D-Glucitol, 1,5-anhydro-1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-, 2,3,4,6-tetraacetate, (1S)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An OECD 471 Ames bacterial study was conducted and it was concluded that BMS 587172-01 did not induce mutation in four histidine-requiring strains of Salmonella typhimurium and one tryptophan-requiring strain of e-coli when tested under conditions of this study. These conditions included treatment concentrations up to 5000 ug/plate in the absence and in the presence of a rat liver metabolic activation system (S-9)

A chromosome aberration study was also carried out with BMS-587172-01 according to OECD 473 guideline in cultured Chinese Hamster Ovary (CHO-WBL) cells in two independent experiments. It is concluded that BMS-587172-01 is not clastogenic in CHO-WBL cells.

In a OECD 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene study that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line the test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test or in any of the three exposure groups (4 hours with S9, 4 Hours with S9 (2%) and 24 hr without S9.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 April 2016 - 23 October 2016

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:

26-Sept-2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) guideline S2(R1)

Version / remarks:

09-Nov-2011

Deviations:

no

GLP compliance:

yes

Remarks:

This study is conducted in compliance with U.S. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) regulations; CFR Title 21, Part 58 with the following exception. Concentration analysis of the dose formulations will not be conducted.

Type of assay:

other: In vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Genetic Toxicology Laboratory at Pfizer,Inc.
- Number of passages if applicable: Cells were used at passage 22 for the range-finding assay, passage 7 for the aberration assay, passage 19 and 22 for the repeat aberration assay, passage 3 for the 2nd repeat aberration assay, passage 19, 22, and 5 for the 3rd repeat aberration assay
- Modal number of chromosomes: 21
- Normal (negative control) cell cycle time: 12 to 14 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy’s medium supplemented with a final concentration of 10% (v/v) heat-inactivated fetal bovine serum, 2mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin. All cultures were incubated in a humidified atmosphere of 4% to 6% CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254.

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 3hr exposure; 20 hr fixation: 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, and 500 μg/mL.
Without S9-mix, 20 hour continuous exposure: 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, and 500 μg/mL.

The highest concentration chosen for evaluation of chromosome aberrations were from the lowest concentration tested at which precipitates were observed or a concentration at which 50% - 60% cytotoxicity was observed.

Repeat aberration assay:
Without S9-mix, 3 h exposure time, 20 h fixation time: 34.4, 57.4 and 87.5 μg/mL. (87.5 µg/ mL: lowest precipitating dose)
With S9-mix, 3 h exposure, 20 h fixation time: 70.9, 78.7 and 87.5 µg/ mL. (87.5 µg/ mL: lowest precipitating dose)
Without S9-mix, 20 h continuous exposure: 2.41, 9.62 and 34.4 μg/mL. (34.4 µg/ mL: 54% cytotoxicity)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9, in DMSO: 0.75 µg/mL for a 3 h exposure period and 0.10 µg/mL for a 24 h exposure period

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 in DMSO: 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
- Trail 1: 1.0-1.5 × 10^6 CHO cells per 75 cm^2 flask
- Trial 2, 3 and 4: 0.9 – 1.2 × 10^6 CHO cells per 75 cm^2 flask

DURATION
- Exposure duration: for short incubations 3 hours and for long incubations 20 hours for Trial 1 or 24 hours for Trial 2, 3 and 4
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours for Trail 1 or 24 hours for Trial 2,3 and 4

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (approximately 2 hours prior to harvest)
STAIN (for cytogenetic assays): DiffQuik solution

NUMBER OF REPLICATIONS: duplicates in two independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were trypsinized, centrifuged, resuspended in hypotonic solution (0.075 M KCL) and fixed twice using an approximately 3:1 methanol:glacial acetic acid mix. The final concentrated cell suspension was dropped on clean, cold wet glass slides, dried prior to staining with DiffQuik solution at room temperature, and coverslipped.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE:
- 300 metaphase cells or ≥ 50 aberrant cells except for the 3-hour treatment with metabolic activation, for which 600 metaphase cells were scored

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling using cell counts

OTHER EXAMINATIONS:
- Determination of polyploidy: yes (from 400 cells)
- Determination of endoreplication: yes

Rationale for test conditions:

Concentrations tested were based on the range-finding assay.

