Mouse Micronucleus Research Articles

Toxicity Assessment of Thiodiglycol

Sulfur mustard (HD) undergoes hydrolysis to form various products such as thiodiglycol (TG) in biological and environmental systems. TG is a precursor in the production of HD and it is also considered as a "Schedule 2" compound (dual-use chemicals with low to moderate commercial use and high-risk precursors). Several toxicological studies on TG were conducted to assess environmental and health effects. The oral LD(50) values were >5000 mg/kg in rats. It was a mild skin and moderate ocular irritant and was not a skin sensitizer in animals. It was not mutagenic in Ames Salmonella, Escherichia coli, mouse lymphoma, and in vivo mouse micronucleus assays, but it induced chromosomal aberrations in Chinese hamster ovarian (CHO) cells. A 90-day oral subchronic toxicity study with neat TG at doses of 0, 50, 500, and 5000 mg/kg/day (5 days/week) in Sprague-Dawley rats results show that there are no treatment-related changes in food consumption, hematology, and clinical chemistry in rats of either sex. The body weights of both sexes were significantly lower than controls at 5000 mg/kg/day. Significant changes were also noted in both sexes in absolute weights of kidneys, kidney to body weight ratios, and kidney to brain weight ratios, in the high-dose group. The no-observed-adverse-effect level (NOAEL) for oral toxicity was 500 mg/kg/day. The developmental toxicity conducted at 0, 430, 1290, and 3870 mg/kg by oral gavage showed maternal toxicity in dams receiving 3870 mg/kg. TG was not a developmental toxicant. The NOAEL for the developmental toxicity in rats was 1290 mg/kg. The provisional oral reference dose (RfD) of 0.4 mg/kg/day was calculated for health risk assessments. The fate of TG in the environment and soil showed biological formation of thiodiglycalic acid with formation of an intermediate ((2-hydroxyethyl)thio)acetic acid. It was slowly biodegraded under anaerobic conditions. It was not toxic to bluegill sunfish at 1000 mg/L and its metabolism and environmental and biochemical effects are summarized.

Chemopreventive Effects of Aloe Against Genotoxicity Induced by Benzo[a]pyrene

Chemopreventive effects of aloe against benzo[a]pyrene (BaP) mutagenicity were investigated in the Salmonella typhimurium bacterial mutation assay, the chromosome aberration assay using Chinese hamster ovary (CHO) cells, and the mouse micronuclei test using bone-marrow cells. In the bacterial assay, aloe produced a concentration-dependent decrease in the number of mutant colonies induced by BaP. The chromosome-damaging responses of BaP in CHO cells were abolished by treatment with aloe, approximately to the level seen in control. In the in vivo mouse bone-marrow micronuclei test, pretreatment of aloe 24 h prior to BaP treatment reduced the frequency of micronucleated polychromatic erythrocytes. In the cells of CHO and bone marrow treated with aloe, glutathione (GSH) levels were shown to be higher and extracellular discharge rate increased as incubation time with aloe rose. MDR1 and MRP2 gene were more expressed in Hepa c cells than in NTCC cells, but there was no change in BCRP gene expression. The antimutagenic effects of aloe were statistically significant and concentration dependent. These results demonstrated that aloe might exert chemopreventive effects against BaP-induced mutagenicity.

A subchronic toxicity study in rats and genotoxicity tests with an aqueous ethylcellulose dispersion

