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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:
experimental study
Adequacy of study:
key study
Study period:
May 27, 1998 - July 20, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 92/69
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Liver S9-mix from rats induced with ß-naphthoflavone
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 33, 100, 333, 1000, 2500, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Solvent: DMSO
Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose level

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test article can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.



Rationale for test conditions:
According to guideline
Evaluation criteria:
A test article is considered positive if either a reproducible duse related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.

A test article producing neither a reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A biologically relevant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice on TA 1535 and TA 1537.

Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether ihe highest dose induced the criteria described above or not.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occured.

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100 eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Since no cytoxicity occured up to the highest concentration (5000 µg/plate), this concentration was chosen as maximum concentration for the main experiment. Applying an adequate spacing factor the following concentrations were tested: 33, 100, 333, 1000, 2500 and 5000 µg/plate.

HISTORICAL CONTROL DATA
- Positive historical control data:
- S9 mix: TA1535: 68-814; TA1537: 42-191; TA98: 86-450; TA100: 460-910; TA102: 751-1395

- Negative (untreated) historical control data:
- S9 mix: TA1535: 9-29; TA1537: 5-28; TA98: 15-57; TA100: 77-189; TA102: 121-293

- Solvent historical control data:
- S9 mix: TA1535: 9-25; TA1537: 4-28; TA98: 14-58; TA100: 77-225; TA102: 121-295

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study according to OECD Guideline 471 was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and III) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The experiments were performed with and without liver microsomal activation. In experiment II, no unambiguous positive response was reached in strain TA 100 with S9 mix (factor 2.1) and in strain TA 102 with (factor 2.2) and without S9 mix (factor 2.4) Therefore, these result were dismissed and an additional experiment was performed with strains TA 100 with S9 mix and TA 102 with and without S9 mix. The results of this additional experiment are reported as experiment II. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

33, 100, 333, 1000, 2500, and 5000 µg/plate.

No relevant toxic effects, evident as a reduction in the number of revenants, occurred in the test groups with and without metabolic activation.

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

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

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.