The alkaline (pH>13) SCG assay, with post-lysis treatment of DNA with proteinase K (PK), to discriminate between DNA-protein and DNA-DNA crosslinks was evaluated using mouse lymphoma L5178Y tk^+/- cells exposed in vitro to formaldehyde or cisplatinum. Formaldehyde specifically induces DNA-protein crosslinks, whereas cisplatinum induces inter- and intra-strand DNA crosslinks. In the absence of treatment with PK after lysis, formaldehyde induced a dose-dependent significant decrease in DNA migration. The use of PK after lysis to remove residual protein prevented the reduction in DNA migration. In contrast, cisplatinum induced a decrease in tail moment, with and without PK treatment after lysis. These results indicate that post-lysis treatment with PK can be expected as a supplementary method in the SCG assay to discriminate between DNA-protein and DNA-DNA crosslinks.

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Global new perspectives on genotoxicity, i.e., threshold and germ cell mutagenicity, are summarized. On the aspect of threshold, proposal of a flow scheme toward risk assessment and standard setting for chemical carcinogens from European Academy, guideline on the limits of genotoxic impurities in pharmaceutical from the European Medicines Agency, and the concept of thresholds of toxicological concern (TTC) are introduced. On germ cell mutagenicity, health hazard classification criteria by a system of Globally Harmonized System of Classification and Labeling of Chemicals (GHS) and examples of classification by EU or Germany MAK Commission are also explained. These give major impacts to genotoxicity evaluation, risk assessment and hazard classification of chemicals.

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This paper discusses, from statistical viewpoint, about the methodology to examine whether substances have threshold on tumorgenecity or genotoxicity based on experiment data. When we try to discriminate models with threshold from those without threshold through in vivo experiment, the necessary sample size n will be too huge to be practical. In vitro assays seem to be more promising. Current methodologies utilized in in vitro assays, however, are not suitable for our purpose because they are involved in the determination of the positivity/negativity of test chemicals, while the methodologies we require are those for estimating dose-response relationships. Collaborative studies among toxicologists and statisticians are desirable to establish reasonable methodologies.

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The concept of a “biological threshold” is attracting interest as an evaluation criterion for the mutagenic activity of DNA-targeting mutagens. In this context, the concept is defined as “a concentration of a chemical which does not produce any damage through its inability to perform the necessary biochemical reactions, even though present at the target in finite amount”. To clarify whether this criterion is indeed applicable to DNA-targeting mutagens, we re-evaluated the reverse mutation assay data using DNA repair-deficient bacterial strains, such as S. typhimurium strains lacking the O⁶-methylguanine DNA methyltransferase genes (ada_ST and ogt_ST), the nucleotide excision repair gene (uvrB) or the 8-hydroxyguanine DNA glycosylase gene (mutM_ST), and E. coli strains lacking the nucleotide excision repair gene (uvrA). Mutagenic responses of 20 test chemicals including alkylating and non-alkylating agents were compared between the repair-deficient and their wild-type strains.
All the alkylating agents, such as MNNG, ENNG, EMS, ENU, DMN and DEN, exhibited more sensible mutagenic responses in strains YG7108 (Δada_ST, Δogt_ST) and YG7113 (same as YG7108 but containing the plasmid pKM101) than in the parental strains TA1535 and TA100 (same as TA1535 but containing the pKM101), respectively. Upon applying MNNG, YG7108 showed about 2-100 fold increase in the number of His⁺ revertants above the spontaneous level over the range of 0.00025-0.25 μg/plate, whereas TA1535 did not show any significant increase in the number of His⁺ revertants over the same dose range. On TA1535, an increasing tendency of the number of revertants was observed at 0.5 μg/plate or above. This indicates an approximate 2,000-fold difference at the mutagenic concentration level between the wild-type and the repairdeficient strains. Other alkylating agents also showed significant differences in mutagenic responses between YG7108 and TA1535, or between YG7113 and TA100 respectively, with some variations among test chemicals. On the other hand, non-alkylating agents, such as 4-NQO, AF-2, 2-NF and MX, did not show any differences in the dose-response relationships between YG7113 and TA100. When non-alkylating agents, such as 4-NQO, 2-NF and MX were applied to TA1535 (ΔuvrB), TA1538 (ΔuvrB) and WP2uvrA (ΔuvrA), clearly different mutagenic responses, i.e. about 30- to 60-fold, were observed between the repair-deficient and the parental strains (TA1975, TA1978 and WP2, respectively). 4-NQO showed different mutagenic responses between YG3002 (ΔmutM_ST) and TA1975 (about 10-fold), though the application of other oxidative agents such as hydrogen peroxide resulted in less than 10-fold differences. The present results indicate that the wild-type strains having normal repair capacity show no gene mutation induction at the concentrations at which gene mutations are clearly induced in the repair-deficient strains through DNA damage. Thus, the present results suggest the existence of a “biological threshold” below which no mutagenic response is induced by DNA-targeting mutagenic substances.

