Disodium 2,2'-(9,10-dioxoanthracene-1,4-diyldiimino)bis(5-methylsulphonate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

RA on similar substance 01 - OECD TG 471 - with and without S9 - negative (non genotoxic)

Link to relevant study records

Reference

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 October 03, 2002 to November 11, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: rat-liver post mitochondrial supernatant (S9 fraction) was prepared from male rats (HanBrl:WIST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland.
- Method of preparation of S9 mix: the animals were treated with Aroclor 1254 (Analabs lnc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80°C for no longer than one year.
- Concentration or volume of S9 mix and S9 in the final culture medium: the amount of S9 supernatant was 10% v/v in the mixture.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator.

Test concentrations with justification for top dose:

312.5, 625.0, 1250.0, 2500.0 and 5000.0 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO; Merck, Germany,99.5%).

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

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate.
- Number of independent experiments: two indipendent experiments.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not reported.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: plate incorporation method (first experiment); pre-incubation method (second experiment with S9).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes.
- Exposure duration/duration of treatment: 48 hours.
- Harvest time after the end of treatment (sampling/recovery times): not specified.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
- Any supplementary information relevant to cytotoxicity: to evaluate the toxicity of the test item a range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations of the test item. The concentrations applied were 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000.0 µg/plate. One plate was prepared per test item concentration, negative and positive controls. The plates were inverted and incubated for about 48 hours at 37+2°C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY:
Colonies were counted electronically using an Accucount 1000 (Biologics, Gainsville, Virginia, USA), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have intedered with automating counting. The results were sent online to a computer. The operator checked them on a random basis. Observations indicating precipitates of the test item in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated and included in the results section. ln case of an increased number of revertant colonies, reaching the level for a positve response, is obtained at any test item concentration, ten colonies of each respecitive plate are tested on selective agar plates for their mutant properties, in order to avoid false positive or false negative results.
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, and WP2 uvrA) or thrice (strains TA 1535 and 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 solvent controls such an increase is not considered biologically relevant.

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, and WP2 uvrA) or thrice (strains TA 1535 and 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 solvent controls such an increase is not considered biologically relevant.

Statistics:

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

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The substance was tested following the OECD TG 471 (bacterial reverse mutation assay). ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S.Typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvrA. The test was performed in two independent experiments both with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The test item was dissolved in DMSO and tested at five concentrations: 312.5, 625, 1250, 2500 and 5000 µg/plate.
ln order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay.
Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control (reference item). Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S.Typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000.0 µg/plate.
Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.
From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.
ln the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with the test substance at any concentrations.
ln the second mutagenicity test carried out with and without metabolic activation, again treatment of the tester strains with the test item did not lead to an increase in the number of revertant colonies at any concentrations.
ln both mutagenicity tests normal background growth was observed with all strains at all concentrations. Due to a growth inhibiting effect of the test item in the pre-incubation assay on strain TA 1537, the number of revertant colonies was reduced at the concentration of 5000 µg/plate. No similar effect was observed with the other strains. The test item did not precipitate on the surface of the agar plates.
ln the experiments negative (solvent) and positive control (reference items) treatments were included for all strains. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The reference items induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.

ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used.
Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as "In vitro" mutagenicity tests such as these indicated in 3.5.2.3.8:

- In vitro mammalian chromosome aberration test;

- In vitro mammalian cell gene mutation test;

- Bacterial reverse mutation tests.

The substance did not create gene mutations in the strains of Salmonella typhimurium and E. Coli under the performed test, therefore according to the 3.5. of the CLP Regulation EC n.1272/2008, it cannot be classified as mutagenic for germ cells.