1,1'-(4-methyl-1,3-phenylene)bis-1H-pyrrole-2,5-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria: negative in S. typhimurium TA1535, TA 1537, TA98, TA100 and E. coli WP2 uvrA (with and without metabolic activation); OECD TG 471; RL 1; GLP

- Cytogenicity/in vitro micronucleus test: negative in human lymphocytes (with and without metabolic activation); OECD TG 487; RL 1; GLP

- Gene mutation in mammalian cells: negative in L5178Y lymphoma cells (with and without metabolic activation); OECD TG 476; RL 1; GLP

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-01-18 to 2008-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

published June 8, 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: his operon
E. coli: trp operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

DNA polymerase A deficient

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of the dose range finding test, the substance was tested in the first mutation assay at a concentration range of 3 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains.
In an independent repeat of the assay with additional parameters, the substance was tested at a concentration range of 3 to 333 µg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA 1535 and TA 100, at a concentration range of 1 to 100 µg/plate in the absence of S9-mix and at 3 to 333 µg/plate in thepresence of S9-mix in tester strains TA 1537 and TA 98 and at a concentration range of 10 to 1000 µg/plate in the absence and presence of S9-mix in the tester strain WP2uvrA. Toxicity was observed in all tester strains.

Vehicle / solvent:

dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Saline, Milli-Q water or DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable):1E+9 cells/mL

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of background lawn, reduction of number of revertants or an increase in the size of the microcolonies

Rationale for test conditions:

The procedures described in this study were based on the most recent OECD and EEC guidelines.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the
concurrent control, and the total number of revertants in tester strains TA1535, TA1537,
TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case an positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:In the dose range finding test, no increase in the number of revertants was observed upon treatment with the substance under all conditions tested. Cytotoxicity by means of a reduced backgroud lawn and in the number of revertant colonies were found for TA 100 and WP2uvrA at the concentrations summarized in Table 1.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Table 2
- Negative (solvent/vehicle) historical control data: Table 3

Table 1: Toxicity of the test item in the dose range finding test

Strain	without S9-mix			with S9-mix
	Dose (µg/plate)	Bacterial backgroud lawn	Revertant colonies	Dose (µg/plate)	Bacterial backgroud lawn	Revertant colonies
TA 100	100	moderate	-^1	100	moderate	-^2
	333-5000	absent	complete	333	extreme	microcolonies
				1000-5000	absent	complete
WP2uvrA	1000-3330	extreme	microcolonies	1000-3330	extreme	microcolonies
	5000	absent	complete	5000	absent	complete

-¹Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range

-²No reduction in the number of revertant colonies

Table 2: Historical positive control data

Strain		Minimum value	Maximum value	Mean	± 3 x S.D.
TA1535	- S9-mix	231	1923	1145	819
	+ S9-mix	58	538	183	233
TA1537	- S9-mix	79	927	315	415
	+ S9-mix	57	833	334	433
TA98	- S9-mix	147	1790	1053	748
	+ S9-mix	154	1703	648	966
TAI OO	- S9-mix	452	1593	1038	596
	+ S9-mix	223	2061	1094	996
WP2uvrA	- S9-mix	67	1280	643	565
	+ S9-mix	56	887	254	343

Table 3: Historical negative control data

Strain		Minimum value	Maximum value	Mean	± 3xS.D.
TA 1535	- S9-mix	3	28	13		14
	+ S9-mix	3	29	12		14
TA1537	- S9-mix	3	17	6		8
	+ S9-mix	3	21	6		9
TA98	- S9-mix	12	45	20		19
	+ S9-mix	12	51	25		21
TAI OO	- S9-mix	63	194	126		79
	+ S9-mix	60	195	116		83
WP2uvrA	- S9-mix	4	39	14		17
	+ S9-mix	4	44	15		18

Conclusions:

In the present study conducted according to OECD guideline 471, all bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, thus, the substance does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-04-20 to 2017-01-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 26 September 2014

Deviations:

yes

Remarks:

The recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487. The optimum in responses was found with the time schedule stated in the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Three young healthy donors, non-smoking and not receiving medication aged 22-34 years
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Sex, age and number of blood donors if applicable: One male and two female donors
- Whether whole blood or separated lymphocytes were used if applicable: whole blood samples
- Methods for maintenance in cell culture if applicable: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Supplementation with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).

