Sodium 4-(2-hydroxynaphth-1-ylazo)-3-methylbenzenesulphonate

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, test procedure in accordance with OECD 471 methods, meets generally accepted scientific principles, acceptable for assessment and GLP compliant. The test has been performed on only four Salmonella strains.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Remarks:

migrated information: powder

Method

Target gene:

Salmonella typhimurium and Escheria coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 6

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rats

Test concentrations with justification for top dose:

For both mutagenicity test the treatment-levels are:
Strain TA100/TA102/TA1535 with and without S9 mix: 312,5; 625; 1250; 2500 and 5000 µg/plate
Strain TA98 and TA1537 with S9 mix: 156,3; 312,5; 625; 1250; 2500 and 5000 µg/plate

Vehicle / solvent:

water for injections (Batch EVB03A, Frenesius Kabi, Sevres, France)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 an TA1535 without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

Remarks:

TA1537 wihtout S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 4-nitro-o-phenylenediamine (NPD)

Remarks:

TA98 without S9 mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA102 without S9 mix

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2 uvrA without S9 mix

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

TA98, TA100, TA102, TA1535, TA1537 and WP2 uvrA with S9 mix

Details on test system and experimental conditions:

BACTERIAL STRAINS INFORMATION:
The five strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were supplied by B.N. Ames' Laboratory (University of Califomia, Berkeley, USA).
The strain of Escherichia coli: WP2uvrA was supplied by S. Venitt's Laboratory (ICR, Sutton, England).
They are stored in a cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethylsulfoxide) in liquid nitrogen.
The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximatel 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.

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine. The strain of Escherichia coli contains one mutation in the tryptophan operon, resulting in a requirement for tryptophan.
In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
- the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall,
- the uvr (uvrB for Salmonella typhimurium and uvrA for Escherichia coli) mutation is a deletion of a gene coding for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
- the addition of the plasmid pKM 101 to strains TA 98, TA 100 and TA 102 enhances their sensitivity of detection to some mutagens,
- in the case of TA 102, the histidine mutation is located on the multicopy plasmid pAQ 1.

METHOD OF APPLICATION: in agar (plate incorporation)

PREPARATION OF S9 mix: 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 the S9 mix was:
5mM Glucose-6-phosphate
4 mM NADP
33 mM KCl
8mM MgCl2
100mM Sodium phosphate buffer pH 7.4
10% (v/v) S9 fraction, batch Nos. 1475 and 1565, protein concenfrations: 43 and 38.8 mg/mL, respectively.
to volume water

Evaluation criteria:

A test article is considered as positive if either a significant dose-related 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 significant dose-related 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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
The test item was freely soluble in the vehicle (water for injections) at 50 mg/mL.
Consequently, with a treatment volume of 100 pL/plate, the dose-levels were 10, 100, 500, 1000,2500 and 5000 µg/plate.
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 sfrains used, with and without S9 mix, except a decrease in the number of revertants at
dose-levels > 1000 µg/plate, for the TA 98 sfrain with S9 mix.

Mutagenicity experiments:
The number of revertants for the vehicle and positive confrols was as specified in the acceptance criteria. The study was therefore considered valid.
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 international guidelines.
The selected freatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/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.
No precipitate was observed in the Petri plates when scoring flie revertants at all dose-levels.
A moderate to marked toxicity was noted in the second experiment with S9 mix (preincubation method), in tiie TA 1535, TA 1537, TA 98 and TA 100 sfrains, 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 sfrains.

Remarks on result:

other: strain/cell type: TA98

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results
negative

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 sfrains.

Executive summary:

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 Acid Orange 8 is considered to be non-mutagenic in this Bacterial reverse mutation assay based on the result on similar substance.