Trisodium [2-[[α-[[2-hydroxy-5-sulpho-3-[(2,5,6-trichloro-4-pyrimidinyl)amino]phenyl]azo]benzyl]azo]-5-sulphobenzoato(5-)]cuprate(3-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

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 peer-reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Ames Salmonella/mammalian-microsome testing of peptides and peptide synthesis reagents

Author:

Jane S. Allen and Jennifer Panfili

Year:

1986

Bibliographic source:

Mutation Research, 170 (1986) 23-29

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:

Salmonella/reverse mutation assay was performed to evaluate the mutagenic potential of the test material 1-Hydroxybenzotriazole monohydrate.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L-Phenylalaninamide, L-tryptophyl-L-methionyl-L-alpha-aspartyl-

Cas Number:

1947-37-1

Molecular formula:

C37H42N6O8S

IUPAC Name:

L-Phenylalaninamide, L-tryptophyl-L-methionyl-L-alpha-aspartyl-

Details on test material:

- Name of test material: L-TryptophyI-L-methionyI-L-aspartyl-L-phenylalaninamide
- Molecular Formula: C37H42N6O8S
- Molecular Weight: 596.7054 g/mol
- Substance type: Organic
- Purity: No data available
- Impurities (identity and concentrations): No data available

Specific details on test material used for the study:

- Name of test material: L-TryptophyI-L-methionyI-L-aspartyl-L-phenylalaninamide (TMAP)
- Molecular Formula: C37H42N6O8S
- Molecular Weight: 596.7054 g/mol
- Substance type: Organic
- Purity: No data available
- Impurities (identity and concentrations): No data available

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from aroclor 1254-induced male Sprague-Dawley rat livers

Test concentrations with justification for top dose:

With S9:
TA1535/ TA1537/ TA98: 0, 100, 500, 1000, or 5000 µg/plate
TA100: 0, 100, 500, 1000, 3000, 4000 or 5000 µg/plate

Without S9
TA1535/ TA1537/ TA98: 0, 100, 500, 1000, or 5000 µg/plate
TA100: 0, 100, 500, 1000, 1250, 2500, 4000 or 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The chemical was soluble in water.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (TA100, TA1535; without S9), 2-aminoanthracene (2-AA) (All strains, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- 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): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Each compound was tested at least twice using duplicate plates per dose level.

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Toxicity was estimated by examining background bacterial lawns using a stereoscope and the mean number of revertants were noted

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: No data available

Rationale for test conditions:

No data available

Evaluation criteria:

Increase in the number of revertants/plates

Statistics:

No data available

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Mean numbers of revertants per plate determined from all positive control test plates in the presence and absence of S9 were 1535 and 1055 for TA100, 183 and 1190 for TA1535, 175 and 322 for TA1537, and 1137 and 959 for TA98, respectively.

Additional information on results:

No data

Remarks on result:

other: no mutagenic potential

Any other information on results incl. tables

Table: AMES TEST RESULTS FOR PEPTIDE SYNTHESIS REAGENTS USING S9

Strain	Amount tested (µg/plate)	Mean number of colonies per plate
TA1535	5000	12
	1000	12
	500	13
	100	15
	0	15
TA1537	5000	7
	1000	9
	500	9
	100	6
	0	8
TA98	5000	18
	1000	18
	500	19
	100	23
	0	27
TA100	5000	111
	4000	-
	3000	-
	1000	95
	500	92
	100	87
	0	99

 

Table: AMES TEST RESULTS FOR PEPTIDE SYNTHESIS REAGENTS WITHOUT S9

Strain	Amount tested (µg/plate)	Mean number of colonies per plate
TA1535	5000	20
	1000	18
	500	16
	100	19
	0	15
TA1537	5000	4
	1000	5
	500	6
	100	5
	0	5
TA98	5000	7
	1000	15
	500	16
	100	12
	0	12
TA100	5000	101
	4000	104
	2500	86
	1250	84
	1000	90
	500	-
	100	-
	0	87

 

Applicant's summary and conclusion

Conclusions:

L-TryptophyI-L-methionyI-L-aspartyl-L-phenylalaninamide (TMAP) did not show any mutagenic activity at concentrations up to 5000 μg/plate in S.typhimurium strains TA1535, TA1537, TA98, TA100 in the presence and absence of S9 metabolic activation system and hence, according to CLP criteria, it can be concluded that L-TryptophyI-L-methionyI-L- aspartyl-L-phenylalaninamide (TMAP) is non genotoxic.

Executive summary:

The Ames Salmonella/mammalian-microsome assay was used to evaluate the bacterial mutagenicity of L-TryptophyI-L-methionyI-L-aspartyl-L-phenylalaninamide (TMAP), a chemical reagent used in peptide synthesis. The study was performed using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 in the presence and absence of S9 metabolic activation system. Plate incorporation assay was performed and the plates were incubated for 2 days. The plates were observed for a dose depnedent increase in the number of revertants/plate. Two replicates per dose level and concurrent positive and solvent controls were used. Each assay was performed at least twice. L-TryptophyI-L-methionyI-L-aspartyl-L-phenylalaninamide (TMAP) did not show any mutagenic activity at concentrations up to 5000 μg/plate in S.typhimurium strains TA1535, TA1537, TA98, TA100 in the presence and absence of S9 metabolic activation system and hence, according to CLP criteria, it can be concluded that L-TryptophyI-L-methionyI-L- aspartyl-L-phenylalaninamide (TMAP) is non genotoxic.