Cytokinesis block (if used):

Cytochalasin B ( 4µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

6.5, 11.4, 19.9, 34.8, 60.9, 107, 187, 327, 571, 1000 µg/mL during short term (4 h) exposure
0.6, 1.1, 2.0, 3.5, 6.1, 10.7, 18.7, 32.7, 57.1, 100 µg/mL during long term (20 h) exposure
With regard to the solubility properties of the test item, the highest applied dose in this study was not 2000 µg/mL, but 1000 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period: no
- Exposure duration: Either 4 h pulse incubation and 16 h recovery or 20 h continuous incubation
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 µg/mL)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cells were spun down by gentle centrifugation for 5 minutes. The cells were re-suspended in 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 cells/culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

DETERMINATION OF CYTOTOXICITY
- Method: other: cytokinesis-block proliferation and cytostasis [%]

Rationale for test conditions:

A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487. The optimum in responses was found with the time schedule stated in the 'Any other information on materials and methods incl. tables' section.

Evaluation criteria:

The substance can be classified as non-clastogenic and non-aneugenic if:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval).
The substance can be classified as clastogenic and aneugenic if:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% confidence interval).

Statistics:

Statistical significance was confirmed by the Chi square test and a linear regression was performed to assess a possible dose dependent increase of micronuclei frequencies.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

micronucleated cells (0.65% and 1.68%) at 19.9 µg/mL and 34.8 µg/mL test item after 4hr incubation without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

47.5 % cytostasis at 1000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

at a concentration of 61.7 µg/mL an increase in the number of micronucleated cells was observed after 4hr incuabtion without metabolic acitvation/within the historical control data range

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

25.3 % cytostasis at 61.7 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

continuous increase of 0.45 % & 0.55 % at 18.7 µg/mL and 32.7 µg/mL/ within the historical data range

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: above 1000 µg/mL
- Definition of acceptable cells for analysis: The criteria for the evaluation of micronuclei are based on the description in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

RANGE-FINDING/SCREENING STUDIES: yes, the pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

CYTOKINESIS BLOCK (if used)
Cytochalasin B

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
Exp. IA; w/o met.act.: Solvent control: 9/2000; Positive control: 184/2000; Test item 6.5µg/mL: 5/2000, Test item 11.4 µg/mL: 6/2000, Test item 19.9 µg/mL: 13/2000, Test item 34.8 µg/mL: 67/4000
Exp. IA; w met.act.: Solvent control: 4/2000; Positive control: 83/2000; Test item 19.9 µg/mL: 2/2000, Test item 34.8 µg/mL: 8/2000, Test item 60.9 µg/mL: 5/2000
Exp. IB; w/o met.act.: Solvent control: 16/2000; Positive control: 176/2000; Test item 35.4 µg/mL: 15/2000, Test item 46.8 µg/mL: 5/2000, Test item 61.7 µg/mL: 4/2000
Exp. II; w/o met.act.: Solvent control: 1/2000; Positive control: 64/2000; Test item 18.7 µg/mL: 9/2000, Test item 32.7 µg/mL: 11/2000, Test item 57.1 µg/mL: 3/2000

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Positive control; pulse treatment; MMC (w/o met.act.): 95 % Ctrl limit : 1.48 - 21.85, mean 11.66, 1 x SD: 5.09
Positive control; pulse treatment; Demecolcin (w/o met.act.): 95 % Ctrl limit : 1.69 - 5.41, mean 3.55, 1 x SD: 0.93
Positive control; pulse treatment; CPA (w met.act.): 95 % Ctrl limit : 0.88 - 8.73, mean 4.80, 1 x SD: 1.96

