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

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 233101-01. The bacterial strains were exposed to concentrations of 50, 150, 500, 1500 and 5000 ug/plate substance per plate, both with and without metabolic activation. This study validly found the test substance to be non-mutagenic according to the specified guidelines.

Mouse lymphoma assay

The results of a genotoxicity study, performed according to OECD guideline 490 and GLP principles, showed that BMS-233101-01 was positive in the 3-hour treatment with S9 and negative in the 3-hour and 24-hour treatments without S9 in the In Vitro Mammalian Cell Gene Mutation Test (L5178Y TK+/- Microwell Assay).

In Vitro Chromosomal Aberration study

A study was performed to assess the ability of BMS 233101-01 to induce chromosomal aberrations in human lymphocytes cultured in vitro. It is concluded that BMS 233101-01 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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.

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 / cell type:

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

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.

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

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

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

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

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

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.

Conclusions:

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2017 - 24 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Target gene:

- Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells (heterozygous at the normally diploid TK locus)
- Source of cells: from The European Collection of Authenticated Cell Cultures (ECACC) of Public Health England (Salisbury, UK).
- Suitability of cells: recommended test system in international guidelines
- Modal number of chromosomes: 40
- Number of passages: 6 (dose-rang finding study), 16 (mutagenicity assay), 13, 15 and 9 (first, second and third repeat assay respectively)
- Storage of cells: frozen in liquid nitrogen

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (100 U/mL and 10 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
* Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Exposure medium: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium);
* Cloning medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate lyophilized with NADPH-regenerating system cofactors and phosphate buffer) from male rats that have been induced with Aroclor™ 1254

Test concentrations with justification for top dose:

Dose-range finding study:

3-hour treatment with and without S9: 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg/mL

24-hours treatment without S9: 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg/mL

The highest concentration evaluated approximated the limit dose for this assay (2000 μg/mL or 10 mM).

Mutagenicity assays:

3-hour treatment, without S9: 6.25, 12.5, 25.0, 50.0, 87.5, 125, and 250 μg/mL

3-hour treatment, with S9: 6.25, 12.5, 25.0, 37.5, 50.0,100, 125, and 150 μg/mL

24-hour treatment, without S9: 1.56, 3.13, 6.25, 12.5, 25.0, 37.5, 50.0, 75.0, 100 and 150.0 μg/mL.

The highest concentrations evaluated was that expected to be minimally insoluble at the end of treatment.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for vehicle: the test item was shown to be soluble in DMSO, which is a recommended vehicle according to international guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1 × 10^6 cells/mL

DURATION
- Exposure duration: 3 hours and 24 hours

SELECTION AGENT: trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 for the test item and negative controls, single cultures were evalutated in the positive controls

NUMBER OF CELLS EVALUATED: 1.1 × 10^6

CRITERIA FOR SCORING: Small colony mutants are defined as those covering < 25% of the well diameter and large colony mutants are those that cover > 25% of the well diameter.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (see 'additional information on material and methods')

Evaluation criteria:

EVALUATION CRITERIA:
- A test article is considered to be to be clearly positive for inducing mutations if it induces a dose-dependent increase in mutant frequency (p≤ 0.05), and the increase exceeds the global evaluation factor (GEF) of 126 × 10^-6.
- A test article is considered to be clearly negative for inducing mutations if no dose-dependent increases in mutant frequency are observed, and none of the observed mutant frequencies exceed the global evaluation factor (GEF) of 126 × 10^-6.
- Responses that do not clearly fit into the positive or negative category may be judged equivocal (e.g., a response that meets only 1 of the 2 criteria above). In these cases the Study Director, based on sound scientific judgment, may take additional factors, including further investigations, into consideration in evaluating the test results. In rare cases, the data set may preclude making a definitive assessment and the result may be concluded to be equivocal.

ACCEPTABILITY CRITERIA:
Each treatment condition (i.e., 3-hour ± S9, 24-hour –S9) is considered independent with regard to assay acceptance and can be repeated independently as necessary to satisfy the acceptance criteria. Acceptance criteria are as recommended by the IWGT MLA Workgroup.
- The average absolute cloning efficiency of vehicle controls must be 65 to 120%, and the average mutant frequency must be 50 to 170 × 10^-6. In addition, the suspension growth must be 8 to 32 for the 3-hour treatments ±S9 and 32 to 180 for the 24-hour treatment –S9. These parameters were calculated separately for the various treatment conditions.
- The positive control articles must induce an increase in total MF (an induced MF, or IMF) ≥ 300 × 10^-6, with ≥ 40% small colony MF; and/or an increase in the small colony MF (a small colony IMF) ≥ 150 × 10^-6.
- Four acceptable concentrations are required for a valid assay. Fewer concentrations may be justified for test articles which clearly show mutagenic activity.

