Mouse Lymphoma Tk Assay Research Articles

Genotoxicity assessment of cellulose nanofibrils using a standard battery of in vitro and in vivo assays

Cellulose nanofibrils (CNFs) are identified as novel nanomaterials with many potential applications. Since CNFs are fibrous manufactured nanomaterials, their potential carcinogenic effects and mesothelial toxicity raise some concerns. In this study, we conducted a standard battery of in vitro and in vivo assays to evaluate the genotoxicity of two CNF types using different manufacturing methods and physicochemical properties. Namely, one was CNF produced via chemical modification by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation, while the other was CNF produced via mechanical defibrillation using needle bleached kraft pulp. A bacterial reverse mutation test and a mouse lymphoma TK assay revealed that CNFs at 100 μg/mL did not induce bacterial reverse mutations and in vitro mammalian cell gene mutation. Further, in vitro chromosomal aberration tests demonstrated that CNFs at 100 μg/mL did not induce chromosomal aberration in Chinese hamster lung fibroblasts. From the mammalian erythrocyte micronucleus test, no statistically significant increase was observed in the proportion of micronucleated polychromatic erythrocytes in the bone marrow cells of rats intratracheally instilled with any concentration of CNFs (0.25–1.0 mg/kg) compared with values from respective negative control groups. Therefore, this battery of in vitro and in vivo assays illustrated that the CNFs examined in this study did not induce genotoxicity, suggesting our results provide valuable insight on the future use of these materials in various industrial applications.

Absence of genotoxicity of purified Aloe vera whole leaf dry juice as assessed by an in vitro mouse lymphoma tk assay and an in vivo comet assay in male F344 rats.

Hydroxyanthracene derivatives (HAD) are naturally present in the latex layer of Aloe vera leaf, predominantly as aloins A, B and aloe-emodin. HAD are typically removed from commercial ingestible aloe products through activated charcoal filtration (decolorization). Current research aimed to evaluate genotoxic potential of a purified aloe whole leaf dry juice containing 0.3 ppm of total aloins and non-detectable aloe-emodin (LOD =0.01 ppm) in the L5178Y mouse lymphoma assay (MLA; OECD 490) and in vivo comet assay (OECD 489). No marked increases in mutant frequency at the tk locus were observed in the MLA at concentrations up to 5000 μg/mL for 3 h and 24 h (-S9), and up to a precipitating concentration of 3000 μg/mL for 3 h (+S9) compared to concurrent vehicle control. Relative total growth at the highest analyzable concentrations at 3 h (±S9) and 24 h (-S9) ranged from 64 to 133 %. In the comet assay, no statistically significant increases in DNA strand breaks were detected in the colon or kidney following oral gavage of 500, 1000 or 2000 mg/kg/day in male F344 rats for 2 days compared to concurrent vehicle control. Overall, these findings demonstrated the test article containing minimal HAD is not genotoxic under the described experimental conditions.

Safety evaluation of collagenase from Streptomyces violaceoruber

A safety assessment was conducted for microbial collagenase (COL) enzyme expressed in Streptomyces violaceoruber. The acute oral toxicity of COL was examined in Sprague-Dawley rats and the LD50 of COL via single oral administration to rats was higher than 2000 mg/kg body weight. A 13-week oral gavage study of COL showed no adverse effects due to the enzyme up to a dose of 234.9 mg total organic solids (TOS)/kg body weight per day (NOAEL). A bacterial reverse mutation test showed no mutagenic activity at the highest dose (4698 μg TOS per plate). In the mouse lymphoma TK assay, a positive result was observed at the highest dose of 4698 μg TOS/mL although it had low reproducibility. To confirm the chromosome aberration potential, an in vivo micronucleus test was conducted that demonstrated the lack of mutagenic potential on the bone marrow of rats at doses up to 1879 mg TOS/kg body weight per day. The results of the genotoxicity studies and acute and subchronic rat studies support the safe use in food production of collagenase produced from S. violaceoruber.

