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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted according to internationally accepted testing guidelines and performed according to GLP. The OECD recommended combination of strains was not respected: none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Salmonella typhimurium TA102 or Escherichia coli-WP2 uvrA strain not tested; 2-aminoanthracene was the only compound used to test the efficacy of the S9 fraction.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
His-locus.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment: 1, 3, 10, 33, 100, 333, 1000 and 5000 µg/plate
Experiment: 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate


Vehicle / solvent:
- Vehicle used: H2O bidest
- Justification for choice of vehicle: the vehicle was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
H2O bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine (4-NPD) and 2 -aminoanthracen (2-AA)e
Remarks:
S9(-): TA1535 ans TA 100 SA; TA 1537, TA 1538 and TA 98 4-NPD. S9(+): all strais 2-AA.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar.

DURATION
- Exposure duration: 72 hours at 37 °C

NUMBER OF REPLICATIONS: 3 plates per test; no independent repeat experiment performed.
Evaluation criteria:
A test substance is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high with two concentrations of the substance, and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher with two concentrations of the test substance as compared to the spontaneous reversion rate. If in a repeated experiment there is a clear increase in the reversion rate by factors as indicated even in only one strain, then this also leads to the substance being classified as a mutagen.
Also, a continuous dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic properties of the test substance, regardless whether the highest dose induces the above described enhancement factors or not. Such findings would perhaps not be sufficient to declare the substance mutagenic. However, it recommends further testing to exclude mutagenicity.
Statistics:
Revertant colony numbers were calculated as average and standard deviation. No statistical analysis test was performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test article precipitated weakly at 5000.0 µg/plate. No toxic effects were observed in all strains. No reduction of revertant numbers for more than 70 % was found in any strain used. The plates incubated with the test substance showed normal background growth up to 5000.0 µg/plate in the strains used with and without S9 mix. The test article did not induce an increase in revertant colony numbers in the absence and in the presence of S9 mix in all the strains used.

Results:

Dose µg/plate metabolic activation TA 35 TA 1537 TA 1538 TA 98 TA 100
0 - 16 16 14 27 154
10 - 12 6 11 26 192
100 - 13 19 10 22 205
333.3 - 20 13 16 21 182
1000 - 18 21 10 25 187
5000 - 14 18 13 25 169
Sodium azide - 1542 1599
4-NOPD - 323 1963 1761
0 + 16 19 23 51 139
10 + 12 21 23 36 150
100 + 11 19 22 46 150
333.3 + 14 22 23 51 173
1000 + 10 22 26 41 179
5000 + 11 12 25 43 154
2-Aminoanthracene + 1025 373 2144 1928 1550

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance is considered to be non-mutagenic in the current Salmonella typhimurium reverse mutation assay.
Executive summary:

Method

The study was performed to investigate the potential of test item to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA1538; the test was performd according to the OECD guideline 471, except for fact that the OECD recommended combination of strains was not respected, because none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested.

The assay was performed in both with and without liver microsomal activation. The test article was tested at the following concentrations:

Pre-Experiment: 1, 3, 10, 33, 100, 333, 1000 and 5000 µg/plate

Experiment: 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate

Results

The test item did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in the current Salmonella typhimurium reverse mutation assay.