Reaction mass of 1,5-diamino-4,8-dihydroxy(4-hydroxyphenyl)anthraquinone and 1,5-diamino-4,8-dihydroxy-2-(4-methoxyphenyl)anthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

An ames test is running and expected negative.

As no data on genotoxic potential of Disperse Blue 073 was available, a read across approach was followed, in order to complete the assessment. Details on the read across procedure are reported in section 13 .

The following in vitro tests were considered to assess genotoxicity of the substance:

 

Chromosome aberration test was available on Similar Substance 03 according to OECD guideline 473 (1997) by using V79 Chinese hamster lung fibroblasts.

In the pre-test, concentrations up to 2000 µg/ml without S9 mix and 1800 µg/l with S9 mix were tested and no relevant toxicity, in terms of reduced cell numbers, was noted. Consequently, test item was tested up to concentrations exhibiting clear precipitation in the main study.

In the main study, two independent experiments (I and II) were performed, differing in chromosome preparation interval and exposure duration. Two parallel cultures were set up in each experimental group. Top concentrations of 40 µg/ml and 10 µg/ml where used in exp. I and exp. II, respectively. In both experiments, it was noted that: mitotic indices and cell numbers were not substantially reduced; there was no biologically relevant increase in the rate of polyploid methaphases; there were no biologically nor statistically relevant increase in cells with structural aberrations, based on 200 cells scored per concentration. Notably, current version of OECD guideline 473 (2014) is more detailed than version followed in the study (1997). In particular, it would require to score 300 cells per concentration for structural chromosomal aberrations. Moreover, the recommended cytotoxicity parameters for cell lines, as V79 cell line, would be the relative population doubling (RPD) or the relative increase in cell count (RICC). In the present study, 200 metaphases were examined and the mitotic index and polyploidy were used as indicators of cytotoxicity in the main test, while the number of cells was taken in the pre-test. However, since aberration rates of treated cells were within the solvent control and the historical control data and no indications of cytotoxicity were found with respect to as indicative of a lack of mutagenic potential of the substance in this test.

 

A further in vitro study on mammalian cells was available. HPRT assay was conducted on Similar Substance 02 following OECD guideline 476 (1997). A pre-test on cytotoxicity was run to select the concentrations. Two experiments were run in the main assay using Chinese Hamster V79 cells. In particular:

1. exp. I with and without metabolic activation, concentration up to 0.25 mM and 4 -hour shorth-term exposure

2. exp. II with metabolic activation, concentration up to 0.25 mM and 4 -hour short-term exposure;

exp. II without metabolic activation, concentration up to 0.20 mM and 20 -hour long-term exposure.

Toxicity was evaluated in terms of number of cells per flask and cloning efficiency. A biologically relevant growth inhibition (reduction of relative growth below 70 %) was noted in exp. I without and in exp. II without and with metabolic activation at the highest concentrations adopted. Mutagenicity was evaluated by considering the number of mutant colonies. No significant differences were found with respect to the controls, thus the substance was considered as non mutagenic in this assay. As for the HPRT assay, current version of OECD guideline 476 (2015) contains more details on duration of exposure and post-exposure treatment, number of cells; moreover, it lowers the highest concentration useable if there is no cytotoxicity or precipitation. Such differences do not affect the validity of the available study.

 

 

A micronucleus assay in bone marrow cells of mouse was also available for Similar Substance 02 according to OECD guideline 474 (1983), which is not significantly different from current version (2014).

Therefore this test was used as key study in order to fully assess the mutagenicity endpoint and the annex VII as requested by the guideline and in order to define a definitive and precise classification of the substance.

Test substance was formulated in polyethylene glycol 400 (PEG 400) and administered orally by gavage. Pre-experiment on toxicity was run and the dose of 2000 mg/kg bw was selected as highest dose.

The following dose levels of the test substance were investigated in the main experiment:

- 24 h preparation interval: 200, 670 and 2000 mg/kg bw

- 48 h preparation interval: 2000 mg/kg bw

Bone marrow cells of the mouse were collected after 24 h and 48 h for micronucleus analysis. Ten animals (5/sex) per test group were evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. Cytotoxicity was estimated by the ratio of polychromatic (PCE) and normochromatic erythrocytes (NCE) in the same sample and reported as the number of NCE per 1000 PCE. Animals showed slight toxic reactions; no increase in mean number of NCE was seen, thus indicating no cytotoxicity of test substance.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the mouse. Therefore, test substance is considered to be non-mutagenic in this micronucleus assay.

 

 

 

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), Annex I, Part 3, substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans are classified in Category 2. This classification is based on positive evidence obtained in:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

In vitro mutagenicity tests are the following:

— in vitro mammalian chromosome aberration test;

— in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

 

The overall assessment on the genotoxic potential of test substance was based on negative result in in vivo micronucleus test in bone marrow cells of mouse. Further tests was used as support, the negative outcome in in vitro chromosome aberration assay and the negative result in an in vitro HPRT assay.

All studies were conducted according to OECD guidelines and have a high reliability. Differences with respect to current versions of guidelines, do not significantly influence the overall evaluation, which was thus taken as valid and reliable.

All the studies were available on similar substances and the read across justification is deeply discussed in section 13.

Based on these results, which fulfil the mutagenicity testing strategy required by Annex VII of the REACH Regulation (EC 1907/2006), the test susbstance was considered as non genotoxic and it was not classified within the CLP Regulation (EC 1272/2008).