3-acetamido-5-amino-4-hydroxybenzenesulphonic acid

Genetic toxicity: in vitro

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

Study period:

07.12.1993 - 21.01.1994

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-acetamido-5-amino-4-hydroxybenzenesulphonic acid

EC Number:

254-879-2

EC Name:

3-acetamido-5-amino-4-hydroxybenzenesulphonic acid

Cas Number:

40306-75-0

Molecular formula:

C8H10N2O5S

IUPAC Name:

3-acetamido-5-amino-4-hydroxybenzenesulfonic acid

Specific details on test material used for the study:

Test material: FAT 92333/A (2 Acetamido-6-Aminophenol-4-sulfosäure (Laborgetrocknet))
Batch No.: K37 19.5.92
Purity: 97.2 %
Stability: See expiry date
Appearance: Gray paste
Expiry date: June 1997
Storage: Room temperature

Material submitted by:
CIBA-GEIGY Limited
Basle, Switzerland
Dyestuffs Division
Dr. J. Maldacker-Kurth


The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 102, TA 1535, TA 1537) were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M.Schüpbach, Hoffrnann-La Roche Limited., Basel, Switzerland.

Method

Target gene:

This test permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium. When the Salmonella strains are exposed to a mutagen, some of the bacteria in the treated population, through chemical interaction with the compound or its metabolites, undergo genetic changes which cause them to revert to a non-histidine-requiring state and thus to grow in the absence of exogenous histidine. Mutagenic effects of the test substance are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone reverse-mutation to histidine prototrophism. Different tester strains are used because of differing sensitivities to known mutagens.

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 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix Aroclor 1254-induced

Test concentrations with justification for top dose:

range finding test: 20.6 to 5000 ug/plate
Mutagenicity test: 61.73 to 5000 ug/plate

Vehicle / solvent:

DMSO and bidistilled water

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

sodium azide

cyclophosphamide

mitomycin C

other: 2-Aminoanthracene, 2-Nitrofluorene, 4-Nitroquinoline

Details on test system and experimental conditions:

Preparation of the bacterial cultures
Inoculates from frozen master copies were set up monthly. They were grown in liquid nutrient broth medium (NB-medium) overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.

Control of the genotype of the strains
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. The strain TA 102 was additionally checked for tetracycline resistance (presence of multicopy plasmid pAQl). The presence of the uvr+ gene (TA 102) was demonstrated by the resistance against UV light. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).

Preparation of the metabolic activation mixture
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male RAI rats (Tif: RAIf [SPF]), reared at the Animal Farm of CIBA-GEIGY Limited, Sisseln, Switzerland. The animals were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 33.37 mg/ml.

Solubilisation of the test substance
FAT 92333/A was suspended in DMSO at room temperature. In the toxicity test, the two highest concentrations and in the mutagenicity test the three highest concentrations were weighed separately. Lower concentrations of the test substance were obtained by appropriate dilution with DMSO. Due to a weighing error, in the original mutagenicity test the concentrations of 555.0 µg/plate and lower are approximately 10% less than intended. This has, however, no effect on the test results.

Setting up of the test plates
0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20.0 ml of minimal agar (1.5 % agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2 % glucose). The top agar was composed of 0.6% agar and 0.6 % NaCl and was supplemented with 10 % of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water.

Preliminary range finding test
A range finding test was carried out with strain TA 100 with and without metabolic activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 ug/plate. The five lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

Mutagenicity test
The mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, TA 1537 with and without metabolic activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration and controls. The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Negative and positive controls
The solvent alone was used as the negative control. The positive controls were the following reference mutagens:
Experiment with methabolic activation:

Strain Mutagen Solvent Concentration
TA 100 2-Aminoanthracene DMSO 2.5 µg/plate
TA 1535 Cyclophosphamide Bidistilled water 400 µg/plate
TA 102 2-Aminoanthracene DMSO 20 µg/plate
TA 98 2-Aminoanthracene DMSO 2.5 µg/plate
TA 1537 2-Aminoanthracene DMSO 2.5 mg/plate

Experiment without methabolic activation:
Strain Mutagen Solvent Concentration
TA 100 Sodium azide Bidistilled water 5.0 µg/plate
TA 1535 Sodium azide Bidistilled water 5.0 µg/plate
TA 102 Mitomycin-C Bidistilled water 2.0 µg/plate
TA 98 2-Nitrofluorene DMSO 20.0 µg/plate
TA 1537 9-Aminoacridine DMSO 150.0 µg/plate

Colony counting and scoring of the plates
Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates might have interfered with automating counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated and included in the Results section.

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response
The test substance will be considered to be positive in the test system if the following condition is met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the strains tested.
Generally a concentration-related effect should be demonstrable.

Evaluation criteria:

A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Statistics:

A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Range finding test (Tables 1-2)
Six concentrations of FAT 92333/A (2 Acetamido-6-Aminophenol-4-sulfosäure (Laborgetrocknet)) ranging from 20.6 to 5000.0 ug/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiment was
performed with and without metabolic activation. Normal background growth was observed in both experimental parts. The numbers of revenant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 ug/plate with and without metabolic activation.

Mutagenicity test, original experiment
In the experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with FAT 92333/A did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment
In the experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with FAT 92333/A no increase in the incidence of either histidine-prototrophic mutants was observed in comparison with the negative control.
In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Executive summary:

FAT 92333/A (2 Acetamido-6-Aminophenol-4-sulfosäure (Laborgetrocknet)), identified as gray paste, purity 97.2%, batch no. K37 19.5.92, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in DMSO and tested at five concentrations in the range of 61.73 to 5000.00 µg/plate in the presence and absence of a metabolic activation system. In order to

confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 92333/A led to an increase in the incidence of histidine-prototrophic mutants by

comparison with the negative control.