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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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.