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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:
23 February 1988 - 18 April 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study report does not cite specific official test guidelines, however the report is thorough and the test conditions are well documented. It may be determined that the methodology is broadly consistent with modern test methods and the report does include a claim that the study was conducted according to the General Principles of Good Laboratory Practice; on this basis the study is considered reliable with restrictions.

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar 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:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): MB-3000, N,N'-(4,4'-diphenylenemethylene) bismaleimide
- Physical state: Pale yellow powder
- Lot/batch No.: CX-1074
- Storage condition of test material: Room temperature

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzyme fraction
Test concentrations with justification for top dose:
2.5, 5, 10 and 20 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 24 hours (without S-9) and 6 hours (with S-9) + 18 hours in treatment free media

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (colcemid 0.1μg/mL) 2 hours before required harvest time
STAIN (for cytogenetic assays): 2% Gurrs Giemsa R66

NUMBER OF REPLICATIONS: 2 per treatment with S-9 and 2 without S-9

NUMBER OF CELLS EVALUATED: 100 consecutive well spread metaphases from each culture

DETERMINATION OF CYTOTOXICITY
- Method: count of cells % of control

COUNT OF CELLS with POLYPLOIDY: yes
Evaluation criteria:
KUEMS Guidelines for mutagenicity Testing (1983)

Results and discussion

Test results
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:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: CHO-K1 BH4cell line
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Cytotoxicity

Cell counts for the treatment without S-9 24 hour treatment

 

Without S-9

Culture A

Culture B

Mean %

Concentration of MB-3000 (μg/mL)

No of cells

X105

% of control

No of cells

X105

% of control

0

3.75

100

2.86

100

100

2.5

2.96

79

3.06

107

93

5.0

2.35

63

2.69

94

78.5

10.0

1.86

50

1.69

59

54.5

20.0

1.34

36

1.59

56

46.0

EMS 1 mg/mL

1.84

49

1.78

62

55.5

Cell counts for the treatment with S-9 6 hour treatment

 

With S-9

Culture A

Culture B

Mean %

Concentration of MB-3000 (μg/mL)

No of cells

X105

% of control

No of cells

X105

% of control

0

1.80

100

1.52

100

100

2.5

1.91

106

1.45

95

100.5

5.0

1.66

92

1.61

106

99

10.0

1.23

68

1.37

90

79

20.0

1.07

59

0.68

45

52

CP 25 ug/mL

1.69

94

1.63

107

100.5

Result of Chromosome aberration assay

Treatment without S-9

Treatment group

Treatment time

(hr)

Con-centration

No of diploid cells scored

Cells with polyploidy

No of cells with observed structural aberrations

N

%

Judge-ment

GAPS

Chromatid

Chromosome

Others

Total No Cells with aberration

Judge-ment

g

ctb

cte

csb

cse

X

Z

-g

+g

Solvent control

24

0

100

10

(9.1)

-

1

0

0

0

0

0

0

2

3

-

100

11

(9.9)

2

0

0

0

0

0

0

0

2

200

21

(9.5)

3 (1.5)

0(0)

0(0)

2(1)

0(0)

0(0)

0(0)

2(1)

5(2.5)

MB-3000

24

2.5

100

12

(10.7)

-

1

2

0

2

1

0

0

5

6

+

100

8

(7.4)

5

0

0

4

1

0

0

5

10

200

20

(9.1)

6 (3)

2 (1)

0(0)

6(3)

2(1)

0(0)

0(0)

10(5)

16 (8)

5.0

100

17

(14.5)

-

4

0

0

1

0

0

0

1

5

+

100

6

(5.7)

4

0

0

5

0

2

1

7

10

200

23

(10.3)

8 (4)

0(0)

0(0)

6(3)

0(0)

2(1)

1(0.5)

8(4)

15(7.5)

10.0

100

11

(9.9)

-

7

0

1

9

0

0

1

10

15

+

100

8

(7.4)

