3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 24 April 2009 and 26 May 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/08 Date of Signature: 04/03/09

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-{[1,1'-biphenyl]-4-yl}-6-(4-chlorophenyl)-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

EC Number:

700-341-1

Cas Number:

105194-34-1

Molecular formula:

C24H15ClN2O2

IUPAC Name:

3-{[1,1'-biphenyl]-4-yl}-6-(4-chlorophenyl)-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

Constituent 2

Reference substance name:

3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

IUPAC Name:

3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

Details on test material:

Sponsor's identification : PDE-28
Description : red powder
Chemical name : 3-(4-chlorophenyl)-6-(biphenyl)-2.5-dihydropyrrole-1,4-dione
Purity : 96.9%
Batch number : 1RF-8017
Date received : 23 March 2009
Storage conditions : Room temperature in the dark

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate.
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in-house. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2007 and 2008 in Appendix 2 (please see attachment)
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 2007 and 2008 are presented in Appendix 2.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.
- Precipitation: The precipitate of the test material was observed above 500µg/plate.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). There were no revertant colonies observed for WP2uvrA- at 5000 µg/plate in the absence of S9 only. This observation was not reproduced in either of the main experiments and was, therefore, considered spurious and of no biological relevance. The test material formulation and S9-mix used in this experiment were both shown to be sterile. (See table in any other information and results section for the preliminary test).

COMPARISON WITH HISTORICAL CONTROL DATA:
A history profile of vehicle and positive control values for 2007 and 2008 is presented in Appendix 2.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test:

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	85	86	92	89	90	92	76	71	92P	77P	93P
+	TA100	91	79	90	70	87	76	76	75	81P	92P	78P
-	WP2uvrA^-	26	19	25	20	29	21	24	18	23P	18P	0P
+	WP2uvrA^-	37	31	29	31	34	22	37	23	36P	38P	29P

Mutation Test:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pink colour with associated precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

Table1              Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
100		20		23		29		11
107	(97)	18	(18)†	27	(25)	16	(22)	19	(14)
85		17		26		20		12

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
87		19		27		17		5
96	(97)	16	(17)†	38	(35)	8	(15)	7	(5)
109		15		40		21		4

†

†         Experintal procedure perford at later dates (with and without S9) due to excessive contamination in the original tests

 

Table2              Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 27 April 2009 From: 01 May 2009†					To: 30 April 2009 To: 04 May 2009†
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535†		WP2uvrA^-			TA98		TA1537
-	0	113 122 120	(118) 4.7#	13 16 26	(18) 6.8	24 21 20		(22) 2.1	19 18 23	(20) 2.6	14 11 14		(13) 1.7
-	50	98 91 125	(105) 18.0	19 20 29	(23) 5.5	20 25 29		(25) 4.5	19 14 19	(17) 2.9	14 9 10		(11) 2.6
-	150	111 97 110	(106) 7.8	27 13 26	(22) 7.8	26 24 23		(24) 1.5	21 18 23	(21) 2.5	11 8 10		(10) 1.5
-	500	90 P 119 P 117 P	(109) 16.2	25 P 18 P 17 P	(20) 4.4	24 P 25 P 18 P		(22) 3.8	16 P 22 P 20 P	(19) 3.1	10 P 9 P 12 P		(10) 1.5
-	1500	99 P 97 P 112 P	(103) 8.1	26 P 19 P 26 P	(24) 4.0	23 P 18 P 24 P		(22) 3.2	19 P 20 P 21 P	(20) 1.0	10 P 13 P 10 P		(11) 1.7
-	5000	101 P 95 P 111 P	(102) 8.1	18 P 11 P 15 P	(15) 3.5	20 P 24 P 24 P		(23) 2.3	21 P 16 P 22 P	(20) 3.2	12 P 8 P 10 P		(10) 2.0
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		372 404 389	(388) 16.0	178 174 131	(161) 26.1	1402 1296 1518		(1405) 111.0	135 112 143	(130) 16.1	457 539 882	(626) 225.5

 