Surelease ® Aqueous Ethylcellulose Dispersion is an excipient used as a modified release coating for beads, granules, non-pariels, drug crystals and tablets and for taste masking applications for drug products and dietary supplement products. A study was conducted to assess the toxicity of spray-dried Surelease when administered orally, via dietary admixture, to Sprague-Dawley CD ® rats (20/sex/group) at dose levels of 0, 2000, 3500, and 5000 mg/kg/day for a period of at least 3 months. After 3 months of treatment, all rats scheduled for terminal sacrifice were killed and selected organs were weighed. Complete macroscopic examinations and histopathological evaluation of selected tissues were conducted on all animals. Neuropathological evaluations were performed on 5 animals/sex/group. No mortality occurred during the study. Clinical observations, ophthalmology, body weight and food consumption, hematology, coagulation, clinical chemistry, urinalysis, functional observational assessments, motor activity, organ weights and ratios and macroscopic and microscopic observations did not reveal any significant, consistent, dose-dependent test article-related adverse effects. The NOAEL (no-observed-adverse-effect-level) is 5000 mg/kg/day, the highest dose tested. A series of genotoxicity tests were conducted with Surelease. Surelease showed no evidence of mutagenic activity in the bacterial reverse mutation test with and without metabolic activation and in the in vitro cell mutation assay under the experimental conditions employed. Surelease did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by gavage in the mouse micronucleus in vivo test procedure. These findings support the safety of Surelease for use as an excipient.

In Vitro and In Vivo Biocompatibility of Nano-Hydroxyapatite/Collagen Composites

A nano-grade hydroxyapatite/collagen composite was prepared by an in situ synthesis technique from calcium nitrate, diammoniun hydrogen phosphate, and a cowhide collagen sol at low temperature. XRD and TEM analyses of the composite indicated that crystals formed in the collagen fibril matrix were nanohydroxyapatite with low crystallinity. Biocompatibility of the composite was evaluated by in vitro cytotoxicity test and in vivo genotoxicity and sensitization test. No mutagenic activity of the composite was observed in mouse micronucleus tests. No evidence of dermal sensitization of the composite was found in guinea pig maximization tests. The results from a filter diffusion test indicated that the composite did not induce a cytotoxic behavior. All these results suggest that the composite has excellent biocompatibility.

고선량 조사된 시판 분말죽의 유전독성학적 안전성평가

분말죽의 위생화 및 물리적 특성 개선을 위해 감마선조사 기술의 이용 가능성이 높아짐에 따라 이들의 안전성을 확보할 목적으로 30 ㎏y 고선량 감마선조사 분말죽의 유전독성학적 안전성 평가를 실시하였다. 감마선 조사 및 비조사 분말죽의 S. Typhimurium TA 98, TA100, TA1535 및 TA1537에 대한 복귀변이 집락수를 조사한 결과, 대사활성계 도입 및 부재시 모두 시험적용 농도인 0.625～10 ㎎/plate의 범위에서 복귀변이 집락수의 농도 의존적인 증가 혹은 감소를 보이지 않아 감마선 조사 분말죽(30 ㎏y)은 돌연변이원성이 없는 것으로 판단되었다. 또한, 설치류 망상적혈구를 이용하여 감마선 조사된 분말죽의 소핵 형성시험을 수행한 결과, 감마선 조사 분말죽은 시험적용 용량인 625～5,000 ㎎/㎏의 범위에서 소핵을 가진 망상적혈구의 출현율이 음성대조군과 유의한 차이를 나타내지 않아 소핵을 유발하지 않음을 확인하였다. 포유류 배양세포를 이용한 염색체 이상시험에서도 감마선 조사 분말죽(30 ㎏y)은 시험적용 용량에서 염색체이상 유발능이 5% 미만이었다.

Evaluation of γ‐Hydroxybutyric Acid for Genotoxicity in the Mouse Micronucleus Assay

Gamma-hydroxybutyric acid (GHB) is an endogenous compound found in the brain and other tissues of mammals. Neurotransmitter/neuromodulator functions have been ascribed to GHB, which has lately become a drug of abuse. In this study, we tested GHB for genotoxicity by measuring its ability to induce micronuclei in polychromatic erythrocytes (reticulocytes) in the peripheral blood of mice. Intraperitoneal injection with a dose of 25 mg/kg/day for 3 days or 50 mg/kg/day x 3 days resulted in a significant (by Dunnett's test) increase of 1.9- to 2.1-fold in micronuclei. However, because increases were small and because no consistent dose-dependent increase in induced micronuclear frequency could be demonstrated, our results do not conclusively show that GHB is an in vivo genotoxicant in mammals.

