The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns. In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis. All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium. Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

The \'Organization for Economic Co-operation and Development (OECD) guidelines for the Testing of Chemicals\' aims to identify whether a chemical is a genotoxic hazard, and these guidelines \'are periodically reviewed in the light of scientific progress, changing regulatory needs and animal welfare considerations\'. OECD published a mammalian erythrocyte micronucleus test guideline for testing chemicals (1) that proposes: \'Animals are treated with the test chemical once…Samples of peripheral blood are taken at least twice (from the same group of animals), starting not earlier than 36 h after treatment, with appropriate intervals following the first sample, but not extending beyond 72 h\'. This guidelines are base on the report by the Collaborative Study Group for the Micronucleus Test (CSGMT), which was based on the sampling of mice peripheral blood every 24 h We investigated the kinetics of micronucleus induction by taking samples prior to administration and every 8 or 10 h after single treatment. The comparisons suggest that 24-h sampling could cause not only an underestimation of the responses to various agents but also a misestimation of the time of maximal induction. We proposed that samples of peripheral blood must be collected at two different times during an optimal 25-h sampling range (from 25 to 50 h). Besides, we hypothesize that the time of maximal EPC-MN induction depends on the time required for the mechanisms involved in micronucleus production; and we suggest that a kinetic analysis of MN-PCE induction by several agents with well-known mechanisms of micronuclei induction would allow derivation of a specific relationship between the kinetics of MN-PCE induction and some process of DNA breaks and/or micronuclei induction.

Genotoxicity tests are designed to detect the genetic damage by various mechanisms. Several guidelines have provided various tests to be conducted for testing the genotoxicity and each of the regulatory agencies around the world have developed their own requirements for mutagenicity, without realizing that the products developed or registered in one country are also going to be registered and marketed around the world. The ICH guideline of genotoxicity helps to optimize the standard battery for genetic toxicology and to provide guidance on interpretation of results. These suggested standard set of tests does not imply that other genotoxicity tests are inadequate or inappropriate, but they help in improving risk characterization for carcinogenic effects that have their basis in changes in the genetic material.

The exposure to arsenic, a potential genotoxic carcinogen in humans, via drinking water is a serious worldwide health hazard. The arsenic content of 10 μg L(-1) in drinking water, however, has been established as its guideline standard (maximum contaminant limit) that has been estimated to pose minimum risk to cancer. Since micronucleus induction in the erythrocytes of fish is a sensitive indicator of genotoxic agents in water, the piscine micronucleus assay was used in the present experiment to assess the genotoxic potential of arsenic at its various exposure levels including the guideline value for drinking water. The experiments were conducted in two different species of fishes, the pond murrel (Channa punctatus) and the goldfish (Carassius auratus). Significant increases in the frequency of micronucleated erythrocytes were documented in a dose-dependent manner in both Channa and Carassius. The fishes, however, exhibited variations in inter-specific sensitivity to micronucleus induction following arsenic exposure. The exposure level of arsenic at its guideline value for drinking water, therefore, exhibited marked genotoxicity in fishes.

There is a pressing requirement to define a hazard identification and risk management strategy for nanomaterials due to the rapid growth in the nanotechnology industry and their promise of life-style revolutions through the development of wide-ranging nano-containing consumer products. Consequently, a battery of well defined and appropriate in vitro assays to assess a number of genotoxicity endpoints is required to minimise extensive and costly in vivo testing. However, the validity of the established protocols in current OECD recognised genotoxicity assays for nanomaterials is currently being questioned. In this report, we therefore consider the in vitro OECD genotoxicity test battery including the Ames, micronucleus and HPRT forward mutation assays, and their potential role in the safety assessment of nanomaterial induced DNA damage in vitro.

The OECD guideline for the in vitro mammalian cell micronucleus test (OECD 487) was recently adopted in July 22, 2010. Since its publication, it has become apparent that the guidance for testing chemicals where solubility is a limiting factor can be interpreted in a variety of ways. In this communication, we provide clarification for testing insoluble chemicals. The intent of the OECD 487 guideline is for the high dose to be the lowest precipitating concentration even if toxicity occurs above the solubility limit in tissue culture medium. Examination of precipitation can be done by the unaided eye or microscopically. Precipitation is examined at the onset or end of treatment, with the intent to identify precipitate present during treatment.

