[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 March 2020 - 18 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline

Version / remarks:

adopted June 2012
(ICH S2(R1) Federal Register.Adopted 2012; 77:33748-33749)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine

IUPAC Name:

Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine

Test material form:

liquid

Details on test material:

Molecular weight: 357.269
Appearance: light amber viscous liquid
Storage conditions: room temperature, in the dark

Method

Target gene:

Salmonella strains - histidine locus
E coli strain - tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Strain:
TA1537
Genotype: his C 3076; rfa-; uvrB-:
Type of mutations indicated: frame shift mutations

TA98
Genotype: his D 3052; rfa-; uvrB-;R-factor
Type of mutations indicated: frame shift mutations

TA1535
Genotype: his G 46; rfa-; uvrB-:
Type of mutations indicated: base-pair substitutions

TA100
Genotype: his G 46; rfa-; uvrB-;R-factor
Type of mutations indicated: base-pair substitutions

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules.
A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin.
In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000).

CELLS USED
The bacteria used in the test were obtained from:
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
- Trinova Biochem GmbH on 27 June 2017
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. On a regular basis (approximately monthly), batches of culture from master stocks are prepared and coded, these are then routinely tested for appropriate characteristics, viability and mutation frequency to ensure acceptability criteria is met.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2547490 expiry date 07/2024) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Strain:
WP2uvrA
Genotype: trp-; uvrA-
Type of mutations indicated: base-pair substitution

In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

CELLS USED

The bacteria used in the test were obtained from:
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
- Trinova Biochem GmbH on 27 June 2017
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. On a regular basis (approximately monthly), batches of culture from master stocks are prepared and coded, these are then routinely tested for appropriate characteristics, viability and mutation frequency to ensure acceptability criteria is met.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2547490 expiry date 07/2024) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Metabolic activation:

with and without

Metabolic activation system:

TYPE AND COMPOSITION OF METABOLIC ACTIVATION SYSTEM
Source of S9:
The Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No. 4146 and the protein level was adjusted to 20 mg/mL.

Method of preparation of S9 mix :
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
5.0 mL - S9
1.0 mL - 1.65 M KCl/0.4 M MgCl2
2.5 mL - 0.1 M Glucose-6-phosphate
2.0 mL - 0.1 M NADP
25.0 mL - 0.2 M Sodium phosphate buffer (pH 7.4)
14.5 mL - Sterile distilled water
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Quality controls of S9:
A copy of the S9 Quality Control and Production Certificate is presented in Appendix 2 (appended to attached background material).

Test concentrations with justification for top dose:

Experiment 1 – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 – Pre-Incubation Method: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 – Pre-Incubation Method (repeat): Salmonella strain TA100 (absence of S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate

Vehicle / solvent:

Vehicle/solvent used: Tetrahydrofuran
Justification for choice of solvent/vehicle: In solubility checks performed in–house, the test item was noted as immiscible in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully miscible in tetrahydrofuran at 200 mg/mL. Tetrahydrofuran was therefore selected as the vehicle.
Justification for percentage of solvent in the final culture medium: Tetrahydrofuran is toxic to the bacterial cells at and above 50 µL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 µL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All test item preparation and dosing was performed under yellow safety lighting.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

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:

NUMBER OF REPLICATIONS:
Number of cultures per concentration: triplicate
Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate. The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate

METHOD OF TREATMENT/ EXPOSURE:


The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in high purity tetrahydrofuran by mixing on a vortex mixer and sonication for 10 minutes at 40 °C.

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Without Metabolic Activation
A 0.025 mL aliquot of the appropriate concentration of test item or solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).


Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.025 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Method: background growth inhibition. The plates were viewed microscopically for evidence of thinning (toxicity).

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Method: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system.

Rationale for test conditions:

According to OECD guidelines

Evaluation criteria:

Evaluation Criteria
There are several criteria for determining a positive result. 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 (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item will be considered non-mutagenic (negative) 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 judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s 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 results

Test results 1

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment 1 (plate incorporation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

absence and presence of metabolic activation (S9-mix) initially from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 1 (plate incorporation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

absence and presence of metabolic activation (S9-mix) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 1 (plate incorporation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

absence and presence of metabolic activation (S9-mix) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment 1 (plate incorporation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

absence and presence of metabolic activation (S9-mix) initially from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 1 (plate incorporation)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

absence and presence of metabolic activation (S9-mix) at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 10

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 11

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns were noted from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 12

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns were noted from 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 13

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns were noted from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 14

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns were noted from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 15

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 2 (pre-incubation)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weakened bacterial background lawns were noted from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile.
The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) and viability are presented in Table 1 (appended to attached background material) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation (S9-mix), are presented in Table 2 and Table 3 (appended to attached background material) for Experiment 1 and Table 4 and Table 5 (appended to attached background material) for Experiment 2.
A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1 (appended to attached background material).

Experiment 1 (plate incorporation) – Table 2 and Table 3
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.
Toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure to all tester strains in the absence and presence of metabolic activation (S9-mix) initially from 1500 µg/plate to TA98 and TA1537 and at 5000 µg/plate to TA100, TA1535 and WP2uvrA.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no 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).

Experiment 2 (pre-incubation) – Table 4 and Table 5
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate) or the toxic limit depending on bacterial strain type and absence or presence of S9-mix.
The test item induced a stronger toxic response following the pre-incubation procedure with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate to all of the tester strains. In the presence of S9-mix, weakened bacterial background lawns were noted from 50 µg/plate to TA100, 500 µg/plate to TA98, 1500 µg/plate to TA1537 and at 5000 µg/plate to TA1535 and 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.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no 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).

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine was considered to be non-mutagenic.

Executive summary:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test. Study was conducted under GLP conditions. 

 

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 up to nine 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 (plate incorporation) was based on OECD TG 471 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 0.015 and 5000 µg/plate depending on bacterial strain type and absence or presence of S9-mix.  Nine test item concentrations were selected in Experiment 2 in order to ensure the study achieved both four non-toxic dose levels as required by the test guideline, the cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

 

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 and 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 OECD TG 471 recommended dose level of 5000 µg/plate.  Toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure to all tester strains in the absence and presence of metabolic activation (S9-mix) initially from 1500 µg/plate. Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) or the toxic limit was employed in the second mutation test (pre-incubation method).  The test item induced a stronger toxic response following the pre-incubation modification with weakened bacterial background lawns noted in the absence and presence of S9-mix from 50 and 150 µg/plate, respectively.

 

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2. There were no 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 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).

 

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix).  Under the conditions of this test Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine was considered to be non-mutagenic.