4,4'-methylenedianiline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The extensive data base on bacterial mutagenicity assays (Ames) available for 4,4’-MDA clearly shows that the substance has a mutagenic potential in Salmonella typhimurium strains TA 98 (frame shift mutations) and TA 100 (base pair substitutions) (key study BASF, 2018). Mutagenicity in bacteria occurs only in the presence of metabolic activation and thus, the reaction with cellular macromolecules resulting in DNA damage and mutagenicity requires biotransformation of the substance. An Ames study assessing the mutagenic potential of various steric isomers of MDA in the presence of metabolic activation showed that o,m’-MDA, m,m’-MDA, p,p’-MDA were mutagenic in TA 98 and TA 100, o,p’-MDA was only mutagenic in TA 98 and o,o’-MDA or m,p’-MDA did not lead to mutagenicity in any of the tested strains (Ross et al., 1983). A bacterial mutagenicity study evaluating metabolites of 4,4’-MDA in TA 98 and TA 100 showed that mono- and diacetylated derivatives were not mutagenic in the absence and presence of metabolic activation. The nitroso and hydroxylamine derivatives of mono-acetylated MDA were weakly mutagenic in the absence and presence of metabolic activation in both strains, but showed severe toxic effects in the absence of metabolic activation (Tanaka et al., 1985). The hydroxamic derivative showed weak mutagenic potential only in strain TA 100 in the presence of metabolic activation. Other published results investigating the mutagenic potency of N-acetylated metabolites of 4,4’-MDA report confirmed the non-mutagenicity of the acetylated derivatives (Morgott, 1982).

A number of results on DNA damage and repair in mammalian cell cultures obtained in in vitro indicator test systems (UDS, Comet assay, SCE, determination of DNA adducts) is available, but the results are in many cases not sufficiently well reported for a final assessment and thus need to be regarded with reservation (for details on the individual studies see section “additional information”). Most importantly, information on cytotoxicity – which can be a crucial confounding factor in genotoxicity studies – is often scarce.

Unscheduled DNA synthesis (UDS) was reported in primary rat hepatocytes by Mori et al., 1988, Shaddock et al., 1989 (only after pretreatment of the animals with Aroclor 1254 or phenobarbital), and Martelli et al., 2002, but it was not confirmed in primary human hepatocytes (Martelli et al., 2002). It should be noted that the UDS test is an outdated test system, and since it is an indicator test, its reliability is questionable.

Indication of DNA fragmentation was observed in an in vitro Comet assay in primary rat and human hepatocytes and thyreocytes, but not in primary cell cultures of kidney (rat, human), urinary bladder mucosa (rat, human) and brain (rat) (Martelli et al., 2002). DNA fragmentation was also observed in Chinese hamster V79 cells in the presence of metabolic activation, although the extent of DNA fragmentation was not reported, and a confounding influence of cytotoxicity cannot be excluded (Swenberg J., 1981). Gulati et al., 1989, reported a slight increase in sister chromatid exchanges (SCE) in CHO cells in the absence and presence of metabolic activation, but as no details on cytotoxicity were reported, the results are not suitable for final assessment. Investigations in human skin equivalents showed increases in DNA adducts. The evidence of peroxidase activity in the skin suggests a peroxidase-mediated metabolism of the substance to be involved in the formation of DNA adducts (Kenyon et al., 2004).

The potential to induce micronucleus formation was investigated in two separate studies:

In an in vitro study in chinese hamster lung fibroblasts (V79), 4,4’-MDA induced a concentration-dependent increase in the frequency of micronuclei in the absence of any metabolic activation system. As the majority of the micronuclei were negative with respect to anti-kinetochore antibody binding, the authors concluded that MDA-induced micronuclei formation was due to clastogenic chromosomal damage (Zhong et al., 2001). In contrast, in primary rat and human kidney cells, no increased incidences in cells carrying micronuclei were observed after MDA treatment in the absence of metabolic activation (Robbiano et al., 1999). It is striking that in the study by Zhong et al., 2001, a positive response was found despite the lack of a metabolic activation system – this is inconsistent with the bacterial mutagenicity data base and also many of the assessable in vitro indicator tests in mammalian cells in which MDA only elicited a genotoxic response when a metabolic activation system was present. Furthermore, the finding by Zhong et al. was not confirmed in the most relevant and reliable availablein vitrocytogenicity study: 4,4’-MDA did not cause chromosomal aberrations in the presence or absence of metabolic activation in human peripheral blood lymphocytes in a state-of-the-art GLP-compliant study according to OECD TG 473 (key study BASF, 2019). It must be pointed out that Zhong et al. did not specify the purity of the MDA used in their study, and so it is possible that micronuclei formation was caused by contaminants in the test material and not by MDA itself.

A cell transformation assay in baby hamster kidney cells did not reveal any transformed colonies in the presence of metabolic activation (ICI Central Toxicology Laboratory, 1982).

In conclusion, the available in vitro genotoxicity data of 4,4’-MDA clearly show the mutagenicity in bacteria after biotransformation of the substance. The outcome of in vitro indicator tests in freshly isolated primary cells show that the induction of DNA damage occurs in cells isolated from tissues which are the target for the development of non-neoplastic and neoplastic lesions (liver, thyroid) in rats and mice, whereas no mutagenicity was observed in cells isolated from other tissues. Most importantly, no chromosomal aberrations were detected in human peripheral blood lymphocytes in the most reliable study (state-of-the-art GLP-compliant study according to OECD TG 473).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

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

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.

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 / 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.

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)

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)

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)

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)

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.

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

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

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

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

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

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

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

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

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

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.

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

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 September 2018 - 08 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: 4,4'-Methylenediphenyldiamine
BASF Test substance number: 17/0409-1
Batch: B1187
Content: 98.0 g/100 g
Identity: confirmed
Homogeneity: given
Expiry date: 17 July 2019 (The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility)
Storage conditions: At +2 to +8 °C, light protected
Physical state/appearance: Solid, melt, beige to brown
Molecular weight: 198.27 g/mol
Stability in solvent:
Additional information: Fusing of the test item at ca. 127 °C

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Blood samples were drawn from healthy non-smoking donors not receiving medication. Blood was collected from one single donor for each experiment. For this study, blood was collected from a male donor (29 years old) for Experiment I, from a female donor (28 years old) for Experiment IIA, IIB and IID and from a male donor (35 years old) for Experiment IIC. 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 chromosomal aberrations in their peripheral blood lymphocytes.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Envigo CRS GmbH SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation used for this study was 34.3 mg/mL.

Test concentrations with justification for top dose:

With regard to the molecular weight and the content (98%) of the test item, 2023 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 13.1 to 2023 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 1156 μg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using reduced mitotic indices as an indicator for toxicity in the pre-test, no clear toxic effects were observed after 4 hours treatment in the absence and presence of S9 mix.
Dose selection of Experiment IIA was influenced by the occurrence of precipitation. Therefore, 2023 μg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix. This experiment was repeated (Exp. IIB) with a top dose of 800 μg/mL and narrow concentration spacing due to lack of evaluable concentrations in a cytotoxic range.
Two confirmatory experiments (IIC and IID) were performed with a top dose of 800 μg/mL and narrow concentration spacing due to a significant increase in chromosome aberration in Experiment IIB.

Vehicle / solvent:

DMSO (dimethyl sulfoxide)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent controls (culture medium with 0.5 % DMSO) were performed. DMSO had a purity of 99.93%.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

The dilutions of the stock solutions were prepared on the day of the experiment. Stability of the positive control substance in solution is unknown but a mutagenic response in the expected range is a sufficient biological evidence for chemical stability.

Details on test system and experimental conditions:

Reason for the choice of human lymphocytes:
Human lymphocytes have been used successfully for a long time in in vitro experiments. It has been shown that they are an extremely sensitive indicator of in vitro induced chromosome structural changes. These changes in chromosome structure offer readily scored morphological evidence of damage to the genetic material. A proportion of the lymphocytes can be stimulated by mitogens to undergo mitosis in culture; they are easy to culture and thus provide a ready source of dividing cells for the scoring of chromosome aberrations. Furthermore human lymphocytes have a low spontaneous chromosome aberration frequency.

Culture conditions:
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). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Test Item Preparation:
Prior to the stock solution preparations the test item was melted and several aliquots were prepared.
Stock formulations of the test item and serial dilutions were made in DMSO. The final concentration of DMSO in the culture medium was 0.5 %. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
The osmolarity and pH were determined in Experiment I:
Solvent control: 400 mOsm, pH 7.6; Test item: 373 mOsm, pH 7.6

Analysis of Test Substance Preparation:
In addition to guideline requirements, the stability of the test item in the application vehicle was demonstrated.
The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was verified analytically. The analyses were carried out as a separate study at the Competence Center Analytics of BASF SE (BASF study code 18L00316).

Pre-experiment:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the main experiment. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure.

Cytogenetic Experiment:
Pulse exposure:
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation, 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose •H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. After washing, the cells were resuspended in complete culture medium (with 10% FBS) and cultured until preparation of the cells.
Continuous exposure (without S9 mix):
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. The culture medium was not changed until preparation of the cells.

Preparation of metaphases:
Cultures were treated with the metaphase-arresting substance colcemid (final concentration: 0.2 μg/mL) approximately three hours before the requested harvest time. The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g), the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, and, after drying covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

Evaluation of cytotoxicity and cytogenetic damage:
Evaluation of the slides was performed according to the standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" using microscopes with 100 x oil immersion objectives. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate.
At least 150 well-spread metaphases were evaluated per culture for structural aberrations. Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established.

Acceptability Criteria:
In summary, the chromosomal aberration assay is considered acceptable if it meets the following criteria (OECD guideline 473 (2016)):
a) The number of aberrations found in the solvent controls falls within the 95% control limits of the distribution of the laboratory’s historical negative control database. If they fall outside those limits, they are acceptable as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ and technical or human failure can be excluded.
b) The rate of chromosomal aberrations in the positive controls was statistically significantly increased compared with the concurrent negative control and compatible with those generated in the historical positive control data base.
c) Cell proliferation criteria in the solvent control were fulfilled by ensuring sufficient number of cells have reached mitosis and cytotoxicity levels were acceptable. For primary lymphocyte cultures the mitotic index (MI) is an appropriate measure of cytotoxicity.
d) The test item is tested with and without metabolic activation for 4 h, and sampled to a time equivalent to about 1.5 normal cell cycle length after the beginning of treatment, as well as a long treatment experiment without metabolic activation was performed. For the long treatment the cells were exposed to the test item continuously until sampling for a time equivalent to 1.5 normal cell cycle length. All three experimental conditions will be tested unless one part turns out positive.
e) At least three test concentrations that meet the acceptability criteria were evaluated and at least 300 well-spread metaphases were counted.

Evaluation criteria:

A test substance is classified as non-clastogenic if:
a. none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. there is no concentration-related increase when evaluated with an appropriate trend test,
c. all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

A test substance is classified as clastogenic if all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions could be useful.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.
An increase in the number of polyploid cells may indicate that the test substances have the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substances have the potential to inhibit cell cycle progress. Therefore incidence of polyploid cells and cells with endoreduplicated chromosomes should be recorded separately.

Statistics:

The statistical significance is confirmed by the Fisher’s exact test (modified) (p < 0.05) using a validated test script of “R”, a language and environment for statistical computing and graphics.
A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance were considered together.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item 4,4’-Methylenediphenyldiamine, dissolved in DMSO, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Five independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment IIA, IIB, IIC and IID, the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2023 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the content (98%) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, precipitation of the test item in the culture medium was observed at 1156 μg/mL and above in the absence and presence of S9 mix at the end of treatment. In Experiment IIA, precipitation of the test item in the culture medium was observed at 2023 μg/mL. In Experiment IIB, IIC and IID no precipitation was observed at the end of treatment.
No relevant influence on osmolarity or pH was observed.
In Experiment I in the absence of S9 mix, cytotoxicity (MI of 53.7%) was observed at the highest evaluated concentration, which showed precipitation. In Experiment I in the presence of S9 mix, no cytotoxicity (MI of 84.7%) was observed up to the highest evaluated concentration, which showed precipitation. In Experiment IIB, IIC and IID in the absence of S9 mix after continuous treatment, cytotoxicity (MI of 51.4% in Experiment IIB and 50.3% in Experiment IIC) and no cytotoxicity (MI of 69.3% in Experiment IID) were observed at the highest evaluated concentrations. An evaluation of the next higher concentrations, which were separated by a factor smaller than 2, was not possible due to strong cytotoxicity (MI of 14.1%, 34.5% and 33.3%, respectively). Experiment IIA was not evaluated for the genotoxic parameter, due to lack of evaluable concentrations in a cytotoxic range.
In Experiment I, either with or without metabolic activation neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.
In Experiment IIB, in the absence of S9 mix after continuous treatment, all values (2.2, 2.7 and 2.0 % aberrant cells, excluding gaps) exceeded the range of the historical control data (0.0 – 1.9% aberrant cells, excluding gaps). In addition, the value of 2.7 % aberrant cells, excluding gaps after treatment with 300 μg/mL was statistically significantly increased. No dose-dependency, tested by a trend test was observed.
In the first and second confirmatory experiments (IIC and IID), in the absence of S9 mix after continuous treatment, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. Taken together, the findings in Experiment IIB after continuous treatment were not confirmed in the confirmatory experiments (IIC and IID) and therefore they can be regarded as biologically irrelevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Either EMS (550, 660, 770 and 825 μg/mL) or CPA (7.5 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table 3. Toxicity - Experiment I.

