International Workshop On Genotoxicity Testing Research Articles

Epigenotoxicity: Decoding the epigenetic imprints of genotoxic agents and their implications for regulatory genetic toxicology.

Regulatory genetic toxicology focuses on DNA damage and subsequent gene mutations. However, genotoxic agents can also affect epigenetic marks, and incorporation of epigenetic data into the regulatory framework may thus enhance the accuracy of risk assessment. Additionally, epigenetic alterations may identify non-genotoxic carcinogens that are not captured with the current battery of tests. Epigenetic alterations could also explain long-term consequences and potential transgenerational effects in the absence of DNA mutations. Therefore, at the 2022 International Workshops on Genotoxicity Testing (IWGT) in Ottawa (Ontario, Canada), an expert workgroup explored whether including epigenetic endpoints would improve regulatory genetic toxicology. Here we summarize the presentations and the discussions on technical advancements in assessing epigenetics, how the assessment of epigenetics can enhance strategies for detecting genotoxic and non-genotoxic carcinogens and the correlation between epigenetic alterations with other relevant apical endpoints.

Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

Interpretation of in vitro concentration-response data for risk assessment and regulatory decision-making: Report from the 2022 IWGT quantitative analysis expert working group meeting.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.

Analysis of chemical structures and mutations detected by Salmonella TA98 and TA100

As part of an analysis performed under the auspices of the International Workshop on Genotoxicity Testing (IWGT) in 2017, we and others showed that Salmonella frameshift strain TA98 and base-substitution strain TA100 together + /- S9 detected 93% of the mutagens detected by all the bacterial strains recommended by OECD TG471 (Williams et al., Mutation Res. 848:503081, 2019). We have extended this analysis by identifying the numbers and chemical classes of chemicals detected by these two strains either alone or in combination, including the role of S9. Using the Leadscope 2021 SAR Genetox database containing > 21,900 compounds, our dataset containing 7170 compounds tested in both TA98 and TA100. Together, TA98 and TA100 detected 94% (3733/3981) of the mutagens detected using all the TG471-recommended bacterial strains; 39% were mutagenic in one or both strains. TA100 detected 77% of all of these mutagens and TA98 70%. Considering the overlap of detection by both strains, 12% of these mutagens were detected only by TA98 and 19% only by TA100. In the absence of S9, sensitivity dropped by 31% for TA98 and 29% for TA100. Overall, 32% of the mutagens required S9 for detection by either strain; 9% were detected only without S9. Using the 2021 Leadscope Genetox Expert Alerts, TA100 detected 18 mutagenic alerting chemical classes with better sensitivity than TA98, whereas TA98 detected 10 classes better than TA100. TA100 detected more chemical classes than did TA98, especially hydrazines, azides, various di- and tri-halides, various nitrosamines, epoxides, aziridines, difurans, and half-mustards; TA98 especially detected polycyclic primary amines, various aromatic amines, polycyclic aromatic hydrocarbons, triazines, and dibenzo-furans. Model compounds with these structures induce primarily G to T mutations in TA100 and/or a hotspot GC deletion in TA98. Both TA98 and TA100 + /- S9 are needed for adequate mutagenicity screening with the Salmonella (Ames) assay.

Assessing the quality and making appropriate use of historical negative control data: A report of the International Workshop on Genotoxicity Testing (IWGT).