4

4

1

4

1

0

6

8

12

200

19

(8.7)

11 (5.5)

4(2)

2(1)

13(6.5)

1(0.5)

0(0)

7(3.5)

18(9)

27(13.5)

20.0

100

9

(8.3)

-

3

5

2

5

2

0

4

13

15

+

100

9

(8.3)

6

2

1

5

0

1

5

7

13

200

18

(8.3)

9 (4.5)

7(3.5)

3(1.5)

10(5)

2(1)

1(0.5)

9(10.5)

20

28(14)

EMS

24

1000

50

6

(10.7)

-

22

17

21

14

3

4

0

43

44

-

50

3

(5.7)

14

12

15

19

3

3

0

33

39

100

9

(8.3)

36 (36)

29(29)

36 (36)

33(33)

6(6)

7(7)

0(0)

76(76)

83(83)

 

Treatment with S-9

Treatment group

Treatment time

(hr)

Con-centration

No of diploid cells scored

Cells with polyploidy

No of cells with observed structural aberrations

N

%

Judge-ment

GAPS

Chromatid

Chromosome

Others

Total No Cells with aberration

Judge-ment

g

ctb

cte

csb

cse

X

Z

-g

+g

Solvent control

6

0

100

14

(12.2)

-

6

1

1

3

0

0

0

5

11

-

100

2

(2.0)

4

1

1

3

1

0

0

6

10

200

16

(7.4)

10 (5)

2(1)

2(1)

6(3)

1(0.5)

0(0)

0(0)

11(5.5)

21(10.5)

MB-3000

6

2.5

100

10

(9.1)

-

1

0

0

1

1

0

0

2

3

+

100

8

(7.4)

1

0

0

0

0

0

0

0

1

200

18

(8.3)

2 (1)

0(0)

0(0)

1(0.5)

1(0.5)

0(0)

0(0)

2(1)

4 (2)

5.0

100

6

(5.7)

-

2

0

2

1

0

0

2

3

5

+

100

6

(5.7)

6

1

1

0

0

0

0

1

6

200

12

(5.7)

8 (4)

1(0.5)

3(1.5)

1(0.5)

0(0)

0(0)

2(1)

4(2)

11(5.5)

10.0

100

11

(9.9)

-

7

6

3

2

0

0

4

11

16

+

100

8

(7.4)

3

2

4

5

1

0

2

9

11

200

19

(8.7)

10 (5)

8(4)

7(3.5)

7(3.5)

1(0.5)

0(0)

6(3)

20(10)

27(13.5)

20.0

100

4

(3.8)

-

7

8

2

4

0

0

8

11

17

+

100

6

(5.7)

6

3

7

4

2

0

9

15

19

200

10

(4.8)

13 (6.5)

11(3.5)

9(4.5)

8(4)

2(1)

0(0)

17(8.5)

26(13)

36(18)

CP

6

25

50

8

(13.8)

-

9

9

14

29

2

0

0

38

40

-

50

8

(13.8)

8

11

7

33

1

0

0

42

43

100

16

(13.8)

17 (17)

20(20)

21(21)

62(62)

3(3)

0(0)

0(0)

80(80)

83(83)

 

Applicant's summary and conclusion

Conclusions:
It was concluded that the test substance showed evidence of cytogenic and clastogenic activities in this in vitro mamalian cell system under the test conditions employed.
Executive summary:

A test was conducted (Safe Pharm Laboratories, 1988) in vitro in Chinese Hamster Ovary cells to assess the clastogenic potential of BMI in mammalian cells. The test was conducted using a 6-hour exposure period in the presence of metabolic activation (achieved by adding induced rat liver homogenate metabolising system) and a 24-hour exposure period in the absence of metabolic activation.

Significant dose-related increases in both toxicity and the frequency of aberrations were observed under both regimes of treatment with the test material. BMI was concluded to be clastogenic to CHO cells in vitro.