- Negative (solvent/vehicle) historical control data:
Solvent control; pulse treatment (w/o met.act.): 95 % Ctrl limit : 0.02 - 1.15, mean 0.61, 1 x SD: 0.27
Solvent control; continuous treatment (w/o met.act.): 95 % Ctrl limit : 0.05 - 1.05, mean 0.55, 1 x SD: 0.25
Solvent control; pulse treatment (w met.act.): 95 % Ctrl limit : 0.08 - 1.20, mean 0.64, 1 x SD: 0.28


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Remarks on result:

other: Experiment IA

Table 3: Summary of results

Exp.	Preparation interval	Test item concentration in µg/mL	Proliferation index CBPI in %*	Cytostasis in %	Micronucleated cells in %**
Exposure period 4 hr without S9 mix
IA	40 hrs	Solvent control^[1]^#	2.10		0.23
		Positive control^[2]	1.83	24.9	9.20^S
		6.5	1.95	13.7	0.25
		11.4	1.94	14.7	0.30
		19.9	1.79	27.9	0.65^S
		34.8^#	1.58	47.5	1.68^S
IB	40 hrs	Solvent control^1	2.01		0.80
		Positive control^2	1.60	40.7	8.80^S
		35.4	1.83	18.0	0.75
		46.8	1.77	23.4	0.25
		61.7	1.75	25.3	0.20
Exposure period 20 hr without S9 mix
II	40 hrs	Solvent control^1	1.88		0.05
		Positive control^[3]	1.69	21.9	3.20^S
		18.7	1.83	5.8	0.45^S
		32.7	1.82	7.1	0.55^S
		57.1	1.78	11.4	0.15
Exposure period 4 hr with S9 mix
IA	40 hrs	Solvent control^1	2.06		0.20
		Positive control^[4]	1.60	43.4	4.15^S
		19.9	2.07	n.c.	0.10
		34.8	1.95	10.0	0.40
		60.9	1.97	7.7	0.25

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^#The number of micronucleated cells was determined in a sample of 4000 binucleated cells

^SThe number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1 DMSO                    0.5 % (v/v)

2 MMC                      1.0 µg/mL

3 Demecolcin           50 ng/mL

4 CPA                       17.5 µg/mL

Table 4: Toxicity of evaluated cultures:

Exp. IA
Concentration (µg/mL)	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix
6.5	4 hrs	40 hrs	1.95	13.7
11.4	4 hrs	40 hrs	1.94	14.7
19.9	4 hrs	40 hrs	1.79	27.9
34.8	4 hrs	40 hrs	1.58	47.5
With S9 mix
19.9	4 hrs	40 hrs	2.07	n.c.
34.8	4 hrs	40 hrs	1.95	10.0
60.9	4 hrs	40 hrs	1.97	7.7
Exp. IB
Concentration (µg/mL)	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix
35.4	4 hrs	40 hrs	1.83	18.0
46.8	4 hrs	40 hrs	1.77	23.4
61.7	4 hrs	40 hrs	1.75	25.3
Exp. II
Concentration (µg/mL)	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix
18.7	20 hrs	40 hrs	1.83	5.8
32.7	20 hrs	40 hrs	1.82	7.1
57.1	20 hrs	40 hrs	1.78	11.4

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

n.c. Not calculated as the CBPI was equal or higher than solvent control value

 

 

 

 

Conclusions:

The results of a study conducted according to OECD test guideline 487 (adopted September 26, 2014) revealed that the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, 2,4-Bismaleimidotoluene is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentrations. Thus, the test item does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-04-26 to 2016-07-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 30, 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Target gene:

The autosomal thymidine kinase (TK) locus of heterozygous lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK.
- Suitability of cells: high proliferation rate and cloning efficiency
- Cell cycle length, doubling time or proliferation index: 10-12 h in stock cultures
- Methods for maintenance in cell culture if applicable: Thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured two times prior to treatment. The cell cultures were incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.
- Modal number of chromosomes: 40 ± 2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:RPMI 1640 medium supplemented with 15 % horse serum (HS) (3 % HS during 4 hour
treatment), 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal agent.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 Mix

Test concentrations with justification for top dose:

Experiment I: 4h without activation: 0.025; 0.05; 0.1; 0.2; 0.4; 0.6; 0.8; and 1.0 µg/mL
Experiment I: 4h with activation: 0.125; 0.25; 0.5; 1.0; 1.2; 1.4; 1.6 and 1.8 µg/mL
Experiment II: 24h without activation: 0.06; 0.13; 0.25; 0.5; 1.0; 1.5; 2.0 and 3.0 µg/mL
Experiment II: 4h with activation: 0.22; 0.44; 0.9; 1.8; 2.7; 3.5; 6.2 and 7.0 µg/mL
Experiment III: 4h with activation: 1.8; 3.5; 7.0; 10.5; 14.0; 21.0; 28.0 and 42.0 µg/mL
The top dose was determined in preliminary tests and chosen due to solubility and cytotoxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1E+7 (3E+6 during 24 h exposure) cells/flask (80 cm² flasks)

DURATION
- Exposure duration: 4 and 24 h

SELECTION AGENT (mutation assays):
RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT (Trifluorothymidine).

DETERMINATION OF CYTOTOXICITY
- Any supplementary information relevant to cytotoxicity: RSG (Relative Suspension Growth, pre-experiment) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20 %, if possible.

Rationale for test conditions:

In order to establish a concentration effect relationship, at least four concentrations of the test item are tested in two parallel cultures. These concentrations should yield a concentration related toxic effect. The highest concentration level should produce a low level of cell culture growth. Reference mutagens are tested in parallel to the test item in order to demonstrate the sensitivity of the test system and the activity of the metabolic activation system.

Evaluation criteria:

A test item is classified as mutagenic
- if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 1E+006 cells above the corresponding solvent control.
- A relevant increase of the mutation frequency should be dose-dependent.
- A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay
- if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent. However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects.
If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 1E+006 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological relevance and statistical significance were considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: solubility of the test item up to 1800 µg/mL

RANGE-FINDING/SCREENING STUDIES: Two pre-tests were performed in order to determine the concentration range of the mutagenicity experiments. The highest applied concentration (1800 µg/mL) was limited by the solubility properties of the test item in DMSO and aqueous medium.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see Table 3

- Negative (solvent/vehicle) historical control data: see Table 3

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RSG (Relative Suspension Growth, pre-experiment) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20 %, if possible.

