Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds

Despite recent improvements in genotoxicity protocols, we have observed an increase in the occurrence of positive results, particularly in chromosomal aberration tests in vitro, yet very few of these are accompanied by positive responses in vivo. Thus, the positive results may not be biologically relevant either for rodents or humans in vivo, but how should we determine "biological relevance"? Chemicals that produce thresholded dose-responses may well not pose a genotoxic risk at low (relevant to human) exposures, but thresholds should not just be "seen"; there must be an explanation and understanding of the underlying mechanism. In addition to extremes of pH, ionic strength and osmolality, as have been identified previously, such mechanisms include indirect genotoxicity resulting from interaction with non-DNA targets, chemicals/metabolites which are inherently genotoxic but which, at low concentrations, are effectively conjugated and unable to form adducts, and production of specific metabolites under in vitro conditions that are not formed in rodents or humans in vivo. If such thresholded mechanisms can be identified at exposures which are well in excess of expected human exposure, then there may be a strong argument that the positive results are not biologically relevant.     

How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop

Workshop participants agreed that genotoxicity tests in mammalian cells in vitro produce a remarkably high and unacceptable occurrence of irrelevant positive results (e.g. when compared with rodent carcinogenicity). As reported in several recent reviews, the rate of irrelevant positives (i.e. low specificity) for some studies using in vitro methods (when compared to this "gold standard") means that an increased number of test articles are subjected to additional in vivo genotoxicity testing, in many cases before, e.g. the efficacy (in the case of pharmaceuticals) of the compound has been evaluated. If in vitro tests were more predictive for in vivo genotoxicity and carcinogenicity (i.e. fewer false positives) then there would be a significant reduction in the number of animals used. Beyond animal (or human) carcinogenicity as the "gold standard", it is acknowledged that genotoxicity tests provide much information about cellular behaviour, cell division processes and cellular fate to a (geno)toxic insult. Since the disease impact of these effects is seldom known, and a verification of relevant toxicity is normally also the subject of (sub)chronic animal studies, the prediction of in vivo relevant results from in vitro genotoxicity tests is also important for aspects that may not have a direct impact on carcinogenesis as the ultimate endpoint of concern. In order to address the high rate of in vitro false positive results, a 2-day workshop was held at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy in April 2006. More than 20 genotoxicity experts from academia, government and industry were invited to review data from the currently available cell systems, to discuss whether there exist cells and test systems that have a reduced tendency to false positive results, to review potential modifications to existing protocols and cell systems that might result in improved specificity, and to review the performance of some new test systems that show promise of improved specificity without sacrificing sensitivity. It was concluded that better guidance on the likely mechanisms resulting in positive results that are not biologically relevant for human health, and how to obtain evidence for those mechanisms, is needed both for practitioners and regulatory reviewers. Participants discussed the fact that cell lines commonly used for genotoxicity testing have a number of deficiencies that may contribute to the high false positive rate. These include, amongst others, lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g. Aroclor-induced rat S9), impaired p53 function and altered DNA repair capability. The high concentrations of test chemicals (i.e. 10 mM or 5000 microg/ml, unless precluded by solubility or excessive toxicity) and the high levels of cytotoxicity currently required in mammalian cell genotoxicity tests were discussed as further potential sources of false positive results. Even if the goal is to detect carcinogens with short in vitro tests under more or less acute conditions, it does not seem logical to exceed the capabilities of cellular metabolic turnover, activation and defence processes. The concept of "promiscuous activation" was discussed. For numerous mutagens, the decisive in vivo enzymes are missing in vitro. However, if the substrate concentration is increased sufficiently, some other enzymes (that are unimportant in vivo) may take over the activation-leading to the same or a different active metabolite. Since we often do not use the right enzyme systems for positive controls in vitro, we have to rely on their promiscuous activation, i.e. to use excessive concentrations to get an empirical correlation between genotoxicity and carcinogenicity. A thorough review of published and industry data is urgently needed to determine whether the currently required limit concentration of 10mM or 5000 microg/ml, and high levels of cytotoxicity, are necessary for the detection of in vivo genotoxins and DNA-reactive, mutagenic carcinogens. In addition, various measures of cytotoxicity are currently allowable under OECD test guidelines, but there are few comparative data on whether different measures would result in different maximum concentrations for testing. A detailed comparison of cytotoxicity assessment strategies is needed. An assessment of whether test endpoints can be selected that are not intrinsically associated with cytotoxicity, and therefore are less susceptible to artefacts produced by cytotoxicity, should also be undertaken. There was agreement amongst the workshop participants that cell systems which are p53 and DNA-repair proficient, and have defined Phase 1 and Phase 2 metabolism, covering a broad set of enzyme forms, and used within the context of appropriately set limits of concentration and cytotoxicity, offer the best hope for reduced false positives. Whilst there is some evidence that human lymphocytes are less susceptible to false positives than the current rodent cell lines, other cell systems based on HepG2, TK6 and MCL-5 cells, as well as 3D skin models based on primary human keratinocytes also show some promise. Other human cell lines such as HepaRG, and human stem cells (the target for carcinogenicity) have not been used for genotoxicity investigations and should be considered for evaluation. Genetic engineering is also a valuable tool to incorporate missing enzyme systems into target cells. A collaborative research programme is needed to identify, further develop and evaluate new cell systems with appropriate sensitivity but improved specificity. In order to review current data for selection of appropriate top concentrations, measures and levels of cytotoxicity, metabolism, and to be able to improve existing or validate new assay systems, the participants called for the establishment of an expert group to identify the in vivo genotoxins and DNA-reactive, mutagenic carcinogens that we expect our in vitro genotoxicity assays to detect as well as the non-genotoxins and non-carcinogens we expect them not to detect.     

Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds?

Today reconstructed skin models that simulate human skin, such as Episkin, are widely used for safety or efficacy pre-screening. Moreover, they are of growing interest for regulatory purposes in the framework of alternatives to animal testing. In order to reduce and eventually replace results of in vivo genotoxicity testing with in vitro data, there is a need to develop new complementary biological models and methods with improved ability to predict genotoxic risk. This can be achieved if these new assays do take into account exposure conditions that are more relevant than in the current test systems. In an attempt to meet this challenge, two new applications using a human reconstructed skin model for in vitro genotoxicity assessment are proposed. The skin is the target organ for dermally exposed compounds or environmental stress. Although attempts have been made to develop genotoxicity test procedures in vivo on mouse skin, human reconstructed skin models have not been used for in vitro genotoxicity testing so far, although they present clear advantages over mouse skin for human risk prediction. This paper presents the results of the development of a specific protocol allowing to perform the comet assay, a genotoxicity test procedure, on reconstructed skin. The comet assay was conducted after treatment of Episkin with UV, Lomefloxacin and UV or 4-nitroquinoline-N-oxide (4NQO). Treatment with the sunscreen Mexoryl was able to reduce the extent of comet signal. A second approach to use reconstructed epidermis in genotoxicity assays is also proposed. Indeed, the skin is a biologically active barrier driving the response to exposure to chemical agents and their possible metabolites. A specific co-culture system (Figure 1) using Episkin to perform the regular micronucleus assay is presented. Micronucleus induction in L5178Y cells cultured underneath Episkin was assessed after treatment of the reconstructed epidermis with mitomycin C, cyclophosphamide or apigenin. This second way of using human reconstructed skin for genotoxicity testing aims at improving the relevance of exposure conditions in in vitro genotoxicity assays for dermally applied compounds.     

Thresholds in genotoxicity responses

It has been commonly accepted that risk assessments of genotoxic chemicals are based on linear extrapolation methods. However, there is substantial evidence that some chemicals may be genotoxic only at high doses by mechanisms that do not occur at low doses, or only under specific conditions in genotoxicity assays, but are inactive at concentrations within the range of human exposure levels. There are a variety of possible mechanisms of thresholded genotoxicity, including disruption of cell division and chromosome segregation, inhibition of DNA synthesis, overloading of oxidative defence mechanisms, metabolism or plasma binding capacity, disturbances of metal homeostasis, cytotoxicity and physiological perturbations in in vivo assays. The degrees of evidence supporting the proposed mechanisms are variable and not all are sufficiently robust to be universally accepted as yet by the scientific community. However, a survey of industrial companies indicated that data have been accepted by some regulatory authorities indicating thresholds contributing to genotoxicity responses.     

ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact.

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces. Two ICH guidances regarding genotoxicity testing have been issued: S2A, 'Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, 'Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA. These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches. Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing. Although these guidances were previously released within the participating regions in their respective regulatory communiqués, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described. The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort. This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay. This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication. The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary.     

ICH-Harmonised guidances on genotoxicity testing of pharmaceuticals: evolution,reasoning and impact

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces. Two ICH guidances regarding genotoxicity testing have been issued: S2A, `Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, `Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA. These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches. Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing. Although these guidances were previously released within the participating regions in their respective regulatory communiqués, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described. The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort. This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay. This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication. The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary.     

Relevance and follow-up of positive results in in vitro genetic toxicity assays: an ILSI-HESI initiative

In vitro genotoxicity assays are often used to screen and predict whether chemicals might represent mutagenic and carcinogenic risks for humans. Recent discussions have focused on the high rate of positive results in in vitro tests, especially in those assays performed in mammalian cells that are not confirmed in vivo. Currently, there is no general consensus in the scientific community on the interpretation of the significance of positive results from the in vitro genotoxicity assays. To address this issue, the Health and Environmental Sciences Institute (HESI), held an international workshop in June 2006 to discuss the relevance and follow-up of positive results in in vitro genetic toxicity assays. The goals of the meeting were to examine ways to advance the scientific basis for the interpretation of positive findings in in vitro assays, to facilitate the development of follow-up testing strategies and to define criteria for determining the relevance to human health. The workshop identified specific needs in two general categories, i.e., improved testing and improved data interpretation and risk assessment. Recommendations to improve testing included: (1) re-examine the maximum level of cytotoxicity currently required for in vitro tests; (2) re-examine the upper limit concentration for in vitro mammalian studies; (3) develop improved testing strategies using current in vitro assays; (4) define criteria to guide selection of the appropriate follow-up in vivo studies; (5) develop new and more predictive in vitro and in vivo tests. Recommendations for improving interpretation and assessment included: (1) examine the suitability of applying the threshold of toxicological concern concepts to genotoxicity data; (2) develop a structured weight of evidence approach for assessing genotoxic/carcinogenic hazard; and (3) re-examine in vitro and in vivo correlations qualitatively and quantitatively. Conclusions from the workshop highlighted a willingness of scientists from various sectors to change and improve the current paradigm and move from a hazard identification approach to a "realistic" risk-based approach that incorporates information on mechanism of action, kinetics, and human exposure..     

International Commission for Protection Against Environmental Mutagens and Carcinogens. ICPEMC publication No. 19. The classification of carcinogens identified in the rodent bioassay as potential risks to humans: what type of substance should be tested next?

The relevance of rodent cancer bioassay data to humans is discussed in relation to the needs of regulatory agencies. The usefulness of in vivo and in vitro genotoxicity testing in this connection is also discussed. In the case of rodent carcinogens that do not elicit genotoxicity, it is suggested that homeostatic imbalance, cell proliferation, and other processes may play a major role in tumor development and its importance to the possible ability of the test agent to induce human cancer. These possibilities need to be evaluated on a case by case basis. The methods by which chemicals are selected for the rodent cancer bioassay are also discussed and it is pointed out that naturally-occurring constituents of human foods should in future receive greater priority as a consequence of anticipated changes resulting from biotechnology.     

The classification of carcinogens identified in the rodent bioassay as potential risks to humans: What type of substance should be tested next?

The relevance of rodent cancer bioassay data to humans is discussed in relation to the needs of regulatory agencies. The usefulness of in vivo and in vitro genotoxicity testing in this connection is also discussed. In the case of rodent carcinogens that do not elicit genotoxicity, it is suggested that homeostatic imbalance, cell proliferation, and other processes may play a major role in tumor development and its importance to the possible ability of the test agent to induce human cancer. These possibilities need to be evaluated on a case by case basis. The methods by which chemicals are selected for the rodent cancer bioassay are also discussed and it is pointed out that naturally-occurring constituents of human foods should in future receive greater priority as a consequence of anticipated changes resulting from biotechnology.     

