Tetrasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(2-methyl-4-sulphonatophenyl)azo]naphthalene-2-sulphonate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1975

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

pre GLP

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Direct Orange 118

IUPAC Name:

Direct Orange 118

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Details on mammalian cell type (if applicable):

- Storage: at 4 °C in deep soft nutrient agar.
- Preparation of inoculum: the day before the test, fresh cultures were prepared by inoculating 10 ml of nutrient broth with each strain, which were incubated overnight at 37 °C. - Checked for histidine requirementt: perfromed.
- Checked for biotin requirement: perfromed.
- Checked for rfa character: perfromed.
- Checked for uvrB character: perfromed.
- Checked for presence or absence of the pKM101 plasmid: perfromed.
- Checked for spontaneous reversion rate to histidine prototrophy: perfromed.
Mutagenesis tests were not taken into consideration, if any check was outside the limits of the expected range

Metabolic activation:

with

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

In view of the lack of antibacterial activity at the 1000 µg/plate level, test item was tested for mutagenic activity at the doses of 1000, 100 and 10 µg/plate.

Vehicle / solvent:

The substance was dissolved 10 mg/ml in sterile distilled water and dimethylsulfoxide (DMSO) 1:1 V/V and further diluted in the same solvent according to the need. DMSO, which does not interfere with the result of Ames' test, was choosen as a solvent because it gives obviously sterile solutions.

Controls

Untreated negative controls:

yes

Remarks:

spontaneous reversion rate

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 2-aminofluorene and diazoacetylglycinamide

Details on test system and experimental conditions:

METHOD OF APPLICATION
0.1 ml of DMSO containing the required amount of dye, 0.1 ml of an overnight culture of each tester strain containing 10^7 bacteria and 0.5 ml of S9-mix were added in sequence to each top agar tube. The contents of each tube were quickly mixed and poured on the surface of a VB plate.

INCUBATION PERIOD: revertant colonies were counted after 2 days of incubation at 37 °C and compared with revertants obtained from controls prepared in the same way, dye omitted.

REPLICATE: tests were carried out in triplicate.

MEDIA
Vogel-Bonner E agar medium (VB) has the following composition: MgSO4 × 7H2O 0.2 g/l, citric acid × H2O 2 g/l, K2PO4 10 g/l, NaNH4HPO4 × 4 H2O, glucose 2 g/l, Agar 15 g/l.
It was prepared by mixing 750 ml of 2 % molten autoclave sterilized water agar with 250 ml of 4 times concentrated salt solution and 10 ml of 20 % glucose solution autoclaved separately. For the antibacterial test histidine was also added to give a final concentration.of 10 µg/ml.
10 cm plastic petri dishes were prepared containing 20 ml each of VB agar and used within 3 days from preparation.
Top agar was prepared by mixing 100 ml of autoclave sterilized 0.6 % agar and 0.5 % NaCl in water with 10 ml of a millipore filter sterilized 0.5 mM histidine, 0.5 mM biotin solution. Top agar was dispensed in 2 ml amounts into 16 × 100 mm glass tubes kept at 42 °C.

CYTOTOXICITY TEST
0.1 ml of DMSO containing 1000 µg of test item, 0.1 ml of a 10^-5 dilution of an overnight culture of TA1535 or TA98 containing between 100 and 500 colony forming units (CFU) and 0.5 ml of S9-mix were added in sequence to each top agar tube.
The contents of each tube were quickly mixed and poured on the surface of a histidine containing VB plate. Controls received exactly the same except that dye was omitted.
Colonies were counted after 24 hours of incubation at 37 °C. Tests were carried out in triplicate.

S9 mix
A liver supernatant fraction was prepared from Wistar male rats (170-250 g). Three rats each received a single i.p. injection of Aroclor 1254 (200 mg/ml in corn oil, 500 mg/kg). Five days after injection the rats were killed by cervical dislocation having been deprived of food for the last 16 h of life. The livers washed with 0.15 M KCl ice cold solution were weighed and transferred to a beaker containing 3 ml of KCl 0.15 M solution per gram of net weight of liver, minced with sterilesgssors and homogenized in a Potter-Evenh-jem apparatus with a teflon pestle. The homogenate was centrifuged for 10 min at 9000 g and the supernatant decanted and stored at -60 °C.
100 ml of S9-mix were prepared by mixing 10 ml of liver homogenate, 4 ml of nicotinamide adenine dinucleotide phosphate (NADP) 0.1 M, 0.5 ml of glucose-6-phosphate (G6P) 1 M, 2 ml of MgCl2 0.4 M, 2 ml of KCl 1.65 M, 50 ml of phosphate buffer pH 7.4 0.2 M, 31.5 ml of sterile distilled water NADP and G6P stock solutions were millipore filter sterilized and stored at -20 °C. The stock salt solutions were autoclave sterilized and stored at 4 °C.
Each batch of S9-mix was tested for sterility and activity.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

It can be concluded that test item is without any mutagenic activity within the limits of the test system used.
For a better evaluation of the limits of validity of the above statement, it may be taken into consideration that 1 µg of 2-aminofluorene gave a relevant increase of revertants with strains TA1538 and TA98, while no revertants were caused by 1000 µg of test item.

Applicant's summary and conclusion

Conclusions:

It can be concluded that test item is without any mutagenic activity within the limits of the test system used.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100. The assay was performed with liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10, 100 and 1000 μg/plate.

No toxic effects occurred in the preliminary investigation. The test item resulted to be without any mutagenic activity within the limits of the test system used.

For a better evaluation of the limits of validity of the above statement, it may be taken into consideration that 1 µg of 2-aminofluorene gave a relevant increase of revertants with strains TA1538 and TA98, while no revertants were caused by 1000 µg of test item.

Conclusion

It can be concluded that test item is without any mutagenic activity within the limits of the test system used.