tert-butyl {(1S)-1-[(2R)-oxiran-2-yl]-2-phenylethyl}carbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation study

A bacterial gene mutation study was carried out for BMS 217947-01. The bacterial strains were exposed to concentrations of 50 to 5000 μg substance per plate, both with and without metabolic activation.

A dose-related, reproducible and statistically significant increase in revertant colony frequency was observed in tester strains TA 100 and TA 1535 both with and without metabolic reaction. The first indication of a mutagenic response was observed at 150µg/plate in tester strain TA 1535 without metabolic reaction. No mutagenic response was observed in Salmonella typhimurium strains TA98 and TA1537 at the any of the doses used.

Mouse lymphoma assay

A mouse lymphoma assay test in L5178Y cells was carried out for BMS 217947-01, according to Directive 88/302/EEC, Part B, OECD Guideline No.476 and US EPA (1998) Health Effects Test Guidelines OPPTS 870.5300. In-vitro mammalian cell gene mutation tets EPA 712-C-98-221. Three tests were carried out with different exposure concentrations and exposure periods (3h and 24h), both with and without metabolic activation.

Statistically significant increases in mutant frequency outside the historical control range with a Day 0 survival of more than 10%, were observed in Test 1 at 135µg/ml and in Test 3 at 25µg/ml in the absence of S-9 mix. As these increases were outside the historical control range with a Day 0 survival of more than 10% they were considered to be of biological significance.

Statistically significant increases in mutant frequency were observed in all of the tests at 200µg/ml in the presence of S-9 mix. These increases were outside the historical control range with a Day 0 survival of more than 10%. Therefore, they were considered to be of biological significance.

In vivo micronucleus

An in vivo micronucleus assay was carried out on BMS 217947-01 according to EEC Directive 92/69/EEC, Method B12 and OECD Guidelines for testing of Chemicals No. 474 "Micronucleus Test". The test was carried out on albino, CD-1 (ICR)BR mice. The material was administered orally to groups of 7 rats at doses of 250, 500 and 1000 mg/kg, dispersed in arachis oil.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups.

In vivo Comet Assay

This study was conducted to determine the potential genotoxicity of BMS-217947-01, when given to rats for a 2-day period by evaluating DNA damage in the duodenum and liver using the alkaline comet assay. BMS-217947-01 was administered by oral gavage at doses of 0(vehicle), 100, 500, or 1000mg/kg/day to groups of 6 male rats at dose volumes of 10, 1, 5, and 10mL/kg, respectively. The vehicle/carrier consisted of peanut oil. The positive control rats were dosed with ethyl methanesulfonate (EMS) at a dose of 200 mg/kg via intraperitoneal injection3 to 4 hours prior to necropsy. BMS-217947-01 induced a statistically significant dose-related increase in duodenum mean %TI at 1000mg/kg/day, and statistically significant dose-related increases in mean and median liver %TI at 500 and 1000mg/kg/day. Anatomically, there were dose-related randomly distributed minimal to mildly increased hepatocytic mitotic figures at ≥ 500 mg/kg/day, which suggests increased DNA repair. In conclusion, BMS-217947-01was genotoxic in liver at 500mg/kg/day and in duodenum at 500 and 1000 mg/kg/day.

Conclusion

Under a weight of evidence assessemnt of the in-vitro and in-vivo studies, the material was considered mutagenic and genotoxic in bacterial and mammalian cell systems in vitro in the absence and presence of hepatic S9 fractions.

Link to relevant study records

Reference

Reference 1

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:

29 October 1998 - 13 November 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: done under OECD method and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was conducted according to Safepharm Standard Method 700.04. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

Liver homogenate derived from Aroclor 1254 induced rats (S-9 mix),

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethyl sulphoxide

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

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

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

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

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535 pSK1002

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
A dose-related, reproducible and statistically significant increase in revertant colony frequency was observed in tester strains TA 100 and TA 1535 both with and without metabolic reaction. The first indication of a mutagenic response was observed at 150µg/plate in tester strain TA 1535 without metabolic reaction. No mutagenic response was observed in Salmonella typhimurium strains TA98 and TA1537 at the any of the doses used.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

The test material was considered to be mutagenic under the conditions of the test.

Executive summary:

A bacterial gene mutation study was carried out for BMS 217947-01. The bacterial strains were exposed to concentrations of 50 to 5000 μg substance per plate, both with and without metabolic activation.

