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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given, acceptable for assessment

Data source

Reference Type:
Salmonella Mutagenicity Tests: IV. Results From the Testing of 300 Chemicals
Zeiger E, et al.
Bibliographic source:
Environ. Mol. Mutagen. 11, Suppl. 12, 1-158 (1988)

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
only four strains tested; no strain included to identify cross-linking mutagens
Principles of method if other than guideline:
Standard plate incorporation according to Ames et al. (1975). Results of 300 tested chemicals were listed in tabular form.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): 2,6-Xylidine
- Supplier: Ethyl Corporation,
- Analytical purity: 99.1 % (Analyzed by Midwest Research Institute, Kansas City, MO .)
- no further data.


Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat and hamster liver S9 mix (Aroclor-induced)
Test concentrations with justification for top dose:
10-3333 µg/plate
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: see details on test system and conditions
Details on test system and experimental conditions:
Tester strains
Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831 . Cultures were grown overnight with shaking at 37° C in Oxoid No.2 broth, and their phenotypes were analyzed prior to their use for mutagenicity assays.

Preparation of Liver S-9 Fractions
The S9 (9,000g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described previously [Haworth et al, 1983]. The S9 mixes were prepared immediately prior to use and contained either 10 % or 30 % S9; occasionally, other levels were used. All chemicals were tested in the absence of
metabolic activation and with rat and hamster S-9 fractions.

Test Protocol
The preincubation assay was performed as described previously [Haworth et. al., 1983], with some differences, as described below. The test chemical (0.05 ml), Salmonella culture (0.1 ml), and S-9 mix or buffer (0.5 ml) were incubated at 37°C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium [Vogel and Bonner, 1956]. The histidine-independent (his+) colonies arising on these plates were counted following two days incubation at 37°C. Plates were
machine counted (New Brunswick, Edison, NJ; Artek, Farmingdale, NY) unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand. Variations in the protocol among the tested chemicals reflect the evolution of the protocol originally described by Haworth et al. [1983] . Four protocol variations are evident from the data in Appendix 2 .
1) Testing in strains TA97, TA98, TA100, and TA1535, with some additional testing in strain TA1537 ; 10% S-9 was used.
2) The first test of a chemical was without activation and with 10 % S-9 in the S-9 mix. If a positive result was obtained the test was repeated. If the tests were negative they were repeated without S-9 and with 30 % S-9.
3) The order of use of 10% and 30% S9 was reversed.
4) Initial testing was in strains TA98 and TA 100 without activation and with 30 % rat and hamster S9s. If a positive result was obtained in one of these two strains it was repeated and the other strains were not used. If the tests were negative, the other strains were used with 30 % and10 % S9. A chemical was not designated non-mutagenic unless it had been tested in strains TA98, TA100, TA1535, and TA97 and/or TA 1537, without activation and with 10 % and 30 % rat and hamster S9. Occasionally, 5 % S9 was also used in all protocol variations. All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay . The toxicity assay was performed using TA 100 or the system developed by Waleh et al . [1982]. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both. Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary toxicity test. Subsequent trials occasionally used narrower dose increments and may not have included doses in the toxic range. Chemicals that were not toxic were tested, with few exceptions, to a maximum dose of 10 mg/plate. Chemicals that were poorly soluble were tested up to doses defined by their solubilities. At least five doses of each chemical were tested in triplicate. Experiments were repeated at least one week following the initial trial. A maximum of 0.05 ml solvent was added to each plate. Concurrent solvent and positive controls were run with each trial. The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA1537), and 4-nitro-o-henylenediamine
(TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
Occasionally, a laboratory used a single solvent and/or positive control for more than one chemical tested on the same day. Each laboratory made its own determination regarding positive control dose levels.
Evaluation criteria:
The data were evaluated as described previously [Zeiger et al. 1987]. Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+W), questionable (?), or non-mutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of "+W," if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a non-mutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach twofold over background for a chemical to be judged mutagenic. A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a "+W" response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged non-mutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or non-mutagenicity.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium; TA 97, 98, 100, 1535, 1537
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
Untreated negative controls validity:
not examined
Positive controls validity:
Remarks on result:
other: other: Salmonella typhimurium; TA 97, 98, 100, 1535, 1537
Migrated from field 'Test system'.

Any other information on results incl. tables

2,6-xylidine produced only weak positve results with the tester strains investigated. Results of 3 independent laboratories judged the test substance as weakly positive ( Microbiological Associates, Inc . (MIC) and SRI International (SRI)) or negative (Case Western Reserve University (CWR) ).

Applicant's summary and conclusion