A cross-sectional clinical study in women to investigate possible genotoxicity and hematological abnormalities related to the use of black cohosh botanical dietary supplements.

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.

Elevated Arsenic and Lead Concentrations in Natural Healing Clay Applied Topically as a Treatment for Ulcerative Dermatitis in Mice.

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.

Black cohosh extracts and powders induce micronuclei, a biomarker of genetic damage, in human cells.

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.

Effect of cell phone radiofrequency radiation on body temperature in rodents: Pilot studies of the National Toxicology Program's reverberation chamber exposure system.

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.

A Black Cohosh Extract Causes Hematologic and Biochemical Changes Consistent with a Functional Cobalamin Deficiency in Female B6C3F1/N Mice.

Black cohosh rhizome, available as a dietary supplement, is most commonly marketed as a remedy for dysmenorrhea and menopausal symptoms. A previous subchronic toxicity study of black cohosh dried ethanolic extract (BCE) in female mice revealed a dose-dependent ineffective erythropoiesis with a macrocytosis consistent with the condition known as megaloblastic anemia. The purpose of this study was to investigate potential mechanisms by which BCE induces these particular hematological changes. B6C3F1/N female mice (32/group) were exposed by gavage to vehicle or 1,000 mg/kg BCE for 92 days. Blood samples were analyzed for hematology, renal and hepatic clinical chemistry, serum folate and cobalamin, red blood cell (RBC) folate, and plasma homocysteine and methylmalonic acid (MMA). Folate levels were measured in liver and kidney. Hematological changes included decreased RBC count; increased mean corpuscular volume; and decreased reticulocyte, white blood cell, neutrophil, and lymphocyte counts. Blood smear evaluation revealed increased Howell-Jolly bodies and occasional basophilic stippling in treated animals. Plasma homocysteine and MMA concentrations were increased in treated animals. Under the conditions of our study, BCE administration caused hematological and clinical chemistry changes consistent with a functional cobalamin, and possibly folate, deficiency. Further studies are needed to elucidate the mechanism by which BCE causes increases in homocysteine and MMA.

Dose-response assessment of the dermal toxicity of Virginia cedarwood oil in F344/N rats and B6C3F1/N mice.

Virginia cedarwood oil is widely used as a fragrance material in household and personal products and as a naturally derived pesticide alternative. Due to conflicting literature on dermal exposures in animals and humans, concern for safe levels of human exposure remains. The present study evaluated the toxicity of cedarwood oil applied dermally to F344/N rats and B6C3F1/N mice for 13 weeks. Groups of 10 male and female rats and mice received no treatment (untreated control) or were administered cedarwood oil in 95% aqueous ethanol dermally at concentrations ranging from 0% (vehicle control), 6.25%, 12.5%, 25%, 50%, and 100% (undiluted). Rats and mice developed extensive skin lesions at the site of application. Benchmark dose modeling (BMD) was performed for the significantly increased skin lesions observed in the rat, to provide perspective for risk assessment applications. Benchmark dose modeling levels (BMDL) of 0.65 to 2.1% and 1.2 to 4.4% (equivalent to 13 to 42 mg/kg and 24 to 48 mg/kg, respectively) cedarwood oil were calculated for the most sensitive endpoint of epidermal hyperplasia in female rats and chronic active inflammation in male rats, respectively. These BMDL levels coincide with reported use levels in cosmetics and pesticides, raising the concern for human exposure.

The naturally occurring luteinizing hormone surge is diminished in mice lacking estrogen receptor Beta in the ovary.

Female ESR2-null mice (betaERKO) display defects in ovarian function and are subfertile. Follicular maturation is impaired and explains smaller litters, but betaERKO also produce fewer litters, which may be partially due to inadequate ovulatory signals. To test this, the amplitude and timing of the naturally occurring luteinizing hormone (LH) surge was measured in individual intact betaERKO and wild-type (WT) mice. Vaginal cytology was evaluated daily, and blood samples were taken from mice in proestrus. The amplitude of the LH surge was severely blunted in betaERKO mice compared to WT, but pituitary LH levels revealed no differences. The betaERKO mice did not produce a preovulatory estradiol surge. To determine if the smaller LH surges and the reduced number of litters in betaERKO were due to the lack of ESR2 in the hypothalamic-pituitary axis or due to the absence of ESR2 in the ovary, ovaries were transplanted from WT into betaERKO mice and vice versa. The size of the LH surge was reduced only in mice lacking ESR2 within the ovary, and these mice had fewer litters. Fertility and size of the LH surge were rescued in betaERKO mice receiving a WT ovary. These data provide the first experimental evidence that the LH surge is impaired in betaERKO females and may contribute to their reduced fertility. ESR2 is not necessary within the pituitary and hypothalamus for the generation of a normal LH surge and for normal fertility, but ESR2 is essential within the ovary to provide proper signals.

Toxicity and carcinogenicity studies of Ginkgo biloba extract in rat and mouse: liver, thyroid, and nose are targets.