Statistics:

The Cochran-Armitage test was used to assess dose dependency.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 50 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING STUDY:
The minimum relative suspension growths observed were 28.3% (3-hour treatment -S9; 125 μg/mL), 38.2% (3-hour treatment +S9; 250 μg/mL), and 37.6% (24-hour treatment –S9; 62.5 μg/mL). In addition, the test article was found to be incompletely soluble at the end of treatment at concentrations ≥ 125 μg/mL ±S9 (3-hour treatments) or ≥ 31.3 μg/mL –S9 (24-hour treatment).

MUTAGENICITY ASSAYS:
Trial 1 (3-hour, with and without S9): the test item was found to be incompletely soluble at concentrations ≥ 100 μg/mL +S9 (3-hour treatment) and ≥ 87.5 μg/mL -S9 (3-hour treatment). However, the RSG for all treatments was below acceptable limits (data will not be reported) and the assay was terminated prior to scoring.
Trial 2 (3-hour without S9): the test item was found to be incompletely soluble at concentrations ≥ 87.5 μg/mL. Those cultures treated at concentrations of 250 μg/mL were discarded due to excessive precipitation. The average RTG at the highest remaining concentrations evaluated was 71.9%.
Trial 2 (24-hour without S9): The average RTG at the highest remaining concentrations evaluated was 60.2%.
Trial 2 (3-hour with S9): Due to a technical error in the 3-hour treatment with S9, this part of the experiment was repeated (trial 3)
Trial 3 (3-hour with S9): the test item was found to be incompletely soluble at concentrations ≥ 100 μg/mL. Those cultures treated at concentrations of 150 μg/mL were discarded due to excessive precipitation. The average RTG at the highest remaining concentrations evaluated was 97.9%.

No dose-dependent increases in average mutant frequencies were observed under any treatment condition, and none of the average mutant frequencies exceeded the global evaluation factor (GEF) of 126 mutants/10^6 clonable cells. Thus, the test item was clearly negative in the 3-hour treatment –S9. However, the average mutant frequency of the vehicle control cultures in the 3-hour treatment +S9 (Trial 3) exceeded acceptable values, and there was no precipitate observed at any concentration evaluated in the 24-hour treatment –S9, and there was insufficient cytotoxicity for a valid assay. Therefore, neither treatment condition met the criteria for an acceptable assay, and both were subsequently repeated.

Trial 4 (3-hour with S9): the test item was found to be incompletely soluble at concentrations ≥ 100 μg/mL. Those cultures treated at concentrations of 150 μg/mL were discarded due to excessive precipitation. Average RTG value at the highest remaining concentration evaluated was 40.5%
Trial 4 (24-hour without S9): the test item was found to be incompletely soluble at concentrations ≥ 37.5 μg/mL. Those cultures treated at concentrations of ≥100 μg/mL were discarded due to excessive precipitation. Average RTG value at the highest remaining concentration evaluated was 87.7%.

No dose-dependent increases in average mutant frequency were observed in the 24-hour treatment –S9, and none of the observed mutant frequencies exceeded the GEF. In contrast, dose-dependent increases in average mutant frequencies were observed in the 3-hour treatment +S9 (p< 0.05), and all of those observed at concentrations ≥ 50.0 μg/mL exceeded the GEF.
In addition, these increases were predominantly due to increases in small colony mutants, indicating a probable clastogenic mechanism of action. Therefore, the test item was clearly positive in the 3-hour treatment +S9, but negative in the 24-hour treatment –S9.

HISTORICAL DATA FOR THE CONTROLS: see attached illustration

ACCEPTABILITY:
Except for the trials that ultimately were repeated, all positive and negative control values were within acceptable ranges and all criteria for a valid assay ultimately were met.

Remarks on result:

other: 3-hour exposure period

Conclusions:

The results of a genotoxicity study, performed according to OECD guideline 490 and GLP principles, showed that BMS-233101-01 was positive in the 3-hour treatment with S9 and negative in the 3-hour and 24-hour treatments without S9 in the In Vitro Mammalian Cell Gene Mutation Test (L5178Y TK+/- Microwell Assay).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 12 1999 to July 19 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

off white solid, 99.15% purity

Species / strain / cell type:

lymphocytes:

Remarks:

Human Lymphocytes in whole blood culture

Metabolic activation:

with and without

Metabolic activation system:

S9 Fraction

Test concentrations with justification for top dose:

On dosing at 1% v/v into aqueous tissue culture medium, giving a final concentration of 2500 µg/ml, a cloudy white precipitate was observed. At a final concentration of 156 µg/ml the medium was very slightly cloudy, and at 78 µg/ml it was clear. Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway el al. 1987). Therefore, concentrations greater than 5000 µg/ml or 10 mM are not used in this test system. In this study, the highest final concentration used for subsequent testing was 320 µg/ml, therefore, providing at least two precipitating dose levels.

Vehicle / solvent:

Prior to commencing testing, the solubility of the test substance was assessed.
BMS 233101-01 was found to form a dosable suspension in dimethyl sulphoxide at 250 mg/ml.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

TREATMENT OF CELLS WITH TEST SUBSTANCE- FIRST TEST
Approximately 48 hours after initiation of the cultures, 50 µl aliquots of BMS 233101-01 were added to one set of duplicate cultures to give final concentrations of 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/ml. Dimethyl sulphoxide, the solvent control, in 50 µl aliquots, was added to two cultures. Mitomycin C, at final concentrations of 0.4 and 0.8 µg/ml, was added to duplicate cultures.

