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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
secondary source
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
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): TP-90B RUBBER CHEMICAL
- Analytical purity: no data

Method

Target gene:
his- or trp-
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.
Test concentrations with justification for top dose:
25 - 800 ug/plate
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, sodium aside, 9-amino-acridine, 2-nitrofluorene, methylmethanesulfonate
Details on test system and experimental conditions:
In Main Assay I, using the plate incorporation method, the test item was assayed at a maximum dose-level of 5000 µg/plate and at four lower dose-levels, separated by two-fold dilutions: 2500, 1250, 625 and 313 µg/Plate.
As no increases in revertant numbers were observed, all treatments of Main Assay II included a pre-incubation step and used the same dose-range employed in Main Assay I. This dose range proved to be too toxic with all tester strains, in the absence and presence of metabolism, with the exception of TA1535. Moreover, moderate microbial contamination was observed on the plates relative to the treatment with TA98 in the absence of metabolism.
Therefore, in order to assay the test item at adequate less-toxic concentrations, a Main Assay III was performed using the following dose-levels:

Tester Strain S9 Dose Levels (µg/plate)
TA 1537 +/- 400, 200, 100, 50, 25
TA 98 + 400, 200, 100, 50, 25
TA 98 - 400, 200, 100, 50, 25, 12.5, 6.25, 3.13
TA 100 & WP2uvrA +/- 800, 400, 200, 100, 50

- Number of replicates: 3 replicate plates were used at each test point
- Application: The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate, and allowed to solidify prior to incubation.
The overlay mixture was composed as follows:
a) Overlay agar (held at 45 °C) 2 ml
b) Test or control item solution 0.1 ml
c) S9 mix or phosphate buffer pH 7.4, 0.1M) 0.5 ml
d) Bacterial suspension 0.1 ml

The second and third experiments were performed using a pre-incubation method. The components were added in turn to an empty test tube:

a) Bacterial suspension 0.1 ml
b) Test or control item solution 0.05 ml
c) S9 mix or phosphate buffer (pH 7.4, 0.1M) 0.5 ml

The incubate was vortexed and placed at 37 °C for 30 minutes. 2 ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

Positive control substance:
- With S-9 mix: 2- Aminoanthracene for the strains TA 100, TA 98, TA 1537, TA 1535, and E. coli WP2 uvrA.
- Without S-9 mix: Sodium aside (strains TA 100 and TA 1535); 2-Nitrofluorene (strain TA 98); 9-Amino-acridine (strain 1537) and Methylmethanesulphonate (strain E. coli WP2 uvrA).
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
Statistical methods beyond the calculation of the mean and standard deviation are not considered necessary for the interpretation of this study.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
- With metabolic activation: In Main Assay II, toxicity was observed at all dose levels for all strains except TA1535. - Without metabolic activation: In Main Assay I, slight toxicity was observed in TA1537 at "higher dose levels". In Main Assay II, toxic
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

It was concluded that the test item, TP-90B rubber chemical, did not induce reverse mutation in Salmonella typhimurium and Escherichia under the reported experimental conditions.

Applicant's summary and conclusion