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Kirkland D.,Kirkland Consulting | Zeiger E.,Errol Zeiger Consulting
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2014

A Workshop sponsored by EURL ECVAM was held in Ispra, Italy in 2013 to consider whether the in vitro mammalian cell genotoxicity test results could complement and mitigate the implications of a positive Ames test response for the prediction of in vivo genotoxicity and carcinogenicity, and if patterns of results could be identified. Databases of Ames-positive chemicals that were tested for in vivo genotoxicity and/or carcinogenicity were collected from different sources and analysed individually (Kirkland et al., in this issue). Because there were overlaps and inconsistent test results among chemicals in the different databases, a combined database which eliminated the overlaps and evaluated the inconsistencies was considered preferable for addressing the above question. A database of >700 Ames-positive chemicals also tested in vivo was compiled, and the results in in vitro mammalian cell tests were analysed. Because the database was limited to Ames-positive chemicals, the majority (>85%) of carcinogens (103/119) and in vivo genotoxins (83/88) were positive when tested in both in vitro gene mutation and aneugenicity/clastogenicity tests. However, about half (>45%) of chemicals that were not carcinogenic (19/28) or genotoxic in vivo (33/73) also gave the same patterns of positive mammalian cell results. Although the different frequencies were statistically significant, positive results in 2 in vitro mammalian cell tests did not, per se, add to the predictivity of the positive Ames test. By contrast, negative results for both in vitro mammalian cell endpoints were rare for Ames-positive carcinogens (3/119) and in vivo genotoxins (2/88) but, were significantly more frequent for Ames-positive chemicals that are not carcinogenic (4/28) or genotoxic in vivo (14/73). Thus, in the case of an Ames-positive chemical, negative results in 2 in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential. © 2014 The Authors.

Fowler P.,Colworth Science Park | Smith R.,Covance | Smith K.,Covance | Young J.,Quotient Bioresearch Ltd. | And 4 more authors.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2014

We have demonstrated previously that the seemingly high rate of "false" or "misleading" positive results from in vitro micronucleus assays (MNvit) was greater when rodent derived cell lines and certain toxicity measures, such as relative cell count or replication index, were used. These studies suggested that the use of a human cell type with functional p53 and a toxicity measure that included a function of cell proliferation could dramatically reduce the detection of misleading positive results. A reduced "false positive rate" should not be at the expense of a loss of sensitivity of the assay. Therefore, we have investigated the sensitivity of the MNvit assay to known genotoxic agents using three cell types shown previously to be less prone to misleading positives, namely human lymphocytes (HuLy), TK6 and HepG2 cells. The 17 chemicals are well characterised and are from a list of chemicals known to produce positive results in in vitro mammalian cell assays.These data demonstrated a high sensitivity of the assay in which TK6 and HuLy cells were employed, such that 15 out of the 17 chemicals were correctly identified. By contrast, the use of HepG2 cells resulted in far fewer than expected positive responses. In conclusion, using TK6 and HuLy cells in preference to long established rodent cell lines in order to improve specificity does not compromise the sensitivity of the MNvit to detect known genotoxic agents. © 2014 Elsevier B.V.

Kirkland D.,Kirkland Consulting | Edwards J.,DSM Nutritional Products AG | Woehrle T.,DSM Nutritional Products AG | Beilstein P.,DSM Nutritional Products AG
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2015

The phenolic anti-oxidant 3-hydroxytyrosol (HT) is a major constituent of olives and olive oil. Published data showed it was negative in the Ames test at concentrations up to 5. μL per plate, but did induce chromosomal aberrations in human lymphocytes. HIDROX, an olive extract containing approximately 2.4% HT, was reported as both positive and equivocal in an Ames test in different papers from the same laboratory. Negative results for micronucleus induction in vivo in both an acute study and as part of a 90-day rat toxicity study were also reported for HIDROX.Given the widespread use and consumption of olives, olive oil and olive extracts, it was important to obtain more data. Here we confirm that pure HT, and an olive extract containing 15% HT, both induced micronuclei in cultured cells in vitro, but show that these responses were either due to high levels of cytotoxicity or to reaction of HT with culture medium components to produce hydrogen peroxide. Another extract (H40) containing 40% HT also induced micronuclei in vitro, probably via the same mechanism. However, both extracts were negative in robust Ames tests.The 15% HT formulated extract did not induce micronuclei in rat bone marrow after 4 weeks of dosing up to 561. mg HT/kg/day. H40 produced increased rat bone marrow micronucleus frequencies at 250 and 500. mg HT/kg/day in a 90-day toxicity study, but the results were questionable for various reasons. However, when two different batches of this extract were tested in acute micronucleus studies at doses up to 2000. mg HT/kg, giving plasma exposures that exceeded those in the 90-day study, negative results were obtained. Based on weight of evidence it is concluded that the olive extracts tested are not genotoxic at high doses in vivo, and any genotoxic risks for human consumers are negligible. © 2014 Elsevier B.V.

Kirkland D.,Kirkland Consulting | Reeve L.,Covance | Gatehouse D.,Old Barn | Vanparys P.,ALTOXICON BVBA
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2011

In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames. +. MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames. +. MNvit - benzyl acetate, toluene, morphine and thiabendazole - and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames. +. MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames. +. MNvit. © 2011 Elsevier B.V.

