Disodium 5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

To evaluate not only the mutagenicity of the D&C Red No. 33 themselves but also that of the reduction products. In the present study, we have evaluated the mutagenic activity of D&C Red No. 33 that are commonly used in foods, drugs and cosmetics, prior to and following reduction by azo reductase-producing bacteria isolated from the human gastrointestinal tract, using the Salmonella typhimurium plate incorporation Assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

To evaluate not only the mutagenicity of the D&C Red No. 33 themselves but also that of the reduction products. In the present study, we have evaluated the mutagenic activity of D&C Red No. 33 that are commonly used in foods, drugs and cosmetics, prior to and following reduction by azo reductase-producing bacteria isolated from the human gastrointestinal tract, using the Salmonella typhimurium plate incorporationAssay.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 98,TA 100

Metabolic activation:

with and without

Metabolic activation system:

RAT LIVER S-9, AROCLOR 1254

Test concentrations with justification for top dose:

50 and 200 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 1,6-dinitropyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period:- Exposure duration: No data available.- Expression time (cells in growth medium): No data available.- Selection time (if incubation with a selection agent): No data available.- Fixation time (start of exposure up to fixation or harvest of cells): No data available.SELECTION AGENT (mutation assays):SPINDLE INHIBITOR (cytogenetic assays):STAIN (for cytogenetic assays):NUMBER OF REPLICATIONS: No data available.NUMBER OF CELLS EVALUATED:DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data available.OTHER EXAMINATIONS:- Determination of polyploidy:- Determination of endoreplication:- Other:OTHER: Reduction of D&C Red No. 33 dye was done by using azo reductase-producing bacteria isolated from the human gastrointestinal tract, naming Clostridium strain.Cultures of Clostridium in BHI, PRAS were incubated with either 0.5 mg or 2 mg of one of the dyes per ml at 37°C until the dyes were completely decolorized. 5 mg sodium dithionite (Sigma Chemical Co. St Louis, MO, USA) was added to each of the cultures, which were centrifuged at 3180g for 30 min. The supernatants were filtered through a 0.2µm Acrodisc filter from Gelman Science and 100/A of each culture filtrate was used directly as the source of metabolites for the mutagenicity assay. A culture of Clostridium in BHI, PRAS, grown without azo dyes but otherwise treated the same way, was used as a negative control.

Evaluation criteria:

The evaluation of mutagenicity, the numbers of revertants for S. typhimurium with 1,6-dinitropyrene, benzo[a]pyrene and the azo dyes before reduction were compared with the numbers with DMSO. The numbers of revertants for S. typhimurium with azo dye metabolites were compared with those with the culture filtrates from dye-free Clostridium cultures. There was a positive mutagenic response due to the controls (l,6-dinitropyrene and benzo[a]pyrene) compared with DMSO.

Statistics:

No data available.

Species / strain:

S. typhimurium, other: TA 98,TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

D&C Red No. 33 and its reduced metabolites in the concentration of 50 and 200 do not cause genetic mutation(s) when Salmonella typhimurium Strain-TA 98, TA 100 exposed to the test chemical in the presence and absence of metabolic activation (S9).

Conclusions:

Interpretation of results (migrated information):negativeD&C Red No. 33 in the concentration of 50 and 200 µg/plate did not show any evidence of gene toxicity when Salmonella typhimurium Strain-TA 98, TA 100 were exposed to the test chemicals.

Executive summary:

In a gene toxicity test, Salmonella typhimurium Strain-TA 98, TA 100 were exposed to D&C Red No. 33 in the concentration of 50 and 200 µg/plate with and without metabolic activation. In addition D&C Red No. 33 metabolites were also prepared by treating with azo reductase -producing bacteria namely Clostridium strain isolated from the human gastrointestinal tract. The results showed that there was no evidence of gene toxicity after treatment with D&C Red No. 33 in the concentration of 50 and 200 µg/plate in Salmonella typhimurium Strain-TA 98, TA 100. Independently of tested D&C Red No. 33 reduced metabolite in the concentration of 50 and 200 µg/plate showed that there was no evidence of gene toxicity. Therefore, it is considered that D&C Red No. 33 and its reduced metabolites in the concentration of 50 and 200 µg/plate do not cause genetic mutation(s) when Salmonella typhimurium Strain-TA 98, TA 100 exposed to the test chemical in the presence and absence of metabolic activation (S9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity in vitro:

