Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed OECD and GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Aminolon - TTR

Method

Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0 - 4 - 20 - 100 - 500 - 2500 - 5000 - 10000 µg per plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: TA 100, TA 1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9

Migrated to IUCLID6: TA 1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9

Migrated to IUCLID6: TA 98, TA 1538
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) WP2uvrA
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation

Migrated to IUCLID6: TA98 , TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: TA98 , TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA
Remarks:
with metabolic activation
Details on test system and experimental conditions:
BACTERIA
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2 /liter) at 37°C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at -80°C, are used. The compound is tested with the strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E. coli WP2uvrA.

TOXICITY AND DOSE RANGE FINDING
Preliminary toxicity tests were performed with all tester strains using two plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically.
In combination with the second experiment, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

MUTAGENICITY TEST
Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.5 % NaC1) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coli histidine is replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients are added (in order) to 2 ml of molten top ager at 45°C:

0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml S-9 Mix (if required) or buffer

After mixing, the liquid is poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 48 to 72 hours at 37°C in the dark, colonies (his+ revertants) are counted.

Evaluation criteria:
After incubation for 48 to 72 hours at 37°C in the dark, colonies (his+ revertants) are counted.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(tested up to 10000µg per plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Summarizing, it can be stated that Aminolon TTR is not mutagenic in these bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.
Executive summary:

Aminolon TTR was tested for mutagenicity with the strains TA 100, TA 1535, TA1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 7 different doses from 4 µg/plate to 10 000 µg/plate was used.

Control plates without mutagen showed that the number of, spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains. 10 000 µg/plate was chosen as top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Aminolon TTR did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Aminolon TTR is not mutagenic in these bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.