Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer-reviewed scientific journal

Data source

Reference

Reference Type:

publication

Title:

Mutagenic activity of the given test chemical in the Salmonella/microsome and mouse lymphoma TK +/- assays

Author:

T.P. Cameron et al.

Year:

1987

Bibliographic source:

Mutation Research

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: AMES ASSAY

Principles of method if other than guideline:

Mutagenicity testing was performed by the standard plate-incorporation assay for the test chemical on Salmonella typhimurium by using TA1535, TA1537, TA1538, TA98 and TA100 strains.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

EC Number:

240-474-8

EC Name:

Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Cas Number:

16423-68-0

Molecular formula:

C20H8I4O5.2Na

IUPAC Name:

disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Details on test material:

- Name of test material (as cited in study report):
D&C Red 3
- Molecular formula (if other than submission substance): C20-H6-I4-O5.2Na
- Molecular weight (if other than submission substance): 879.8424 g/mol
- Substance type: Organic
- Physical state: Solid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Each chemical was tested without metabolic activation and with S9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters.
- source of S9
- method of preparation of S9 mix : The S9 mix was prepared according to the procedure described by Ames et al. (1975) and contained 0.1 ml S9 per ml of S9 mix; an aliquot of 0.5 ml of S9 mix was added per plate.
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

33 .0µg/plate,100 .0µg/plate,333.0 µg/plate,1000.0µg/plate,3333.0µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: The solvents used were water, dimethyl sulfoxide, and acetone (exact solvent was not mention)

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: The positive control for all strains with metabolic activation was 2-aminoanthracene (2.5µg/plate for TA1535 and TA1537, 1.0µg/plate for TA1538 and TA98, and 5.0 µg/plate for TA100).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : TRIPLICATES
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk : PLATE INCORPORATION

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

Evaluation criteria:

A response was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537.

Statistics:

No data available

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: The range of concentrations for testing was based on preliminary toxicity tests in which the viability of the bacterial cells on complete medium was measured at concentrations up to 10 mg/plate or to the limit of solubility. When solubility and toxicity were not limiting factors, the maximum concentration tested was 10 mg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test: The test chemical was negative in the Salmonella mutagenicity assays conducted.

Remarks on result:

other: negative

Any other information on results incl. tables

Results of the Salmonella Mutagenicity study using the Plate incorporation assay.

Dose(µg/plate)	Revertants per plate
	TA1535
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	30±7	16±6	20±6
Positive Cont.^b	380±6	198±17^2	119±7
33.00	21±3	19±3	18±2
100.00	28±3	21±6	21±4
333.00	19±4	15±2	16±2
1000.00	15±4	9±1	10±1
3333.00	14±2	13±3	11±3

Dose(µg/plate)	Revertants per plate
	TA1537
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	5±2	9±5	9±3
Positive Cont.^b	199±30	36±28^2	79±6
33.00	5±4	7±2	9±8
100.00	8±3	10±3	5±2
333.00	6±3	6±3	9±1
1000.00	5±1	5±2	7±6
3333.00	5±1	4±4	7±2

Dose(µg/plate)	Revertants per plate
	TA98
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	21±3	34±5	34±3
Positive Cont.^b	196±34^2	1086±74^2	991±25
33.00	18±4	25±11	24±2
100.00	17±5	27±5	26±6
333.00	19±4	31±2	25±9
1000.00	15±5	23±6	20±6
3333.00	13±4	12±2	20±7

Dose(µg/plate)	Revertants per plate
	TA1538
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	15±3	19±4	22±3
Positive Cont.^b	191±16^1	1040±33^2	525±15
33.00	16±4	22±5	14±9
100.00	12±5	16±4	14±9
333.00	12±2	21±7	15±4
1000.00	9±2	14±6	19±4
3333.00	11±1	14±1	10±5

Dose(µg/plate)	Revertants per plate
	TA100
	(-S)S9                                   (R)S9                            (H)S9
Solvent Cont.	171±14	156±13	191±26
Positive Cont.^b	571±7	1630±63^2	1214±61
33.00	179±21	138±2	160±21
100.00	168±11	136±10	189±21
333.00	106±10	163±20	179±34
1000.00	117±35	164±14	175±7
3333.00	102±33	138±5	171±9
^aMean and standard deviation. ^bin assays without metabofic activation, the positive controls were sodium azide for TA1535 and TA100, 1.0 µg/plate, 9-aminoacridine for TA1537, 50.0 µg/plate; 2-nitrofluorene for TA1538, 1.0 or 5.0 µg/plate, and for TA98, 2.0 or 5.0 µg/plate. In those assays in which the lower dose of 2-nitrofluorene was used, the positive control value is followed by a superscript indicating the dose level used (either^1or^2). In assays with rat-liver S9, the positive control for all strains was 2-aminoanthracene, 2.5 µg/plate except for those positive control values, followed by a numerical superscript. This number is the dose used as a positive control. In assays with hamster-fiver S9, the positive control for all strains was 2-aminoanthracene, 1.0 µg/plate, except for those positive controls followed by the superscript "c". In these cases 2.5µg/plate was used. 1 Dose level was 1.0 µg/plate. 2 Dose level was 2.0 µg/plate. 5 Dose level was 5.0 µg/plate. c Dose level was 2.5 µg/plate. d Only 2 plates

Applicant's summary and conclusion

Conclusions:

The test chemical was negative in the Salmonella mutagenicity assays conducted.

Executive summary:

Mutagenicity testing was performed by the standard plate-incorporation assay for the test chemical on Salmonella typhimurium by using TA1535, TA1537, TA1538, TA98 and TA100 strains. These strains were kindly supplied by Dr. Bruce Ames, University of California at Berkeley. Strain checks were performed immediately after receipt of the cultures and on a routine basis thereafter. The test chemical was tested without metabolic activation and with S9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters. The S9 mix was prepared according to the procedure described by Ames et al. (1975) and contained 0.1 ml S9 per ml of S9 mix; an aliquot of 0.5 ml of S9 mix was added per plate. The test chemical was either dissolved in water, DMSO or acetone and tested at concentrations of 33.0µg/plate, 100.0µg/plate, 333.0µg/plate, 1000.0µg/plate, 3333.0µg/plate in triplicates. The range of concentrations for testing was based on preliminary toxicity tests in which the viability of the bacterial cells on complete medium was measured at concentrations up to 10 mg/plate or to the limit of solubility. When solubility and toxicity were not limiting factors, the maximum concentration tested was 10 mg/plate.In tests without metabolic activation the positive controls were sodium azide for strains TA1535 and TA100 (1.0 µg/plate), 9-aminoacridine for TA1537 (50.0 µg/plate), and 2-nitrofluorene for TA1538 and TA98 (1.0, 2.0 or 5.0 µg/plate). The positive control for all strains with metabolic activation was 2-aminoanthracene (2.5µg/plate for TA1535 and TA1537, 1.0µg/plate for TA1538 and TA98, and 5.0 µg/plate for TA100). A response was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537. The test chemical was negative in the Salmonella mutagenicity assays conducted.