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Until recently it has been generally considered that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged with regard to assessment of cancer risk to humans. In the present study we show that food-related genotoxic hepatocarcinogens, heterocyclic amines and N-nitroso compounds at low doses do not induce preneoplastic lesions and cancer-related markers in rat medium-term carcinogenicity bioassay. The results imply existence of a threshold, at least practical one, for carcinogenicities of genotoxic carcinogens.

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A threshold represents the theoretically defined dose, below which no abnormal increase in response is observed. Genotoxic carcinogens are known to have an irreversible effect on the genetic cellular structure. Even in small amounts, genotoxic carcinogens are assumed to have additive effects and therefore to subject individuals exposed to them to an incremental risk of developing cancer.
Based on this assumption, the no-threshold concept was introduced exclusively for genotoxic carcinogens and has been adopted in Japan as a basis for the regulatory risk assessment in case of such chemicals. The current regulatory policy adheres to the fundamental principle of food safety i.e. to the precedence of protecting people's health.
Dose-response studies recently conducted on various genotoxic agents suggest the existence of a threshold. If confirmed, such findings may provide sufficient scientific evidence to substantiate the adoption of a threshold concept as the principle of the regulatory assessment of the risks of genotoxic carcinogens and their impact on health.
It should however be emphasized that the limitations of a threshold approach must be clearly understood and presented to lend credence to the proposition of a paradigm shift from the current regulatory policy: A threshold is not a value that can be determined directly and precisely from dose-response data, but one that can be estimated from analytical data by means of a logically-elaborated mathematical model calculation.
Scientific efforts in support of the adoption of a threshold in this context should therefore be focused on the development of appropriate mathematical models, and on the establishment of toxicological concepts that substantiate their application.
A realistic first step towards a paradigm shift from the no-threshold concept is to seek general consensus on the introduction of an appropriately estimated“virtually safe dose”, instead of a threshold.

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Non-coding tandem repeat DNA sequences have high rates of mutation that facilitate the measurement of induced mutation in small sample sizes. It has been suggested that these loci may be useful biomarkers for heritable genetic mutation induced by exposure to genotoxic agents. Significant induction of mutation is quantifiable in the germline of mice exposed to mutagens. The primary focus of this work has been on exposure to radiation. The data suggest that meiosis or DNA replication/repair may be required for induction of mutation in the germline at tandem repeats. Mutations arise via indirect mechanisms rather than by direct damage to the repeat locus itself, therefore reflecting genomic instability rather than targeted DNA damage. These markers have also been used to measure induced germline mutations in animals exposed to ambient levels of urban air pollution. The mutagenicity is associated with particulate matter in the air but the exact chemical nature of the mutagens is unknown. Lack of knowledge of the relationship between ESTR instability and gene mutation, and lack of understanding of the mechanisms resulting in instability prevent inference on the health-related implications of induced tandem repeat mutation. We have developed single-molecule PCR approaches to study ESTR instability in vitro. This method circumvents the requirement of sub-cloning and allows for many more individual ESTR alleles to be examined. These types of laboratory-based experiments will be crucial in clarifying the types of chemicals that can generate tandem repeat instability and thereby provide insight into the mechanisms of action and the putative mutagens found in complex environmental matrices.

