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Opinion: regulatory genotoxicity: past, present and future

Opinion: regulatory genotoxicity: past, present and future I will reflect on the role of genotoxicity in the regulation of chemical safety, summarizing the past and current situ‑ ation, and giving personal views for the future. This includes how genotoxicity information has been, and is being, used in the evaluation of the safety of chemical substances including pharmaceuticals, pesticides, food additives and industrial chemicals before they are introduced into the market for sale. In Japan, the Industrial Safety and Health Act, enacted in 1972, assures workers’ safety by including safety assessment of chemicals to which workers may be exposed in the workplace. The law firstly included the bacterial gene mutation assay with rat liver microsome fraction (Ames test) for the evaluation of chemical mutagenicity to predict carcino‑ genic potential, which was the forerunner of requiring a genotoxicity test by law. Since then, genotoxicity, especially the Ames test and the in vitro chromosomal aberration test using cultured mammalian cells (especially Chinese hamster cells) have been incorporated into several laws to assess the safety of various chemicals. Many test systems for different endpoints have been developed, improved, and used in practice. The battery strategy, combining several test systems to detect as many genotoxic chemicals as possible, was implemented because no one test system can detect all genotoxic agents with different mechanisms of genetic damage. In general, the standard battery consists of the Ames test, in vitro chromosomal aberration test and the in vivo rodent erythrocyte micronucleus test as a representative in vivo assay. Many other test systems have been used for supplementary testing as well as for research studies. Important keywords for regulatory science include 1) guidelines, 2) Good Laboratory Practice, 3) evaluation and interpretation of test results. Here, I discuss on these key points, and give personal opinions for the future. Keywords: Genotoxicity, Regulatory science, Evaluation and interpretation, Statistical evaluation Introduction with the rat liver microsome fraction (Ames test) [1], was Regulatory genotoxicology is defined as genotoxic studies introduced into law: The Industrial Safety and Health to support regulatory decision-making related to chemi- Act, required mandatory testing when a new chemi- cal safety. I discuss briefly the necessity of guidelines, cal was proposed to be manufactured as part of safety Good Laboratory Practice (GLP), and more deeply the assessment especially for prediction of the carcinogenic evaluation and interpretation of genotoxic assay results potential of the chemical. Based on the good correla- for supporting rational judgement. In Japan, mutagenic- tion between chemical carcinogenicity and mutagenicity, ity testing started when the bacterial gene mutation assay the mutation assay had been expected to be a good bio- marker of potential carcinogenicity. Subsequently, chro- mosomal aberration and DNA damage were added as *Correspondence: makoto_hayashi@wine.ocn.ne.jp additional endpoints of genotoxicity and became widely makoto international consulting, 4‑23‑3‑1, Ebina, Kanagawa 243‑0431, used for genotoxic evaluation [2, 3]. Japan This manuscript is written for the proceeding of the key‑note lecture at Genotoxicity test systems started mainly with in  vitro the 50th anniversary meeting of Japanese Environmental Mutagen and microorganisms ( bacteria and yeast in/on agar in plates) Genome Society at Yokosuka, Kanagawa on November 1, 2021. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visithttp:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hayashi Genes and Environment (2022) 44:13 Page 2 of 6 and cultured mammalian cells, and in  vivo test systems chemical substances. The above body of testing guide - such as the rodent micronucleus test using bone marrow lines has become an indispensable part of the implemen- or blood cells were subsequently introduced [4, 5]. Sev- tation of safety tests. eral test systems were included the OECD test guidelines, of which many have been revised and some were deleted Evaluation and interpretation of genotoxicity data and archived separately. Genotoxicity information has been widely used for the safety risk assessment of chemicals together with gen- Past and present eral toxicity, carcinogenicity, reproductive toxicity, and In the first era, as I mentioned above, the Ames test was other endpoints. The different feature of genotoxicity thought to be a good tool for prediction of chemical car- from many other toxicities is visibility of toxicological cinogenicity. This assay was followed by the in vitro chro - phenomena. Although many genetic diseases and chro- mosomal aberration assay, using mainly Chinese hamster mosomal anomalies are well known, most of them are cultured cells to detect chromosomal aberrations as caused by heredity or other factors, e.g., aging, rather another major endpoint of mutagenicity. than from exposure to genotoxic chemicals. Genotoxic- The terms “genotoxicity” and “mutagenicity” have been ity may contribute to many other toxicity because it can not well defined and have been used interchangeably affect at early stage of the adverse outcome pathway of sometimes and defined differently at other times. Most various endpoints. Generally other toxicity is visible, we frequently, “mutagenicity” is meant to apply only to gene can see the outcomes of toxicity and also suffer from such mutation and chromosomal aberration while “genotoxic- diseases. While outcomes of genotoxic effects are gen - ity” has a wider coverage including DNA damage, sister- erally silent, such genotoxic damage can affect people chromatid exchange, and other genetic events in addition indirectly, for example they might cause cancer, which to mutagenicity [6]. Here I use mainly “genotoxicity” affects patients much later. Therefore, genotoxicity test - to refer to a wider range of meaning, while I would like ing, which can be engaged short-term and cheaply, was to use “mutagenicity” to restrict the meanings of gene designed to identify potential carcinogenic chemicals, mutation and chromosomal structural aberration. which is very time consuming and costly determine Many studies have been conducted to detect genotoxic experimentally using lifetime cancer bioassays or human chemicals in order to predict carcinogenicity. There - epidemiology. Following data accumulation and a greater fore, the sensitivity