Chromosome aberrations in Chinese hamster and human cells: a comparison using compounds with various genotoxicity profiles

Chromosome aberrations (Cabs) can be induced in vitro by non-DNA damaging compounds, often associated with cytotoxicity and DNA synthesis inhibition, and under conditions that would not be relevant in vivo. Such misleading positive results are reported both in Chinese hamster cell lines and in human peripheral blood lymphocytes (HL). We assessed the response of HL to compounds with varied genetic toxicity profiles, all of which induced Cabs in CHO cells Seven of 10 compounds were negative or equivocal in HL. Results in purified lymphocytes for four verified that the difference was not due to the presence of blood in cultures. Two compounds that were weakly positive in the Ames test and one that induced DNA adducts were negative or equivocal in the HL assay; their overall mutagenic potential in vivo is not clear. Of four Ames-negative compounds, three of which inhibited DNA synthesis in CHO cells, three were negative and one was equivocal in the HL assay. A potent Cab inducer, which also induced micronuclei in vivo (but was negative in the Ames test) was clearly positive in the HL assay. Two compounds were clearly positive in HL only when the mitotic indices (MI) were below 50% of control. These are genotoxic in other assays but our evidence suggests that Cab induction is related more to toxicity than to primary DNA damage. For this limited set of 10 compounds, HL were more likely than CHO cells to give negative or equivocal results. It is likely that more stringent checkpoint controls in human cells prevent damaged cells reaching mitosis, and may also influence the reported greater sensitivity to induction of aneuploidy and polyploidy of normal rodent compared with human cells. In the studies reported here, two strong inducers of polyploidy in CHO cells gave weaker increases in HL. Human lymphocytes have disadvantages as a routine screening assay (finding donors, known individual variability, increased time required and the inadequacy of the MI as a toxicity measure), but may be useful in follow-up testing to assess weight of evidence about genotoxic risk to humans, for compounds that are positive in the Chinese hamster cell Cabs assays.     

Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: an enabling study for independent validation as an alternative method

Sixteen coded compounds were blind-tested at 4 laboratories using the recently described GADD45a-GFP genotoxicity assay. The compounds were chosen to include non-genotoxic compounds as well as weak and strong genotoxins. None of the compounds required metabolic activation in order to exhibit genotoxic effects. The participating laboratories included 2 global pharmaceutical companies, a global consumer goods company and the Gentronix laboratory in Manchester. Each compound was tested 4 times on different days following a protocol previously described. The tests were carried out after a 3-day training period from the parent lab (Manchester). Following the exclusion of data from tests with positive control failures and data series with 'spikes', 92% of assays gave the correct result: non-genotoxins giving negative results and genotoxins giving positive results. There were no randomly distributed problems suggesting that differences between the results from different sites reflected the use of different instruments, procedural differences and operator experience. In na?ve operator laboratories the quality of data improved with operator practice. It was concluded that simple clarification of the protocol would provide the level of reliability required for widespread use of the assay in hazard assessment.     

Protein precipitation In Vitro as a measure of chemical-induced cytotoxicity: an EDIT sub-programme

As a priority area of the Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT) programme, an in vitro protein precipitation (PP) assay was used on the 50 reference chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to confirm and extend the MEIC results. Dose-response curves were generated for only 30 of the chemicals, and the concentrations causing 10% (EC10) and 50% (EC50) protein precipitation versus the positive control were chosen as endpoints. The number of chemicals with a positive response increased to 46 when a new endpoint, the minimum effect concentration (MEC) that induces protein precipitation with respect to the negative control, was used. When the results were correlated with in vitro cytotoxicity in human cell lines, a similarly good correlation was found between the various endpoints of the PP assay at 5 hours and the 24-hour IC50 average cytotoxicity in human cell lines, even though the number of chemicals included in the correlation was larger for the MEC. Using the prediction error, the endpoint that gave the best correlation between the PP assay and human cell cytotoxicity was once more found to be the 5-hour MEC, and this was chosen for the PP assay. The sensitivity of the PP assay is lower than that of the in vitro cell-line cytotoxicity assay, possibly due to its shorter exposure period and because precipitation is the ultimate event in the sequence of a protein disturbance. It is expected that earlier denaturation steps would give better sensitivity. However, this simple, inexpensive and rapid assay could be useful in the early stages of testing chemicals.     