In vitro and in vivo genotoxicity studies on monosodium L-glutamate monohydrate

In response to the lack of authenticated mutagenicity/genotoxicity studies on MSG monohydrate, a series of genotoxicity studies conducted under GLP and according to globally accepted test guidelines (e.g., OECD) was performed. A bacterial reverse mutation test using Salmonella typhimurium (TA100, TA1535, TA98 and TA1537) and Escherichia coli (WP2 uvrA) at concentrations up to 5000 μg/plate, an in vitro chromosomal aberration test in CHL/IU cells at concentrations up to 10 mmol/L (1.9 mg/mL), a mouse lymphoma tk assay at concentrations up to 10 mmol/L (1.9 mg/mL), an in vitro micronucleus test in human peripheral blood lymphocytes at concentrations up to 10 mmol/L (1871 μg/mL), and an in vivo micronucleus test in bone marrow of rats that were gavaged with up to 2000 mg/kg bw were investigated. MSG monohydrate did not cause mutagenicity in any bacterial strain, did not induce chromosomal aberrations in CHL/IU cells or gene mutation in mouse lymphoma cells, was not clastogenic or aneugenic to human lymphocytes, and did not induce micronuclei in erythrocytes of rats when compared with vehicle controls. These results show that MSG is not mutagenic or genotoxic under the study conditions.

Genotoxic and mutagenic potential of camphorquinone in L5178/TK+/− mouse lymphoma cells

ObjectivesCamphorquinone (CQ) is the most important photoinitiator used in dental composite resins. Sparse data indicate a mutagenic potential of CQ. Therefore, it was aim of this study to evaluate the cytotoxicity, genotoxicity, and mutagenicity of CQ in L5178Y TK+/− mouse lymphoma cells. MethodsL5178Y/TK+/− cells were exposed to different concentrations of non-irradiated CQ (0.25–2.5mM). Cytotoxicity was evaluated by propidium iodide assay, determination of suspension growth rate, relative total growth and the mitotic index. Intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) were quantified by 2′,7′-dichlorofluoresceine diacetate (DCFH-DA). Early induction of DNA strand breaks and oxidative DNA base lesions was assessed using the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified alkaline comet assay, whereas mutagenicity of CQ was determined in the mouse lymphoma TK assay (MLA), according to OECD Guideline No. 490. ResultsCQ (0.5–2.5mM) induced concentration- and time-dependent inhibition of cell growth associated with increased ROS/RNS production, amounting to 2342%±1108% of controls after 90min at 2.5mM. Additionally, CQ concentration-dependently caused direct DNA-damage, i.e. formation of DNA strand breaks and 8-hydroxy-2′-deoxyguanosine. Whereas the MLA indicated lack of mutagenicity of CQ after a 4h of treatment, CQ concentration-dependently increased total mutant frequency (MF) after 24h (about 2-fold at 2.5mM). But, based on the global evaluation factor concept, increase in MF did not reach biologically relevance. SignificanceCQ induced concentration-dependent, cytotoxic and genotoxic effects in L5178Y/TK+/− cells, most likely due to oxidative stress, but without mediating obvious biological relevant mutagenicity.

3D-human small intestinal tissue to study ligand-induced acute and chronic inflammation in the gastrointestinal tract

Camphorquinone (CQ) is the most important photoinitiator used in dental composite resins. Sparse data indicate a mutagenic potential of CQ. Therefore, it was aim of this study to evaluate the cytotoxicity, genotoxicity, and mutagenicity of CQ in L5178Y TK+/− mouse lymphoma cells.L5178Y/TK+/− cells were exposed to different concentrations of non-irradiated CQ (0.25–2.5 mM). Cytotoxicity was evaluated by propidium iodide assay, determination of suspension growth rate, relative total growth and the mitotic index. Intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) were quantified by 2′,7′-dichlorofluoresceine diacetate (DCFH-DA). Early induction of DNA strand breaks and oxidative DNA base lesions was assessed using the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified alkaline comet assay, whereas mutagenicity of CQ was determined in the mouse lymphoma TK assay (MLA), according to OECD Guideline No. 490.CQ (0.5–2.5 mM) induced concentration- and time-dependent inhibition of cell growth associated with increased ROS/RNS production, amounting to 2342% ± 1108% of controls after 90 min at 2.5 mM. Additionally, CQ concentration-dependently caused direct DNA-damage, i.e. formation of DNA strand breaks and 8-hydroxy-2′-deoxyguanosine. Whereas the MLA indicated lack of mutagenicity of CQ after a 4 h of treatment, CQ concentration-dependently increased total mutant frequency (MF) after 24 h (about 2-fold at 2.5 mM). But, based on the global evaluation factor concept, increase in MF did not reach biologically relevance.CQ induced concentration-dependent, cytotoxic and genotoxic effects in L5178Y/TK+/− cells, most likely due to oxidative stress, but without mediating obvious biological relevant mutagenicity.

Toxicological assessment of kretek cigarettes Part 6: The impact of ingredients added to kretek cigarettes on smoke chemistry and in vitro toxicity

Mainstream smoke (MS) from experimental kretek cigarettes with three ingredient mixes at low (typical use level) and high (2.5 or 3 times that level) inclusion rates was compared to a control kretek cigarette of identical construction (cloves and humectants), but without the addition of ingredients. 350 ingredients, commonly used in various combinations and in a limited number in a given brand in the manufacture of marketed kretek cigarettes were assessed. The MS composition of the kretek cigarettes was characterized by a comprehensive set of analytes (55 smoke constituents). Furthermore, the smoke was assessed in vitro for its cytotoxicity in the Neutral Red Uptake assay (particle phase and gas/vapor phase separately) in mouse embryo BALB/c 3T3 cells, and for mutagenicity/genotoxicity in the Salmonella typhimurium reverse mutation assay and the mammalian cell mouse lymphoma TK assay in L5178Y cells, the latter with and without metabolic activation. There were some statistically significant differences in the yield of smoke constituents (increases as well as decreases, nearly all of them less than ±20%) as a result of the addition of the ingredient mixes. However, the addition of the three different mixes of ingredients to the experimental kreteks did not change the in vitro cytotoxicity and mutagenicity/genotoxicity of the smoke, when compared to the control kretek cigarette.

Safety Study of Culinary-Medicinal Royal Sun Agaricus, Agaricus brasiliensis S. Wasser et al. KA21 (Higher Basidiomycetes) Assessed by Prokaryotic as well as Eukaryotic Systems

As part of the safety evaluation of culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis KA21, for human consumption, we performed the bacterial reverse mutation test, the mouse micronucleus test, and mouse lymphoma test using A. brasiliensis KA21 as the test substance. The reverse mutation test utilized five bacterial strains, including Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2 uvrAO. For the micronucleus test we used mice. For the mouse lymphoma test, we used one of the most commonly used mammalian cell mutagenesis systems; the L5178YTK +/- mouse lymphoma-TK assay detects the mutations at the thymidine kinase locus caused by base-pair changes, frameshift, and small deletions. All the tests were conducted according to the guidelines for genotoxicity testing of drugs by the Ministry of Health, Labor, and Welfare, Japan. In the bacterial reverse mutation test, no toxicity was observed up to a dose of 5,000 μg/plate. In the mouse micronucleus test, no toxicity was noted up to a dose of 1 g/kg body weight. In the mouse lymphoma test, frequency of the mutation was equal both in the presence or absence of KA21. Supporting the long history of human consumption of A. brasiliensis, the data shown in this study strongly indicate the safety of this mushroom.

The ability of the mouse lymphoma TK assay to detect aneugens

There is some evidence that the mouse lymphoma TK assay (MLA) can detect aneugens, and this is accepted in the current International Conference on Harmonisation guidance for testing pharmaceuticals. However, whether or not it can be used as a reliable screen for aneugenicity has been the subject of debate. Consequently, aneugens with diverse mechanisms of action were tested in the MLA using 24-h exposure. No evidence of increased mutant frequency was seen with noscapine, diazepam or colchicine and increases were seen with taxol, carbendazim, econazole and chloral hydrate only at high levels of toxicity (for all but one taxol concentration survival reduced to ≤10% of control). None of these agents would be unequivocally classified as positive using currently accepted criteria. The largest increases in mutant number were seen with taxol and carbendazim; therefore, trifluorothymidine (TFT)-resistant clones resulting from treatment with them were cultured and analysed for chromosome 11 copy number using fluorescent in situ hybridisation (FISH) and loss of heterozygosity (LOH). High concentrations of these aneugens induced LOH at all loci examined indicating only one chromosome 11 was present but, perhaps surprisingly, all were found to have two copies of chromosome 11 using FISH. This would be consistent with loss of the tk(+) chromosome 11b with concomitant duplication of chromosome 11a, which has been proposed as a likely mechanism for induction of TFT-resistant clones. However, it was also surprising that analysis of centromere size showed that almost all the clones had both small and large centromeres, i.e. suggesting the presence of both chromosomes 11a and 11b. In conclusion, it appears that the TFT-resistant mutants resulting from treatment with toxic concentrations of some aneugens such as taxol and carbendazim have undergone complex genetic changes. However, these data show that the MLA cannot be used as a routine screen to detect aneugens.

