Sodium 4(-2-hydroxy-1-naphthylazo)naphthalenesulphonate

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:

key study

Study period:

from 20th Jun. 2003 to 1st Aug. 2003

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 1

Justification for type of information:

The read across justification is detailed in section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

8 June 2000

Deviations:

no

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, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: S. typhimurium strains supplied by B.N. Ames' Laboratory (University of Califomia, Berkeley, USA); E. coli strain supplied by S. Venitt's Laboratory (ICR, Sutton, England).
- Storage: in a cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethylsulfoxide) in liquid nitrogen.
- Preparation: the day before treatment, cultures were inoculated from frozen permanents; a scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: S. typhimurium strains supplied by B.N. Ames' Laboratory (University of Califomia, Berkeley, USA).
- Storage: in a cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethylsulfoxide) in liquid nitrogen.
- Preparation: the day before treatment, cultures were inoculated from frozen permanents; a scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions.

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal fraction (S9)

Test concentrations with justification for top dose:

CONCENTRATIONS
312.5, 625, 1250, 2500 and 5000 µ/plate, for both mutagenicity experiments with and without S9 mix, except for the first experiment with the TA98 and TA 1537 strains with S9 mix where the following dose-levels were tested: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate.

TOP DOSE SELECTION
Since the test item was freely soluble and generally non-toxic, the highest dose-level was 5000 pg/plate, according to the criteria specified in the intemational guidelines.

Vehicle / solvent:

- Vehicle used: water
- Justification for choice of vehicle: the test item was freely soluble in the vehicle (water for injections) at 50 mg/ml.

Controls

Controls 1

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

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method.
The second experiment with S9 mix was performed according to the preincubation method.

Plate incorporation method
The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45 °C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

Preincubation method
The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter. Perceptive Instmments, Suffolk CB9 7 BN, UK).

NUMBER OF REPLICATIONS
Two independent experiments, using three plates/dose-level.

EVALUATION OF THE RESULTS
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), were stated.

DETERMINATION OF CYTOTOXICITY
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

METABOLIC ACTIBVATION SYSTEM
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats freated with Aroclor 1254 (500 mg/kg) by the infraperitoneal route.
The S9 fraction was preserved in sterile tubes at -80 °C, until use. The S9 mix was prepared at +4 °C immediately before use and maintained at this temperature until added to the overlay agar.
The composition of S9 mix was as follows: Glucose-6-phosphate 5 mM, NADP 4 mM, KCl 33 mM, MgCl2 8mM, Sodium phosphate buffer pH 7.4 100 mM, S9 fraction, batch Nos. 1475 and 1565, protein concenfrations: 43 and 38.8 mg/mL, respectively, 10 % (v/v); water to volume.

The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

ACCEPTANCE CRITERIA
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100, TA 102 and WP2 uvrA strains) or 3-foId increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account m the evaluation of the data obtained.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from moderate to marked generally at dose-levels > 2500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Remarks:

and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MAIN TESTS
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
A moderate to marked toxicity was noted in the second experiment with S9 mix (preincubation method), in the TA 1535, TA 1537, TA 98 and TA 100 strains, generally at dose-levels > 2500 pg/plate.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six strains.

CONTROLS
The number of revertants for the vehicle and positive confrols was as specified in the acceptance criteria. The study was therefore considered valid.

PRELIMINARY TOXICITY  TEST
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
An orange coloration of agar was observed in all test item treated plates at dose-levels > 100 µg/plate.
No noteworthy toxicity was noted towards the four strains used, with and without S9 mix, except a decrease in the number of revertants at dose-levels > 1000 µg/plate, for the TA 98 strain with S9 mix.

Applicant's summary and conclusion

Conclusions:

The test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli under the experimental conditions.

Executive summary:

In order to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium and Escherichia coli, an AMES study was performed according to the OECD Guideline No. 471 (1997).A preliminary toxicity test was performed to define the dose-levels of test substance to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system (S9 mix). The preliminary test and the first experiment were performed according to the direct plate incorporation method; while the second experiment with S9 mix was performed according to the preincubation method. Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA98, TA100 and TA 102 and one strain of Escherichia coli: WP2 uvrA were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level) from. After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolved in water for injections. All concentrations and dose-levels were expressed as active item, taking into account the purity. Also the positive controls were included in the assay both with and without S9 mix.

The acceptance criteria were met. Since the test item was freely soluble and generally non-toxic in the preliminary test, the highest dose-level selected for the main test was 5000 µg/plate. The selected treatment-levels were from 312.5 to 5000 µg/plate, for both mutagenicity experiments, with and without S9 mix, except for the first experiment with the TA 98 and TA 1537 strains with S9 mix where the dose-levels were from 156.3 to 5000 µg/plate. No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. A moderate to marked toxicity was noted in the second experiment with S9 mix (preincubation method), in the TA 1535, TA 1537, TA 98 and TA 100 strains, generally at dose-levels > 2500 µg/plate. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six strains.

The test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli under the experimental conditions.