1,1'-(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 (Bacterial Reverse Mutation Assay/Ames test): the substance 1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione) (CAS No. 3006-93-7) was not mutagenic in the strains S. typhimurium TA98, TA100, TA1535 and TA1537, and E. coli WP2 uvr A in the presence and absence of phenobarbital and 5,6-benzoflavone-induced rat liver S9 metabolic activation. (OECD 471/GLP).

Chromosome aberration (in vitro mammalian chromosome aberration): the substance 1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione)

(CAS No. 3006-93-7) was concluded to be negative for the induction of chromosome aberrations in the presence and absence of phenobarbital and 5,6-benzoflavone-induced rat liver S9 metabolic activation in Chinese hamster lung fibroblasts (CHL/IU) cells. (OECD 473/GLP)

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in Chinese hamster V79 lung fibroblasts exposed to N,N′-m-phenylenedimaleimide (CAS No. 3006-93-7) in the presence or absence of mammalian metabolic activation (PCB-induced rat liver S9) (OECD 476/GLP).  

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12.12.2016 to 22.05.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Xianyang Sanjing Technology Co., ltd./160612
- Expiration date of the lot/batch: June 18, 2018
- Purity: > 99.6 % w/w


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, in a cool and dry place, away from light

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: European Collection of Cell Cultures (ECACC). Lot. No.: 10H016.
- Suitability of cells: Recommended in guideline
- Number of passages if applicable: Cells underwent maximum 5 passages after thawing the original culture delivered from cell collection before using for testing.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1 °C, moistened).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed in experiment without metabolic activation, because of bad growth of cells in HAT medium.

Metabolic activation:

with and without

Metabolic activation system:

PCB-induced rat liver S9

Test concentrations with justification for top dose:

Cytotoxicity – S9 : 0.0001, 0.0005, 0.0010 0.005, 0.01 and 0.02 mg/mL.
Mutagenicity – S9: 0.0001, 0.0005, 0.001, 0.002, 0.004 mg/mL

In the cytotoxicity test without S9, at the concentration 0.005 mg per mL, survival was 11.2 %. To ensure cytotoxicity between 10 and 20% the concentration 0.004 mg/mL was used in mutagenicity experiment without S9.

Cytotoxicity + S9: 0.0005, 0.001, 0.002, 0.003 mg/mL and 0.0075, 0.0150, 0.03, 0.06 mg/mL
Mutagenicity +S9: 0.0025, 0.005, 0.01, 0.02, and 0.04 mg/mL

In the first cytotoxicity with S9, the concentrations of 0.0005, 0.001, 0.002, 0.003 mg per mL were used, but they were completely non-toxic. The tests was repeated with concentrations of 0.075, 0.150, 0.03 and 0.06 mg/mL. 0.02 and 0.04 mg/mL were selected as the top doses for the mutagenicity test with S9 with expected cytotoxicity of 80 and 100% respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle:
The test substance was soluble in DMSO up to a concentration of 133 mg per mL- a red brown solution arose. After addition to DMEM medium, precipitation occurred in all concentrations used; in lower concentrations, precipitation was visible at observation by microscope. Upon further examination, where a concentration row in DMEM was prepared starting with a concentration of 1.33 mg per mL, the first concentration with precipitation not observable with the naked eye was 0.022 mg per mL. This concentration was used as the second highest in the cytotoxicity testing. The highest concentration (the first with visible precipitation) was 0.044 mg per mL.
The test substance was also soluble in acetonitrile in ca half concentration than that in DMSO. After adding to DMEM, precipitation also occurred. As DMSO is more usual solvent with known no mutagenic effect, DMSO was used for the experiments.

Untreated negative controls:

yes

Remarks:

Complete cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 3 hr and 24 hr
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 9 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF CELLS EVALUATED:
Mutants: 2.2 x 10+5
Cloning efficiency: 300 cells

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency (survival)

Rationale for test conditions:

In the cytotoxicity test without S9, at the concentration 0.005 mg per mL, survival was 11.2 %. To ensure cytotoxicity between 10 and 20% the concentration 0.004 mg/mL was used in mutagenicity experiment without S9 (Table 2).

In the first cytotoxicity with S9, the concentrations of 0.0005, 0.001, 0.002, 0.003 mg per mL were used, but they were completely non-toxic. The tests was repeated with concentrations of 0.075, 0.150, 0.03 and 0.06 mg/mL. 0.02 and 0.04 mg/mL were selected as the top doses for the mutagenicity test with S9 with expected cytotoxicity of 80 and 100% respectively (Table 4).

