity, were significantly elevated in all autoclaved diets compared with the irradiated diet. Institutions that autoclave their animal diets should carefully consider the temperatures necessary to achieve feed sterilization and the type of studies in which these autoclaved diets are used.

Despite documented adverse effects, limits for rodent exposure to vibration in the laboratory animal facility have not been established. This study used female C57BL/6 mice to determine the frequencies of vibration at which mice were most sensitive to behavioral changes, the highest magnitude of vibration that would not cause behavioral changes, the behavioral changes that occur in response to vibration, and the extent to which mice habituate to vibration. Mice were exposed to frequencies of vibration between 20 and 190 Hz at accelerations of 0.05 to 1.0 m/s2. Behavioral responses were videorecorded and subsequently scored. Mice showed the most behavioral responses at 1.0 m/s2. At intermediate accelerations of 0.5 and 0.75 m/s2, behavioral responses were most prevalent at frequencies of 70 to 100 Hz. In contrast, at an acceleration of 0.05 m/s2, mice did not show any discernible behavioral response. Behavioral responses induced by the initiation of vibration were transient, generally lasting only 2 to 10 s. Behaviors in awake mice included abrupt freezing of motion, hunched posture, and surveying the cage environment. In mice that were asleep, responses consisted of lifting the head suddenly with or without prior shifting of body position. When exposed to multiple periods of vibration over a short time, responses seemed to decrease. In summary, mice were particularly sensitive to vibration between 70 to 100 Hz, did not respond to the slowest acceleration (0.05 m/s2), and exhibited transient responses at the initiation of vibration.

Radiofrequency radiation (RFR) causes heating, which can lead to detrimental biological effects. To characterize the effects of RFR exposure on body temperature in relation to animal size and pregnancy, a series of short‐term toxicity studies was conducted in a unique RFR exposure system. Young and old B6C3F1 mice and young, old, and pregnant Harlan Sprague‐Dawley rats were exposed to Global System for Mobile Communication (GSM) or Code Division Multiple Access (CDMA) RFR (rats = 900 MHz, mice = 1,900 MHz) at specific absorption rates (SARs) up to 12 W/kg for approximately 9 h a day for 5 days. In general, fewer and less severe increases in body temperature were observed in young than in older rats. SAR‐dependent increases in subcutaneous body temperatures were observed at exposures ≥6 W/kg in both modulations. Exposures of ≥10 W/kg GSM or CDMA RFR induced excessive increases in body temperature, leading to mortality. There was also a significant increase in the number of resorptions in pregnant rats at 12 W/kg GSM RFR. In mice, only sporadic increases in body temperature were observed regardless of sex or age when exposed to GSM or CDMA RFR up to 12 W/kg. These results identified SARs at which measurable RFR‐mediated thermal effects occur, and were used in the selection of exposures for subsequent toxicology and carcinogenicity studies. Bioelectromagnetics. 39:190–199, 2018. © 2018 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

This article describes data related to the research article entitled “Carcinogenic activity of pentabrominated diphenyl ether mixture (DE-71) in rats and mice” (Dunnick et al., 2018). PBDE-induced hepatocellular tumors harbored Hras and Ctnnb1 mutations and the methods for these studies are provided. Tissue levels of PBDE congeners in rats and mice after oral exposure to PBDE mixture increased with increasing dose of PBDE. There was no correlation between AhR status and the incidence of hepatocellular tumors in female Wistar Han rats. This manuscript provides additional information on the methods for conducting mutational analysis, PBDE tissue level determinations, and AhR genotyping.

Black cohosh extract (BCE) is a widely used dietary supplement marketed to women to alleviate symptoms of gynecological ailments, yet its toxicity has not been well characterized. The National Toxicology Program (NTP) previously reported significant increases in micronucleated erythrocytes in peripheral blood of female Wistar Han rats and B6C3F1/N mice administered 15–1,000 mg BCE/kg/day by gavage for 90 days. These animals also developed a dose‐dependent nonregenerative macrocytic anemia characterized by clinical changes consistent with megaloblastic anemia. Both micronuclei (MN) and megaloblastic anemia can arise from disruption of the folate metabolism pathway. The NTP used in vitro approaches to investigate whether the NTP's test lot of BCE, BCEs from various suppliers, and root powders from BC and other cohosh species, were genotoxic in general, and to gain insight into the mechanism of action of BCE genotoxicity. Samples were tested in human TK6 lymphoblastoid cells using the In Vitro MicroFlow® MN assay. The NTP BCE and a BC extract reference material (XRM) were tested in the MultiFlow® DNA Damage assay, which assesses biomarkers of DNA damage, cell division, and cytotoxicity. The NTP BCE and several additional BCEs were tested in bacterial mutagenicity assays. All samples induced MN when cells were grown in physiological levels of folic acid. The NTP BCE and BC XRM produced activity patterns consistent with an aneugenic mode of action. The NTP BCE and five additional BCEs were negative in bacterial mutagenicity tests. These findings show that black cohosh preparations induce chromosomal damage and may pose a safety concern. Environ. Mol. Mutagen. 59:416–426, 2018. © 2018 Published 2018. This article is a US Government work and is in the public domain in the USA.