Evaluation criteria:

Acceptance criteria assay and controls
Each treatment condition (e.g., in the absence or in the presence of metabolic activation, short or long incubations) is considered independent with regard to assay acceptance. The positive control results for each treatment condition must be statistically significant (p ≤ 0.05) when compared with the relevant vehicle controls. The vehicle and positive control results should be comparable to relevant historical control data generated at Charles River Skokie. Additionally, a minimum population doubling of approximately 1.5 should be observed in the vehicle control cultures.

Criteria positive response
A test substance was considered positive (clastogenic) in the chromosome aberration test if a significant increase (p ≤ 0.05) in the mean percent of cells with chromosomal aberrations is observed at 1 or more dose levels. If a significant increase is seen at 1 or more dose levels, a dose-response should be observed. A response would be considered statistically significant for dose-response trend in the Cochran-Armitage test if p ≤ 0.05. At least 1 concentration should be associated with an increase that is outside the historical control range of previous vehicle control treated cultures.

Criteria negative response
A test substance was considered negative (not clastogenic) in the chromosome aberration test if no statistically significant increase (p ≤ 0.05) is observed in the mean percent of cells with chromosomal aberrations at any of the test concentrations and there is no concentration-related increase when evaluated in the Cochran-Armitage test.

Statistics:

After completion of microscopic analyses, data were decoded and a One-tailed Fisher’s Exact test was performed on the total number of cells with structural aberrations and the total number of cells with more than one chromosome aberration comparing the treated cells to the results obtained from the relevant control group. The detection of dose-response trends was carried out using the Cochran-Armitage test. The same statistical analysis was performed for cells exhibiting polyploidy and/or endoreduplication.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3-Hour Treatment without Metabolic Activation 13% (lowest precipitating dose); 24-Hour Treatment without Metabolic Activation 54%; 3-Hour Treatment with Metabolic Activation 18% (lowest precipitating dose).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at the end of test article treatment at ≥ 87.5 μg/mL in the 3-hour treatments with and without metabolic activation, and at ≥ 57.4 μg/mL in the 24-hour treatment without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
- Cytotoxicity was observed at precipitating concentrations; at ≥ 250 μg/mL in the 3-hour treatments with and without metabolic activation and at ≥ 15.6 μg/mL in the 20-hour treatment without metabolic activation. Changes in cell morphology were noted at ≥ 125 μg/mL in the 3-hour treatment with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
Data from the vehicle and positive controls were comparable to expected historical control ranges and yielded expected results with the following exceptions. For the vehicle control for the 3-hour treatment without metabolic activation the percent of cells with aberrations was higher than the upper limit of the historical control range (5.3% versus a range of 0%-4%). This results was considere acceptable since there was only a limited historical control database, the response in all test article treated cultures were within the historical control range and the positive control treatment produced a statistically significant response. The positive control for the 24-hour treatment without metabolic activation induced a higher frequency of chromosomal aberrations that was not within the upper limit of the historical control range (71.4% versus a range of 4% to 65.2%). This response is considered acceptable as there was a longer exposure 24-hours in stead of 20-hours) and the scientific purpose of the positive control treatment was valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RPD
- Changes in cell morphology were noted at ≥ 108 μg/mL in the 3-hour treatment with metabolic activation at harvest.
- In the 3-hour treatment with metabolic activation, three additional trials were conducted to determine reproducibility of the minor elevation in chromosomal aberrations at 87.5 μg/mL. Since optimal cytotoxicity was not achieved in these treatments, additional cells from the repeat aberration assay were scored, which provided enough data to make a definitive conclusion of the clastogenic potential of the substance.