of the test system was considered understanding the mechanisms of carcinogenicity, it important in order to avoid false-negative results while became less simple to make a direct connection between false-positive results often received too little attention. genotoxicity and carcinogenicity. The use of genotoxicity The fair balancing of these two “errors” made difficult to data for predicting carcinogens was diminished but re- construct a rational battery system for the detection of directed to understanding mechanisms of carcinogenic- genotoxicity. ity and also for evaluating heritable adverse effects to the In the second era, attempts were made to standard- next generation. The term “MCR: Mutagenicity, Carci - ize the methods and established international and nogenicity, Reproductive toxicity” has been introduced domestic guidelines for assessing genotoxicity. This and become well known; it means these three endpoints included establishing OECD test guidelines as the inter- are related but are independent events. Genotoxicity is national standard for test protocols. Most of the voices important in regulatory science because it is often the demanded transparency and fairness when evaluat- key contributor to the understanding of carcinogenicity ing the test results, and we believed that the guidelines and other genetic diseases. It is also a key determinant were authoritative and well-constructed. Nevertheless, of whether the subsequent disease outcome behaves as a it is equally important to conduct the tests according to threshold event, with levels of exposure that do not lead the guidelines. The same discussion was held regarding to the disease. GLP, which was triggered by the identification of regula - tory studies at contract laboratories that were faked or Statistical evaluation of test data not clearly documented, and many countries, including Many researchers as well as regulatory people have used Japan, decided to introduce GLP’s for international har- statistical comparison tests for evaluating genotoxicity monization (for example; OECD GLP [7]). This resulted test results. Statistical tests give the probability of the in the Safety Guidelines established through the Interna- expected events occurring. Nevertheless, it is important tional Conferences on Harmonization (ICH) for human to emphasize that statistical tests are based on many use of pharmaceutical drugs [8–10]. These guidelines assumptions, for example, population distribution of the and GLP have played important roles producing test data event; null and alternative hypothesis; nominal probabil- of high quality for regulatory use to judge the safety of ity of errors (type I and type II); etc. Often, toxicologists Ha yashi Genes and Environment (2022) 44:13 Page 3 of 6 do not pay sufficient attention to such assumptions. In ated polychromatic erythrocytes. Another criterion the statistical comparison tests, the null hypothesis is may be based upon detection of a reproducible and that the data of the treatment group(s) are located inside statistically significant positive response for at least the data distribution of the negative control group within one of the test points” [12]. a pre-determined p-value (e.g., 0.05 or 0.01). In toxicolog- TG-474 adopted on 21st July 1997 said “There are ical studies, if the data is not met the null hypothesis then several criteria for determining a positive result, we call the result is positive, although it means only that such as a dose-related increase in the number of the data have not met the negative control distribution micronucleated cells or a clear increase in the num- under the pre-set condition (not negative). Nominal type ber of micronucleated cells in a single dose group at I and type II error rates should be fixed by experiment - a single sampling time. Biological relevance of the ers or sometimes by regulatory persons according to the results should be considered first. Statistical meth - purpose of the test. Generally, people use 0.05 (one event ods may be used as an aid in evaluating the test occurs among 20 trials) or 0.01 (one event occurs among results [9, 10]. Statistical significance should not be 100 trials) as the nominal type I error rate, however, such the only determining factor for a positive response. numbers have been based on the specialists’ intuition but Equivocal results should be clarified by further test - not been established on a scientific basis. We just think ing preferably using a modification of experimental one over 20 looks rare and one over 100 is a very rare conditions… A test substance for which the results case. Moreover, the type II error rate, which is detection do not meet the above criteria is considered non- power of the test, is often not taken into account. These mutagenic in this test” [13]. p-values should be determined in advance of designing TG-474 adopted on 29 July 2016 said “Providing the particular assay. For example, in the in vivo micronu- that all acceptability criteria are fulfilled, a test cleus assay it should be determined in advance how many chemical is considered clearly positive if: a) At least cells should be analyzed and how many animals should one of the treatment groups exhibits a statistically be assigned to each control and treatment group in order significant increase in the frequency of micronu - to achieve an optimal experimental condition [11]. It is cleated immature erythrocytes compared with the most important that the “positive” outcome is defined in concurrent negative control, b) i Th s increase is dose- advance. The Ames assay used doubling as the positive related at least at one sampling time when evalu- definition without any detailed discussion on this point, ated with an appropriate trend test, and c) Any of and regulation used the judgement on this criterion for a these results are outside the distribution of the his- long time. If we assume that a 10% increase from the neg- torical negative control data (e.g. Poisson-based ative control is positive, we must design the assay proto- 95% control limits). If only the highest dose is exam- col using more animals or more cells should be analyzed. ined at a particular sampling time, a test chemical is considered clearly positive if there is a statistically Biological relevance vs statistical significance significant increase compared with the concurrent Another important factor for evaluating data is biological negative control and the results are outside the dis- or toxicological relevance. This factor includes the dose– tribution of the historical negative control data (e.g. response relationship, the level of increase from the con- Poisson-based 95% control limits) [14]. Recommen- trol (concurrent/historical), and reproducibility. These dations for the most appropriate statistical methods factors are often set based on the knowledge and expe- can be found in the literature [15–18]. rience of experts and originally such intuitional factors There are differences among these versions on descrip - should be supported by statistics. We can understand and tion of data evaluation. The 1983 and the 2016 versions accept these approaches as a general idea, and therefore look rather similar and both suggested primarily sta- these intuitive guides appear less transparent and more tistical analyses for the data evaluation. The latest 2016 subjective than the outcomes of statistical tests. Here, I version provides a more precise description and rec- compare the descriptions of OECD test guidelines (as an ommends the appropriate statistical methods as refer- example referring TG-474, the in vivo micronucleus test) ences. In contrast, the 1997 version was different from chronologically. the others. It recommended that biological relevance of TG-474 adopted on 26 May 1983 said “Data should the results should be considered first. It noted that sta - be evaluated by using appropriate statistical meth- tistical methods may be used for helping or supporting ods…There are several criteria for determining a pos - of evaluating the test results. Moreover, it concluded that itive result, one of which is a statistically significant statistical significance should not be the only determin - dose-related increase in the number of micronucle- ing factor for a positive response. I, personally, prefer Hayashi Genes and Environment (2022) 44:13 Page 4 of 6 the 1997 version to others because I believe biological potential carcinogenic activity. The regulatory bodies relevance should be considered first using, if possible, a also tended not to accept the release of such Ames posi- graphic image on which all data are plotted and under- tive chemicals into the market, especially chemical sub- stood at a glance. If we understand statistics well and stances for direct human use. Then, after a lot of data design the experiment properly, and also apply the proper were accumulated and showed that genotoxicity was statistical test method, then, of course, we can appreci- not a prerequisite for all carcinogenicity, some chemi- ate the outcomes of statistical test to endorse biological cals without mutagenic potential were recognized to be relevancy. carcinogenic and, vice versa, some Ames test positive chemicals did not show carcinogenic potential. The Ames test has been most popular and is in wide - Qualitative evaluation and quantitative evaluation spread routine use. The in  vitro chromosomal aber - At the early stage, even now, for establishing mutagenic- ration test is also frequently performed, especially in ity tests, for example, the Ames test and in vitro chromo- Japan using Chinese Hamster Lung cells. The in  vitro somal aberration test using Chinese hamster lung cells, chromosomal aberration test has been shown to give especially in Japan, we used simple criteria for calling the positive results by some non-specific factors, such as tests. We use a doubling of number of revertant colonies osmolality and pH, that is by un-physiological culture for the Ames test and equal to or more than 10% of cells conditions. Such positive results are not considered analyzed having chromosomal aberrations. These criteria to be a genotoxic event because such extreme experi- have been accepted by regulatory bodies as the proper mental conditions are unrealistic and not expected in judgement without any trouble for a long time. humans in  vivo. Some in  vitro chromosomal aberra- Genotoxicity has been evaluated qualitatively but not tion positive chemicals did not induce micronuclei as quantitatively in many countries. In Japan, a little differ - an indicator of chromosomal aberrations in erythro- ently from other countries, we introduced semi-quan- poietic cells in vivo. Hence the positive outcomes of the titative evaluation for the genotoxicity judgement for in  vitro chromosomal aberration test are considered regulatory purposes. For example, we have used a dou- sometimes ambiguous for genotoxicity evaluation. As bling as a criterion for the Ames test and in addition we an example, the genotoxicity and carcinogenicity of have used the mutation rate defined as number of rever - pesticides evaluated from 2012 to 2016 by the Agri- tant (rev) colonies per 1  mg (rev/mg) of a test material. cultural Chemicals Expert Committee of the Cabinet We regard an agent as a strong mutagen if the rate is Office Food Safety Commission in Japan was analyzed more than 1000 rev/mg; the Industrial Safety and Health [19]. The study aimed to see how much the in  vitro Act requires an additional assay, for example, an in vitro chromosomal aberration test contributed to the final chromosomal aberration test. In the Chemical Substance safety judgment of pesticide. A total of 183 chemicals Control law, a chemical is classified as a designated were evaluated during this 5-year period and all have chemical when showing more than 1000 rev/mg in the been approved as pesticides. More than 1/4 chemi- Ames test (nowadays, the law has been revised and the cals were positive in the in  vitro chromosomal aberra- criterion is not used). In the in vitro chromosomal aber- tion test. And, as expected, there were few chemicals ration assay, we have introduced the D value that was that showed a clear Ames test positive result because defined as the predicted concentration (µg/mL) at which developers might not put in applications for Ames test 20% of analyzed metaphases cells had one or more chro- positive chemicals. Moreover, surprisingly, although it mosomal aberration. In the Chemical Substance Con- was not the main theme of this manuscript, nearly half trol law mentioned above, the chemical was classified as were positive in the long term carcinogenesis assay, in a designated chemical when the D value was 0.01  mg/ at least one species and one target organ. mL or less (as same as the Ames test). Moreover, we fre- The most important role for genotoxicity information quently discussed using the D as an indicator of poten- in chemical safety assessment has been the determina- tial clastogenicity. tion of whether or not genotoxicity is a key initiating event in carcinogenesis. A carcinogen that is genotoxic, Interpretation of chemical genotoxicity especially at the carcinogenesis