Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames Test with the test item on 5 Salmonella strains gave throughout negative results.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
mutant histidine gene
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:
S9 mix was made from the livers of Aroclor 1254-induced male Sprague Dawley rats. The S9 mix comprised 10 % S9 fraction.
Test concentrations with justification for top dose:
The test item is a 40 % solution in ethyl acetate. This content was taken into consideration for the calculation of the doses tested:
plate incorporation assay:
0, 10, 25, 50, 160, 500, 1600, 5000 µg/plate with and without S9 mix
pre-incubation assay:
0, 10, 25, 50, 160, 500, 1600, 5000 µg/tube with and without S9 mix
The test was performed up to and including the limit dose of 5000 µg/plate and tube. No bacteriotoxicity but substance precipitation occurred at this dose.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethyl acetate
- Justification for choice of solvent/vehicle: test item is already a solution in ethyl acetate
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
cumene hydroperoxide
mitomycin C
other: 4-nitro-o-phenylenediamine for TA 1537; 1-Anthracene-2-amine for TA 102 and S9-mix activity
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); pre-incubation

DURATION
- Pre-incubation period: 20 min
- Exposure duration: 48 hours (TA102) or 72 hours (TA1535, TA100, TA1537, TA98)

NUMBER OF REPLICATIONS: all plates were prepared in triplicate

DETERMINATION OF BACTERIOTOXICITY
- Method: gross appraisal of background growth

Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100, TA 1537 and TA 98 this increase should be about twice that of negative controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
no statistics perfomed; evaluation based on criteria mentioned above
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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
Positive controls validity:
valid
Additional information on results:
Test item precipitation occurred at 5000 µg per plate/tube. No bacteriotoxic effects were seen up to and including 5000 µg/plate and tube. Bacteriotoxic effects were observed at 5000 µg/plate and tube.
No indication of mutagenic effects could be found for the test item at doses of up to and including 5000 µg per plate in any of the Salmonella typhimurium strains used, without and with metabolic activation, under the experimental conditions applied.
Remarks on result:
other: precipitation occured at the highest dose tested
Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102, performed according to OECD TG 471. Doses of up to and including 5000 µg per plate did not produce bacteriotoxic effects. Substance precipitation occurred at 5000 µg per plate. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the pre-incubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The technical form of the registered substance is a solution in ethyl acetate (ca. 40 % w/w). The solvent free substance solidifies as a glassy but still sticky material. It is not possible to obtain a powdered sample in a mortar for testing and thus, testing of the isolated (pure) registered substance is technically not feasible. Therefore, it was decided to apply the technical product for toxicological tests. In the in vitro testing for skin and eye irritation the solvent ethyl acetate was tested in parallel to distinguish potential toxicity of the registered substance from solvent toxicity.

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102, performed according to OECD TG 471. Doses of up to and including 5000 µg per plate did not produce bacteriotoxic effects. Substance precipitation occurred at 5000 µg per plate. The test material was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. Positive controls increased the mutant counts to well over those of the negative controls, thus demonstrated the system´s sensitivity.

Justification for classification or non-classification

Based on the available information (Ames test in bacteria) no classification is warranted for the test substance.