Summary of structural aberrations

Exposure	Concentration (μg/mL)	Structural Aberrations (%)	Polyploid cells (%)	Endoreplicated cells (%)
3 hours -S9	0	5.3	8.5	0.0
	34.4	3.7	8.3	0.0
	57.4	1.3	6.0	0.0
	87.5^a	2.3	7.0	0.0
	MMC 0.75	68.5**	6.0	0.0
3 hours +S9	0	3.0	6.5	0.5
	70.9	3.7	6.8	0.0
	78.7	2.7	5.8	0.5
	87.5^a	4.8	9.0	0.0
	CP 7.5	79.4**	4.5	0.3
24 hours -S9	0	3.0	4.8	0.0
	2.41	3.0	4.5	0.0
	9.62	4.7	5.0	0.0
	34.4	3.3	4.8	0.0
	MMC 0.10	71.4**	5.3	0.0

** p ≤ 0.01 using one-tailed Fisher’s Exact Test.

^a Precipitates present at end of test article treatment

Conclusions:

A chromosome aberration study with BMS-587172-01 was performed according to OECD 473 guideline and GLP principles, in cultured Chinese Hamster Ovary (CHO-WBL) cells in two independent experiments. It is concluded that BMS-587172-01 is not clastogenic in CHO-WBL cells.

Executive summary:

In a chromosome aberration study, cultured Chinese Hamster Ovary (CHO-WBL) cells were exposed to different concentrations of BMS-587172-01 (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473/ICH S2(Rl) guidelines and GLP principles. In the range-finding assay, BMS-587172-01 was tested up to and including 500 μg/mL for a 3 hour exposure time in the presence or absence of metabolic activation with a 20 hour fixation time and for a 20 hour continuous exposure time without metabolic activation. In the repeat aberration assay, BMS-587172-01 was tested up to and including precipitating concentrations for a 3 hour exposure time in the presence or absence of metabolic activation with a 24 hour fixation time and for a 24 hour continuous exposure time without metabolic activation. BMS-587172-01 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of metabolic activation, in either of the two independently repeated experiments. No effects of BMS-587172-01 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of metabolic activation. Adequate positive and vehicle controls were included. Therefore it can be concluded that BMS-587172-01 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy when evaluated under conditions and at the maximum concentrations recommended by international guidelines for in vitro cytogenetic studies.

Reference 2

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:

March 23 to April 28th, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

four histidine-requiring strains and one tryptophan-requiring strain

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Range finding experiment and main experiment carried out at concentrations from 0 to 5000 ug/plate with 10 concentrations tested. For the main study doses of 50, 150, 500, 1500 and 5000 ug/plate.

Vehicle / solvent:

Test solutions were prepared by dissolving BMS 587172-01 in sterile anhydrous grade dimethyl sulphoxide (DMSO).

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

use strain TA98 without S-9)

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6:

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

use strain TA 1537 without S-9

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

use strain TA98 with S-9

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

use strain WP2uvrA without S-9

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

use strain TA100,TA1535,TA1537, WP2uvrA with S-9

Details on test system and experimental conditions:

A preliminary range finding experiment was conducted with strain TA100 and WP2uvrA at 10 concentrations from 15 to 5000 ug/plate plus negative (solvent) controls. Manual counts were performed above 1500 ug/plate due to test material film. No evidence of toxicity was observed. This test was acceptable.Experiment 1 and 2 involved the five strains with and without metabolic activation using the same concentrations as preliminary. Normal plating treatment procedures followed. No evidence of toxicity was observed following this treatment. Negative and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates were all acceptable. There was a greasy, patchy film precipitate observed at and above 150 ug/plate but it didn't prevent scoring the revertant colonies.

Evaluation criteria:

Acceptance criteria for validity of assay : the mean negative control counts fell within normal ranges, the positive control chemical induced clear increases in revertant numbers confirming discrimination between different strains and an active S-9 preparation, and no more than 5 % of the plates were lost through contamination or some other unforeseen event. Evaluation criteria: test article would be considered mutagenic if: the assay was valid, and an increase in frequency in revertant colonies in excess of 2 or 3 fold depending on the tester starin was noted verses the concurrent solvent controls.

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:

>5000 ug/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:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

(> 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

Conclusions:

It was concluded that BMS 587172-01 did not induce mutation in four histidine-requiring strains of Salmonella typhimurium and one tryptophan-requiring strain of e-coli when tested under conditions of this study. These conditions included treatment concentrations up to 5000 ug/plate in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Mouse Lymphoma Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Septmeber 2017 - 09 October 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro gene mutation study in mammalian cells

Specific details on test material used for the study:

BMS 587172-01 is a white powder with 100% purity and molecular weight of 577.03 g/mol. Sample stored at room temoerature in the dark

Target gene:

thymidine kinase, TK +/-, locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the
MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were
originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and
were frozen in liquid nitrogen at that time

