Rodent Carcinogenicity Research Articles

Comparison of hepatocarcinogen-induced gene expression profiles in conventional primary rat hepatocytes with in vivo rat liver

At present, substantial efforts are focused on the development of in vitro assays coupled with "omics" technologies for the identification of carcinogenic substances as an alternative to the classical 2-year rodent carcinogenicity bioassay. A prerequisite for the eventual regulatory acceptance of such assays, however, is the in vivo relevance of the observed in vitro findings. In the current study, hepatocarcinogen-induced gene expression profiles generated after the exposure of conventional cultures of primary rat hepatocytes to three non-genotoxic carcinogens (methapyrilene hydrochloride, piperonyl butoxide, and Wy-14643), three genotoxic carcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-nitrofluorene), and two non-carcinogens (nifedipine and clonidine) are compared with previously obtained in vivo data after oral administration for up to 14 days of the same hepatocarcinogens to rats. In addition to the comparison of deregulated genes and functions per compound between in vivo and in vitro models, the major discriminating cellular pathways found in vivo in livers of exposed rats were examined for deregulation in vitro. Further, in vivo-derived gene signatures for the identification of genotoxic versus non-genotoxic carcinogens are used to classify in vitro-tested hepatocarcinogens and non-carcinogens. In the primary hepatocyte cultures, two out of the three tested genotoxic carcinogens mimicked the in vivo-relevant DNA damage response and were correctly assessed. Exposure to the non-genotoxic hepatocarcinogens, however, triggered a relatively weak response in the in vitro system, with no clear similarities to in vivo. This study contributes to the further optimization of toxicogenomics predictive tools when applied in in vitro settings.

DEHP: Genotoxicity and potential carcinogenic mechanisms—A review

Di(ethylhexyl) phthalate (DEHP) is a manufactured chemical commonly added to plastics: it is a ubiquitous environmental contaminant to which humans are exposed through multiple routes. DEHP is a rodent carcinogen with an extensive data base on genotoxicity and related effects spanning several decades. Although DEHP has been reported to be negative in most non-mammalian in vitro mutation assays, most studies were performed under conditions of concurrent cytotoxicity, precipitation, or irrelevant metabolic activation. However, a number of in vitro rodent tissue assays have reported DEHP to be positive for effects on chromosomes, spindle, and mitosis. A robust database shows that DEHP increases transformation and inhibits apoptosis in Syrian hamster embryo cells. In a transgenic mouse assay, in vivo DEHP exposure increased the mutation frequency only in the liver, which is the target organ for cancer. In vitro exposure of human cells or tissues to DEHP induced DNA damage; altered mitotic rate, apoptosis, and cell proliferation; increased proliferation, tumor mobility, and invasiveness of tumor cell lines; and activated a number of nuclear receptors. DEHP has been shown to be an agonist for CAR2, a novel constitutive androstane receptor occurring only in humans. Environmental exposures of humans to DEHP have been associated with DNA damage. After taking into account study context and relevant issues affecting interpretation, in vitro studies reported that a similar DEHP concentration range induced both mutagenic and non-mutagenic effects in human tissues and, using a much more limited rodent database, transformation of embryonic rodent tissues. The human and rodent data suggest that DEHP induces cancer through multiple molecular signals, including DNA damage. The analyses presented here may provide guidance for similar data sets used in structure-activity relationships, computational-toxicology extrapolations, and attempts to extrapolate in vitro results to predict in vivo effects for hazard characterization.