Reference genotoxic compounds 2-aminoanthracene, diethylstilboestrol and vinblastine were tested in the in vitro micronucleus assay using Chinese hamster V79 derived cells in the laboratories of British American Tobacco in the UK. The work was conducted in support of the cytotoxicity measures recommended in the 2007 version of the OECD Test Guideline 487. The three compounds were positive in the assay in the presence and absence of the cytokinesis blocking agent cytochalasin B at concentrations that did not exceed the recommended cytotoxic limits determined by relative population doubling, relative increase in cell counts, relative cell counts and cytokinesis block proliferation index. Consequently, this work supports the hypothesis that relative population doubling, relative increase in cell counts and relative cell counts are appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay.

The development of an environmental health and safety risk management system for nanoscale particle-types requires a base set of hazard data. Accurate determination of health and environmental risks of nanomaterials is a function of the integration of hazard and exposure datasets. Recently, a nanoparticle risk assessment strategy was promulgated and the components are described in a document entitled “Nanorisk framework” (www.nanoriskframework.com). A major component of the hazard evaluation includes a proposed minimum base set of toxicity studies. Included in the suggested studies were substantial particle characterization, a variety of acute hazard and environmental tests, concomitant with screening-type genotoxicity studies. The implementation of well-accepted genotoxicity assays for testing nanomaterials remains a controversial issue. This is because many of these genotoxicity tests were designed for screening general macroparticle chemicals and might not be suitable for the screening of nanomaterials (even of the same compositional material). Furthermore, no nanoparticle-type positive controls have been established or universally accepted for these tests. Although it is the comparative results of the test material vs. the negative or vehicle control that forms the basis for interpreting the results and potency of test materials in genetic toxicology assays, the lack of a nanoparticle-type positive control questions the suitability of the tests to identify nanomaterials with genotoxic properties. It is also not possible to establish historical positive control ranges that would confirm the sensitivity of the tests. Although several genetic toxicology tests have been validated for chemicals according to the Organisation for Economic Co-operation and Development (OECD) test guidelines, the relevance of these assays for nanoparticulate materials remains to be determined. In an attempt to remedy this issue, the OECD has established current projects designed to evaluate the relevance and reproducibility of safety hazard tests for representative nanomaterials, including genotoxicity assays (i.e., Steering Group 3 – Safety Testing of Representative Nanomaterials). In this article, we discuss our past approaches and experience in conducting genotoxicity assays (1) for a newly developed ultrafine TiO₂ particle-type; and (2) in a recent inhalation study, evaluating micronucleus formation in rat erythrocytes following exposures to engineered amorphous nanosilica particles. It seems clear that the development of standardized approaches will be necessary in order to determine whether exposures to specific nanoparticle-types are associated with genotoxic events. The appropriateness of available genotoxicity test systems for nanomaterials requires confirmation and standardization. Accordingly, it seems reasonable to conclude that any specific regulatory testing requirements for nanoparticles would be premature at this time.

The reference genotoxic agents 2-aminoanthracene (a metabolism dependent weak clastogen), 5-fluorouracil (a nucleoside analogue, characterised by a steep dose response profile), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation), cadmium chloride (an inorganic carcinogen), and cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair) were tested in the in vitro micronucleus assay using the Chinese hamster ovary (CHO) cell line at Covance Laboratories, Harrogate, UK. All chemicals were treated in the absence and presence of cytokinesis block (via addition of cytochalasin B) with this work forming part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 on the In vitro Mammalian Cell Micronucleus Test (MNvit). The toxicity measures used, detecting a possible combination of both cytostasis and cell death (though not cell death directly), were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index in the presence of cytokinesis block. All of the chemicals tested either gave marked positive increases in the percentage of micronucleated cells with and without cytokinesis block, or did not induce micronuclei at concentrations giving approximately 50-60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcome from this series of tests supports the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay.