In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances.

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.

Report from the In Vitro Micronucleus Assay Working Group.

At the Washington International Workshop on Genotoxicity Test Procedures (March 25-26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA-specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one-third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis-block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 microg/ml [lymphocytes cultured from whole blood cells] and 3-6 microg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages.

Threshold effects in genetic toxicity: perspective of chemicals regulation in Germany.

In the regulation of chemical substances, it is generally agreed that there are no thresholds for genotoxic effects of chemicals, i.e. , that there are no doses without genotoxic effects. When classifying and labelling chemicals, dangerous properties of chemicals are to be identified. In this context, in general, the mode of action (threshold or not) is not considered for genotoxic substances. In the process of quantitative risk assessment, however, determination of the type of dose-effect relationships is decisive for the outcome and the type of risk management. The presence of a threshold must be justified specifically in each individual case. Inter alia, the following aspects may be discussed in this respect: aneugenic activity, indirect modes of action, extremely steep dose-effect relationships in combination with strong toxicity, specific toxicokinetic conditions which may lead to 'metabolic protection' prior to an attack of DNA. In the practice of the regulation of chemical substances with respect to their genotoxic effects, the discussion of thresholds has played a minor role. For notified new substances, there are, in general, no data available that would allow a reasonable discussion. Concerning substances out of the European programme on existing substances, so far 29 have been assessed in our institute with respect to genetic toxicity. Eight out of these have shown considerable evidence for genotoxicity. For two of them, a possible threshold is discussed: one substance is an aneugen, the other one is metabolised to an endogenic compound with genotoxic potential. In the practice of risk assessment of genotoxic substances, the discussion of the mode of action for genotoxicity is frequently associated with the evaluation of potential carcinogenic effects. Here, tissue-specific genotoxic effects in target organs for carcinogenicity are to be discussed. Moreover, the contribution of genotoxicity to the multifactorial process of tumour development should be assessed.

Chemically induced micronucleus formation in V79 cells--comparison of three different test approaches.

The in vitro micronucleus test (MNT) is a useful assay for the detection of mutagenic events on both the chromosomal and the genomic level. The main disadvantage for introducing the in vitro MNT into official test guidelines seems to be the disparity of existing protocols. To contribute to the aim of standardisation, three different methodological approaches of the in vitro MNT with V79 cells were compared: the standard assay using an asynchronically growing mixed cell population, the cytokinesis block (CB) assay and a modified MNT, the so-called mitotic shake-off (MSO) method. V79 cells were thus treated with two known aneugens (colcemide and griseofulvin) and two clastogens (mitomycin C and cyclophosphamide) over various time periods. The cultures of the CB assay were additionally exposed to cytochalasin B (Cyt-B), an inhibitor of cell, but not of nucleus division. After treatment, the cells were harvested and analysed for the appearance of micronuclei (MN). All three assays yielded positive results for all test substances. These results support the suitability of the MNT with V79 cells with regard to the ability to detect the genotoxic potential of both clastogens and aneugens independent of the test protocol applied. Thus, all three methods are appropriate for MN detection, but due to the fact that the application of Cyt-B has no advantages for a cell line like V79 in which nearly all cells undergo a normal cell cycle, its use is not recommended.

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in V79 cells by enantiomeric epoxides.

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate the influence of the stereoisomeric forms of epoxides in mammalian genotoxicity tests. The SCE-inducing potency of 12 pairs of (R)- and (S)-enantiomeric epoxides which differed in the degree of substitution of the oxirane ring was determined. Of these, 2 pairs of epoxides failed to induce SCE. Different SCE-inducing potencies between the (R)- and (S)-enantiomers were shown for 5 epoxides. This study demonstrates that stereoselectivity might play an important role in genotoxicity testing of chemicals with asymmetric C atoms.

Report of a comparative study of DNA damage and repair assays in primary rat hepatocytes with five coded chemicals.

We report the results of a collaborative study for the detection of chemical-induced DNA damage in primary cultures of rat hepatocytes. The methods include the detection of unscheduled DNA synthesis (UDS) with either autoradiography (5 laboratories) or liquid scintillation counting (2 laboratories) and the assessment of DNA single-strand breaks with the alkaline elution assay (1 laboratory). Interlaboratory standardization was omitted in order to prove the agreement of the assays under routine conditions. Five coded chemicals were tested. For 4 chemicals (2-acetylaminofluorene, thiourea, glycerine and potassium chloride) the UDS data were consistent in all laboratories, thus indicating a high consensus of the test systems applied in the different laboratories. Those 3 chemicals that were not expected to elicit genotoxic activity (thiourea, glycerine, and potassium chloride) yielded negative results in all laboratories. 2-Acetylaminofluorene, a known DNA-damaging agent in hepatocytes, gave strongly positive responses in all laboratories. In contrast, N-nitrosodiphenylamine led to equivocal responses.

