1,7-Naphthalenedisulfonic acid, 2-[[4-chloro-6-(cyanoamino)-1,3,5-triazin-2-yl]amino]-5-hydroxy-6-[2-[2-methoxy-5-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, lithium sodium salt (1:?:?)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Nov. 17, 1999 to Apr. 10, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, L133, pp. 61 - 63, March 1987

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

440-050-4

EC Name:

-

Cas Number:

243857-97-8

Molecular formula:

Hill formula: C23H(19-x-y)ClLixN8NayO14S4 (x+y=3), x and y > 0

IUPAC Name:

lithium(1+) disodium 2-{[4-chloro-6-(cyanoamino)-1,3,5-triazin-2-yl]amino}-5-hydroxy-6-(2-{2-methoxy-5-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reaktiv Orange DYPR 1466

Method

Target gene:

HRPT locus (V79 Chinese hamster cells)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Source: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
Cell culture medium: MEM (minimal essential medium) with Hankssalts and 25 mM Hepes-buffer
pH values and osmolality of the treatment media: Determined before treatment

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Main mutation experiment (First):
- without S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000* µg/mL
- with S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

* = because of high toxicity in the first main experiment no mutant selection was performed

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle:

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: Seeded in each well of a microtiter plate

DURATION (Preliminary toxicity)
- Preincubation period: 12 h (overnight)
- Exposure duration: 4 h
- Incubation period: 24 h
- Fixation and staining: Crystal violet
NUMBER OF REPLICATIONS: 6 (For each concentration at least 6 wells)

DURATION (Main experiment)
- Preincubation period: 24 h
- Exposure duration: 4 h (Medium+ test substance+ buffer/S9-mix)
- Incubation period: 24 h
- Fixation and staining: Crystal violet

Evaluation criteria:

The test item was considered positive if
(a) It reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
(b) There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.
(c) Survival of the responding dose group is at least 30 %.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

- The biometry of the results for the test compound is performed off-line with the Mann-Whitney- U-Test

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects


RANGE-FINDING/SCREENING STUDIES:
Evaluation of the solubility of the suspension in cell culture medium showed that 5000 µg/mL was the highest concentration at which no visible precipitation was observed.

Preliminary toxicity study: Was carried out using a maximum concentration of 5000 µg/mL and a range of lower dose levels down to 10 µg/mL.

Based on the preliminary study results, 5000 µg/mL was selected as the maximum dose level for the main mutation experiments in both the absence and in the presence of S9-mix. Seven lower concentrations down to 100 µg/mL were also included.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Any other information on results incl. tables

The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

Mutation results:

Main experiment: A significant increase of the mutation frequency was observed in the presence of metabolic activation at the concentrations of 250, 2500 and 3750 µg/mL.

These results were not reproducible and not three fold higher than the corresponding controls and were therefore considered to be of no biological relevance.

No relevant reproducible increase in the mutant colonies or mutant frequency over the range of the solvent control was found with any other concentration, either with or without metabolic activation by S9-mix.

Applicant's summary and conclusion

Conclusions:

The test substance is considered to be non-mutagenic in the HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of test substance to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro according to OECD Guideline 476, EPA OPPTS 870.5300 and EEC Directive 87/302, L133 in compliance with GLP.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). The test substance was dissolved in cell culture medium and tested at the following concentrations:

- without S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

- with S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

Due to the limitation of the OECD guideline 5000 µg/mL is the highest tolerated dose.

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed toxic effects with and without metabolic activation.

Test substance produced no macroscopical precipitation. Up to the highest investigated dose no relevant and reproducible increase in mutant colony numbers was obtained in two independent experiments.

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

Test substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system.

Under the test conditions, the test substance is considered to be non-mutagenic in the HPRT-test with V79 Chinese hamster cells, either in the presence or absence of metabolic activation.