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
Study period:
6 July - 31 October 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 471) and in compliance with GLP.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
GLP compliance:
yes (incl. certificate)
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): TH 1165 I
- Physical state: yellow liquid
- Analytical purity: 99.01 % (GC)
- Purity test date: 6. march 2000
- Lot/batch No.: 14
- Expiration date of the lot/batch: can be requeste from sponsor
- Stability: can be request from sponsor
- Storage condition of test material: in original container, at room temperature

Method

Target gene:
TA 98: his D 3052; rfa; uvrB; R-factor; frame shift mutations
TA 100: his G46; rfa; uvrB; R-factor; base-pair substitutions
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation) and preincubation

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 each per concentration level and control
Evaluation criteria:
Number of revertant colonies produced.
The colony number for control, positive control and the test plates were determined, the mean values and appropriate standard deviations were calculated.
The mutation factor (MF) was calculated by dividing the mean value of the revertant colonies by the mean values of the solvent control (the exact and not rounded values were used for this calculation).
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occur and/or
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or withour metabolic activation.

A biologically relevant increase is described as follows:
- if in strain TA 100 the number of reversions is at least twice as high when compared to the spontaneousreversion rate of the solvent control plates
- if in strains TA 98, TA 100 the number of reversions is at least three times higher than those of the control plates.
Statistics:
not regarded as neccessary according to the OECD guidelines.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only at the highest level of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only at the highest level of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test item TH 1165 I was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using Salmonella typhimurium strains TA 100 and TA 98.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation (S9 -mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6; 100.0; 316.2; 1000.0; 2500.0; and 5000.0 µg/plate

Slight toxic effects (indicated by a reduction of the background lawn or by a reduction of the spontaneous rate) of the test item were observed at the hightest concentration (5000.0 µg/plate) in experiment I without and in exp. II with and without metabolic activation (S9 -mix) in both tester strains.

No substantial increase in revertant colony numbers of any of the two tester strains were observed following treatment with TH 1165 I at any concentration level, either in the presence or absence of metabolic activation (S9 -mix) in experiment I and II. There was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance in both experiments independently performed.The reference mutagens showed a distinct increase of induced revertant colonies.

Detailed test results are attached as pdf.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.