Statistical analysis of in vivo rodent micronucleus assay

The in vivo rodent micronucleus assay (MNC) is widely used as a cytogenetic assay to detect the clastogenic activity of a chemical in vivo. MNC is one of three tests in a battery recommended by the fourth International Conference on Harmonization (ICH4) of Genotoxicity Guidelines. As such it has been accepted by many regulatory authorities. However, the determination of a positive result in a genotoxicity test, including MNC, has been an issue of debate among toxicologists and biometricians. In this presentation we compare several statistical procedures that have been suggested for the analysis of MNC data and indicate which one is the most powerful. The standard protocol of MNC has at least three dose levels plus the control dose and uses at least four animals per group. For each animal, 2000 polychromatic erythrocytes (PCE) are counted. Two statistical procedures can be employed, either alone or jointly, for the analysis of the MNC dose-response curve. These are the Cochran-Armitage (C-A) trend test and the Dunnett type test. For performing Dunnett type tests, toxicologists often use negative historical control rate for the estimate of the concurrent negative control rate. Some toxicologists emphasize the reproducibility of assay results instead of the dose-response relationship for the important criterion [J. Ashby, H. Tinwell, Mutat. Res. 327 (1995) 49-55; for the rebuttal see M. Hayashi, T. Sofuni, Mutat. Res. 331 (1995) 173-174]. The following three procedures are currently employed in toxicology labs for the evaluation of MNC result. The assay response is deemed positive if it is detected by (i) the C-A trend test alone, (ii) both the C-A trend test and the Dunnett type test and (iii) either the C-A trend test or the Dunnett type test. Using Monte Carlo simulation, we first find for each procedure, sizes of tests which yield the experiment-wise type I error rate of 0.05 and show that the procedure (ii) is the most powerful against the alternatives of monotone increase. The procedure (ii) which originated from Hayashi's three-step procedure was coded in C and termed 'MNC'. The MNC software program is available in the public domain through the ftp.     

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.     

The genotoxicity study of garlic and pasipy herbal drops by peripheral blood micronucleus test

The in vivo rodent micronucleus test is widely used as a genotoxic assay to detect the clastogenic activity of chemicals. In this research the genotoxic effects of herbal drops of garlic and pasipy were evaluated using the micronucleus test. Maximum Tolerated Dose (MTD) was determined by a dose-response test. For each medicine three treatment groups were considered with doses of MTD, 1/2 MTD and 1/4 MTD according to the CSGMT protocol (1995 Japan). Drugs were administered orally to mice (test groups). Mitomicin C was used as a known genotoxic agent in positive control group. The peripheral blood samples before treatment (zero time samples) were considered as negative control. The appearance of a micronucleus is used as an index for genotoxic potential. The results obtained indicated that the herbal drops showed genotoxicity effect and it was dose-dependent compared to the negative control group. This genotoxicity was significant (p < 0.05) but the genotoxic effects of garlic and pasipy were "not significant" compared to the historical negative control group (p > 0.05). Therefore our results if compared to the negative control group is significant and it is worthy of consideration.     

Validation of an automatic comet assay analysis system integrating the curve fitting of combined comet intensity profiles.

In recent years, the single-cell gel electrophoresis (comet) assay has become a reference technique for the assessment of DNA fragmentation both in vitro and in vivo at the cellular level. In order to improve the throughput of genotoxicity screening, development of fully automated systems is clearly a must. This would allow us to increase processing time and to avoid subjectivity brought about by frequent manual settings required for the 'classical' analysis systems. To validate a fully automatic system developed in our laboratory, different experiments were conducted in vitro on murine P388D1 cells with increasing doses of ethyl methanesulfonate (up to 5 mM), thus covering a large range of DNA damage (up to 80% of DNA in the tail). The present study (1) validates our 'in house' fully automatic system versus a widely used semi-automatic commercial system for the image-analysis step, and versus the human eye for the image acquisition step, (2) shows that computing tail DNA a posteriori on the basis of a curve fitting concept that combines intensity profiles [G. Dehon, P. Bogaerts, P. Duez, L. Catoire, J. Dubois, Curve fitting of combined comet intensity profiles: a new global concept to quantify DNA damage by the comet assay, Chemom. Intell. Lab. Syst. 73 (2004) 235-243] gives results not significantly different from the 'classical' approach but is much more accurate and easy to undertake and (3) demonstrates that, with these increased performances, the number of comets to be scored can be reduced to a minimum of 20 comets per slide without sacrificing statistical reliability.     

