Registration Dossier

Administrative data

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

Referenceopen allclose all

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
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).
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:
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. certificate)
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

 9.20S

 

 

6.5

1.95

13.7

0.25

 

 

11.4 

1.94

14.7

0.30

 

 

19.9 

1.79

27.9

 0.65S

 

 

34.8# 

1.58

47.5

 1.68S

IB

40 hrs

Solvent control1

2.01

 

0.80

 

 

Positive control2

1.60

40.7

 8.80S

 

 

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 control1

1.88

 

0.05

 

 

Positive control[3]

1.69

21.9

 3.20S

 

 

18.7

1.83

5.8

 0.45S

 

 

32.7

1.82

7.1

 0.55S

 

 

57.1

1.78

11.4

0.15

Exposure period 4 hr with S9 mix

IA

40 hrs

Solvent control1

2.06

 

0.20

 

 

Positive control[4]

1.60

43.4

 4.15S

 

 

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).
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:
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:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 30, 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
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

106cells

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

106cells

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.