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other: Study available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium in a modified version of the traditional Ames test, i.e. the Ames II assay (microtiter version). The test is carried out based on the description of Gee, P. et al (1998).
GLP compliance:
no
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: 2,6-Dimethyl-4-tert.-butylbenzylcyanid
- Appearance, consistency: white powder
- Storage conditions: room temperature

Method

Target gene:
S. typhimurium
Species / strain
Species / strain:
other: TA 98, TA Mix (Mixed strains TA 7001 - TA 7006)
Additional strain characteristics:
other: See "any other information on materials and methods"
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
4 µg - 5000 µg/mL (1st experiment)
1000 µg - 6000 µg/mL (2nd experiment)
Vehicle:
Due to the Iimited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
Remarks:
without metabolic activation
Details on test system and conditions:
EXPERIMENT 1:
- Strains: TA 98, TA Mix
- Doses: 0; 4; 20; 100; 500; 2,500 and 5,000 µg/mL
- Type of test: Liquid fluctuation test with and without S-9 mix
- Number of microtiter plates: Triplicate plates per dose, control chemical or vehicle

EXPERIMENT 2:
- Strains: TA 98
- Doses: 0; 1,000; 2,000; 3,000; 4,000; 5,000 and 6,000 µg/mL
- Type of test: Liquid fluctuation test with S-9 mix
- Number of microtiter plates: Triplicate plates per dose, control chemical or vehicle

- Optical density (OD600 values): The determination of the OD600 values is carried out by adding 100 µL aliquots from overnight cultures of each strain in 1 mL spectrophotometer cuvettes containing 900 µL growth medium. The OD600 values are at least 2.0 for the Mixed strains or TA 98 and ~0.0 for the negative control.
- Liquid exposure: 5 mL of the overnight cultures are added in culture tubes containing 19 mL Ames II Exposure medium (Xenometrix) and are gently mixed. After thorough pipetting of the content the following components are added in 24-well exposure plates using a 8-channel pipettor to a final volume of 250 µL/well: 240 µL exposure medium & tester strain (in tests without S-9 mix); 200 µL exposure medium & tester strain (in tests with S-9 mix); 40 µL S-9 mix (final concentration of 4.8% S-9 fraction); 10 µL Test solution, control chemicals or vehicle. The 24-well exposure plates are then incubated at 37°C for 90 minutes, with shaking at 250 rpm using an environmental shaker.
- Prototrophic selection: After the 90-minute incubation, 2.8 mL Ames II Reversion indicator medium (Xenometrix) (containing bromocresol purple as an essential constituent) is pipetted to each well of the 24-well plate. The histidine-deficient Indicator medium which selects for prototrophic reversion is mixed gently several times. When adequately mixed, the contents of each well of a 24-well microtiter plate are distributed in 50 µL aliquots over 48 wells of a 384-well Revertant Colony Selection Plate (RCSP) by Eppendorf pipettes. After sealing the RCSP in plastic bags to prevent evaporation and after incubation at 37°C tor about 48 hours in the dark each 48-well section of the 384-well plates are scored and the number of positive wells (yellow) are counted.
- Toxicity detected by a decrease in the number of positive wells and/or clearing or diminution of the background lawn (= reduced his- background growth) leading from turbid to non-turbid purple wells.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of positive wells by a factor of about 2 (calculated primarily on the basis of baseline data) in at least one tester strain either withaut S-9 mix or after adding a metabalizing system.
A test substance is generally considered non-mutagenic in this test if the number of revertant wells for all tester strains were within the historical negative control range under all experimental conditions.

Results and discussion

Test results
Species / strain:
other: TA 98, TA Mix (Mixed strains TA 7001 - TA 7006)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No precipitation of the test substance was found.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

According to the results of the present study, the test substance is not mutagenic in the Ames II Assay (Salmonella typhimurium reverse mutation assay) under the experimental conditions chosen here.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium in a modified version of the traditional Ames test, i.e. the Ames II assay (microtiter version) (based on the description of Gee, P. et al). The Salmonella typhimurium strains (TA 98, and mixed strains TA 7001 – TA 7006) were used to test the mutagenic potential, both with and without metabolic activation. In the first experiment TA98 and TA mix were exposed to 4 -5000µg/mL test substance. In the second experiment, TA98 was exposed to 1000 -6000 µg/mL test substance. No precipitation of the test substance was found and no bacteriotoxic effect was observed. An increase in the number of positive wells (his+ revertants) was not observed either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance is not mutagenic in the Ames II Assay under the experimental conditions chosen here.