Reaction products of 6-amino-4-hydroxynaphthalene-2-sulphonic acid and 7-amino-4-hydroxynaphthalene-2-sulphonic acid, condensed with Formaldehyde and Sodium hydrogensulfite, coupled with diazotised Sodium 2-amino-4,6-dinitrophenoxide, metalized with Sodium Dichromate, sodium salts

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)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed on the analogue substance; the read across justification is detailed in section 13. The Reliability of the Source Study is 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26, 1983

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: hisG46 base-pair substitution

Remarks:

Additional mutations: rfa, gal, chl, bio, uvrB

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: hisC3076 frameshift

Remarks:

Additional mutations: rfa, gal, chl, bio, uvrB

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: hisD3052/R-factor frameshift

Remarks:

Additional mutations: rfa, gal, chl, bio, uvrB

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: hisG46/R-factor base-pair substitution

Remarks:

Additional mutations: rfa, gal, chl, bio, uvrB

Metabolic activation:

with and without

Metabolic activation system:

Rat microsomal fraction S9 mix

Test concentrations with justification for top dose:

-Experiment concentrations: 100, 333, 1000, 3330, 5000 µg/plate
Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix. Nine concentrations have been tested in duplicate for toxicity. The highest concentration of test substance used in the subsequent mutagenesis assay was that which gave a reduced survival on the non-selective plates. If no toxicity was observed, the highest dose level used in the mutagenesis assay was 5 mg/plate unless the test substance exhibited limited solubility or was not uniformly dispersible in the solvent of choice.

Vehicle / solvent:

Milli-Q water (Millipore Corp.,Bedford, Mass., USA)

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: daunomycine and methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

TEST SYSTEM
-Source: Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (1987)
-Storage: stock cultures of the four strains in liquid nitrogen (-196°C).
-Check: strains regularly checked for their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

CELL CULTURE
-Preparation of bacterial cultures: samples of frozen stock cultures of bacteria transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking bath (37 °C, 150 spm), until the cultures reached an O.D. of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain used for a test.
-Agar plates: 0 9 cm, contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid, code L28) in Vogel-Bonner Medium E (Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218, 1956, 97-106), 10 g glucose, 0.5 mg biotin and 0.6 mg histidine.
-Top agar: Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 120 °C. Prior to the viability determination the top agars were supplemented with 1.55 mg histidine per top agar.
-Environmental conditions: all incubations have been carried out in the dark at 37°C. The temperature was monitored during the experiment.

METABOLIC ACTIVATION SYSTEM
-Preparation of S9-homogenate: rat liver microsomal enzymes were routinely prepared from adult male Wistar or Sprague Dawley rats, which were obtained from Charles River Wiga, Sulzfeld, F.R.G. The animals were housed at RCC NOTOX in a special room under standard laboratory conditions. The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C).
-Preparation of S9-mix: prepared immediately before use and kept on ice during the test. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml aqua bidest; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution, and 0.5 ml S9. The above solutions were mixed and filter (0.22 µm)-sterilised (apart from the S9-fraction, which was added after filter-sterilisation of the S9-mix components).

Evaluation criteria:

No formal hypothesis testing has been done.
A test substance was considered:
-negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
-positive (mutagenic) in the Ames test if:
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA100 bacteria

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY OF THE TEST SUBSTANCE
The survival of the TA100 culture is determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plate. Both in the absence and presence of S9-mix the survival of strain TA100 is not reduced up to test substance concentrations of 5000 µg/plate. Based on these data, the test substance was tested up to a concentration of 5000 µg/plate in the absence and presence of S9-mix.

THE AMES SALMONELLA/MICROSOME PLATE TEST
All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values fell within laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

The test substance can be considered as not mutagenic in this Ames Salmonella assay.

Executive summary:

The test substance was tested for mutagenic effects with an in vitro bacterial reverse mutation assay, according to the OECD Guideline 471 (1983). The test was performed without and with metabolic activation in the range of concentration of 100 - 5000 μg/plate, using strains of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). Each concentration and controls were tested in triplicate. The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the tester strains. These results were confirmed in an independently repeated experiment. Therefore, test substance can be considered as not mutagenic in this test system.