target site, is regarded As mentioned before, we regarded genotoxic chemicals as a “genotoxic carcinogen” that has no threshold [20, as potential carcinogens based on the good correlation 21]. This means under the current assessment strategy, between genotoxic and carcinogenic outcomes at the genotoxic carcinogenicity is a key factor for chemical first era. Pharmaceutical, agricultural, and other chemi - safety to human beings. It indicates, in this sense, the cal developers stopped developing their chemicals genotoxicity information plays an important role for when the Ames test was positive, due to concern about the final regulatory judgement. Ha yashi Genes and Environment (2022) 44:13 Page 5 of 6 Future—Conclusion and proposals and unrealistic high dose-levels in in vivo may be mean- We have to think more deeply about the final aim of ingless for extrapolation to the actual safety risk. I have chemical genotoxicity information in human health risk to note this situation is not only for genotoxicity but also assessment. If genotoxicity is exclusively important for for other toxicology endpoints including carcinogenicity. identifying genotoxic carcinogens that have no thresh- Sometimes we see TV news on the serious adverse effects old and not acceptable putting into market, then geno- of carcinogens to humans. However, almost all these were toxic assays are not needed for every chemicals, but only the result of accidents, improper uses/handling, or expo- for carcinogenic chemicals. It is, however, true that the sures to naturally existing local contaminants like some current long-term bioassay for carcinogenicity is time heavy metals. Most of these occasions can be explained consuming and costly and not practical to perform on by the extremely high exposure levels in comparison to all chemicals being developed. Nevertheless, genotoxic- the actual situation. ity information alone does not determine human risk. Considering all, I would like to propose to change the For example, if a chemical was negative in the long-term strategy for chemical safety assessment from the cur- carcinogenicity bioassay, showed a no observed-adverse- rent system that requires all information should be gath- effect level greater than 1000 mg/kg/day, no reproductive ered according to the guideline before application to the adverse effect but showed positive result in the Ames test regulatory body and then the safety assessment start and the in  vitro chromosomal aberration test, then how using the full data set. It may be reasonable if we have shall we treat it for human safety risk assessment? For the enough resource, time and budget, but it is inefficient. final decision, we have to take the expected benefit of the ILSI/HESI proposed “Risk 21” [22–24], and several years chemical for our lives into account to balance risk and later another report has been published [25]. The Risk 21 benefit. is based on a step by step approach for chemical safety The most important feature of genotoxicity is the evaluation. Firstly, “Problem formulation” is followed by absence of a threshold. This is the reason why genotox - “Estimation of human exposure”, “Estimate of human icity information is considered important because it is toxicity” and then evaluating the “Intersection of expo- necessary to identify genotoxic carcinogens. Regulatory sure and toxicity on a RISK21 matrix”. Then a judgement people tend to believe that genotoxicity does not have is made whether there is sufficient information to make threshold. Nevertheless, data have been accumulated a decision. If “YES”, then we make a conclusion but if that show thresholds, at least apparent or biological “NO” we start again from the “Problem formulation” for thresholds even for direct DNA acting genotoxic chemi- a second cycle based on the results of the first one. We cals. If we accept the existence of thresholds, then the repeat the cycle until we have enough confidence to make level of exposure to humans becomes critical for assess- a decision. This is a good idea for the evolution of the ment of safety risk. I believe it is time to stop discussion whole strategy. We should make decisions based on the about existence of thresholds and to accept practical minimum essential information/data we need, leading to thresholds and to start considering levels of exposure for the most efficient and accurate assessment. Nevertheless, genotoxicity. we must consider that the assessment should be accept- It is difficult to estimate chemical exposure to humans able for regulatory purposes. Who, when, and how we except for pharmaceutical drugs for human use. In engage each step is a key issue, so that sound conclusions human use pharmaceuticals drugs, we know the expo- are based on the essential facts that determine safety and sure level and benefit of the treatment, thus we can con - risk. Such a new approach should be used not only for trol the risk–benefit balance. Nevertheless, for example, genotoxicity, but also for all kinds of expected toxicities. in the case of industrial chemicals it is difficult to make In other words, we have to make a rational assessment of human exposure assessments, because we generally do risk based on relevant and sound information to avoid not know the volume of chemical that was released into over- or under-estimation of risk. our environment and how much reaches us through Acknowledgements water, air and through other routes. And it is more dif- The author would like to thank Dr. James T. MacGregor for his critical review ficult to quantify the benefit of the chemical at a given and kind suggestions and also to thank Dr. David P. Lovell for his kind review especially the part of statistical issues and invaluable comments and sugges‑ level of exposure. Because the actual quantity of the tions. The author also like to thank Dr. Masami Yamada for her giving me an chemical we intake into our body is very small, I am curi- opportunity to deliver the key‑note talk. ous whether we can assess safety of the chemical using Authors’ contributions existing test systems which evaluate their toxic effects The author reads and approved the final manuscript. using up to very high exposure levels and extrapolation to the actual human exposure levels. Outcomes of tests Funding Not applicable. evaluated up to extremely high concentrations in in vitro Hayashi Genes and Environment (2022) 44:13 Page 6 of 6 Availability of data and materials Mutagenicity Testing, Report, Part III. 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Opinion: regulatory genotoxicity: past, present and future