A Linear Prediction Rule Based on Ensemble Classifiers for Non-Genotoxic Carcinogenicity

This article presents a novel approach for developing a linear prediction rule to predict the non-genotoxic carcinogenicity potential of a new compound in the drug development pipeline. We construct the approach using data from 24-hour microarray experiments on rats treated with the compound. This method was developed to address an actual problem that we were presented with by scientists in mechanistic toxicology. Short-term, preclinical assays for non-genotoxic carcinogenicity, a toxicity commonly observed in long-term rodent carcinogenicity studies, have proven difficult to develop. A quick and early preclinical assay, such as this, is of particular interest and urgency. The linear prediction rule is derived using an Ensemble Linear Discriminant classifier. This is a hybrid approach which leverages the advantages of ensemble approaches for addressing over-fitting (a problem endemic to microarray data), and that of LDA for interpretability. In a limited comparison with some other classifiers, including random forest, we show that our approach has good predictive performance in addition to being interpretable.

Evaluation of the Threshold of Toxicological Concern (TTC) – Challenges and approaches

Thresholds of Toxicological Concern (TTCs) have been used in the risk assessment of chemicals to which humans are exposed at very low levels. TTC values were developed using data from rodent cancer bioassays and from oral chronic and sub-chronic toxicity studies for non-cancer effects. The workshop assessed the adequacy and fitness for purpose of the TTC approach and the potential for future modifications of critical aspects. The current TTC value for cancer was considered adequate and fit for purpose because it is derived by linear extrapolation from the lowest TD50 for each compound in the largest available rodent carcinogenicity database. The database on non-cancer endpoints was considered adequate and fit for purpose because the chemical domain, the distributions of NOAELs and the calculated TTC values are comparable across different databases. Application of the TTC approach gives conclusions compatible with the risk assessment approaches currently used by international advisory committees. The workshop recognised the desirability of developing better tools to assess the comparability of the chemical domain covered in different toxicological databases, and the need to develop an internationally acceptable framework and databases for updating the aspects critical to application of the TTC approach.

Ochratoxin A and Mitotic Disruption: Mode of Action Analysis of Renal Tumor Formation by Ochratoxin A

The mycotoxin and food contaminant ochratoxin A (OTA) is a potent renal carcinogen in rodents, but its mode of action (MoA) is still poorly defined. In 2006, the European Food Safety Authority concluded that there is a "lack of evidence for the existence of OTA-DNA adducts" and thus insufficient evidence to establish DNA reactivity as a MoA for tumor formation by OTA. In reviewing the available database on OTA toxicity, a MoA for renal carcinogenicity of OTA is developed that involves a combination of genetic instability and increased proliferative drive as consequences of OTA-mediated disruption of mitosis, whereby the organ- and site-specificity of tumor formation by OTA is determined by selective renal uptake of OTA into the proximal tubule epithelium. The proposed MoA is critically assessed with respect to concordance of dose-response of the suggested key events and tumor formation, their temporal association, consistency, and biological plausibility. Uncertainties, data gaps and needs for further research are highlighted.

First report on development of quantitative interspecies structure–carcinogenicity relationship models and exploring discriminatory features for rodent carcinogenicity of diverse organic chemicals using OECD guidelines

Different regulatory agencies in food and drug administration and environmental protection worldwide are employing quantitative structure–activity relationship (QSAR) models to fill the data gaps related with properties of chemicals affecting the environment and human health. Carcinogenicity is a toxicity endpoint of major concern in recent times. Interspecies toxicity correlations may provide a tool for estimating sensitivity towards toxic chemical exposure with known levels of uncertainty for a diversity of wildlife species. In this background, we have developed quantitative interspecies structure–carcinogenicity correlation models for rat and mouse [rodent species according to the Organization for Economic Cooperation and Development (OECD) guidelines] based on the carcinogenic potential of 166 organic chemicals with wide diversity of molecular structures, spanning a large number of chemical classes and biological mechanisms. All the developed models have been assessed according to the OECD principles for the validation of QSAR models. Consensus predictions for carcinogenicity of the individual compounds are presented here for any one species when the data for the other species are available. Informative illustrations of the contributing structural fragments of chemicals which are responsible for specific carcinogenicity endpoints are identified by the developed models. The models have also been used to predict mouse carcinogenicities of 247 organic chemicals (for which rat carcinogenicities are present) and rat carcinogenicities of 150 chemicals (for which mouse carcinogenicities are present). Discriminatory features for rat and mouse carcinogenicity values have also been explored.

