According to Organisation for Economic Co-operation and Development test guideline no. (TG) 487 for the in vitro mammalian cell micronucleus test (MNT), short (pulse) treatment of primary human lymphocytes should be followed immediately by a sampling period of 1.5-2.0 cell cycle lengths in the presence of cytochalasin B. However, TG 487 allows extending the sampling period "if it is known or suspected that the test chemical affects the cell cycling time." It is often unknown in advance whether a test substance induces cell cycle delays, and therefore whether an extension of the sampling period is warranted. Here, we investigate whether extending the sampling time after pulse treatment-by including a recovery period prior to cytochalasin B treatment-affects the sensitivity of the in vitro MNT. Primary human lymphocytes were exposed to 11 mutagens and assessed using two protocols ("recovery" and "no recovery") in parallel from the same treatment culture. Including a recovery period after pulse treatment generally allowed testing of higher concentrations without excessive cytotoxicity. In addition, cultures subjected to a recovery period showed generally higher micronucleus rates at concentrations with acceptable levels of cytotoxicity, compared to cultures not having undergone a recovery period. To compare the sensitivity of both protocols at non-cytotoxic concentrations, benchmark concentration analysis was performed. Here, the "recovery method" proved to be at least as sensitive as the "no recovery method." The reproducibility of these findings was demonstrated in an interlaboratory ring trial using a subset of up to five mutagens. In conclusion, the inclusion of a recovery period after pulse treatment per se covers potential substance-induced cell cycle delays without any loss in sensitivity toward the induced mutagenic effect. The "recovery" protocol can be used for assessing the mutagenic potential of test substances affecting and not affecting the cell cycle.

The in vitro micronucleus (IVMN) assay is a genetic toxicity assay routinely conducted in early drug development to evaluate clastogenicity and aneugenicity. At this discovery stage, synthetic routes for potential active pharmaceutical ingredients (API) are not optimized, which can lead to incomplete purification or solvation. API in genetic toxicity screens often contain residual solvent(s) in higher quantities than will be present in the GMP material. Understanding when solvents used either in API synthesis or as a vehicle may interfere with genetic toxicity screening results of APIs is important. In this work, twenty solvents commonly used in API synthesis were analyzed in the IVMN assay. These twenty solvents include acetonitrile, formic acid, anisole, dichloromethane, heptane, isopropyl amine, dimethyl sulfoxide, sodium hexafluorophosphate, N,N-Dimethylethylamine, N,N-Dimethylformamide, cyclopentyl methyl ether, 2-propanol, 1-butanol, 1-Methyl-2-pyrrolidinone, methanol, tetrahydrofuran, 2-Methyltetrahydrofuran, ethyl acetate, trifluoroacetic acid, and 2,6-Di-tert-butyl-4-methylphenol. When the in vitro data was correlated to available in vivo genotoxicity data published in the literature, only 5 of the 14 positively correlated. However, when a 10 mM limit of exposure was applied to the solvents, 13 of the 14 correlated to the in vivo literature results. While the results do not consider vehicle interaction within the assay, the data demonstrates that residual solvent carryover from API or other sources is generally not of concern. The 10 mM limit also reduces the false-positive risk of this assay. Baseline cytotoxicity and micronucleus formation from the solvents in this system provide a ranking on which solvents may produce confounding results in the assay.

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.

It is often assumed that genotoxic substances will be detected more easily by using in vitro rather than in vivo genotoxicity tests since higher concentrations, more cytotoxicity and static exposures can be achieved. However, there is a paucity of data demonstrating whether genotoxic substances are detected at lower concentrations in cell culture in vitro than can be reached in the blood of animals treated in vivo. To investigate this issue, we compared the lowest concentration required for induction of chromosomal damage in vitro (lowest observed effective concentration, or LOEC) with the concentration of the test substance in blood at the lowest dose required for biologically relevant induction of micronuclei in vivo (lowest observed effective dose, or LOED). In total, 83 substances were found for which the LOED could be identified or estimated, where concentrations in blood and micronucleus data were available via the same route of administration in the same species, and in vitro chromosomal damage data were available. 39.8 % of substances were positive in vivo at blood concentrations that were lower than the LOEC in vitro, 22.9 % were positive at similar concentrations, and 37.3 % of substances were positive in vivo at higher concentrations. Distribution analysis showed a very wide scatter of > 6 orders of magnitude across these 3 categories. When mode of action was evaluated, the distribution of clastogens and aneugens across the 3 categories was very similar. Thus, the ability to detect induction of micronuclei in bone marrow in vivo regardless of the mechanism for micronucleus induction, is clearly not solely determined by the concentration of test substance which induced chromosomal damage in vitro.

