Sodium 2-[[2-amino-8-hydroxy-6-[(methylanilino)sulphonyl]-1-naphthyl]azo]-5-(chloroacetamido)benzenesulphonate

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:

18 October 1995 to 12 January 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-[[2-amino-8-hydroxy-6-[(methylanilino)sulphonyl]-1-naphthyl]azo]-5-(chloroacetamido)benzenesulphonate

EC Number:

274-395-5

EC Name:

Sodium 2-[[2-amino-8-hydroxy-6-[(methylanilino)sulphonyl]-1-naphthyl]azo]-5-(chloroacetamido)benzenesulphonate

Cas Number:

70209-97-1

Molecular formula:

C25H22ClN5O7S2.Na

IUPAC Name:

sodium 2-({2-amino-8-hydroxy-6-[(2-methylphenyl)sulfamoyl]-1-naphthyl}diazenyl)-5-[(chloroacetyl)amino]benzenesulfonate

Test material form:

other: solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 1.56
- Expiration date of the lot/batch: June, 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 8° C, protected from light
- Stability under storage conditions: stable until June 2000

Method

Target gene:

histidine (his)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Füllinsdorf, weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, FR.G.) in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenized. The homogenate, was diluted 1+3 in KCl and centrifuged at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to several weeks before use. The standardization of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6 (6).
The protein concentration in the S9 preparation was 31.2 mg/ml (lot 170795) in the pre-experiment and experiment I or 32.4 mg/ml (lot 271195) in experiment II.

S9Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix:
8mM MgCl2
33 mM KCl
5mM Glucose-6-phosphate
5mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate. According to the results of the pre-experiment the concentrations applied in the main experiments were chosen. The maximum concentration was 5000 µg/plate. The concentration range included two logarithmic decades. In this study six adequately spaced concentrations were tested. Two independent experiments were performed.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation: TA 1535, TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation: TA 1537, TA 98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation: TA 1535, TA 1537, TA 98, TA 100

Details on test system and experimental conditions:

Pre-Experiment for Toxicity:
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test). Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. According to the dose selection criteria the test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate

Experimental Performance:
For each strain and dose level, including the controls three plates were used as a minimum. The following materials were mixed in a test tube and poured onto the selective agar plates: 100 µl Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 µl Bacteria suspension (cf. test system, pre-culture of the strains), 2000 µl overlay agar. In the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Data Recording:
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben; F.R.G.). The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Since the test article precipitated at concentrations of 1000 µg/plate and above the corresponding plates were evaluated manually.

Evaluation criteria:

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A significant response is described as follows: A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strain TA 100 or thrice on TA 1535, TA 1537, and TA 98.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Statistics:

According to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, 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

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

FAT 20075/B is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 20075/B to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The study was carried out according to OECD guideline 4711 and EU method B.14. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at concentration 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate.

 

Precipitation of the test article occurred at concentrations of 1000 µg/plate and above. Toxic effects evident as a reduction in the number of revertants occurred in strains TA 1535 and TA 1537 at 5000 µg/plate without metabolic activation in the first experiment. The plates incubated with the test article showed normal background growth up to 5000 µg/plate in all experiments. No relevant increases in revertant colony numbers of any of the four tester strains were observed following treatment with FAT 20075/B at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 20075/B is considered as non-mutagenic in the Salmonella typhimurium reverse mutation assay.