A dose-related, reproducible and statistically significant increase in revertant colony frequency was observed in tester strains TA 100 and TA 1535 both with and without metabolic reaction. The first indication of a mutagenic response was observed at 150µg/plate in tester strain TA 1535 without metabolic reaction. No mutagenic response was observed in Salmonella typhimurium strains TA98 and TA1537 at the any of the doses used.

Therefore, under the conditions of the test, the material was considered to be mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 1999 - 4th Oct 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: done under OECD method and GLP

Qualifier:

according to guideline

Guideline:

other: 1.Directive 88/302/EEC, Part B;OECD Guideline No.476 2.US EPA (1998) Health Effects Test Guidelines OPPTS 870.5300. In-vitro mammalian cell gene mutation tets EPA 712-C-98-221

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mammalian cell line, other: Mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50 ... 250 µg/ml
Concentration range in the main test (with metabolic activation): 30 ... 200 µg/ml
Concentration range in the main test (with metabolic activation): 30 ... 250 µg/ml
Concentration range in the main test (without metabolic activation): 25 ... 135 µg/ml
Concentration range in the main test (without metabolic activation): 1 ... 25 µg/ml
Concentration range in the main test (without metabolic activation): 10 ... 30 µg/ml

Vehicle / solvent:

Dimethylsulphoxide (DMSO)

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 3 hours

Expression time:
48 hours

Selection time:
10-14 days for mutant frequency. Selective agent TFT

Fixation time:
Exposure period without metabolic activation:

First test:3 hours

Second test: 24 hours

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 125 µg/ml)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 15 µg/ml)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 150 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 10 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
Statistically significant increases in mutant frequency
outside the historical control range with a Day 0 survival
of more than 10%, were observed in Test 1 at 135µg/ml and in
Test 3 at 25µg/ml in the absence of S-9 mix. As these
increases were outside the historical control range with a
Day 0 survival of more than 10% they were considered to be
of biological significance.

Statistically significant increases in mutant frequency were
observed in all of the tests at 200µg/ml in the presence of
S-9 mix. These increases were outside the historical control
range with a Day 0 survival of more than 10%. Therefore,
they were considered to be of biological significance.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

it was concluded that BMS 247947-01 did demonstrate mutagenic potential in this in vitro gene mutation assay

Executive summary:

A mouse lymphoma assay test in L5178Y cells was carried out for BMS 217947-01, according to Directive 88/302/EEC, Part B, OECD Guideline No.476 and US EPA (1998) Health Effects Test Guidelines OPPTS 870.5300. In-vitro mammalian cell gene mutation tets EPA 712-C-98-221. Three tests were carried out with different exposure concentrations and exposure periods (3h and 24h), both with and without metabolic activation.

Statistically significant increases in mutant frequency outside the historical control range with a Day 0 survival of more than 10%, were observed in Test 1 at 135µg/ml and in Test 3 at 25µg/ml in the absence of S-9 mix. As these increases were outside the historical control range with a Day 0 survival of more than 10% they were considered to be of biological significance.

Statistically significant increases in mutant frequency were observed in all of the tests at 200µg/ml in the presence of S-9 mix. These increases were outside the historical control range with a Day 0 survival of more than 10%. Therefore, they were considered to be of biological significance.

Therefore, under the conditions of the test, the material was considered to be mutagenic.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In Vivo Micronucleus Assay

An in vivo micronucleus assay was carried out on BMS 217947-01 according to EEC Directive 92/69/EEC, Method B12 and OECD Guidelines for testing of Chemicals No. 474 "Micronucleus Test". The test was carried out on albino, CD-1 (ICR)BR mice. The material was administered orally to groups of 7 rats at doses of 250, 500 and 1000 mg/kg, dispersed in arachis oil.

In the range finding toxicity study, the toxicity observed at 2000 mg/kg was considered to be excessive, therefore, the maximum tolerated dose (MTD) of the test material, 1000 mg/kg, was selected for use in the main study, with 500 and 250 mg/kg as the lower dose levels. In animals dosed with the test material via the oral route one premature death occurred at 2000 mg/kg, and clinical signs were observed at and above 1000 mg/kg as follows: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration, tonic convulsions, increased salivation, splayed gait, pilo-erection, tiptoe gait, emaciation, ptosis and distended abdomen. These clinical signs indicate that systemic absorption had taken place.