Endometrial Stromal Polyps in Rodents: Biology, Etiology, and Relevance to Disease in Women

Endometrial stromal polyps (ESP) are a common spontaneous reproductive tract lesion in the female rat. However, there is limited information concerning the etiology, biology, and significance of these polyps as an end point in toxicology and carcinogenicity studies. This paper reviews relevant literature to address these aspects of ESP with respect to potential relevance to human uterine tumors. Endometrial stromal polyps in rodents appear as age-related lesions. There are only a few chemicals tested for carcinogenicity in rat and mouse cancer bioassays associated with increased incidence of ESP with no common characteristics or mechanism of action. Uterine endometrial polyps that occur in women and the uterine stromal polyps that occur in rodents have distinct characteristics, although both types of uterine lesions are common, benign, and noncancerous. Human endometrial polyps develop from both endometrial and stromal components, whereas rodent polyps develop from the stromal component of the uterus. Endometrial polyps in women are hormone sensitive, but there is no scientific or experimental evidence to date that suggests that uterine stromal polyps in rodents are hormone sensitive. Therefore, based on differences in their etiology and biology, endometrial stromal polyps observed in rodent toxicity and carcinogenicity studies appear to have limited relevance to human endometrial polyps occurring in women.

Insulin glargine: a reevaluation of rodent carcinogenicity findings.

The 1995 to 1997 lifetime carcinogenicity studies of insulin glargine in rats and mice were reanalyzed and reassessed for their validity according to current guidelines. In 2-year studies, 50 animals per sex and per group were used. Survival rates between weeks 80 and 90 in female mice and rats were greater than 20 animals in all groups, fulfilling current Food and Drug Administration requirements that enough animals lived long enough to provide adequate exposure to glargine and to be at risk of forming late-developing tumors. Exposure to 5 or 12.5 IU/kg glargine was similar to or 2 to 3 times greater than 5 IU/kg neutral protamine Hagedorn insulin, respectively. Using statistical methods recommended by current guidelines, no significant effect of glargine on mammary gland neoplastic lesions in female rodents was found, confirming earlier results. Thus, both studies can be considered valid according to contemporary standards. Insulin glargine does not present a carcinogenic risk.

Evaluation of In Vivo Mutagenicity by 2,4-Diaminotoluene and 2,6-Diaminotoluene in Liver of F344 gpt delta Transgenic Rat Dosed for 28 Days: A Collaborative Study of the gpt delta Transgenic Rat Mutation Assay

The transgenic rodent (TGR) assay has been widely used to study in vivo gene mutations by chemicals or radiation; however, an optimal protocol has not yet been established to assess unknown genotoxic potential. The International Workshop on Genotoxicity Testing (IWGT) strongly recommends a repeated-dose regimen for the TGR assay protocol for regulatory safety assessment as follows: a treatment period of 28 days and a sampling time of 3 days following the final treatment. In this study, TGR assays using F344 gpt delta transgenic rats were conducted at three laboratories to evaluate the validity of the IWGT protocol, as part of a collaborative study of the transgenic rat mutation assay. Male F344 gpt delta transgenic rats were orally treated with 2,4-diaminotoluene (2,4-DAT; hepatic carcinogen in rodents; 10 and 30 mg/kg/day) or 2,6-diaminotoluene (2,6-DAT; non-carcinogen in rodents; 60 mg/kg/day) once daily for 28 days. Rats were euthanized 3 days after the last dosing, and then mutant frequencies (MFs) of the gpt gene in the livers were studied. As a result, a significant increase in the MF was observed at 30 mg/kg in the 2,4-DAT-treated group, but not in the 2,6-DAT-treated group. These results were commonly observed among the three laboratories. In addition, the overall results from the three laboratories were in general agreement. These results indicate that 2,4-DAT induces gene mutation in the liver of gpt delta rats, but 2,6-DAT does not. These results also indicate that the F344 gpt delta transgenic rat mutation assay can distinguish differences in the in vivo mutagenic potential between a hepatic carcinogen and a non-carcinogen. Results from one laboratory showed more variability than those from the other two laboratories, and this appearance was due to the smaller number of colonies scored. Thus, these results demonstrate that the IWGT protocol for the TGR assays is valid, and show that consistent results are obtained among different laboratories.

