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Toxicological information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Experimental Procedures: Date Started: 10 October 1992; Date Completed: 10 November 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across study therefore categorised as Klimisch 2.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
No
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Mixture containing zinc 3,5-bis(α-methylbenzyl)salicylate
- Physical state: white opaque liquid
- Analytical purity: 38%
- Storage condition of test material: room temperature
- Other:
- Date received: 24 July 1992
- Container: white opaque plastic jar

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
EXPERIMENT 1: 0, 8, 40, 200, 1000, 5000 µg/plate +/- S9 mix

EXPERIMENT 2: 0, 312.5, 625, 1250, 2500, 5000 µg/plate +/- S9 mix
Vehicle / solvent:
water
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 4-nitro-o-phenylenediamine and 2 aminoanthracene
Details on test system and experimental conditions:
EXPERIMENT 1:

Test Material and Negative Controls: A 0.1 ml aliquot of one of the bacterial suspensions was placed in sets of sterile test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar at 45°C. These sets comprised of two test tubes for each bacterial tester strain. 0.1 ml of the appropriately diluted test material or negative control was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 ml of the S9 liver microsome mix; in the other tube 0.5 ml of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.

Positive Controls
1) Without Activation: 0.1 ml of one of the positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar and 0.1 ml of the appropriate bacterial suspension. Finally 0.5 ml of pH 7.4 buffer was added to the test tube. This procedure was then repeated, in triplicate, for each of the positive controls.

2) With Activation: 0.1 ml of 2AA or BP solution was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar and 0.1 ml of one of the test bacterial suspensions. Finally 0.5 ml of S9 mix was added to the test tube. The procedure was then repeated, in triplicate, for each tester strain.

The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37°C for approximately 48 hours and the number of revertant colonies counted.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained then a third experiment may be used to confirm the correct response.
Statistics:
All data are statistically analysed using the methods recommended by the UKEMS*. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

*Kirkland, D.J., (Ed). Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report - Part III (1989) - Cambridge University Press.

Results and discussion

Test results
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
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Study:
- The dose range of the test material used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate (dose levels expressed as weight of pure test material).Test material was non-toxic in strain of Salmonella used (TA100).
- The mean numbers of revertant colonies for the toxicity were:

Dose (µg/plate)
Strain: 0 312.5 625 1250 2500 5000
TA100: 175.0 184.5 168.0 167.0 187.5 164.5
- Mutation Study: The results for the checks on characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
- No toxicity was exhibited to the bacterial background lawn of any of the strains of Salmonella used although in some strains a reduction in the numbers of revertant bacterial colonies was observed at the upper dose levels.
- No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level either with or without metabolic activation.
- The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

(See Tables 1 to 4 of attached report for detailed results: Individual plate counts together with the mean number of revertant colonies obtained for each tester strain following incubation with the test material, with and without metabolic activation, are given in Tables 1 and 2 for experiment 1 and Tables 3 and 4 for experiment 2. The results for the positive controls are given in Tables 1 to 4).
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

The test material was found to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA9B and TA100 were treated with the test material by the plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system at 10% in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. In this case the dose range of the test material was 312.5 to 5000 µg/plate.

 

The solvent (sterile water) control plates gave counts of revertant colonies within the normal range.

 

All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system.

 

The test material caused no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. The test material was, therefore tested up to the maximum recommended dose of 5000 µg/plate (an appropriate allowance was made for the 38% purity of the test material).

 

No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the test material

either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test.