Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assy no mutagenic activity was detected.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals", Section 4, No. 471: "Bacterial Reverse Mutation Test", adopted July 21 , 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate.
The top dose is the limit dose given by the guideline

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

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

cyclophosphamide

other: 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

8.3.4 Selective Agar
The plates with the selective agar (minimal agar plates) were made in-house. Each Petri dish contained about 20.0 ml of minimal agar (1.5% agar supplemented with 2% salts of the VogelBonner Medium E and 2% glucose). The agar used was Select AGAR, GIBCO BRL, Switzerland. Glucose was delivered from Merck, Germany [D(+)glucose, anhydrous]. The Vogel-Bonner Medium E was prepared in-house.
8.3.5 Overlay Agar
The overlay agar contained per litre:
6.0 g GIBCO BRL Select Agar
6.0 g NaCI (Merck, Germany)
Sterilisation was performed at 121 a C in an autoclave, cooled down to 50°C and dispensed into glass bottles. On day of test peformance the agar was molten in a water bath and 1 0%
aliquotes (vlv) of 0.5 mM L-histidine I 0.5 mM d-biotin for Salmonella strains or 0.5 mM tryptophan, dissolved in bidistilled water, for E. coli strains were added sterile filtered.
8.4 Mammalian Microsomal Fraction S9 Mix The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
8.4.1 S9 (Preparation in-house)
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats [HanBri:WIST (SPF)], delivered by RCC Ltd, Animal Breeding and Biotechnology, FOIIinsdorf,
Switzerland. The animals were treated with Aroclor 1254 (Analabs Inc., delivered by Antechnika, Karslruhe, Germany), 500 mglkg, i.p. 5 days prior to sacrifice. The livers were
homogenized with 3 volumes of 150 mM KCI. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -aoac for no longer than one year. The protein content of the S9 fraction was 42.5 mglml.

8.4.2 S9 Mix
On day of experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 1 O% v/v in the mixture. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCI2
33 mM KCI
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium phosphate-buffer, pH 7.4.
Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator. The S9 mix preparation was performed according to Ames et al..

Rationale for test conditions:

These are standard conditions

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
However, whenever the colony counts remain within the historical range of negative controls (solvent) such an increase is not considered biologically relevant.
A reduction in the number of revertant colonies in the groups treated with the test item by >50%, compared with the respective solvent control, is reported as toxic effect.

Statistics:

A statistical analysis was not required. At present the use of statistical methods concerning this particular test system is not generally recommended

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

During the described mutagenicity test according to OECD TG 471 and under the given experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (first experiment with and without metabolic activation, second experiment without activation) and the pre-incubation test (second experiment with metabolic activation) using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, the negative (solvent) and the positive controls were tested in triplicate. The test item was tested at the following concentrations:

312.5, 625, 1250, 2500 and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants occurred at any concentration tested.

No precipitation of the test item was seen on the surface of the agar plates.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sanolin Violett E2R at any concentration level, neither in the presence nor in the absence of a metabolic activation system (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the given experimental conditions reported, Sanolin Violett E2R did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No classification

In a bacterial reverse mutation assy no mutagenic activity was detected.