Table 1: Summary of results Experiment I and II

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	1E06 cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with DMSO		-	100.0	154	280	100.0	180	306
Pos. control with MMS	19.5	-	37.6	544	280	62.5	960	306
Test item	0.025	-	culture was not continued^#			culture was not continued^#
Test item	0.05	-	culture was not continued^#			culture was not continued^#
Test item	0.1	-	culture was not continued^#			culture was not continued^#
Test item	0.2	-	94.9	162	280	108.3	121	306
Test item	0.4	-	95.9	176	280	98.5	153	306
Test item	0.6	-	83.9	217	280	68.2	120	306
Test item	0.8	-	71.8	191	280	36.4	197	306
Test item	1.0	-	34.3	340	280	30.6	187	306
Solv. control with DMSO		+	100.0	92	218	100.0	61	187
Pos. control with CPA	3.0	+	27.8	615	218	19.3	607	187
Pos. control with CPA	4.5	+	2.7	9319	218	5.0	3562	187
Test item	0.125	+	culture was not continued^#			culture was not continued^#
Test item	0.25	+	culture was not continued^#			culture was not continued^#
Test item	0.5	+	culture was not continued^#			culture was not continued^#
Test item	1.0	+	178.6	96	218	118.8	50	187
Test item	1.2	+	141.7	96	218	168.2	43	187
Test item	1.4	+	135.1	97	218	117.6	66	187
Test item	1.6	+	108.4	134	218	117.6	63	187
Test item	1.8	+	122.1	105	218	92.2	119	187
Experiment II / 24 h treatment			culture I			culture II
Solv. control with DMSO		-	100.0	147	273	100.0	82	208
Pos. control with MMS	13.0	-	19.7	546	273	7.2	784	208
Test item	0.063	-	culture was not continued^#			culture was not continued^#
Test item	0.13	-	culture was not continued^#			culture was not continued^#
Test item	0.25	-	159.0	102	273	91.5	138	208
Test item	0.5	-	149.6	90	273	44.2	179	208
Test item	1.0	-	39.6	106	273	19.7	126	208
Test item	1.5	-	7.5	104	273	2.5	127	208
Test item	2.0	-	0.5	130	273	0.4	53	208
Test item	3.0	-	culture was not continued^##			culture was not continued^##
Experiment II / 4 h treatment			culture I			culture II
Solv. control with DMSO		+	100.0	107	233	100.0	84	210
Pos. control with CPA	3.0	+	55.6	226	233	34.1	439	210
Pos. control with CPA	4.5	+	39.9	368	233	15.0	1148	210
Test item	0.22	+	culture was not continued^#			culture was not continued^#
Test item	0.44	+	culture was not continued^#			culture was not continued^#
Test item	0.9	+	culture was not continued^#			culture was not continued^#
Test item	1.8	+	119.2	89	233	110.2	101	210
Test item	2.7	+	143.3	95	233	110.9	79	210
Test item	3.5	+	73.5	86	233	114.5	90	210
Test item	6.2	+	82.2	105	233	87.6	60	210
Test item	7.0	+	158.1	85	233	58.9	7	210

#   culture was not continued as a minimum of only four analysable concentrations is required

## culture was not continued due to exceedingly severe cytotoxic effects the bold printend values are not considered valid based on exceedingly severe cytotoxicity

Table 2: Summary results Experiment II

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	1E06 cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment III / 4 h treatment			culture I			culture II
Solv. control with DMSO		+	100.0	115	241	100.0	139	265
Pos. Control with CPA	3.0	+	46.7	324	241	102.6	183	265
Pos. Control with CPA	4.5	+	43.3	395	241	43.4	310	265
Test item	1.8	+	84.9	104	241	117.1	92	265
Test item	3.5	+	71.6	117	241	91.7	98	265
Test item	7.0	+	45.9	164	241	76.8	125	265
Test item	10.5	+	3.8	175	241	4.1	136	265
Test item	14.0	+	culture was not continued^#			culture was not continued^#
Test item	21.0	+	culture was not continued^#			culture was not continued^#
Test item	28.0	+	culture was not continued^#			culture was not continued^#
Test item	42.0	+	culture was not continued^#			culture was not continued^#

#        culture was not continued due to exceedingly severe cytotoxic effects

Table 3: Historical control data

	Number of mutant colonies per 1E+006 cells
	4 h treatment / without metabolic activation
	Solvent control(medium, DMSO, water, ethanol, acetone, THF)	Positive control (MMS)
range:	31 - 204	182 - 1488
Mean value:	84	407
Standard deviation:	30	181
Number of studies:	119	119
95% Confidence Interval	25 - 143	45 - 769
	4 h treatment / with metabolic activation
	Solvent control (medium, DMSO, water, ethanol, acetone, THF)	Positive control (CPA)
range:	38 - 180	181 - 3513
Mean value:	85	488
Standard deviation:	31	341
Number of studies:	120	120
95% Confidence Interval	24 - 146	0 - 1175
	24 h treatment / without metabolic activation
	Solvent control (medium, DMSO, water, ethanol, acetone, THF)	Positive control (MMS)
range:	41 - 174	206 - 1904
Mean value:	81	486
Standard deviation:	29	238
Number of studies:	72	72
95% Confidence Interval	23 - 138	9 - 962