Exposure‐Based Validation List for Developmental Toxicity Screening Assays

Validation of alternative assays requires comparison of the responses to toxicants in the alternative assay with in vivo responses. Chemicals have been classified as “positive” or “negative” in vivo, despite the fact that developmental toxicity is conditional on magnitude of exposure. We developed a list of positive and negative developmental exposures, with exposure defined by toxicokinetic data, specifically maternal plasma C_max. We selected a series of 20 chemicals that caused developmental toxicity and for which there were appropriate toxicokinetic data. Where possible, we used the same chemical for both positive and negative exposures, the positive being the C_max at a dose level that produced significant teratogenicity or embryolethality, the negative being the C_max at a dose level not causing developmental toxicity. It was not possible to find toxicokinetic data at the no‐effect level for all positive compounds, and the negative exposure list contains C_max values for some compounds that do not have developmental toxicity up to the highest dose level tested. This exposure‐based reference list represents a fundamentally different approach to the evaluation of alternative tests and is proposed as a step toward application of alternative tests in quantitative risk assessment     

Genotoxicity evaluation of selenium sulfide in in vivo and in vivo/in vitro micronucleus and chromosome aberration assays

Selenium monosulfide (Ses) was reported to be carcinogenic to livers of male and female rats and livers and lungs of female mice. However, its genotoxicity profile in short-term assays is somewhat equivocal. A multiple endpoint/multiple tissue approach to short-term genetic toxicity testing has been developed in our laboratory. In the present paper, the effect of SeS in in vivo and in vivo/ in vitro micronucleus and chromosome aberration assays in rat bone marrow and spleen are reported. In the in vivo assay, small but statistically significant increases in bone marrow micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment of rats with 50 mg/kg SeS and 48 h after treatment with 12.5 mg/kg. A significant decrease in the PCE/total erythrocyte (TE) ratio, indicative of cytotoxicity, was observed at the 50 mg/kg dose at the 24-h timepoint. In spleen, no increases in MNPCEs or decreases in the PCE/TE ratios were observed. No evidence of a significant increase in aberrations was observed in bone marrow or spleen. In the in vivo/in vitro assay, no increase in micronucleated binucleated cells or cells with aberrations was observed in SeS-treated rats. The small but statistically significant increases in MN observed in the in vivo study are considered likely not to be biologically significant since no dose-response was observed and all the values obtained were within historical control range in our laboratory. Given the overall genetic toxicity profile of SeS, it appears that SeS may be a weak mutagen and that differences between testing protocols may be very important in determining whether or not it is found to be negative or positive. Histological evidence was obtained in this study that suggests that the liver is the acute target organ of SeS in rats. Given the fact that SeS is selectively hepatocarcinogenic, we are currently testing the hypothesis that the genotoxicity of SeS in rats may be more readily detectable in liver than in bone marrow or spleen.     

Historical vehicle and positive control micronucleus data in mice and rats

The rodent bone marrow micronucleus (MN) assay has been widely used as part of an in vivo genotoxicity test battery in product safety evaluation. In this assay, the historical vehicle and positive control data form an important component in the assay performance and data interpretation. Also, in light of minimizing animal use in research and still obtain required data from a study, the routine use of positive control in every MN assay has been questioned by the scientific community, especially in laboratories which have demonstrated assay reproducibility and conduct studies under Good Laboratory Practice regulations. In this paper, mouse and rat vehicle and positive control MN data, collected manually, are described as a reference for a period of 12 years (1987-1998) in our laboratory. The vehicles generally included a variety of aqueous solutions and suspensions and cyclophosphamide dosed intraperitoneally at 20mg/kg (rats) or 40 mg/kg (mice) served as positive control, in all studies. Based on combined sex data (430 animals), for CD(1) mice, the vehicle control MN polychromatic erythrocyte (PCE) range was 0.9-3.1 with a mean of 1.75 per 1000 PCE and the positive control range (220 animals) was 8.8-42.1 with a mean of 23.1 MNPCE per 1000 PCE. Similarly, for Wistar rats, the vehicle control range (360 animals) was 1.3-5.3 with a mean of 2.6 MNPCE per 1000 PCE and the positive control range (240 animals) was 10.4-33.8 MNPCE per 1000 PCE. Vehicle control ranges reported here are comparable to the literature database and the positive control response was > or = 4-fold over vehicle control, in all studies. These data demonstrate the reproducibility of positive control response in MN assay in our laboratory and support the MN Assay Expert Panel's view that the use of positive control may not be necessary in every study.     