Genotoxicity testing of a fenugreek extract

Fenugreek seeds have been used in traditional medicines as a remedy for diabetes. Rich in protein, fenugreek seeds contain the unique major free amino acid 4-hydroxyisoleucine (4-OH-Ile), which has been characterized as one of the active ingredients for blood glucose control. Current use of fenugreek in foodstuff has been limited to its role as a flavoring agent, and not as an ingredient to help mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a fenugreek seed extract (THL), containing a minimum of 40% 4-OH-ILE, was evaluated using the standard battery of tests (reverse mutation assay; mouse lymphoma forward mutation assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. THL was determined not to be genotoxic under the conditions of the tested genetic toxicity battery. The negative assay results provide support that addition of THL to foodstuffs formulated for people with diabetes is expected to be safe. A wide safety margin is established, as anticipated doses are small compared to the doses administered in the assays.

Antimutagenicity of selenium-enriched rice on mice exposure to cyclophosphamide and mitomycin C

The in vivo antimutagenicity of Se-enriched rice was evaluated by bone marrow micronucleus and testicle chromosomal aberrations assay in mice exposed to cyclophosphamide (CP) and mitomycin C (MMC). Regular rice did not alter the occurrence of chemical-induced mutation. However, the addition of Se-enriched rice or selenite significantly inhibited the incidence of CP-induced micronuclei and MMC-induced chromosomal aberration in mice and the effect was dose-dependent. Providing selenite or Se-enriched rice also significantly increased the activity of glutathione peroxidase in liver and the selenium concentration in blood compared to regular rice. No significant differences were found in mice body weight gain. These results revealed the antimutagenic potential of Se-enriched rice against chemical-induced mutation.

Frequency of Tk and Hprt lymphocyte mutants and bone marrow micronuclei in mice treated neonatally with zidovudine and didanosine.

The nucleoside analog zidovudine (3'-azido-3'-deoxythymidine, AZT), by itself or in combination with other anti- retroviral drugs, is used perinatally to prevent mother to child transmission of human immunodeficiency virus type 1. AZT is mutagenic in vitro and mutagenic and carcinogenic when administered to neonatal mice. A previous study indicated that the anti-retroviral agent didanosine (2',3'-dideoxyinosine, ddI) potentiated the mutagenicity of AZT in the thymidine kinase (TK) gene of cultured human TK6 lymphoblastoid cells. We have evaluated whether or not ddI affects the in vivo genotoxicity of AZT by breeding C57Bl/6N/Tk+/- female mice with C3H/HeNMTV male mice and treating the offspring daily on postnatal days 1-8 with 200 mg/kg ddI alone or in combination with 200 mg/kg AZT. One day after the last dose, bone marrow polychromatic erythrocytes (PCEs) were obtained to assess the induction of micronuclei; 3 weeks following treatment, the induction of mutants was determined in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) and Tk genes of splenic T lymphocytes from B6C3F1/Tk+/- mice. The mixture of AZT and ddI, but not ddI alone, caused a significant increase in micronucleated PCEs. When assessed 3 weeks after dosing, ddI did not induce mutations in the Hprt or Tk genes. The mixture of AZT and ddI also did not induce mutations in the Hprt gene, but did induce a significant increase in Tk mutants, similar to that observed previously with AZT alone. The induction of mutations in the Tk gene by the mixture of AZT and ddI was associated with loss of the wild-type Tk+ allele. These data indicate that, under the conditions of this experiment, ddI is not mutagenic in neonatal B6C3F1/Tk+/- mice and that it does not potentiate the mutagenicity of AZT.

A comparative toxicologic and genotoxic study of the herbicide arsenal, its active ingredient imazapyr, and the surfactant nonylphenol ethoxylate

The herbicide arsenal 250 NA, its technical-grade active ingredient imazapyr, and the surfactant nonylphenol ethoxylate (NP) were evaluated through genotoxicity and toxicity studies in different organisms. A comparative study of these three compounds was carried out to assess how the addition of surfactant components may pose the highest toxicological risk to pesticide formulations. The results showed that arsenal, imazapyr, and NP did not cause chromosome aberration in Allium cepa nor increase the frequency of micronuclei in mice. However, toxicological evaluations showed that NP was the most toxic compound to mice, A. cepa, Drosophila melanogaster, and Biomphalaria tenagophila. In this evaluation, it was observed that the adverse effects were produced by the surfactant additive of the pesticide formulation.