Ginkgo biloba extract (GBE) is a popular herbal supplement that is used to improve circulation and brain function. In spite of widespread human exposure to relatively high doses over potentially long periods of time, there is a paucity of data from animal studies regarding the toxicity and carcinogenicity associated with GBE. In order to fill this knowledge gap, 3-month and 2-year toxicity and carcinogenicity studies with GBE administered by oral gavage to B6C3F1/N mice and F344/N rats were performed as part of the National Toxicology Program's Dietary Supplements and Herbal Medicines Initiative. The targets of GBE treatment were the liver, thyroid, and nose. These targets were consistent across exposure period, sex, and species, albeit with varying degrees of effect observed among studies. Key findings included a notably high incidence of hepatoblastomas in male and female mice and evidence of carcinogenic potential in the thyroid gland of both mice and rats. Various nonneoplastic lesions were observed beyond control levels in the liver, thyroid gland, and nose of rats and mice administered GBE. Although these results cannot be directly extrapolated to humans, the findings fill an important data gap in assessing risk associated with GBE use.

The estrogenic content of rodent diets, bedding, cages, and water bottles and its effect on bisphenol A studies.

The lowest observed adverse effect level for bisphenol A (BPA) in mice and rats is currently poorly defined due to inconsistent study designs and results in published studies. The objectives of the current study were to (1) compare the estrogenic content of rodent diets, bedding, cages, and water bottles to evaluate their impact on the estrogenic activity of BPA and (2) review the literature on BPA to determine the most frequently reported diets, beddings, cages, and water bottles used in animal studies. Our literature review indicated that low-dose BPA animal studies have inconsistent results and that factors contributing to this inconsistency are the uses of high-phytoestrogen diets and the different routes of exposure. In 44% (76 of 172) of all reports, rodents were exposed to BPA via the subcutaneous route. Our literature review further indicated that the type of diet, bedding, caging, and water bottles used in BPA studies were not always reported. Only 37% (64 of 172) of the reports described the diet used. In light of these findings, we recommend the use of a diet containing low levels of phytoestrogen (less than 20 µg/g diet) and metabolizable energy (approximately 3.1 kcal/g diet) and estrogen-free bedding, cages, and water bottles for studies evaluating the estrogenic activity of endocrine-disrupting compounds such as BPA. The oral route of BPA exposure should be used when results are to be extrapolated to humans.

Mechanistic insights from the NTP studies of chromium.

Hexavalent chromium (Cr(VI)) is a contaminant of water and soil and is a human lung carcinogen. Trivalent chromium (Cr(III)), a proposed essential element, is ingested by humans in the diet and in dietary supplements such as chromium picolinate (CP). The National Toxicology Program (NTP) demonstrated that Cr(VI) is also carcinogenic in rodents when administered in drinking water as sodium dichromate dihydrate (SDD), inducing neoplasms of the oral cavity and small intestine in rats and mice, respectively. In contrast, there was no definitive evidence of toxicity or carcinogenicity following exposure to Cr(III) administered in feed as CP monohydrate (CPM). Cr(VI) readily enters cells via nonspecific anion channels, in contrast to Cr(III), which cannot easily pass through the cell membrane. Extracellular reduction of Cr(VI) to Cr(III), which occurs primarily in the stomach, is considered a mechanism of detoxification, while intracellular reduction is thought to be a mechanism of genotoxicity and carcinogenicity. Tissue distribution studies in additional groups of male rats and female mice demonstrated higher Cr concentrations in tissues following exposure to Cr(VI) compared to controls and Cr(III) exposure at a similar external dose, indicating that some of the Cr(VI) escaped gastric reduction and was distributed systemically. The multiple potential pathways of Cr-induced genotoxicity will be discussed.

Two-year toxicity and carcinogenicity studies of Panax ginseng in Fischer 344 rats and B6C3F1 mice.

Ginseng is one of the most popular herbal supplements on the US market. Numerous reports of adverse effects from products containing ginseng have been filed with the US Food and Drug Administration (FDA) and the literature documents a "ginseng abuse syndrome" among regular users. However, the chronic toxic effects of ginseng are not well characterized. Because of its significant human exposure and the fact that little information on its toxicity is available, Panax ginseng was nominated by the US National Institutes of Health (NIH) to the US National Toxicology Program (NTP) to assess its carcinogenic potential. In this paper, we reported the results of NTP chronic toxicity and tumorigenicity bioassay. It shows that, under these experimental conditions, Panax ginseng is not toxic or tumorigenic.

Investigating the potential for toxicity from long-term use of the herbal products, goldenseal and milk thistle.

Two-year toxicity studies were conducted on the widely used herbal products, goldenseal and milk thistle, in male and female F344/N rats and B6C3F1 mice. With goldenseal root powder, the primary finding was an increase in liver tumors in rats and mice, and with milk thistle extract, a decrease in spontaneous background tumors including mammary gland tumors in female rats and liver tumors in male mice. Increased tumorigenicity in rodents exposed to goldenseal root powder may be due in part to the topoisomerase inhibition properties of berberine, a major alkaloid constituent in goldenseal, or its metabolite, berberrubine. In the clinic, use of topoisomerase-inhibiting agents has been associated with secondary tumor formation and inhibition in DNA repair processes. In contrast, the radical-scavenging and antioxidant properties of silibinin and other flavonolignans in milk thistle extract may have contributed to the decrease in background tumors in rodents in the present studies. The fate of the active constituents of goldenseal and milk thistle is similar in humans and rodents; therefore, the modes of action may translate across species. Further studies are needed to extrapolate the findings to humans.