Conclusions:

Under the experimental conditions of a test conducted according to OECD test guideline 490 (adopted July 28, 2015), the test item did not induce gene mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. Therefore, 2,4-Bismaleimidotoluene is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay using the cell line L5178Y.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse gene mutation assay

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA98, TA100, TA1537 and TA1535 and E.coli strain WP2 uvrA were exposed to 2,4 -Bismaleimidotoluene in DMSO in concentrations of 0 (control), 3, 10, 33, 100, 333 and 1000 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method.

The test substance was tested up to cytotoxic concentrations. Cytotoxic effects were noted in all tester strains with and without metabolic activation. Precipitation was not observed. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA1537,TA1 535 or E.coli strain WP2 uvrA) examined at dose levels up to 10 µg/plate in the absence of a metabolic activation source (S9) or at dose levels up to 50 µg/plate in the presence of S9. Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA1537, TA 1535 or E.coli strain WP2 uvrA

under the conditions employed (plate incorporation assay).

There was no evidence of induced mutant colonies over background. Under the conditions of the study, the test substance was negative for mutagenic potential.

Mammalian cell gene mutation assay

In a mammalian cell gene mutation assay according to OECD guideline 476, adopted July 21, 1997 (thymidine kinase (TK)), L5178Y mouse lymphoma cells cultured in vitro were exposed to 2,4 -Bismaleimidotoluene in DMSO in the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix):

Experiment I: 4h without activation: 0.025; 0.05; 0.1; 0.2; 0.4; 0.6; 0.8; and 1.0 µg/mL

Experiment I: 4h with activation: 0.125; 0.25; 0.5; 1.0; 1.2; 1.4; 1.6 and 1.8 µg/mL

Experiment II: 24h without activation: 0.06; 0.13; 0.25; 0.5; 1.0; 1.5; 2.0 and 3.0 µg/mL

Experiment II: 4h with activation: 0.22; 0.44; 0.9; 1.8; 2.7; 3.5; 6.2 and 7.0 µg/mL

Experiment III: 4h with activation: 1.8; 3.5; 7.0; 10.5; 14.0; 21.0; 28.0 and 42.0 µg/mL

(dissolved in DMSO (final concentration 0.5% in culture medium)

2,4 -Bismaleimidotoluene was tested up to cyctotoxic concentrations. The positive controls induced the appropriate response.There was no evidence of induced mutant colonies over background.

In vitro Mammalian Cell Micronucleus Test

In a mammalian cell Micronucleus test according to OECD guideline 487, adopted September 26, 2014, primary human lymphocytes were exposed to 2,4 -Bismaleimidotoluene in DMSO in the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix):

Experiment I: 6.5, 11.4, 19.9, 34.8, 60.9, 107, 187, 327, 571, 1000 µg/mL with and without met. act.

Experiment II: 11.7, 15.4, 20.3, 26.8, 35.4, 46.8, 61.7, 81.5, 108, 142 µg/mL

Experiment III: 0.6, 1.1, 2.0, 3.5, 6.1, 10.7, 18.7, 32.7, 57.1, 100 µg/mL

2,4 -Bismaleimidotoluene was tested up to cyctotoxic concentrations. The positive controls induced the appropriate response. There was no evidence of an increased number of micronucleated cells, however, there was an increase of micronucleated cells without metabolic activation but this increase was considered to be not biologically relevant.

Justification for classification or non-classification

Based on reliable, relevant and adequate data on 2,4 -Bismaleimidotoluene, the substance is considered to be not mutagenic. According to Regulation EC No 1272/2008 (CLP) and the Globally System for Classification and Labelling of Chemicals (GHS) no classification and labelling for mutagenicity is required.