In vivo Approaches to Identify Mutations and in vitro Research to Reveal Underlying Mechanisms of Genotoxic Thresholds

In regulatory toxicology, it is assumed that genotoxic carcinogens, which induce cancer through genotoxic mechanisms, have no threshold for their action. However, humans possess a number of defense mechanisms against DNA damaging agents, which may reduce the genotoxic and cancer risk at low doses to the spontaneous levels. The defense mechanisms may constitute practical thresholds for genotoxic carcinogens. In fact, accumulating evidence with rodent carcinogenicity and genotoxicity assays suggest that some genotoxic compounds clearly exhibit threshold-like dose responses in vivo. These results challenge the paradigm that cancer risk induced by genotoxic compounds at high doses can be linearly extrapolated into low doses where people are exposed in daily life (linear non-threshold model). Here, we discuss two issues regarding the practical thresholds for genotoxic carcinogens. The first issue is how to define “genotoxicity” of chemicals. There are a number of genotoxicity assays in vitro and in vivo. Therefore, it is unclear what genotoxicity assay(s) should be employed to define whether the compound is genotoxic or not. The second issue is possible mechanisms underlying the practical thresholds. In particular, we emphasize the importance of DNA repair and translesion DNA synthesis as the underlying mechanisms of the practical thresholds. Finally, we discuss issues associated with low dose exposure to genotoxic carcinogens, i.e., risk assessment of exposure to multiple genotoxic chemicals.

Estimating Human Cancer Risk from Rodent Carcinogenicity Studies: The Changing Paradigm for Pharmaceuticals

Estimating Human Cancer Risk from Rodent Carcinogenicity Studies: The Changing Paradigm for Pharmaceuticals

Genotoxicity of Acrylamide and Glycidamide: A Review of the Studies by HPRT Gene and TK Gene Mutation Assays

Acrylamide (AA), a proven rodent carcinogen, is found in a variety of commonly consumed human foods, which has raised public health concerns. AA is largely oxidized to the chemically reactive epoxide, glycidamide (GA), by cytochrome P450 2E1. The genotoxic effects of AA and GA have been extensively evaluated. However, the results in mammalian gene mutation tests were inconsistent, especially the genotoxic effects at the HPRT gene and TK gene. In this article, the relevant mutations induced by AA and GA on both gene loci in various test systems involving in vivo and in vitro tests are reviewed. It is confirmed that AA acts directly as a clastogen and produces weakly mutagenic effects at the HPRT gene probably by metabolic conversion of AA to GA. On the other hand, GA is a strong mutagen with high reactivity to DNA, inducing predominantly point mutations. The molecular mutation spectra of AA and GA at the HPRT and TK genes are also compared and summarized here, for better clarifying the mechanisms of mutation induced by these two compounds. These data would help to understand the mutagenicity of AA and its contribution to human cancers.

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0 for assessment of carcinogenic potential of chemicals

The European Centre for the Validation of Alternative Methods (ECVAM) has organised an interlaboratory prevalidation study on the Syrian hamster embryo (SHE) cell transformation assay (CTA) at pH 7.0 for the detection of rodent carcinogens. The SHE CTA at pH 7.0 has been evaluated for its within-laboratory reproducibility, transferability and between-laboratory reproducibility. Four laboratories using the same basic protocol with minor modifications participated in this study and tested a series of six coded-chemicals: four rodent carcinogens (benzo(a)pyrene, 3-methylcholanthrene, 2,4-diaminotoluene and o-toluidine HCl) and two non-carcinogens (anthracene and phthalic anhydride). All the laboratories found the expected results with coded chemicals except for phthalic anhydride which resulted in a different call in only one laboratory. Based on the outcome of this study, it can be concluded that a standardised protocol is available that should be the basis for future use. This protocol and the assay system itself are transferable between laboratories and the SHE CTA at pH 7.0 is reproducible within- and between-laboratories.