Historical negative control data (HCD) have played an increasingly important role in interpreting the results of genotoxicity tests. In particular, Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines recommend comparing responses produced by exposure to test substances with the distribution of HCD as one of three criteria for evaluating and interpreting study results (referred to herein as "Criterion C"). Because of the potential for inconsistency in how HCD are acquired, maintained, described, and used to interpret genotoxicity testing results, a workgroup of the International Workshops for Genotoxicity Testing was convened to provide recommendations on this crucial topic. The workgroup used example data sets from four in vivo tests, the Pig-a gene mutation assay, the erythrocyte-based micronucleus test, the transgenic rodent gene mutation assay, and the in vivo alkaline comet assay to illustrate how the quality of HCD can be evaluated. In addition, recommendations are offered on appropriate methods for evaluating HCD distributions. Recommendations of the workgroup are: When concurrent negative control data fulfill study acceptability criteria, they represent the most important comparator for judging whether a particular test substance induced a genotoxic effect. HCD can provide useful context for interpreting study results, but this requires supporting evidence that (i) HCD were generated appropriately, and (ii) their quality has been assessed and deemed sufficiently high for this purpose. HCD should be visualized before any study comparisons take place; graph(s) that show the degree to which HCD are stable over time are particularly useful. Qualitative and semi-quantitative assessments of HCD should also be supplemented with quantitative evaluations. Key factors in the assessment of HCD include: (i) the stability of HCD over time, and (ii) the degree to which inter-study variation explains the total variability observed. When animal-to-animal variation is the predominant source of variability, the relationship between responses in the study and an HCD-derived interval or upper bounds value (i.e., OECD Criterion C) can be used with a strong degree of confidence in contextualizing a particular study's results. When inter-study variation is the major source of variability, comparisons between study data and the HCD bounds are less useful, and consequentially, less emphasis should be placed on using HCD to contextualize a particular study's results. The workgroup findings add additional support for the use of HCD for data interpretation; but relative to most current OECD test guidelines, we recommend a more flexible application that takes into consideration HCD quality. The workgroup considered only commonly used in vivo tests, but it anticipates that the same principles will apply to other genotoxicity tests, including many in vitro tests.

Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT)

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a “Use of 3D Tissues in Genotoxicity Testing” working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.

Revisiting the bacterial mutagenicity assays: Report by a workgroup of the International Workshops on Genotoxicity Testing (IWGT)

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing “proficiency compounds” to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.

Are all bacterial strains required by OECD mutagenicity test guideline TG471 needed?

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to seek consensus on difficult or conflicting approaches to genotoxicity testing based upon experience, available data, and analysis techniques. At the 2017 IWGT meeting in Tokyo, one working group addressed the sensitivity and selectivity of the bacterial strains specified in the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471 to recommend possible modification of the test guideline. Three questions were posed: (1) Although TA100 is derived from TA1535, does TA1535 detect any mutagens that are not detected by TA100? (2) Among the options of Salmonella TA1537, TA97 or TA97a, are these strains truly equivalent? (3) Because there is a choice to use one of either E. coli WP2 uvrA, E. coli WP2 uvrA pKM101, or Salmonella TA102, are these strains truly equivalent? To answer these questions, we analyzed published bacterial mutation data in multiple strains from large (>10,000 compound) databases from Leadscope and Lhasa Limited and anonymized data for 53 compounds tested in TA1535 and TA100 provided by a pharmaceutical company. Our analysis involved (1) defining criteria for determining selective responses when using different strains; (2) identifying compounds producing selective responses based upon author calls; (3) confirming selective responses by visually examining dose-response data and considering experimental conditions; (4) using statistical methods to quantify the responses; (5) performing limited additional direct-comparison testing; and (6) determining the chemical classes producing selective responses. We found that few mutagens would fail to be detected if the test battery did not include Salmonella strains TA1535 (8/1167), TA1537 (2/247), TA102 (4/46), and E. coli WP2 uvrA (2/21). Of the mutagens detected by the full TG471 strain battery, 93% were detected using only strains TA98 and TA100; consideration of results from in vitro genotoxicity assays that detect clastogenicity increased this to 99%.