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Oxygen radicals are produced through normal cellular metabolism, and formation of such radicals is further enhanced by ionizing radiation and by various chemicals. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is the most abundant, and appears to play important roles in mutagenesis and carcinogenesis. Enzymatic activities that may be responsible for preventing 8-oxoG-evoked mutations were identified in mammalian cells. We have focused on following the two enzymes. MTH1 (Mth1) protein is the mammalian counterpart of E. coli MutT protein, which hydrolyzes 8-oxo-dGTP to its monophosphate form in the nucleotide pool, thereby preventing the incorporation of the mutagenic substrate into DNA. On the other hand, MUTYH (Mutyh) protein, a counterpart of E. coli MutY protein, having adenine/2-hydroxyadenine DNA glycosylase activity, is expected to prevent G:C to T:A transversions, by excising adenine from G:A mismatches induced by 8-oxoG and 2-OH-A. To analyze the function of the mammalian Mth1 and Mutyh proteins in vivo, we established gene-knockout mice for these two enzymes by gene targeting, and investigated spontaneous tumorigenesis as well as mutagenesis. Here we discuss our recent progress on spontaneous and oxidative stress-induced mutagenesis with these mutant mouse lines.

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Ionizing radiation induces genomic instability, which is transmitted through many generations after irradiation in the progeny of surviving cells. We have hypothesized that radiation-induced large deletion causes potentially unstable chromosome regions, which are involved in delayed induction of radiation-induced genomic instability. Using phosphorylation-specific antibodies against ATM and histone H2AX, whose phosphorylation is induced by DNA double strand breaks, we detected delayed induction of phosphorylated ATM and H2AX foci in the progeny of X-ray-surviving cells, which indicated delayed induction of DNA double strand breaks. Furthermore, we found delayed chromosomal instability in X chromosomes in clones which contain large deletion involving the HPRT loci. It is suggested that large deletion involving —Mb region causes unstable chromatin structure, and it results in delayed rearrangement of chromosomes involved. These findings provide the possibility that manifestation of radiation-induced genomic instability results from delayed DNA breaks, i.e., the breaks lead to delayed chromosome rearrangements, delayed cell death etc., many generations after irradiation.

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This article aims to introduce the outline of issue and phenomena of welfare of laboratory animals which is enacted in Law for Welfare and Proper Management of Animals, and to clarify the difference of conception and method between welfare of laboratory animals and experiment using laboratory animals, in anticipation of contributing to its improvement in practice.

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The most recent and international movement regarding to the alternative methods to animal testing is the 7th amendment of EU Cosmetic Directive. By this Directive, testing ban and marketing ban enter into a phased prohibition in consideration of scientific progress. However, the development of the alternative method that has been left is extremely difficult compared with the one that has been developed so far. A global collaboration is necessary now for accomplishment of the goal.

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The OECD established the Guidelines for Testing of Chemicals in 1981, which is the basis of the mutual acceptance of data (MAD) system among the member countries to prevent unnecessary repetition of toxicity tests, and consequently to reduce the number of animals used. The Guidelines was soon subjected to revision from the viewpoint of animal welfare besides its periodical updating with the state-of-art in the toxicological sciences. Revision of the Test Guidelines is in progress according to the 3Rs principle, reduction, refinement and replacement. The acute toxicity test and the skin and eye irritation/corrosion tests were assumed to be the most problematic ones among the animal tests in animal welfare aspect. Three different test methods have been adopted for the alternative to acute oral toxicity test (Test Guideline (TG) 401), namely, the fixed dose procedure (TG420), acute toxic class method (TG423) and up-and-down procedure (TG425), and the reduction of animals was accomplished. Then the traditional acute oral toxicity test (TG401) has been deleted from the Guidelines. Procedures for irritation/corrosion tests for skin (TG404) and eye (TG405) were reorganized into tier-test system in order to prevent any corrosion or strong irritation to take place. The tier system consists of survey of toxicities of the test chemical, structure-activity relationship, pH, and testing with in vitro methods. Moreover, at the final tier animal testing should proceed by one animal. Three kinds of in vitro corrosivity tests have been adopted in the Guidelines.

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In the Pharmaceuticals Affairs Law, development, manufacturing and marketing of drugs, quasi-drugs, cosmetics and medical devices are regulated from the point of view of their quality, efficacy and safety. Non-clinical safety data are required not only to consider relationship between test substances and adverse events occurred in clinical trials at review of the application but also to evaluate safety and ethical appropriateness of protocols of clinical trials during development of them. Alternative tests to animal testing are quite important in the regulation of drugs, quasi-drugs and cosmetics from two viewpoints: one is the animal welfare, and the other is the sufficient evaluation of safety of products. From the administrative point of view, objectives and usages of results of alternative tests for drugs, quasi-drugs and cosmetics should be considered on development, evaluation and validation of alternative tests.

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