Evaluation criteria:

Each experiment is evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987 (2).

The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to the negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).

There is no requirement for verification of a clearly positive or negative response.

In cases when the response is neither clearly negative nor clearly positive than a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.

Statistics:

For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.02 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.004 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Addition of the test substance to cultivation medium did not change pH values of treatment solutions. It slightly decreased the pH in the highest dose (not used in mutagenicity experiments). Differences approximately 0.05 pH were observed between results at the start and in the end of 3 hours period. This change has not such extent which could produce artifactual positive results. No pH adjustment was needed. (Table 6)

- Effects of osmolality: As the test substance is not soluble in DMEM, results could be taken with caution. Measuring was performed two times. Firstly, the test substance/solvent was dosed in 100 μL of DMSO. So each solution for osmolality measuring contained 9.90 mL of DMEM and 100 μL of the test substance in appropriate concentration in DMSO. Samples were very unstable, gas on thermistor released at formation of drops, which affects ofshape and size of drops. The highest change was observed between negative and solvent control. Osmolality of samples was more closely to the solvent than medium itself. The test substance is well soluble in DMSO, so volume of application form was decreased to 50 μL and the second osmolality measuring (as well as mutagenicity testing) was performed in solutions containing 9.95 mL of DMEM and 50 μL of DMSO/application form of the test substance. Such measured samples had better stability, nevertheless results were analogic (Table 7).

- Precipitation: After addition to DMEM medium, precipitation occurred in all concentrations used; in lower concentrations, precipitation was visible at observation by microscope. Upon further examination, where a concentration row in DMEM was prepared starting with a concentration of 1.33 mg per mL, the first concentration with precipitation not observable with the naked eye was 0.022 mg per mL. This concentration was used as the second highest in the cytotoxicity testing. The highest concentration (the first with visible precipitation) was 0.044 mg per mL.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
EMS 50 µL: 17.53; historical control range (95% confidence interval) is 9.89 – 17.59 mutants per 105cells;
EMS 100 µL: 36.01; historical control range (95% confidence interval) is 19.67 – 36.26 mutants per 105cells;
DMBA: 33.85 and 28.21 what is an evidence of good function of the test system. Historical control range (95% confidence interval) is 9.92 – 33.99 mutants per 105cells.

- Negative (solvent/vehicle) historical control data:
Mutation frequencies of negative control were 2.22 and 2.27 mutants per 105 plated cells. Historical control range (97.5% confidence interval) is 0.76-2.31 mutants per 105cells.
Mutation frequencies of solvent control range varied from 2.00 – 3.23 mutants per 105 plated cells. Historical control range (97.5% confidence interval) is 0.85 – 3.45 mutants per 105cells.

Conclusions:

Under the above-described experimental design the test substance, N,N′-m-phenylenedimaleimide, was non-mutagenic in Chinese hamster V79 lung fibroblasts with and without metabolic activation.

Executive summary:

In a mammalian cell gene mutation assay (HPRT; 17-331), Chinese hamster V79 lung fibroblasts cultured in vitro were exposed to N,N′-m-phenylenedimaleimide (99.6%), in DMSO at concentrations of  0.0001, 0.0005, 0.001, 0.002, 0.004 mg/mL in the absence and 0.0025, 0.005, 0.01, 0.02, and 0.04 mg/mL in the presence of mammalian metabolic activation (PCB-induced rat liver S9).  

N,N′-m-phenylenedimaleimide was tested up to cytotoxic concentrations The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.  

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21.09.2010 to 31.03.2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity: 99.3%

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone-induced rat liver S9

Test concentrations with justification for top dose:

-S9 mix; 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains for dose-finding study)
+S9 mix; 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains for dose-finding study)

-S9 mix; 0, 0.469, 0.938, 1.88, 3.75, 7.50, 15.0, 30.0 μg/plate (all strains for main study)
+S9 mix; 0, 0.938, 1.88, 3.75, 7.50, 15.0, 30.0, 60.0 μg/plate (all strains for main study)

-S9 mix; 0, 3.38, 4.51, 6.01, 8.01, 10.7, 14.2, 19.0 μg/plate (TA100, TA1535, TA98 and TA1537 for confirmative study)
-S9 mix; 0, 6.01, 8.01, 10.7, 14.2, 19.0, 25.3, 33.8 μg/plate (WP2uvrA for confirmative study)
-S9 mix; 0, 2.67, 3.56, 4.75, 6.33, 8.44, 11.3, 15.0 μg/plate (TA1537 for confirmative study (2nd trial))

