tert-butyl {(1S)-2-[(1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hex-2-yl]-1-[3-hydroxyadamantan-1-yl]-2-oxoethyl}carbamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2002

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch number ZD48725 LOT002

Method

Target gene:

four histidine-requiring strains and one tryptophan-requiring strain

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Obtained from the UK NCTC

Species / strain 2

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Obtained from the UK NCTC

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

An initial range finding experiment was carried out in strain TA100 only, using final concentrations of BMS 528235-01 at 1.6, 8, 40, 200, 1000 and 5000 ug/plate, plus negative (solvent) adn positive contols. Experiment 1 treatments of the remained tester strains retained teh same dose series. Experiment 2 treatments of all the tester strains retained 5000 ug/plate as the max test dose. A narrowed dsoe range was employed for treatment of all strains (78.125 to 5000 ug/plate), in order to more closely examine the highest concentration considered most likely to provide evidence of any mutagenic activity.

Vehicle / solvent:

Sterile anhydrous analytical grade dimethyl solphoxide (DMSO)

Controls

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

in DMSO

Positive controls:

yes

Remarks:

Treatments with the appropriate stock positive control solution

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

A preliminary range finding experiment was conducted with strain TA100 at 5 concentrations from 1.6 to 5000 ug/plate plus negative (solvent) controls. No evidence of toxicity was observed following this treatment. These data were considered acceptable for mutation assessment and were used to comprise the TA100 mutagenticity data for Experiment 1. Experiment 1 treatments fo the remaining tester strains retained the same dose series as used in teh Range-finder experiment. No evidence of toxicty was observed following any Experiment 1 treatments.
Experiment 2 treatments of all teh teser strains retained 5000 ug/plate as the max test dose. A narrowed dsoe range was employed for treatment of all strains (78.125 to 5000 ug/plate), in order to more closely examine the highest concentration considered most likely to provide evidence of any mutagenic activity. In addition all treatments in teh presence of S-9 were further modified by teh inclusion of a pre-incubation step. In this way it was hoped to increase the range of mutagenic chemicals that cold be detected using this assy system. Evidence of toxicisty was observed following the top dose treatments of some of the strains in teh presnced ofn S-9 only. Preciipitation of the test article was observed followign teh top dose treatment of S. tryphimurium strains in teh presenced of S-9 only.
Negative (solvent) and poistive control treatments were included fora ll strains in both experiments. Teh mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly evelvated by postive control treatments

Evaluation criteria:

The assay was considered valid if the following crieteria were met:
1. The mean negative control counts fell within the normal ranges
2. The positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation.
3. No more than 5% of the plates were lost through contamination or some other unforseen event.

The test article was considered to be mutagenic if:
1. The assay was valid (see above)
2. Dunnett's test gave a significant response (p< 0.01) and the data set(s) showed a significant dose correlation
3. The positive responses described above were reproducible.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

u ot 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

No statistically significant, dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absenced or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any BMS 528235-01 mutagenic activity.
It was concluded that BMS 528235-01 did not induce mutation in four histidine-requiring strains of S. typhimurium (TA98, TA1000, TA1535 adn tA1537), adn one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 ug/plate in teh absence and in the prsence of a rat liver metabolic activation sysatem (S-9)