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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26-Sept-2014

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}oxan-2-yl]methyl acetate

EC Number:

930-010-9

Cas Number:

461432-25-7

Molecular formula:

C29H33ClO10

IUPAC Name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}oxan-2-yl]methyl acetate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White crystalline solid
Storage conditions: At room temperature protected from light

Method

Species / strain

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

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.