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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016 - 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl
Molecular formula:
[(CnH2n+1)NH3+].[(CxH2x+1)-C6H4-S03-]
IUPAC Name:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl
Test material form:
liquid: viscous
Details on test material:
Chemical registery number : 953-378-2
Chemical name : Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

Method

Target gene:
histidine for S. typhimurium strains, tryptophan for E. coli strains
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-mix
Test concentrations with justification for top dose:
The maximum concentration was 5000 µg/plate.
Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed
Vehicle / solvent:
Tetrahydrofuran
Controls
Untreated negative controls:
yes
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:
Top agar was prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd.

The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. Evaluation of the properties of these strains regarding membrane permeability, ampicillin resistance, biotin/tryptophan dependences as well as the number of normal background spontaneous revertant colonies is routinely performed.
Evaluation criteria:
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:

TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.15 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix.
Eight test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.


The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 150 μg/plate in the absence of S9-mix and 500 μg/plate in the presence S9-mix. Consequently the toxic limit or the maximum recommended dose level (5000 μg/plate) of the test item was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. In the second mutation test (pre-incubation method), the test item again induced a toxic response with reductions in the growth of bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA100, TA98 and TA1537), 150 μg/plate (TA1535) and 1500 μg/plate (WP2uvrA). In the presence of S9-mix toxicity was initially noted from 500 μg/plate (TA100 and TA1537), 1500 μg/plate (TA1535 and TA98) and at 5000 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA100 revertant colony frequency were observed in the first mutation test at 50 μg/plate (TA100) in the presence of S9-mix only and in the second mutation test at 15 and 50 μg/plate in the presence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.4 times the concurrent vehicle controls.


The test item was considered to be non-mutagenic under the conditions of this test.