Study on the genetic damage in mice induced by the volatile organic compounds of decoration materials

To study genetic damage of mice caused by the volatile organic compounds (VOC) of decoration materials. Fifty-five hotel guest rooms newly decorated within 6 months and 18 hotel guest rooms not decorated within 3 years were selected to determine the concentrations of 6 main VOC (benzene, methylbenzene, dimethylbenzene, ethyl acetate, butyl acetate, formaldehyde) in the air. Mice were exposed to VOC with the concentrations of 5, 10, 20, 40 times respectively as high as those present in the newly decorated rooms in an exposure cabinet for 15 days. DNA damage of peripheral lymphocytes of the mice was determined by single cell gel electrophoresis (SCGE) and bone marrow micronucleus test. The concentrations of benzene, methylbenzene, dimethylbenzene, ethyl acetate, butyl acetate and formaldehyde in the rooms newly decorated within 6 months (6.50, 3.00, 6.70, 41.33, 1.70 and 0.14 mg/m(3) respectively) were significantly higher than those in rooms not decorated within 3 years (0.08, 0.94, 1.38, 0.25, 0.25, 0.01 mg/m(3), P < 0.01). DNA damage rates of peripheral lymphocytes in the concentrations of 10, 20, 40 times of exposure groups were significantly higher than those in the control groups (P < 0.05 or P < 0.01), and the frequencies of micronucleus in the mice exposed to 40 times of concentration was significantly higher than that in control group. High concentrations of the volatile organic chemical compounds may cause genetic damage in mice. SCGE test is more sensitive than micronucleus test.

2-Hydroxy-1,4-naphthoquinone, the natural dye of Henna, is non-genotoxic in the mouse bone marrow micronucleus test and does not produce oxidative DNA damage in Chinese hamster ovary cells

2-Hydroxy-1,4-naphthoquinone (HNQ) has been found positive in a previous chromosome aberration test in Chinese hamster ovary (CHO) cells and in a mouse bone marrow micronucleus test at 72 h after oral administration (vehicle: DMSO). However it was negative at 24 and 48 h sampling times, and in subsequent micronucleus tests that used 0.5% aqueous methyl cellulose (MC) as vehicle. We performed a bone marrow micronucleus test in male and female NMRI BRL/BR mice at oral doses of 75, 150 and 300 mg/kg in two vehicles (DMSO and 0.5% aqueous MC), evaluated micronuclei at 24, 48 and 72 h, plasma levels of HNQ at 0.5, 1 and 4 h, and haematology parameters at 72 h after administration. The mechanism of in vitro clastogenic activity of HNQ was investigated by evaluation of the potential of HNQ to produce oxidative DNA damage after treatment of CHO with 10 mM HNQ, followed by quantification of DNA fragments using the comet assay. In the micronucleus test, HNQ at 300 mg/kg produced mortality and clinical signs at similar incidence and severity for both vehicles. Levels of HNQ in the plasma of treated mice were dose-related, of similar magnitude for both vehicles, but higher in females than in males. Maximum concentrations were found at 0.5 or 1 h. At 300 mg/kg, HNQ slightly affected RBC parameters suggesting haematotoxicity. No increase in the frequency of micronuclei was observed for any dose, vehicle or time point, whereas the positive control substance (CPA) produced a clear positive response. No evidence of HNQ-induced oxidative DNA damage was found at clastogenic concentrations in vitro, whereas the positive control substance (H 2O 2) produced a clear increase. In conclusion, HNQ was negative for induction of bone marrow micronuclei in mice up to 72 h after administration in two different vehicles, and its in vitro clastogenicity was not due to oxidative damage. These results confirm that HNQ poses no or negligible genotoxic risk.