Genotoxicity study for D&C Red No. 33 (F. RAFII et al.1997) was conducted in Salmonella typhimurium strain-TA 98, TA 100, when they were exposed in the concentration of 50 and 200 µg/plate with and without metabolic activation. In addition D&C Red No. 33 metabolites were also prepared by treating with azo reductase -producing bacteria namely Clostridium strain isolated from the human gastrointestinal tract. The results showed that there was no evidence of gene toxicity after treatment with D&C Red No. 33 in the concentration of 50 and 200 µg /plate in Salmonella typhimurium Strain-TA 98, TA 100. Independently of tested D&C Red No. 33 reduced metabolite in the concentration of 50 and 200 µg/plate showed that there was no evidence of gene toxicity. Therefore, it is considered that D&C Red No. 33 and its reduced metabolites in the concentration of 50 and 200 µg/plate do not cause genetic mutation(s) when Salmonella typhimurium Strain-TA 98, TA 100 exposed to the test chemical in the presence and absence of metabolic activation (S9).

In the supporting studiesScientific Committee on Consumer Safety report 2007, the genotoxicity study for D&C Red 33 was investigatedin Salmonella typhimurium and Escherichia coli. The test chemical investigated for the induction of gene mutations in Salmonella typhimurium and Escherichia coli (Ames test). Liver S9 fraction from Syrian golden hamsters was used as exogenous metabolic activation system. Test concentrations were based on the level of toxicity in a pre-experiment with all Salmonella and Escherichia strains. Toxicity was evaluated on the basis of a reduction in the number of revertant colonies and/or a clearing of the bacterial background lawn. Since D&C Red 33 was freely soluble and non toxic in this preliminary toxicity test, it was tested up to the prescribed maximum concentration of 5000 µg/plate. The pre-experiment is reported as main experiment I. Both experiments were performed with the preincubation method. Negative and positive controls were in accordance with the OECD guideline. No substantial and biological relevant increase in revertant colonies of any of the five tester strains was observed in both experiments following treatment with D&C Red 33 at any dose level neither in the absence nor in the presence of metabolic activation. Under the experimental conditions used D&C Red 33 was not genotoxic (mutagenic) in the gene mutation tests in bacteria.

In another study by R. B. Havelandsmith* and R. D. Combes, 1980, the gene toxicity test was observed in Escherichia Coli strains WP2. When they were exposed to Red 10B in the concentration of 10000µg/ml with and without metabolic activation by Rec assay.The repair-proficient (WP2) cells were treated with the activated dye for 3.5 hr and the repair-deficient cells were treated for 5.5 hr before being serially diluted and plated onto nutrient agar. The mutagenicity was evaluated by observing CS - Coefficient of survival, defined as percentage survival of treated repair-deficient cells/percentage survival of treated repair-proficient cells.The result was found to be positive without S9 and negative with S9 for Red 10B by Rec assay.

In another study by Scientific Committee on Consumer Safety report, 2007: the genotoxicity test was observed in L5178Y Mouse lymphoma cells when they were exposed to D&C Red No. 33 in the concentration of 300 - 4800 μg/ml with and without metabolic activation. D&C Red 33 was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. Test concentrations were based on the results of a pre-test measuring relative suspension growth. The cells were treated for 4 hour followed by an expression period of 72 h to fix the DNA damage into a stable tk mutation. Toxicity was measured as percentage relative survival and total growth of the treated cultures relative to the survival of the solvent control cultures. Therefore, it is considered that D&C Red No. 33 in the concentration of 300 - 4800 μg/ml do not cause genetic mutation at the tk locus of L5178Y Mouse lymphoma cells.

In another study by Scientific Committee on Consumer Safety report, 2007: the genotoxicity test was observed in L5178Y Mouse lymphoma cells. When they were exposed to D&C Red No. 33 in the concentration of 150 - 4800 μg/ml without metabolic activation. D&C Red 33 was assayed for gene mutations at the tk locus of mouse lymphoma cells in the absence of S9 metabolic activation. Test concentrations were based on the results of a pre-test measuring relative suspension growth. The cells were treated for 24 hour followed by an expression period of 48 hour to fix the DNA damage into a stable tk mutation. Toxicity was measured as percentage relative survival and total growth of the treated cultures relative to the survival of the solvent control cultures. Therefore, it is considered that D&C Red No. 33 in the concentration of 150 - 4800 μg/ml do not cause genetic mutation at the tk locus of L5178Y Mouse lymphoma cells , when they exposed to the test chemical in the absence of metabolic activation (S9).

In another study by R. B. Havelandsmith* and R. D. Combes 1980 was conducted in E coli strain. In a gene toxicity test, Escherichia Coli strains WP2 uvr A were exposed to Red 10B in the concentration of 10000µg/ml with and without metabolic activation by fluctuation assays. The dyes were incubated with or without for 28 hr with E. coli strain WP2 uurA and then observed for decolourization using a Beckman Model 25 spectrophotometer. The result was found to be positive without S9 and negative with S9 for Red 10B by fluctuation assays.

