Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Study period:
16-01-2013 to 19-03-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to the guidelines, under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Name of test material (as cited in study report): Fatty acids, C12-18 , reaction products with ethanol, 2-amino, reaction products with ammonia, by products from"
Substance type: UVCB
Purity : 100%
Storage conditions : Room temperature in the dark

Method

Target gene:
Salmonella typhimurium: histidine
E-coli: tryptophan
Species / strainopen allclose all
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:
Salmonella species: 1.5, 5, 15, 50, 150, 500 and 1500 ug/plate with and without metabolic activation in preincubation and plate incorporation assay
E. coli: 50, 150, 500, 1500 and 5000 ug/plate with and without metabolic activation in plate incorporation assay; 15, 50, 150, 500, 1500 and 5000 ug/plate with and without metabolic activation in preincubation assay
Vehicle / solvent:
acetone (100 mg/mL)
Controlsopen allclose all
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 TA1537 without metabolic activation
Positive control substance:
9-aminoacridine
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
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
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 + presence of bacterial background lawn

COLONY COUNT:
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

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Remarks:
reduction of bacterial background lawn at 1500 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:
presence of greasy precipitate did not hinder the evaluation at 1500 and 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results
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 of 1.5 to 1500 ug/plate (cytotoxicity obeserved) in the salmonella strains TA98, TA100, TA1535 and TA1537. The concentrations in the test on E. coli WP2 uvr A were 15 -5000 ug/plate. At 1500 and 5000 ug/plate a greasy precipitate was observed, that did not hamper the evaluation. In a plate incorporation and a pre-incubation assay (both performed in triplicate) with and without 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.