Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Genetic toxicity: in vitro

Administrative data

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)

Rationale for reliability incl. deficiencies:

other: data from peer- reviewed journals

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity Testing of the Food Colours Amaranth and Tartrazine

Author:

Aparajita Das and Anita Mukherjee

Year:

2004

Bibliographic source:

Int J Hum Genet, 4(4): 277-280 (2004)

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Ames mutagenicity assay was performed to evaluate the mutagenic nature of the test compound amaranth.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

EC Number:

213-022-2

EC Name:

Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Cas Number:

915-67-3

Molecular formula:

C20H14N2O10S3.3Na

IUPAC Name:

trisodium 3-hydroxy-4-[(4-sulfonato-1-naphthyl)diazenyl]naphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

trisodium (4E)-3-oxo-4-[(4- sulfonato-1- naphthyl)hydrazono]naphthalene- 2,7-disulfonate

IUPAC Name:

trisodium (4E)-3-oxo-4-[(4- sulfonato-1- naphthyl)hydrazono]naphthalene- 2,7-disulfonate

Constituent 3

Reference substance name:

Amaranth dye

IUPAC Name:

Amaranth dye

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name of test material (as cited in study report): Amaranth [Food Red 2]
Molecular formula (if other than submission substance): C20-H11-N2-O10-S3.3Na
C20-H14-N2-O10-S3.3Na
Molecular weight (if other than submission substance): 604.4789 g/mole
Substance type: Organic
Physical state: Solid

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: Salmonella typhimurium TA97a, TA98 and TA100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

no metabolic activation system used

Test concentrations with justification for top dose:

10,100,250,500 and 1000 μg /plate

Vehicle / solvent:

Vehicle
Vehicle(s)/solvent(s) used: Sterile double distilled water
Justification for choice of solvent/vehicle: No data available

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

for TA97a and TA98, 20 μg/plate nitro phenylene diamine and for TA100, 1.5 μg/plate sodium azide

Positive control substance:

other: for TA97a and TA98, 20 μg/plate nitro phenylene diamine and for TA100, 1.5 μg/plate sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
Preincubation period: No data available
Exposure duration: 48⁰C
Expression time (cells in growth medium): 48⁰C
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

NUMBER OF REPLICATIONS: triplicate

Evaluation criteria:

Increase in the number of mutagenic revertants

Statistics:

ANOVA test was performed at 0.05 level

Results and discussion

Test results

Species / strain:

S. typhimurium, other: Salmonella typhimurium TA97a, TA98 and TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not 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

Table 1: Mutagenicity of food colours in tester strains of Salmonella typhimurium

 

Dose           μg/plate	Mean of the No. Revertant Colonies ± S.D.
	TA97a	TA98	TA100
10	120.33 ± 15.50	222.0 ± 49.79	103.7 ± 9.07
100	115.67 ±  17.78	205.7 ±  4.04	92.7 ± 16.17
250	92.33 ±  12.50	170.7 ± 68.72	92.0 ±  27.73
500	120.67 ± 22.94	178.3 ± 30.29	83.7 ±  7.095
1000	122.33± 24.82	195.7± 36.23	109.0 ± 10.19
10	134.00 ± 42.51	49.3 ± 13.7	98.0  ± 7.55
Solvent control	122.30 ± 9.61	21.3 ± 9.02	121.0  ±  3.61
NPD	827.70 ± 106.53	522.0 ± 50.48	-
SA	-	-	1624.67 ±  89.76

 

Where:

NPD= Nitrophynylenediamine

SA= Sodium azide

S.D.= Standard Deviation

Anova Value of TA97a (Amaranth —2.11, ns)

TA 98 (Amaranth –10.19*)

TA100 (Amaranth —2.196, ns)

P< 0.01 (5.06)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

The test compound amaranth [Food Red 2] was not mutagenic in the study conducted using Salmonella typhimurium TA97a, TA98 and TA100 without metabolic activation system.

Executive summary:

Ames mutagenicity assay was performed to evaluate the mutagenic nature of the test compound Amaranth in plate incorporation assay.

 

The test material was tested at a concentration of 10,100,250,500 and 1000 μg /plate. The plates were inverted within an hour and placed in a dark vented incubator at 37⁰C for 48 hours. Positive controls (for TA97a and TA98, 20 μg/plate nitro phenylene diamine and for TA100, 1.5 μg/plate sodium azide) and negative controls were maintained concurrently for all the experiments. Three plates were used for each set. After 48 hours of incubation, the revertant colonies were counted. ANOVA test was performed at 0.05 level.

 

Amaranth [Food Red 2] was determined to be non - mutagenic under the study conditions.