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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
For the test substance the Ames test with Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA1537, as well as E-coli is negative with and without metabolic activation (Harlan 2012k). The substance did not induce mutations in the Mouse lymphoma assay (Harlan 2013i). No chromosome aberrations were found in a chromosome aberration test(Harlan 2013h) with and without metabolic activation
Link to relevant study records
Reference
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:
29-02-2012 to 09-06-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to the guidelines, under GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium: histidine
E-coli: tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 from rats induced with Phenobarbitone/ß-Naphthoflavone
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
acetone (100 mg/ml)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA100, TA1535 and WP2uvrA without metabolic activation
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA100, TA1535, TA 1537 and WP2uvrA with metabolic activation; for TA1537 without metabolic activation
Positive control substance:
2-acetylaminofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylformamide
Positive controls:
yes
Remarks:
for TA98 without metabolic acivation
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA98 with metabolic activation
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st test in agar (plate incorporation); 2nd test preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: growth rate bacterial bachground lawn

COLONY COUNT:
Domino colony counter

Evaluation criteria:
A test item will be considered mutagenic (positive) in the test system if one or more of these criteria are met
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
Statistics:
Not specified according to UKEMS
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitate at 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitate at 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the second test a slight but significant increase (p≤0.05) of the number of mutants was seen in E. coli WP2 uvr A with metabolic activation at 5000 ug/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

The test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation.
Executive summary:

The test substance was tested in an Ames test at concentrations upto the preciptation level (5000 ug/plate). In a plate incorporation and a pre-incubation assay (both performed in triplicate), the test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation.

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

Additional information

Additional information from genetic toxicity in vitro:

The test substance was tested in an Ames test at concentrations upto the preciptation level (5000 ug/plate). In a plate incorporation and a pre-incubation assay (both performed in triplicate), the test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation.

(Harlan 2012k).

The substance is considered to be non mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y (Harlan 2013i) and no chromosomal changes were observed in a chromosome aberration test in Chinese hamster lung fibroblasts (V79) (Harlan2013h). Both tests were performed in presence and absence of metabolic activation.

Based on the negative outcome of the three tests, it is concluded that it is not expected that the test substance will induce mutagenic or clastogenic effects. No additional testing is considered necessary.

Justification for classification or non-classification

The test substance does not need to be classified for mutagenicity.