Evaluation of the clastogenicity of isoprothiolane and pyroquilon using the mouse micronuclei test

We estimated the clastogenicity of isoprothiolan (diisopropyl 1,3-dithiolan-2-ylidenemalonate) and pyroquilon (1,2,5,6-tetrahydropyrrolo [3.2.1-ij] quinolin-4-one), using an in vivo mouse peripheral blood micronuclei assay to measure their effects on gene toxicities. The present data showed that purified samples of isoprothiolane and pyroquilon were not clastogenic data in this assay, whereas the commercial compounds we purchased had demonstrated clastogenic reactions. Our study showed that we must evaluate the pesticides that have been purified in parallel with that of commercial products containing impurities that are used in the field.

Subchronic toxicity study in rats and genotoxicity tests with polyvinyl alcohol

The potential systemic and neurotoxicity of polyvinyl alcohol (PVA) was assessed when fed in the diet to male and female Sprague–Dawley rats for 90 days at doses of 2000, 3500 and 5000 mg/kg/day. Control rats received untreated standard laboratory diet. Assessments included clinical observations, ophthalmology, body weight and food consumption, hematology, coagulation, clinical chemistry, urinalyses, motor activity and functional observational battery evaluations and gross and microscopic pathology. The only test-article-related finding observed during the study was unformed stool with brown/black anogenital staining in rats fed 3500 and 5000 mg/kg/day. This finding was attributed to the high levels of test article being consumed and subsequently excreted in the stool. It was not accompanied by macroscopic or microscopic changes in these rats. No test-article-related changes were seen in mortality, ophthalmology, body weight and food consumption data, hematology, clinical chemistry, urinalysis data, functional observational assessments, motor activity, organ weight data and macroscopic and microscopic examinations. Doses of 2000, 3500 and 5000 mg/kg/day of PVA administered as a dietary admixture to male and female Sprague–Dawley rats for up to 90 days did not result in any adverse, toxicological effects. The no-observed-adverse-effect level (NOAEL) was determined to be 5000 mg/kg/day. PVA showed no evidence of mutagenic activity in the Ames test, mouse lymphoma assay and the mouse micronucleus test. (A critical evaluation of the available information on PVA will appear in a review to be published in Food and Chemical Toxicology 2003, 41, 319–326)

Safety evaluation of an α-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I

BD5088 α-amylase derived from archaeal sources has characteristics of pH and temperature tolerance that are well suited to hydrolysis of starch in food processing applications. The production microorganism recipient strain, Pseudomonas fluorescens biovar I, strain MB101, was avirulent after oral administration to mice and does not represent an infectious threat to humans. Repeated dose gavage studies with BD5088 enzyme preparation, up to 13 weeks in duration, showed no systemic toxicity due to the oral route with an NOAEL of 890 mg/kg/day as Total Organic Solids. Some irritation occurred in the respiratory tract, which was considered to be a consequence of reflux and aspiration of test material that contained lipopolysaccharide from the Pseudomonas production strain. A 2-week dietary study (0 and 310 mg/kg/day) confirmed that there were no respiratory tract effects related to oral ingestion. There was no genotoxic activity based on Ames, mouse lymphoma, mouse micronucleus, and rat lymphocyte chromosomal aberration tests. There was no evidence of allergenic potential based on a comparison of the primary sequence of BD5088 with sequences in an allergen database. The enzyme was labile to pepsin digestion. Based on these data, BD5088 α-amylase preparation may be considered safe for use in food production such as corn wet milling.