Cytochrome P450-mediated metabolism and DNA binding of 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline and its carcinogenic isomer 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in mice.

2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx) is a recently discovered heterocyclic aromatic amine (HAA) that is formed during the cooking of meats. MeIgQx is an isomer of 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), a rodent carcinogen and possible human carcinogen that also occurs in cooked meats. MeIgQx is a bacterial mutagen, but knowledge about its metabolism and carcinogenic potential is lacking. Metabolism studies on MeIgQx and MeIQx were conducted with human and mouse liver microsomes, and recombinant human P450s. DNA binding studies were also investigated in mice to ascertain the genotoxic potential of MeIgQx in comparison to MeIQx. Both HAAs underwent comparable rates of N-oxidation to form genotoxic N-hydroxylated metabolites with mouse liver microsomes (0.2-0.3 nmol/min/mg protein). The rate of N-oxidation of MeIQx was 4-fold greater than the rate of N-oxidation of MeIgQx with human liver microsomes (1.7 vs 0.4 nmol/min/mg protein). The rate of N-oxidation, by recombinant human P450 1A2, was comparable for both substrates (6 pmol/min/pmol P450 1A2). MeIgQx also underwent N-oxidation by human P450s 1A1 and 1B1 at appreciable rates, whereas MeIQx was poorly metabolized by these P450s. The potential of MeIgQx and MeIQx to form DNA adducts was assessed in female C57BL/6 mice given [(14)C]-MeIgQx (10 μCi, 9.68 mg/kg body wt) or [(14)C]-MeIQx (10 μCi, 2.13 mg/kg body wt). DNA adduct formation in the liver, pancreas, and colorectum was measured by accelerator mass spectrometry at 4, 24, or 48 h post-treatment. Variable levels of adducts were detected in all organs. The adduct levels were similar for both HAAs, when adjusted for dose, and ranged from 1 to 600 adducts per 10(7) nucleotides per mg/kg dose. Thus, MeIgQx undergoes metabolic activation and binds to DNA at levels that are comparable to MeIQx. Given the high amounts of MeIgQx formed in cooked meats, further investigations are warranted to assess the carcinogenic potential of this HAA.

Trapping of cis-2-butene-1,4-dial to measure furan metabolism in human liver microsomes by cytochrome P450 enzymes.

Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.

Mechanistic considerations for human relevance of cancer hazard of di(2-ethylhexyl) phthalate

Di(2-ethylhexyl) phthalate (DEHP) is a peroxisome proliferator agent that is widely used as a plasticizer to soften polyvinylchloride plastics and non-polymers. Both occupational (e.g., by inhalation during its manufacture and use as a plasticizer of polyvinylchloride) and environmental (medical devices, contamination of food, or intake from air, water and soil) routes of exposure to DEHP are of concern for human health. There is sufficient evidence for carcinogenicity of DEHP in the liver in both rats and mice; however, there is little epidemiological evidence on possible associations between exposure to DEHP and liver cancer in humans. Data are available to suggest that liver is not the only target tissue for DEHP-associated toxicity and carcinogenicity in both humans and rodents. The debate regarding human relevance of the findings in rats or mice has been informed by studies on the mechanisms of carcinogenesis of the peroxisome proliferator class of chemicals, including DEHP. Important additional mechanistic information became available in the past decade, including, but not limited to, sub-acute, sub-chronic and chronic studies with DEHP in peroxisome proliferator-activated receptor (PPAR) α-null mice, as well as experiments utilizing several transgenic mouse lines. Activation of PPARα and the subsequent downstream events mediated by this transcription factor represent an important mechanism of action for DEHP in rats and mice. However, additional data from animal models and studies in humans exposed to DEHP from the environment suggest that multiple molecular signals and pathways in several cell types in the liver, rather than a single molecular event, contribute to the cancer in rats and mice. In addition, the toxic and carcinogenic effects of DEHP are not limited to liver. The International Agency for Research on Cancer working group concluded that the human relevance of the molecular events leading to cancer elicited by DEHP in several target tissues (e.g., liver and testis) in rats and mice can not be ruled out and DEHP was classified as possibly carcinogenic to humans (Group 2B).