Current and future needs for developmental toxicity testing

A review is presented of the use of developmental toxicity testing in the United States and international regulatory assessment of human health risks associated with exposures to pharmaceuticals (human and veterinary), chemicals (agricultural, industrial, and environmental), food additives, cosmetics, and consumer products. Developmental toxicology data are used for prioritization and screening of pharmaceuticals and chemicals, for evaluating and labeling of pharmaceuticals, and for characterizing hazards and risk of exposures to industrial and environmental chemicals. The in vivo study designs utilized in hazard characterization and dose-response assessment for developmental outcomes have not changed substantially over the past 30 years and have served the process well. Now there are opportunities to incorporate new technologies and approaches to testing into the existing assessment paradigm, or to apply innovative approaches to various aspects of risk assessment. Developmental toxicology testing can be enhanced by the refinement or replacement of traditional in vivo protocols, including through the use of in vitro assays, studies conducted in alternative nonmammalian species, the application of new technologies, and the use of in silico models. Potential benefits to the current regulatory process include the ability to screen large numbers of chemicals quickly, with the commitment of fewer resources than traditional toxicology studies, and to refine the risk assessment process through an enhanced understanding of the mechanisms of developmental toxicity and their relevance to potential human risk. As the testing paradigm evolves, the ability to use developmental toxicology data to meet diverse critical regulatory needs must be retained.     

A review of the genotoxicity of marketed pharmaceuticals

Information in the 1999 Physician's Desk Reference as well as from the peer-reviewed published literature was used to evaluate the genotoxicity of marketed pharmaceuticals. This survey is a compendium of genotoxicity information and a means to gain perspective on the inherent genotoxicity of structurally diverse pharmaceuticals. Data from 467 marketed drugs were collected. Excluded from analysis were anti-cancer drugs and nucleosides, which are expected to be genotoxic, steroids, biologicals and peptide-based drugs. Of the 467 drugs, 115 had no published gene-tox data. This group was comprised largely of acutely administered drugs such as antibiotics, antifungals, antihistamines decongestants and anesthetics. The remaining 352 had at least one standard gene-tox assay result. Of these, 101 compounds (28.7%) had at least one positive assay result in the pre-ICH/OECD standard four-test battery (bacterial mutagenesis, in vitro cytogenetics, mouse lymphoma assay (MLA), in vivo cytogenetics). Per assay type, the percentage of positive compounds was: bacterial mutagenesis test, 27/323 (8.3%); in vitro cytogenetics 55/222 (24.8%); MLA 24/96 (25%); in vivo cytogenetics 29/252 (11.5%). Of the supplemental genetic toxicology test findings reported, the sister chromatid exchange (SCE) assay had the largest percentage of positives 17/39 (43.5%) and mammalian mutagenesis assays (excluding MLA) had the lowest percentage of positives 2/91 (2.2%). The predictive value of genetic toxicology findings for 2-year bioassay outcomes is difficult to assess since carcinogenicity can occur via non-genotoxic mechanisms. Nevertheless, the following survey findings were made: 201 drugs had both gene-tox data and rodent carcinogenicity data. Of these, 124 were negative and 77 were equivocal or positive for carcinogenicity in at least 1 gender/1 species. Of the 124 non-carcinogens, 100 had no positive gene-tox findings. Of the remaining 24, 19 were positive in in vitro cytogenetics assays. Among the 77 compounds that exhibited equivocal or positive effects in carcinogenesis studies, 26 were positive in gene-tox assays and 51 were negative. Of the 51 negatives, 47 had multiple negative gene-tox assay results suggesting that these are probably non-genotoxic carcinogens. Statistical analyses suggested that no combination of gene-tox assays provided a higher predictivity of rodent carcinogenesis than the bacterial mutagenicity test itself.     