The incidence of positive results in the mouse lymphoma TK assay (MLA) in pharmaceutical screening and their prediction by MultiCase MC4PC

There are published data indicating that the mouse lymphoma TK assay (MLA) has an unacceptably high incidence of positive results, hence it was decided to review the MLA data generated in this laboratory for potential drug candidates. Of the 355 compounds tested, only 52 (15%) gave positive results so, even if it is assumed that all of these are non-carcinogens, the incidence of 'false positive' predictions of carcinogenicity is much lower than the 61% apparent from analysis of the literature. Furthermore, only 19 compounds (5%) were positive by a mechanism that could not be associated with the compounds primary pharmacological activity or positive responses in other genotoxicity assays. It should be noted that the majority of these compounds were not bacterial mutagens so, in most cases, the positive results were an additional indicator of genotoxicity. However, data are not available to assess any risk they might present. At least for pharmaceuticals, it appears that the MLA does not generate as many positive results as is commonly believed, and it is against this incidence that the performance of other in vitro genotoxicity tests should be compared. The predictive accuracy of the program MultiCase MC4PC was also examined using these results. The sensitivity and specificity were found to be 62 and 38%, respectively; in fact, 62% of all compounds were predicted to be positive irrespective of whether they were actually positive or negative. It was concluded that, in its current state of development, M4PC cannot be considered sufficiently accurate to be used to predict the activity of pharmaceuticals in the MLA.

A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins

In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames + MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames + MNvit – benzyl acetate, toluene, morphine and thiabendazole – and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames + MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames + MNvit.

Multi-endpoint genotoxic assay using L5178Y (Tk+/- -3.7.2c) cells

When the mouse lymphoma Tk assay (MLA) provides a positive result, its cause can be roughly estimated by examining colony sizes. An increase in the number of large colonies means that the compound tested has point mutational potential, while an increase in small colonies indicates the potential for chromosome aberration. However, it was found to be difficult to clearly judge this in the case of caffeine known as a clastogen lacking the potential of point mutation. In our study, caffeine significantly increased the thymidine kinase (Tk) mutation frequencies derived from large colonies as well as those from small colonies in the standard protocol, although the frequencies derived from a small colony were higher than those from large colonies at higher doses. Therefore, we prolonged the expression period from 2 days, a standard period, to 6 days after treatment and then examined the Tk and Hprt mutations simultaneously. The result showed that caffeine gave a completely negative result on a mutation test for both Tk and Hprt. On the other hand, ethyl methanesulfonate (EMS), a genotoxic carcinogen, showed a positive result for both. Moreover, caffeine and EMS significantly increased the frequencies of micronucleated cells. In conclusion, when MLA gives a positive result and the cause is ambiguous, in order to identify the exact cause of the positive response, it is helpful to perform a confirmatory test investigating the potential of Tk and Hprt gene mutation simultaneously after 6-day expression and to perform an in vitro micronucleus assay during the expression period.

Testing strategies in mutagenicity and genetic toxicology: An appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, 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] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, 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] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.

Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24 h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320 mg/kg in mice and 2000 mg/kg in rats: Muta™Mouse assay in colon and liver (5×320 mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [ 14 C ]-AMP397; comet assay (1×2000 mg/kg) in jejunum and liver of rats, sampling times 3 and 24 h after administration; micronucleus test (2×320 mg/kg) in the bone marrow of mice, sampling 24 h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.

