Melanie Guérard

IWGT report on quantitative approaches to genotoxicity risk assessment II. Use of point-of-departure (PoD) metrics in defining acceptable exposure limits and assessing human risk ☆

This is the second of two reports from the International Workshops on Genotoxicity Testing (IWGT) Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (the QWG). The first report summarized the discussions and recommendations of the QWG related to the need for quantitative dose-response analysis of genetic toxicology data, the existence and appropriate evaluation of threshold responses, and methods to analyze exposure-response relationships and derive points of departure (PoDs) from which acceptable exposure levels could be determined. This report summarizes the QWG discussions and recommendations regarding appropriate approaches to evaluate exposure-related risks of genotoxic damage, including extrapolation below identified PoDs and across test systems and species. Recommendations include the selection of appropriate genetic endpoints and target tissues, uncertainty factors and extrapolation methods to be considered, the importance and use of information on mode of action, toxicokinetics, metabolism, and exposure biomarkers when using quantitative exposure-response data to determine acceptable exposure levels in human populations or to assess the risk associated with known or anticipated exposures. The empirical relationship between genetic damage (mutation and chromosomal aberration) and cancer in animal models was also examined. It was concluded that there is a general correlation between cancer induction and mutagenic and/or clastogenic damage for agents thought to act via a genotoxic mechanism, but that the correlation is limited due to an inadequate number of cases in which mutation and cancer can be compared at a sufficient number of doses in the same target tissues of the same species and strain exposed under directly comparable routes and experimental protocols.

Assessment of the Genotoxic Potential of Azidothymidine in the Comet, Micronucleus, and Pig-a Assay

The genotoxic potential of azidothymidine (Zidovudine, AZT), chosen as a model compound for nucleotide analogs, was comprehensively assessed in vivo for gene mutation, clastogenicity, and DNA breakage endpoints. Male Wistar rats were treated by oral gavage over 7 days with AZT at dose levels of 2×0 (control), 2×250, 2×500, and 2×1000mg/kg/day with a final single dose given on day 8. DNA damage was then evaluated with the comet assay in liver, stomach, and peripheral blood and with the micronucleus test in bone marrow and peripheral blood (by flow cytometry) in the same animals. After a treatment-free period of upto 42 days, the Pig-a gene mutation assay was performed in peripheral blood of the high-dose animals. In the comet assay as well as the micronucleus test, AZT caused a considerable dose-dependent increase in DNA damage in all tissues evaluated and was highly cytotoxic to bone marrow and peripheral blood cells. These data are well in line with published results. Surprisingly, AZT did not significantly increase the number of Pig-a mutant cells. We speculate that two factors likely contributed to this negative result: a predominance of large deletions caused by AZT, and the relatively low statistical power of the first-generation scoring method used for this study.

Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay: Multiplexed High Information Content Assay

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent‐liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho‐histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow® DNA Damage Kit—p53, γH2AX, Phospho‐Histone H3. For these experiments, seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and nongenotoxicants. TK6 cells were exposed to chemicals in 96‐well plates over a range of concentrations for 24 hr. At 4 and 24 hr, cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96‐well plate via a robotic walk‐away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all interlaboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or nongenotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. Environ. Mol. Mutagen. 58:146–161, 2017.

Quantitative assessment of the dose–response of alkylating agents in DNA repair proficient and deficient ames tester strains

Mutagenic and clastogenic effects of some DNA damaging agents such as methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) have been demonstrated to exhibit a nonlinear or even “thresholded” dose–response in vitro and in vivo. DNA repair seems to be mainly responsible for these thresholds. To this end, we assessed several mutagenic alkylators in the Ames test with four different strains of Salmonella typhimurium: the alkyl transferases proficient strain TA1535 (Ogt+/Ada+), as well as the alkyl transferases deficient strains YG7100 (Ogt+/Ada−), YG7104 (Ogt−/Ada+) and YG7108 (Ogt−/Ada−). The known genotoxins EMS, MMS, temozolomide (TMZ), ethylnitrosourea (ENU) and methylnitrosourea (MNU) were tested in as many as 22 concentration levels. Dose–response curves were statistically fitted by the PROAST benchmark dose model and the Lutz‐Lutz “hockeystick” model. These dose–response curves suggest efficient DNA‐repair for lesions inflicted by all agents in strain TA1535. In the absence of Ogt, Ada is predominantly repairing methylations but not ethylations. It is concluded that the capacity of alkyl‐transferases to successfully repair DNA lesions up to certain dose levels contributes to genotoxicity thresholds. Environ. Mol. Mutagen. 55:15–23, 2014.

In vitro genotoxicity of neutral red after photo-activation and metabolic activation in the Ames test, the micronucleus test and the comet assay.