Refinement and reduction of acute oral toxicity testing: a critical review of the use of cytotoxicity data

Acute oral toxicity testing is still required for the classification and labelling of chemicals, agrochemicals and related formulations. There have been increasing efforts over the last two decades to reduce the number of animals needed for this testing, according to the Three Rs concept. To evaluate the utility of an in vitro cytotoxicity test in our routine testing for acute oral toxicity, we have implemented in our laboratory the neutral red uptake (NRU) method, with Balb/c 3T3 fibroblasts after a 48-hour exposure, which was recommended in ICCVAM Report 07-4519, 2006. Initially, we tested 16 substances that had existing in vivo and in vitro data available, to prove our technical proficiency with the in vitro test. Then, testing was performed with 187 test substances, including a broad variety of chemicals, agrochemicals and formulations. The starting dose for acute oral systemic toxicity assays in rats (LD50) was estimated by using the prediction model presented in the ICCVAM validation study, and subsequently compared to the results obtained by in vivo testing performed according to, or similar to, OECD Test Guideline 423. Comparison of all of the 203 predicted LD50 values that were deduced from the in vitro IC50 values, with the in vivo results from oral toxicity studies in rats, resulted in a low overall concordance of 35%. The in vitro cytotoxicity assay achieved a good concordance of 74%, only for the weakly toxic substances (EU-GHS Cat. 4). However, it must be noted that 71% of the substances tested (i.e. 145/203) were classified as being weakly toxic in vitro. We further analysed the utility of the in vitro test for predicting the starting dose for an in vivo study, and the potential reduction in animal usage that this would engender. In this regard, the prediction by the cytotoxicity test was useful for 59% of the substances. However, the use of a standard starting dose of 300 mg/kg bw by default (without previous cytotoxicity testing) would have been almost as useful (50%). In contrast, the prediction by an experienced toxicologist was correct for 95% of the substances. However, this was only performed for 40% of the substances, mainly those of no to low toxicity. Calculating the theoretical animal numbers needed in several scenarios supported these results. The additional analysis, considering some physicochemical data (solubility, molecular weight, log POW), substance class and mode of action, revealed no specific applicability domains. In summary, the use of the 3T3 NRU cytotoxicity data alone did not sufficiently contribute to refinement and reduction in the acute oral toxicity testing of the substance portfolio tested routinely in our laboratory.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

SFTG international collaborative study on in vitro micronucleus test V. Using L5178Y cells

In this report, results are presented from an international study of the in vitro micronucleus assay using mouse lymphoma L5178Y cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, 5-fluorouracil, colchicine and griseofulvin. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in mouse lymphoma L5178Y cells. Mannitol was the only exception, being tested in only one laboratory. Mannitol was negative, while bleomycin induced a concentration-dependent increase in micronucleated cells. Equivocal results were obtained for 5-fluorouracil, colchicine and griseofulvin. High levels of cytotoxicity interfered with the assessment of aneuploidy for colchicine and griseofulvin, preventing the ability to obtain clear results in all the treatment schedules. Experiments with 5-fluorouracil, colchicine and griseofulvin showed that both short and long treatment times are required as each compound was detected using one or more treatment protocol. No clear differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. It was also found that a recovery period may help to detect compounds which induce a genotoxicity associated to a reduction in cell number or cell proliferation. Overall, the results of the present study show that mouse lymphoma L5178Y cells are suitable for the in vitro micronucleus assay.     

