4,4'-methylenedianiline

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:

18 June 2018 - 28 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenedianiline

EC Number:

202-974-4

EC Name:

4,4'-methylenedianiline

Cas Number:

101-77-9

Molecular formula:

C13H14N2

IUPAC Name:

4,4'-methylenedianiline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: B1187
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (under light exclusion)
- Stability under test conditions: The stability of the test substance under storage conditions is guaranteed by the sponsor.

Method

Target gene:

Salmonella typhimurium: The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined.
Escherichia coli: The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix from Wistar rat liver (phenobarbital and β-naphthoflavone induced)

Test concentrations with justification for top dose:

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment.
1st Experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (Standard plate test with and without S9 mix, TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA)
2nd Experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (Standard plate test, TA 1537 with and without S9 mix); 0; 0.33; 1.0; 3.3; 10; 33 and 100 μg/plate (Standard plate test, TA 100 and TA 98 both with S9 mix)

Reason for 2nd Experiment: Increase of revertants was observed in the standard plate test using the tester strains TA 100 and TA 98 with S9 mix. The Increase was observed from the 1st tested concentration onward, therefore the experiment was confirmed with adjusted dose levels. Furthermore, due to technical reason, the experiment with TA 1537 was not performed, therefore the part was repeated in the 2nd Experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

2.5 μg/plate (TA 1535, TA 100, TA 1537, TA 98), 60 μg/plate (Escherichia coli WP2 uvrA)

Controls 2

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

100 μg/plate (TA 1537)

Controls 3

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

5 μg/plate (E. coli WP2 uvrA)

Controls 4

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

5 μg/plate (TA 1535, TA 100)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Remarks:

10 μg/plate (TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity detected by a decrease in the number of revertants (factor ≤ 0.6) and clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments.

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect (slight decrease in the number of trp+ revertants) was observed in the standard plate test at 5000 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test at 5000 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect (slight decrease in the number of trp+ revertants) was observed in the standard plate test at 5000 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data: see table 1
- Positive historical control data: see table 2

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies compatible with the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at 5000 μg/plate.

Any other information on results incl. tables

Table 1: Historical Negative Controls (Feb 2016 - Feb 2017)

Strain	S9 mix	Vehicle	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without	(All)	414	152	7	16	10	2.0
	With	(All)	414	152	6	18	10	2.0
TA 100	Without	(All)	426	152	71	132	100	11.4
	With	(All)	417	153	70	147	107	13.7
TA 1537	Without	(All)	411	152	5	13	8	1.7
	With	(All)	411	152	5	16	9	2.1
TA 98	Without	(All)	420	152	14	34	21	3.3
	With	(All)	413	152	12	38	28	4.5
E.coli	Without	(All)	396	152	15	34	24	3.9
	With	(All)	399	152	17	36	25	4.4

Table 1: Historical Positive Controls (Feb 2016 - Feb 2017)

Strain	S9 mix	Positive control	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without	MNNG	312	110	1541	6171	3967	1253.9
	With	2 -AA	312	110	105	520	197	56.3
TA 100	Without	MNNG	315	110	1126	5557	3298	1109.6
	With	2 -AA	309	111	272	3021	1748	587.2
TA 1537	Without	AAC	309	110	253	2190	1044	404.3
	With	2 -AA	312	110	50	399	141	50.3
TA 98	Without	NOPD	309	110	324	1746	863	206.8
	With	2 -AA	315	110	493	3096	1524	529.1
E.coli	Without	4 -NQO	306	110	164	1721	860	431.9
	With	2 -AA	306	110	61	537	133	61.8

Table 3: Without metabolic activation (Standard plate test)

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony Counts
TA 1535	DMSO	-	10.0	1.7	-	9, 9, 12
	Test item	33	8.0	3.5	0.8	4, 10, 10
		100	11.0	1.7	1.1	10, 13, 10
		333	11.0	5.3	1.1	15, 5,13
		1000	8.3	2.5	0.8	8,6,11
		2500	7.3	2.1	0.7	8,9,5
		5000	8.7	1.2	0.9	10,8,8
	MNNG	5.0	4972.7	63.7	497.3	4925,4948,5045
TA 100	DMSO	-	113.0	16.5	-	114,96,129
	Test item	33	98.0	7.0	0.9	90, 103, 101
		100	108.0	15.7	1.0	91,111,122
		333	116.7	16.3	1.0	111,104,135
		1000	138.3	9.5	1.2	148, 138, 129
		2500	156.0	29.9	1.4	190,134,144
		5000	127.3	3.2	1.1	125, 126, 131
	MNNG	5.0	3406.0	612.2	30.1	3967,3498, 2753
TA 1537	DMSO	-	-	-	-	-T,-T,-T
	Test item	33	-	-	-	- T, - T, - T
		100	-	-	-	-T,-T,-T
		333	-	-	-	-T,-T,-T
		1000	-	-	-	-T,-T,-T
		2500	-	-	-	-T,-T,-T
		5000	-	-	-	-T,-T,-T
	AAC	100	-	-	-	-T,-T,-T
TA 98	DMSO	-	19.0	5.3	-	15, 25, 17
	Test item	33	24.3	5.9	1.3	31, 22, 20
		100	18.3	1.2	1.0	17, 19, 19
		333	18.7	5.1	1.0	13, 23, 20
		1000	23.0	5.3	1.2	17, 27, 25
		2500	22.7	7.6	1.2	14, 28, 26
		5000	22.0	4.6	1.2	27, 18, 21
	NOPD	10	774.7	51.1	40.8	725,772, 827
E. coli	DMSO	-	23.0	6.2	-	18, 21, 30
	Test item	33	27.7	7.5	1.2	20, 28, 35
		100	20.0	4.4	0.9	15, 23, 22
		333	23.3	6.1	1.0	22, 18, 30
		1000	22.7	4.7	1.0	19, 28, 21
		2500	15.7	6.7	0.7	14, 23, 10
		5000	9.3	2.1	0.4	11,7, 10
	4-NQO	5	632.7	88.6	27.5	535,708, 655