The IWGT in vitro Mammalian Cell Gene Mutation (MCGM) assays working group—Introductory remarks & consensus statements

Assays for gene mutations in cultured mammalian cells, i.e., Mammalian Cell Gene Mutation (MCGM) assays, are widely used in genetic toxicology laboratories worldwide; over the past four decades they have been commonly employed in safety assessment studies, and studies designed to address hypothesis-driven research questions. Despite many advances in the fields of cellular and molecular biology over the past four decades, the MCGM assays commonly used for regulatory evaluations continue to be those developed in the 1970s, including assays that enumerate induced mutations at the hprt or tk loci of several commonly used cell lines. Consequently, the Steering Committee of the 7th International Workshops on Genotoxicity Testing (IWGT) convened a working group (WG) to critically assess the state-of-the-science in regards to the current and emerging tools for the detection of mutagens using cultured mammalian cells. The WG was divided into four sub-groups that evaluated the state-of- the-science with respect to the: (1) in vitro Pig-a gene mutation assay, (2) in vitro assays based on cells from transgenic rodents, (3) technologies and innovations to improve MCGM assays using TK6 cells, and (4) novel and emerging technologies and approaches for detection and enumeration of gene mutations in mammalian cells. Each of these sub-groups critically reviewed the scientific literature, along with other unpublished data, to develop consensus statements on the status of the test systems in their respective focus areas. These reviews, with their associated consensus statements, are presented in the accompanying works by Bemis and Heflich., White et al., Honma et al., and Evans et al. The MCGM assay WG, in consultation with the entire IWGT, formulated consensus statements regarding the overall utility of MCGM assays for identification and assessment of mutagenic hazard.

In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT)

The working group reached complete or majority agreement on many issues.Results from TGR and in vivo comet assays for 91 chemicals showed they have similar ability to detect in vivo genotoxicity per se with bacterial mutagens and Ames-positive carcinogens.TGR and comet assay results were not significantly different when compared with IARC Group 1, 2 A, and unclassified carcinogens. There were significantly more comet assay positive responses for Group 2B chemicals, and for IARC classified and unclassified carcinogens combined, which may be expected since mutation is a sub-set of genotoxicity.A liver comet assay combined with the bone marrow/blood micronucleus (MNviv) test would detect in vivo genotoxins that do not exhibit tissue-specific or site-of-contact effects, and is appropriate for routine in vivo genotoxicity testing.Generally for orally administered substances, a comet assay at only one site-of-contact GI tract tissue (stomach or duodenum/jejunum) is required.In MNviv tests, evidence of target tissue exposure can be obtained in a number of different ways, as recommended by ICH S2(R1) and EFSA (Hardy et al., 2017).Except for special cases the i.p. route is inappropriate for in vivo testing; for risk evaluations more weight should be given to data from a physiologically relevant administration route.The liver MN test is sufficiently validated for the development of an OECD guideline. However, the impact of dosing animals >6 weeks of age needs to be evaluated. The GI tract MN test shows promise but needs more validation for an OECD guideline.The Pig-a assay detects systemically available mutagens and is a valuable follow-up to in vitro positive results. A new freeze-thaw protocol provides more flexibility. Mutant reticulocyte and erythrocyte frequencies should both be determined. Preliminary data are available for the Pig-a assay in male rat germ cells which require validation including germ cell DNA mutation origin.

In vitro mammalian cell mutation assays based on transgenic reporters: A report of the International Workshop on Genotoxicity Testing (IWGT)

Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets contained in shuttle vectors. In order to improve in vitro mutagenicity assessment, and to substantially reduce animal use, in vitro assays using transgenic reporters have been developed. These assays are based on cells derived from TGRs, or cells transfected with transgenic shuttle vectors containing a mutation target. As part of the 7th International Workshop on Genotoxicity Testing, an In Vitro Mammalian Cell Gene Mutation Assay working group reviewed all published information pertaining to in vitro transgene mutagenicity assays; the utility, advantages and disadvantages of the assays were evaluated and discussed. The review revealed that over 20 TGR-based in vitro assays have been used to assess the mutagenic activity of over 150 agents. Overall, the Working Group considered in vitro transgene mutagenicity assays pragmatic tools for the safety evaluation of new and existing substances. A formal SWOT (strengths, weaknesses, opportunities, threats) analysis revealed advantages including the use of established scoring protocols, avoidance of laborious clone isolation and enumeration, ability to use metabolically competent primary cells, ability to detect different types of genetic damage, large dynamic range, and complementarity to in vivo TGR endpoints. Disadvantages include lack of validation and little consistency in protocols, the use of specialised reagents, the time and effort required for mutant enumeration, the use of some cell lines that lack metabolic capacity, and the need for multiple assays to cover all mutational mechanisms. Several assays have been partially validated, indicating promising reliability, reproducibility and applicability domain. Once in vitro transgene mutagenicity assays have been more thoroughly validated, they are well placed to augment or replace existing in vitro mammalian cell mutagenicity assays, particularly in cases where the in vivo TGR mutation assay is intended for follow-up.

In vitro mammalian cell mutation assays based on the Pig-a gene: A report of the 7th International Workshop on Genotoxicity Testing (IWGT) Workgroup

Pig-a gene mutation assays enumerate cells with the glycosylphosphatidylinositol (GPI) anchor-deficient phenotype as a reporter of mutation in the endogenous Pig-a gene. Methods for measuring mutation in this gene are quite well established for in vivo systems. This approach to mutagenicity assessment has now been extended to in vitro mammalian cell-based systems. An expert workgroup from the 7th International Workshop on Genotoxicity Testing tasked with assessing the status of in vitro mammalian cell mutation assays has investigated the merits and limitations of in vitro Pig-a gene mutation assays. A review of the current status of these developing methodologies and the formation of consensus statements on the utility and application of these assays were performed to provide guidance for their potential use in genotoxicity hazard identification and risk assessment.

High information content assays for genetic toxicology testing: A report of the International Workshops on Genotoxicity Testing (IWGT)

We live in an era of ‘big data’, where the volume, velocity, and variety of the data being generated is increasingly influencing the way toxicological sciences are practiced. With this in mind, a workgroup was formed for the 2017 International Workshops on Genotoxicity Testing (IWGT) to consider the use of high information content data in genetic toxicology assessments. Presentations were given on adductomics, global transcriptional profiling, error-reduced single-molecule sequencing, and cellular phenotype-based assays, which were identified as methodologies that are relevant to present-day genetic toxicology assessments. Presenters and workgroup members discussed the state of the science for these methodologies, their potential use in genetic toxicology, current limitations, and the future work necessary to advance their utility and application. The session culminated with audience-assisted SWOT (strength, weakness, opportunities, and threats) analyses. The summary report described herein is structured similarly. A major conclusion of the workgroup is that while conventional regulatory genetic toxicology testing has served the public well over the last several decades, it does not provide the throughput that has become necessary in modern times, and it does not generate the mechanistic information that risk assessments ideally take into consideration. The high information content assay platforms that were discussed in this session, as well as others under development, have the potential to address aspect(s) of these issues and to meet new expectations in the field of genetic toxicology.

Horizon scanning for novel and emerging in vitro mammalian cell mutagenicity test systems