Implementation of the seventh amendment to the EU Cosmetics Directive has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006, further development occurred and a standard protocol was published in 2011. To evaluate and optimise the assay at Covance Laboratories, we tested nine chemicals [4-nitrophenol (4-NP), cyclohexanone (CH), 2-ethyl-1,3-hexanediol (2-EHD), methyl methansulfonate (MMS), mitomycin C (MMC), ethyl nitrosourea (ENU), benzo[a]pyrene (BaP), cyclophosphamide (CPA) and vinblastine (VIN)] using the EpiDerm™ 3D skin model (MatTek Corporation®, IVLSL, Bratislava, Slovakia) and compared the data using the standard 48-h treatment regimen and also an emerging 72-h treatment protocol. The EpiDerm™ tissue has reportedly some metabolic capacity but data using 48-h treatments has provided mixed results. Our investigations demonstrate that the two chemicals requiring metabolic activation (BaP and CPA) were negative following the 48-h protocol but were clearly positive following 72-h treatment. Furthermore, Replication Index (RI) data showed higher RI values in vehicle control treatments (indicating increased cell division) across the treatment set following 72-h treatments. A general greater magnitude of micronucleus (MN) induction was also observed following test chemical treatment. These data suggest that the 72-h treatment protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. For further assay optimisation, we compare the statistical power of scoring cells from duplicate or triplicate cultures per treatment concentration and provide recommendations.

The in vitro micronucleus (IVMN) test was endorsed for regulatory genotoxicity testing with adoption of the Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 487 in 2010. This included two equally acceptable options for extended treatment in the absence of metabolic activation: a treatment for 1.5-2.0 cell cycles with harvest at the end of treatment (Option A) or treatment for 1.5-2.0 cell cycles followed by recovery for 1.5-2.0 cell cycles prior to harvest (Option B). Although no preferences were discussed, TG 487 cautions that Option B may not be appropriate for stimulated lymphocytes where exponential growth may be declining at 96 h after phytohaemagglutinin (PHA) stimulation. Following revision of TG 487 in 2014 and 2016, emphasis has been placed on using Option A. Given the purpose of the IVMN assay is to determine both clastogenic and aneugenic potential, the authors believe the assay is compromised if an extended treatment with recovery is not included for sensitive detection of certain classes of chemical. In this study, average generation time (via bromodeoxyuridine incorporation) of human peripheral blood lymphocytes (HPBL) was measured up to 144 h after PHA stimulation. In addition, the HPBL micronucleus (MN) assay was performed using Option A and B treatment schedules. Cytotoxicity (replication index) and MN induction were determined following treatment with 14 chemicals. The data demonstrate that lymphocytes actively divide beyond 96 h after PHA stimulation. Furthermore, MN induction was only observed with some aneugenic chemicals and nucleoside analogues in HPBLs following extended treatment with a recovery period. For the majority of chemicals tested the magnitude of MN induction was generally greater and MN induction was observed across a wider concentration range following the Option B treatment schedule. In addition, steep concentration-related toxicity following treatment without recovery is more common, making selection of suitable concentrations (within regulatory toxicity limits) for MN analysis challenging.

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity.

Accumulated evidence has shown that in vitro mammalian cell genotoxicity assays produce high frequencies of “misleading” positive results, i.e. predicted hazard is not confirmed in in vivo and/or carcinogenicity studies [1], raising the question of relevance to human risk assessment. A recent study of micronucleus (MN) induction [2] showed that commonly used p53-deficient rodent cell lines (CHL, CHO and V79) gave a higher frequency of “misleading” positive results with 9 non-DNA reactive, Ames-negative and in vivo negative chemicals [3] than human p53-competent cells (blood lymphocytes, TK6 and HepG2 cell lines). This raised the question of whether these differences were due to p53 status or species origin. This present study compared human versus mouse and p53-competent versus p53-mutated function. The same 9 chemicals were tested for induction of MN in mouse lymphoma L5178Y (mutated p53), human TK6 (functional p53) and WIL2-NS (TK6 related, with mutated p53) cells. Six chemicals provided clear positive increases in MN frequency in at least one cell type. L5178Y cells yielded clear positive responses with more chemicals than either TK6 or WIL2-NS, indicating origin rather than p53 functionality was most relevant. Apoptosis induction (measured via caspase-3/7) was also investigated with clear differences in the timing and extent of apoptosis induction between mouse and human cells noted. With curcumin in TK6 cells, induction of caspase-3/7 activity coincided with MN induction, whereas for L5178Y cells, MN induction occurred in the absence of increased caspase activity. By contrast, with MMS in TK6 cells, MN induction preceded increased caspase-3/7 activity. These data suggest that MN induction by “misleading positive” genotoxins in p53-competent human cell lines may result from apoptosis, whereas in p53-defective rodent cells such as L5178Y, MN induction may be independent of apoptosis.

