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Genetic toxicity in vitro

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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.
Reason / purpose for cross-reference:
reference to other study
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
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
Negative solvent / vehicle controls:
yes
Remarks:
DMEM containing 1% DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
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
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'.

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.

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28.1.-22.3.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with internationally valid GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT - hypoxantine-phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbecco´s Modified Eagle´s medium (DMEM) with glucose, with L-glutamine supplemented with fetal bovine serum (FBS), penicilin and 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
Negative solvent / vehicle controls:
yes
Remarks:
DMEM containing 1% DMSO
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMEM containing 1% DMSO
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
PLATING EFFICIENCY:
Cells were treated with test substance for 3 h. After this the cells were trypsinized, diluted a nd plated in Petri dishes. Seven days later the colonies grown, they were stained and the number of viable cells were determined.

ESTIMATION OF MUTANT FREQUENCY:
V79 cells were cultivated for 24 h in medium with serum. The medium was removed and cells were treated with anthraquinone for 3 h in medium without serum. After treatment, cells were washed with medium and trypsinized. The cells were plated into Petri dishes. After expression period of 7 days, cells were trypsinized and sampled for resistance to TG. Surviving colonies were counted 7 days after plating.

NUMBER OF REPLICATIONS: For each experimental condition two independent experiments were performed (2 without S9 fraction and 2 with S9 fraction).

NUMBER OF CELLS EVALUATED: 200000 cells

SELECTION AGENT (mutation assays): MEM with 10% FBS and TG (6-thioguanine)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
- mutant frequency (plating efficiency, mean mutants/100000 survivors)
Statistics:
Multiple sample comparisn of treated and untreated cell sets was processed applying Kruskal-Wallis test. The P-value outcoming from the test was considered to draw the relevant conclusion about statistical significance. Significance level of 0.01 was taken into account.
Multiple sample comparison was followed bz two sample test applying Mann-Whitney W test to compare the medians of the two samples. The statistical significance between the medians was indicated for P-values smaller than 0.01 (Diem, 1982; Arlett, 1989).
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
- Precipitation: Anthraquinone was tested in test PE (plating efficiency) in concentartion range up to maximum dose 30 µg/mL, limited by precipitation of test article.

RANGE-FINDING/SCREENING STUDIES: On the base of cytotoxicity test results, the concentrations ranging from 1.25 to 20 µg/mL, for in vitro mammalian cell gene mutation tests with and wihout metabolic activation were chosen.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

RESULTS AND DISCUSSION

EVALUTION OF CYTOTOXIC EFFECTS

Preliminary cytotoxicity test was undertaken to define the dose range for V79/HPRT gene mutation test. The evaluation of the cytotoxic effect of Anthraquinone on V79 cells was based on their colony forming ability due to the fact that only viable cells are able to grow, proliferate and form colonies in medium (PE). Anthraquinone was tested in test PE in concentration range up to maximum dose 30 µg/ml, limited by precipitation of test article. The cytotoxic effect wasn´t manifested at any concentration. In this experiment test article at concentration of 30 µg/ml didn´t produce reduction of the colony forming ability (PE) (127.14%).

On the base of these results, the concentrations ranging from 1.25 to 20 µg/ml, for in vitro mammalian cell gene mutation tests with and without metabolic activation were chosen.

 

EVALUATION OF MUTATION FREQUENCY

The mutagenic effect of Anthraquinone was evaluated as resistance to TG (6-thioguanine). Resistance to the purine analogue TG is widely used genetic marker in a number of mammalian mutagenesis systems. Mutants resistant to TG arise mainly by alterations of the gene encoding the salvage pathway enzyme hypoxanthine-guanine phosphoribosyl-transferase (HPRT), which normally enables cells to utilize exogenous guanine, hypoxanthine, or their analogues for nucleotide synthesis. Since a functional enzyme is not essential for DNA synthesis. TG mutants may aarise through base pair substitution, frameshift mutation, or deletion of the HPRT gene.

According to the cytotoxicity of the test substance (first-pilot experiment), the concentration range was adjusted.

Five concentrations of Anthraquinone (1.25, 2.5, 5, 10, 20 µg/ml) in tests with S9, both in two independent experiments. In all experiments negative (solvent)control (1% DMSO in medium) for determination f spontaneous mutations was need. For the verification of cell line mutability positive controls FMS (0.4 mg/ml) in tests without S9and DMBA (0.0003 mg/ml) in tests with S9 were used.

