5-(benzoylamino)-4-hydroxy-3-[[1-sulpho-6-[[2-(sulphooxy)ethyl]sulphonyl]-2-naphthyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

Endpoint summary

Additional information

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guidelines for testing of chemicals 471 Genetic Toxicology : Salmonella typhimurium, Reverse Mutation Assay, Adopted: May 26th, 1983 and EEC Directive 79/831 Annex V, 4.3.1. This study was conducted in compliance with the principles of good laboratory practice (GLP).

Reactive Red 180 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 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 6 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. 5 000 microgram/plate was chosen as top dose level for the mutagenicity study.

Ames Test:

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 rat liver activation system (10 %), treatment of the cells with Reactive Red 180 did not result in relevant increases in the number of revertant colonies.

Prival Test:

In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival Reactive Red 180 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 Red 180 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

In the In Vitro Rat Primary Hepatocyte Unscheduled DNA Synthesis (UDS) Assay, the test substance did not induce significant increases in UDS. In the assay described in this report, freshly prepared rat hepatocytes were exposed to the test substance at concentrations ranging from 5000 µg/mL to 0.500 µg/mL in the presence of 10 µCi/mL 3HTdR (47 Ci/mmole). The test material was

soluble in media at all concentrations tested. Treatments from 5000 µg/ml to 500 µg/ml were not analyzed for nuclear labeling due to high toxicity.

Treatments from 250 µg/mL to 1.00 µg/mL covered a good range of toxicity (69.2% to 96.5% survival) and were selected for analysis of nuclear labeling.

None of the criteria used to indicate UDS were approached by any of the analyzed treatments and no dose-related response was observed. The test substance was therefore evaluated as inactive in the In Vitro Rat Primary Hepatocyte UDS Assay.

The test substance was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced a significant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 1000 µg/mL up to a concentration of 9357 µg/mL (= 10 mM, which is the highest dose level tolerated for the test system). A significant cytotoxic effect was also observed with metabolic activation from 1500 up to the concentration of 9357 µg/mL. For mutagenicity testing two independent cell cultures with and without metabolic activation (S9-mix) were used.

For main experiment dose levels of 75, 375 and 750 µg/mL in the absence and 100, 500 and 1000 µg/mL in the presence of S9-mix metabolic activation were used.

The test substance induced a significant increase in the number of chromosome aberrations 7 h after treatment with 750 µg/mL without S9-mix inclusive and exclusive gaps. At the same preparation time in the highest dose group with metabolic activation the mutation rate was enhanced inclusive gaps. Also at preparation time 18h and 28h the aberration rate increased after treatment with 750 µg/mL inclusive and exclusive gaps in the absence of S9-mix.

In addition an increased number of cells with aberrations at 28h after treatment in the highest dose group with S9-mix exclusive gaps was observed.

In conclusion, the test substance induced chromosome aberrations in V79 Chinese hamster cells in the absence of a metabolic activation system, under the experimental conditions described in this report.

The test substance was administered 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. 5 males and 5 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, the test substance 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, the test substance is not mutagenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.

Short description of key information:
The test substance is not genotoxic. The only unambigious positive test result was found in the in-vitro chromosome aberration assay without metabolic activation system, which is a known false positive result for vinyl-sulphone dyes in in vitro clastogenicity tests. However, the same endpoint was negative in the in vivo test system.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Justification for classification or non classification

The above studies have all been ranked reliability 1 or 2 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP and/or in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. 

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for genetic toxicity is therefore required.