We have demonstrated previously that the seemingly high rate of "false" or "misleading" positive results from in vitro micronucleus assays (MNvit) was greater when rodent derived cell lines and certain toxicity measures, such as relative cell count or replication index, were used. These studies suggested that the use of a human cell type with functional p53 and a toxicity measure that included a function of cell proliferation could dramatically reduce the detection of misleading positive results. A reduced "false positive rate" should not be at the expense of a loss of sensitivity of the assay. Therefore, we have investigated the sensitivity of the MNvit assay to known genotoxic agents using three cell types shown previously to be less prone to misleading positives, namely human lymphocytes (HuLy), TK6 and HepG2 cells. The 17 chemicals are well characterised and are from a list of chemicals known to produce positive results in in vitro mammalian cell assays. These data demonstrated a high sensitivity of the assay in which TK6 and HuLy cells were employed, such that 15 out of the 17 chemicals were correctly identified. By contrast, the use of HepG2 cells resulted in far fewer than expected positive responses. In conclusion, using TK6 and HuLy cells in preference to long established rodent cell lines in order to improve specificity does not compromise the sensitivity of the MNvit to detect known genotoxic agents.

The aim of this study was to examine the concordance between human in vivo and in vitro skin irritation classifications of cosmetic products and to evaluate the correlations between the different parameters. For that purpose, 22 formulations from product development test series, covering the full range of in vivo scores and representing different cosmetic product classes, were tested in vivo (modified Frosch-Kligman Soap Chamber Patch Test with repetitive occlusive application) and in vitro using two epidermis equivalents commercially available as kits (EpiDerm™ and EPISKIN™) and one in-house model (Cosmital). In vivo, skin reactions (erythema, dryness and fissures) were visually evaluated and, in addition, skin redness and transepidermal water loss (TEWL) were measured by means of technical instruments. The parameters measured in vitro were the percent cell viability in the MTT reduction assay, with ET50 determination, and the extracellular release of the pro-inflammatory mediator IL-1α and of the cytosolic enzyme lactate dehydrogenase (LDH), into the culture medium collected after topical application of the products for different exposure times (time-course assay). In general, good Spearman rank correlations could be observed between the different in vivo parameters (with the exception of TEWL and dryness at day 2). Furthermore, high correlation coefficients were obtained by comparing the different in vitro parameters (except for LDH release) and different models, which allowed to conclude that the results obtained with the different reconstructed epidermis models were very similar. A comparison between in vivo and in vitro parameters resulted in the best rank correlation for ET50, then in decreasing order, for the percent MTT viability at 16 h, the IL-1α release and finally, for LDH release, where the correlation was generally low. A direct comparison of the mean total scores (sum of erythema, dryness and fissures at day 5) of the 22 products with the best predictor, ET50 obtained with the three reconstructed epidermis models, using simple linear regression analysis resulted in a coefficient of correlation R=0.94 for EpiDerm, R=0.90 for Cosmital and R=0.84 for EPISKIN. Multivariate descriptive statistics showed that the in vitro parameters, MTT viability evaluated after the 16-h exposure and ET50, as well as the in vivo parameters, sum of visual scores at day 5 and chromameter value, were the best endpoints to discriminate between irritant and non-irritant products. Using the in vivo mean total scores at day 5 with a cut-off value at 2 and the in vitro percent MTT viability after the 16-h exposure with a cut-off value at 50% to classify the products, the same two-by-two contingency table was obtained for all the three reconstructed epidermis models with sensitivity=92%, specificity=100% and observed concordance=95% (κ=0.91; 95% confidence interval 0.74–1.08). This classification system was a satisfactory and relevant approach to discriminate the “irritant” from the “non-irritant” cosmetic products in this study. In conclusion, this study demonstrated the usefulness of reconstructed human epidermis equivalents for the in vitro assessment of the irritation potential of a series of cosmetic products. These models allow the measurement of quantifiable and objective endpoints relevant to in vivo irritative phenomena.