In the first and second experiments without metabolic activation Anthraquinone did not induce statistically significant increase in mutation frequency in Chinese hamster lung cells at any concentrations form range 1.25 µg/ml - 20 µg/ml.

In these experiments test article at the top concentration of 20 µg/ml did not produce reduction of the colony forming ability (PE1) (82.81%, 98.54%).

Responsiveness of the test system was verified by exposing the cells to the direct acting mutagen ethylmethanesulfonate (EMS). The well know genotoxic agents produced a high frequency of HPRT mutations (18.53 and 17.69 mutants/105cells).

In the first experiment with external metabolic activation system (S9) statistically significant increases in mutant frequency were found at concentrations of 5 and 20 µg/ml. Mutant frequencies (6.65, 5.22 mutants/105cells) were found to be 3.25- and 2.55- higher than concurrent negative control (2.04 mutants/105cells).

In the second test with external metabolic system (S9) statistically significant increase in mutant frequency was found at concentration of 2.5 µg/ml. Mutant frequency (4.66 mutants/105cells)was found to be 1.51- higher than the corresponding negative control rate (3.08 mutants/105cells).

In some cases, the small differences between the test groups at given concentrations vs. control group both for mutants´ count as well as frequency of survival were identified as statistically significant at p< 0.01. These differences being of a negligible biological meaning can be explained by low variability of the data in component sample groups.

In these experiments test article at the top concentration of 20 µg/ml did not produced reduction of the colony forming ability (PE1) (105.89% and 114.35%).

Responsiveness of the test system as well as metabolic activity of S9-mix were verified by exposing the cells to the 7,12-dimethyl-benz(a)anthracene (DMBA). The positive control induced a high frequency of HPRT mutations (46.01 mutants/105cells and 51.74 mutants/105cells).

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

Anthraquinone in the mammalian cell gene mutation (V79/HPRT) test did not induce statistically significant increase in the mutation frequency at concentration range from 1.25 to 20 µg/ml in Chinese hamster lung cells V79 in the absence of metabolic system.
In the mammalian cell gene mutation (V79/HPRT) test in the presence of the metabolic activation Anthraquinone induced statistically significant increase in the frequency of mutation at concentrations of 5 and 20 µg/ml in the first test and at the concentration of 2.5 µg/ml in the second test. There was no evidence of relationship between dose and mutant frequency. A reproducible increase in mutation frequencies wasn´t observed. The mutation frequency in cells V79 was higher than 3 times the negative control value only at concentration of 5 µg/ml. It was therefore considered a chance event of no biological relevance (Heidemann, 1996).
Laboratory results showed that Anthraquinone did no induce a concentration-related increase in mutation frequency at the HPRT locus in V79 cells, both in the absence and presence of metabolic activation.
Executive summary:

Anthraquinone was examined for mutagenicity in Chinese hamster V79 cells at concentrations from 1.25 to 20 µg/mL in the absence and in the presence of methylcholanthrene-induced rat liver S9, with cofactors for NADP generation. Incubation time with test article was 3 h in both test systems. The protocol examined the induction of HPRT - hypoxantine-phosphoribosyl transferase – deficient mutants and the mutagenicity was measured as 6-thioguanine (TG) resistance.

Validity of the test method was ascertained by positive controls: ethylmethanesulfonate (EMS) in experiment without exogenous activation and 7,12-dimethyl-benz(a)anthracene (DMBA) in experiments with exogenous activation. For each experimental condition two independent experiments were performed (2 without S9 fraction and 2 with S9 microsomal fraction) to show the response to induce HPRT mutations.

Anthraquinone in the mammalian cell gene mutation (V79/HPRT) test did not induce statistically significant increase in the mutation frequency at concentration range from 1.25 to 20 µg/ml in Chinese hamster lung cells V79 in the absence of metabolic system.

In the presence of exogenous activation Anthraquinone induced statistically significant increase in the frequency of mutation at concentrations of 5 and 20 µg/ml in the first test and at the concentration of 2.5 µg/ml in the second test. There was no evidence of relationship between dose and mutant frequency. Reproducible increase to mutation frequencies wasn´t observed. The mutation frequency in cells V79 was higher than 3 times the negative control value only at concentration of 5 µg/ml. It was therefore considered a chance event of no biological relevance (Heidemann, 1996).