Concentration (μg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent control
Without S9 mix
Solvent control	4 hrs	22 hrs	23.9	100.0
13.1	4 hrs	22 hrs	n.d.	n.d.
23.0	4 hrs	22 hrs	n.d.	n.d.
40.2	4 hrs	22 hrs	n.d.	n.d.
70.4	4 hrs	22 hrs	n.d.	n.d.
123	4 hrs	22 hrs	21.7	91.0
216	4 hrs	22 hrs	17.8	74.4
377	4 hrs	22 hrs	19.9	83.4
661	4 hrs	22 hrs	18.0	75.5
1156^P	4 hrs	22 hrs	12.8	53.7
2023^P	4 hrs	22 hrs	n.p.	n.p.
With S9 mix
Solvent control	4 hrs	22 hrs	19.3	100.0
13.1	4 hrs	22 hrs	n.d.	n.d.
23.0	4 hrs	22 hrs	n.d.	n.d.
40.2	4 hrs	22 hrs	n.d.	n.d.
70.4	4 hrs	22 hrs	n.d.	n.d.
123	4 hrs	22 hrs	21.4	110.6
216	4 hrs	22 hrs	20.3	104.9
377	4 hrs	22 hrs	17.2	89.1
661	4 hrs	22 hrs	12.4	64.0
1156^P	4 hrs	22 hrs	16.4	84.7
2023^P	4 hrs	22 hrs	n.p.	n.p.

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

P Precipitation occurred at the end of treatment by the unaided eye

n.p. Not prepared

n.d. Not determined

Table 4. Toxicity - Experiment IIA.

Concentration (μg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent control
Without S9 mix
Solvent control	22 hrs	22 hrs	22.8	100.0
87.0	22 hrs	22 hrs	17.5	76.7
152	22 hrs	22 hrs	17.1	75.2
266	22 hrs	22 hrs	15.3	67.0
400	22 hrs	22 hrs	8.1	35.4
599	22 hrs	22 hrs	4.8	21.1
899	22 hrs	22 hrs	0.0	0.0
1349	22 hrs	22 hrs	0.0	0.0
2023^P	22 hrs	22 hrs	0.0	0.0

* Mean value of two cultures in %

P Precipitation occurred at the end of treatment by the unaided eye

Table 5. Toxicity - Experiment IIB.

Concentration (μg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent control
Without S9 mix
Solvent control	22 hrs	22 hrs	18.5	100.0
50.0	22 hrs	22 hrs	n.d.	n.d.
100	22 hrs	22 hrs	n.d.	n.d.
150	22 hrs	22 hrs	17.9	96.8
200	22 hrs	22 hrs	18.8	101.6
300	22 hrs	22 hrs	12.5	67.6
400	22 hrs	22 hrs	10.2	55.1
500	22 hrs	22 hrs	9.5	51.4
800	22 hrs	22 hrs	2.6	14.1

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

n.d. Not determined

Table 6. Toxicity - Experiment IIC.

Concentration (μg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent control
Without S9 mix
Solvent control	22 hrs	22 hrs	25.4	100.0
50.0	22 hrs	22 hrs	20.8	81.9
100	22 hrs	22 hrs	22.9	90.1
150	22 hrs	22 hrs	15.1	59.4
200	22 hrs	22 hrs	13.4	52.7
300	22 hrs	22 hrs	14.2	55.8
400	22 hrs	22 hrs	12.8	50.3
500	22 hrs	22 hrs	8.8	34.5
800	22 hrs	22 hrs	0.0	0.0

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

Table 7. Toxicity - Experiment IID.

Concentration (μg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells*	% of solvent control
Without S9 mix
Solvent control	22 hrs	22 hrs	13.1	100.0
50.0	22 hrs	22 hrs	16.0	122.6
100	22 hrs	22 hrs	13.1	100.4
150	22 hrs	22 hrs	13.3	101.9
200	22 hrs	22 hrs	9.7	73.9
300	22 hrs	22 hrs	9.1	69.3
400	22 hrs	22 hrs	4.4	33.3
450	22 hrs	22 hrs	4.8	36.4
500	22 hrs	22 hrs	4.0	30.7
550	22 hrs	22 hrs	0.0	0.0
600	22 hrs	22 hrs	0.0	0.0
800	22 hrs	22 hrs	0.0	0.0

Experimental groups evaluated for cytogenetic damage are shown in bold characters

* Mean value of two cultures in %

Table 8. Mitotic index; preparation interval 22 hrs with and without S9 mix, Experiment I.

Treatment			Conc.					S9				Exposure					Mitotic indices*
group		per mL			mix				period/		Absolute				Mean			%**
Recovery						1								2
Solv. control#	0.5 %			-			4 / 18 hrs			25.5			22.2			23.9			100.0
Pos. control##	825 μg			-			4 / 18 hrs			14.4			19.1			16.8			70.2
Test item	377 μg			-			4 / 18 hrs			21.7			18.1			19.9			83.4
″	661 μg			-			4 / 18 hrs			18.6			17.4			18.0			75.5
″	1156 μg			-			4 / 18 hrs			11.4			14.2			12.8			53.7
Solv. control#	0.5 %			+			4 / 18 hrs			17.6			21.0			19.3			100.0
Pos. control###	7.5 μg			+			4 / 18 hrs			11.1			10.9			11.0			57.0
Test item	377 μg			+			4 / 18 hrs			15.3			19.1			17.2			89.1
″	661 μg			+			4 / 18 hrs			14.7			10.0			12.4			64.0
″	1156 μg			+			4 / 18 hrs			16.2			16.5			16.4			84.7

* The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

** For the positive control groups and the test item groups, the relative values of the mitotic index are related to the solvent controls

# DMSO

## EMS

### CPA

Table 9. Structural chromosome aberrations Experiment I; preparation interval 22 hrs without S9 mix: exposure period 4 hrs.

Slide				Cells						% Aberrant cells						Aberrations**
no.	scored			incl.			excl.			carrying ex-	Gaps			Chromatid type				Chromosome type					Other
gaps*	gaps*	changes		g	ig		b		f	d	ex	ib		if		id			cx		ma			cd
Without S9 mix
Solvent control: DMSO 0.5 %
1	150	1.3	1.3	0.0		0		0	2	0	0	0	0		0		0			0		0			0
2	150	1.3	1.3	0.0		0		0	2	0	0	0	0		0		0			0		0			0
1 + 2	300	1.3	1.3	0.0		0		0	4	0	0	0	0		0		0			0		0			0
Positive control: EMS 825μg / mL
1	150	12.0	12.0	5.3		0		0	10	1	0	10	0		0		0			0		0			0
2	150	10.0	10.0	1.3		0		0	5	3	0	2	1		4		0			0		2			0
1 + 2	300	11.0	11.0	3.3		0		0	15	4	0	12	1		4		0			0		2			0
Test item: 377 μg / mL
1	150	0.7	0.7	0.0		0		0	1	0	0	0	0		0		0			0		0			0
2	150	2.0	2.0	0.0		0		0	1	0	0	0	0		1		0			0		1			0
1 + 2	300	1.3	1.3	0.0		0		0	2	0	0	0	0		1		0			0		1			0
Test item: 661 μg / mL
1	150	0.7	0.7	0.0		0		0	1	0	0	0	0		0		0			0		0			0
2	150	0.7	0.7	0.0		0		0	0	0	0	0	1		0		0			0		0			0
1 + 2	300	0.7	0.7	0.0		0		0	1	0	0	0	1		0		0			0		0			0
Test item: 1156 μg / mL
1	150	2.0	2.0	0.0		0		0	4	3	0	0	0		0		0			0		0			0
2	150	2.7	2.7	0.0		0		0	5	0	0	0	0		0		0			0		0			0
1 + 2	300	2.3	2.3	0.0		0		0	9	3	0	0	0		0		0			0		0			0

* Including cells carrying exchanges

** Note: multiple aberrations may occur in a single cell, therefore, the numbers in these columns may not (nor are they intended to) correlate with the number in the columns of % Aberrant cells.

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

Table 10. Structural chromosomal aberrations Experiment I; preparation interval 22 hrs with S9 mix: exposure period 4 hrs.

Slide				Cells										% Aberrant cells										Aberrations**
no.	scored			incl.			excl.				carrying ex-						Gaps				Chromatid type					Chromosome type					Other
gaps*	gaps*	changes		g	ig		b		f			d				ex			ib		if		id				cx		ma			cd
With S9 mix
Solvent control: DMSO 0.5 %
1	150	1.3	1.3	0.0		0		0		2			0		0			0		0		0			0			0		0			0
2	150	1.3	1.3	0.0		0		0		1			0		0			0		0		1			0			0		0			0
1 + 2	300	1.3	1.3	0.0		0		0		3			0		0			0		0		1			0			0		0			0
Positive control: CPA 7.5μg / mL
1	150	8.0	8.0	0.7		0		0		9			1		0			1		3		0			0			0		0			0
2	150	8.7	8.7	0.7		1		0		7			0		0			1		3		1			0			0		1			0
1 + 2	300	8.3	8.3	0.7		1		0		16			1		0			2		6		1			0			0		1			0
Test item: 377 μg / mL
1	150	2.0	2.0	0.0		0		0		2			1		0			0		0		0			0			0		0			0
2	150	1.3	1.3	0.7		0		0		2			0		0			1		0		0			0			0		0			0
1 + 2	300	1.7	1.7	0.3		0		0		4			1		0			1		0		0			0			0		0			0
Test item: 661 μg / mL
1	150	1.3	1.3	0.7		0		0		1			0		0			0		0		0			0			1		0			0
2	150	2.0	2.0	0.0		0		0		0			1		0			0		0		2			0			0		0			0
1 + 2	300	1.7	1.7	0.3		0		0		1			1		0			0		0		2			0			1		0			0
Test item: 1156 μg / mL
1	150	2.7	2.7	1.3		0		0		2			1		0			2		0		0			0			0		0			0
2	150	3.3	3.3	0.0		0		0		4			0		0			0		0		0			0			0		1			0
1 + 2	300	3.0	3.0	0.7		0		0		6			1		0			2		0		0			0			0		1			0

* Including cells carrying exchanges

** Note: multiple aberrations may occur in a single cell, therefore, the numbers in these columns may not (nor are they intended to) correlate with the number in the columns of % Aberrant cells.

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

Table 11. Mitotic index; preparation interval 22 hrs without S9 mix, Experiment IIB.

Treatment			Conc.					S9				Exposure					Mitotic indices*
group		per mL			mix				period/		Absolute				Mean			%**
Recovery						1								2
Solv. control#	0.5 %			-			22 / 0 hrs			20.0			17.0			18.5			100.0
Pos. control##	660 μg			-			22 / 0 hrs			13.3			12.0			12.7			68.4
Test item	150 μg			-			22 / 0 hrs			19.7			16.1			17.9			96.8
″	300 μg			-			22 / 0 hrs			9.6			15.4			12.5			67.6
″	500 μg			-			22 / 0 hrs			8.3			10.7			9.5			51.4

* The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

** For the positive control groups and the test item groups, the relative values of the mitotic index are related to the solvent controls

# DMSO

## EMS

Table 12. Structural chromosome aberrations Experiment IIB; preparation interval 22 hrs without S9 mix: exposure period 22 hrs.

Slide				Cells										% Aberrant cells										Aberrations**
no.	scored			incl.			excl.				carrying ex-						Gaps				Chromatid type					Chromosome type					Other
gaps*	gaps*	changes		g	ig		b		f			d				ex			ib		if		id				cx		ma			cd
Without S9 mix
Solvent control: DMSO 0.5 %***
1	300	1.0	1.0	0.0		0		0		2			0		0			0		1		0			0			0		0			0
2	300	1.3	1.3	0.0		0		0		3			1		0			0		0		0			0			0		0			0
1 + 2	600	1.2	1.2	0.0		0		0		5			1		0			0		1		0			0			0		0			0
Positive Control: EMS 660μg / mL
1	150	14.0	13.3	3.3		1		0		14			0		0			5		0		2			0			0		0			0
2	150	14.0	12.7	1.3		3		0		14			0		0			2		1		2			0			0		0			0
1 + 2	300	14.0	13.0	2.3		4		0		28			0		0			7		1		4			0			0		0			0
Test Item: 150 μg / mL***
1	300	2.3	2.3	0.0		0		0		5			2		0			0		0		0			0			0		0			0
2	300	2.7	2.0	0.3		2		0		1			3		0			1		0		0			0			0		1			0
1 + 2	600	2.5	2.2	0.2		2		0		6			5		0			1		0		0			0			0		1			0
Test Item: 300 μg / mL***
1	300	3.0	3.0	0.0		0		0		8			0		0			0		0		0			0			0		1			0
2	300	2.3	2.3	0.0		1		0		7			1		0			0		0		0			0			0		0			0
1 + 2	600	2.7	2.7	0.0		1		0		15			1		0			0		0		0			0			0		1			0
Test Item: 500 μg / mL***
1	300	3.0	2.3	0.0		2		0		7			0		0			0		0		0			0			0		0			0
2	300	1.7	1.7	0.0		0		0		5			0		0			0		0		1			0			0		0			0
1 + 2	600	2.3	2.0	0.0		2		0		12			0		0			0		0		1			0			0		0			0

* Including cells carrying exchanges

** Note: multiple aberrations may occur in a single cell, therefore, the numbers in these columns may not (nor are they intended to) correlate with the number in the columns of % Aberrant cells.

*** Evaluation of 300 metaphases per culture

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

Table 13. Mitotic index; preparation interval 22 hrs without S9 mix, Experiment IIC.

Treatment			Conc.					S9				Exposure					Mitotic indices*
group		per mL			mix				period/		Absolute				Mean			%**
Recovery						1								2
Solv. control#	0.5 %			-			22 / 0 hrs			26.0			24.7			25.4			100.0
Pos. control##	770 μg			-			22 / 0 hrs			12.1			12.8			12.5			49.1
Test item	50.0 μg			-			22 / 0 hrs			19.9			21.6			20.8			81.9
″	150 μg			-			22 / 0 hrs			13.9			16.2			15.1			59.4
″	400 μg			-			22 / 0 hrs			13.0			12.5			12.8			50.3

* The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

** For the positive control groups and the test item groups, the relative values of the mitotic index are related to the solvent controls

# DMSO

## EMS

Table 14. Structural chromosome aberrations Experiment IIC; preparation interval 22 hrs without S9 mix: exposure period 22 hrs.

Slide				Cells										% Aberrant cells										Aberrations**
no.	scored			incl.			excl.				carrying ex-						Gaps				Chromatid type					Chromosome type					Other
gaps*	gaps*	changes		g	ig		b		f			d				ex			ib		if		id				cx		ma			cd
Without S9 mix
Solvent control: DMSO 0.5 %
1	150	1.3	1.3	0.7		0		0		1			0		0			1		0		0			0			0		0			0
2	150	0.7	0.7	0.0		0		0		1			0		0			0		0		0			0			0		0			0
1 + 2	300	1.0	1.0	0.3		0		0		2			0		0			1		0		0			0			0		0			0
Positive control: EMS 770μg / mL
1	150	25.3	25.3	7.3		1		0		41			0		0			13		2		0			0			0		1			0
2	150	18.0	17.3	5.3		1		1		18			1		0			7		7		1			0			1		2			0
1 + 2	300	21.7	21.3	6.3		2		1		59			1		0			20		9		1			0			1		3			0
Test item: 50.0 μg / mL
1	150	0.7	0.0	0.0		1		0		0			0		0			0		0		0			0			0		0			0
2	150	0.7	0.7	0.0		0		0		0			0		0			0		0		0			0			0		1			0
1 + 2	300	0.7	0.3	0.0		1		0		0			0		0			0		0		0			0			0		1			0
Test item: 150 μg / mL
1	150	0.0	0.0	0.0		0		0		0			0		0			0		0		0			0			0		0			0
2	150	1.3	1.3	0.0		0		0		1			0		0			0		0		1			0			0		0			0
1 + 2	300	0.7	0.7	0.0		0		0		1			0		0			0		0		1			0			0		0			0
Test item: 400 μg / mL
1	150	0.7	0.7	0.0		0		0		1			0		0			0		0		0			0			0		0			0
2	150	0.7	0.7	0.0		0		0		0			0		0			0		0		1			0			0		0			0
1 + 2	300	0.7	0.7	0.0		0		0		1			0		0			0		0		1			0			0		0			0

* Including cells carrying exchanges

** Note: multiple aberrations may occur in a single cell, therefore, the numbers in these columns may not (nor are they intended to) correlate with the number in the columns of % Aberrant cells.

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

Table 15. Mitotic index; preparation interval 22 hrs without S9 mix, Experiment IID.

Treatment			Conc.					S9				Exposure					Mitotic indices*
group		per mL			mix				period/		Absolute				Mean			%**
Recovery						1								2
Solv. control#	0.5 %			-			22 / 0 hrs			11.5			14.6			13.1			100.0
Pos. control##	550 μg			-			22 / 0 hrs			9.0			8.9			9.0			68.6
Test item	100 μg			-			22 / 0 hrs			15.0			11.2			13.1			100.4
″	200 μg			-			22 / 0 hrs			7.8			11.5			9.7			73.9
″	300 μg			-			22 / 0 hrs			9.2			8.9			9.1			69.3

* The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

** For the positive control groups and the test item groups, the relative values of the mitotic index are related to the solvent controls

# DMSO

## EMS

Table 16. Structural chromosome aberrations Experiment IID; preparation interval 22 hrs without S9 mix: exposure period 22 hrs.

Slide				Cells										% Aberrant cells										Aberrations**
no.	scored			incl.			excl.				carrying ex-						Gaps				Chromatid type					Chromosome type					Other
gaps*	gaps*	changes		g	ig		b		f			d				ex			ib		if		id				cx		ma			cd
Without S9 mix
Solvent control: DMSO 0.5 %***
1	300	2.3	2.3	0.0		0		0		4			2		0			0		0		2			0			0		0			0
2	300	0.3	0.3	0.0		0		0		1			0		0			0		0		0			0			0		0			0
1 + 2	600	1.3	1.3	0.0		0		0		5			2		0			0		0		2			0			0		0			0
Positive control: EMS 550μg / mL
1	150	20.0	20.0	5.3		4		0		22			1		0			9		2		1			0			0		0			0
2	150	21.3	21.3	9.3		1		0		24			3		0			17		3		1			0			0		0			0
1 + 2	300	20.7	20.7	7.3		5		0		46			4		0			26		5		2			0			0		0			0
Test item: 100 μg / mL***
1	300	1.3	1.0	0.3		1		0		1			0		0			1		1		0			0			0		0			0
2	300	2.3	2.0	0.0		1		0		3			1		0			0		1		3			0			0		0			0
1 + 2	600	1.8	1.5	0.2		2		0		4			1		0			1		2		3			0			0		0			0
Test item: 200 μg / mL***
1	300	1.0	1.0	0.0		0		0		3			0		0			0		0		0			0			0		0			0
2	300	2.0	2.0	0.3		0		0		4			0		0			0		0		1			0			1		1			0
1 + 2	600	1.5	1.5	0.2		0		0		7			0		0			0		0		1			0			1		1			0
Test item: 300 μg / mL***
1	300	1.7	1.3	0.0		1		0		1			1		0			0		0		1			0			0		1			0
2	300	2.3	2.0	0.0		1		0		3			1		0			0		1		1			0			0		0			0
1 + 2	600	2.0	1.7	0.0		2		0		4			2		0			0		1		2			0			0		1			0

* Including cells carrying exchanges

** Note: multiple aberrations may occur in a single cell, therefore, the numbers in these columns may not (nor are they intended to) correlate with the number in the columns of % Aberrant cells.

*** Evaluation of 300 metaphases per culture

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, 4,4’-Methylenediphenyldiamine is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or precipitating concentrations.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

no metabolic activation (S9 mix) was used.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Aldrich (Milwaukee, WI, USA).

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. C.C. Chang (Michigan State University, East Lansing, MI, USA)
- Cell cycle length: 12 - 14 h
- Methods for maintenance in cell culture: Cells were maintained in 75 cm² flasks with 15 mL Minimum Essential Medium (MEM), 10 % FBS, 2mM L-glutamine, 100 U penicillin/mL, 100 µg streptomycin/mL and sub-cultured every 3 – 4 days by treatment with trypsin–EDTA solution in PBS. Cultures were incubated at 37 °C in a humidified atmosphere of 5 % CO2 for all experiments.

MEDIA USED
- Type and identity of media including CO2 concentration: Minimum Essential Medium (MEM) at 37 °C in a humidified atmosphere of 5 % CO2.
- Properly maintained: yes

Cytokinesis block (if used):

cytochalasin B (Cyt B)

Metabolic activation:

without

Test concentrations with justification for top dose:

62.5 ,125, 250 or 500 µg/mL
The exposure concentration levels were selected from a prior study.

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: vincristine sulfate (VCR), mitomycin C (MMC)

Remarks:

Cysteine and GSH (500 µg/mL) were used as thiol controls.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 2 x 10E5 cells/mL

DURATION
- Preincubation period: 26 h
- Exposure duration: 3 h
- Expression time (cells in growth medium): 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 23 h

SPINDLE INHIBITOR (cytogenetic assays):
cytochalasin B (Cyt B)

STAIN (for cytogenetic assays):
Slides for immunofluorescent staining of kinetochore in binucleated cells were rinsed with PBS, treated with Tween 20–PBS, and cells were labeled with anti-kinetochore antibody and goat anti-human IgG antibody (FITC). Propidium iodide (PI) was used to stain the cells followed by treatment with anti-fade solution.

NUMBER OF REPLICATIONS:
1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cultures were terminated after incubation with Cyt B by rinsing with PBS, and treated with hypotonic 0.075 M KCl. After fixation of the cells in cold methanol (−20 °C) for 15 min, the cells were air dried at room temperature for a few minutes. Cell-cycle kinetic analysis (nuclear division index, NDI) to determine the cytotoxicity of chemical exposures was performed on two slides/exposure.

NUMBER OF CELLS EVALUATED:
The number of micronuclei at each dose level was determined by scoring 2000 binucleated cells per culture and two experiments were run with a total of 4000 cells counted per treatment.

DETERMINATION OF CYTOTOXICITY
- Method: other: nuclear division index
- Any supplementary information relevant to cytotoxicity: NDI was determined by scoring the number of cells with one to four nuclei in 1000 V79 cells per group:

Statistics:

Frequency of MN was calculated per 1000 cells. Statistical analysis was performed using the Cochran and Armitage trend test and variance t-test for group comparisons. Percentages of KC+ and KC− were compared by the Chi-square test. The statistical analysis of NDI was performed using the t-test. All statistical comparisons are made in reference to the DMSO control cultures.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1: Frequency of MN and percentage of KC+ in cytokinesis-blocked V79 cells after treatment

Chemical treatment	Concentration (µg/mL)	Number of MN cells (‰)a			KC+(%)	NDIb ± S.D.
		Mean c ± S.D.	KC+ ± S.D.	KC− ± S.D.
DMSO	Vehicle	17.3 ± 4.6	6.3 ± 2.5	11.0 ± 2.9	36	2.1 ± 0.1
VCR	0.04	113.5 ± 6.4∗∗	104.5 ± 7.8∗∗	9.0 ± 1.2	92∗∗	2.3 ± 0.0
MMC	0.04	115.0 ± 12.7∗∗	24.0 ± 1.4∗∗	91.0 ± 14.1∗∗	21∗∗	1.9 ± 0.0
MDA	62.5	48.3 ± 2.2∗∗	7.8 ± 2.6	49.5 ± 2.6∗∗	14∗∗	2.0 ± 0.1
MDA	125	67.8 ± 10.9∗∗	7.3 ± 2.9	60.5 ± 8.7∗∗	11∗∗	2.0 ± 0.1
MDA	250	74.8 ± 14.7∗∗	15.0 ± 3.7	59.8 ± 18.2∗∗	20∗∗	1.9 ± 0.1
MDA	500	80.5 ± 9.7∗∗	13.8 ± 5.9	66.8 ± 4.9∗∗	17∗∗	1.9 ± 0.1
a The results shown are the means of two experiments based on 4000 cells scored. b Nuclear division index=[M1+(2×M2)+(3×M3)+(4×M4)]/N, where M1–M4 are the number of cells with one to four nuclei andNis the total number of cells (2000). cA concentration–response relationship. Trend testZ=12.174;P <0.01. ∗∗P <0.01 by Chi-square test and variance t-test.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

GLP compliance:

not specified

Type of assay:

other: unscheduled DNA synthesis

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Nippon Kayaku Co., Tokyo, Japan

Species / strain / cell type:

hepatocytes: from male ACI/N rats

Metabolic activation:

without

Test concentrations with justification for top dose:

1 to 1000 µmol/L (1-100 µmol = 19.8 - 19800 µg/mL)

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-2-fluorenylacetamide

Key result

Species / strain:

hepatocytes: from male ACI/N rats

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

clear dose dependency

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 19800 µg/mL totally cytotoxic

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No information on the number of cells counted were given. Hepatocyte viability was not indicated.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

GLP compliance:

not specified

Type of assay:

other: unscheduled DNA synthesis

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Radian Corporation, Austin, TX, USA

Species / strain / cell type:

hepatocytes: adult male Sprague-Dawley rats

Metabolic activation:

without

Test concentrations with justification for top dose:

0.5 - 250 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

Pretreatment: Aroclor1254 was administered intraperitoneally (i.p.) at 500 mg/kg body weight in sterile corn oil 1 day prior to killing while Phenobarbital was administered i.p. at a dose of 80 mg/kg body weight in sterile saline for 3 days with killing occurring on day 4.

Isolation/culture: Hepatocyte primary cultures were established from the livers of untreated and pretreated adult male Sprague- Dawley rats, ranging from 215 to 346 g body weight, via the in situ collagenase perfusion technique. Yields of 6.96 x 10E8 to 12.8 x 10E8 hepatocytes with 81-95.3% viability were obtained. Monolayer cultures were established by plating 1.25 x 10E6 viable hepatocytes well in six-well Costar tissue culture dishes; each well contained a 22 mm round Thermonox (Miles Laboratories) coverslip. Following a 1-2 hr attachment period in Medium containing 10% FBS, cells were incubated overnight at 37°C, 95% air/5% CO2 humidified atmosphere in serum-free Medium containing 10 pCi/mL methyl-[3H]thymidine and an appropriate dose of test chemical delivered in DMSO. Treatment was performed in triplicate, and the solvent concentration did not exceed 1%.

Autoradiography and measurement of UDS: Following incubation in treatment medium with meth-yI-|3H]thymidine, the hepatocytes were washed three times in 1x Hank's Balanced Salt Solution, fixed in 3:1 methanol:glacial acetic acid for two 15 min periods, dehydrated in 70% and 95% ethanol, washed twice with cold 5% trichloroacetic acid for 30 min, dehydrated in 70% and 95% ethanol, and air dried. The cell-coated coverslips were then mounted on glass slides with a gelatin mounting medium and thinly coated with a gelatin/glycerin subbing solution to ensure uniform coating of the coverslips with photographic emulsion. The slides were dipped in a 1:2 mixture of Kodak NTB-2 photographic emulsion:distiIled deionized water, air dried, sealed in light-tight canisters containing desiccant, and autoexposed at 5°C for 6 days. After autoexposure the emulsion was developed with Kodak developer (D-19) for 2 min, fixed with Kodak Rapid Fix Hardener for 5 min, washed in cold running water for 5 min, and dehydrated for 5 min in 95% ethanol. After drying, the hepatocytes were stained with Giemsa stain.

Evaluation criteria:

Fifty viable cells per slide were chosen at random and examined. Developed emulsion grains in an appropriate area over the nucleus and cytoplasm of each cell were counted using an Artek 880 electronic counter. The data in this study were reported for the 150 counts/dose as the mean and standard error of the mean of the NNG counts. Percentage in repair (%IR) data were obtained by identifying the number of NNG counts of three or more over the concurrent solvent controls and reported as the mean %IR per dose.This study reports that a response is evaluated as positive with 95% confidence with a false-positive error rate of less than 1% if the NNG count is three NNGs higher than the solvent control for the same animal. Additionally, when a response is evaluated as marginal, the %IR is used as an adjunct evaluation to the number of NNGs and, depending on the number of cells in repair, is used to identify a positive response or the necessity to repeat an experiment. All experiments were performed at least twice, exhibiting similar responses on each occasion.

Key result

Species / strain:

hepatocytes: from male Sprague-Dawley rats

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/mL cytotoxic

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

hepatocytes: from Sprague-Dawley rats

Genotoxicity:

positive

Remarks:

from 25 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/mL cytotoxic

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: tested without pretreatment

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

Number of donors was limited to 1; evaluation criteria not clearly stated

GLP compliance:

no

Type of assay:

other: unscheduled DNA synthesis

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Sigma Chimica (Milan, Italy)
- Purity: >97 %

Species / strain / cell type:

hepatocytes: rat

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Sprague Dawley rats
- Sex: male
- Methods for isolation of cell culture: Williams et al. (1977)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Williams medium E (WME) supplemented with 10% fetal bovine serum and 50 µg/mL gentamicin

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

hepatocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: discarded surgical material
- Sex, age and number of blood donors if applicable: 72-year old female; 67-year old male
- Methods for isolation of cell culture: Strom et al. (1982)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Williams medium E (WME) supplemented with 10% fetal bovine serum and 50 µg/mL gentamicin

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

hepatocytes:
10, 18, 32, 56, 100, 180 µM (rat)
10, 18, 32, 56 µM (human)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Details on test system and experimental conditions:

Method
Aliquots of the various cell suspensions were plated in 35-mm dishes coated with rat tail collagen (10E6 cells per dish) for determination of cytotoxicity and DNA repair synthesis. After an attachment period of 3 h at 37 °C in an atmosphere of 95 % air–5 % CO2, cell cultures were washed and incubated for 4 or 20 h with serial concentrations of the test compound in serum-free medium. [methyl-3H]Thymidine (10 Ci/mL) was added to hepatocyte culture to be used for the DNA repair assay. At the end of treatment, cells were immediately examined for cytotoxicity by quantitative autoradiography.

Determination of DNA repair
DNA repair synthesis was evaluated according to the autoradiographic method developed by Williams (1977), as described previously by Brambilla et al. (1989). The data are expressed as the mean ±SD of the 100 net nuclear counts obtained from two autoradiographs prepared from each rat or human donor. Cytoplasmic labeling was also considered to evaluate a possible effect of MDA on mitochondrial DNA.

Evaluation criteria:

Not given

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

hepatocytes: human

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A 20-h exposure to the tested concentrations elicited a modest amount of DNA repair synthesis in primary hepatocytes from five rats and two human donors, as indicated by the increase in both the number of net nuclear grains (NNGs) and the percentage of repairing cells. In rat hepatocytes the average increase over controls of NNGs was dose dependent over at least two consecutive concentrations (10 and 18 µM) and exceeded the lab-specific threshold (NNGs treated – NNGs control >= 5), thus matching the criteria of international guidelines for a positive response, but dose dependence was absent at concentrations ranging from 32 to 180 µM. In cultures of human hepatocytes, the above criteria for a positive response were not fulfilled, even if an increase over controls of both NNGs and the percentage of repairing cells was observed at all concentrations tested.

A preliminary cytotoxicity assay provided evidence that a 20-h exposure to test item concentrations ranging from 10 to 180 µM produced a modest dose-dependent reduction in the fraction of viable tryphan blue-excluding rat hepatocytes and thyreocytes; at 180 µM test item the average fraction of viable cells was 72 % of that observed in controls for hepatocytes.

Table 1: DNA Repair Synthesis in Primary Cultures of Rat and Human Hepatocytes Exposed to the test item for 20 h

Species	Test concentrations [µM]	Nuclear grain count (NG)	SD	Cytoplasmatic grain count (CG)	SD	Net nuclear grains (NNG)	SD	Cell with ≥ NNG (%)	SD
Rat	0	21.5	6.0	19.8	6.2	1.7	1.8	31.0	9.2
	10	27.0	3.3	18.7	2.3	8.3	1.0	65.0	1.4
	18	25.5	6.9	16.7	3.9	8.8	3.4	70.0	17.4
	32	20.4	8.8	12.3	4.9	8.1	4.6	65.0	18.7
	56	19.3	7.8	11.1	2.9	8.2	6.3	63.0	27.4
	100	15.1	0.4	10.0	0.9	5.1	0.5	47.0	5.7
	180	12.4	-	6.3	-	6.1	-	57.0	-
	NDMA (5 mM)	66.6	18.5	19.2	6.1	47.4	13.7	99.8	0.4
Human	0	9.3	6.3	6.3	5.2	3.0	1.1	33.0	12.7
	10	14.8	-	5.9	-	8.9	-	83.0	-
	18	11.4	6.0	4.0	3.0	7.4	2.9	70.0	18.4
	32	10.9	2.3	3.0	1.8	7.9	0.6	74.0	5.7
	56	12.2	4.5	4.4	4.7	7.8	0.1	79.0	7.1
	NDMA (5 mM)	38.2	7.8	3.9	4.5	34.3	3.3	100.0	-

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The increases of UDS after treatment with 4,4’-MDA observed in vitro were not confirmed by mouse and rat liverin vivodata (Mirsalis et al., 1989). Increases in DNA fragmentation was reported in the liver, stomach, kidney, bladder, lung, brain and (slightly) colon of mice, however only at doses that likely exceeded the MTD (Sasaki et al., 1999).

Formation of DNA adducts with MDA (investigated using the 32P-postlabeling assay) was observed in the livers of orally treated rats (Vock et al., 1996). The DNA binding potency of MDA was investigated in rat liver and was considered to be low by the authors, in the range of a weak genotoxic carcinogen (Schuetze et al., 1996).

Formation of micronuclei after short-term treatment (single to triple application) of mice or rats with 4,4’-MDA was investigated in three different studies (Shelby et al., 1993; Morita et al., 1997; Suzuki et al., 2005). While reporting of these studies was limited (e.g., rationale for selection of dose levels unclear, clinical/target organ toxicity insufficiently reported, route of application partly unclear), overall they gave no clear indication of relevant increases in micronucleus frequencies of bone marrow cells or peripheral blood of both species and rat liver.

Relevant increases in the incidences of micronucleated cells were, however, observed in rats after 14-day repeated dosing in liver and bone marrow cells and after 28-day repeated dosing in liver, but not in the bone marrow. The positive findings in the liver were accompanied by hepatocyte hypertrophy, single cell necrosis, inflammatory cell infiltration and bile duct proliferation for both treatment schedules. In addition, increased mitotic figures were observed after 28-day repeated dosing (Hamada et al., 2015; Sanada et al., 2015). These findings are in line with hepatic lesions observed after 90-day repeated dosing in rats at comparable dose levels (see Repeated dose toxicity section of the IUCLID).

Further data on mutagenicity available from anin vivogene mutation assay in erythrocytes (Pig-a-assay) and reticulocytes (PIGRET assay): Administration of single doses of 4,4’-MDA did not lead to an increase in mutation frequency in erythrocytes or reticulocytes, whereas repeated dosing showed increased mutation frequencies in erythrocytes after 14 and 28 days of dosing and in reticulocytes after 14 days of dosing. The increased incidences in mutation frequencies were accompanied by hepatic toxicity and hematological findings, i.e. reduction in red blood cells, increase in reticulocytes, neutrophils, monocytes and basophils (Sanada et al., 2014). The increased incidences in mutation frequencies were accompanied by hepatic toxicity and hematological findings, i.e. reduction in red blood cells, increase in reticulocytes, neutrophils, monocytes and basophils (Sanada et al., 2014).

NTP (1986) reported structural chromosomal aberrations and increases in SCE in mouse bone marrow, but these results are considered “not assignable” due to extremely limited reporting.

A weight-of-evidence evaluation of the studies by Hamada et al. (2015) and Sanada et al. (2015), Shelby et al. (1992), Morita et al. (1997) and Suzuki et al. (2005) suggests that the liver is the more relevant target tissue for genotoxicity than the bone marrow. While mutagenicity on erythropoietic cells was reported by Sanada et al. (2014), the statistical significance of the findings is not clearly reported, and the authors themselves could not exclude that the observed changes in hematopoietic activities induced by repeated MDA dosing may have contributed to the positive results. The hypothesis of the liver being the primary target organ for genotoxicity is supported by a publication of Robbiano (1999), where single to triple treatment with 4,4’-MDA did not lead to an increase in micronucleated cells or in DNA fragmentation in the kidney of rats.
The data described above indicate that 4,4’-MDA requires repeated administration to elicit genotoxicity in the liver as the relevant target organ. However, it cannot be unequivocally excluded that the increased incidences in micronucleated cells reported by Hamada et al. (2015) and Sanada et al. (2015) were unspecific findings secondary to the observed liver toxicity.

 

In conclusion, the whole of the available data indicate that MDA is genotoxicin vivoin the liver, where it also causes severe hepatotoxicity after acute and repeated dosing. There are some indications of other target organs in which MDA may cause genotoxicity, but the reports are inconclusive.

Since liver tissue is known to be highly regenerative, the accumulation of damaged cells and an increase in cellular proliferation would subsequently reduce the time available for the repair of DNA damage and increase the probability for the manifestation of a mutation.

Increased cell proliferation due to severe cytotoxicity is considered a tumor promoting effect, which might be also relevant in the mechanism of carcinogenicity of 4,4’-MDA. The tumor promoting effect of 4,4’-MDA has been demonstrated in a rat carcinogenicity model using an initiation/promotion protocol and is further described in the carcinogenicity section of the IUCLID.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

too little information on selection of dose levels, target organ toxicity unclear, uncertain why one animal died during the first experiment (substance-related death or not?)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Only 4 instead of 5 animals were analysed in Experiment I; 200 cells per animal were scored for PCE (instead of 500); 2000 cells per animal were scored for MN (instead of 4000); No information on historical control data were given.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Radian Corporation, Austin, TX

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Toxicology Program production facility at Taconic Farms
- Age at study initiation: 9-14 weeks
- Weight at study initiation: 25-33 g

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

All treatments were by intraperitoneal injection at a volume of 0.4 mL per mouse.

Duration of treatment / exposure:

3 injections (once daily)

Frequency of treatment:

3 consecutive days

Post exposure period:

Mice were euthanized with CO2 24 hr after the third treatment.

Dose / conc.:

9.3 mg/kg bw/day (actual dose received)

Dose / conc.:

18.5 mg/kg bw/day (actual dose received)

Dose / conc.:

37 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylbenzanthracene
- Route of administration: intraperitoneal
- Doses / concentrations: 12.5 mg/kg bw in corn oil

Tissues and cell types examined:

The frequency of MN-PCE among 2000 PCE and the percentage of PCE among 2000 erythrocytes were determined

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of the maximum dose to be tested for MN induction was based on either mortality, administration characteristics (ability to be administered as a homogeneous suspension in corn oil or dissolved in PBS), depression in the percentage of bone marrow PCE (no less than 15% of the erythrocytes).

SAMPLING TIMES: 24 h after treatment

DETAILS OF SLIDE PREPARATION: Bone marrow smears (two slides/mouse) were prepared, fixed in absolute methanol, and stained with acridine orange.

Evaluation criteria:

no data

Statistics:

The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives. The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group were by an unadjusted one-tailed Pearson chisquared test which incorporated the calculated variance inflation factor for the study.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- The initial test gave a trend P = .054 with MN-PCE frequencies ranging from 1.7 in control to 2.9 at the high dose of 37 mg/kg bw. The repeat test using the same doses gave significant trend test (P = .019) from 1.9 in control to 3.5 at 37 mg/kg bw.

Table 1: MN Data analysis - Trial 1 and 2

Dose (mg/kg bw)	MN-PCE/1.000 (No. animals)	Pair-wise	Survival	% PCE	Trend P value
0	1.70+/-0.20(5)	.	5/5	51.3	0.054
9.3	2.30+/-0.41(5)	0.1711	5/5	44.5
18.5	2.50+/-0.84(4)	0.1195	5/5	43.3
37	2.88+/-0.97(4)	0.0481	4/5	36.9
0	1.90+/-0.19(5)	.	5/5	35.4	0.019
9.3	2.70+/-0.44(5)	0.1188	5/5	45.9
18.5	2.40+/-0.33(5)	0.2226	5/5	54.7
37	3.50+/-0.32(5)	0.0146	5/5	62.0

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

liver and bone marrow micronucleus assays

Type of information:

other: Review article

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

In vivo repeated dose micronucleus (MN) assay in rats. Examined tissues: bone marrow and liver

GLP compliance:

yes

Type of assay:

other: liver and bone marrow micronucleus assays

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot No of test material: Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Lot No LAR7254

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 6 weeks old
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle: methyl cellulose, 0.5 %

Duration of treatment / exposure:

14 or 28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

14 and 28 days treatment

Dose / conc.:

4.4 mg/kg bw/day (actual dose received)

Remarks:

28 days treatment

Dose / conc.:

13 mg/kg bw/day (actual dose received)

Remarks:

28 days treatment

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Remarks:

28 days treatment

Dose / conc.:

120 mg/kg bw/day (actual dose received)

Dose / conc.:

15.6 mg/kg bw/day (actual dose received)

Remarks:

14 days treatment

Dose / conc.:

31.3 mg/kg bw/day (actual dose received)

Remarks:

14 days treatment

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Remarks:

14 days treatment

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Remarks:

14 days treatment

Positive control(s):

Diethylnitrosamine (DEN) for the liver MN assay. Throughout the study of the liver MN assay, DEN was dissolved in distilled water and given daily by oral gavage to 5 rats at dose levels of 0, 3.13, 6.25, and 12.5 mg/kg bw/day for 14 days.

Tissues and cell types examined:

Hepatocytes, bone marrow cells

Statistics:

Differences in the incidences of MNHEPs and MNIMEs between the test and vehicle control groups were analyzed by the conditional binomial test reported by Kastenbaum and Bowman (1970) at upper-tailed significance levels of 5% and 1%. We determined positive results based mainly on the statistical analysis, and we also considered biological relevance, i.e., the historical control at the laboratory where the study was conducted, as well as the dose-response relationship. The other quantitative data were analyzed for their statistical significance by the multiple comparison procedure. Namely, the homogeneity of variance was examined using Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied; otherwise, Kruskal–Wallis test was applied. When statistical significance was demonstrated between the groups, the difference was assessed using Dunnett’s test or the Dunnett-type multiple comparison test.

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

liver MN,

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not specified

Remarks on result:

other: after 14 and 28 repeated dose treatment

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

BM MN

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not specified

Remarks on result:

other: after 14 but not 28 days repeated dose treatment

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Only 1000 cells instead of 4000 cells were analysed for micronuclei. No information was given on the proportion of mature/immature cells and on historical control data. No separate controls were analysed for the different time points.

Principles of method if other than guideline:

The test substance was injected intraperitoneally.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot No. of test material: Wako, JPG9015
- Purity: >98 %

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-10 weeks
- Diet (e.g. ad libitum): Food pellets
- Water (e.g. ad libitum): tap water

Route of administration:

intraperitoneal

Vehicle:

olive oil

Duration of treatment / exposure:

no data

Frequency of treatment:

Although the number of administrations was not specified, intervals between them were 24 h

Post exposure period:

no data

Remarks:

A single dose of 56 and 112 mg/kg bw in one study, and those doses plus 140 mg/kg bw in a second study and two daily doses of 22, 45 and 90 mg/kg bw in a third study

No. of animals per sex per dose:

5 males

Control animals:

other: sample from each animal immediately before treatment (0-h sample) was used as the negative control

Positive control(s):

mitomycin C
- Route of administration: single intraperitoneal treatment
- Doses / concentrations: 0.5 mg/kg bw
- sampled at 24 h or 48 h for bone marrow and peripheral blood, respectively

Tissues and cell types examined:

peripheral blood reticulocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Acute toxicity tests following Lorke's method or a minor modification of it, were performed to determine the toxic dose. Toxicity was monitored for a period of about 4 days. The highest dose was based on mortality. Micronucleus assay was performed at three levels - the highest dose and 1/2 and 1/4 of the highest dose.

METHOD OF ANALYSIS: 1000 cells/animal assessed at 0, 24, 48 and 72 hours after treatment.

Evaluation criteria:

no data

Statistics:

no data

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

One dose experiments: a weak but dose-dependent increase was observed in one experiment and a marginal increase in the other; Two daily doses experiment: negative

Toxicity:

yes

Remarks:

The highest dose was near the LD50

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1. 4,4'-Methylenedianiline [101-77-9]

Dose (mg/kg)	0h	24h		48h		72h
	Mean   SD	Mean   SD	p	Mean   SD	p	Mean   SD	p
28	0.14 +/- 0.05	0.14 +/- 0.11	0.605	0.22 +/- 0.08	0.240	0.28 +/- 0.15	0.095
56	0.10 +/- 0.07	0.24 +/- 0.15	0.072	0.30 +/- 0.14	0.021	0.28 +/- 0.26	0.032
112	0.16 +/- 0.11	0.22 +/- 0.15	0.324	0.42 +/- 0.08	0.012	0.12 +/- 0.13	0.788
.	.	.	(0.043)	.	(0.000)	.	(0.239)
28	0.16 +/- 0.11	0.18 +/- 0.08	0.500	0.24 +/- 0.11	0.252	0.22 +/- 0.08	0.324
56	0.12 +/- 0.08	0.18 +/- 0.18	0.304	0.20 +/- 0.07	0.227	0.18 +/- 0.04	0.304
112	0.14 +/- 0.11	0.18 +/- 0.15	0.402	0.26 +/- 0.05	0.132	0.28 +/- 0.08	0.095
140	0.14 +/- 0.11	0.26 +/- 0.05	0.132	0.26 +/- 0.18	0.132	0.24 +/- 0.11	0.180
.	.	.	(0.051)	.	(0.015)	.	(0.016)

Table 2. 4,4'-Methylenedianiline [101-77-9]

Dose (mg/kg)	0h	24h		48h		72h
	Mean   SD	Mean   SD	p	Mean   SD	p	Mean   SD	p
22.5	0.16 +/- 0.11	0.18 +/- 0.11	0.500	0.12 +/- 0.04	0.788	0.16 +/- 0.15	0.598
45	0.14 +/- 0.05	0.24 +/- 0.09	0.180	0.24 +/- 0.17	0.180	0.22 +/- 0.13	0.240
90	0.12 +/- 0.11	0.24 +/- 0.17	0.119	0.12 +/- 0.08	0.613	0.18 +/- 0.15	0.304
.	.	.	(0.038)	.	(0.348)	.	(0.160)

LD50 Maximum tolerance dose: Table 1 = 141mg/kg / Table 2 = 113 mg/kg

No of treatments at 24 hr intervals: Table 1 = 1 / Table 2 = 2

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

According to the NTP standard protocols. No guideline is in place for this study type. When compared with the OECD 479 guideline for in vitro SCE analysis (deleted in 2014) and current OECD guidelines for cytogenetic damage in vivo there are a few methodological deficiencies. The number of metaphases is considered to be low with regard to the number of metaphases which is evaluated nowadays in cytogenetic studies (200 metaphases per animal for chromosomal aberrations). In addition, only 4 or 3 animals instead of 5 animals were evaluated. No information on toxicity is available, there are no clear evaluation criteria reported and no information on the historical control data range is given. The application of the substance via intraperitoneal injection is not recommended by current cytogenetic guidelines.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in the cold throughout the study

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Kodak Company (Rochester, NY, USA), Lot No: A6A, A8
- Expiration date of the lot/batch:
- Purity: A6A: 102 %; A8: 98.6 %

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO

Frequency of treatment:

no data

Remarks:

Doses / Concentrations:
Trial 1, 2 and 4: 50, 100 and 200 mg/kg bw; Trial 3: 35, 70 and 140 mg/kg bw