Genes and Environment , Volume 44 (1) – Apr 22, 2022

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Abstract

I will reflect on the role of genotoxicity in the regulation of chemical safety, summarizing the past and current situ‑ ation, and giving personal views for the future. This includes how genotoxicity information has been, and is being, used in the evaluation of the safety of chemical substances including pharmaceuticals, pesticides, food additives and industrial chemicals before they are introduced into the market for sale. In Japan, the Industrial Safety and Health Act, enacted in 1972, assures workers’ safety by including safety assessment of chemicals to which workers may be exposed in the workplace. The law firstly included the bacterial gene mutation assay with rat liver microsome fraction (Ames test) for the evaluation of chemical mutagenicity to predict carcino‑ genic potential, which was the forerunner of requiring a genotoxicity test by law. Since then, genotoxicity, especially the Ames test and the in vitro chromosomal aberration test using cultured mammalian cells (especially Chinese hamster cells) have been incorporated into several laws to assess the safety of various chemicals. Many test systems for different endpoints have been developed, improved, and used in practice. The battery strategy, combining several test systems to detect as many genotoxic chemicals as possible, was implemented because no one test system can detect all genotoxic agents with different mechanisms of genetic damage. In general, the standard battery consists of the Ames test, in vitro chromosomal aberration test and the in vivo rodent erythrocyte micronucleus test as a representative in vivo assay. Many other test systems have been used for supplementary testing as well as for research studies. Important keywords for regulatory science include 1) guidelines, 2) Good Laboratory Practice, 3) evaluation and interpretation of test results. Here, I discuss on these key points, and give personal opinions for the future. Keywords: Genotoxicity, Regulatory science, Evaluation and interpretation, Statistical evaluation Introduction with the rat liver microsome fraction (Ames test) [1], was Regulatory genotoxicology is defined as genotoxic studies introduced into law: The Industrial Safety and Health to support regulatory decision-making related to chemi- Act, required mandatory testing when a new chemi- cal safety. I discuss briefly the necessity of guidelines, cal was proposed to be manufactured as part of safety Good Laboratory Practice (GLP), and more deeply the assessment especially for prediction of the carcinogenic evaluation and interpretation of genotoxic assay results potential of the chemical. Based on the good correla- for supporting rational judgement. In Japan, mutagenic- tion between chemical carcinogenicity and mutagenicity, ity testing started when the bacterial gene mutation assay the mutation assay had been expected to be a good bio- marker of potential carcinogenicity. Subsequently, chro- mosomal aberration and DNA damage were added as *Correspondence: makoto_hayashi@wine.ocn.ne.jp additional endpoints of genotoxicity and became widely makoto international consulting, 4‑23‑3‑1, Ebina, Kanagawa 243‑0431, used for genotoxic evaluation [2, 3]. Japan This manuscript is written for the proceeding of the key‑note lecture at Genotoxicity test systems started mainly with in  vitro the 50th anniversary meeting of Japanese Environmental Mutagen and microorganisms ( bacteria and yeast in/on agar in plates) Genome Society at Yokosuka, Kanagawa on November 1, 2021. © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hayashi Genes and Environment (2022) 44:13 Page 2 of 6 and cultured mammalian cells, and in  vivo test systems chemical substances. The above body of testing guide - such as the rodent micronucleus test using bone marrow lines has become an indispensable part of the implemen- or blood cells were subsequently introduced [4, 5]. Sev- tation of safety tests. eral test systems were included the OECD test guidelines, of which many have been revised and some were deleted Evaluation and interpretation of genotoxicity data and archived separately. Genotoxicity information has been widely used for the safety risk assessment of chemicals together with gen- Past and present eral toxicity, carcinogenicity, reproductive toxicity, and In the first era, as I mentioned above, the Ames test was other endpoints. The different feature of genotoxicity thought to be a good tool for prediction of chemical car- from many other toxicities is visibility of toxicological cinogenicity. This assay was followed by the in vitro chro - phenomena. Although many genetic diseases and chro- mosomal aberration assay, using mainly Chinese hamster mosomal anomalies are well known, most of them are cultured cells to detect chromosomal aberrations as caused by heredity or other factors, e.g., aging, rather another major endpoint of mutagenicity. than from exposure to genotoxic chemicals. Genotoxic- The terms “genotoxicity” and “mutagenicity” have been ity may contribute to many other toxicity because it can not well defined and have been used interchangeably affect at early stage of the adverse outcome pathway of sometimes and defined differently at other times. Most various endpoints. Generally other toxicity is visible, we frequently, “mutagenicity” is meant to apply only to gene can see the outcomes of toxicity and also suffer from such mutation and chromosomal aberration while “genotoxic- diseases. While outcomes of genotoxic effects are gen - ity” has a wider coverage including DNA damage, sister- erally silent, such genotoxic damage can affect people chromatid exchange, and other genetic events in addition indirectly, for example they might cause cancer, which to mutagenicity [6]. Here I use mainly “genotoxicity” affects patients much later. Therefore, genotoxicity test - to refer to a wider range of meaning, while I would like ing, which can be engaged short-term and cheaply, was to use “mutagenicity” to restrict the meanings of gene designed to identify potential carcinogenic chemicals, mutation and chromosomal structural aberration. which is very time consuming and costly determine Many studies have been conducted to detect genotoxic experimentally using lifetime cancer bioassays or human chemicals in order to predict carcinogenicity. There - epidemiology. Following data accumulation and a greater fore, the sensitivity of the test system was considered understanding the mechanisms of carcinogenicity, it important in order to avoid