Observations:

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the presence of clinical signs indicated that systemic absorption had occurred.

The test material, BMS 217947-01, tested negative in the test and was thus considered to be non-genotoxic under the conditions of the test.

A InVivo Mammalian Alkaline Comet Assay was conducted in accordance with OECD (2016), Test No. 489

A Comet assay was conducted determine the potential genotoxicity of BMS-217947-01, when given to rats for a 2-day period by evaluating DNA damage in the duodenum and liver using the alkaline comet assay. BMS-217947-01 was administered by oral gavage at doses of 0 (vehicle), 100, 500, or 1000mg/kg/day to groups of 6 male rats at dose volumes of 10, 1, 5, and 10mL/kg, respectively. The vehicle/carrier consisted of peanut oil. The positive control rats were dosed with ethyl methanesulfonate(EMS) at a dose of 200 mg/kg via intraperitoneal injection3 to 4 hours prior to necropsy.

BMS-217947-01 was well tolerated with no recordable clinical observations or mortality throughout the dosing period.

BMS-217947-01 induced a statistically significant dose-related increase in duodenum mean %TI at 1000mg/kg/day, and statistically significant dose-related increases in mean and median liver %TI at 500 and 1000mg/kg/day.

In conclusion, BMS-217947-01was genotoxic in liver at 500mg/kg/day and in duodenum at 500 and 1000 mg/kg/day

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Nov 1998 - 11 March 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: done under OECD method and GLP

Qualifier:

according to guideline

Guideline:

other: EEC Directive 92/69/EEC, Method B12, OECD Guidelines for testing of Chemicals No. 474 "Micronucleus Test".

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Remarks:

albino, (ICR)BR

Route of administration:

oral: unspecified

Vehicle:

Arachis oil

No. of animals per sex per dose:

Male: 1000 mg/kg; No. of animals: 7; Sacrifice time: 48 hours
Male: 1000 mg/kg; No. of animals: 7; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 7; Sacrifice time: 24 hours
Male: 250 mg/kg; No. of animals: 7; Sacrifice time: 24 hours

Additional information on results:

Doses producing toxicity:
In the range finding toxicity study, the toxicity observed at 2000 mg/kg was considered to be excessive, therefore, the maximum tolerated dose (MTD) of the test material, 1000 mg/kg, was selected for use in the main study, with 500 and 250 mg/kg as the lower dose levels. In animals dosed with the test material via the oral route one premature death occurred at 2000 mg/kg, and clinical signs were observed at and above 1000 mg/kg as follows: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration, tonic convulsions, increased salivation, splayed gait, pilo-erection, tiptoe gait, emaciation, ptosis and distended abdomen. These clinical signs indicate that systemic absorption had taken place.

Observations:
There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the presence of clinical signs indicated that systemic absorption had occurred.

Conclusions:

Interpretation of results (migrated information): negative
The test material, BMS 217947-01, was considered to be non-genotoxic under the conditions of the test.

Executive summary:

An in vivo micronucleus assay was carried out on BMS 217947-01 according to EEC Directive 92/69/EEC, Method B12 and OECD Guidelines for testing of Chemicals No. 474 "Micronucleus Test". The test was carried out on albino, CD-1 (ICR)BR mice. The material was administered orally to groups of 7 rats at doses of 250, 500 and 1000 mg/kg, dispersed in arachis oil.

In the range finding toxicity study, the toxicity observed at 2000 mg/kg was considered to be excessive, therefore, the maximum tolerated dose (MTD) of the test material, 1000 mg/kg, was selected for use in the main study, with 500 and 250 mg/kg as the lower dose levels. In animals dosed with the test material via the oral route one premature death occurred at 2000 mg/kg, and clinical signs were observed at and above 1000 mg/kg as follows: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration, tonic convulsions, increased salivation, splayed gait, pilo-erection, tiptoe gait, emaciation, ptosis and distended abdomen. These clinical signs indicate that systemic absorption had taken place.

Observations:

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the presence of clinical signs indicated that systemic absorption had occurred.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

Two in vitro mutagenicity tests (bacterial mutation and mouse lymphoma assay) returned positive results on potential for mutagenicity of BMS 217947-01. One in vivo test on mice (micronucleus assay), showed negative results and a 2 day oral COMET assay in rats resulted in a positive result. Based on a weight of evidence approach the material has the potential to be genotoxic.