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7 for assessment of carcinogenic potential of chemicals

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is an important in vitro method that is highly predictive of rodent carcinogenicity. It is a key method for reducing animal usage for carcinogenicity prediction. The SHE assay has been used for many years primarily to investigate and identify potential rodent carcinogens thereby reducing the number of 2-year bioassays performed in rodents. As for other assays with a long history of use, the SHE CTA has not undergone formal validation. To address this, the European Centre for the Validation of Alternative Methods (ECVAM) coordinated a prevalidation study. The aim of this study was to evaluate the within-laboratory reproducibility, test method transferability, and between-laboratory reproducibility and to develop a standardised state-of-the-art protocol for the SHE CTA at pH 6.7. Formal ECVAM principles for criteria on reproducibility (including the within-laboratory reproducibility, the transferability and the between-laboratories reproducibility) were applied. In addition to the assessment of reproducibility, this study helped define a standard protocol for use in developing an Organisation for Economic Co-operation and Development (OECD) test guideline for the SHE CTA. Six compounds were evaluated in this study: benzo(a)pyrene, 3-methylcholanthrene, o-toluidine HCl, 2,4-diaminotoluene, phthalic anhydride and anthracene. Results of this study demonstrate that a protocol is available that is transferable between laboratories, and that the SHE CTA at pH 6.7 is reproducible within- and between-laboratories.

Chromosomal damage and polymorphisms of metabolic genes among 1, 3-butadiene-exposed workers in a matched study in China

1, 3-Butadiene (BD) is a high-efficiency carcinogen in rodents and was classified as a human carcinogen in 2008 by the International Agency for Research on Cancer. However, its ability to induce genetic damage and the influence of metabolic polymorphisms to such damage in humans are both controversial claims. This study was conducted to investigate the relationships between exposure to BD, the polymorphisms of metabolic genes and the chromosomal damage in 45 pairs of occupationally exposed workers in a BD product workshop and matched control workers in an administrative office and circulatory water workshop in China. Exposure to BD was evaluated by personal sampling and stationary sampling. Different chromosomal damage endpoints in peripheral blood lymphocytes were determined using the cytokinesis-blocked micronucleus (CBMN) cytome assay; polymorphisms of metabolic genes [cytochrome P450 2E1 (CYP2E1), glutathione S-transferases (GST) and microsomal epoxide hydrolase (mEH)] in BD-exposed group were detected by polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism analysis. The results show that the average BD measurements of the exposed group were significantly higher than those for the control group (a personal sampling and stationary sampling, respectively). The BD-exposed workers exhibited increased frequencies of micronuclei (MNi) (8.00 ± 3.78‰ versus 5.62 ± 2.41‰) and nucleoplasmic bridges (NPBs) (2.58 ± 2.79‰ versus 1.13 ± 1.34‰) and a decreased nuclear division index (2.20 ± 0.14 versus 2.35 ± 0.27) when compared subjects in the control group. Meanwhile, BD-exposed workers carrying CYP2E1 c1c2/c2c2 or mEH intermediate (I)/high (H) group had a significantly higher NPB frequency than those carrying CYP2E1 c1c1 [frequency ratio (FR) = 2.60, 95% confidence interval (CI) 1.72-3.93; P < 0.0001) or the mEH low(S) group (FR = 2.06, 95% CI% 1.17-3.62; P < 0.05), respectively. Our study suggests that MNi and NPB frequency in CBMN cytome assay could be potential genotoxic biomarkers for BD exposure in humans. The polymorphism of CYP2E1 and mEH could also affect the chromosomal instability of BD workers.

