1,4-dihydroxyanthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several in vitro assays (Ames tests, a chromosome aberration test, a HGPRT assay, 2 SCE assays and several UDS/DNA repair assays) are published. Additionally an in-vitro micronuleus test in chinese hamster V79 cells according to OECD TG 487 was conducted.
No in vivo assay is available.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

The mutagenic potential of quinizarine was examined in the Salmonella/microsome test. Bacteria of 4 histidine auxotrophic Salmonella typhimurium strains (TA 98, TA 100, TA 1535, and TA 1537) and Escherichia coli WP2uvrA were exposed to doses up to 5000 µg per plate.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

no data

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 1.22, 4.88, 19.5, 78.1, 156, 313, 625, 1250, 2500 or 5000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 2-aminoanthracene

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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:

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Additional information on results:

No data.

Remarks on result:

other: Quinizarine was positive in this Ames test on S. typhimurium strain TA 1537 with and without metabolic activation, whereas quinizarine was negative in S. typhimurium strains TA 98, TA100, TA1535 and Eschrichia coli stain WP2uvrA.

Quinizarine (1,4-dihydroxyanthraquinone) was positive in this Ames test on S. typhimurium strain TA 1537 with and without metabolic activation, wheras in quinizarine was negative in S. typhimurium strains TA 98, TA100, TA1535 and Eschrichia coli stain WP2uvrA.

Conclusions:

Interpretation of results: positive

Executive summary:

The mutagenic potential of quinizarine was examined in the Salmonella/microsome test. Bacteria of 4 histidine auxotrophic Salmonella typhimurium strains (TA 98, TA 100, TA 1535, and TA 1537) and Escherichia coli WP2uvrA were exposed to doses up to 5000 µg per plate.

Quinizarine was positive in this Ames test on S. typhimurium strain TA 1537 with and without metabolic activation, whereas quinizarine was negative in S. typhimurium strains TA 98, TA100, TA1535 and Eschrichia coli stain WP2uvrA.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

A chromosomal aberration test in CHL/IU Cells was carried out in doses up to 2.5 mg/ml.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

no data

Species / strain / cell type:

mammalian cell line, other: CHL/IU cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

short treatment: 0.31, 0.63, 1.3, 2.5 mg/ml
continuous treatment: 0.0013, 0.0025, 0.0050, 0.010 mg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Key result

Species / strain:

mammalian cell line, other: CHL/IU cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Quinizarine was positive in this CHL/IU test with and without metabolic activation.

Conclusions:

Interpretation of results: positive

Executive summary:

A chromosomal aberration test in CHL/IU cells was carried out in doses up to 2.5 mg/ml. Quinizarine was positive in this CHL/IU test with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Principles of method if other than guideline:

The test item 1,4-Dihydroxy-9,10-anthraquinone, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Only one experiment was performed. The exposure period was 4 hours with and without S9 mix. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Purity: 99.3%

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (obtained from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

With regard to the solubility properties of the test item, 150 μg/mL of 1,4-Dihydroxy-9,10-anthraquinone were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 3.9 to 150 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 29.6 μg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

Vehicle / solvent:

Solubility experiments under non-GLP condition were performed. DMSO, Ethanol, Acetone and THF were tested as adequate solvent. DMSO was chosen since the highest solubility of the test item was achieved with DMSO compared to the other solvents tested. Thus, stock formulations of the test item and serial dilutions were made in DMSO. The final concentration of DMSO in the culture medium was 1.0 %. The top dose of the test item was 150 μg/mL, due to the solubility properties in the solvent and in culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Culture conditions
Thawed stock cultures were propagated at 37°C in 80 cm² plastic flasks. About 5 x 10E5 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week.
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37°C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. Per culture approximately 5.0 – 6.0 x 105 cells were seeded into 25 cm2 plastic flasks.
All incubations were done at 37°C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Dose Selection
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. The highest test item concentration should be 10 mM, 2 mg/mL or 2 μL/mL, whichever is the lowest. At least three test item concentrations should be evaluated for cytogenetic damage.

In case of test item induced cytotoxicity, measured by a reduced cytokinesis-block proliferation index (CBPI) and expressed as cytostasis, or precipitation (observed at the end of test item exposure by the unaided eye) the dose selection should reflect these properties of the test item. Where cytotoxicity occurs the applied concentrations should cover a range from no to approximately 55 ± 5 % cytostasis (inhibition of cell growth). For poorly soluble test items that are not cytotoxic at concentrations lower than the lowest insoluble concentration, the highest concentration analyzed should produce turbidity or visible precipitation (phase separation for liquid test items).
Furthermore, test item induced changes in osmolarity influence the dose selection.

