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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

229-175-3

EC Name:

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

Cas Number:

6422-83-9

Molecular formula:

C15H10N2O4

IUPAC Name:

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

Test material form:

solid: particulate/powder

Method

Target gene:

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

Species / strain

Species / strain 1

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 2

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

Controls

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).