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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)

Justification for classification or non-classification

Based on the test results and according to the EC criteria for classification and labelling requirements for dangerous substances and mixtures the test substance Anthraquinone does not have to be classified for mutagenicity.