BACKGROUND:Continued efforts to phase out bisphenol A (BPA) from consumer products have been met with the challenges of finding safer alternatives. OBJECTIVES:This study aimed to determine whether early-life exposure to BPA and its related analogues, bisphenol AF (BPAF) and bisphenol S (BPS), could affect female pubertal mammary gland development and long-term mammary health in mice. METHODS:Timed pregnant CD-1 mice were exposed to vehicle, BPA (0.5, 5, 50 mg/kg), BPAF (0.05, 0.5, 5 mg/kg), or BPS (0.05, 0.5, 5 mg/kg) via oral gavage between gestation days 10–17. Mammary glands were collected from resulting female offspring at postnatal day (PND) 20, 28, 35, and 56, and at 3, 8, and 14 months for whole mount, histopathological evaluation, and quantitative real-time polymerase chain reaction (qPCR); serum steroid concentrations were also measured at these time points. RESULTS:In the bisphenol-exposed mice, accelerated mammary gland development was evident during early puberty and persisted into adulthood. By late adulthood, mammary glands from bisphenol-exposed female offspring exhibited adverse morphology in comparison with controls; most prominent were undifferentiated duct ends, significantly more lobuloalveolar hyperplasia and perivascular inflammation, and various tumors, including adenocarcinomas. Effects were especially prominent in the BPAF 5 mg/kg and BPS 0.5 mg/kg groups. Serum steroid concentrations and mammary mRNA levels of Esr1, Pgr, Ar, and Gper1 were similar to controls. CONCLUSIONS:These data demonstrate that prenatal exposure of mice to BPAF or BPS induced precocious development of the mammary gland, and that siblings were significantly more susceptible to spontaneous preneoplastic epithelial lesions and inflammation, with an incidence greater than that observed in vehicle- and BPA-exposed animals. https://doi.org/10.1289/EHP3189.

Purpose of Review Uterine fibroids are common benign tumors of women in the USA and worldwide, yet the biological nature and pathogenesis of these tumors remain largely unknown. This review presents our view of the stages in the life cycle of a subset of uterine fibroid myocytes, introduces hypothetical concepts and morphological data to explain these changes, and relates these changes in individual myocytes to the phases of fibroid tumor development. Recent Findings The observations gained from light and electron microscopic, immunohistochemical, and morphometric studies in our laboratory have led to the hypothesis that fibroid changes over time may relate to the excessive production of collagen by phenotypically transformed myocytes. This accumulation of collagen results in decreased microvessel density, followed by myocyte injury and atrophy, with eventual senescence and involution through ischemic cellular degeneration and inanition. Summary Uterine leiomyomas, or fibroids, are characterized by two histologic features-proliferation of myocytes and production of an extracellular collagenous matrix. In the larger tumors, the collagenous matrix is often abundant. Within those regions in which the accumulating collagen is excessive, the myocytes are progressively separated from their blood supply, resulting in myocyte atrophy and eventually cell death. It is within these hypocellular, hyalinized areas that the complete lifecycle of the fibroid myocyte is realized. It begins with the phenotypic transformation of a contractile cell to one characterized by proliferation and collagen synthesis, progresses through an intermediate stage of atrophy related to interstitial ischemia, and eventuates in cell death due to inanition. Lastly, resorption of inanotic cells appears to occur by a non-phagocytic, presumably enzymatic process of degradation and recycling that we refer to as reclamation.

Despite documented adverse effects, limits for rodent exposure to vibration in the laboratory animal facility have not been established. This study used female C57BL/6 mice to determine the frequencies of vibration at which mice were most sensitive to behavioral changes, the highest magnitude of vibration that would not cause behavioral changes, the behavioral changes that occur in response to vibration, and the extent to which mice habituate to vibration. Mice were exposed to frequencies of vibration between 20 and 190 Hz at accelerations of 0.05 to 1.0 m/s(2). Behavioral responses were videorecorded and subsequently scored. Mice showed the most behavioral responses at 1.0 m/s(2). At intermediate accelerations of 0.5 and 0.75 m/s(2), behavioral responses were most prevalent at frequencies of 70 to 100 Hz. In contrast, at an acceleration of 0.05 m/s(2), mice did not show any discernible behavioral response. Behavioral responses induced by the initiation of vibration were transient, generally lasting only 2 to 10 s. Behaviors in awake mice included abrupt freezing of motion, hunched posture, and surveying the cage environment. In mice that were asleep, responses consisted of lifting the head suddenly with or without prior shifting of body position. When exposed to multiple periods of vibration over a short time, responses seemed to decrease. In summary, mice were particularly sensitive to vibration between 70 to 100 Hz, did not respond to the slowest acceleration (0.05 m/s(2)), and exhibited transient responses at the initiation of vibration.

Early deaths of young or juvenile animals (before sexual maturation is achieved) in routine regulatory safety studies present pathologists and toxicologists with the challenge of interpreting findings in the male reproductive tract. Additionally, the advent of toxicity testing regulations has resulted in a growing need for the use of juvenile animals in toxicology studies. Here, we present the reproductive toxicity findings from a 13-week inhalation toxicity study with ortho-phthalaldehyde (OPA) in male rats and mice as a case example for working through this challenging task. In this study with OPA, survival was significantly reduced in the two highest exposure concentrations of OPA tested. Early deaths and histopathological lesions in the testes and epididymides were generally also limited to these two highest exposure groups. Therefore, there was concern that peripubertal morphological features could be a confounding factor for the histopathological evaluation of exposure-related testicular and epididymal findings. Although it can be difficult to differentiate exposure-related effects from the normal morphological features defining peripubertal changes in the testes and epididymides in animals that die early in a toxicity study, the use of age-matched controls in this case study with OPA provided a reference and aided in the differentiation of these effects.