Risk assessment of coumarin using the bench mark dose (BMD) approach: Children in Norway which regularly eat oatmeal porridge with cinnamon may exceed the TDI for coumarin with several folds

Coumarin is a naturally occurring flavouring substance in cinnamon and many other plants. It is known that coumarin can cause liver toxicity in several species, and it is considered a non-genotoxic carcinogen in rodents. By using the bench mark dose approach we re-assessed coumarin toxicity and established a new TDI for coumarin of 0.07mg/kgbw/day. Oral intake of coumarin is related to consumption of cinnamon-containing foods and food supplements. Cinnamon is a widely used spice in Norway, and can be used as topping on oatmeal porridge. Based on analyses of coumarin in Norwegian foods, intake calculations for children and adults were conducted, and a risk assessment of coumarin in the Norwegian population was performed. Intake estimates of coumarin show that small children eating oatmeal porridge several times a week sprinkled with cinnamon could have a coumarin intake of 1.63mg/kgbw/day and may exceeding the TDI with several folds. Adults drinking cinnamon-based tea and consuming cinnamon supplements also can exceed TDI. The coumarin intake could exceed the TDI by 7- to 20-fold in some intake scenarios. Such large daily exceedances of TDI, even for a limited time period of 1–2weeks, cause concern of adverse health effects.

Reduction of misleading (“false”) positive results in mammalian cell genotoxicity assays. I. Choice of cell type

Current in vitro mammalian cell genotoxicity assays show a high rate of positive results, many of which are misleading when compared with in vivo genotoxicity or rodent carcinogenicity data. P53-deficiency in many of the rodent cell lines may be a key factor in this poor predictivity. As part of an European Cosmetics Industry Association initiative for improvement of in vitro mammalian cell assays, we have compared several rodent cell lines (V79, CHL, CHO) with p53-competent human peripheral blood lymphocytes (HuLy), TK6 human lymphoblastoid cells, and the human liver cell line, HepG2. We have compared in vitro micronucleus (MN) induction following treatment with 19 compounds that were accepted as producing misleading or “false” positive results in in vitro mammalian cell assays [6]. Of these, six chemicals (2-ethyl-1,3-hexandiol, benzyl alcohol, urea, sodium saccharin, sulfisoxazole and isobutyraldehyde) were not toxic and did not induce any MN at concentrations up to 10 mM. d,l-Menthol and ethionamide induced cytotoxicity, but did not induce MN. o-Anthranilic acid was not toxic and did not induce MN in V79, CHL, CHO, HuLy and HepG2 cells up to 10 mM. Toxicity was induced in TK6 cells, although there were no increases in MN frequency up to and above the 55% toxicity level. The other 10 chemicals (1,3-dihydroxybenzene, curcumin, propyl gallate, p-nitrophenol, ethyl acrylate, eugenol, tert-butylhydroquinone, 2,4-dichlorophenol, sodium xylene sulfonate and phthalic anhydride) produced cytotoxicity in at least one cell type, and were evaluated further for MN induction in most or all of the cell types listed above. All these chemicals induced MN at concentrations <10 mM, with levels of cytotoxicity below 60% (measured as the replication index) in at least one cell type. The rodent cell lines (V79, CHO and CHL) were consistently more susceptible to cytotoxicity and MN induction than p53-competent cells, and are therefore more susceptible to giving misleading positive results. These data suggest that a reduction in the frequency of misleading positive results can be achieved by careful selection of the mammalian cell type for genotoxicity testing.