The dose-response relationship at very low doses of acrylamide is linear in the flow cytometer-based mouse micronucleus assay

Acrylamide (AA) is genotoxic and has been classified as a probable human carcinogen. Human exposure to AA may be high by the consumption of starch-based food that has been treated at high temperature, e.g. potato chips and crisps. For risk assessment, extrapolation to the expected low doses to humans will be more reliable when data from low experimental doses can be used. We have registered the effects of a series of low doses in the sensitive flow cytometer-based micronucleus assay in mice, paying special attention to deviations from the expected linear dose-response function. Two experiments were performed with CBA mice, injected i.p. with different doses of AA. In one experiment the effects of 22 doses (two mice per dose) ranging from 0 to 100 mg/kg b.w. were studied. In the second experiment seven doses (five mice per dose) ranging from 0 to 30 mg/kg b.w. were used. In both experiments, a clear increase of the frequency of micronucleated erythrocytes was seen, already at the lowest doses used. The dose-response function was found to be linear with a tendency to have a steeper rise at the lowest doses. The low DNA content of the micronuclei indicated an absence of whole chromosomes, i.e. no aneugenic effect of AA.

Genetic Toxicity of Naphthalene: A Review

Results of five previously unpublished studies of the genotoxicity of naphthalene are presented and extensively discussed in relation to the large database that exists in the published literature. According to the published literature, naphthalene has not induced gene mutations in bacterial assays or in a metabolically competent human cell line. However, naphthalene has caused cytotoxicity in some cell lines, and induced clastogenicity in Chinese hamster ovary (CHO) cells, in a human lymphoblastoid cell line, and in preimplantation mouse embryos. Some naphthalene metabolites were cytotoxic, but only naphthoquinones produced chromosomal damage in vitro. No chromosomal damage was observed in vivo in bone marrow erythrocytes from treated mice; however, a positive response was reported in a Drosophila assay for wing somatic mutation and recombination. The five unpublished studies of naphthalene genotoxicity include three studies in vitro (two Ames bacterial assays and an in vitro unscheduled DNA synthesis assay) and two in vivo (mouse micronucleus and in vivo unscheduled DNA synthesis). Naphthalene was inactive in all five studies, in agreement with reports in the published literature. Chronic inhalation of naphthalene over 2 yr induced an increased incidence of benign alveolar/bronchial adenomas in female mice, and nasal epithelial tumors in both sexes of rats. Inflammation, tissue damage, and subsequent regenerative hyperplasia at target organ sites occurred in both species. Results of standard genetic toxicity assays suggest that naphthalene is not likely to be genotoxic in vivo. Since the in vitro results come primarily from assays utilizing liver-mediated activation systems, and the in vivo results come from rodent organs that are not targets for tumors, tests using naphthalene-sensitive rodent tissues would determine the applicability of current data in addressing the mechanisms of these species and site-specific cancers. The standard assays reported here may be useful in predicting potential health hazard in other species, or in humans, in whom there are few reported instances of naphthalene-induced cancer, especially as more data on species-specific differences in naphthalene metabolism become available. Despite present data limitations, a threshold mechanism for tumorigenesis can be proposed. The absence of naphthalene-induced gene mutation and the presence of cytotoxicity and some chromosomal events in vitro are consistent with a threshold-related mechanism of tumor induction, driven by cytotoxicity and cell regeneration, followed by genetic events, or by accumulation of naphthalene at specific target sites to allow in situ formation of a genotoxic metabolite to trigger or enhance spontaneous tumor development.

Mouse bone marrow micronucleus test results do not predict the germ cell mutagenicity of N-hydroxymethylacrylamide in the mouse dominant lethal assay.

N-Hydroxymethylacrylamide (NHMA), a mouse carcinogen inactive in the Salmonella assay and mouse micronucleus (MN) assay, was tested for reproductive effects in a mouse continuous breeding study. In that study, increased embryonic deaths were observed after 13 weeks exposure of parental animals to NHMA via drinking water (highest dose, 360 ppm); the results indicated the possible induction of chromosome damage in germ cells of treated males. An additional mouse MN test was conducted using a 31-day treatment period to better match the dosing regimen used in the breeding study; the results were negative. Additional studies were conducted to explore the germ cell activity of NHMA. A male mouse dominant lethal study was conducted using a single intraperitoneal injection of 150 mg/kg NHMA; the results were negative. A follow-up study was conducted using fractionated dosing, 50 mg/kg/day for 5 days; again, no increase in dominant lethal mutations was observed. NHMA (180-720 ppm) was then administered to male mice in drinking water for 13 weeks, during which three sets of matings occurred. Two weeks after mating, females were killed and the uterine contents were analyzed. Large, dose-related increases in dominant lethal mutations were observed with increasing length of exposure. The magnitude of the increases stabilized after 8 weeks of treatment. However, the frequency of micronucleated peripheral blood erythrocytes was not elevated in mice treated for 13 weeks with NHMA in drinking water. Thus, NHMA appears to be unique in inducing genetic damage in germ cells but not somatic cells of male mice.

Genotoxicity and toxicity of the potential cancer-preventive agent polyphenon E.

The potential health benefits of green tea continue to attract public and scientific interests and are attributed in part to polyphenolic catechin constituents. Polyphenon E (Poly E) is a decaffeinated green tea catechin mixture containing about 50% epigallocatechin gallate and 30% other catechins. We evaluated the toxicity and genotoxicity of Poly E by using two in vitro assays: bacterial mutagenesis in a Salmonella typhimurium-E. coli assay and the L5178Y mouse lymphoma cell thymidine kinase (Tk) gene mutation assay. In addition, we used two in vivo genotoxicity assays: the mouse micronucleus assay and the Big Blue cII transgenic mouse mutation assay. Repeat-dose toxicity evaluations were performed in mice in parallel with the Big Blue transgenic mutation assays. No significant increases in the revertant colonies were found in the bacterial mutagenesis assay, but a significant increase in the mutant frequency (MF) at the Tk locus was observed in the mouse lymphoma test system. We observed toxicity in mice when Poly E was administered at doses of 2,000 mg/kg/day. Lower doses produced no significant increases in micronucleated erythrocytes in the bone marrow of Swiss-Webster mice and no significant increases in cII transgene MF in the liver, lung, or spleen compared with controls. These results indicate that Poly E, although toxic at high doses (2,000 mg/kg/day), poses minimal genotoxic concern. In addition, these studies highlight the importance of using both in vitro and in vivo systems in genetic toxicity screening of pharmaceuticals before they are administered to humans.

Lack of in vivo clastogenic activity of grape seed and grape skin extracts in a mouse micronucleus assay

Meganatural™ brand grape seed extract (GSE) and grape skin extract (GSKE), containing proanthocyanidin polyphenolic compounds, are intended for use in food as functional ingredients exhibiting antioxidant activity. Proanthocyanidins, as well as the minor constituent phenolic compounds in GSE and GSKE, are present naturally in many foods such as fruits, vegetables, chocolate, tea, etc., and on average people consume 460–1000 mg/day of these combined substances. While some polyphenolic compounds, tested individually, have demonstrated antitumorigenic or antipromotional activity, at least one minor component of GSE and GSKE, quercitin, has exhibited positive activity in Salmonella and other in vitro mutagenicity assays. As part of a program to investigate the safety of GSE and GSKE, these products were tested for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1®(ICR) BR mouse bone marrow. The appropriate test article was dissolved in 0.5% carboxymethylcellulose and dosed by oral gavage to five males/test article/dose level/harvest time point. Animals were dosed at 500, 1000 and 2000 mg/kg. Five animals dosed with either test article at 500, 1000 and 2000 mg/kg dose levels and five animals dosed with the cyclophosphamide (80 mg/kg) positive control were euthanized approximately 24 h after dosing for extraction of bone marrow. Five animals dosed with either test article at the 2000 mg/kg dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 h after dosing for extraction of bone marrow. At least 2000 PCEs per animal were analyzed for frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal. For both GSE and GSKE, no statistically significant increase in micronucleated PCEs was observed at any dose level or harvest time point. GSE produced indication of cytotoxicity (decreased PCE:NCE ratio) at the 2000 mg/kg dose level for the 48-h harvest time point, confirming that the test article reached the target bone marrow in significant amount. Meganatural™ GSE and Meganatural™ GSKE were evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.