Fowler P.,Colworth Science Park | Fowler P.,Covance | Smith R.,Covance | Smith K.,Covance | And 5 more authors.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2012

In a previous publication, Fowler et al. . [4] demonstrated that the seemingly high rate of false or misleading positive results obtained in . in vitro cytogenesis assays for genotoxicity - when compared with . in vivo genotoxicity or rodent carcinogenicity data - was greater when rodent cell lines were used that were also reported to have mutant or non-functional p53.As part of a larger project for improvement of . in vitro mammalian cell assays, we have investigated the impact of different toxicity measures, commonly used in . in vitro cytogenetic assays, on the occurrence of misleading positive results.From a list of 19 chemicals that produce " false" positive results in . in vitro mammalian cell assays . [10], six substances that had given positive responses in CHO, CHL and TK6 cells . [4], were evaluated for micronucleus induction . in vitro, with different measures of toxicity for selection of the top concentration.The data show that estimating toxicity by relative cell count (RCC) or replication index (RI) consistently underestimates the toxicity observed by other measures (Relative Population Doubling, RPD, or Relative Increase in Cell Count, RICC). RCC and RI are more likely to lead to selection of concentrations for micronucleus scoring that are highly cytotoxic and thus could potentially lead to artefacts of toxicity being scored (elevated levels of apoptosis and necrosis), generating misleading positive results.These results suggest that a further reduction in the frequency of misleading positive results in . in vitro cytogenetic assays can be achieved with this set of chemicals, by avoiding the use of toxicity measures that underestimate the level of toxicity induced. © 2012 Elsevier B.V.

Fowler P.,Colworth Science Park | Fowler P.,Covance | Smith K.,Covance | Young J.,Covance | And 4 more authors.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2012

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. © 2011 Elsevier B.V.

Fowler P.,Covance | Whitwell J.,Covance | Jeffrey L.,Covance | Young J.,Covance | And 2 more authors.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2010

The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the human lymphoblastoid cell line TK6. Cadmium chloride (an inorganic carcinogen), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, capable of detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used.The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in the in vitro micronucleus assay. © 2010 Elsevier B.V.

Bowen D.E.,JSC International Ltd. | Whitwell J.H.,Covance | Lillford L.,Covance | Henderson D.,Covance | And 5 more authors.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2011

With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals.Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow ® flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays.A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[. a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45. h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays.All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[. a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint.The data generated from these investigations demonstrate the suitability of the multi-endpoint design. © 2011 Elsevier B.V.

In the analysis by Parry et al. [Parry, J. M., Parry, E., Phrakonkham, P. and Corvi, R. (2010) Analysis of published data for top concentration considerations in mammalian cell genotoxicity testing. Mutagenesis, 25, 531-538], 24 rodent carcinogens that were negative in the Ames test were identified that were only positive in mammalian cell tests at concentrations between 1 and 10 mM. These carcinogens can be subdivided into four groups as follows: (1) probable non-genotoxic (non-mutagenic) carcinogens, tumour promoters or negative for genotoxicity in vivo (n=10); (2) questionable carcinogens (n=4); (3) carcinogens with a probable genotoxic mode of action (n=5); (4) compounds where carcinogenicity or in vivo genotoxicity is unknown or unclear (n=5). It is not expected that in vitro mammalian cell tests should give positive results with Group 1 chemicals. Within Groups 2-4, five chemicals were considered a low priority because they could be detected using modified conditions because genotoxicity was associated with precipitate or pH shifts or because non-standard metabolism was required. The remaining nine chemicals were therefore considered most critical in terms of detection of genotoxic activity in mammalian cells. Daminozide was also included because it may have given positive responses between 1 and 10 mM. Many of the reported studies could have given positive results only at >1 mM because 'old' protocols were followed. These 10 chemicals have therefore been retested using modern protocols. Some were negative even up to 10 mM. Others were positive at concentrations <1 mM. Only methylolacrylamide was positive at a concentration >1 mM (2 mM = 202 μg/ml). Low-molecular weight substances may therefore require concentrations >1 mM, but further work is needed. Based on this analysis, it is concluded that the 10 mM upper limit in mammalian cell tests can be lowered without any loss of sensitivity in detecting genotoxic rodent carcinogens. A new limit of 1 mM or 500 μg/ml, whichever is the higher, is proposed. © The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.

Kirkland D.,Kirkland Consulting | Gatehouse D.,Old Barn
Food and Chemical Toxicology | Year: 2015

Aspartame is a methyl ester of a dipeptide of aspartic acid and phenylalanine. It is 200× sweeter than sucrose and is approved for use in food products in more than 90 countries around the world. Aspartame has been evaluated for genotoxic effects in microbial, cell culture and animal models, and has been subjected to a number of carcinogenicity studies. The in vitro and in vivo genotoxicity data available on aspartame are considered sufficient for a thorough evaluation. There is no evidence of induction of gene mutations in a series of bacterial mutation tests. There is some evidence of induction of chromosomal damage in vitro, but this may be an indirect consequence of cytotoxicity. The weight of evidence from in vivo bone marrow micronucleus, chromosomal aberration and Comet assays is that aspartame is not genotoxic in somatic cells in vivo. The results of germ cell assays are difficult to evaluate considering limited data available and deviations from standard protocols. The available data therefore support the conclusions of the European Food Safety Authority (EFSA) that aspartame is non-genotoxic. © 2015 .

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