+S9 mix; 0, 8.01, 10.7, 14.2, 19.0, 25.3, 33.8, 45.0, 60.0 μg/plate (TA100 for confirmative study)
+S9 mix; 0, 10.7, 14.2, 19.0, 25.3, 33.8, 45.0, 60.0 μg/plate (TA1535, TA98, TA1537 and WP2uvrA for confirmative study)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle:
DMSO: ≥ 100 mg / mL (Test facility data). No change such as heat generation, foaming, smoking, etc. was observed up to 2 hours after preparation (Test facility data)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene (+S9); 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: 4

Rationale for test conditions:

The doses for the main studies were chosen based on range-finding tests up to 5000 μg/plate with and without S9.
Doses were adjusted for several confirmatory assays.

Evaluation criteria:

Results were judged to be positive when the number of revertant mutant colonies increased more than twice that of the negative control group and dose dependence or reproducibility was recognized in the increase.

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

15 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

15 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

30 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, E. coli WP2uvrA

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

60 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium TA98, TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

14.2 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium TA1535, TA1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10.7 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

25.3 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

11.3 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, E. coli WP2uvrA

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

45 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was noted in the dose-finding study without S9 at 2000 and 5000 μg/plate and with S9 at 800, 2000, 5000 μg/plate. No precipitation was noted in the main or confirmatory studies (Tables 1 & 2).


RANGE-FINDING/SCREENING STUDIES:
In the dose-finding study without S9, growth inhibition was noted at 20.5 μg/plate and with S9 at 51.2 μg/plate. The maximum doses for the mutagenicity assays were 30 (-S9) and 60 (+S9) μg/plate (Tables 1 & 2)..


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Historical control data from negatrive
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Remarks on result:

other: Main assay

Conclusions:

In the reverse bacteria mutation assay, 1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione) is not mutagenic to S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvr A in the presence and absence of metabolic activation.

Executive summary:

In a reverse gene mutation assay (C542 (314 -023)), strains TA98, TA100, TA1535 and TA1537 of S. typhimurium and E. coli WP2 uvr A were exposed to 1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione) (99.3%) at concentrations of 0 – 30 µg/plate and varying concentrations in confirmatory assays (pre-incubation method) in the absence of metabolic activation (Phenobarbital and 5,6-benzoflavone-induced rat liver S9). The same strains were exposed to concentrations of 0 – 60 μg/plate (pre-incubation method) in the presence of metabolic activation (Phenobarbital and 5,6-benzoflavone-induced rat liver S9).

1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione) was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of revertant colonies over background in any assay for strains of TA98, TA100, TA1535 S. typhimurium and E. coli WP2 uvr A. In the absence of metabolic activation, there was a two or more increase in revertant colonies as compared with those in the negative control in the S. typhimurium TA1537 strain. However this increase was not repeated in either of the two subsequent confirmatory studies.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21-09-2010 to 31-03-2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: In Vitro Chromosomal Aberration Test

Specific details on test material used for the study:

Purity: 993%

Species / strain / cell type:

other: Chinese hamster lung fibroblast cell line (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: National Institute of Hygienic Experiments (now National Institute of Food Hygiene)


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:MEM with inactivated FCS, 5% Co2.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test 1 (±S9, 6h and 24 hr): 0, 5.24, 10.5, 21.0, 41.9, 83.8, 168, 335, 671, 1341, 2682 μg/mL
Preliminary toxicity test 2 (±S9, 6h and 24 hr): 0, 1.21, 1.73, 2.47, 3.53, 5.04, 7.20, 10.3, 14.7, 21.0, 30.0 μg/mL


Main test (6hr, -S9): 0, 3.36, 4.19, 5.24, 6.55, 8.19, 10.2, 12.8 μg/mL
Main test (6hr, +S9): 0, 5.24, 6.55, 8.19, 10.2, 12.8, 16 μg/mL
Main test (24hr, -S9): 0, 4.19, 5.24, 6.55, 8.19, 10.2 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: The substance is poorly soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 6 and 24hrs
- Expression time (cells in growth medium): 24h
- Following treatment: Colchicine for 2 hrs
STAIN (for cytogenetic assays): Giemsa staining method
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 metaphase spreads


