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Toxicological information

Genetic toxicity: in vitro

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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:
22 Sep 2014 to 25 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guidelines study under GLP, positive and vehicle controls do not fall clearly within historical control ranges in part of the experiments

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): EnvaMul™200
- Substance type: UVCB

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Source: Genetic Toxicology Laboratory at Pfizer, Inc., Groton, CT, USA
- Type and identity of media: McCoy’s complete media
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, 12-14 hour cycling time, average 21 chromosomes

Each culture was prepared by seeding with 1.4 × 10E6 CHO cells per 75 cm2 flask in McCoy’s complete media and incubated at 37 ± 1°C in vented flasks in a humidified atmosphere of 5 ± 1% CO2 for approximately 24 hours prior to the initiation of treatment.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor™ 1254-induced rat liver S9 fraction
Test concentrations with justification for top dose:
Concentrations are based on the results of 3 pre-tests

Exp 1 (3 h without metabolic activation) : stock solution of 150 mg/mL further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL
Exp 2 (20 h without metabolic activation) : stock solution of 50 mg/mL further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL
Exp 3 (3 h with metabolic activation) : stock solution of 50 mg/L further diluted with DMSO/ethanol(1/1) to:
6.25, 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 225, 300, and 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO/Ethanol (1/1)
- Justification for choice of solvent/vehicle: pre-test at 500 mg/L
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO/Ethanol (1/1)
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
tests without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO/Ethanol (1/1)
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
test with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: exp 1 3 hours without metabolic activation, exp 2 20 hours without metabolic activaton, exp3 3 hours with metabolic activation
- Expression time: until 20 hours

SPINDLE INHIBITOR: Colcemid (0.1 µg/mL) 2 hours before harvesting
FIXATIVE: 3:1 methanol:glacial acetic acid

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases per replicate (+ 15 aberrant cells per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (with Coulter counter) + mitotic index (1000 cells)+ visible signs of cytotoxicity

OTHER EXAMINATIONS:
- Determination of polyploidy: 200 cells/replicate
- Determination of endoreplication: 200 cells/replicate

Evaluation criteria:
POSITIVE RESPONSE:
The test item would be considered positive for inducing chromosomal aberrations if a significant increase (p ≤0.01) in the mean percent of cells with chromosomal aberrations is observed at one or more dose levels, or if a significant increase (p ≤0.05) in the mean percent of cells with chromosomal aberrations is observed at two or more dose levels. If a significant increase is seen at one or more dose levels, a dose-response should be observed.
NEGATIVE RESPONSE
The test item would be considered negative for inducing chromosomal aberrations 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.
Statistics:
One-tailed Fisher’s Exact test on cell numbers with aberrations
A response would be considered statistically significant for dose-response trend in the Cochran-Armitage test if p ≤0.05.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
no dose response
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 150 ug/mL and above
Vehicle controls validity:
other: within historical control ranges
Positive controls validity:
other: MMC (0.75 ug/mL) within historica control values
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 225 ug/mL and above; precipitate at 125 ug/mL and above
Vehicle controls validity:
other: outside historical control ranges
Positive controls validity:
other: MMC (0.10 ug/mL) within historical control ranges
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
dose response
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 75 ug/mL and above
Vehicle controls validity:
other: at the upper range of historical control values
Positive controls validity:
other: CP (0.75 ug/mL) at the lower range of historical control values
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Precipitation: see above

RANGE-FINDING/SCREENING STUDIES: performed, but only cytotoxicity part reported

COMPARISON WITH HISTORICAL CONTROL DATA: see above
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Exp 1Cell Count Reduction and Aberration Summary: 3-Hour Incubation without Metabolic Activation

Treatment

 

% Mitotic

Reduction

% Cytotoxicity

RPD

% Cells

w/Abs

% Cells

w/>1 Abs

% Endo

Cells

% Polyploid

Cells

Vehicle (1%)

0.00

0.00

0.0

0.0

0.0

4.8

MMC 0.75 µg/mL

35.39

10.69

66.7*

46.7*

0.0

4.5

37.5 µg/mL

0.00

0.33

2.5**

0.0

0.0

2.8

75.0 µg/mL

0.00

0.67

2.5**

0.0

0.3

3.3

150 µg/mLa

 

0.00

7.17

2.5**

0.0

15.0*

3.3

 

Exp 2 Cell Count Reduction and Aberration Summary: 20-Hour Incubation without Metabolic Activation

Treatment

 

% Cytotoxicity

RPD

% Cells

w/Abs

% Cells

w/>1 Abs

% Endo

Cells

% Polyploid

Cells

Vehicle (1%)

0.00

4.0

0.0

0.0

5.0

MMC 0.10 µg/mL

15.21

50.8*

27.1*

0.0

4.8

75.0 µg/mL

13.14

1.0

0.0

0.0

5.3

100 µg/mL

23.02

2.0

0.0

0.0

6.5

125µg/mLa

 

33.15

3.5

0.0

0.0

3.3

 

Exp 3 Cell Count Reduction and Aberration Summary: 3-Hour Incubation with Metabolic Activation

Treatment

 

% Cytotoxicity

RPD

% Cells

w/Abs

% Cells

w/>1 Abs

% Endo

Cells

% Polyploid

Cells

Vehicle (1%)

0.00

2.0

0.0

8.5

4.8

CP 7.5 µg/mL

19.40

15.5*

3.5*

5.3

4.3

12.5 µg/mL

6.71

1.5

0.0

9.8

4.5

25.0 µg/mL

30.33

6.0**

1.0

9.8

5.0

75.0

µg/mLa

 

54.04

12.5*

3.0**

9.0

3.0

Endo -Endoreduplicated cells

Abs -Aberrations

MMC -Mitomycin C

CP -CycloPhosphamide

RPD- Relative Population Doubling

Vehicle- 50% Dimethylsulfoxide 50% Ethanol

Percent Aberrant cells: *p=0.01, **p=0.05 using Fisher's Exact Test

aPrecipitates observed at the end of treatment

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous

The substance was considered positive for inducing structural aberrations in CHO-WBL cells with metabolic activation and equivocal for inducing structural aberrations in CHO-WBL cells in the short-treatment without metabolic activation under the conditions of this test system. A significant test item-related increase in numerical aberrations (endoreduplication) was observed in the short-treatment without metabolic activation
Executive summary:

In a chromosome aberration test in CHO-WBL cells, structural (with metabolic activation) and numerical (without metabolic activation) aberrations were reported after 3-hour treament at concentrations upto cytotoxicity. Prolonged testing without metabolic activation did not induce any effects. Therefore it cannot be excluded that the substance exhibits clastogenic effects.