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in Chinese hamster V79 cells.

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate structure-activity relationships of epoxides in mammalian cells. For this purpose the SCE-inducing potency of 58 epoxides was determined. Of these, 16 failed to induce SCE in V79 cells. According to the substitution of the oxirane ring the results show general agreement with results obtained in the Ames test. Mono-substituted epoxides had the highest genotoxic potency compared to di- and tri-substituted epoxides. In detail, there are differences in genotoxic potency between bacteria and mammalian cells which can be explained by differences in the cellular uptake of the compounds and by detoxification reactions.

Induction of DNA-repair synthesis in primary rat hepatocytes by epoxides.

The genotoxicity of 10 epoxides was investigated in the UDS test with primary rat hepatocytes. The sensitivity of the assay was demonstrated using 2-acetylaminofluorence. The epoxides 1,2-epoxyoctane, 1,2-epoxydecane, epoxycyclooctane, epoxycyclododecane, (+)-limoneoxide, alpha-pinaneoxide, transstilbeneoxide, and cis-2,3-epoxysuccinic acid, which are known to be non-mutagenic in the Ames test, as well as the bacterial mutagen, 1,2-epoxyphenoxypropane did not induce UDS in primary hepatocytes of the rat. However, a positive UDS response obtained with glycidyltrimethylammonium chloride showed that metabolic inactivation of the oxirane ring in hepatocytes is influenced by further structural substituents.

Epoxides: comparison of the induction of SOS repair in Escherichia coli PQ37 and the bacterial mutagenicity in the Ames test.

The genotoxicity of 51 epoxides is studied with the SOS-Chromotest using Escherichia coli PQ37 as tester strain. The results obtained with this test system are compared with results of the Ames test. Out of 51 epoxides, 39 are shown to be mutagenic in Salmonella typhimurium whereas only 27 mutagenic epoxides induced the SOS response in Escherichia coli PQ37.

Evaluation of the SOS chromotest.

In the present investigation, the SOS chromotest with E. coli PQ37 was evaluated. The potential to identify different kinds of bacterial mutagens was examined. 124 chemicals of different chemical classes were tested. Their responses in the SOS chromotests were compared to reported test results obtained with the Ames test.

Enhancement and inhibition of benzo[a]pyrene-induced SOS function in E. coli by synthetic antioxidants.

8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo[a]pyrene-induced SOS function. None of the antioxidants leads to increased beta-galactosidase activity by itself. Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo[a]pyrene-induced SOS function at benzo[a]pyrene concentrations between 10(-6) M and 3 X 10(-5) M. Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo[a]pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene. Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo[a]pyrene action. In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo[a]pyrene. Preliminary data suggest that the apparent suppression of benzo[a]pyrene-induced SOS function is not due to an effect on the formation of benzo[a]pyrene metabolites by the metabolizing system used.

Screening of the food additive propionic acid for genotoxic properties.

Genotoxic properties of the food additive propionic acid were analysed using the Escherichia coli DNA repair assay, the SOS chromotest, the Salmonella/microsome mutagenicity test, the sister chromatid exchange test in vitro and the micronucleus test in vivo. All tests except the DNA repair assay with E. coli yielded negative results. These data support other evidence that propionic acid is not mutagenic and that genotoxic events are unlikely to be the cause of forestomach lesions in rats fed propionic acid in the diet (Griem, Bundesgesundheitsblatt 1985, 28, 322).

Genotoxicity of three-carbon compounds evaluated in the SCE test in vitro.

Three-carbon chemicals (chlorinated and nonchlorinated, saturated and unsaturated, hydroxy- and oxo-hydrocarbons) were assayed for genotoxicity. The sister chromatid exchange test in vitro served as the test system. Without S9 mix, the nonchlorinated solvents 1-propanol, 2-propanol, and 2-propanone (acetone) did not increase the SCE frequencies. All chlorinated 3-C hydrocarbons, except 1,2,3-trichloropropane, proved to be potent SCE inducers in V79 cells without S9 mix. In the presence of S9 mix, the results obtained with the nonchlorinated solvents were also negative, whereas 1,2,3-trichloropropane was transformed to SCE-inducing metabolites. The addition of S9 mix resulted in an increased SCE rate for 2,3-dichloropropanol, whereas genotoxicity of 2,3-dichloropropene, 1,2-dichloropropane, 1,3-dichloropropene, and 1,3-dichloropropanone was reduced. 1,3-dichloropropanol, 1,3-dichloropropene, and epichlorohydrin were substantially inactivated by S9 mix in the V79/SCE test. It can be concluded that the reactivity of the saturated dichloro compounds in the SCE test depends on the degree of oxidation. There is no general difference between the reactivity of alpha, beta-dichloro and alpha, omega-dichloro compounds.

