Sodium 9,10-dihydro-9,10-dioxoanthracene-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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The potential of Anthrachinon-2-sulfonsäure, Na-Salz Monohydrat (CAS153277-35-1) to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) was investigated, using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

EC Number:

205-009-5

EC Name:

Sodium 9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Cas Number:

131-08-8

Molecular formula:

C14H8O5S.Na

IUPAC Name:

sodium 9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Specific details on test material used for the study:

Identification: Anthrachinon-2-sulfonsäure, Na-Salz Monohydrat CAS 153277-35-1
CAS No.: 153277-35-1
EINECS No. / EC No.: 205-009-5
Empirical formula: C14H7O5SNa x H2O
Molecular weight: 310.3 (Anhydrate), 328.3 (Hydrate) .
Purity: ca. 81% (purity of active ingredient)
Correction factor: 1.235
Physical State /
Appearance: Yellowish powder
Storage Conditions: At room temperature
Stability in Solvent: Stable and homogenous in DMSO for 4 and 24 hours

The dose selection was adjusted to purity.

Method

Target gene:

The dose selection was adjusted to purity.

Species / strain

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:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

DMSO (Dimethyl sulfoxide)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine , 4-NOPD; 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

Test Item Preparation
On the day of the experiment, the test item Anthrachinon-2-sulfonsäure, Na-Salz Monohydrat CAS153277-35-1 was dissolved in DMSO (purity > 99 %). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within two hours of preparation.

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, since the following criteria were met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre­experiment is reported as experiment I. Since no toxic effects were observed 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 µg/plate.

Experimental Performance
For each strain and dose level, including the controls, three plates were used.

Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar

Experiment II (Pre-Incubation)
In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre­ incubation 2.0 mL overlay agar (45°C) was added to each tube.

The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 µL of the stock solution, 500 µl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Rationale for test conditions:

Since no toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

Evaluation criteria:

Acceptability of the Assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• regular background growth in the negative and solvent control;
• the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
• the positive control substances should produce an increase above the thres hold 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;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation of 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 negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Anthrachinon -2-sulfonsäure, Na-Salz Monohydrat CAS153277-35-1 at an dos level, neither in the presence nor absence of metabolic activation (S9 rnix). 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 and showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

Characterisation of the Salmonella typhimurium Strains and Escherichia coli Strain

The histidine dependent strains are derived from Salmonella typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the 11 deep rough11  (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.

Strain Escherichia coli WP2 and its derivatives carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

Regular checking of the properties of the Salmonella  typhimurium  and Escherichia  coli strains regarding the membrane permeability, ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontaneous mutation rates is performed in Envigo CRS GmbH according to B. Ames et al. and D. Maron and B. Ames. In this way it is ensured that the experimental conditions set down by Ames are fulfilled.

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Storage

The strain cultures are stored as stock cultures in ampoules with nutrient broth plus 5% DMSO in liquid nitrogen.

Precultures

The thawed bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 50 mL nutrient medium. A solution of 50 µL ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:

8 g Nutrient Broth

5 g NaCl

The bacterial cultures were incubated in a shaking water bath for 4 hours at 37°C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated  that the bacteria  were harvested  at the late exponential  or early stationary phase (10E8-10E9 cells/mL).

Selective Agar

Plates with selective agar (without histidine/tryptophan) were used.

Overlay Agar

The overlay agar contains per litre: for  Salmonella typhimurium:

7.0 g  Agar Agar

6.0 g NaCl

10.5 mg L-Histidine x HCl x H2O

12.2 mg Biotin

for Escherichia coli:

7.0 g Agar Agar

6.0 g NaCl

10.2 mg Tryptophan

Sterilisations were performed at 121 °C in an autoclave.

Applicant's summary and conclusion

Conclusions:

Anthrachinon-2-sulfonsäure, Na-Salz Monohydrat was considered to be negative (non-mutagenic) under the conditions of this test.

Executive summary:

The potential of Anthrachinon-2-sulfonsäure, Na-Salz Monohydrat CAS153277-35-1 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

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

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333;  1000;  2500;  and 5000 µg/plate

Experiment II: 33;  100; 333; 1000; 2500; and  5000 µg/plate

No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers  of any of the five tester strains was observed following treatment with Anthrachinon-2-sulfonsäure, Na-Salz Monohydrat CAS153277-35-1 at  any  dose  level,  neither  in  the  presence  nor  absence  of  metabolic activation (S9 rnix). 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 and showed a distinct increase of induced revertant colonies.

Conclusion

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

Therefore, Anthrachinon-2-sulfonsäure, Na-Salz Monohydrat CAS153277-35-1 is considered to be negative (non-mutagenic) in this Salmonella typhimurium and  Escherichia coli reverse mutation assay.