Tetrasodium 4-amino-6-[[2,5-dimethoxy-4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]-5-hydroxy-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

Endpoint summary

Additional information

Reactive Blue 203 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 of Salmonella typhimurium. Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. For both studies, the compound was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels. Concentrations for both studies were 50, 160, 500, 1600 and 5000 µg/plate.

The test compound did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range with most strains. The positive control compounds showed a significant Increase in the number of revertant colonies. The number of revertant colonies of the positive controls with the strains TA 98 and TA 102 in the absence and in the presence of S9-mix and also the number of revertant colonies of the solvent control with the strain TA 100 and TA 102 in the absence and presence of metabolic activation were slightly out of the historical control data range, but the criteria for the positive/negative response were succeeded.

Toxicity: In both independent mutagenicity tests, toxicity was not observed with and without metabolic activation.

Mutagenicity: In the presence and in the absence of the metabolic activation system Reactive Blue 203 did not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated that Reactive Blue 203 was not mutagenic in this bacterial mutation test at any dose level either in the absence or presence of an exogenous metabolic activation.

 

The bacterial mutation test for the structural analogue Reactive Black 5 was tested in the strains TA100, TA1535, TA1537, TA98 of Salmonella typhimurium. The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of six different doses from 4 µg/plate to 5000 µg/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 item proved to be not toxic to the bacterial strains. Consequently, 5000 µg/plate was chosen as top dose level for the mutagenicity study.

a) Ames test:

Mutagenicity: In the absence of the metabolic activation system, the test item 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 Black 5 did not result in relevant increases in the number of revertant colonies.

b) Prival Test:

In the presence of hamster Liver S9 using the preincubation method according to Prival, Reactive Black 5 did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

Summarizing, it can be stated that Reactive Black 5 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Structural Analogue 01 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For both studies, the compound was dissolved in DMSO, and each bacterial strain was exposed to 5 dose levels. Doses for both mutagenicity studies ranged from 50 to 5000 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive controls gave the expected increase in the number of revertant colonies.

In the preincubation test the number of revertant colonies of the solvent controls with the strain TA 98 in the absence of S9-mix and the number of revertant colonies of the negative controls with the strain TA 1537 in the presence of S9-mix were out of the historical control data range, but the criteria for the negative response were fulfilled.

Toxicity: In the plate incorporation test toxicity was not observed either with or without metabolic activation. Structural Analogue 01 did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate.

Plate incorporation test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v», treatment of the cells with Structural Analogue 01 did not result in relevant increases in the number of revertant colonies.

Preincubation test:

In the absence and in the presence of hamster liver S9-mix (30 % (v/v» using the preincubation method according to Prival Structural Analogue 01 did not result in relevant increases in the number of revertant colonies with any of the tester strains.

Summarizing, it can be stated that Structural Analogue 01 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival (8) at the dose levels investigated.

 

The test item (structural analogue 04) 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	-	525.0	1050	2100	3150	4200
4 hours	+	1050	2100	4200	6300	8400
		Experiment II
24 hours	-	134.2	268.4	536.9	805.3	1073.8
4 hours	+	268.4	536.9	1073.6	2147.5	4295.0

No precipitation of the test item was observed up to the maximum concentration in any of the experiments.

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 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL 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. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls, the induction factor did not reach or exceed the threshold of 3.0. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.5 up to 23.4 mutants per 10⁶cells; the range of the groups treated with the test item was from 4.5 up to 35.4 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL in experiment I and 2.2 µg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.

The structural analogue Reactive Black 5 was tested for clastogenicity/aneugenicity in a mouse micronucleus test and a chromosome aberration test in vivo.

In the micronucleus test in mice, Reactive Black 5 was administered once orally by gavage as single doses of 0 (vehicle control), 2000 and 10000 mg/kg bodyweight to male and female NMRI mice (5 mice per sex and killing time point). 10000 mg/kg bw had been shown in a preliminary study to be the maximum feasible dose. A positive control group received Endoxan at an oral dose of 50 mg/kg body weight. Animals were killed 12, 24, or 48 hours after treatment by carbon dioxide asphyxiation. The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained. 1000 poly- and 1000 normochromatic erythrocytes were screened for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined.

Under the conditions of the present study, Reactive Black 5 caused no significant increase in the number of cells with micronuclei in the bone marrow cells of treated animals as compared with the control group. Endoxan however produced a marked increase in number of cells with micronuclei.

The results indicate that, under the conditions of the present study, Reactive Black 5 is not clastogenic or aneugenic in the in vivo micronucleus test.

In the chromosome aberration test in the Chinese hamster, Reactive Black 5 was administered once orally by gavage in a single dose of 5000 mg/kg bodyweight to male and female Chinese hamsters. This dose had been shown in a preliminary study to be the maximum tolerated dose. A positive control group, induced exactly 24 hours later to run parallel with the negative control and the dose group, received Endoxan in an oral dose of 50 mg/kg bodyweight. Animals from each group were killed 12, 24 and 48 hours after treatment by carbon dioxide asphyxiation. Five males and five females from each group were killed at each of these times. The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained, after which 50 metaphases per animal were evaluated. The completeness in the number of chromosomes and the various chromatic and chromosomal aberrations were assessed.

Under the conditions of the present study, Reactive Black 5 caused no significant increase in the aberration rate in the bone marrow cells of the treated animals as compared with the control group. Endoxan however produced a marked increase in the aberration rate in the test animals.

The results indicate that, under the conditions of the present study, Reactive Black 5 is not clastogenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.

A micronucleus test was carried out Structural Analogue 01. The test compound was suspended in sesame oil and was given twice at an interval of24 hours as an oral dose of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley), based on the results of a previous acute oral toxicity study. At study start the animals were 6 weeks of age and had mean body weights of 179.7 g (M) and 140.2 g (F). According to the test procedure the animals were killed 24 hours after the last administration. Endoxan® was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Structural Analogue 01 and differed less than 20% from the control value.

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei and a small, but significant reduction of polychromatic erythrocytes, indicating the sensitivity of the test system.

Under the conditions of the present study the results indicate that Structural Analogue 01 is not clastogenic in the micronucleus test in vivo.

 

 

Based on the data from Reactive Blue 203 and its structural analogues, it is concluded that Reactive Blue 203 is not genotoxic.

Short description of key information:
Based on the data from Reactive Blue 203 and its structural analogues, it is concluded that Reactive Blue 203 is not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Mutation and clastogenicity tests negative - No classification necessary