3-amino-2,4,6-triiodobenzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 October 2002 to 31 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-amino-2,4,6-triiodobenzoic acid

EC Number:

221-493-0

EC Name:

3-amino-2,4,6-triiodobenzoic acid

Cas Number:

3119-15-1

Molecular formula:

C7H4I3NO2

IUPAC Name:

3-amino-2,4,6-triiodobenzoic acid

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 37055008
- Expiration date of the lot/batch: report states "stable according to 03219859" and no date provided

Method

Target gene:

N/A

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: B. Ames (University of California, 94720 Berkeley, USA)

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

N/A

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsomal fraction S9 mix

Test concentrations with justification for top dose:

3, 10, 33; 100; 333; 1000; 2500; and 5000 µg/plate

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Concurrent untreated

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Controls 2

Untreated negative controls:

yes

Remarks:

Concurrent untreated

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): N/A

DURATION
- Preincubation period: 48 hours
- Exposure duration: N/A
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µLTest solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 µLS9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µLBacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µLOverlay agar

NUMBER OF CELLS EVALUATED: N/A

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: N/A
- Any supplementary information relevant to cytotoxicity: N/A

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: N/A

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Summary of Results:

Test substance without S9 metabolic activation:

Concentration µg/plate	Revertants/plate mean fromthreeplates
	TA1535	TA 1537	TA 98	TA 100	TA 102
Negative control	10	5	25	147	192
Solvent control	7	6	20	140	157
Positive control	716	54	142	912	939
3	7	5	21	131	188
10	8	5	22	144	159
33	8	5	24	140	178
100	8	5	23	143	179
333	7	5	22	134	171
1000	8	5	20	140	164
2500	6	5	20	139	144
5000	7	5	23	146	157

Test substance with S9 metabolic activation:

Concentration µg/plate	Revertants/plate mean fromthreeplates
	TA1535	TA 1537	TA 98	TA 100	TA 102
Negative control	10	6	27	135	167
Solvent control	10	5	26	147	158
Positive control	93	76	542	738	828
3	10	6	28	160	145
10	8	5	26	166	155
33	8	6	25	147	142
100	9	5	25	165	158
333	7	5	26	151	149
1000	7	5	24	168	159
2500	9	6	28	154	146
5000	9	5	28	166	172

Applicant's summary and conclusion

Conclusions:

The test substance did not induce gene mutations by base pair changes or frameshifts in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 with or without activation.

Executive summary:

The test item ZK 2218 was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102, with and without metabolic activation testing each strain in triplicate according to OECD guideline 471. The test item was tested at the following concentrations: 3, 10, 33, 100; 333; 1000; 2500; and 5000 µg/plate.

No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains used. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with ZK 2218 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. 

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.