2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(1H-imidazol-5-yl)propanoyl]amino]-2-methyl-propanoic acid Trifluoroacetic acid salt (1:1)

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:

22/03/2019 - 4/07/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(1H-imidazol-5-yl)propanamido]-2-methylpropanoic acid; trifluoroacetic acid

EC Number:

815-122-7

Cas Number:

1446013-08-6

Molecular formula:

C27H27F3N4O7

IUPAC Name:

2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(1H-imidazol-5-yl)propanamido]-2-methylpropanoic acid; trifluoroacetic acid

Test material form:

solid: particulate/powder

Details on test material:

Target Substance Molecular Formula: C27H27F3N4O7
Target Substance Molecular Weight: 576.51 g/mol
Batch Number: 160004
Physical form and colour: Solid, White powder
Storage: Store at 2÷8°C

Specific details on test material used for the study:

Batch: 160004
Preparation date: 30/11/2016
Expiration date: 09/02/2020
Storage conditions: Refrigerated (2-8 °C)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

The concentration of S9 used in both phase A preliminary cytotoxicity and phase B Micro-Ames test, was 10 %.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;

- Justification for choice of solvent/vehicle: based on a solubility test. The test substance was dissolved in ddH2O at the highest allowed test concentration, i.e. 50 mg/ml (corresponding to the final concentration of 0.5 mg/well). The solution resulted insoluble. Since the test substance was insoluble in ddH2O, it was dissolved in DMSO at the highest allowed test concentration (i.e. 50 mg/ml). The solution resulted soluble and this concentration was considered the first to use in the preliminary cytotoxicity.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

ddH2O and DMSO were used

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

The Bacterial Reverse Mutation test exploits amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of a few DNA base pairs. Hence, the principle of this test is the detection of mutations present in the test strains which revert and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino-acid required by the parent test strain.
In this study the plate incorporation method was applied. The study was divided into 2 main phases:
1) Preliminary cytotoxicity, to determine whether the test item was cytotoxic or not. For this preliminary phase, Salmonella typhimurium TA100, one of the 5 strains that were employed in the second phase of the test (Micro-Ames test), was exposed to 5 concentrations of the test substance, in presence and absence of exogenous metabolic activation system (S9 mix). The survival of treated cultures was determined by verifying the presence of a normal bacterial background. Absent or abnormal background was indicative of a cytotoxic response.
2) Micro-Ames test. 5 concentrations of the test substance were placed in plates of minimal medium then suspensions of 5 different bacteria strains mixed with an overlay agar were added to the plates, in the presence and in the absence of an exogenous metabolic activation system (S9 mix).
After 72 hours of incubation, revertant colonies were compared to the number of spontaneous revertant colonies on solvent control plates. In parallel the phenotypic characteristics proper of each strain were confirmed repeating the controls (histidine requirement, tryptophane requirement, ampicillin resistance, sensitivity to crystal violet, sensitivity to UV light)

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:

cytotoxicity

Remarks:

at the concentration 0.158 mg/well +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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

True 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

True 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:

cytotoxicity

Remarks:

at the concentration 0.158 mg/well

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In all strains the induction factor (IF) did not exceed 2 for all strains, hence the test substance is considered not to be mutagenic under the test conditions applied. Notably, it was not necessary to verify the dose-concentration correlation, as no IF increase ≥ 2 was observed.

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained, it can be concluded that under the test conditions applied, the test item FMOC-HIS-AIB-OH TFA LS; N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-histidyl-2-methylalanine and trifluoroacetic acid (1:1)”, CAS 1446013-08-6, batch n° 160004, is considered not to be mutagenic up to the concentration 0.050 mg/well. Notably, the concentration 0.158 mg/well was not considered, as it resulted cytotoxic for strains TA100 and TA1535.

Executive summary:

The Bacterial Reverse Mutation test exploits amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of a few DNA base pairs. Hence, the principle of this test is the detection of mutations present in the test strains which revert and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino-acid required by the parent test strain.

In this study the plate incorporation method was applied. The study was divided into 2 main phases:

1) Preliminary cytotoxicity, to determine whether the test item was cytotoxic or not. For this preliminary phase, Salmonella typhimurium TA100, one of the 5 strains that were employed in the second phase of the test (Micro-Ames test), was exposed to 5 concentrations of the test substance, in presence and absence of exogenous metabolic activation system (S9 mix). The survival of treated cultures was determined by verifying the presence of a normal bacterial background. Absent or abnormal background was indicative of a cytotoxic response.

2) Micro-Ames test. 5 concentrations of the test substance were placed in plates of minimal medium then suspensions of 5 different bacteria strains mixed with an overlay agar were added to the plates, in the presence and in the absence of an exogenous metabolic activation system (S9 mix).

After 72 hours of incubation, revertant colonies were compared to the number of spontaneous revertant colonies on solvent control plates. In parallel the phenotypic characteristics proper of each strain were confirmed repeating the controls (histidine requirement, tryptophane requirement, ampicillin resistance, sensitivity to crystal violet, sensitivity to UV light)

The following 5 strains of bacteria were used:

Salmonella typhimurium TA98

Salmonella typhimurium TA100

Salmonella typhimurium TA1535

Salmonella typhimurium TA1537

Escherichia coli WP2 uvrA pKM101

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli.

Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.

On the basis of the results obtained, it can be concluded that under the test conditions applied, the test item FMOC-HIS-AIB-OH TFA LS; N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-histidyl-2-methylalanine and trifluoroacetic acid (1:1)”, CAS 1446013-08-6, batch n° 160004, is considered not to be mutagenic up to the concentration 0.050 mg/well. Notably, the concentration 0.158 mg/well was not considered, as it resulted cytotoxic for strains TA100 and TA1535