Synthetic resin, a reaction product of 4,4'-bis(N-maleimido)diphenylmethane with 3-aminobenzoic acid hydrazide

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

not reported

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

1,1'-(methylenedi-p-phenylene)bismaleimide

EC Number:

237-163-4

EC Name:

1,1'-(methylenedi-p-phenylene)bismaleimide

Cas Number:

13676-54-5

Molecular formula:

C21H14N2O4

IUPAC Name:

1,1'-[methylenedi(4,1-phenylene)]di(1H-pyrrole-2,5-dione)

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.