Ethylene thiourea (ETU). A review of the genetic toxicity studies

Ethylene thiourea (ETU) is a common contaminant, metabolite and degradation product of the fungicide class of ethylene bisdithiocarbamates (EBDCs); as such, they present possible exposure and toxicological concerns to exposed individuals. ETU has been assayed in many different tests to assess genotoxicity activity. While a great number of negative results are found in the data base, there is evidence that demonstrates ETU is capable of inducing genotoxic endpoints. These include responses for gene mutations (e.g. Salmonella), structural chromosomal alterations (e.g. aberrations in cultured mammalian cells as well as a dominant lethal assay) and other genotoxic effects (e.g. bacterial rec assay and several yeast assays).It is important to consider the magnitude of the positive responses as well as the concentrations/doses used when assessing the genotoxicity of ETU. While ETU induces a variety of genotoxic endpoints, it does not appear to be a potent genotoxic agent. For example, it is a weak bacterial mutagen in the Salmonella assay without activation in strain TA1535 at concentrations generally above 1000 μg/plate. Weak genotoxic activity of this sort is usually observed in most of the assays with positive results. Since ETU does not appear very potent and is not extremely toxic to test cells and organisms, it is not surprising to find that ETU does not produce consistent effects in many of the assays reviewed. Consequently, in many instances, mixed results for the same assay type are reported by different investigators, but as reviewed herein, these results may be dependent upon the test conditions in each individual laboratory. A primary shortcoming with many of the reported negative results is that the concentrations or doses used are not high enough for an adequate test for ETU activity. There are also problems with many of the negative assays generally in protocol or reporting, particularly with the in vivo studies (e.g. inappropriate sample number and/or sampling times; inadequate top dose employed).Overall, while ETU does not appear to be a potent genotoxic agent, it is capable of producing genotoxic effects (e.g. gene mutations, structural chromosomal aberrations). This provides a basis for weak genotoxic activity by ETU. Furthermore, based on a suggestive dominant lethal positive result, there may be a concern for heritable effects. Due to the many problems with the conduct and assessment of the in vivo assays, it is worth repeating in vivo     

Influence of cytotoxicity and compound precipitation on test results in the alkaline comet assay

We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug candidates. A dataset of more than 250 tests carried out with 75 drug candidates of various chemical classes was analyzed to elucidate the influence of cytotoxicity and compound precipitation on DNA migration in the comet assay. Using a V79 Chinese hamster cell line, 38 of the compounds were negative and 37 were positive in the comet assay. The reproducibility of test results between repeat experiments was 85%. Data on 72 tests with a negative call in which the compounds were tested up to highly cytotoxic concentrations demonstrated that cytotoxicity, as determined by Trypan blue dye exclusion and occurrence of cells with completely fragmented chromatin, did not lead to false positive test results. The majority (64.2%) of compounds with a positive call induced elevated DNA migration in the absence of excessive cytotoxicity. Compound precipitation was observed in 84 tests. In 88.1% of these cases, the test result at the precipitating concentration did not differ from that found at the highest soluble concentration. Half of the remaining 11.9% of contrary results (most of them weak effects) were not reproducible in the respective repeat experiment, indicating no or only a negligible influence of precipitation on test results. The data indicate that using V79 cells, the comet assay specifically detects genotoxic effects and is not confounded by cytotoxicity or compound precipitation under the conditions used.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles

One of the consequences of the low specificity of the in vitro mammalian cell genotoxicity assays reported in our previous paper [D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] is industry and regulatory agencies dealing with a large number of false-positive results during the safety assessment of new chemicals and drugs. Addressing positive results from in vitro genotoxicity assays to determine which are "false" requires extensive resources, including the conduct of additional animal studies. In order to reduce animal usage, and to conserve industry and regulatory agency resources, we thought it was important to raise the question as to whether the protocol requirements for a valid in vitro assay or the criteria for a positive result could be changed in order to increase specificity without a significant loss in sensitivity of these tests. We therefore analysed some results of the mouse lymphoma assay (MLA) and the chromosomal aberration (CA) test obtained for rodent carcinogens and non-carcinogens in more detail. For a number of chemicals that are positive only in either of these mammalian cell tests (i.e. negative in the Ames test) there was no correlation between rodent carcinogenicity and level of toxicity (we could not analyse this for the CA test as insufficient data were available in publications), magnitude of response or lowest effective positive concentration. On the basis of very limited in vitro and in vivo data, we could also find no correlation between the above parameters and formation of DNA adducts. Therefore, a change to the current criteria for required level of toxicity in the MLA, to limit positive calls to certain magnitudes of response, or to certain concentration ranges would not improve the specificity of the tests without significantly reducing the sensitivity. We also investigated a possible correlation between tumour profile (trans-species, trans-sex and multi-site versus single-species, single-sex and single-site) and pattern of genotoxicity results. Carcinogens showing the combination of trans-species, trans-sex and multi-site tumour profile were much more prevalent (70% more) in the group of chemicals giving positive results in all three in vitro assays than amongst those giving all negative results. However, single-species, single-sex, single-site carcinogens were not very prevalent even amongst those chemicals giving three negative results in vitro. Surprisingly, when mixed positive and negative results were compared, multi-site carcinogens were highly prevalent amongst chemicals giving only a single positive result in the battery of three in vitro tests. Finally we extended our relative predictivity (RP) calculations to combinations of positive and negative results in the genotoxicity battery. For two out of three tests positive, the RP for carcinogenicity was no higher than 1.0 and for 2/3 tests negative the RP for non-carcinogenicity was either zero (for Ames+MLA+MN) or 1.7 (for Ames+MLA+CA). Thus, all values were less than a meaningful RP of two, and indicate that it is not possible to predict outcome of the rodent carcinogenicity study when only 2/3 genotoxicity results are in agreement.     