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Test concentrations with justification for top dose:

A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range was set at 7.81 to 2000 µg/mL
in all three exposure groups. The dose levels were selected to avoid excessive precipitate. The cultures were incubated at 37°C with 5% CO2 in air and sub-culured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the
MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were
originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and
were frozen in liquid nitrogen at that time.
Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were
routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM)
supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate
(1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at
approximately 37 o C with 5% CO2 in air. The cells have a generation time of approximately
12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20),
10% donor horse serum (R10), and without serum (R0), are used during the course of the
study. Master stocks of cells were tested and found to be free of mycoplasma.
Lot No. PB/βNF S9 20/08/17 was used in this study, and was pre-prepared in-house (outside
the confines of the study) following standard procedures. Prior to use, each batch of S9 is
tested for its capability to activate known mutagens in the Ames test and a certificate of S9
efficacy is presented in Appendix 2.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction
(20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM),
glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed
at a 10% volume of S9-mix into culture media, was 2%.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but
significant rate. Before the stocks of cells were frozen they were cleansed of homozygous
(TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained
Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine
(22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e.
THMG without Methotrexate) before being returned to R10 medium.

Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and
formulated in DMSO prior to serial dilutions being prepared. The test item had a molecular
weight of 577.03g/mol; therefore the maximum recommended dose level was initially set at
2000µg/mL. There was no marked change in pH when the test item was dosed into media
and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). The pH and
osmolality readings are presented in the following table.

Rationale for test conditions:

A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range was set at 7.81 to 2000 µg/mL
in all three exposure groups. The dose levels were selected to avoid excessive precipitate. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 105 cells/mL.
The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 2000 µg/mL or 10 mM, whichever is the lowest concentration.
ii) The presence of precipitate regardless of where test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical packge used indicates the presence of
statistically significant increases and linear trend events. The Delta building monitoring system was used during the course of the study.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and 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 valid

Positive controls validity:

valid

In the 4-hour –S9 exposure, there was no evidence of marked test item induced toxicity, as indicate by the %RSG and RTG values. There was evidence of marked toxicity following exposure to the test item in the 4-hour +S9 and 24-hour-S9 exposure groups, as indicated by the %RSG and RTG values. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred. Optimum levels of toxicity were achieved in in the 4-hour+S9 and 24-hour –S9 exposures. The dose levels of 62.5 and 125 µg/mL in the 4-hour –S9 exposure were not plated out for 5-TFT resistance and viability due to excessive precipitate, leaving only one precipitating dose level for analysis. The dose levels of 125 µg/mL in the 4-hour +S9 exposure and 93.75 and 125 µg/mL in the 24-hour – S9 exposure were not plated out for 5-TFT resistance and viability due to excessive toxicity and precipitate. Acceptable levels of toxicity were seen with both positive control substances.

The test item did not induce any toxicologically significant increases in the mutant frequency x 10-6 per viable cell in either of the three exposure groups. The GEF value of the test item dose levels were not exceeded in any of the three exposure groups.A precipitate of the test item was observed at and above 31.25 µg/mL in the 4-hour -S9. A precipitate of the test item was observed at and above 93.75 µg/mL in the 4-hour +S9. Also a precipitate of the test item was observed at and above 62.5 µg/mL in the 24-hour –S9 exposure.

Conclusions:

The test item, BMS-587172-01 did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6. consequently it is considered to be non-mutagenic in this assay

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17.

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at up to 8 dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. A precipitate of the test item was observed at and above 31.25 µg/mL in the 4-hour -S9 (absence of metabolic activation). A precipitate of the test item was observed at and above 93.75 µg/mL in the 4-hour +S9 (presence of metabolic activation). Also a precipitate of the test item was observed at and above 62.5 µg/mL in the 24-hour –S9 exposure. The dose levels plated for viability and expression of mutant colonies were as follows:

4-hour without S9 0.98, 1.95, 3.91, 7.81, 15.63, 31.25 µg/mL

4-hour with S9 (2%) 3.91, 7.81, 15.63, 31.25, 62.5, 93.75 µg/mL

24-hour without S9 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 µg/mL

The maximum dose level used was limited by a combination of test item induced toxicity and precipitate. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. . The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)