Reaction mass of sodium 7-hydroxy-8-((E)-(4-(1-(4-((E)-(4-hydroxyphenyl)diazenyl)phenyl)cyclohexyl)phenyl)diazenyl)naphthalene-1,3-disulfonate and sodium 8,8'-(1E,1'E)-(4,4'-(cyclohexane-1,1-diyl)bis(4,1-phenylene))bis(diazene-2,1-diyl)bis(7-hydroxynaphthalene-1,3-disulfonate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Remarks:

the tested substance is one of the two components of the substance under registration

Adequacy of study:

key study

Study period:

From February 01st to March 07th, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The tested substance is one of the two components of the substance under registration.

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:

May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

July 13, 1987

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Orange 033_constituent 1

IUPAC Name:

Acid Orange 033_constituent 1

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Source of cells: except for strain TA 100, all strains from Prof. B. Ames, Berkeley. Strain TA 100 from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basle.
- Suitability of cells: 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. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).
- 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.

Metabolic activation:

with and without

Metabolic activation system:

rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

61.73, 185.19, 555.56, 1666.67 and 5000.00 µg/plate

Vehicle / solvent:

Test item was dissolved in bidistilled water at room temperature.
The test substance was soluble up to the concentration of 50 mg/ml. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with bidistilled water. No precipitates or aggregates were noted.

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclohexylamine

other: 2-Aminoanthracene in DMSO for TA100, TA 98 and TA 1537 with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
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.

NUMBER OF REPLICATIONS: two experiments, three plates per test substance concentration and controls.

CELLS EVALUATION
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.

VALIDITY 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.

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 testing laboratory. 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 35.81 / 40.63 mg/ml.

The activation mixture contained: rat liver S9 fraction: 100.0 µl/ml, NADP: 4.0 µmol/ml, MgCl2: 8.0 µmol/ml, KCl: 33.0 µmol/ml, Na-phosphate-buffer, pH 7.4: 100.0 µmol/ml, Glucose-6-phosphate: 5.0 µmol/ml.

RANGE FINDING
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 a Standard Operating Procedure of Genetic Toxicology. The highest concentration applied was 5000 µg/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.

Evaluation criteria:

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.

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight toxic effect

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight toxic effect

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight toxic effect

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight toxic effect

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

not specified

Genotoxicity:

other: n.a.

Cytotoxicity / choice of top concentrations:

other: n.a.

Remarks:

slight toxic effect

Vehicle controls validity:

other: n.a.

Untreated negative controls validity:

other: n.a.

True negative controls validity:

other: n.a.

Positive controls validity:

valid

Remarks:

n.a.

Additional information on results:

In the first experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with test item did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

In the second experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with test item 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 occasionally slightly reduced at the highest concentration of 5000 µg/plate. The test substance exerted a slight 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.

COMPARISON WITH HISTORICAL CONTROL DATA
Arithmetic Mean and Standard Deviation (SD) of colony counts obtained in 75 separate experiments over the period of January 01, 1993 to December 31, 1993 and acceptable ranges for mean colony counts of spontaneous revenants.

RANGE-FINDING TEST
Six concentrations of test item) ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and 5000.0 µg/plate without metabolic activation.

Applicant's summary and conclusion

Conclusions:

It is concluded that the substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Executive summary:

Method

The substance 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 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 dissolved in bidistilled water and tested at five concentrations in the range of 61.7 to 5000.0 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 five concentrations in the range of 61.7 to 5000.0 µg/plate. 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.

Results

In both experiments, performed with and without metabolic activation, none of the tested concentrations of test item led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.