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase locus, but not in the Na+/K+ ATPase locus in V79 hamster cells.

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase (HPRT) locus as measured by 6-thioguanine resistance (TGr) in V79 hamster cells. Alu I does not induce mutations in the Na+/K+ ATPase locus as measured by ouabain resistance (OUAr). The data are interpreted to mean that most if not all Alu I-induced TGr mutations represent chromosomal aberrations.

Bacterial mutagenicity of the tranquilizing constituents of Valerianaceae roots.

The valepotriates valtrate/isovaltrate and dihydrovaltrate are considered to be the main tranquilizing constituents of drugs derived from the roots of several Valerianaceae. The decomposition products of valtrate and isovaltrate include the metabolites baldrinal and homobaldrinal, respectively, whereas the decomposition products of dihydrovaltrate do not include baldrinal-like metabolites. Purified valtrate/isovaltrate, dihydrovaltrate, baldrinal and homobaldrinal were investigated for their genotoxic activity in the Salmonella/microsome test and the SOS-chromotest. The valepotriates developed mutagenic activity in these test systems only in the presence of S9 mix, whereas both baldrinals showed mutagenic effects in both tests with and without metabolic activation.

[The mutagenicity of caramel colors]. / Untersuchungen zur Mutagenität von Zuckercouleuren.

In the present study commercially available caustic and ammoniated caramel colours were tested for their mutagenic potential using the Ames assay. The test was performed using the standard test strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without a metabolic activation system (S9-mix). Furthermore, a special preincubation procedure without metabolic activation system was applied. None of the tested caramel colours showed any mutagenic effect in the Ames test.

[Mutagenicity of meat flavorings with natural and/or nature-identical flavorings]. / Untersuchungen zur Mutagenität von Fleischaromen mit natürlichen und/oder naturidentischen Aromastoffen.

The mutagenic potential of twenty commercially available meat flavours from three different producers was determined using the Ames test. The flavours contained natural and/or nature-identical components as recorded on their label. Twelve flavours showed mutagenic activity with at least one of the four employed test strains (TA1535, TA100, TA97, and TA98). Flavours containing natural components yielded positive results in this mutation test in all cases but one. On the other hand flavours produced from nature-identical substances with one exception did not demonstrate mutagenic effects.

In vitro mutagenicity of valepotriates.

Valepotriates are epoxide-bearing triesters of the monoterpene alcohol 4,7-dimethylcyclopenta-(c)-pyrane isolated from the roots of several Valerianacae species. They are regarded as the main tranquilizing constituents of these drugs. Although the valepotriates valtrate/isovaltrate (VAL) and dihydrovaltrate (DH-VAL) showed a strong alkylating activity against the nucleophilic agent 4-(p-nitrobenzyl)-pyridine (NBP), they were not clearly mutagenic for the strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium or for the strains WP2 and WP2 uvrA- of Escherichia coli in the absence of a metabolic activation system (S9-mix). However, the valepotriates were mutagenic for TA100, WP2 and WP2 uvrA- at concentrations up to about 1.0 mumole/plate when S9-mix was added to the test system. With more than 1 mumole/plate the valepotriates were toxic in the presence of a metabolic activation system for all strains tested. The mutagenicity of the valepotriates was inversely related to the protein content of the S9-mix used. The mutagenicity and toxicity of the valepotriates could be inhibited when the S9-mix was preincubated with the esterase inhibitor paraoxon (1 mM) for 5 min before the test compounds and bacteria were added. Therefore, bioactivation of the valepotriates by an enzymatic hydrolysis of their ester groups is considered. This could be proven by activating the valepotriates with purified esterase.

On the mutagenicity of metabolites derived from the mushroom poison gyromitrin.

The hepatotoxic and carcinogenic hydrazine N-methyl-N-formyl hydrazine (MFH), which is formed from the mushroom poison gyromitrin by hydrolytic cleavage in vivo and in vitro during food processing is much more mutagenic for the strain TA 100 of Salmonella typhimurium in the presence of a metabolic activation system than in its absence. On the other hand, acetylated MFH (Ac-MFH) was not mutagenic for TA 100 in both test conditions. For the strain TA 98 neither MFH nor Ac-MFH were mutagenic both with and without metabolic activation. Therefore, a metabolic conversion of the free NH2-moiety of MFH into a genotoxic metabolite of MFH is postulated.