false-negative results while became less simple to make a direct connection between false-positive results often received too little attention. genotoxicity and carcinogenicity. The use of genotoxicity The fair balancing of these two “errors” made difficult to data for predicting carcinogens was diminished but re- construct a rational battery system for the detection of directed to understanding mechanisms of carcinogenic- genotoxicity. ity and also for evaluating heritable adverse effects to the In the second era, attempts were made to standard- next generation. The term “MCR: Mutagenicity, Carci - ize the methods and established international and nogenicity, Reproductive toxicity” has been introduced domestic guidelines for assessing genotoxicity. This and become well known; it means these three endpoints included establishing OECD test guidelines as the inter- are related but are independent events. Genotoxicity is national standard for test protocols. Most of the voices important in regulatory science because it is often the demanded transparency and fairness when evaluat- key contributor to the understanding of carcinogenicity ing the test results, and we believed that the guidelines and other genetic diseases. It is also a key determinant were authoritative and well-constructed. Nevertheless, of whether the subsequent disease outcome behaves as a it is equally important to conduct the tests according to threshold event, with levels of exposure that do not lead the guidelines. The same discussion was held regarding to the disease. GLP, which was triggered by the identification of regula - tory studies at contract laboratories that were faked or Statistical evaluation of test data not clearly documented, and many countries, including Many researchers as well as regulatory people have used Japan, decided to introduce GLP’s for international har- statistical comparison tests for evaluating genotoxicity monization (for example; OECD GLP [7]). This resulted test results. Statistical tests give the probability of the in the Safety Guidelines established through the Interna- expected events occurring. Nevertheless, it is important tional Conferences on Harmonization (ICH) for human to emphasize that statistical tests are based on many use of pharmaceutical drugs [8–10]. These guidelines assumptions, for example, population distribution of the and GLP have played important roles producing test data event; null and alternative hypothesis; nominal probabil- of high quality for regulatory use to judge the safety of ity of errors (type I and type II); etc. Often, toxicologists Ha yashi Genes and Environment (2022) 44:13 Page 3 of 6 do not pay sufficient attention to such assumptions. In ated polychromatic erythrocytes. Another criterion the statistical comparison tests, the null hypothesis is may be based upon detection of a reproducible and that the data of the treatment group(s) are located inside statistically significant positive response for at least the data distribution of the negative control group within one of the test points” [12]. a pre-determined p-value (e.g., 0.05 or 0.01). In toxicolog- TG-474 adopted on 21st July 1997 said “There are ical studies, if the data is not met the null hypothesis then several criteria for determining a positive result, we call the result is positive, although it means only that such as a dose-related increase in the number of the data have not met the negative control distribution micronucleated cells or a clear increase in the num- under the pre-set condition (not negative). Nominal type ber of micronucleated cells in a single dose group at I and type II error rates should be fixed by experiment - a single sampling time. Biological relevance of the ers or sometimes by regulatory persons according to the results should be considered first. Statistical meth - purpose of the test. Generally, people use 0.05 (one event ods may be used as an aid in evaluating the test occurs among 20 trials) or 0.01 (one event occurs among results [9, 10]. Statistical significance should not be 100 trials) as the nominal type I error rate, however, such the only determining factor for a positive response. numbers have been based on the specialists’ intuition but Equivocal results should be clarified by further test - not been established on a scientific basis. We just think ing preferably using a modification of experimental one over 20 looks rare and one over 100 is a very rare conditions… A test substance for which the results case. Moreover, the type II error rate, which is detection do not meet the above criteria is considered non- power of the test, is often not taken into account. These mutagenic in this test” [13]. p-values should be determined in advance of designing TG-474 adopted on 29 July 2016 said “Providing the particular assay. For example, in the in vivo micronu- that all acceptability criteria are fulfilled, a test cleus assay it should be determined in advance how many chemical is considered clearly positive if: a) At least cells should be analyzed and how many animals should one of the treatment groups exhibits a statistically be assigned to each control and treatment group in order significant increase in the frequency of micronu - to achieve an optimal experimental condition [11]. It is cleated immature erythrocytes compared with the most important that the “positive” outcome is defined in concurrent negative control, b) i Th s increase is dose- advance. The Ames assay used doubling as the positive related at least at one sampling time when evalu- definition without any detailed discussion on this point, ated with an appropriate trend test, and c) Any of and regulation used the judgement on this criterion for a these results are outside the distribution of the his- long time. If we assume that a 10% increase from the neg- torical negative control data (e.g. Poisson-based ative control is positive, we must design the assay proto- 95% control limits). If only the highest dose is exam- col using more animals or more cells should be analyzed. ined at a particular sampling time, a test chemical is considered clearly positive if there is a statistically Biological relevance vs statistical significance significant increase compared with the concurrent Another important factor for evaluating data is biological negative control and the results are outside the dis- or toxicological relevance. This factor includes the dose– tribution of the historical negative control data (e.g. response relationship, the level of increase from the con- Poisson-based 95% control limits) [14]. Recommen- trol (concurrent/historical), and reproducibility. These dations for the most appropriate statistical methods factors are often set based on the knowledge and expe- can be found in the literature [15–18]. rience of experts and originally such intuitional factors There are differences among these versions on descrip - should be supported by statistics. We can understand and tion of data evaluation. The 1983 and the 2016 versions accept these approaches as a general idea, and therefore look rather similar and both suggested primarily sta- these intuitive guides appear less transparent and more tistical analyses for the data evaluation. The latest 2016 subjective than the outcomes of statistical tests. Here, I version provides a more precise description and rec- compare the descriptions of OECD test guidelines (as an ommends the appropriate statistical methods as refer- example referring TG-474, the in vivo micronucleus test) ences. In contrast, the 1997 version was different from chronologically. the others. It recommended that biological relevance of TG-474 adopted on 26 May 1983 said “Data should the results should be considered first. It noted that sta - be evaluated by using appropriate statistical meth- tistical methods may be used for helping or supporting ods…There are several criteria for determining a pos - of evaluating the test results. Moreover, it concluded that itive result, one of which is a statistically significant statistical significance should not be the only determin - dose-related increase in the number of micronucle- ing factor for a positive response. I, personally, prefer Hayashi Genes and Environment (2022) 44:13 Page 4 of 6 the 1997 version to others because I believe biological potential carcinogenic activity. The regulatory bodies relevance should be considered first using, if possible, a also tended not to accept the release of such Ames posi- graphic image on which all data are plotted and under- tive chemicals into the market, especially chemical sub- stood at a glance. If we understand statistics well and stances for direct human use. Then, after a lot of data design the experiment properly, and also apply the proper were accumulated and showed that genotoxicity was statistical test method, then, of course, we can appreci- not a prerequisite for all carcinogenicity, some chemi- ate the outcomes of statistical test to endorse biological cals without mutagenic potential were recognized to be relevancy. carcinogenic and, vice versa, some Ames test positive chemicals did not show carcinogenic potential. The Ames test has been most popular and is in wide - Qualitative evaluation and quantitative evaluation spread routine use. The in  vitro chromosomal aber - At the early stage, even now, for establishing mutagenic- ration test is also frequently performed, especially in ity tests, for example, the Ames test and in vitro chromo- Japan using Chinese Hamster Lung cells. The in  vitro somal aberration test using Chinese hamster lung cells, chromosomal aberration test has been shown to give especially in Japan, we used simple criteria for calling the positive results by some non-specific factors, such as tests. We use a doubling of number of revertant colonies osmolality and pH, that is by un-physiological culture for the Ames test and equal to or more than 10% of cells conditions. Such positive results are not considered analyzed having chromosomal aberrations. These criteria to be a genotoxic event because such extreme experi- have been accepted by regulatory bodies as the proper mental conditions are unrealistic and not expected in judgement without any trouble for a long time. humans in  vivo. Some in  vitro chromosomal aberra- Genotoxicity has been evaluated qualitatively but not tion positive chemicals did not induce micronuclei as quantitatively in many countries. In Japan, a little differ - an indicator of chromosomal aberrations in erythro- ently from other countries, we introduced semi-quan- poietic cells in vivo. Hence the positive outcomes of the titative evaluation for the genotoxicity judgement for in  vitro chromosomal aberration test are considered regulatory purposes. For example, we have used a dou- sometimes ambiguous for genotoxicity evaluation. As bling as a criterion for the Ames test and in addition we an example, the genotoxicity and carcinogenicity of have used the mutation rate defined as number of rever - pesticides evaluated from 2012 to 2016 by the Agri- tant (rev) colonies per 1  mg (rev/mg) of a test material. cultural Chemicals Expert Committee of the Cabinet We regard an agent as a strong mutagen if the rate is Office Food Safety Commission in Japan was analyzed more than 1000 rev/mg; the Industrial Safety and Health [19]. The study aimed to see how much the in  vitro Act requires an additional assay, for example, an in vitro chromosomal aberration test contributed to the final chromosomal aberration test. In the Chemical Substance safety judgment of pesticide. A total of 183 chemicals Control law, a chemical is classified as a designated were evaluated during this 5-year period and all have chemical when showing more than 1000 rev/mg in the been approved as pesticides. More than 1/4 chemi- Ames test (nowadays, the law has been revised and the cals were positive in the in  vitro chromosomal aberra- criterion is not used). In the in vitro chromosomal aber- tion test. And, as expected, there were few chemicals ration assay, we have introduced the D value that was that showed a clear Ames test positive result because defined as the predicted concentration (µg/mL) at which developers might not put in applications for Ames test 20% of analyzed metaphases cells had one or more chro- positive chemicals. Moreover, surprisingly, although it mosomal aberration. In the Chemical Substance Con- was not the main theme of this manuscript, nearly half trol law mentioned above, the chemical was classified as were positive in the long term carcinogenesis assay, in a designated chemical when the D value was 0.01  mg/ at least one species and one target organ. mL or less (as same as the Ames test). Moreover, we fre- The most important role for genotoxicity information quently discussed using the D as an indicator of poten- in chemical safety assessment has been the determina- tial clastogenicity. tion of whether or not genotoxicity is a key initiating event in carcinogenesis. A carcinogen that is genotoxic, Interpretation of chemical genotoxicity especially at the carcinogenesis target site, is regarded As mentioned before, we regarded genotoxic chemicals as a “genotoxic carcinogen” that has no