Benzenesulfonic acid, 4-chloro-2-[2-[4,5-dihydro-3-methyl-5-oxo-1-(3-sulfophenyl)-1H-pyrazol-4-yl]diazenyl]-5-methyl-, ammonium salt (1:2)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

26 May 1983

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Yellow powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: soluble in DMSO; stability not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolved in dimethyl sulphoxide

Method

Target gene:

his / trp

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rodent liver microsomes fractions

Type and composition of metabolic activation system:
- source of S9 : livers of rats (induced with Aroclor 1254) and hamsters
- method of preparation of S9 mix: Mixed function oxidase systems in the livers of a group of rats were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed, and their livers aseptically removed.The rat livers were placed in 0.15 M KCl (3 ml KCl : 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at —80°C until required. Rat S-9 mix contained: S-9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM), NADH (4 mM). All the cofactors were filter-sterilised before use.

Following an overnight starvation, the hamsters were killed, and their livers aseptically removed. The following steps were carried out at 0-4°C under aseptic conditions. The livers were placed in 0.1 M sodium phosphate buffer (pH 7.4) (3 ml buffer : 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for
10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at —80°C until required. Hamster S-9 mix contained: S-9 fraction (30% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer (pH 7.4) (100 mM), glucose-6-phosphate (20 mM), NADP (4 mM), NADH (2 mM), flavin mononucleotide (2 mM), glucose-6-phosphate dehydrogenase (2.8 units/ml). All the cofactors were filter-sterilised before use.

- concentration or volume of S9 mix and S9 in the final culture medium: An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml of rat or hamster S-9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added and the mixture incubated at 30°C for 30 minutes. 2 ml of histidine/tryptophan deficient agar was then added and the mixture thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The efficacy of each batch of rat S-9 fraction was tested with the carcinogens 7,12-dimethylbenzanthracene and
2-aminoanthracene before use. The efficacy of each batch of hamster S-9 fraction was tested with the carcinogens Congo Red and 2-aminoanthracene before use.

Test concentrations with justification for top dose:

5000, 2500, 1250, 625, 312.5 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)

- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test substance was assessed at 50 mg/ml in water and dimethyl sulphoxide. The test substance formed a poor suspension in water but dissolved in dimethyl sulphoxide after warming to ca 50°C. Therefore dimethyl sulphoxide was chosen as the solvent for this study.

- Justification for percentage of solvent in the final culture medium: not specified

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

In the absence of S-9 mix, in DMSO; for TA1535, TA100 and WP2 uvrA

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S-9 mix, in DMSO; for TA1538 and TA98

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S-9 mix, in DMSO; for TA1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

In the presence of S-9 mix, in DMSO; for TA1538 and TA98

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene; in the presence of S-9 mix, in DMSO; for TA1535, TA100 and WP2 uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments. 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 min
- Exposure duration/duration of treatment: 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: reduction in revertant colony counts or absence of a complete background bacterial lawn

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance was assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9
mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Water solubility: 0.6 g/L
- Precipitation and time of the determination: Precipitation was observed from test substance concentrations of 625 µg/plate

RANGE-FINDING/SCREENING STUDIES (if applicable): Four concentrations of test substance were assessed for toxicity using the six tester strains. The highest concentration was 50 mg/ml of test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Three 10-fold serial dilutions of the highest concentration were also tested. The chosen solvent, dimethyl sulphoxide, was used as the negative control. The test substance was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests.

Applicant's summary and conclusion

Conclusions:

It is concluded that, when tested in dimethyl sulphoxide, the test item shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test article, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test substance, diluted in dimethyl sulphoxide which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats and un-induced Syrian hamsters. A 30 minutes at 30°C preincubation stage was included in both tests.

In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, 312.5 µg/plate.

No evidence of mutagenic activity was seen at any dose level in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in dimethyl sulphoxide the test substance was not mutagenic in this bacterial system.