Ciprofloxacin: in vivo genotoxicity studies

The fluoroquinolone ciprofloxacin is widely used in antimicrobial therapy. It inhibits the bacterial gyrase and in high concentrations in vitro also the functionally related eukaryotic topoisomerase-II, which resulted in genotoxic effects in several in vitro tests. In order to evaluate the relevance of these findings, ciprofloxacin was tested in vivo for genotoxic activity using the following test systems: micronucleus test in bone marrow of mice, cytogenetic chromosome analysis in Chinese hamster, dominant lethal assay in male mice and UDS tests in primary rat and mouse hepatocytes in vivo. These results are compared with already published in vitro and in vivo studies with ciprofloxacin. All in vivo genotoxicity revealed no genotoxic effect for ciprofloxacin. In addition, ciprofloxacin was found to be non-carcinogenic in two rodent long-term bioassays. Therefore, ciprofloxacin is considered to be safe for therapeutic use.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper No. 5. Genotoxicity tests as predictors of carcinogens: an analysis

Differences between the results of numerical validation studies comparing in vitro and in vivo genotoxicity tests with the rodent cancer bioassay are leading to the perception that short-term tests predict carcinogenicity only with uncertainty. Consideration of factors such as the pharmacokinetic distribution of chemicals, the systems available for metabolic activation and detoxification, the ability of the active metabolite to move from the site of production to the target DNA, and the potential for expression of the induced lesions, strongly suggests that the disparate sensitivity of the different test systems is a major reason why numerical validation is not more successful. Furthermore, genotoxicity tests should be expected to detect only a subset of carcinogens, namely genotoxic carcinogens, rather than those carcinogens that appear to act by non-genetic mechanisms. Instead of relying primarily on short-term in vitro genotoxicity tests to predict carcinogenic activity, these tests should be used in a manner that emphasizes the accurate determination of mutagenicity or clastogenicity. It must then be determined whether the mutagenic activity is further expressed as carcinogenicity in the appropriate studies using test animals. The prospects for quantitative extrapolation of in vitro or in vivo genotoxicity test results to carcinogenicity requires a much more precise understanding of the critical molecular events in both processes.     

Ciprofloxacin: in vivo genotoxicity studies.

The fluoroquinolone ciprofloxacin is widely used in antimicrobial therapy. It inhibits the bacterial gyrase and in high concentrations in vitro also the functionally related eukaryotic topoisomerase-II, which resulted in genotoxic effects in several in vitro tests. In order to evaluate the relevance of these findings, ciprofloxacin was tested in vivo for genotoxic activity using the following test systems: micronucleus test in bone marrow of mice, cytogenetic chromosome analysis in Chinese hamster, dominant lethal assay in male mice and UDS tests in primary rat and mouse hepatocytes in vivo. These results are compared with already published in vitro and in vivo studies with ciprofloxacin. All in vivo genotoxicity revealed no genotoxic effect for ciprofloxacin. In addition, ciprofloxacin was found to be non-carcinogenic in two rodent long-term bioassays. Therefore, ciprofloxacin is considered to be safe for therapeutic use.     

Genotoxicity and carcinogenicity studies of antihypertensive agents

This survey is a compendium of genotoxicity and carcinogenicity information of antihypertensive drugs. Data from 164 marketed drugs were collected. Of the 164 drugs, 65 (39.6%) had no retrievable genotoxicity or carcinogenicity data; this group was comprised largely of drugs marketed in a limited number of countries. The remaining 99 (60.4%) had at least one genotoxicity or carcinogenicity test result. Of these 99, 48 (48.5%) had at least one positive finding: 32 tested positive in at least one genotoxicity assay, 26 in at least one carcinogenicity assay, and 10 gave a positive result in both at least one genotoxicity assay and at least one carcinogenicity assay. In terms of correlation between results of the various genotoxicity assays and absence of carcinogenic activity in both mice and rats 2 of 44 non-carcinogenic drugs tested positive in the in vitro bacterial mutagenesis assay, 2 of 9 tested positive in the mouse lymphoma assay, none of 14 tested positive for gene mutation at the hprt locus, 5 of 25 tested positive in in vitro cytogenetic assays, none of 31 in in vivo cytogenetic assays, and none of 14 in inducing DNA damage and/or repair in in vitro and/or in vivo assays. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, 75 drugs had both genotoxicity and carcinogenicity data; of these 37 (49.3%) were neither genotoxic nor carcinogenic, 14 (18.7%) were non-carcinogens which tested positive in at least one genotoxicity assay, 14 (18.7%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, and 10 (13.3%) were both genotoxic and carcinogenic. Only 42 of the 164 marketed antihypertensives (25.6%) had all data required by the guidelines for testing of pharmaceuticals.     

Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: determination of No-Observed-Genotoxic-Effect-Levels

Nowadays, there is clear progress in using the threshold concept in genetic toxicology, but its demonstration and acceptance in risk assessment is still under debate. Although it has been accepted for some non-DNA-reactive agents for which mechanisms of action were demonstrated, there is a growing weight of evidence to also support the existence of thresholded dose-responses for DNA-reactive agents. In this context, we have recently shown in human TK6 lymphoblastoid cells, that DNA-oxidizing agents [potassium bromate, bleomycin and hydrogen peroxide (via glucose oxidase)] produced non-linear dose-responses in the in vitro micronucleus test, thus allowing the determination of No-Observed-Genotoxic-Effect-Levels (NOGELs). Therefore, the aim of the present study was to focus on the analysis of thresholded dose-response curves in order to further investigate the existence of NOGELs for these same directly DNA-damaging agents, by use of other genotoxicity endpoints. Mutation frequency was determined after a 1-h treatment in the thymidine kinase (TK) gene-mutation assay. Primary DNA damage, especially oxidative DNA damage, was also assessed after 1h of treatment, followed - or not - by a 23-h recovery period, with the modified version of the comet assay (i.e. with the glycosylases Fpg and hOgg1). Overall, our analysis demonstrates that there is convincing evidence to support the existence of thresholded dose-responses for DNA-oxidizing agents. The determination of NOGELs depends on the genotoxic endpoint studied and consequently requires different genotoxicity assays performed concurrently. NOGELs could only be defined for the induction of chromosomal aberrations and gene mutations, i.e. for an effect-endpoint but not for primary DNA damage, i.e. for an exposure-endpoint. Further statistical analyses of these data are now required in order to draw conclusions on the exact level of the thresholds.     

Silica nanoparticles administered at the maximum tolerated dose induce genotoxic effects through an inflammatory reaction while gold nanoparticles do not

While the collection of genotoxicity data and insights into potential mechanisms of action for nano-sized particulate materials (NPs) are steadily increasing, there is great uncertainty whether current standard assays are suitable to appropriately characterize potential risks. We investigated the effects of NPs in an in vivo Comet/micronucleus (MN) combination assay and in an in vitro MN assay performed with human blood. We also incorporated additional endpoints into the in vivo study in an effort to delineate primary from secondary mechanisms. Amorphous silica NPs (15 and 55 nm) were chosen for their known reactivity, while gold nano/microparticles (2, 20, and 200 nm) were selected for their wide size range and lower reactivity. DNA damage in liver, lung and blood cells and micronuclei in circulating reticulocytes were measured after 3 consecutive intravenous injections to male Wistar rats at 48, 24 and 4h before sacrifice. Gold nano/microparticles were negative for MN induction in vitro and in vivo, and for the induction of DNA damage in all tissues. Silica particles, however, caused a small but reproducible increase in DNA damage and micronucleated reticulocytes when tested at their maximum tolerated dose (MTD). No genotoxic effects were observed at lower doses, and the in vitro MN assay was also negative. We hypothesize that silica NPs initiate secondary genotoxic effects through release of inflammatory cell-derived oxidants, similar to that described for crystalline silica (quartz). Such a mechanism is supported by the occurrence of increased neutrophilic infiltration, necrosis, and apoptotic cells in the liver, and induction of inflammatory markers TNF-α and IL-6 in plasma at the MTDs. These results were fairly consistent between silica NPs and the quartz control, thereby strengthening the argument that silica NPs may act in a similar, thresholded manner. The observed profile is supportive of a secondary genotoxicity mechanism that is driven by inflammation.     