Fourteen-week toxicity study of green tea extract in rats and mice.

The toxicity of green tea extract (GTE) was evaluated in 14-week gavage studies in male and female F344/NTac rats and B6C3F1 mice at doses up to 1,000 mg/kg. In the rats, no treatment-related mortality was noted. In the mice, treatment-related mortality occurred in male and female mice in the 1,000 mg/kg dose groups. The cause of early deaths was likely related to liver necrosis. Treatment-related histopathological changes were seen in both species in the liver, nose, mesenteric lymph nodes, and thymus. In addition, in mice, changes were seen in the Peyer's patches, spleen, and mandibular lymph nodes. The no adverse effect level (NOAEL) for the liver in both species was 500 mg/kg. In the nose of rats, the NOAEL in males was 62.5 mg/kg, and in females no NOAEL was found. No NOAEL was found in the nose of female or male mice. The changes in the liver and nose were considered primary toxic effects of GTE, while the changes in other organs were considered to be secondary effects. The nose and liver are organs with high metabolic enzyme activity. The increased susceptibility of the nose and liver suggests a role for GTE metabolites in toxicity induction.

Variations in phytoestrogen content between different mill dates of the same diet produces significant differences in the time of vaginal opening in CD-1 mice and F344 rats but not in CD Sprague-Dawley rats.

BACKGROUND: The optimum test diet and rodent species/strain for evaluating endocrine-disrupting compounds (EDCs) are critical. OBJECTIVES: We conducted studies to evaluate rodent species sensitivity and the effects of diets varying in phytoestrogen content on the time of vaginal opening (VO) in CD-1 mice, Fischer 344 (F344) rats, and CD Sprague-Dawley (S-D) rats. METHODS: Mice were weaned on postnatal day (PND) 15 and rats on PND19 and randomly assigned to control or test diets. Body weights, food consumption, and time of VO were recorded. RESULTS: The time of VO was significantly advanced in F344 rats fed diets containing daidzein and genistein, whereas these same diets did not advance VO in S-D rats. When animals were fed the AIN-76A diet spiked with genistein, time of VO was significantly advanced at all doses in CD-1 mice, at the two highest doses in F344 rats, and at the highest dose in S-D rats. The time of VO in F344 rats was more highly correlated with the phytoestrogen content than with the total metabolizable energy (ME) of 12 diets. CONCLUSIONS: The S-D rat is less sensitive to dietary phytoestrogens compared with the F344 rat or the CD-1 mouse, suggesting that the S-D rat is not the ideal model for evaluating estrogenic activity of EDCs. The profound effects of dietary phytoestrogens on the time of VO, an estrogen-sensitive marker, indicate that a standardized open-formula phytoestrogen-free diet containing a low ME level should be used to optimize the sensitivity of estrogenic bioassays.

Immunohistochemical analysis of expressions of hepatic cytochrome P450 in F344 rats following oral treatment with kava extract.

Kava (Piper methysticum), used for relaxation and pain relief, has been one of the leading dietary supplements and several reports linking hepatic functional disturbances and liver failure to kava have resulted in a ban on sales in Europe and Canada and the issuance of warnings by the US FDA. The National Toxicology Program conducted 14-week rat studies to characterize the toxicology of kava exposure in Fischer 344 rats [National Toxicity Program. 90 day gavage toxicity studies of KAVA KAVA EXTRACT in Fischer rats and B6C3F1 mice. Research Triangle Park, NC; 2005a; National Toxicity Program. Testing status of agents at NTP (KAVA KAVA EXTRACT M990058). Research Triangle Park, NC; 2005b. (http://ntp.niehs.nih.gov/index.cfm?objectid=071516E-C6E1-7AAA-C90C751E23D14C1B)]. Groups of 10 male and 10 female rats were administered kava extract by gavage at 0, 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg/day. Increased gamma-glutamyl-transpeptidase (GGT) activities were observed in the 2.0 g/kg males and 1.0 and 2.0 g/kg females, as well as increased serum cholesterol levels in males and females at 0.5 g/kg and higher. Increases in incidence and severity of hepatocellular hypertrophy (HP) were noted in males at 1.0 g/kg and females at 0.5 g/kg and higher, as well as increased liver weights. Immunohistochemical analyses of the expression of cytochrome-P450 (CYP) enzymes in liver of the control and 1.0- and 2.0-g/kg-treated groups indicated decreased expression of CYP2D1 (human CYP2D6 homolog) in 2.0 g/kg females and increased expression of CYP1A2, 2B1, and 3A1 in 1.0 and 2.0 g/kg groups of both sexes. The no observed adverse effect levels were decided as 0.25 g/kg in both genders, based on neurotoxic effects, increases in GGT, cholesterol, liver weight, and HP and decreases in body weight. Kava-induced hepatic functional changes in the F344 rat might be relevant to human clinical cases of hepatotoxicity following exposure.