DETERMINATION OF CYTOTOXICITY
- Method: relative cell growth

Evaluation criteria:

Incidence of cells with chromosome aberration;
10 %=<, and reproducibility or dose-dependency; positive
=>5 %, <10 %, and reproducibility; equivocal
<5 %; negative

Species / strain:

other: Chinese hamster lung fibroblast cell line (CHL/IU)

Remarks:

3 hrs

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10.2 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: Chinese hamster lung fibroblast cell line (CHL/IU)

Remarks:

3 hrs

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

8.19 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: Chinese hamster lung fibroblast cell line (CHL/IU)

Remarks:

24 hrs

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Toxic at 8.19 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipiitation was observed during the preliminary toxicity test 1 (Tables 1-2) but not test 2 at the lower dose of 30 μg/mL (Tables 3-4) .None was observed during the main tests (Tables 5-7).

HISTORICAL CONTROL DATA
Historical control data (structural aberrations, polyploid cells) with and without metabolic activation for positive and negative controls were provided from pooled data (January 8, 2009 to December 24, 2010) in Appendix 4.

Conclusions:

In an in vitro chromosome abberration assay in Chinese hamster lung fibroblasts (CHL/IU), 1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione), did not induce any chromosomal abnormalities in the absence or presence of metabolic activation.

Executive summary:

In a mammalian cell cytogenetics assay (543(314-024)), Chinese hamster lung fibroblasts (CHL/IU) cell cultures were exposed to 1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione) (99.3%) in DMSO at concentrations of up to 0 – 12. 8 µg/mL (without Phenobarbital and 5,6-benzoflavone-induced rat liver S9 metabolic activation) and 0 - 16µg/mL µg/mL (with metabolic activation).

1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione) was tested up to the highest dose not limited by toxicity or solubility. Positive controls induced the appropriate response. There was no evidence of chromosome aberrations induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline [In vitro mammalian cytogenetics [Chromosome aberration]] OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance N,N′-m-phenylenedimaleimide was evaluated in 3 in vitro mutagenicity assays.

In a reverse gene mutation assay (OECD 471/GLP), strains TA98, TA100, TA1535 and TA1537 of S. typhimurium and E. coli WP2 uvr A were exposed to 1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione) (99.3%) at concentrations of 0 – 30 µg/plate and varying concentrations in confirmatory assays (pre-incubation method) in the absence of metabolic activation (Phenobarbital and 5,6-benzoflavone-induced rat liver S9). The same strains were exposed to concentrations of 0 – 60 μg/plate (pre-incubation method) in the presence of metabolic activation (Phenobarbital and 5,6-benzoflavone-induced rat liver S9). 1,1’-(1,3-phenylene)bis(1H -pyrrole-2,5-dione) was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of revertant colonies over background in any assay for strains of TA98, TA100, TA1535 S. typhimurium and E. coli WP2 uvr A. In the absence of metabolic activation, there was a two or more increase in revertant colonies as compared with those in the negative control in the S. typhimurium TA1537 strain. However this increase was not repeated in either of the two subsequent confirmatory studies.

In a mammalian cell cytogenetics assay (OECD 473/GLP), Chinese hamster lung fibroblasts (CHL/IU) cell cultures were exposed to N,N′-m-phenylenedimaleimide  (99.3%) in DMSO at concentrations of up to 0 – 12. 8 µg/mL (without Phenobarbital and 5,6-benzoflavone-induced rat liver S9 metabolic activation) and 0 - 16µg/mL µg/mL (with metabolic activation). N,N′-m-phenylenedimaleimide was tested up to the highest dose not limited by toxicity or solubility. Positive controls induced the appropriate response. There was no evidence of chromosome aberrations induced over background.

In a mammalian cell gene mutation assay (OECD 476/GLP), Chinese hamster V79 lung fibroblasts cultured in vitro were exposed to N,N′-m-phenylenedimaleimide (99.6%), in DMSO at concentrations of  0.0001, 0.0005, 0.001, 0.002, 0.004 mg/mL in the absence and 0.0025, 0.005, 0.01, 0.02, and 0.04 mg/mL in the presence of mammalian metabolic activation (PCB-induced rat liver S9).  N,N′-m-phenylenedimaleimide was tested up to cytotoxic concentrations. The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

No in vivo studies were necessary as all three in vitro studies were negative. These studies are suitable for use in the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance N,N′-m-phenylenedimaleimide  (CAS No. 3006-93-7) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.