Induction of chromosomal aberrations by phenolic compounds: possible role of reactive oxygen species

Phenolic molecules are widely present in the environment and some of them are well known carcinogens. Some phenolic molecules are also genotoxic but the mechanisms involved in this process are not fully understood. We have studied the induction of chromosomal aberrations by phenol, catechol and pyrogallol in V79 cells at different pH values (6.0, 7.4 and 8.0). At the same pH values, the production of hydroxyl radicals was assessed by measuring the degradation of deoxyribose. Apart from phenol, which only induces a non-significant increase in chromosomal aberration in this experimental system, catechol and pyrogallol showed clear clastogenic effect in a pH-dependent way. Experiments carried out at pH 7.4 in the presence of S9 Mix, SOD, catalase and catalase + SOD suggest that the formation of reactive oxygen species is not the main mechanism involved in the genotoxicity of catechol. However, concerning pyrogallol, our results suggest that its genotoxicity is almost exclusively mediated by reactive oxygen species. Taken together, these results suggest that, in spite of the structural similarity between the different molecules studied, the mechanisms of genotoxicity of these molecules could be considerably different. The existence of several mechanisms of genotoxicity, partially shared by this class of compounds, could explain the synergistic effects observed between these compounds in several genotoxicity test systems. Accurate knowledge of their mechanisms of genotoxicity could improve considerably the assessment of their relevance to human health, since these compounds, once absorbed, are subject to a wide range of pH values in vivo.     

Correcting for toxic inhibition in quantification of genotoxic response in the umuC test.

An improved procedure for quantification of results from the umuC tests for genotoxicity is presented. The calculation method better separates toxic growth inhibition (cytotoxicity) from genotoxic effects than currently used methods and therefore, greatly extends the applicability of genotoxicity tests on environmental samples. The basic principle is to normalize the genotoxic response compensating for both decreasing biomass and growth rate reduction that results from cytotoxicity. The improved method and the currently used method was compared for umuC tests on the pure compounds: methylmethanesulfonate (MMS), N-methyl-N'-nitro-N-nitroguanidine (MNNG), sodium azide (NaN3), and 4-nitroquinoline-1-oxide (4-NQO). For compounds with no or low cytotoxicity, the two calculation methods gave practically identical results, while for highly cytotoxic compounds, the traditional method overestimated genotoxicity. umuC tests were also carried out on leachate polluted groundwater sampled downgradient of a landfill (Grindsted, Denmark). All polluted samples showed high cytotoxicity concomitant with high genotoxicity when the results were quantified in the traditional way. The new method showed that these results were in fact false positive, as the apparent genotoxicity was a result of cytotoxicity. Based on the mathematical analysis leading to the improved procedure for correction for cytotoxicity, it is suggested to alter the present test design of the umuC test in order to obtain well-defined exposure concentrations as well as mathematical consistency in the quantification of genotoxicity.     

Extrapolation from in vitro tests to human risk: experience with sodium fluoride clastogenicity

Genotoxic effects observed in vitro, only at high doses or high levels of cytotoxicity, will be false positives if such conditions are not achieved or cannot be tolerated in vivo. However, for such effects to be disregarded there must be a threshold dose or level of cytotoxicity below which genotoxicity is absent. Sodium fluoride (NaF) has previously been shown to be clastogenic in vitro in Syrian hamster cells and human fibroblasts. We have extended these studies in human fibroblasts and included a positive control (mitomycin C, MMC) which is clastogenic in vivo and carcinogenic, and a chemically related control (NaCl). Cytotoxicity was measured as mitotic inhibition and cell death (loss of clonogenicity). The results are used to illustrate the problems associated with quantitative extrapolation from in vitro tests to human risk, as follows. (1) There appears to be a threshold response (clastogenicity vs. dose) with NaF at around 10 micrograms/ml (48 h exposure) but a more definitive conclusion must await elucidation of the mechanisms of clastogenicity. (2) NaCl is weakly clastogenic at 1000 times the threshold dose for NaF. The mechanisms are unlikely to be similar. (3) No clastogenicity was detected with NaF below about 30% mitotic inhibition but the relationship between clastogenicity and mitotic inhibition was similar for NaF and MMC. (4) There was no obvious threshold in the relationship between clastogenicity and cell killing with NaF. MMC was less clastogenic than NaF at equitotoxic doses. Observations 3 and 4 preclude the possibility of regarding the clastogenicity of NaF as a false positive by virtue of associated cytotoxicity.