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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, restriction in design (no E.coli WP2 or S.typhimurium TA 102 used and only one positive control used to test efficacy of the S9-mix), but adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
, no E.coli WP2 or S.typhimurium TA 102 used and only one positive control used to test efficacy of the S9-mix
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 84/449, B14. Mutageniticy (Salmonella typhimurium - reverse mutation assay)
Deviations:
yes
Remarks:
, no E.coli WP2 or S.typhimurium TA 102 used and only one positive control used to test efficacy of the S9-mix
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of the test substance used in the study report: Asta-C6-Acetal
- Physical state: colorless liquid
- Storage: refrigerator

Method

Target gene:
His-locus
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:
Aroclor-1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
1st and 2nd experiment: 0, 100, 500, 2500, 5000 µg/plate
3rd experiment: 500, 1000, 1500, 2000 µg/plate
Vehicle / solvent:
Vehicle used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 µg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains tested with metabollic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
TA 100, TA 1535; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
TA 98; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
100 µg/plate
Positive control substance:
9-aminoacridine
Remarks:
TA 1537; without metabolic activation
Details on test system and experimental conditions:
--> 1st EXPERIMENT

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: Reduces his- background growth, decrease in the number of his+ revertants


--> 2nd and 3rd EXPERIMENT

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: Reduces his- background growth, decrease in the number of his+ revertants
Evaluation criteria:
In general, a substance to be characterized as positve 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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed in the standard plate test at doses ≥ 2500 µg/plate. In the preincubation test bacteriotoxicity was observed from about 1000 µg - 1500 µg onward.

TEST-SPECIFIC CONFOUNDING FACTORS
No test substance precipitation was found.
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

Under the experimental conditions, it is concluded that the test substance is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

The substance was tested for its mutagenic potential, in an OECD 471 guideline study (compliant with GLP), based on the ability to induce back mutation in selected loci in several strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA 98) in the Ames test. Bacteria were exposed to 20 - 5000 μg/plate in a standard plate test and a preincubation test both with and without metabolic activation (Aroclor-induced rat liver S9 mix). No precipitation of the test substance was found. A bacteriotoxic effect was observed at doses ≥ 2500 µg in the standard plate test and from about 1000 - 1500 µg/plate onward in the preincubation assay. An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of this metabolizing system. The test substance is not mutagenic in the Ames test under these experimental conditions.