Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
- Histidine mutation: hisG46
- Additional Mutation Repair LPS-Coat: uvrB- rfa
- Mutation type detected: Base Pair
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
- Histidine mutation: hisD3052
- Additional Mutation Repair LPS-Coat: uvrB- rfa
- Affecting R-Factor: pKM101
- Mutation type detected: Frameshift
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
- Histidine mutation: hisC3076
- Additional Mutation Repair LPS-Coat: uvrB- rfa
- Mutation type detected: Frameshift
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
- Histidine mutation: hisAG8476 rfa, hisG428 on plasmid pAQ1
- Affecting R-Factor: pKM101, + pAQ1
- Mutation type detected: Base Pair Substitution
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
- Histidine mutation: hisG46
- Additional Mutation Repair LPS-Coat: uvrB-
- Affecting R-Factor: pKM101
- Mutation type detected: Substitution
Metabolic activation:
with and without
Metabolic activation system:
liver homogenates (S9)
Test concentrations with justification for top dose:
1.5 - 1500 µg/plate in presence of S9 and 1.5 - 5000 µg/plate in the absence of S9
Vehicle / solvent:
- Vehicle: ethanol
Controls
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoantracene
Remarks:
9-Aminoacridine, mitomycin C, and sodium azide were dissolved in distilled water. 2-Aminoanthracene, and 2-nitrofluorene were dissolved in DMSO.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation): Vogel-Bonner minimal plates enriched with histidine (264M), biotin (3p.M) and ampicillin.

DURATION
- Exposure duration: Plates were kept for 48 to 72 h at 37°C in the dark

EXAMINATION:
- counting of the number of revertant colonies (his+revertants)
- normal background lawn and/or precipitates
- microscopically: microcolony growth
- genotype of revertant colonies are spot-checked by picking and streaking on histidine free plates, if relevant.

OTHER: The experiment was repeated in full after an interval of at least 3 days.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation at 500 µg/plate, with at 1500 µg/plate
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with and without metabolic activation at 500 µg/plate
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation at 1500 µg/plate, with at 500 µg/plate
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation at 5000 µg/plate, with at 1500 µg/plate
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1500 µg/plate
Positive controls validity:
valid
Additional information on results:
The results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.

Applicant's summary and conclusion

Conclusions:
The results indicate that test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system, under the experimental conditions described. No precipitation was observed.
Executive summary:

In the present study, the substance was investigated using the standard plate incorporation procedure according to Ames et al., (1975) (OECD 471). The number of revertant colonies on the plates, with and without the test compound, were compared to evaluate mutagenicity.

The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system.

The test item was dissolved in ethanol and tested in concentrations of 1.5 to 1500 µg per plate in the presence of S9 and 1.5 to 5000 µg per plate in the absence of S9.

In the absence of S9-mix the test item was bacteriotoxic towards the strains TA100 (500 µg/plate), TA102 (500 µg/plate), TA1537 (1500 µg/plate) and TA98 (5000 µg/plate).

In the presence of S9-mix the substance was bacteriotoxic towards the strains TA1537 (500 µg/plate), TA102 (500 µg/plate), TA1535 (1500 µg/plate), TA98 (1500 µg/plate), and TA100 (1500 µg/plate).

Precipitation of the test compound on the plates was not observed.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the test strains in the presence and absence of a metabolic activation system.

In conclusion, these results indicate that the test substance, under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.