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

dimethylbenzanthracene;
- Doses / concentrations: 2.5 - 50 mg/kg bw

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

SAMPLING TIMES:
Trail 1, 2 and 4: 23 h
Trial 3: 42 h

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Trail 1

	Dose (mg/kg bw)	n	SCE / Cell (Mean ± SEM)	Pairwise P
Dimethylsulfoxide	0	4	4.25 ±  .398	.0000
Methylenedianiline	50	4	6.72 ±  .730	.0171
	100	4	4.70 ±  .715	.3017
	200	4	4.17 ±  .170	.4396
Dimethylbenzanthracene	2.5	4	7.82 ±  .612	.0021

 

Trial 2:

	Dose (mg/kg)	n	SCE / Cell (Mean ± SEM)	Pairwise P
Dimethylsulfoxide	0	4	3.90 ±  .774	.0000
Methylenedianiline	50	4	4.95 ±  .319	.1399
	100	4	5.28 ±  .186	.0859
	200	4	6.35 ±  .792	.0343
Dimethylbenzanthracene	2.5	4	8.73 ±  .984	.0046

 

Trial 3:

	Dose (mg/kg)	n	SCE / Cell (Mean ± SEM)	Pairwise P
Dimethylsulfoxide	0	4	4.97 ±  .517	.0000
Methylenedianiline	35	4	3.73 ±  .230	.0450
	70	4	3.41 ±  .349	.0252
	140	4	6.44 ±  .372	.0334
Dimethylbenzanthracene	50	4	16.33 ±  1.202	.0005

 

Trial 4:

	Dose (mg/kg)	n	SCE / Cell (Mean ± SEM)	Pairwise P
Dimethylsulfoxide	0	4	4.39 ±  .575	.0000
Methylenedianiline	50	4	4.71 ±  .287	.3211
	100	4	5.15 ±  .598	.1996
	200	4	7.98 ±  .370	.0016
Dimethylbenzanthracene	2.5	4	11.43 ±  1.203	.0025

 

Reference 5

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

A second sampling time (> 24 hours) is missing. No information on toxicity and historical control data is given. The evaluation criteria are not clearly defined. Only 50 metaphases instead of 200 per animal were evaluated for cytogenetic damage.

Principles of method if other than guideline:

According to the NTP standard protocols. The test substance was injected intraperitoneally. 8 animals per dose were treated.

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in the cold throughout the study

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Kodak Company (Rochester, NY, USA), Lot No: A6A, A8
- Expiration date of the lot/batch:
- Purity: A6A: 102 %; A8: 98.6 %

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO and Corn oil

Frequency of treatment:

no data

Dose / conc.:

50 mg/kg bw (total dose)

Dose / conc.:

100 mg/kg bw (total dose)

Dose / conc.:

200 mg/kg bw (total dose)

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Positive control(s):

dimethylbenzanthracene;
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

SAMPLING TIMES:
Trial 1, 2 and 3: 17 h

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Summary of the Results of Trial 1

Test Group		Dose (mg/kg bw)	n	Percent Cells w/ Aberrations (Mean ± SEM)	Pairwise P
Vehicle Control	Dimethylsulfoxide	0	8	1.75 ± 0.59
Test Data	Methylenedianiline	50	8	1.75 ± 0.80	0.5000
		100	8	1.25 ± 0.37	0.7196
		200	8	1.00 ± 0.76	0.8188
Positive Control	Dimethylbenzanthracene	100	8	14.50 ± 1.76	0.0000

 

Table 2: Summary of the Results of Trial 2

Test Group		Dose (mg/kg)	n	Percent Cells w/ Aberrations (Mean ± SEM)	Pairwise P
Vehicle Control	Corn Oil	0	8	3.00 ± 1.00
Test Data	Methylenedianiline	50	8	4.25 ± 1.87	0.1721
		100	8	2.50 ± 0.73	0.6673
		200	8	8.00 ± 1.89	0.0010
Positive Control	Dimethylbenzanthracene	50	8	24.25 ± 4.32	0.0000

 

Table 3: Summary of the Results of Trial 3

Test Group		Dose (mg/kg)	n	Percent Cells w/ Aberrations (Mean ± SEM)	Pairwise P
Vehicle Control	Dimethylsulfoxide	0	8	0.75 ± 0.53
Test Data	Methylenedianiline	100	8	2.00 ± 0.65	0.0645
		150	8	4.25 ± 1.79	0.0008
		200	8	7.50 ± 3.02	0.0000
Positive Control	Dimethylbenzanthracene	50	8	23.00 ± 6.01	0.0000

Reference 6

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

micronucleus test in mouse kidney

GLP compliance:

not specified

Type of assay:

other: in vivo micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material:Sigma Chimica (Milan. Italy)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Nossan (Carezzana, Italy)
- Weight at study initiation: 100-150 g
- Acclimation period: min. one week
- Diet: rat chow (S. Morini. S.Polo d'Enza. ltaly), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 10
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Solvent used: Ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The media containing the test item was freshly prepared from stock solutions in ethanol.

Duration of treatment / exposure:

- single administration
- 3 days repeated dose administration

Frequency of treatment:

daily

Post exposure period:

- single administration: 2 days
- 3 days repeated dose administration: 1 day

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

414 mg/kg bw/day (actual dose received)

Remarks:

single administration

Dose / conc.:

277 mg/kg bw/day (actual dose received)

Remarks:

3 days repeated dose administration

No. of animals per sex per dose:

3 males/dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

potassium bromate (PB)
- single administration: 160 mg/kg bw
- 3 days repeated dose administration: 107 mg/kg bw/day

lead acetate (LA)
- single administration: 117 mg/kg bw
- 3 days repeated dose administration: 78 mg/kg bw/day

Tissues and cell types examined:

kidney cells,

Details of tissue and slide preparation:

see "any other information on materials and methods"

Evaluation criteria:

Micronucleus Assay: Micronucleated kidney cells were counted regardless of the number of micronuclei per cell. Scoring was limited to cells with intact nuclear and cellular membranes: no distinction was made with respect so cellular ploidy. Only small bodies lying in close proximity to the nucleus, with shapes and staining properties like the parent nucleus and a diameter less than a third of that of the main nucleus were identified as micronuclei.

Statistics:

Statistical analysis was performed by using the method of Bailey (1959)

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

kidney cells

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Single dose and 3 days repeated dose.

Table 1: Frequency of Micronucleated Cells in the Kidney of Rats Treated p.o. with the Test Item

Treatment condition (mg/kg bw x No of doses)	No of rats	MicronucleusAssay
		No of cells scored	Micronucleated Cells ( ‰)	S.D.	Binucleated Cells (%)	S.D.
Control	8	15148	0.73	0.49	2.15	0.51
LA, 117 mg/kg x 1	3	5565	6.70	3.22	1.99	0.26
LA, 78 mg/kg x 3	3	5261	2.25	1.04	2.00	0.19
PB 160 mg/kg x 1	3	5520	2.37	0.39	1.83	0.33
PB 107 mg/kg x 3	3	5868	2.81	1.18	1.88	0.67
Test Item, 415 mg/kg x 1	3	5979	1.33	0.25	2.76	0.26
Test Item, 277 mg/kg x 3	3	5983	0.83	0.37	2.56	0.34

Reference 7

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Positive control data are not reported; the authors report that statistical analysis was performed, but statistical significance is not indicated in the mutagenicity result tables.

Qualifier:

no guideline available

Principles of method if other than guideline:

- Short description of test conditions: A 28-day repeated-dose test was performed by orally administering the test item to three animals in 4 different dosage groups. Blood samples were obtained from the rats treated with the test item for 28 days, and hematological parameters were measured. A RBC Pig-a and a PIGRET assay was performed. Cells were analyzed via FACS.

GLP compliance:

not specified

Type of assay:

other: in vivo mammalian somatic cell study: gene mutation

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Wako Pure Chemical Industries, Ltd. (Osaka, Japan)

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Co. Ltd. (Yokohama, Japan)
- Age at study initiation: 6-week-old
- Diet: CRF-1 (Oriental Yeast Co. Ltd., Tokyo, Japan), ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23
- Humidity (%): 37 - 54
- Photoperiod (hrs dark / hrs light): 12/12 (7:00 - 19:00)

Route of administration:

oral: gavage

Vehicle:

- Vehicles used: 0.5 w/v % methyl cellulose (test item), distilled water (N-ethyl-N-nitrosourea, ENU)
- Amount of vehicle: 10 mL/kg bw

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

4.4 mg/kg bw/day (actual dose received)

Dose / conc.:

13 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

120 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 male/dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

N-ethyl-N-nitrosourea
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw, single administration

Tissues and cell types examined:

reticulocytes, erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A 5-day repeated-dose test was performed by orally administering the test item to three animals in 4 different dosage groups; 250 mg/kg bw/day was the highest dosage used. In this study, animal death was only observed at the 250 mg/ kg bw/day dosage, and a decrease in body weight was observed at dosages greater than 125 mg/kg bw/day. Therefore, the maximum tolerable dose of 120 mg/kg bw/day was selected as the highest dosage for this study.

SAMPLING TIMES:
On days 7, 14 and 28 during repeated-dosing, blood samples were obtained from the tail veins of the rats.

METHOD OF ANALYSIS:
RBC Pig-a assay:
Blood samples were suspended in phosphate-buffered saline (PBS) with biotinylated HIS49 and fluorescein isothiocyanate (FITC)-conjugated anti-rat CD59 antibodies to label erythrocytes and GPI anchor proteins, respectively. After incubation for 1 h at room temperature, the samples were washed with PBS, centrifuged and resuspended in PBS. Then, al-lophycocyanin (APC)-conjugated streptavidin was added to the samples, and they were incubated at room temperature for 15 min. After resuspension following centrifugation, the samples were used for Pig-a gene mutation analysis.

PIGRET assay:
Blood samples were incubated with a phycoerythrin (PE)-conjugated anti-rat CD7I antibody as a maker of reticulocytes at 4°C for 15 min. After being washed with IMag™ Buffer and centrifuged, the cells were mixed with PE Particles PIus-DM and incubated at 4°C for 15 min. To enrich for CD71-positive cells, the samples were processed with a BD IMagnet™ magnetic stand according to the manufacturer's instructions. The samples were labeled with HIS49 and anti-CD59 antibodies as described in the methods for the RBC Pig-a assay, except that the incubation time for labeling reticulocytes (RETs) was half that of the RBC Pig-a assay.

Flow cytometric analysis:
A FACSCalibur equipped with 488-nm blue and 635-nm red lasers and CellQuest 3.3 software were used for data acquisition/analysis. The gating for enumerating CD59-negative cells was performed. All gates for the detection of CD59-negative RBCs and RETs were set using unstained and single-stained samples before evaluating the experimental samples. The frequency of CD59-negative cells to 1 million HIS49-positive RBCs and 1 million HIS49- and CD71-positive RETs was calculated as the mutation frequency.

Statistics:

For the hematological examination, the statistical significance of the differences between the test item treated rats and control rats were analyzed using the Dunnett's multiple comparison test (parametric). For the Pig-a gene mutation assays, the statistical significance of the differences between the vehicle control group and each test item treated group was analyzed using the Dunnett-type test for multiple comparison (non-parametric). The level of significance was 5% on both sides.

Sex:

male

Genotoxicity:

positive

Remarks:

at 40 mg/kg bw/day

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Pig-a Assay, on day 14 and 28

Sex:

male

Genotoxicity:

positive

Remarks:

at 40 mg/kg bw/day

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: PIGRET assay, on day 14

Additional information on results:

In histopathological findings, severe hepatic impairments such as hepatic cell necrosis, fibrosis and hyperplasia of bile duct were observed at days 14 and 28 in the 40 and 120 mg/kg/day groups. These toxicities were thought to influence a metabolic activity and reduce an exposure of metabolites of the test item. Therefore, the changes in the exposure might to be responsible for the decreased frequencies observed at days 14 and 28. In this study, hepatic toxicity in the 120 mg/kg/day group was greater than that of lower groups and reduction of erythrocytes as well as hepatic toxicities was observed in the maximum dose.

