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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
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with the OECD guideline 471 adopted in 1983.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
The study was conducted in accordance with the OECD guideline 471 adopted in 1983. Deviations to current guideline (adopted 1997): no co-testing of E. coli and no GLP.
GLP compliance:
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:
Comonomere (1,3-Dioxepan)
Batch Nr. B 4001 A (18. 03. 91)
Purity 99.5 %
Physical state: colorless liquid
Storage: RT


Target gene:
Gene of a histidine requiring strain (his-) to produce a histidine independent strain of this organism (his+)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: defect in the excision repair system (uvrB)
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction from the liver of male sprague-dawley rats treated with AROCLOR 1254, mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
Destilled water
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
For details on control, see below.
Details on test system and experimental conditions:
Standard plate test and preincubation test both with and without metabolic activation (S-9 mix).

Standard plate test
The experimental procedure is based on the method of Ames et al. (Proc Nat Acad Sci USA 70: 2281 - 2285, 1973, and Mut Res 31: 347 - 364, 1975).

Test tubes containing 2 ml portions of soft agar which consisted of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at 45°C, and the remaining components were added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples were poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al. (Mut Res 48: 121 - 130, 1977 and Norpoth KH and Garner RC, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980).

0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix were incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar was added and after mixing, the samples were poured onto the Vogel-Bonner agar plates within approx. 30 seconds. Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) were counted.

Negative control
Parallel with each experiment with and without S-9 mix, a negative control (solvent control, sterility control) was carried out for each tester strain in order to determine the spontaneous mutation rate.

Positive controls
The following positive control substances were used to check the mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix:
10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535
without S-9 mix:
5 µg N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537.

Two test series were conducted:
First experiment: all strains were tested with concentrations ranging from 20 to 5000 µg/plate, with and without S-9 mix; 3 test plates per dose or per control were considered.
Second experiment: because of contaminants, a colony scoring for the strain TA 100 in the first experiment was not possible; therefore, testing of TA 100 under conditions similar as above was repeated within a second experiment.
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either with or without S-9 mix.
No cytotoxic effect was seen up to a test concentration of 5000 µg/plate
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion