Endpoint study record: Genetic toxicity in vitro.AMES 

Benzenesulfonyl chloride LANXESS 41201581

UUID IUC5-96313fff-3c3a-4177-9182-64a3c5fcb43d 

Dossier UUID 0 

Author abowles / Harlan Laboratories Ltd. / Other / other:

Date 2012-11-28 14:05:25 GMT 

Remarks 

 

Administrative Data 

Purpose flag robust study summary 

Study result type experimental result Study 

period 

The experimental phase of this study was performed between 11 July 

2012 and 18 September 2012 

Reliability 1 (reliable without restriction) 

Rationale for reliability 

incl. deficiencies 

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from 

standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the 

relevant results. 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

study 

report 

P W 

Thompson 

2012 Reverse 

Mutation Assay 

“Ames Test” 

using 

Salmonella 

typhimurium 

and Escherichia 

coli 

 Harlan 

Laboratories Ltd 

Shardlow 

Business Park 

Shardlow 

Derbyshire 

DE72 2GD UK 

41201581 Lanxess 

Corporation, 111 

RIDC Park West 

Drive, Pittsburgh, 

PA 15275-1112, 

UNITED 

STATES OF 

AMERICA 

 2012-

11-15 

Materials and methods 

Type of genotoxicity 

gene mutation 

Type of study 

bacterial reverse mutation assay (e.g. Ames test) 

Test guideline 

Qualifier Guideline Deviations 

according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) no 

according to EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) no 

according to JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals no 

equivalent or 

similar to 

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998) ( Meets the requirements of 

the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing 

of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.) 

no 

GLP compliance 

yes (incl. certificate) (See Appenidx 3 attached) 

Test materials 

Test material identity 

Identifier Identity 

Common name Benzenesulfonyl chloride 

Test material form 
other: liquid 

Details on test material 

Sponsor's identification: Benzenesulfonyl chloride

Description: Clear colourless liquid 

Purity: 99.53% 

Batch number: KX120302.A 

Date received: 16 April 2012 

Expiry date: 12 April 2013 

Storage conditions: Room temperature in the dark 

Method 

Target gene 

Histidine for Salmonella. 

Tryptophan for E.Coli 

Species/strain 

Species/strain S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 

Details on 

mammalian 

cell lines (if 

applicable) 

Not applicable. 

Additional 

strain 

characteristics 

not applicable 

Metabolic 

activation 

with and without 

Metabolic 

activation 

system 

phenobarbitone/betanaphthoflavone induced rat liver, S9 

Species/strain E. coli WP2 uvr A 

Details on 

mammalian 

cell lines (if 

applicable) 

Not applicable. 

Additional 

strain 

characteristics 

not applicable 

Metabolic 

activation 

with and without 

Metabolic 

activation 

system 

phenobarbitone/betanaphthoflavone induced rat liver, S9 

Test concentrations 

Preliminary Toxicity Test; 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. 

Expt 1: 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. 

Expt 2: 0 All Salmonella strains (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. 

All Salmonella strains (absence of S9-mix) 

Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate. 

Vehicle 

Vehicle: Acetone 

Justification for choice of solvent/vehicle: The test item was noted to be miscible but unstable in dimethyl sulphoxide at 50 mg/ml (an 

exothermic reaction occurred) but was fully miscible in acetone at 100 mg/ml in solubility checks performed in-house. Acetone was 

therefore selected as the vehicle. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated 

as a potential vehicle in this test system. 

Controls 

Negative 

controls 

yes (Spontaneous mutation rates of TA100) 
Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

other: 2-Aminoanthracene: 1 μg/plate 

Remarks With S9 mix 

Negative 

controls 

yes (Spontaneous mutation rates of TA1535) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

other: 2-Aminoanthracene: 2 μg/plate 

Remarks With S9 mix 

Negative 

controls 

yes (Spontaneous mutation rates of TA1537) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

other: 2-Aminoanthracene: 2 μg/plate 

Remarks With S9 mix 

Negative 

controls 

yes (Spontaneous mutation rates of WP2uvrA) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

other: 2-Aminoanthracene: 10 μg/plate 

Remarks With S9 mix 
Negative 

controls 

yes (Spontaneous mutation rates of TA98) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

benzo(a)pyrene Benzo(a)pyrene: 5 μg/plate 

Remarks With S9 mix 

Negative 

controls 

yes (Spontaneous mutation rates of TA98) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

4-nitroquinoline-N-oxide 4-Nitroquinoline-1-oxide: 0.2 μg/plate 

Remarks without S9 mix 

Negative 

controls 

yes (Spontaneous mutation rates of TA1537) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

9-aminoacridine 9-Aminoacridine: 80 μg/plate 

Remarks without S9 mix 

Negative 

controls 

yes (Spontaneous mutation rates of TA100) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N'-nitro-N-nitrosoguanidine: 3 μg/plate 
Remarks without S9 mix 

Negative 

controls 

yes (Spontaneous mutation rates of TA1535) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no data 

Positive 

controls 

yes 

Positive 

control 

substance 

N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N'-nitro-N-nitrosoguanidine: 5 μg/plate 

Remarks Without S9 mix 

Negative 

controls 

yes (Spontaneous mutation rates of WP2uvrA) 

Solvent / 

vehicle 

controls 

yes (Acetone) 

True 

negative 

controls 

no data 

Positive 

controls 

yes 

Positive 

control 

substance 

N-ethyl-N-nitro-N-nitrosoguanidine N-ethyl-N'-nitro-N-nitrosoguanidine: 2 μg/plate 

Remarks Without S9 mix 

Details on test system and conditions 

METHODS OF APPLICATION: in agar – Experiment 1 (Plate Incorporation) 

- Pre-incubation period for bacterial strains: 10hrs 

- Exposure duration: 48-72hrs 

- Expression time (cells in growth medium): Not applicable 

- Selection time (in incubation with a selective agent): 20 minutes at 37 degrees C 

NUMBER OF REPLICATIONS: Triplicate plating. 

DETERMINATION OF CYTOTOXICITY 

-Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial 

background lawn. 

METHODS OF APPLICATION: in agar (pre-incubation) – Experiment 2 (Pre-Incubation) 

- Pre-incubation period for bacterial strains: 10hrs 

- Exposure duration: 48-72hrs 

- Expression time (cells in growth medium): Not applicable 

- Selection time (in incubation with a selective agent): 20 minutes at 37 degrees C 

NUMBER OF REPLICATIONS: Triplicate plating. 

DETERMINATION OF CYTOTOXICITY 

-Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial 

background lawn. 

Evaluation criteria 

Acceptance Criteria: 

The reverse mutation assay may be considered valid if the following criteria are met: 

All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according 

to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000). All tester strain cultures should exhibit a 

characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Combined historical negative and 
solvent control ranges for 2009 and 2010 are presented in Appendix 3. All tester strain cultures should be in the range of 0.9 to 9 x 

109 bacteria per ml. Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity 

of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study 

should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical 

ranges of the positive controls for 2009 and 2010 are presented in Appendix 3. There should be a minimum of four non-toxic test

item dose levels. There should be no evidence of excessive contamination. 

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. 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 (negative) in the test system if the above criteria are not met. 

Statistics 

Standard Deviation 

Dunnetts Linear Regression Analysis 

Any other information on materials and methods incl. tables 

Tester strains 

 

Salmonella typhimurium

Strains Genotype Type of mutations indicated

TA1537 hisC 3076;rfa-

; uvrB-:

frame shift mutations 

TA98 hisD 3052;rfa-

; uvrB-;R-factor 

TA1535 hisG 46;rfa-

; uvrB-: 

base-pair substitutions TA100 hisG 46;rfa-

; uvrB-;R-factor 

Escherichia coli

Strain Genotype Type of mutations indicated

WP2uvrA trp-

;uvrA-: base-pair substitution 

The strains of bacteria used in the test were obtained from the University of California, Berkeley, on 

culture discs, on 04 August 1995, from Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007 

or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 

1987. All of the strains were stored at approximately -196°C in a Statebourne liquid nitrogen freezer, 

model SXR 34. 

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient 

broth and incubated at 37oC for approximately 10 hours. Each culture was monitored 

spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. 

The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide 

by mixing on a vortex mixer on the day of each experint. All formulations were used within four hours of 

preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and 

stability of the test item formulations is not a requirent of the test guidelines and was, therefore, not 

determined. This is an exception with regard to GLP and has been reflected in the GLP compliance 

statement. 

 

The S9 Microsomal fraction was prepared in-house (01 July 2012) from rats induced with 

Phenobarbitone/b-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 

4. The S9 homogenate was produced by homogenising the liver in a 0.15M KCl solution (1g liver to 3 ml 

KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted 

to 20 mg/ml. Aliquots of the supernatant were frozen and stored at approximately -196o

C. Prior to use, 

each batch of S9 was tested for its capability to activate known mutagens in the Ames test. 
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for 

the duration of the test. 