Laboratory results showed that Anthraquinone did not induce a concentration-related increase in mutation frequency at the HPRT locus in V79 cells, both in the absence and presence of metabolic activation.

The study satisfied the requirements for Test Guideline OECD 476 for in vitro mammalian cell gene mutation test.

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.
Qualifier:
according to guideline
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
Target gene:
gene for synthesis histidine or tryptophan
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 / 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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
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.
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
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
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
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
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'.

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).

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro data

Anthraquinone was tested for the mutagenic potential in vitro, according to the EU Method B.17, Mutagenicity – In vitro Mammalian Cell Gene Mutation Test (analogous to the OECD TG No. 476), using Chinese hamster V79 cells at concentrations from 1.25 to 20.0µg/ml in the absence and in the presence of methylcholanthrene-induced rat liver S9, with cofactors for NADP generation. Incubation time with test article was 3 hours in both test systems. For each experimental condition two independent experiments were performed (2 without S9 fraction and 2 with S9 microsomal fraction) to show the response to induce HPRT mutations. In all experiments negative (solvent) control (1 % DMSO in medium) for determination of spontaneous mutation was used. For the verification of cell line mutability positive controls EMS (0.4 mg/ml) in tests without S9 and DMBA (0.003 mg/ml) in tests with S9 were used. Anthraquinone did not induce statistically significant increase in the mutation frequency at concentration range from 1.25 to 20µg/ml in the absence of metabolite system. In the presence of exogenous activation Anthraquinone induced statistically significant increase in the frequency of mutation at concentration of 5 and 20µg/ml in the first test and at concentration of 2.5µg/ml in the second test. There was no evidence of relationship between dose and mutant frequency. Reproducible increase in mutation frequencies was not observed. The mutation frequency in cells V79 was higher than 3 times the negative control value only at concentration of 5µg/ml. Therefore, the result was considered to be a chance event with no biological relevance. Anthraquinone did not induce a concentration-related increase in mutation frequency at the HPRT locus in V79 cells, both in the absence and presence of metabolic activation (Bednáriková M, Mgr., 2010).

Anthraquinone was tested for the mutagenic potential in vitro, according to the EU Method B.10, Mutagenicity – In vitro Mammalian Chromosome Aberration Test (analogous to the OECD TG No. 473), using Chinese hamster lung V79 cells at concentrations from 1.25 to 20.0µg/ml in the absence and presence of a rat liver exogenous metabolic activation system (S9 mix). Experiments were conducted in duplicate culture. Cells were incubated with test substance and harvested either after 24 hrs (all doses) or incubated for 3 hrs and harvested after 21-hrs (all doses) recovery period. Cells exposed to S9 mix were treated with test substance for 3 hrs and harvested after 21-hrs (all doses) recovery period. In each experiment negative (solvent) control (≤1 % DMSO in medium) and the reference mutagens cyclophosphamide (+S9) and mitomycin C (-S9) were used. Stained chromosome preparations were examined for chromosomal aberrations (100 metaphases per treatment group). Anthraquinone at concentration range of 1.25 - 20µg/ml in the absence and in the presence of metabolic activation system arter 3-h treatment of Chinese hamster lung (V79) cells did not induce significant increases in cells with structural chromosomal aberrations. Extended exposure for 24 h without metabolic activation at concentrations up to 20µg/ml also resulted in a negative response. The results of this study demonstrate that Anthraquinone was not genotoxic under the conditions of thein vitrochromosomal aberration assays in V79 cells (Lazová, J., Ing., 2010).

Anthraquinone was tested for its mutagenic potential in vitro, according to the EU Method B.13/14, Mutagenicity – Reverse Mutation test Using Bacteria (Ames test) (analogous to the OECD TG No. 471). Four indicator Salmonella typhimurium strains (TA98, TA100, 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/plate 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. Anthraquinone was non mutagenic for all the used bacterial strains with, as well as without metabolic activation (Täublová E., M. Sc., 2009c).

According to the open literature, commercial Anthraquinone (AQ) (9, 10-anthracenedione) is produced by at least three different production methods worldwide: oxidation of anthracene (AQ-OX), Friedel–Crafts technology (AQ-FC) and by Diels–Alder chemistry (AQ-DA). AQ-OX begins with anthracene produced from coal tar and different lots can contain various contaminants, particularly the mutagenic isomers of nitroanthracene (The Draft Assessment Report – Anthraquinone, Belgium, 2006).