threshold [20, as potential carcinogens based on the good correlation 21]. This means under the current assessment strategy, between genotoxic and carcinogenic outcomes at the genotoxic carcinogenicity is a key factor for chemical first era. Pharmaceutical, agricultural, and other chemi - safety to human beings. It indicates, in this sense, the cal developers stopped developing their chemicals genotoxicity information plays an important role for when the Ames test was positive, due to concern about the final regulatory judgement. Ha yashi Genes and Environment (2022) 44:13 Page 5 of 6 Future—Conclusion and proposals and unrealistic high dose-levels in in vivo may be mean- We have to think more deeply about the final aim of ingless for extrapolation to the actual safety risk. I have chemical genotoxicity information in human health risk to note this situation is not only for genotoxicity but also assessment. If genotoxicity is exclusively important for for other toxicology endpoints including carcinogenicity. identifying genotoxic carcinogens that have no thresh- Sometimes we see TV news on the serious adverse effects old and not acceptable putting into market, then geno- of carcinogens to humans. However, almost all these were toxic assays are not needed for every chemicals, but only the result of accidents, improper uses/handling, or expo- for carcinogenic chemicals. It is, however, true that the sures to naturally existing local contaminants like some current long-term bioassay for carcinogenicity is time heavy metals. Most of these occasions can be explained consuming and costly and not practical to perform on by the extremely high exposure levels in comparison to all chemicals being developed. Nevertheless, genotoxic- the actual situation. ity information alone does not determine human risk. Considering all, I would like to propose to change the For example, if a chemical was negative in the long-term strategy for chemical safety assessment from the cur- carcinogenicity bioassay, showed a no observed-adverse- rent system that requires all information should be gath- effect level greater than 1000 mg/kg/day, no reproductive ered according to the guideline before application to the adverse effect but showed positive result in the Ames test regulatory body and then the safety assessment start and the in  vitro chromosomal aberration test, then how using the full data set. It may be reasonable if we have shall we treat it for human safety risk assessment? For the enough resource, time and budget, but it is inefficient. final decision, we have to take the expected benefit of the ILSI/HESI proposed “Risk 21” [22–24], and several years chemical for our lives into account to balance risk and later another report has been published [25]. The Risk 21 benefit. is based on a step by step approach for chemical safety The most important feature of genotoxicity is the evaluation. Firstly, “Problem formulation” is followed by absence of a threshold. This is the reason why genotox - “Estimation of human exposure”, “Estimate of human icity information is considered important because it is toxicity” and then evaluating the “Intersection of expo- necessary to identify genotoxic carcinogens. Regulatory sure and toxicity on a RISK21 matrix”. Then a judgement people tend to believe that genotoxicity does not have is made whether there is sufficient information to make threshold. Nevertheless, data have been accumulated a decision. If “YES”, then we make a conclusion but if that show thresholds, at least apparent or biological “NO” we start again from the “Problem formulation” for thresholds even for direct DNA acting genotoxic chemi- a second cycle based on the results of the first one. We cals. If we accept the existence of thresholds, then the repeat the cycle until we have enough confidence to make level of exposure to humans becomes critical for assess- a decision. This is a good idea for the evolution of the ment of safety risk. I believe it is time to stop discussion whole strategy. We should make decisions based on the about existence of thresholds and to accept practical minimum essential information/data we need, leading to thresholds and to start considering levels of exposure for the most efficient and accurate assessment. Nevertheless, genotoxicity. we must consider that the assessment should be accept- It is difficult to estimate chemical exposure to humans able for regulatory purposes. Who, when, and how we except for pharmaceutical drugs for human use. In engage each step is a key issue, so that sound conclusions human use pharmaceuticals drugs, we know the expo- are based on the essential facts that determine safety and sure level and benefit of the treatment, thus we can con - risk. Such a new approach should be used not only for trol the risk–benefit balance. Nevertheless, for example, genotoxicity, but also for all kinds of expected toxicities. in the case of industrial chemicals it is difficult to make In other words, we have to make a rational assessment of human exposure assessments, because we generally do risk based on relevant and sound information to avoid not know the volume of chemical that was released into over- or under-estimation of risk. our environment and how much reaches us through Acknowledgements water, air and through other routes. And it is more dif- The author would like to thank Dr. James T. MacGregor for his critical review ficult to quantify the benefit of the chemical at a given and kind suggestions and also to thank Dr. David P. Lovell for his kind review especially the part of statistical issues and invaluable comments and sugges‑ level of exposure. Because the actual quantity of the tions. The author also like to thank Dr. Masami Yamada for her giving me an chemical we intake into our body is very small, I am curi- opportunity to deliver the key‑note talk. ous whether we can assess safety of the chemical using Authors’ contributions existing test systems which evaluate their toxic effects The author reads and approved the final manuscript. using up to very high exposure levels and extrapolation to the actual human exposure levels. Outcomes of tests Funding Not applicable. evaluated up to extremely high concentrations in in vitro Hayashi Genes and Environment (2022) 44:13 Page 6 of 6 Availability of data and materials Mutagenicity Testing, Report, Part III. 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Journal

Genes and EnvironmentSpringer Journals

Published: Apr 22, 2022

Keywords: Genotoxicity; Regulatory science; Evaluation and interpretation; Statistical evaluation

References