Anthraquinone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9.6-9.8.2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report Date:

2009

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

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

Deviations:

yes

Remarks:

The deviations had no influence on study results.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Batch No.: V 1161
Purity: main component: 9,10-Anthraquinone 98.9 wt.%
Significant impurities: Anthracene 0.01 wt.%, 2,3-Naphthalic anhydride 0.25%, Naphtho[2,3-b]thiphenquinone 0.33%, 1,4- Anthraquinone 0.32%
Appearance: Yellow crystalline powder
Expiration date: 06/2024
Storage: The test substance was stored in the dark place at laboratory temperature during the study.

Method

Target gene:

gene for synthesis histidine or tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine dependent strain

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan dependent strain

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 1000 μg/plate

Vehicle / solvent:

- Solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine hydrochloride monohydrate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

DETERMINATION OF CYTOTOXICITY:
- Method: total growth

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, its using is comparable with using of statistical methods (2, 3). After this rule the result is positive, when reproducible dose-effect and/or doubling of ratio Rt/Rc is reached.

Statistics:

Statistical method:
2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240.
3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Spontaneous reversions, negative controls (solvent), and positive controls were compared with historical controls in our laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Selection of doses/toxicity The test substance was dissolved in DMSO at 45ºC (temperature at which test substance is held in top agar before pouring onto Petri dishes) till the final concentration 150 µg/01 mL. This basic concentration was then diluted to gain a concentration range 1-150 µg/0.1 mL (plate). Because of the highest concentration was more than by digit position lower than the highest dose recommended by the guidelines, two other doses - 750 and 1500 µg per plate - were further prepared as suspensions of test substance in DMSO. Final concentration range 1-1500 µg per plate was then tested for toxicity in TA 100.
The test substance precipitated in top agar in doses 50 and 150 µg per plate. No signs of toxicity were observed. The dose 150 µg/0.1 mL (plate) stayed as maximum for dilution to concentration range 1-150 µg/0.1 mL (plate) in the first mutagenicity experiments. The highest dose of 1000 µg/plate was chosen as a dose between two other doses following according to rules (500 and 1500 µg/plate). It was additional, it was applied as a suspension and it caused cloud in top agar.
The highest dose (1000 µg/plate) used in the first mutagenicity experiments caused minor difficulties at evaluation caused by occurrence of suspension in background so it was not used for the second experiments. Instead, the other lower dose set according to the rules for dilution was added to the concentration range. In the the second mutagenicity experiments, 150 µg/0.1 mL stayed basic (the highest) concentration from what the concentration range was diluted. The solution was prepared by stirring the test substance in DMSO at the temperature of 45ºC approximately for an hour following by heating to 60ºC for approx. 5 minutes.
In the all experiments, the chosen doses were dosed in volume 0.1 mL per plate. Fresh solutions of test substance were prepared before each experiment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

STUDY RESULTS

The results of experiments are summarized in tables IN Final report. The tables contain the dose applied per plate inµg (doses were applied to plates in volume 0.1 mL), amount of S9 per plate in µL numbers of revertants in single plates, average number of revertants per plate x and its standard deviation sd and ratio of revertants at tested dose (Rt) to number of revertants at negative control (Rc, dimethylsulfoxide).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
ambiguous with metabolic activation

Under the above-described experimental design, the test substance Anthraquinone was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without as well as with metabolic activation.

Executive summary:

Test substance Anthraquinone was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria , which is analogous to the OECD Test Guideline No. 471.

Four  indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was diluted in dimethylsulfoxide and assayed in doses of 1.5-1000µg which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Anthraquinone was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.