The induction of gene mutation within a DNA sequence can result in an adverse impact, altering or preventing gene function. Therefore, in vitro evaluation of mutagenicity is an essential component of the toxicological screening process. A variety of mutagen screening tools are routinely used in genetic toxicology, which are based on selected reporter genes. These assays are however typically labour intensive and impractical for high throughput screening. Considering this, the IWGT (International Workshops on Genotoxicity Testing) sub-group on Novel & Emerging in vitro Mammalian Cell Mutagenicity Test Systems undertook a literature search to identify new approaches for mutation detection. This review therefore focused on identifying new approaches for mutation detection that have the potential for use as a future genotoxicity screening tool. A comprehensive literature review identified genome-wide loss-of-function screening tools, next generation sequencing (NGS) mutation characterisation and fluorescence-based mutation detection methods as having significant promise as an emerging in vitro mammalian cell mutagenicity test system. Each of the technologies considered was assessed for its capacity to report on a wide array of heritable mutagenic changes, necessary to cover the full spectrum of genetic events imparted by substances with a broad range of modes of action. Of the technologies evaluated, NGS techniques exhibited the greatest advantages for use in a genotoxicity testing setting. However, it is important to note that the emerging techniques identified could not facilitate routine mutagenicity testing in their current format and require substantial additional optimisation and tailoring before they could be utilised as an in vitro mammalian cell mutagenicity test system. Additionally, new mammalian cell mutation test systems must be able to accurately and reliably detect and quantify rare events; hence any new system would require careful validation. Nevertheless, with further development emerging technologies such as NGS could become important in establishing more predictive and high-throughput regulatory hazard screening tools of the future.

In Vitro Mouse Lymphoma Cell (L5178Y Tk+/- -3.7.2.C) Forward Mutation Assay.

The in vitro mouse lymphoma cell assay (MLA) is one of the most widely practiced assays in genetic toxicology. MLA detects forward mutations at the thymidine kinase (Tk) locus of the L5178Y (Tk+/- -3.7.2C) cell line derived from a mouse thymic lymphoma. This assay is capable of detecting a wide range of genetic events including point mutations, deletions and multilocus, chromosomal rearrangements, mitotic recombination and nondisjunction. There are two equally accepted versions of the assay, one using soft agar cloning and the second method using liquid media cloning in 96-microwell plates. There are two morphologically distinct types of mutant colonies recovered in the MLA; small and large colony mutants. The induction of small colony mutants is associated with chemicals inducing gross chromosomal aberrations, whereas the induction of large mutant colonies is generally associated with chemicals inducing point mutations. The source and karyotype of the cell line as well as the culture conditions are important variables that could influence the assay performance. The assay when performed according to the standards recommended by the International Workshops on Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development Test Guideline 490 is capable of providing valuable genotoxicity hazard information as part of the overall safety assessment process of various classes of test substances.

Evaluation of the PIGRET assay in rats by single oral dosing with azidothymidine

In vivo phosphatidylinositol glycan, class A (Pig-a) gene mutation assay using peripheral blood is known to be a novel and useful tool to evaluate the mutagenicity of compounds. Recently, the rat PIGRET assay which is an improved method for measuring Pig-a mutant cells in reticulocytes with magnetic enrichment of CD71 positive cells has been developed. Several reports showed that the PIGRET assay could detect the increase of Pig-a mutant frequency earlier than the Pig-a assay in total red blood cells (RBC Pig-a assay). Therefore, as part of a collaborative study by the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society, the usefulness of the PIGRET assay in comparison to the RBC Pig-a assay has been assessed for 24 compounds with various mechanisms of action. In the present study, we performed the PIGRET assay and RBC Pig-a assay with a nucleoside analogue, azidothymidine (AZT), and compared the results in these assays. We administered a single dose of AZT to rats by oral gavage up to 2000mg/kg and examined Pig-a mutant frequencies at days 7, 14 and 28 by PIGRET and RBC Pig-a assays. No significant increases in mutant frequency were observed after administration of AZT in both the RBC Pig-a and PIGRET assays and comparable to the previous results of the International Workshop on Genotoxicity Testing (IWGT) workgroup. AZT has been thought to induce not only DNA chain termination as a pharmacological effect but also a large deletion on the genome DNA. The Pig-a assays may be less sensitive to compounds such as AZT which induce large deletions on the genome DNA.