Table 1: Frequencies of CD59-negative cells determined by [he RBCs and PIGRET assays during 28-day dosing

	Mutation Frequencies of pig-a (x 10E-6)
	RBCs assay			PIGRET assay
	Day 7	Day 14	Day 28	Day 7	Day 14	Day 28
Control	0.00	0.00	3.30	6.63	0.00	6.64
	3.32	3.21	3.31	13.29	0.00	6.50
	13.19	10.00	0.00	3.30	3.30	0.00
	3.31	6.55	0.00	9.99	0.00	3.33
	3.30	10.24	3.31	3.32	3.30	0.00
	13.28	0.00	0.00	0.00	3.30	0.00
Ave.	6.06	5.00	1.65	6.09	1.65	2.75
S.D.	5.70	4.65	1.81	4.90	1.81	3.23
TEST ITEM 4.4 mg/kg bw/day	0.00	3.32	3.31	3.32	3.30	0.00
	3.31	16.65	0.00	3.32	3.33	3.33
	3.31	6.64	0.00	19.95	6.65	0.00
	6.63	3.32	0.00	0.00	0.00	3.33
	6.62	3.31	9.91	13.28	0.00	3.33
	3.32	COO	3.31	6.64	3.33	0.00
Ave.	3.87	5.54	2.75	7.75	2.77	1.67
S.D.	2.49	5.83	3.86	7.48	2.50	1.82
TEST ITEM 13 mg/kg bw/day	9.97	10.03	3.05	9.97	9.91	3.33
	6.67	0.00	6.63	23.09	0.00	0.00
	3.32	6.64	0.00	3.32	0.00	6.66
	0.00	29.72	6.55	32.68	9.96	0.00
	3.30	19.89	0.00	3.17	3.32	0.00
	9.98	3.31	5.36	6.64	3.31	3.33
Ave.	5.54	11.60	3.60	13.15	4.42	2.22
S.D.	4.03	11.20	3.07	12.07	4.53	2.72
TEST ITEM 40 mg/kg bw/day	6.61	13.29	13.25	3.30	6.63	3.31
	9.92	9.95	6.65	16.61	6.65	0.00
	13.18	33.22	19.82	6.64	3.29	0.00
	6.64	16.55	9.88	46.67	32.86	3.24
	33.24	23.21	16.22	6.59	3.31	6.51
	16.60	43.07	13.05	43.21	9.92	3.33
Ave.	14.36	23.21	13.15	20.50	10.44	2.73
S.D.	10.02	12.75	4.62	19.48	11.26	2.46
TEST ITEM 120 mg/kg bw/day	3.32	26.31	19.95	3.33	0.00	0.00
	6.66	6.65	9.95	23.29	0.00	3.33
	0.00	6.63	3.32	0.00	3.32	3.33
	0.00	9.96	3.30	23.36	3.31	0.00
	23.27	16.65	3.30	0.00	3.31	3.32
	16.45	39.98	3.32	6.64	3.31	0.00
Ave.	8.28	17.70	7.19	9.44	2.21	1.66
S.D.	9.55	13.24	6.79	11.04	1.71	1.82

Reference 8

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

In vivo repeated dose micronucleus (MN) assay in young adult Sprague Dawley rats. Examined tissues: bone marrow and liver

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Wako Pure Chemical Industries, Ltd. (Osaka, Japan)

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Co. Ltd. (Yokohama, Japan)
- Age at study initiation: 5 weeks
- Housing: two or three rats per cage
- Diet: CRF-1; Oriental Yeast Co. Ltd., Tokyo, Japan; ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 – 23
- Humidity (%): 44 – 65
- Photoperiod (hrs dark / hrs light): 12/12, lights on: 07:00–19:00

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.5% (w/v) Methyl Cellulose 400 Solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan)
- Amount of vehicle: 10 mL/kg bw

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

4.4 mg/kg bw/day (actual dose received)

Dose / conc.:

13 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

120 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5–6 males/group

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

one-third of the left lateral liver lobe, bone marrow cells from femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A 5-day repeated-dose test was performed by orally administering the test item to three animals in 4 different dosage groups; 250 mg/kg bw/day was the highest dosage used. In this study, animal death was only observed at the 250 mg/kg bw/day dosage, and a decrease in body weight was observed at dosages greater than 125 mg/kg bw/day. Therefore, the maximum tolerable dose of 120 mg/kg bw/day was selected as the highest dosage for this study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
On days 7, 14 and 28 after single dosing and during repeated-dosing, blood samples were obtained from the tail veins of the rats and mixed with EDTA after single dosing and during repeated-dosing and used for the Pig-a gene mutation assay.

METHOD OF ANALYSIS:
A FACS Calibur™ (Becton, Dickinson and Company, NJ, USA) equipped with 488-nm blue and 635-nm red lasers and CellQuest 3.3 software (Becton, Dickinson and Company) were used for data acquisition/analysis. The gating for enumerating CD59-negative cells was performed as described previously (12). All gates for the detection of CD59-negative RBCs and RETs were set using unstained and single-stained samples before evaluating the experimental samples. The frequency of CD59-negative cells to 1 million HIS49-positive RBCs and 1 million HIS49- and CD71-positive RETs was calculated as the mutation frequency.

Statistics:

Differences in the incidences of MNHEPs and MNIMEs between the test item treated rats and control rats were analyzed using the conditional binomial test reported by Kastenbaum and Bowman (1970) at the upper-tailed significance levels of 5% and 1%. The other quantitative data were analyzed for statistical significance using the multiple comparison test. Variance was evaluated by Bartlett’s method for equality of variance between groups. Dunnett’s method was used to test the significance between groups when a variance analysis showed equal variance. Additionally, the Dunnett’s rank test was used when a variance analysis showed unequal variance. The level of significance was 5% on both sides.

Sex:

male/female

Genotoxicity:

ambiguous

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Clinical signs and body weight development

A decrease in body weight gain was observed at the maximum dose of the test item in both dosing period groups. There were no changes in body weight gain in the other MDA-treated groups compared with the control animals for both of the dosing period groups. No abnormal clinical signs were observed at any of the doses in either of the dosing periods

Liver and bone marrow MN assay

Statistically significant increases in MNHEPs were observed in the rats treated with the test item at doses of 62.5 mg/kg bw and above and 40 mg/kg bw and above for the 14 and 28-day repeated treatment groups, respectively. The incidences of MNHEPs at the maximum dose in both dosing periods were similar.

Test item treatment induced MNIMEs at doses of 31.3 and 125 mg/kg bw at 14 days after treatment. The increases were statistically significant, but not dose-dependent. In addition, there was no observed significant difference in the frequency of MNIMEs in bone marrow at 28 days after the treatment. There were no significant differences observed in the MIs or the proportion of IMEs at any of the doses or dosing periods.

Histopathological analysis

Hypertrophy of centrilobular hepatocytes was detected in all the test item-treated groups. Necrosis of the centrilobular hepatocytes, proliferation of bile duct cells and infiltration of inflammatory cells were observed at doses of 31.3 mg/kg bw or above for the 14 day treatment group. Proliferation of the bile duct, hepatocyte necrosis and infiltration of inflammatory cells were observed at doses of or above 13 mg/kg for the 28-day treatment group. Additionally, hepatocyte mitosis was noted at doses of or above 40 mg/kg bw.

Reference 9

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

OECD 474 does not apply to MN in liver

Deviations:

yes

Remarks:

One sampling time (48 h); 2000 cells/animal were analysed for MN (instead of 4000); No controls for sampling day 3 and 4 in hepatocytes; No clear evaluation criteria; No information on historical control data, robust control data shown within the trial

Principles of method if other than guideline:

- Short description of test conditions: Male Fischer F344 or SD rats were treated with the test item, solvent substance or positive control substance once intraperitoneally or orally. Each group consisted of four or five animals. Isolated hepatocytes or peripheral blood cells of the rats were stained with DAPI. The prepared cells were evaluated for mirconucleus formation. Genotoxic properties of the test item were evaluated by two laboratories.

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Wako Pure Chemical Industries, Ltd. (Osaka, Japan)

Species:

rat

Strain:

Fischer 344

Remarks:

or SD, not specified

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 4 weeks
- Diet: commercial pellets, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12



Rats were anesthetized with ethylether

Liver micronucleus assay:
Hepatocytes were isolated by the collagenase perfusion method, rinsed with 10% neutral formalin two or three times, centrifuged at 50×g for 1 min, suspended in 10% neutral formalin, and stored under refrigeration.

STAIN (for cytogenetic assays):
10–20 µL of the cell suspension was mixed with an equal volume of acridine orange (AO)–4_6-diamidino-2-phenylindole dihydrochloride (DAPI).

NUMBER OF REPLICATIONS: 1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Approximately 10–20 µL of stained suspension was dropped onto a clean glass slide and covered with a cover slip (24mm×40 mm). Microscopic preparations were evaluated with the aid of a fluorescence microscope (×400 or greater) with UV excitation.

NUMBER OF CELLS EVALUATED:
2000

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Micronucleated hepatocytes (MNHEPs) were defined as hepatocytes with round or distinct micronuclei that stained like the nucleus, with the ≤1/4 diameter of the nucleus.

Peripheral blood micronucleus assay:
A small amount of blood was collected from a tail vessel on Day 2 after treatment, at which time the maximum response was induced.

STAIN (for cytogenetic assays):
The collected blood was stained by either of the following methods: (1) 5 – 10 µL was dropped on to AO-coated slides, covered with cover glasses, and stored in a deep freezer until analysis or (2) 10 µL suspension was mixed with about 30 µL of 10% neutral formalin and stored at room temperature, the samples were mixed with an equal volume of AO solution (500 µg/mL) in the ratio of 1:1 and smeared on a glass slide immediately before analysis.

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED:
reticulocytes (RET): 2000
erythrocytes: 1000

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
micronucleated reticulocytes (MNRETs) were defined as hepatocytes with round or distinct micronuclei that stained like the nucleus, with the ≤1/4 diameter of the nucleus.

Route of administration:

not specified

Vehicle:

- Vehicles: The test item was suspended in olive oil. The positive control substances were dissolved in distilled water.

Frequency of treatment:

single exposure

Post exposure period:

3, 4, or 5 days after administration of the test item.
5 days after administration of the negative or positive control chemicals.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

Remarks:

Lab 1

Dose / conc.:

300 mg/kg bw/day

Remarks:

Lab 1

Dose / conc.:

400 mg/kg bw/day

Remarks:

Lab 1

Dose / conc.:

150 mg/kg bw/day

Remarks:

Lab 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Lab 2

No. of animals per sex per dose:

4 - 5 males/dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

liver microsomal assay: Diethylnitrosamine (DEN),
peripheral blood micronucleus assay: Cyclophosphamide (CP),
- Route of administration: oral, gavage
- Doses / concentrations:
DEN: 40 mg/kg bw
CP: 10 mg/kg bw

Tissues and cell types examined:

hepatocytes, reticulocytes

Evaluation criteria:

Micronucleated hepatocytes (MNHEPs) were defined as hepatocytes with round or distinct micronuclei that stained like the nucleus, with the ≤1/4 diameter of the nucleus.
Micronucleated reticulocytes (MNRETs) were defined as reticulocytes with round or distinct micronuclei that stained like the nucleus, with the ≤1/4 diameter of the nucleus.

Statistics:

The statistical significance of the incidence of micronucleated hepatocytes or reticulocytes was determined by using Kastenbaum and Bowman’s method and that of reticulocytes with the Student t-test.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

hepatocytes:

Toxicity:

yes

Remarks:

Deaths occurred at the 400 mg/kg bw dose group as follows: two animals on Day 3, two on Day 4, and one on Day 5. Thus, the positive response in samples harvested on Day 3 was based on only two animals. At 300 mg/kg bw, one animal died on Day 4.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

reticulocytes

Toxicity:

yes

Remarks:

s.a.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Please refer to table 1 and 2.