In another study (R. B. Haveland-Smith and R. D. Combes ,1979) with similar substances (CAS NO: 3567-69-9), Genotoxicity study was carried out forAcid Red 14. Fluctuation test was performed for the test chemical Carmoisine used at a concentration of 5 mg/mL in liquid medium for 72-96 hrs. The tester strains used wereSalmonella typhimurium1538 both in the presence and absence of metabolic activation system. The assay was performed for each bacterium in three separate experiments. The given test material failed to induce genotoxicity in the bacteriaSalmonella typhimurium1538 and hence is negative for gene mutation in vitro.

In another study (R. B. Haveland-Smith and R. D. Combes ,1979) with similar substances (CAS NO: 3567-69-9), Genotoxicity study was carried out for Acid Red 14.Fluctuation test was performed for the test chemical Carmoisine used at a concentration of 5 mg/mL in liquid medium for 72-96 hrs. The tester strains used were Escherichia coli WP2 uvrA both in the presence and absence of metabolic activation system. The assay was performed for each bacterium in three separate experiments.The given test material failed to induce genotoxicity in the bacteria Escherichia coli WP2 uvrA and hence is negative for gene mutation in vitro.

In another study (R. B. Haveland-Smith and R. D. Combes ,1979) with similar substances (CAS NO: 3567-69-9), Genotoxicity study was carried out for Acid Red 14.Carmoisine was used at a concentration of 5 mg/mL and its genotoxic effect was studied in the bacteriaE. coli strains WP2 trp uvrA and WP100 trp uvrA recAbyrecplate assay in the agar medium. The given test material is negative for genotoxicity in the bacteriaE.Coli strains WP2 trp uvrA and WP100 trp uvrA recAand hence are negative for gene mutation in vitro.

In another study (T.P. Cameron et.al., 1987) with similar substances (CAS NO: 3567-69-9), Genotoxicity study was carried out forAcid Red 14.L5178Y TK +/- mouse lymphoma assay was performed using C. I ACID RED 14 at different concentrations both with and without S9 metabolic activation system. The C. I ACID RED 14 did not induce mutation and hence was negative (with and without S9 mix) inL5178Y TK +/- mouse lymphoma assay.

In another study (T.P. Cameron et.al., 1979) with similar substances (CAS NO: 3567-69-9), Genotoxicity study was carried out forAcid Red 14.Bacterial gene mutation assay was performed for the test material C. I -ACID RED 14 inSalmonella typhimuriumTA1535, TA1537, TA1538, TA100 and TA98 strains at a dose range of 333.0, 1000.0, 3333.0, 6666.0 and 10000.0 µg/plate by Plate-incorporation method. Chemical was tested without metabolic activation and with S9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters. Appropriate positive and solvent controls were also incorporated in the study. The dye. ACID RED 14was found to be negative to induce gene mutation with and without S9 mix inSalmonella typhimuriumstrains TA1535, TA1537, TA1538, TA100 and TA98 by Plate-incorporation assay.

In another study (T.P. Cameron et.al., 1987) with similar substances (CAS NO: 3567-69-9), Genotoxicity study was carried out forAcid Red 14.FMN modified assay was performed in theSalmonella typhimuriumstrains TA98 and TA100 for the test chemical C. I ACID RED 14. The strains were exposed to a concentration of 333.0, 1000.0, 3333.0, 6666.0 and 10000.0 µg/plate in the preincubation protocol. Positive control (Trypan blue) and solvent control were incorporated in the study. All plates were counted on an Artek automated counter, which was calibrated before use.The test material C. I ACID RED 14 failed to induce gene mutationinSalmonella typhimuriumstrains TA98 and TA100 in the FMN- modified assay.

Overall reported genetic toxicity studies of D&C Red No. 33 as well as its read acrossand applying weight of evidence approach, indicate that D&C Red No. 33 is not likely to exhibit genetic toxicity can be classified as 'non-hazardous' as per the CLP classification criteria .

Justification for selection of genetic toxicity endpoint
D&C Red No. 33 in the concentration of 50 and 200 µg/plate did not show any evidence of gene toxicity when Salmonella typhimurium Strain-TA 98, TA 100 were exposed to the test chemicals.

Justification for classification or non-classification

On the basis of majority of available data, the substance can be considered to be non-mutagenic and hence can be classified as non-hazardous as per the CLP criteria.