With regard to the solubility properties of the test item, 150 μg/mL of 1,4-Dihydroxy-9,10-anthraquinone were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 3.9 to 150 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 29.6 μg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

Since 1,4-Dihydroxy-9,10-anthraquinone was considered to be mutagenic after the first experiment, no further experiment was performed.

Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the Cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure.

Cytogenetic Experiment
Pulse exposure
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose ∙ H2O, 192 mg/L Na2HPO4 ∙ 2 H2O and 150 mg/L KH2PO4. The cells were then be cultured in complete medium containing 10 % (v/v) FBS in the presence of Cytochalasin B (1.5 μg/mL) for the remaining culture time of 20 hours.

Preparation of cells
Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvested and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37°C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the supernatant after centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on clean, wet microscope slides and allowed to dry. The mounted cells were Giemsa-stained and, after drying, covered with coverslips. All slides were labeled with a computer-generated random code to prevent scorer bias.

Rationale for test conditions:

The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 ± 2.

Evaluation criteria:

Evaluation of cytotoxicity and cytogenetic damage
Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI (Cytokinesis-block proliferation index) was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Acceptability Criteria
The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range.
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described in section 3.5 were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence and presence of S9 mix, statistically significant increases in the number of micronucleated cells, clearly above the range of the 95% ctrl. limit (without S9 mix: 0.0 – 2.55 %; with S9 mix: 0.0 – 2.08 %) were observed after treatment with all evaluated concentrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.

Therefore, 1,4-Dihydroxy-9,10-anthraquinone is considered to be mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.
Therefore, 1,4-Dihydroxy-9,10-anthraquinone is considered to be mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Executive summary:

The test item 1,4-Dihydroxy-9,10-anthraquinone, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in one experiment. The following study design was performed:

	Without S9 mix Exp. I	With S9 mix Exp. I
Exposure period	4 hrs	4 hrs
Recovery	20 hrs	20 hrs
Preparation interval	24 hrs	24 hrs

In each experimental group two parallel cultures were analyzed. Per culture 1000 cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (150 μg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation.

In the absence and presence of S9 mix, statistically significant increases in the number of micronucleated cells, clearly above the range of the 95% ctrl. limit (without S9 mix: 0.0 – 2.55 %; with S9 mix: 0.0 – 2.08 %) were observed after treatment with all evaluated concentrations.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.

Therefore, 1,4-Dihydroxy-9,10-anthraquinone is considered to be mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The mutagenesis assay with and without the cocultivation of these cells with primary rat hepatocytes was performed as described ba Marquardt et al., Cancer Res 36, 2065-2069 (1976) and Westendorf et al., Cancer Res, 44, 5599-5604 (1984). A test is considered to be positive if a doubling of the background mutation frequency and/or a dose dependency is observed.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

no data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells provided by Dr. E.H.Y. Chu

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data.

1,4-dihydroxyanthraquinone was negative in this HGPRT assay.

Conclusions:

Interpretation of results: negative

Executive summary:

A HGPRT assay with V79 cells was perforemed.1,4-dihydroxyanthraquinone was negative in this HGPRT assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in vivo assay is available.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the Ames test, 1,4-dihydroxyanthraquinoe was positive (with the exception of those assays were only a limited number of S. typhimurium strains were used. 1,4-dihydroxyanthraquinone was also positive in the CA test and the in-vitro micronucleus test in Chinese hamster V79 cells.

In the HGPRT test and the UDS/DNA repair assays, 1,4-dihydroxyanthraquinone was negative, also in the genotoxicity assay in Saccharomyces cervesia. In the SCE assay, 1,4-dihydroxyanthraquinone revealed a weak (and dose independent) effect.

In 2 in vitro DNA binding assays a positive and a negative result was obtained (see section 7.9.3 specific investigations).

Justification for classification or non-classification

In the Ames tests, the chromosome aberration test and the in-vitro MNT a positive result was obtainded. In the HGPRT test and the UDS/DNA repair assays, 1,4-dihydroxyanthraquinone was negative, also in the genotoxicity assay in Saccharomyces cervesia. In the SCE assay, 1,4-dihydroxyanthraquinone revealed a weak (and dose independent) effect.

In 2 in vitro DNA binding assays a positive and a negative result was obtained (see section 7.9.3 specific investigations).

Overall, 1,4-dihydroxyanthraquinone was positive in several in-vitro assays, but no in-vivo studies are available. Based on the inconclusive results a classification according to CLP classification criteria (Regulation (EC) No 1272/2008) is not suitable, however due to the ambigouos results 1,4-dihydroxyanthraquinone is allocated to the medium hazard band.