2-[(4-amino-3-methoxyphenyl)sulphonyl]ethyl hydrogen sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from publication.

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of Azo Dyes Used in Foods, Drugs and Cosmetics Before and After Reduction by Clostridium Species from the

Author:

F. Rafii, J. D. Hall and C. E. Cerniglia

Year:

1997

Bibliographic source:

Food and Chemical Toxicology 35, 897-901, 1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: As mention below

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 incorporation
Assay.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

D&C Red No. 33

IUPAC Name:

D&C Red No. 33

Constituent 2

Reference substance name:

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

EC Number:

222-656-9

EC Name:

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

Cas Number:

3567-66-6

Molecular formula:

C16H13N3O7S2.2Na

IUPAC Name:

disodium 5-amino-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): - D&C Red No. 33
- Molecular formula (if other than submission substance): C16H13N3O7S2.2Na
- Molecular weight (if other than submission substance): 467.3889 g/mol
- Substance type: Organic
- Physical state: No data available.
Purity- No data available.
- Impurities (identity and concentrations): No data available.

Specific details on test material used for the study:

- Name of test material (as cited in study report): - D&C Red No. 33
- Molecular formula (if other than submission substance): C16H13N3O7S2.2Na
- Molecular weight (if other than submission substance): 467.3889 g/mol
- Substance type: Organic

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 98,TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

Number of revertants of Salmonella typhimurium TAI00 withD&C Red No. 33 dyes in the presence and absence of S-9 fraction

Sr no	D&C Red No. 33	50 µg	200µg	50 µg	200µg
	STRAIN	Dye without S-9		Dye with S-9
1	TA 100	149	183±9	107±20	130±2
2	DMSO	105 ± 12	NA^b	99 ± 12	NA
3	1,6-DNP^c	4448	NA	NA	NA
4	Benzo[a]pyrene	44 ±8	NA	503 ± 53	NA

^a300 lag of dye was used .b Not applicable.^C5 µg/plate of 1,6-dinitropyrene or benzo[a]pyrene was used.

Number of revertants of Salmonella typhimurium TA98 with D&C Red No. 33 in the presence and absence of S-9 fraction

Sr no	D&C Red No. 33	50 µg	200µg	50 µg	200µg
	STRAIN	Dye without S-9		Dye with S-9
1	TA 98	14 ± 1	14± 6	38 ± 1	41 ± 5
2	DMSO	20 ± I	NA^a	27 ± 11	NA
3	1,6-DNP^b	1050 ± 43	NA	3112 _+ 33	NA
4	Benzo[a]pyrene	20±1	NA	249±16	NA

^aNot applicable.b5 lag/plate of 1,6-dinitropyrene or benzo[a]pyrene was used.

Number of revertants of Salmonella typhimurium TAI00 with D&C Red No. 33 metabolites after incubation with Clostridium sp. in the presence and absence of S-9 fraction

Sr no	D&C Red No. 33	50 µg	200µg	50 µg	200µg
	STRAIN	Dye without S-9		Dye with S-9
1	TA 100	235± 11	430±23	232±6	359± 18
2	DMSO	169±19	NA	141±6	NA
3	1,6-DNP^b	4448	NA	NA	NA
4	Benzo[a]pyrene	44 ± 8	NA	503 ± 53	NA

^aNot applicable.b5 lag/plate of 1,6-dinitropyrene or benzo[a]pyrene was used.

Number of revertants of Salmonella typhimurium TA98 with D&C Red No. 33 metabolites after incubation with Clostridium sp. in the presence and absence of S-9 fraction

Sr no	D&C Red No. 33	50 µg	200µg	50 µg	200µg
	STRAIN	Dye without S-9		Dye with S-9
1	TA 98	52 ± 28	50 -+ 1	77 ± 4	60 ± 5
2	DMSO	27 + 11	NA	20 _+ 1	NA
3	1,6-DNP^b	1050 ± 43	NA	3112 ± 33	NA
4	Benzo[a]pyrene	20 ± 1	NA	249 ± 16	NA

^aNot applicable.b5 lag/plate of 1,6-dinitropyrene or benzo[a]pyrene was used.

Applicant's summary and conclusion

Conclusions:

D&C Red No. 33 was observed for its mutagenic potential in strain-TA 98, TA 100 . The test substance considered to be negative for mutagenic both in the presence and absence of metabolic activation.

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).