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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
not reported
Qualifier:
according to
Guideline:
other: Abbreviated Japanese MOL/MHW/MITI Metaphase Analysis In CHO Cells In Vitro
Version / remarks:
not specified
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: B.I.B.R.A. (British Industrial Biological Reasearch center)
- Suitability of cells: cells are recommended from OECD guideline
- Cell cycle length, doubling time or proliferation index: doubling time: 12-16 h
- Number of passages if applicable: 31
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: 20


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Nutrient medium - HAMS-F12 Gibco ltd. + 5% foetal bovine serum (Gibco), Lot-No.: 20G3373Y
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Colcemid 0.1 µg/mL
Metabolic activation:
with and without
Metabolic activation system:
S9-Liver Mix
Test concentrations with justification for top dose:
2.5, 5, 10 and 20 µg/mL based on the results from a preliminary study
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item is soluble in the vehicle
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Cell density at seeding (if applicable): 1E+05 cells/mL

DURATION

- Exposure duration: 24h without metabolic activation and 6h with metabolic activation
STAIN (for cytogenetic assays): 2% Gurrs Giemsa R66 for 5 min


SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (0.1 µg/mL) for 2h


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): first 100 consecutive well-spread metaphases from each culture were counted.


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: hyperploidy

Evaluation criteria:
The range of abberation frequencies acceptable for control cultures:
i) Diploid cells with abberations (gaps included)
Range
Untreated controls 3-14
DMSO controls 2-10
Combined 2-14

S9-controls 2-6
S9/DMSO-controls 1-10
Combined 1-10

All controls 1-14

ii) Diploid cells with abberations (gaps included)
Range
Untreated controls 1-10
DMSO controls 0-7
Combined 0-10

S9-controls 1-3
S9/DMSO-controls 0-9
Combined 0-9

All controls 0-10
A positive response was recorded for a particular treatment if the % diploid cells with abberation (gaps excluded) exceeded the maximum historical value. If only the % diploid cells with abberations (gaps included) exceed historical values then +/- reponse was recorded. Positive responses were also recorded if the % cells with abberations (gaps excluded) was greater than twice the concurrent control level, even if it was below historical levels, but only if there was an indication of a dose response.
However, consideration is given to a number of factors, such as the frequency of chromosome exchange events which are comparatively rare in control cultures, and the ultimate designation must rely upon experience and sound scientific judgement (UKEMS Guidelines for Mutagenicity Testing, 1983).

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Results without S9 Mix

Treat-ment group

Treatment time (hr)

Concentration µg/mL

No. diploid cells scored

N

%

Judge-ment

Gaps

Chromatid

Chromosome

others

Total No. cells with abberation

Judgement

 

g

ctb

cte

csb

cse

X

Z

-g

+g

 

Solvent control

24

0

100

10

9.1

-

1

1.5

0

0

0

0

2

1

0

0

0

0

0

0

2

1

3

2.5

-

100

11

9.9

1

0

0

0

0

0

0

0

2

200

21

9.5

3

0

0

2

0

0

0

2

5

Compimide 183

24

2.5

100

12

10.7

-

1

3

2

1

0

0

2

3

1

1

0

0

2

1.5

5

5

6

8

+

100

8

7.4

5

0

0

4

1

0

1

5

10

200

20

9.1

6

2

0

6

2

0

3

10

16

5.0

100

17

14.5

-

4

4

0

0

0

0

1

3

0

0

0

1

0

0.5

1

4

5

7.5

+

100

6

5.7

4

0

0

5

0

2

1

7

10

200

23

10.3

8

0

0

6

0

2

1

8

15

10.0

100

11

9.9

-

7

5.5

0

2

1

1

9

6.3

0

0.5

0

0

1

3.5

10

9

15

13.5

+

100

8

7.4

4

4

1

4

1

0

6

8

12

200

19

8.7

11

4

2

 

1

0

7

18

27

20.0

100

9

8.3

-

3

4.5

5

3.5

2

1.5

5

5

2

1

0

0.5

4

10.5

13

10

15

14

+

100

9

8.3

6

2

1

5

0

1

5

7

13

200

18

8.3

9

7

3

10

2

1

9

20

28

EMS

24

1000

50

6

10.7

-

22

36

17

29

21

36

14

33

3

6

4

7

0

0

43

76

44

83

+

50

3

5.7

14

12

15

19

3

3

0

33

39

100

9

8.3

36

29

36

33

6

7

0

76

83

Table 2: Results with S9 Mix

Treat-ment group

Treatment time (hr)

Concentration µg/mL

No. diploid cells scored

N

%

Judge-ment

Gaps

Chromatid

Chromosome

others

Total No. cells with abberation

Judgement

 

g

ctb

cte

csb

cse

X

Z

-g

+g

 

Solvent control

6

0

100

14

12.2

-

6

5

1

1

1

1

3

3

0

0.5

0

0

0

0

5

5.5

11

10.5

-

100

2

2.0

4

1

1

3

1

0

0

6

10

200

16

7.4

10

2

2

6

1

0

0

11

21

Compimide 183

6

2.5

100

10

9.1

-

1

1

0

0

0

0

1

0.5

1

0.5

0

0

0

0

2

1

3

2

-

100

8

7.4

1

0

0

0

0

0

0

0

1

200

18

8.3

2

0

0

1

1

0

0

2

4

5.0

100

6

5.7

-

2

4

0

0.5

2

1.5

1

0.5

0

0

0

0

2

1

3

2

5

5.5

-

100

6

5.7

6

1

1

0

0

0

0

1

6

200

12

5.7

8

1

3

1

0

00

2

4

11

10.0

100

11

9.9

-

7

5

6

4

3

3.5

2

3.5

0

0.5

0

0

4

3

11

10

16

13.5

+

100

8

7.4

3

2

4

6

1

0

2

9

11

200

19

8.7

10

8

7

 

1

0

6

20

27

20.0

100

4

3.8

-

7

6.5

8

5.5

2

4.5

4

4

0

1

0

0

8

8.5

11

13

17

18

+

100

6

5.7

6

3

7

4

2

0

9

15

19

200

10

4.8

13

11

9

8

2

0

17

26

36

CP

6

25

50

8

13.8

-

9

17

9

20

14

21

29

62

2

3

0

0

0

0

38

80

40

83

+

50

8

13.8

8

11

7

33

1

0

0

42

43

100

16

13.8

17

20

21

62

3

0

0

80

83

Applicant's summary and conclusion

Conclusions:
In the present study conducted according to OECD 473 CHO cells (1E+05 cells/mL) were treated with 2.5, 5.0, 10.0 and 20.0 µg/mL MDAB either 24 h without metabolic activation or 6 h with metabolic activation. MDAB produced significant, dose-related increases in the frequency of chromosome abberations both in the presence and in the absence of a liver enzyme metabolising system. The test item is therefore considered to be clastogenic to CHO cells in vitro.