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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 17 to July 2, 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
A standard NTP (National Toxicology Program) protocol.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1986
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
other: in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methyl ethyl ketoxime

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254-induced male Sprague-Dawley rat liver S9)
Test concentrations with justification for top dose:
Without metabolic activation: 1873, 2497, 3727, 5000 µg/mL
With metabolic activation: 2513, 3750, 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
(Dimethyl Sulfoxide)
Positive controls:
yes
Positive control substance:
mitomycin C
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: In the test without S9, cells were incubated in McCoy’s 5A medium with methyl ethyl ketoxime for 18 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the test with S9, cells were treated with methyl ethyl ketoxime and S9 for 2 hours, after which the treatment medium was removed and the cells incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The harvest time for the test was based on the cell cycle information obtained in the SCE test; because some cytotoxicity and cell cycle delay were anticipated in the absence of S9, the incubation period was extended. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. Up to 200 first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).
Evaluation criteria:
Chromosomal aberration data are presented as percentages of cells with aberrations. To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P>=0.05) difference for one dose point and a significant trend (P>=0.003) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test, in the absence of a statistically significant increase at any one dose resulted in an equivocal call. Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.
Statistics:
Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose. Analyses were conducted on both the dose response curve and individual dose points

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(up to 5000 µg/mL)
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Results of Chromosome Aberration test:

Trial #:1   Activation: No Activation   Date: 07/02/1986   Harvest Time: 20.0 hrs   Trial Call: Negative

Dose (µg/mL)

Total Cells Examined

Total Aberrations

Complex Aberrations

Simple Aberrations

Other Abs.

No. of

Abs

% Cells

No. of

Abs

% Cells

No. of

Abs

% Cells

No. of

Abs

% Cells

Abs.

Per

With

Abs.

Per

With

Abs.

Per

With

Abs.

Per

With

Cell

Abs.

Cell

Abs.

Cell

Abs.

Cell

Abs.

Vehicle Control

Negative (Not Specified)

200

2

0.010

1.0

1

0.005

0.5

1

0.005

0.5

0

0.000

0.0

Vehicle Control

Dimethyl Sulfoxide

200

5

0.025

2.5

1

0.005

0.5

4

0.020

2.0

0

0.000

0.0

Test Chemical

 

 

Methyl ethyl ketoxime

 

 

1873

200

2

0.010

1.0

0

0.000

0.0

2

0.010

1.0

0

0.000

0.0

2497

200

5

0.025

2.5

0

0.000

0.0

5

0.025

2.5

0

0.000

0.0

3727

200

1

0.005

0.5

1

0.005

0.5

0

0.000

0.0

0

0.000

0.0

5000

0

0

0.000

0.0

0

0.000

0.0

0

0.000

0.0

0

0.000

0.0

Positive Control

Mitomycin-C

0.05

200

31

0.155

12.5

16

0.080

7.5

15

0.075

6.0

0

0.000

0.0

0.08

25

9

0.360

36.0

4

0.160

16.0

5

0.200

20.0

0

0.000

0.0

Trend

-1.211

0.069

-1.345

 

Probability

0.887

0.472

0.911

 

Trial #:2   Activation: No Activation   Date: 06/17/1986   Harvest Time: 20.0 hrs   Trial Call: Test Failure  

Dose (µg/mL)

Total Cells Examined

Total Aberrations

Complex Aberrations

Simple Aberrations

Other Abs.

No. of

Abs

% Cells

No. of

Abs

% Cells

No. of

Abs

% Cells

No. of

Abs

% Cells

Abs.

Per

With

Abs.

Per

With

Abs.

Per

With

Abs.

Per

With

Cell

Abs.

Cell

Abs.

Cell

Abs.

Cell

Abs.

Vehicle Control

Dimethyl Sulfoxide

 

200

5

0.025

2.0

0

0.000

0.0

5

0.025

2.0

0

0.000

0.0

Test Chemical

Methyl ethyl ketoxime

2500         

200

7

0.035

3.5

0

0.000

0.0

7

0.035

3.5

0

0.000

0.0

Positive Control

Mitomycin-C

 0.08      

25

11

0.440

28.0

4

0.160

12.0

7

0.280

20.0

0

0.000

0.0

Trend

0.000

 

 

0.000

 

0.000

 

 

 

Probability

0.000

0.000

0.000

 

Trial #:1   Activation: Induced Rat Liver S9   Date: 06/17/1986   Harvest Time: 12.0 hrs   Trial Call: Negative  

Dose (µg/mL)

Total Cells Examined

Total Aberrations

Complex Aberrations

Simple Aberrations

Other Abs.

No. of

Abs

% Cells

No. of

Abs

% Cells

No. of

Abs

% Cells

No. of

Abs

% Cells

Abs.

Per

With

Abs.

Per

With

Abs.

Per

With

Abs.

Per

With

Cell

Abs.

Cell

Abs.

Cell

Abs.

Cell

Abs.

Vehicle Control

Negative (Not Specified)

 

200

6

0.030

2.5

2

0.010

1.0

4

0.020

2.0

0

0.000

0.0

Vehicle Control

Dimethyl Sulfoxide

 

200

6

0.030

2.5

2

0.010

1.0

4

0.020

2.0

0

0.000

0.0

Test Chemical

Methyl ethyl ketoxime

2513         

200

6

0.030

2.5

2

0.010

1.0

4

0.020

2.0

0

0.000

0.0

3750         

200

8

0.040

3.0

4

0.020

2.0

4

0.020

1.5

0

0.000

0.0

5000         

200

4

0.020

2.0

1

0.005

0.5

3

0.015

1.5

0

0.000

0.0

Positive Control

Cyclophosphamide

7.5       

200

32

0.160

11.5

13

0.065

4.0

19

0.095

7.5

0

0.000

0.0

37.5       

25

13

0.520

44.0

4

0.160

16.0

9

0.360

32.0

0

0.000

0.0

 

 

Trend

 

-0.166

 

-0.063

 

-0.493

 

Probability

0.566

0.525

0.689

Applicant's summary and conclusion

Conclusions:
No increase in chromosomal aberrations was observed in cultured Chinese hamster ovary cells treated with up to 5000 μg/mL methyl ethyl ketoxime, with or without S9
Executive summary:

A cytogenetic Chromosome aberration tests with cultured Chinese hamster ovary cells was performed on methyl ethyl ketoxime according to NTP's standard protocol (test method similar to OECD Guideline 473). Chinese hamster ovary cells were exposed to 2513, 3750, 5000 µg/mL test item with metabolic activation (Aroclor 1254-induced male Sprague-Dawley rat liver S9) and to 1873, 2497, 3727, 5000 µg/mL without metabolic activation for a harvest time of 20 and 12 hours respectively. Up to 200 first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Negative and positive controls were performed satisfactorily. No increase in chromosomal aberrations was observed in cultured Chinese hamster ovary cells treated with up to 5000 μg/mL methyl ethyl ketoxime, with or without S9.