S9: 5.0 ml 

1.65 M KCl/0.4 M MgCl2: 1.0 ml 

0.1 M Glucose-6-phosphate: 2.5 ml 

0.1 M NADP: 2.0 ml 

0.2 M Sodium phosphate buffer (pH 7.4): 25.0 ml 

Sterile distilled water: 14.5 ml 

 

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. 

Top agar was prepared using 0.6% Bacto agar (lot number 1297425 09/16) 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 (lot numbers 32515 08/12, 

32765 10/12 and 32773 10/12). 

 

Preliminary Toxicity Test 

In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to 

determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 

500, 1500 and 5000 μg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or 

WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test item 

formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner 

Minimal agar (30 ml/plate). Ten concentrations of the test item formulation and a vehicle control (acetone) 

were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace 

histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to 

assess the sterility of the test item. After approximately 48 hours incubation at 37°C the plates were 

assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on 

the growth of the bacterial background lawn. 

 

Mutation Test – Experiment 1 

Seven concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 μg/plate) were assayed in triplicate 

against each tester strain, using the direct plate incorporation method. 

Additional dose levels and an expanded dose range were selected in order to achieve both four non-toxic 

dose levels and the toxic limit of the test item. 

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed 

by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the vehicle, test item 

formulation or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test 

tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube 

per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration 

of test item both with and without S9-mix. 

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant 

colonies assessed using an automated colony counter. 

 

Mutation Test – Experiment 2 

The second experiment was performed using fresh bacterial cultures, test item and control solutions. The 

dose range was amended slightly following the results of Experiment 1 as follows: 

All Salmonella strains (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. 

All Salmonella strains (absence of S9-mix) and Escherichia coli strain WP2uvrA (absence and presence 

of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate. 

Additional dose levels and an expanded dose range were again selected in order to achieve both four 

non-toxic dose levels and the toxic limit of the test item. 

As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was 
changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were 

therefore dosed as follows: 

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed 

by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test item formulation and incubated 

for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace 

histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally 

distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was 

repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and 

without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation 

method described in Experiment 1. 

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant 

colonies assessed using an automated colony counter.

Results and discussions 

Test results 

Species/strain E. coli WP2 uvr A 

Metabolic 

activation 

with and without 

Test system all strains/cell types tested 

Genotoxicity negative 

Cytotoxicity yes (The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester 

strains, initially from 150 and 500 μg/plate in the absence and presence of S9-mix respectively.) 

Vehicle 

controls valid 

yes 

Negative 

controls valid 

yes 

Positive 

controls valid 

yes 

Species/strain S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 

Metabolic 

activation 

with and without 

Test system all strains/cell types tested 

Genotoxicity negative 

Cytotoxicity yes (The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester 

strains, initially from 150 and 500 μg/plate in the absence and presence of S9-mix respectively.) 

Vehicle 

controls valid 

yes 

Negative 

controls valid 

yes 

Positive 

controls valid 

yes 

Additional information on results 

TEST-SPECFIC CONFOUNDING FACTORS: 

- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of 

S9-mix. 

STERILITY, VEHICLE AND POSITIVE CONTROL DATA: 

- Prior to use, the master 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. These data are not given in the report. 

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. 

These data are for concurrent untreated control plates performed on the same day as the Mutation Test. 

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, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and 

Table 5 for Experiment 2. 
A history profile of vehicle/untreated and positive control values (reference items) for 2010 and 2011 are presented in Appendix 2. 

Any other information on results incl. tables 

RESULTS 

The test item was toxic at and above 500 μg/plate to both of the strains of bacteria used (TA100 and 

WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile. 

 

The numbers of revertant colonies for the toxicity assay were: 

With (+) or 

without (-) 

S9-mix 

Strain 

Dose (μg/plate) 

0 0.15 0.5 1.5 5 15 50 150 500 1500 5000

- TA100 84 79 79 97 91 68 84 91 0V 0V 0T 

+ TA100 89 98 106 101 82 92 107 116 97S 0T 0T 

- WP2uvrA 20 22 21 27 20 34 36 26 0V 0T 0T 

+ WP2uvrA 44 27 35 36 38 32 31 35 0V 0T 0T 

S: Sparse bacterial background lawn 

V: Very weak bacterial background lawn 

T: Toxic, no bacterial background lawn 

Mutation Test 

Table 1: Spontaneous Mutation Rates (Concurrent Negative Control) 

Experiment 1 

Number of revertants (mean number of colonies per plate) 