Table3              Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 27 April 2009 From: 01 May 2009†					To: 30 April 2009 To: 04 May 2009†
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535†		WP2uvrA^-			TA98		TA1537
+	0	131 106 106	(114) 14.4#	21 9 8	(13) 7.2	37 33 31		(34) 3.1	22 26 25	(24) 2.1	13 14 13	(13) 0.6
+	50	113 114 112	(113) 1.0	11 8 17	(12) 4.6	32 29 33		(31) 2.1	21 24 23	(23) 1.5	13 12 13	(13) 0.6
+	150	111 125 124	(120) 7.8	9 13 11	(11) 2.0	31 33 33		(32) 1.2	24 20 20	(21) 2.3	14 14 11	(13) 1.7
+	500	111 P 114 P 120 P	(115) 4.6	12 P 13 P 10 P	(12) 1.5	25 P 27 P 32 P		(28) 3.6	20 P 18 P 20 P	(19) 1.2	13 P 15 P 14 P	(14) 1.0
+	1500	112 P 115 P 121 P	(116) 4.6	10 P 14 P 13 P	(12) 2.1	31 P 27 P 33 P		(30) 3.1	27 P 22 P 23 P	(24) 2.6	15 P 15 P 10 P	(13) 2.9
+	5000	106 P 115 P 128 P	(116) 11.1	9 P 13 P 8 P	(10) 2.6	27 P 32 P 34 P		(31) 3.6	24 P 20 P 26 P	(23) 3.1	12 P 10 P 13 P	(12) 1.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1225 1487 1525	(1412) 163.3	182 252 229	(221) 35.7	564 517 503		(528) 32.0	272 269 244	(262) 15.4	470 349 362	(394) 66.4

Table 4              Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 13 May 2009 From: 23 May 2009†					To: 16 May 2009 To: 26 May 2009†
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535†		WP2uvrA^-			TA98		TA1537
-	0	118 108 116	(114) 5.3#	20 18 18	(19) 1.2	30 28 26		(28) 2.0	25 24 19	(23) 3.2	15 11 11		(12) 2.3
-	50	110 98 118	(109) 10.1	18 15 18	(17) 1.7	26 31 30		(29) 2.6	25 22 22	(23) 1.7	12 12 8		(11) 2.3
-	150	118 102 87	(102) 15.5	16 18 16	(17) 1.2	24 36 36		(32) 6.9	24 17 20	(20) 3.5	8 12 10		(10) 2.0
-	500	108 P 90 P 107 P	(102) 10.1	18 P 18 P 18 P	(18) 0.0	25 P 32 P 24 P		(27) 4.4	20 P 16 P 17 P	(18) 2.1	4 P 11 P 14 P		(10) 5.1
-	1500	95 P 120 P 118 P	(111) 13.9	18 P 20 P 16 P	(18) 2.0	15 P 36 P 36 P		(29) 12.1	19 P 20 P 18 P	(19) 1.0	9 P 10 P 13 P		(11) 2.1
-	5000	98 P 96 P 88 P	(94) 5.3	14 P 16 P 18 P	(16) 2.0	31 P 28 P 31 P		(30) 1.7	16 P 19 P 17 P	(17) 1.5	13 P 10 P 7 P		(10) 3.0
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		394 386 453	(411) 36.6	134 137 142	(138) 4.0	200 214 132		(182) 43.9	77 94 94	(88) 9.8	319 148 125	(197) 106.0

Table 5              Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 13 May 2009 From: 23 May 2009†					To: 16 May 2009 To: 26 May 2009†
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535†		WP2uvrA^-			TA98		TA1537
+	0	102 85 83	(90) 10.4#	10 12 9	(10) 1.5	33 35 30		(33) 2.5	24 26 22	(24) 2.0	9 9 9	(9) 0.0
+	50	85 95 95	(92) 5.8	10 12 11	(11) 1.0	35 35 26		(32) 5.2	24 28 21	(24) 3.5	12 10 6	(9) 3.1
+	150	101 102 85	(96) 9.5	11 10 9	(10) 1.0	15 19 14		(16) 2.6	15 29 20	(21) 7.1	9 12 5	(9) 3.5
+	500	68 P 88 P 102 P	(86) 17.1	8 P 9 P 13 P	(10) 2.6	34 P 34 P 25 P		(31) 5.2	14 P 28 P 32 P	(25) 9.5	10 P 12 P 10 P	(11) 1.2
+	1500	102 P 99 P 87 P	(96) 7.9	12 P 10 P 11 P	(11) 1.0	19 P 35 P 25 P		(26) 8.1	17 P 11 P 24 P	(17) 6.5	7 P 9 P 8 P	(8) 1.0
+	5000	81 P 87 P 83 P	(84) 3.1	10 P 11 P 12 P	(11) 1.0	17 P 35 P 30 P		(27) 9.3	29 P 30 P 20 P	(26) 5.5	11 P 7 P 12 P	(10) 2.6
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1770 1880 1777	(1809) 61.6	381 405 404	(397) 13.6	477 535 514		(509) 29.4	131 224 171	(175) 46.7	391 489 359	(413) 67.7

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

†         Experintal procedure perford at later date due to excessive contamination in the original test

#        Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction:

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirments of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. 

Results:

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pink colour with associated precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion:

The test material was considered to be non-mutagenic under the conditions of this test.