Kirkland Consulting

Tadcaster, United Kingdom

Kirkland Consulting

Tadcaster, United Kingdom
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Brusick D.,Toxicology Consultant | Aardema M.,Marilyn Aardema Consulting LLC | Kier L.,Private Consultant | Kirkland D.,Kirkland Consulting | Williams G.,New York Medical College
Critical Reviews in Toxicology | Year: 2016

In 2015, the International Agency for Research on Cancer (IARC) published a monograph concluding there was strong evidence for genotoxicity of glyphosate and glyphosate formulations and moderate evidence for genotoxicity of the metabolite aminomethylphosphonic acid (AMPA). These conclusions contradicted earlier extensive reviews supporting the lack of genotoxicity of glyphosate and glyphosate formulations. The IARC Monograph concluded there was strong evidence of induction of oxidative stress by glyphosate, glyphosate formulations, and AMPA. The Expert Panel reviewed the genotoxicity and oxidative stress data considered in the IARC Monograph, together with other available data not considered by IARC. The Expert Panel defined and used a weight of evidence (WoE) approach that included ranking of studies and endpoints by the strength of their linkage to events associated with carcinogenic mechanisms. Importantly, the Expert Panel concluded that there was sufficient information available from a very large number of regulatory genotoxicity studies that should have been considered by IARC. The WoE approach, the inclusion of all relevant regulatory studies, and some differences in interpretation of individual studies led to significantly different conclusions by the Expert Panel compared with the IARC Monograph. The Expert Panel concluded that glyphosate, glyphosate formulations, and AMPA do not pose a genotoxic hazard and the data do not support the IARC Monograph genotoxicity evaluation. With respect to carcinogenicity classification and mechanism, the Expert Panel concluded that evidence relating to an oxidative stress mechanism of carcinogenicity was largely unconvincing and that the data profiles were not consistent with the characteristics of genotoxic carcinogens. © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

Kirkland D.,Kirkland Consulting
Expert Opinion on Drug Metabolism and Toxicology | Year: 2011

Introduction: In vitro genotoxicity assays have a high sensitivity to detect rodent carcinogens, but mammalian cell tests have a propensity for misleading positive results (poor specificity). Recent data show a greater risk of misleading positive results in p53-deficient rodent cell lines than in p53-competent human cells. Measures of cytotoxicity, source and stability of cells used are also important. Areas covered: In this review, potential reduction in the top concentration for testing (10 mM) is discussed. Indirect effects on non-DNA targets, which may not be relevant for humans or may exhibit a threshold, have been identified. Expert opinion: The reliability of in vitro genotoxicity tests could be improved by selecting p53-proficient, human cells. The provenance and stability of the cells used should be demonstrated. Measures of cytotoxicity based on cell proliferation should be used. Lowering the top concentration for testing from 10 mM to 4 mM or 2000 μg/ml, whichever is the lower, as proposed by some experts, would seem to be justified. Artefacts that may be caused by reaction of test substance with culture medium should be avoided. Better understanding and investigation of the potential for threshold and irrelevant modes of action are encouraged. © 2011 Informa UK, Ltd.

Kier L.D.,Private Consultant | Kirkland D.J.,Kirkland Consulting
Critical Reviews in Toxicology | Year: 2013

An earlier review of the toxicity of glyphosate and the original Roundup™-branded formulation concluded that neither glyphosate nor the formulation poses a risk for the production of heritable/somatic mutations in humans. The present review of subsequent genotoxicity publications and regulatory studies of glyphosate and glyphosate-based formulations (GBFs) incorporates all of the findings into a weight of evidence for genotoxicity. An overwhelming preponderance of negative results in well-conducted bacterial reversion and in vivo mammalian micronucleus and chromosomal aberration assays indicates that glyphosate and typical GBFs are not genotoxic in these core assays. Negative results for in vitro gene mutation and a majority of negative results for chromosomal effect assays in mammalian cells add to the weight of evidence that glyphosate is not typically genotoxic for these endpoints in mammalian systems. Mixed results were observed for micronucleus assays of GBFs in non-mammalian systems. Reports of positive results for DNA damage endpoints indicate that glyphosate and GBFs tend to elicit DNA damage effects at high or toxic dose levels, but the data suggest that this is due to cytotoxicity rather than DNA interaction with GBF activity perhaps associated with the surfactants present in many GBFs. Glyphosate and typical GBFs do not appear to present significant genotoxic risk under normal conditions of human or environmental exposures. © 2013 Informa UK Ltd.

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.

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.

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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