A positive response was reported in the Ames test performed with Anthraquinone from unknown synthesis (Liberman, 1982) and in a micronucleus assay in Syrian hamster embryo cells in which Anthraquinone from the National Toxicology Program (NTP) was used (Gibsonet al., 1997). In the studies reported by Butterworthet al.(2001), a sample of the AQ-OX used in the NTP bioassay was shown to be mutagenic in the Ames tester strains TA98, TA100 and TA1537. Addition of an S9 metabolic activation system decreased or eliminated the mutagenic activity. In contrast, the purified NTP AQ-OX as well as the technical grade samples AQ-FC and AQ-DA was not mutagenic in the Ames test (The Draft Assessment Report – Anthraquinone, Belgium, 2006).

A recent study reported results of aSalmonellamutation assay using the same aliquot of Anthraquinone that was tested in the 2-year carcinogenicity studies (99.8% pure) (Butterworthet al., 2001). The authors suggested that, although the chemical produced a positive response in strains TA98, TA100, and TA1537 with and without S9, the observed mutagenicity was the result of a low level of 9-nitroanthracene present as a contaminant in the sample at a concentration of 1200ppm, but not in the purified material. To further support this hypothesis, the authors purified the Anthraquinone sample, retested it along with Anthraquinone samples produced by chemical processes believed not to result in appreciable contamination, and observed no indication of mutagenic activity in any of these samples. Therefore, they concluded that the mutagenic activity displayed by the original, 99.8% pure sample was produced by the contaminant 9-nitroanthracene (The National Toxicology Program,NIH Publication No. 05-3953,2005). The presence of 9-nitroanthracene used in the NTP cancer bioassays was responsible for the carcinogenic effects as a result of its genotoxic activity (The Draft Assessment Report – Anthraquinone, Belgium, 2006).

Anthraquinone (97% pure) was mutagenic inS. typhimuriumstrains TA98 and TA100, with and without rat and hamster S9 metabolic activation enzymes. A 100% pure Anthraquinone sample showed no mutagenic activity in strains TA98, TA100, or TA102, with or without rat liver S9 enzymes. A 99.8% pure Anthraquinone (sample A07496), the compound used in the 2-year studies was negative in TA98, TA100, and TA1537, with and without rat S9 metabolic activation enzymes. Samples A65343 (Diels-Alder process) and A54984 (Friedel-Crafts process) were negative in TA98 and TA100, with and without rat S9 metabolic activation enzymes. Sample A40147 (Diels-Alder process) was mutagenic in TA98 and TA100, with and without rat S9 metabolic activation enzymes (The National Toxicology Program,NIH Publication No. 05-3953,2005).

Several substituted Anthraquinones were also tested inSalmonella, and results showed significant mutagenic activity for 2-hydroxyAnthraquinone and 1-, 2-, and 9-nitroanthracene, with and without S9 metabolic activation enzymes. 1-HydroxyAnthraquinone was not mutagenic in Salmonella, with or without S9 metabolic activation enzymes (The National Toxicology Program,NIH Publication No. 05-3953,2005).

Early mutagenicity studies of Anthraquinone inSalmonella typhimurium,most using the plate incorporation assay protocol, reported negative results (Brown and Brown, 1976; Anderson and Styles, 1978; Gibson et al.,1978; Salamone et al.,1979; Tikkanen et al.,1983; Sakaiet al.,1985). Later studies showed clear mutagenic activity for Anthraquinone in TA100 and the frameshift strains TA98, TA1537, and TA1538, in the presence and absence of S9 activation enzymes (Liberman et al., 1982; Zeiger et al., 1988). None of the bacterial mutagenicity assays that reported negative results included the purity of the Anthraquinone samples used for testing. The Zeiger et al.(1988) preincubation assay that produced positive results tested an Anthraquinone sample that was 97% pure. Sample purity, along with dose selection and other protocol variations, may have been critical to the outcome of these mutagenicity assays. A structurally related compound, 9-nitroanthracene was positive in theSalmonellamutation assay over a concentration range of 10 to 1,000 μg/plate using strains TA98 and TA100, with and without 30% hamster or rat liver S9 activation enzymes (Zeiger et al., 1988). A number of investigators have studied the mutagenicity of substituted Anthraquinones in the Salmonella assay and have suggested that certain methyl, nitro, and phenolic substitutions confer enhanced mutagenic activity after metabolic activation (Brown and Dietrich, 1979; Fuet al., 1986; Krivoboket al., 1992). Hydroxylation, up to a maximum of four substitutions, also appears to enhance mutagenic potential (Tikkanen et al., 1983; Matsushima et al., 1986). Thus, particular substituted compounds appear to be more mutagenically active than the parent compound, Anthraquinone (The National Toxicology Program,NIH Publication No. 05-3953,2005).

