Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, sulfo [[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]sulfonyl derivs.

Endpoint summary

Additional information

Reactive Blue 21 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of six different doses from 4 microgram/plate to 5000 microgram/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains. Consequently, 5000 microgram/plate was chosen as top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabolic activation system the test-compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Reactive Blue 21 did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Reactive Blue 21 is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

The test item Reactive Blue 21 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The main experiments were evaluated at the following concentrations:

Exposure period	S9 mix	Concentrations in µg/mL
		Experiment I
4 hours	-	39.1	78.1	156.3	312.5	625.0
4 hours	+	78.1	156.3	312.5	625.0	1250
		Experiment II
24 hours	-	18.8	37.5	75.0	150.0	225.0
4 hours	+	150.0	300.0	600.0	900.0	1200.0

 

No precipitation of the test item was observed up to the maximum concentration with and without metabolic activation.

Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 625.0 µg/mL without metabolic activation and at 1250 µg/mL with metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 150.0 µg/mL and above without metabolic activation and at 900 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control in both cultures of experiment I without metabolic activation at 625.0 µg/mL. However, the absolute mutation frequency of culture I was just 22.9 colonies per 10⁶cells remaining well within the historical range of solvent controls and even within the actual solvent control range of the first experiment (6.8 to 25.3 colonies per 10⁶cells). In the second culture of the first experiment without metabolic activation an isolated substantial increase of the mutation frequency exceeding both, the historical range of solvent controls and the threshold described above, occurred at 625 µg/mL. This isolated increase was judged as irrelevant outlier as it neither was reproduced in the parallel culture under identical conditions nor in the second experiment without metabolic activation. In the first culture of the second experiment with metabolic activation the threshold as described above was exceeded at 150 and 600 µg/mL. However, these increases were based on relative low solvent control and remained well within the historical solvent control data.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in both cultures of the first experiment without metabolic activation. However, both of the significant increases were judged as irrelevant as they are either based on fluctuations within the actual and historical solvent control range (culture I) or on an irrelevant outlier (culture II).

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.1 up to 25.3 mutants per 10⁶cells; the range of the groups treated with the test item was from 4.4 up to 50.4 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (2.2 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Reactive Blue 21 is considered to be non-mutagenic in this HPRT assay.

A study was performed to investigate the potential of Reactive Blue 21 to induce chromosome aberrations in V79 cells of the Chinese hamster in vitro. The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation.

The test article was tested at the following concentrations:

without S9-mix:

18 h: 10, 100, 1000 µg/ml

with S9-mix:

18 h: 10, 100, 1000 µg/ml

The concentration ranges were selected according to the preliminary experiment for solubility and toxicity. The concentrations produced a distinct decrease of the mitotic index.

The highest investigated dose of the test compound Reactive Blue 21 induced a statistically significant and reproducible increase in the number of phases with aberrations and in the number of chromosome aberrations 18h after the start of the treatment without metabolic activation inclusive and exclusive gaps.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in chromosome aberrations, thus indicating the sensitivity of the assay.

In conclusion, Reactive Blue 21 did induce chromosome mutations (=aberrations) in V79 Chinese hamster cells in the absence of a metabolic activation system, under the experimental conditions described.

Therefore, Reactive Blue 21 is considered to be clastogenic in this chromosome aberration assay.

It is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warr TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. As in the currently assessed dye, vinyl sulphone moieties are used in fibre-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in plenty of tests with vinyl sulphone dyes (internal data DyStar). Hence, the in-vivo test produces more reliable data for this kind of substance.

Reactive Blue 21 was tested in the in-vivo mouse micronucleus test. The test compound was dissolved in deionized water and dosed once orally at 5000 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration. Endoxan® was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Reactive Blue 21 and was statistically not different from the control values.

Endoxan® induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

The results indicate that, under the conditions of the present study, Reactive Blue 21 is not clastogenic or aneugenic in the micronucleus test.

Short description of key information:
No evidence for genotoxicity was observed in the bacterial reverse mutation assay and the mammalian gene mutation assay. The clastogenic evidence observed in the in vitro chromosomal aberration assay was not confirmed or reproduced in the in vivo micronucleus assay in rats.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No genotoxicity observed, no classification necessary