Anthraquinone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.1.2010-19.4.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Anthraquinone

EC Number:

201-549-0

EC Name:

Anthraquinone

Cas Number:

84-65-1

Molecular formula:

C14H8O2

IUPAC Name:

9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

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

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: DMEM medium with Glucose and L-Glutamine and with the addition of fetal bovine serum, penicilin, streptomycin

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1.25, 2.5, 5, 10, 20 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMEM containing 1% DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMEM containing 1% DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Cells were incubated with test substance and harvested either after 24 hrs (all doses) ar incubated for 3 hrs and harvested after 21-hrs (all doses) recovery period. Cells exposed to S9 mix were treated with test test substance for 3 hrs and harvested after 21-hrs (all doses) recovery period.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 well-spread metaphases containing 22+/- 2 centromeres were analysed per each concentration, solvent and positive controls equally divided amongs the duplicates (100 metaphases per experiment)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
relative cell growth

Evaluation criteria:

The percentages of aberrant metaphases, total number of aberrations (chroamtid gaps and breaks, isochromatid gaps and breaks, and exchange)

Statistics:

Student´s t-test

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH of the treatment media was neutral
- Precipitation: no precipitate was observed throughout the experiment

RANGE-FINDING/SCREENING STUDIES: Concentrations chosen for chromosomal aberration test were based on preliminary cytotoxicity assay. The results of this assay are stated in Final report "Anthraquinone. Mutagenicity: In Vitro Mammalian Cell Gene Mutation Test (OECD 476)." The highest concentration was limited by Anthraquinone solubility.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

DISCUSSION

Clastogenic effect of Anthraquinone against V79 cells was assessed in independent experiments with and without metabolic activation at 3-h exposure. Because these tests gave negative results, an additional experiment without metabolic activation at 24-h exposure was carried out. Five concentrations of test article, solvent control and positive control were evaluated in each experiment.

In both assays with and without metabolic activation with 3-h exposure to Anthraquinone at concentration range of 0.3125 – 20 µg/mL the concentrations of 1.25, 2.5, 5, 10, 20 µg/mL were evaluated.

Other experiments without presence of metabolic activation were performed with 24-h exposure to Anthraquinone at concentrations of 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 µg/mL.

The results of experiments for induction of structural chromosomal aberrations by Anthraquinone in V79 Chinese hamster lung cells are summarized in tables in Final report.

No precipitate was observed throughout the experiment and the pH of the treatment media was neutral. The negative control cultures showed optimal growth.

The data showed no dose-related increase in the percentages of aberrant metaphases and in the total numbers of aberrations in the Chinese hamster lung (V79) cells in assays either with or without S9.

In experiment without metabolic activation exposure to 0, 1.25, 2.5, 5, 10, 20 µg/mL of Anthraquinone for 3-h resulted in relative cell growth (RCG) of 100, 98.5, 106.3, 88.7, 86.8 and 100.5%, respectively, RCG of 100, 101.2, 60.8, 74.6, 78.8 and 78.5% was determined in cultures treated with 0, 1.25, 2.5, 5, 10, 20 µg/mL of Anthraquinone, respectively, in the presence of S9.

In the short/term treatment (3h), the percentages of aberrant metaphases excluding gaps of Anthraquinone treated groups were less than 3% without S9 and less than 2% with S9, respectively. No evidenc of reduction of MI values was observed in both tests with 3 h exposure to Anthraquinone in the absence as well as in the presence of metabolic activation system compared to MI values of solvent control.

Relative cell growth 100, 96.7, 108.4, 137.4, 107.9 and 89.7% at 24 h treatment with Anthraquinone at concentrations of 0, 1.25, 2.5, 5, 10, 20 µg/mL, respectively.

In the case of continuous treatment (24h), the percentage of aberrant metaphases excluding gaps of Anthraquinone treated groups was similar to that of the solvent control.

No mitotic inhibition by Anthraquinone was observed up to its solubility limit t the concentration of 20 µg/mL.

The percentage of aberrant metaphases excluding gaps of solvent control treated group ranged 0 to 1%. Because significance of gaps Is not clearly understood, they are not included in assessment of chromosome damage.

All solvent control cultures had percentages of aberrant metaphases within the expected range.

The positive control substances, cyclophosphamide (20 µg/mL] and mitomycin C (0.04, 0.4 µg/mL induced statistically significant increases (p<0.001] in the incidence of aberrant metaphases, indicating that the experimental system employed was functioning correctly.

Applicant's summary and conclusion

Conclusions:

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

Anthraquinone at concentartion range 1.25-20 µg/mL in the absence and in the presence metabolic activation system (S9) after 3-h exposure treatment of Chinese hamsted lung(V79) cells did mot induce significant increases in cells with sructural chromosomal aberrations. Extended exposuer for 24-h without metabolic activation at concentration up to 20 µg/mL also resultedin a negative response.
the results of this studz demonstrate that Anthraquinone is not genotoxic under the conditions of the in vitro chromosomal aberration assazs in V79 cells.

Executive summary:

The present study was carried out to investigate to clastogenic effect of Anthraquinone using cultured Chinese hamster V 79 cells in vitro.

Chinese hamster lung V 79 cells were exposed to the test substance with and without exogenous metabolic activation. Experiments were conducted in duplicate culture. Cells were incubated with Anthraquinone at concentration of 1.25, 2.5, 5, 10, 20 µg/mL and harvested either after 24 hrs (all doses) ar incubated for 3 hrs and harvested after 21-hrs (all doses) recovery period. Cells exposed to S9 mix were treated with Anthraquinone at concentration of 1.25, 2.5, 5, 10, 20 µg/mL for 3 hrs and harvested after 21-hrs (all doses) recovery period. The reference mutagens cyclophosphamide (+S9) and mitomycin C (-S9) were used. Stained chromosome preparations were examined for chromosomal aberrations (100 metaphases per treatment group).

Under both test condition - the absence and presence of metabolic activation - Anthraquinone produced no significant increases in cells with structural aberrations (% aberrant metaphases) at any of the dose levels tested. The positive controls showed marked increases in the number of cells with structural chromosome aberrations. Under these test conditions Anthraquinone is not clastogenic in vitro.