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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: GLP study performed according to OECD Guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine
EC Number:
284-664-9
EC Name:
Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine
Cas Number:
84961-74-0
IUPAC Name:
Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine (1:1)
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine (1:1)
- Substance type: technical product (BIO-SOFT 411-E)
- Physical state: viscous liquid
- Analytical purity: 97%
- Lot/batch No.: 50148
- Expiration date of the lot/batch: 30 June 2013
- Storage condition of test material: room temperature in the dark

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 male rat liver microsomal fraction; animals induced with phenobarbitone/b-naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate

Mutation test, Exp. I
all strains with S9: 1.5, 5, 15, 50, 150, 500, 1500 ug/plate
all strains without S9: 5, 15, 50, 150, 500, 1500 and 5000 ug/plate

Mutation test, Exp. II
Salmonella strains TA100 & TA1537 (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 ug/plate
Salmonella strains TA100 & TA1537 (with S9) and TA98 & TA1535 (without S9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate
Salmonella strains TA1535 & TA98 (with S9) and E.coli strain WP2uvrA (without and with S9): 5, 15, 50, 150, 500, 1500, 5000 ug/plate
Vehicle / solvent:
- Vehicle used: tetrahydrofurane
- Justification for choice of vehicle: the test item was insoluble in water, DMSO, dimethyl formamide and acetonitrile at 50 mg/ml and acetone at 100 mg/ml, but fully soluble in tetrahydrofuran at 200 mg/ml
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: exp. I in agar (plate incorporation), exp.II pre-incubation method; according to the OECD Guideline

DURATION
- Preincubation period (exp. II): 20 min
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn


Evaluation criteria:
Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
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).

A test item will be considered non-mutagenic in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
UKEMS (Mahon GAT et al 1989; Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing)

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
The strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.
Precipitation: no precipitation seen at any doses
Preliminary toxicity test: the test material was toxic to TA100 from 500 ug/plate and to WP2urvA from 150 ug/plate onwards

Main tests:
Cytotoxiciy: The test material caused a reduction in the bacterial background lawn of all the tester strains, from 150 and 500 ug/plate with and without S9 mix.

Mutagenicity: no significant increases in the revertant colonies were seen at any dose level, both with and without metabolic activation

Positive and negative control results were considered valid.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Detailed results can be seen in the attached document.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this test the test material (LAS IPA) does not induce any mutations in bacterial strains, when tested up to cytotoxic concentrations, both with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA1535, TA1537, TA98, and TA100 of S. typhimurium and strain WP2urvA of E. coli  were exposed to BIO-SOFT 411-E (97% benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.-, compd. with 2-propanamine) at concentrations ranging between 1.5 to 5000 µg/plate, in the presence and absence of mammalian metabolic activation (S9 microsomal liver fraction, taken from male rats induced with phenobarbitone/b-naphthoflavone.The productwas tested up to cytotoxic concentrations based on a preliminary toxicity test. The results showed a negative response, i.e. no significant increase in the number of revertants was recorded at any dose level tested. The positive controls induced the appropriate responses in the corresponding strainsand the vehicle controls were considered valid.

 

This study is classified asacceptable. It satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.