Evaluation for a mutagenicity of aristolochic acid by Pig-a and PIGRET assays in rats

The Pig-a assay, which uses the endogenous phosphatidylinositol glycan, class A gene (Pig-a) as a reporter of mutation, has been developed as a method for evaluating in vivo mutagenicity. Pig-a gene mutation can be detected by identifying the presence of CD59, the glycosylphosphatidylinositol anchor protein, on the surface of erythrocytes (RBC Pig-a assay) and reticulocytes (PIGRET assay). The International Workshop on Genotoxicity Testing (IWGT) showed the usefulness of the RBC Pig-a assay through the evaluation of several compounds. Aristolochic acid (AA), one of the evaluated compounds in the IWGT workgroup, is a carcinogenic plant toxin that is a relatively strong gene mutagen both in vitro and in vivo, but a weak inducer of micronuclei in vivo. In the present study, we examined the mutagenicity of AA in the peripheral blood of rats treated orally with a single dose of AA using Pig-a assays. Furthermore, we evaluated the advantages of the PIGRET assay compared with the RBC Pig-a assay. The results showed that a statistically significant increase in mutant frequency of the Pig-a gene was detected at day 28 by the RBC Pig-a assay, and at days 7, 14 and 28 by the PIGRET assay. In addition, the mutant frequency by the PIGRET assay was higher than that by the RBC Pig-a assay. These results indicate that the mutagenicity of AA can be detected using the Pig-a assays, as reported by the IWGT, and the PIGRET assay can detect Pig-a mutants at an early time point compared with the RBC Pig-a assay.

Summary of major conclusions from the 6th International Workshop on Genotoxicity Testing (IWGT), Foz do Iguaçu, Brazil

a Novartis Institutes for BioMedical Research, Preclinical Safety, 4002 Basel, Switzerland b Public Interest Incorporated Foundation BioSafety Research Center, Iwata, Japan c National Institute of Health Sciences, Division of Genetics & Mutagenesis, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan d Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany e Exponent, Inc., Center for Toxicology & Mechanistic Biology, Midland, MI 48640, USA f Pharmaceutical Sciences, Roche Innovation Center, F Hoffmann-La Roche AG, CH 4070 Basel, Switzerland g Office of Science Policy, Office of Research and Development, U.S. Environmental Protection Agency, Room 51134 RRB, 1200 Pennsylvania Avenue NW (8104R), Washington DC 20460-0001, USA h Safety Research Laboratories, Mitsubishi Tanabe Pharma Co., Chiba 292-0818, Japan i Kirkland Consulting, PO Box 79, Tadcaster LS24 0AS, UK

Recommended protocols for the liver micronucleus test: Report of the IWGT working group

At the 6th International Workshop on Genotoxicity Testing (IWGT), the liver micronucleus test working group discussed practical aspects of the in vivo rodent liver micronucleus test (LMNT). The group members focused on the three methodologies currently used, i.e., a partial hepatectomy (PH) method, a juvenile/young rat (JR) method, and a repeated-dose (RD) method in adult rodents. Since the liver is the main organ that metabolizes chemicals, the LMNT is expected to detect clastogens, especially those that need metabolic activation in the liver, and aneugens. Based on current data the three methods seem to have a high sensitivity and specificity, but more data, especially on non-genotoxic but toxic substances, would be needed to fully evaluate the test performance. The three methods can be combined with the micronucleus test (MNT) using bone marrow (BM) and/or peripheral blood (PB). The ability of the PH method to detect both clastogens and aneugens has already been established, but the methodology is technically challenging. The JR method is relatively straightforward, but animal metabolism might not be fully comparable to adult animals, and data on aneugens are limited. These two methods also have the advantage of a short testing period. The RD method is also straightforward and can be integrated into repeated-dose (e.g. 2 or 4 weeks) toxicity studies, but again data on aneugens are limited. The working group concluded that the LMNT could be used as a second in vivo test when a relevant positive result in in vitro mammalian cell genotoxicity tests is noted (especially under the condition of metabolic activation), and a negative result is observed in the in vivo BM/PB-MNT. The group members discussed LMNT protocols and reached consensus about many aspects of test procedures. However, data gaps as mentioned above remain, and further data are needed to fully establish the LMNT protocol.

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.