Two identified rat metabolites of Anthraquinone, anthrone and 2-hydroxyAnthraquinone, were reported to be weak mutagens in S. typhimurium(Tikkanen et al., 1983; Molleret al., 1985; Ramdahl, 1985; Matsushima et al., 1986). As with Anthraquinone, anthrone was reported to lack mutagenicity in several studies (Brown and Brown, 1976; Anderson and Styles, 1978; Gibson et al., 1978; Liberman et al., 1982). Thus, protocol characteristics, dose, and purity may all be important factors in the detection of mutagenicity of Anthraquinone and substituted Anthraquinones. In addition, the identity of specific side groups and their spatial orientation to the main ring structure of the Anthraquinone molecule are important to the mutagenic activity of the chemical (The National Toxicology Program,NIH Publication No. 05-3953,2005).

When Anthraquinone was tested for induction of forward mutations in cultured human BT lymphoblastoid cells, a metabolically competent cell line for polycyclic aromatic compounds, no mutagenic activity was detected (Durant et al., 1996) (The National Toxicology Program, NIH Publication No. 05-3953, 2005).

In vivo data

Cesarone et al. (1982) reported in vivo induction of DNA strand breaks by Anthraquinone in liver and kidney cells of CD-1 mice treated with 250 mg Anthraquinone/kg via intraperitoneal injection, and dose-related increases in micronuclei were reported in cultured Syrian hamster embryo cells treated with 3.13 to 25 μg Anthraquinone (99% pure)/mL (Gibson et al.,1997). Butterworth et al. (2001) reported negative results with Anthraquinone produced through a Diels-Alder process in an acute mouse bone marrow micronucleus test and in a mouse lymphoma L5178Y cell forward mutation assay (The National Toxicology Program, NIH Publication No. 05-3953, 2005).

Significant increases in the frequencies of micronucleated normochromatic erythrocytes were observed in peripheral blood samples from M/F mice exposed to Athraquinone (99.8% pure) in feed for 14 weeks. However, results of an acute exposure mouse bone marrow micronucleus test, with Anthraquinone administered by intraperitoneal injection, were negative (The National Toxicology Program,NIH Publication No. 05-3953, 2005).

Short description of key information:
The Anthraquinone was evaluated for its mutagenic and genotoxic potential in vitro and in vivo.
Mutagenic activity of Anthraquinone was demonstrated in both, in vitro and in vivo testing, although much of the observed activity has been attributed to contaminants (mainly structurally related contaminant 9-nitroanthracene), depending upon the method used to produce the Anthraquinone under study.
Description of in vitro and in vivo studies on mutagenicity of Anthraquinone reported in the NTP Technical Report (The National Toxicological Program, NIH Publication No. 05-3953, 2005) was very brief with limited information of the tests conditions. Information is not considered to be sufficient for the assessment of human health.
In vitro studies performed according to current standards and under GLP conditions were available. Based on the results of three in vitro studies provided by registrant, no genotoxic effect for Anthraquinone was observed.
Three in vivo studies on genotoxicity of Anthraquinone reported in the NTP Technical Report (The National Toxicological Program, NIH Publication No. 05-3953, 2005) were available. Studies were only briefly described, with many data gaps about the tests conditions. Information is not considered to be sufficient for the assessment of human health.
The protocol used, dose selection and purity (a low level of 9-nitroanthracene as a contaminant in the sample) may have been critical for the mutagenicity test results. Highly purified Anthraquinone itself was not proved to be a mutagen (The National Toxicology Program, NIH Publication No. 05-3953, 2005). Anthraquinone considered for this registration is 99.21% pure and does not contain 9-nitroanthracene as the contaminant.
Based on the data available from a genotoxicity studies described above and considering uncertainties in the test results, the Anthraquinone can be considered as a non-genotoxic substance.

Endpoint Conclusion: No adverse effect observed (negative). However, it was decided to classify the substance as Muta 1B as a precaution.

Justification for classification or non-classification

As a precaution, the substance is classified as Muta 1B.