Immediately before treatment of the second set of cultures, 1 ml of medium was removed from each culture and discarded. This was replaced with 1 ml of S9 mix, followed by 50 µl aliquots of the various dilutions of BMS 233101-01, giving the same series of final concentrations as above. Dimethylsulphoxide(50 µI) was added to two cultures. Cyclophosphamide was added to duplicate cultures at final concentrations of25 and 30 µg/ml.

Three hours after dosing, the cultures were centrifuged at 500 g for 10 minutes and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours.

SECOND TEST

Cultures were initiated and maintained as previously described. In this second test a continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a three hour treatment was used, as in the first test. The harvest time was at 21 hours for both parts of the test. Concentrations of BMS 233101-01 were as follows:

Without and with S9 mix: 40, 80 and 160 µg/ml.

Duplicate cultures were used for each treatment and two cultures were treated with the solvent control. Positive control cultures were treated as in the first test.
Three hours after dosing, the cultures containing S9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. Cultures treated in the absence of S9 mix were incubated for 21 hours.

All cultures were treated with Colcemid", at a final concentration of 0.1 µg/ml, two hours before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously described. The slides were then examined microscopically as previously described.

HARVESTING AND FIXATION
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® (Sigma) to each culture at a final concentration of 0.1 µg/ml. After 2 hours incubation, each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with 4 ml of a hypotonic solution (0.075M KCI prewarmed at 37°C). After a l O minute period ofhypotonic incubation at 37°C, 4 ml of ice-coldfresh fixative (3 parts methanol: I part glacial acetic acid) was added and then mixed. The cultures were then centrifuged at 500 g for
5 minutes, the supernatant removed, and the cell pellets resuspended in 4 ml fixative. The fixative was replaced further times until it became colourless.

SLIDE PREPARATION
The pellets were resuspended, then centrifuged at 500 g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides, which were then allowed to air-dry. The slides were then stained in l 0% Giemsa, prepared in buffered water (pH 7.2). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.

MICROSCOPIC EXAMINATION
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in mitotic index of at least 50% of the solvent control value or, if there was no decrease, the maximum achievable concentration was used as the highest dose level for the metaphase analysis. The intermediate and low dose levels were also selected.

The concentration of each positive control compound selected for analysis was the lowest concentration dosed unless a preliminary scan of metaphase figures indicated an insufficient level of aberrant cells.

The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of 10000 using an oil inunersion objective. One hundred metaphase figures were examined, where possible, from each culture. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.

Evaluation criteria:

An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met: Statistically significant increases (P<0.01) in the frequency ofmetaphases with aberrant
chromosomes (excluding gaps) are observed at one or more test concentration.

The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.

The increases are reproducible between replicate cultures.
The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.

Statistics:

The incidence of polyploid metaphase cells, out of 500 metaphase cells, was determined quantitatively for negative control cultures and cultures treated with the highest dose level of the test substance used in the analysis for chromosomal aberrations.

The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).

Key result

Species / strain:

lymphocytes: First test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Metaphase analysis first test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Second Test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Second test metaphase analysis

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

It is concluded that BMS 233101-01 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Executive summary:

A study was performed to assess the ability of BMS 233101-01 to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers.  Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid", the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

In order to assess the toxicity of BMS 233101-0 I to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test
Without S9 mix - 3 hours treatment, 18 hours recovery: With S9	160, 80 and 40 µg/ml.
With S9 mix - 3 hours treatment, 18 hours recovery:	160, 80 and 40 µg/ml.
Second test
Without S9 mix - 21 hours continuous treatment:	160, 80 and 40 µg/ml.
With S9 mix - 3 hours treatment, 18 hours recovery:	160, 80 and 40 µg/ml.

In both the absence and presence of S9 mix, BMS 233101-0 I caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control, in either test.

A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. No statistically significant increases in the proportion of polyploid cells were seen.  All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

 It is concluded that BMS 233101-01 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test article BMS-233101-01 was evaluated as negative under the conditions of a mouse micronucleus study.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Sex:

male

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Male rats were dosed at 2000, 600, and 200 mg/kg/day once per day on three consecutive days via oral gavage at a volume of 10 mL/kg. Forty eight hours after a second dose was adminstrated

Duration of treatment / exposure:

48hrs

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Control animals:

yes, concurrent vehicle

other: Positive Control

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The test article BMS-233101-01 was evaluated as negative under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Three in vitro mutagenicity tests (bacterial mutation and mouse lymphoma assay and Chomosomal abberation) returned one positive results on potential for mutagenicity of BMS 233101-01. One in vivo test in rats (micronucleus assay), showed negative results. Based on a weight of evidence approach the material has the potential to be non- genotoxic