2-[(2S,3S)-3-[[(1,1dimethylethoxy)carbonyl]amino]-2-hydroxy-4-phenylbutyl]-2-[[4-(2pyridinyl)phenyl]methyl]-,1,1-dimethylethyl ester

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:

1998-10-29 to 1998-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

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:

tert-butyl 2-{(2S,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-4-phenylbutyl}-2-[4-(pyridin-2-yl)benzyl]hydrazinecarboxylate

EC Number:

606-396-7

Cas Number:

198904-86-8

Molecular formula:

C32H42N4O5

IUPAC Name:

tert-butyl 2-{(2S,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-4-phenylbutyl}-2-[4-(pyridin-2-yl)benzyl]hydrazinecarboxylate

Test material form:

solid

Details on test material:

- Appearance: Off-white powder
- Storage condition of test material: At room temperature protected from light

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and 070W5 H-002-03

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark.
- Solubility and stability of the test substance in the solvent/vehicle: Precipitation was observed at and above 1500 ug/plate

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of test substance in DMSO was prepared after the test substance was dried using silica gel.
- Final dilution of a dissolved solid, stock liquid or gel: Half-log dilutions of the prepared stock solution

FORM AS APPLIED IN THE TEST (if different from that of starting material)\
0, 50, 150, 500, 1500, and 5000 ug/plate with and without metabolic activation.

Method

Target gene:

These mutant strains of Salmonella are incapable of syntheslsing histidine and are, therefore, dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is either added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. A mutant strain of Escherichia coli (WP2uvrA), which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence was used to complement the Salmonella strains. This strain also has a deletion in an excision repair gene (uvrA).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver enzyme mix derived from metabolically activated rats

Test concentrations with justification for top dose:

The top dose was selected as it was the highest concentration that was soluble in the vehicle and non-toxic in a preliminary toxicity study.
Concentrations:
Preliminary range finder 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.
final study 50, 150, 500, 1500 and 5000 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance dissolved in it up to the concentration desired by the procedure.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);
- Cell density at seeding (if applicable):

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

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

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

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

- OTHER:

Rationale for test conditions:

This study was conducted according to Safepharm Standard Method 700.04 and was designed to assess the mutagenic potential of the test material using a
bacterial/microsome test system.

These mutant strains of Salmonella are incapable of syntheslsing histidine and are, therefore, dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is either added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. A mutant strain of Escherichia coli (WP2uvrA), which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence was used to complement the Salmonella strains. This strain also has a deletion in an excision repair gene (uvrA).

Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material
and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a compound known to induce an elevated level of these enzymes

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression^ )) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.

Statistics:

None

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No effects seen up to top dose.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no effects seen up to top dose

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no effects seen up to top dose

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no effects seen up to top dose

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No effects seen up to top dose

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH was measured and found to be acceptable according to guidelines
- Precipitation: Non-confoudning precipitation did occur at 1500 ug/plate concentrations and up

RANGE-FINDING/SCREENING STUDIES:
A range-finding toxcitiy study found no toxicity up to the maximum dose tested.

The concurrent negative controls were considered to be acceptable. These data are for concurrent untreated control plates performed on teh same day as the Mutation study.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

This study validly found the test substance to be non-mutagenic according to the specified guidelines.