Neutral red (Nr) is relatively non-toxic and is widely used as indicator dye in many biological test systems. It absorbs visible light and is known to act as a photosensitizer, involving the generation of reactive oxygen species (type-I reaction) and singlet oxygen (type-II reaction). The mutagenicity of Nr was determined in the Ames test (with Salmonella typhimurium strains TA1535, TA97, TA98, TA98NR, TA100, and TA102) with and without metabolic activation, and with and without photo-activation on agar plates. Similarly to the situation following metabolic activation, photo-mutagenicity of Nr was seen with all Salmonella strains tested, albeit with different effects between these strains. To our knowledge, Nr is the only photo-mutagen showing such a broad action. Since the effects are also observed in strains not known to be responsive to ROS, this indicates that ROS production is not the sole mode of action that leads to photo-genotoxicity. The reactive species produced by irradiation are short-lived as pre-irradiation of an Nr solution did not produce mutagenic effects when added to the bacteria. In addition, mutagenicity in TA98 following irradiation was stronger than in the nitroreductase-deficient strain TA98NR, indicating that nitro derivatives that are transformed by bacterial nitroreductase to hydroxylamines appear to play a role in the photo-mutagenicity of Nr. Photo-genotoxicity of Nr was further investigated in the comet assay and micronucleus test in L5178Y cells. Concentration-dependent increases in primary DNA damage and in the frequency of micronuclei were observed after irradiation.

Performance and data interpretation of the in vivo comet assay in pharmaceutical industry: EFPIA survey results

In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by the ICH S2(R1) guideline. This paper summarizes a survey suggested by the Safety Working Party of European Medicines Agency (EMA), and conducted by the European Federation of Pharmaceutical Industries and Associations (EFPIA) to investigate the experience among European pharmaceutical companies by conducting the in vivo comet assay for regulatory purpose. A special focus was given on the typology of the obtained results and to identify potential difficulties encountered with the interpretation of study data. The participating companies reported a total of 147 studies (conducted in-house or outsourced) and shared the conclusion on the comet assay response for 136 studies. Most of the studies were negative (118/136). Only about 10% (14/136 studies) of the comet assays showed a positive response. None of the positive comet assay results were clearly associated with organ toxicity indicating that the positive responses are not due to cytotoxic effects of the compound in the tissue examined. The number of comet assays with an equivocal or inconclusive response was rare, respectively <1% (1/147 studies) and 2% (3/147 studies). In case additional information (e.g. repeat assay, organ toxicity, metabolism, tissue exposure) would have been available for evaluation, a final conclusion could most probably have been drawn for most or all of these studies. All (46) negative in vivo comet assays submitted alongside with a negative in vivo micronucleus assay were accepted by the regulatory authorities to mitigate a positive in vitro mammalian cell assay following the current ICH S2 guidance. The survey results demonstrate the robustness of the comet assay and the regulatory acceptance of the current ICH S2 guidance.

Influence of experimental conditions on data variability in the liver comet assay

The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post‐mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time‐dependent increase in comet parameters. Environ. Mol. Mutagen. 55:114–121, 2014.

A proposal for a novel rationale for critical effect size in dose-response analysis based on a multi-endpoint in vivo study with methyl methanesulfonate.

Methyl methanesulfonate, a well-known direct-acting genotoxicant, was assessed in a multi-endpoint study in rats using six closely spaced dose levels. The main goal of the study was to investigate the genotoxic response at very low doses and to analyse this response with dedicated statistical tools in order to find a Point of Departure (PoD) and related metrics. Software packages like PROAST or EPA-BMDS require the toxicologist to define a so-called critical effect size (CES) or benchmark response (BMR) and this choice has a large impact on the result of the PoD calculation. Currently, increases of 5%, 10% or 1 standard deviation over concurrent vehicle controls have been proposed for CES/BMR, values that may or may not be suited for all genotoxicity endpoints. Based on the data obtained in this study, we propose an endpoint specific CES approach that reflects the typical evaluation process of a regulatory acceptable genotoxicology study. However, we are aware that this ratio-based CES strategy will need to be more fully developed with additional experimentation and should be mainly seen as a starting point for scientific discussion.

Genotoxicity testing of peptides: folate deprivation as a marker of exaggerated pharmacology.

The incidence of micronucleated-cells is considered to be a marker of a genotoxic event and can be caused by direct- or indirect-DNA reactive mechanisms. In particular, small increases in the incidence of micronuclei, which are not associated with toxicity in the target tissue or any structurally altering properties of the compound, trigger the suspicion that an indirect mechanism could be at play. In a bone marrow micronucleus test of a synthetic peptide (a dual agonist of the GLP-1 and GIP receptors) that had been integrated into a regulatory 13-week repeat-dose toxicity study in the rat, small increases in the incidence of micronuclei had been observed, together with pronounced reductions in food intake and body weight gain. Because it is well established that folate plays a crucial role in maintaining genomic integrity and pronounced reductions in food intake and body weight gain were observed, folate levels were determined from plasma samples initially collected for toxicokinetic analytics. A dose-dependent decrease in plasma folate levels was evident after 4 weeks of treatment at the mid and high dose levels, persisted until the end of the treatment duration of 13-weeks and returned to baseline levels during the recovery period of 4 weeks. Based on these properties, and the fact that the compound tested (peptide) per se is not expected to reach the nucleus and cause DNA damage, the rationale is supported that the elevated incidence of micronucleated polychromatic erythrocytes is directly linked to the exaggerated pharmacology of the compound resulting in a decreased folate level.

Inter-laboratory comparison of the in vivo comet assay including three image analysis systems

To compare the extent of potential inter‐laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed—including independent slide evaluation by image analysis—in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter‐laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide‐stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories. Environ. Mol. Mutagen. 56:788–793, 2015.