Base-pair substitution type Frameshift type 

TA100 TA1535 WP2uvrA TA98 TA1537 

75 17 29 15 17 

82 (80) 28 (20) 17 (21) 18 (15) 7 (11) 

83 16 16 13 10 

 

Experiment 2 

Number of revertants (mean number of colonies per plate) 

Base-pair substitution type Frameshift type 

TA100 TA1535 WP2uvrA TA98 TA1537 

84 13 30 19 10 

94 (88) 8 (13) 20 (25) 15 (15) 9 (8) 

86 18 24 10 5 

 

FOR TABLES OF RESULTS FOR MUTATION TEST: Please see attached in overall remarks, 

attachments 

 

References:
Ames B N, McCann J and Yamasaki E (1975), Methods for detecting carcinogens and mutagens with the 

Salmonella/mammalian microsome mutagenicity test, Mutation Research, 31, 347-364. 

Maron D M and Ames B N (1983), Revised Methods for the Salmonellamutagenicity test, Mutation 

Research, 113, 173 - 215. 

Mortelmans K and Zeiger E (2000), The Ames Salmonella/microsome mutagenicity assay, Mutation 

Research, 455, 29-60. 

Green M H L and Muriel W J (1976), Mutagen Testing Using TRP+ Reversion in Escherichia coli, Mutation 

Research, 38, 3- 32. 

Maron D M, Katzenellenbogen J and Ames B N (1981), Compatibility of organic solvents with the 

Salmonella/Microsome Test, Mutation Research, 88, 343-350. 

De Serres F J and Shelby M D (1979), Recommendations on data production and analysis using the 

Salmonella/microsome mutagenicity assay, Environmental Mutagenesis, 1, 87-92. 

Mahon G A T 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 (Kirkland D J Ed.), 

Cambridge University Press Report, 26-65.

Overall remarks, attachments 

Remarks on results including tables and figures 

41201581-tables.pdf: Summary Data Tables 2-5.

41201581-CoA.pdf: A copy of the CoA (as presented in the report).

41201581-Appendix2.pdf; In-house Historical Ranges 2009-2010.

41201581-Appendix 3-GLP; GLP certificate as presented in the report

Attached background material 

Attached document Remarks 

41201581-tables.pdf / 40.61 KB (application/pdf) Summary Data Tables 2-5 

41201581-COA.pdf / 125.34 KB (application/pdf) A copy of the CoA (as presented in the report) 

41201581-Appendix2.pdf / 20.35 KB (application/pdf) In-house Historical Ranges 2009-2010 

41201581-Appendix3.pdf / 100.12 KB (application/pdf) GLP certificate as presented in the report 

Applicant's summary and conclusion 

Interpretation of results 

negative 

Conclusions 

The test item, Benzenesulfonyl chloride, was considered to be non mutagenic under the conditions of this test. 

Executive summary 

Introduction. 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 and the USA, EPA (TSCA) 

OPPTS harmonised guidelines 40 CFR 799.9510. 

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain 

WP2uvrAwere treated with the test item, Benzenesulfonyl chloride, using both the Ames plate 

incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the 

addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose 

range for the first experiment was determined in a preliminary toxicity assay and was 1.5 to 1500 

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 slightly following the 

results of Experiment 1 and ranged between 0.5 and 1500 μg/plate, depending on bacterial strain type 

and presence or absence of S9-mix. 
Additional dose levels and an expanded dose range were selected in both experiments in order to 

achieve both four non-toxic dose levels and the toxic limit of the test item. 

Results. The vehicle (acetone) control plates gave counts of revertant colonies generally within the 

normal range. All of the positive controls 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 test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester 

strains, initially from 150 and 500 μg/plate in the absence and presence of S9-mix respectively. The test 

item was, therefore, tested up to the toxic limit. No test item precipitate was observed on the plates at any 

of the doses tested in either the presence or absence of S9-mix. 

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 or exposure 

method. Small, statistically significant increases in revertant colony frequency were observed in several 

tester strains in both the absence and presence of S9-mix at sub toxic dose levels in Experiment 1. These 

increases were considered to be of no biological relevance because there was no evidence of a doseresponse relationship or reproducibility in any case. Furthermore, the individual revertant counts at the 

statistically significant dose levels were within the in-house historical untreated/vehicle control range for 

each tester strain and the maximum fold increase was only 1.8 times the concurrent vehicle control. 

Conclusion. The test item, Benzenesulfonyl chloride, was considered to be non-mutagenic under the 

conditions of this test.