Compilation and use of genetic toxicity historical control data

The optimal use of historical control data for the interpretation of genotoxicity results was discussed at the 2009 International Workshop on Genotoxicity Testing (IWGT) in Basel, Switzerland. The historical control working group focused mainly on negative control data although positive control data were also considered to be important. Historical control data are typically used for comparison with the concurrent control data as part of the assay acceptance criteria. Historical control data are also important for providing evidence of the technical competence and familiarization of the assay at any given laboratory. Moreover, historical control data are increasingly being used to aid in the interpretation of genetic toxicity assay results. The objective of the working group was to provide generic advice for historical control data that could be applied to all assays rather than to give assay-specific recommendations. In brief, the recommendations include:     

Establishment and Assessment of a New Human Embryonic Stem Cell‐Based Biomarker Assay for Developmental Toxicity Screening

A metabolic biomarker‐based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery‐phase detection of potential human developmental toxicants. In this study, metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure‐based biomarker assay using these metabolites, along with a cytotoxicity endpoint, was then developed using a 9‐point dose–response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity, an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy, but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity, 100% specificity). The assay had a high concordance (≥75%) with existing in vivo models, demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests.     

The effect of reducing the number of cells scored on the performance of the in vivo rat liver UDS assay

The most labour-intensive feature of the in vivo rat liver UDS assay is the scoring of hepatocyte autoradiograms by microscope. Even with image analyser and computer equipment the scoring phase of a full study might require half of the technical effort applied. Practice recommended by guidelines has been to score 50 cells/slide and two slides per animal. Now sufficient data have been accumulated, an evaluation was made to observe whether this procedure was necessary. An analysis of the accumulated UDS database in our laboratory was made to determine the sources of variability of mean net nuclear grain count, [N - C]. It was observed that the two largest components of variation in negative control animal mean [N - C]. were between-day and interanimal variability. The contribution from sampling error during slide scoring was relatively small. Theoretical calculations showed that the greater sampling error derived from scoring 30 rather than 50 cells/slide would result in only a marginal increase in total assay variation. To test this, 30 cells/slide were randomly selected from the 50 cells scored originally in negative control animals in each of 18 studies over an 18-month period. It was confirmed that reducing the number of cells had a negligible effect on the variation of negative control animal mean [N - C] values. Furthermore, analysis of data from 10 more studies confirmed that within-study variation would be essentially unaffected by scoring 30 cells/slide. The use of 30 rather than 50 cells per slide (a total of 60 cells per animal) has therefore been adopted for all current studies and scoring procedures modified to avoid operator bias during the selection of a smaller number of cells.     

Validation of an automatic comet assay analysis system integrating the curve fitting of combined comet intensity profiles

In recent years, the single-cell gel electrophoresis (comet) assay has become a reference technique for the assessment of DNA fragmentation both in vitro and in vivo at the cellular level.In order to improve the throughput of genotoxicity screening, development of fully automated systems is clearly a must. This would allow us to increase processing time and to avoid subjectivity brought about by frequent manual settings required for the ‘classical’ analysis systems.To validate a fully automatic system developed in our laboratory, different experiments were conducted in vitro on murine P388D1 cells with increasing doses of ethyl methanesulfonate (up to 5 mM), thus covering a large range of DNA damage (up to 80% of DNA in the tail). The present study (1) validates our ‘in house’ fully automatic system versus a widely used semi-automatic commercial system for the image-analysis step, and versus the human eye for the image acquisition step, (2) shows that computing tail DNA a posteriori on the basis of a curve fitting concept that combines intensity profiles [G. Dehon, P. Bogaerts, P. Duez, L. Catoire, J. Dubois, Curve fitting of combined comet intensity profiles: a new global concept to quantify DNA damage by the comet assay, Chemom. Intell. Lab. Syst. 73 (2004) 235–243] gives results not significantly different from the ‘classical’ approach but is much more accurate and easy to undertake and (3) demonstrates that, with these increased performances, the number of comets to be scored can be reduced to a minimum of 20 comets per slide without sacrificing statistical reliability.