T = Technical fault

Table 4: With metabolic activation (Standard plate test)

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony Counts
TA 1535	DMSO	-	12.3	4.0	-	10,17,10
	Test item	33	13.0	3.6	1.1	9,16,14
		100	14.3	4.0	1.2	10,15,18
		333	11.7	3.1	0.9	15,11,9
		1000	14.0	3.6	1.1	15,17,10
		2500	12.3	5.9	1.0	19,10,8
		5000	7.0	1.0	0.6	7,6,8
	2 -AA	2.5	172.3	13.1	14.0	187,168,162
TA 100	DMSO	-	110.0	6.9	-	114,114,102
	Test item	33	354.0	33.2	3.2	389,350,323
		100	525.7	40.4	4.8	549,479,549
		333	746.3	65.9	6.8	674,803,762
		1000	874.0	37.2	7.9	917,852,853
		2500	804.3	72.9	7.3	856,836,721
		5000	533.3	56.9	4.8	586,473,541
	2 -AA	2.5	1269.0	249.5	11.5	1554,1090,1163
TA 1537	DMSO	-	-	-	-	-T,-T,-T
	Test item	33	-	-	-	- T, - T, - T
		100	-	-	-	-T,-T,-T
		333	-	-	-	-T,-T,-T
		1000	-	-	-	-T,-T,-T
		2500	-	-	-	-T,-T,-T
		5000	-	-	-	-T,-T,-T
	2 -AA	2.5	-	-	-	-T,-T,-T
TA 98	DMSO	-	28.0	8.5	-	37,20,27
	Test item	33	66.0	4.6	2.4	70,67,61
		100	66.0	3.0	2.4	63,69,66
		333	114.3	8.3	4.1	105,121,117
		1000	119.3	4.0	4.3	123,120,115
		2500	111.3	8.7	4.0	104,121,109
		5000	80.7	17.0	2.9	98,80,64
	2 -AA	2.5	1005.0	188.4	35.9	860,937,1218
E. coli	DMSO	-	30.0	7.8	-	39,25,26
	Test item	33	28.3	4.5	0.9	28,24,33
		100	28.0	8.2	0.9	21,37,26
		333	39.3	3.1	1.3	36,40,42
		1000	29.7	9.1	1.0	40,26,23
		2500	20.3	9.3	0.7	16,14,31
		5000	13.7	2.1	0.5	12,16,13
	2 -AA	60	91.0	28.8	3.0	117,60,96

T = Technical fault

Table 5: Without metabolic activation (Standard plate test, 2nd Experiment)

Strain	Test group	Dose (µg/mL)	Mean revertants per plate	Standard devisation	Factor	Individual revertant colony counts
	DMSO	-	8.0	1.7	-	7, 10,7
	Test item	33	11.3	5.5	1.4	14, 15,5
TA 1537		100	7. 3	2.5	0.9	10, 5,7
		333	10.7	1.5	1.3	12, 9,11
		1000	6.0	3.6	0.8	9,2,7
		2500	6.0	2.6	0.8	8,3,7
		5000	5.7	2.1	0.7	4, 5,8
	AAC	100	875.0	287.1	109.4	726,693,1206

Table 5: With metabolic activation (Standard plate test, 2nd Experiment)

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony
						counts
TA 100	DMSO	-	102.3	8.6	-	104,93, 110
	Test item	0.33	91.7	3.2	0.9	88, 94,93
		1.0	104.0	10.1	1.0	102, 115, 95
		3.3	147.7	11.9	1.4	134,153, 156
		10	187.7	18.9	1.8	181,173, 209
		33	302.3	40.7	3.0	258,311, 338
		100	466.3	93.0	4.6	374,465, 560
	2-AA	2.5	1943.0	195.5	19.0	1756,1927, 2146
TA 1537	DMSO	-	8.3	1.2	-	9,7,9
	Test item	33	8.7	3.2	1.0	10, 5, 11
		100	9.3	6.0	1.1	10, 15, 3
		333	6.0	3.6	0.7	10, 3, 5
		1000	6.7	2.1	0.8	6, 5,9
		2500	9.0	2.0	1.1	11,9,7
		5000	7.7	4.0	0.9	4,7,12
	2-AA	2.5	115.7	20.5	13.9	133, 93,121
TA 98	DMSO	-	19.0	1.7	-	20, 17, 20
	Test item	0.33	23.3	2.5	1.2	23, 26, 21
		1.0	22.0	4.0	1.2	26, 22, 18
		3.3	29.0	5.0	1.5	34, 24, 29
		'0	39.7	1.5	2.1	38, 40, 41
		33	47.7	3.2	2.5	49, 44, 50
		100	53.3	8.5	2.8	47, 63, 50
	2-AA	2.5	1324.0	128.4	69.7	1390,1176, 1406