Appropriate levels of cytotoxicity for genotoxicity tests using mammalian cells in vitro

Among standard battery genotoxicity assays, the in vitro chromosome aberration test and the mouse lymphoma tk assay (MLA) yield about fourfold higher incidences of positive test results than the bacterial reverse mutation test or in vivo bone marrow tests. This is a result of experience with submissions of 335 new pharmaceuticals to the German Federal Institute for Drugs and Medical Devices. While all of the standard systems have their value in detecting relevant genotoxins, there is no supportive evidence for DNA reactivity for a considerable number of in vitro clastogens and MLA positives. In particular the clastogenic response of such compounds is often associated with high cytotoxicity. This may invoke the need to change the approach to test for clastogenicity in vitro. A combination of measures such as (1) a change in the upper limits of cytotoxicity that are currently given in International Conference on Harmonisation (ICH) and Organization for Economic Co-Operation & Economic Development (OECD) guidelines, (2) the creation of a common ground of understanding for interpretation of in vitro (positive) test results, and (3) lowering the upper limits of test compound concentration irrespective of cytotoxicity may prove useful to ensure a sufficient reliability of genotoxicity testing with mammalian cells in vitro.

Appropriate levels of cytotoxicity for genotoxicity tests using mammalian cells in vitro

Among standard battery genotoxicity assays, the in vitro chromosome aberration test and the mouse lymphoma tk assay (MLA) yield about fourfold higher incidences of positive test results than the bacterial reverse mutation test or in vivo bone marrow tests. This is a result of experience with submissions of 335 new pharmaceuticals to the German Federal Institute for Drugs and Medical Devices. While all of the standard systems have their value in detecting relevant genotoxins, there is no supportive evidence for DNA reactivity for a considerable number of in vitro clastogens and MLA positives. In particular the clastogenic response of such compounds is often associated with high cytotoxicity. This may invoke the need to change the approach to test for clastogenicity in vitro. A combination of measures such as (1) a change in the upper limits of cytotoxicity that are currently given in International Conference on Harmonisation (ICH) and Organization for Economic Co-Operation & Economic Development (OECD) guidelines, (2) the creation of a common ground of understanding for interpretation of in vitro (positive) test results, and (3) lowering the upper limits of test compound concentration irrespective of cytotoxicity may prove useful to ensure a sufficient reliability of genotoxicity testing with mammalian cells in vitro. Environ. Mol. Mutagen. 35:202–205, 2000 © 2000 Wiley-Liss, Inc.

ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact.

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces. Two ICH guidances regarding genotoxicity testing have been issued: S2A, 'Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, 'Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA. These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches. Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing. Although these guidances were previously released within the participating regions in their respective regulatory communiqués, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described. The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort. This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay. This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication. The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary.

Applications and interpretation of data obtained in the mouse lymphoma tk assay

Applications and interpretation of data obtained in the mouse lymphoma tk assay

1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay, but is in mouse liver micronucleus assay.

1,4-Dioxane, an animal carcinogen, was not previously genotoxic in in vitro assays. We reevaluated the compound's genotoxic potential in five in vitro genotoxicity tests in the presence and absence of S9 mix using recommended new protocols. We used the bacterial reverse mutation assay with Salmonella TA and E. coli WP2 strains, including the plate and preincubation methods, the CHO chromosomal aberration assay, including examination of polyploid induction and extended sampling time, the CHO sister-chromatid exchange assay with short and long treatment time, the mouse lymphoma tk assay (microtiter method), including longer treatment time (24 hr), and the CHO micronucleus assay with short and long treatment times. The highest concentration we used was five mg/ml or plate. We also evaluated the genotoxic effect of 1,4-dioxane in vivo by conducting peripheral blood and liver micronucleus assays in the same mice after single oral administration of up to 3,000 mg/kg. All in vitro assays and the peripheral blood micronucleus assay were negative. The mouse liver micronucleus assay, on the other hand, was positive, indicating that 1,4-dioxane might be genotoxic. It is also conceivable that the positive result in mouse liver micronucleus assay was due to a nongenotoxic mechanism, i.e., errors in genetic repair following enhancement of hepatocyte proliferation.

P XIX.2 - P XIX.2 The mouse lymphoma TK assay (MLA): experiences with continuous (24 hour) treatment incubation

P XIX.2 - P XIX.2 The mouse lymphoma TK assay (MLA): experiences with continuous (24 hour) treatment incubation