Table 1: Results of the liver micronucleus assay

Chemical and dose (mg/kg bw)	No. of animals	Sampling time (days)	MNHEP mean (%)	±S.D.	Mitotic cell mean (%)	±S.D.
Lab 1
0	4	5	0.11	0.09	0.38	0.35
200	4	3	0.14	0.11	0.23	0.10
	4	4	0.19	0.13	1.35	0.94
	4	5	0.16	0.09	0.73	0.49
300	4	3	0.10	0.04	0.19	0.18
	3	4	0.08	0.10	0.43	0.40
	4	5	0.10	0.09	0.50	0.19
400	2	3	0.38	0.04	0.23	0.11
	2	4	0.20	0.14	0.28	0.32
	3	5	0.18	0.08	0.20	0.13
DEN	4	5	1.21	0.08	0.25	0.12
Lab 2
0	4	5	0.00	0.00	0.06	0.09
150	4	3	0.00	0.00	0.18	0.12
	4	4	0.05	0.04	0.20	0.12
	4	5	0.03	0.03	0.00	0.00
300	3	3	0.10	0.09	0.13	0.13
	3	4	0.02	0.03	0.07	0.03
	4	5	0.08	0.06	0.08	0.03
DEN	4	5	0.44	0.20	0.18	0.09

Table 2: Results of the peripheral blood micronucleus assay

Chemical and dose (mg/kg bw)	No. of animals	MNRET mean (%)	±S.D.	RET (%)	±S.D.
Lab 1
0	4	0.06	0.06	14.0	1.3
200	4	0.06	0.03	11.6	1.2
400	3	0.15	0.05	14.4	3.3
DEN	4	0.05	0.04	13.0	1.7
CP	4	0.73	0.10	90.	1.0
Lab 2
0	4	0.04	0.03	NT	NT
150	4	0.13	0.03
300	4	0.09	0.08
DEN	4	0.01	0.03
CP	4	0.93	0.42
NT: not tested

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

This section contains individual summaries of the study records for the endpoint “Genetic toxicity”.

 

Bacterial mutagenicity

 

BASF SE, unpublished report, Report No. 40M0409/17M329, 2018:

The GLP-compliant key study was performed according to OECD TG 471 to determine its mutagenic potential based on the ability to induce point mutations in several bacterial strains, i.e. Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA 98) and Escherichia coli (WP2 uvrA) in a standard plate test with and without metabolic activation at concentrations of 0.33 up to 5000 µg/mL. A biologically relevant increase in the number of revertants was not observed in the standard plate test using tester strains TA 1535, TA 1537 and WP2 uvrA with and without S9 mix. Furthermore, using tester strains TA 100 and TA 98 without metabolic activation no relevant increase in the number of revertants was observed in standard plate test. A relevant, reproducible and dose dependent increase in the number of revertants was observed with TA 100 and TA 98 with metabolic activation. Under the experimental conditions of this study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the presence of metabolic activation in the Salmonella typhimurium strain TA 98 (frame shift mutations) and TA 100 (base pair substitutions).

 

BASF SE, unpublished report, Report No: 77/207, 1977:

In a non-GLP screening study using the Salmonella typhimurium strains TA 98, TA 100, TA 1537 and TA 1538 the mutagenic potential of 4,4’-MDA was assessed in the absence and presence of metabolic activation (S9 mix). There are some deviations to the current recommendations of the OECD guideline 471 and documentation deficiencies, however, the mutagenic response in some tester strains provides relevant supporting information. The substance did not show any mutagenic potential in the absence of metabolic activation. Clear mutagenic results were obtained in strain TA 100 in the presence of metabolic activation, whereas only a weak mutagenic effect was found in strain TA 98. The increase was dose-related in both strains and the experimental repeat of the test with strain TA 100 confirmed the positive outcome. The author considered strain TA 1538 also to show weak mutagenicity. However, according to the recent recommendations for the evaluation of results, strain TA 1538 is considered not to show a relevant increase in revertants. Additionally, TA 1537 did not show relevant changes in the number of revertants.

 

BASF SE, unpublished report, Report No: 40M0290/084036, 2008:

A screening study was performed according the current OECD guideline 471 with acceptable restrictions. Only two Salmonella typhimurium strains (TA 98 and TA 100) were used in the study instead of 5 strains. Testing up to the requested maximum concentration of 5000 µg/plate 4,4’-MDA revealed no mutagenic effects in the absence of metabolic activation. A clear mutagenic response was observed in both tester strains in the presence of metabolic activation, showing a slightly higher response in the strain TA 100.

 

National Toxicology Program, Study ID: 320619, 1986:

A bacterial mutagenicity study was conducted according to the Standard NTP protocol (preincubation assay) being comparable to the protocol described by Mortelsman and Zeiger (2000). The Salmonella typhimurium strains TA 98 and TA 100 were treated with 4,4’-MDA in the absence and presence of metabolic activation (rat and hamster S9). In the absence of metabolic activation, no increase in revertants was observed, whereas the substance induced an increase in revertants in the presence of both rat and hamster S9.

 

BASF SE, unpublished report, Report No: 83/389, 1984:

An Ames test performed with strain Salmonella typhimurium TA 98 in the presence of metabolic activation did not show an increase in bacterial mutations in this strain in two independent experiments performed.

 

Rao et al., Arch Toxicol 49:185-190, 1982:

An increase in revertants in the Salmonella typhimurium strains TA 98 and TA 100 in the presence of metabolic activation was reported, with TA 100 showing a stronger effect.

 

Zeiger et al., Environmental and Molecular Mutagenesis Volume 11, Supplement 12: 1-158, 1988:

MDA elicited no mutagenicity in the absence of metabolic activation but caused mutagenic effects with metabolic activation in TA 98 and TA 100.

 

Tanaka et al., Mutation Research, 143, 11 -15, 1985:

This study investigated the potential of 4,4’-MDA and its N-acetyl and N,N’-diacetyl metabolites to induce mutations in the Salmonella typhimurium strains TA 98 and TA 100. The parent 4,4’-MDA showed an increased number in revertants in the presence of a metabolic activation system, whereas both acetylated metabolites did not show mutagenic effects in any of the strains in the absence and presence of metabolic activation.

 

Ross et al., unpublished report, Report No: NASA-CR-166085, 1983:

In an in vitro bacterial reverse mutation assay, the mutagenic potential of six individual isomers of the substance was assessed in the Salmonella typhimurium tester strains TA 98 and TA 100 at concentrations up to 10 mg/plate with metabolic activation (Aroclor induced rat liver S9). An increase of at least twice the revertant number of the solvent control was observed in 4 out of 6 isomers in TA 98 (o,m’-MDA, o,p’-MDA, m,m’-MDA, p,p’-MDA) and in 3 out of 6 isomers in TA 100 (o,m’-MDA, m,m’-MDA, p,p’-MDA). 

 

Darby et al., Toxicology and Applied Pharmacology 46, 449-453, 1978:

A bacterial reverse mutation assay was performed using the Salmonella typhimurium tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 at concentrations up to 0.1 mL/plate with or without metabolic activation (rat S9 mix). After incubation with S9 mix, an increase of at least twice the revertant number of the solvent control was observed in TA 98 and in TA 100 and a definite dose-response relationship was observed.

 

Lavoie et al., Mutation Research, 67, 123-131, 1979:

In this Ames test, Salmonella typhimurium – TA 98 (with S9 mix) and TA 100 (with and without S9 mix) – were exposed to 4,4’-MDA in concentrations up to 500 µg/plate. In addition, heteroatomic analogs of the substance, i.e. bis(4-aminophenyl)sulfide, bis(4-aminophenyl) ether and bis(4-aminophenyl)disulfide, were included in the investigation. Substitution of the methylene group by heteroatoms had a marked impact on the mutagenic activity in TA 100. 4-aminophenyl disulfide was toxic to the bacteria at higher concentrations, all other substances did not reveal any toxic effects. The mutagenic potential of these compounds were as follows: -S- > -0- > -CH2- ≥ -S-S-. This was in line with the polarisability of the atoms connecting the aniline moieties. A similar response was observed in assaying these compounds with TA 98. The reduced mutagenic potential and increased cytotoxicity of the disulfide derivative may be the result of enzymatic cleavage of the disulfide linkage or its reaction with thiol groups in the liver homogenate. MDA showed mutagenic properties under the given testing conditions of the present study.

 

Morgott D., Thesis Progress Report, Project No. KA-AB-27, 1982:

The mutagenic potential of the substance was assessed in the Salmonella typhimurium tester strains TA 98 and TA 100 in concentrations up to 500 µg/plate with or without metabolic activation (rat S9 mix). The substance showed mutagenic properties in both tester strains after metabolic activation with rat S9 mix. The acetyl derivatives of 4,4’-MDA did not show any mutagenic potential under these experimental conditions. The nitroso (N-acetyl-N’=O-MDA) and hydroxylamine (N-acetyl-N’-OH-MDA) derivatives showed weak mutagenic effects without metabolic activation, but were severely toxic to the bacteria. In the presence of S9 mix no potentiation of the mutagenic effects was observed, however, the derivatives displayed lower toxicity. The hydroxamic acid derivative showed weak mutagenicity only in strain TA 100 with S9.

 

McCarthy et al., Cancer Research, 42, 3475 - 3479, 1982:

Salmonella typhimurium strain TA 100 4,4’-MDA was tested for its mutagenic potential at concentrations up to 1000 µg/plate. In the presence of rat S9 mix, the substance showed a strong mutagenic potential (concentration-dependent increased number of revertants). In addition, N,N’-dimethyl-4,4’-methylenedianiline, N,N-dimethyl-4,4’-methylenedianiline, N-methyl-4,4’-methylenedianiline showed also strong mutagenicity in the presence of metabolic activation. 

Rannug et al., lndustrial Hazards of Plastics and Synthetic Elastomers, pages 407- 419; 1984:

The mutagenic potential of the substance was assessed in the Salmonella typhimurium tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in concentrations up to 1000 µg/plate with or without metabolic activation (rat S9 mix). The substance showed mutagenic properties only in strains TA 98 and 1538 with metabolic activation.

 

Messerly et al., Environmental and Molecular Mutagenesis 10, 263-274, 1987:

In the study by Messerly et al. (1987) two tester strains, Salmonella typhimurium TA 98 and TA 100 were incubated with various benzidine analogues including 4,4’-MDA. Increased mutagenicity was observed after treatment with 4,4’-MDA in TA 100 and only slight increases in TA 98 in the presence of metabolic activation. The authors conclude, that substances with a high degree of molecular planarity have a higher mutagenic potential (frameshift mutations). This explanation was based on the following hypothesis: “It has been suggested (Ames et al., 1973) that two factors involved in frameshift mutations are the existence of a flat aromatic moiety in the molecule capable of DNA intercalation and an activated electrophilic side chain capable of reacting covalently with DNA. Further, this intercalation of a flat aromatic molecule into DNA could stabilize a shifted pairing in a repetitive sequence of bases, leading to addition or deletion of base pairs.” 

 

Andersen et al., Scand j work environ health 6, 221-226, 1980:

The mutagenic potential of Toluene diisocyanat (TDI, mixture of 2,4 and 2,6 TDI: ratio 80:20), 4,4’-methylenediphenyldiisocyanate (MDI) and 4,4’-MDA (positive control) was determined in an in vitro bacterial reverse mutation assay similar to OECD guideline 471. In the Salmonella typhimurium strains TA 98 and TA 100 MDI, TDI and MDA showed increases in the number of revertants in the presence of S9 mix.

 

Takemura et al., Mutat Res; 54: 256 (abstract 35), 1978:

4,4’-MDA was tested for its mutagenic potential in the Ames II assay, a miniaturized microplate assay using a TA-mix and strain TA 98 with or without metabolic activation (rat S9 mix). The results with the Ames II were concordant with the results obtained in a classic Ames assay. The test substance showed a positive mutagenicity response in both strains with S9 mix. (Kamber et al., Mutagenesis, 24 (4): 359-366, 2009)

In the published abstract of Takemura et al. (1978) the mutagenic potential of 4,4’-MDA in the Salmonella typhimurium strain TA 100 with metabolic activation (rat S9 mix) is reported. The substance was not mutagenic in TA 98.

 

Shimizu et al., Jpn. J. Ind. Health. Vol. 24, 1982:

Various epoxy resin hardeners were tested in the bacterial reverse mutation assay (Ames) in the Salmonella typhimurium tester strains TA 98 and TA 100. Mutagenicity was observed after treatment with 4,4’-MDA in both tester strains after metabolic activation with rat liever S9 mix.

 

Chromosomal aberrations (structural/numerical) in mammalian cells

 

BASF SE, unpublished report, Report No: 30M0409/17X353, 2019:

4,4’-MDA was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in a GLP-compliant study performed according to OECD TG 473. After 4 hrs of treatment, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed, neither with nor without metabolic activation. In the absence of S9 mix after continuous treatment for 22 hrs, all percentages of aberrant cells (excluding gaps) exceeded the range of the historical control data and, at one concentration, were statistically significantly increased. No dose-dependency, tested by a trend test, was observed. These findings after continuous treatment were not confirmed in two confirmatory experiments and therefore they can be regarded as biologically irrelevant.

 

Zhong et al., Mutation Research, 497, 29–37, 2001:

The induction of micronuclei (MN) in Chinese hamster lung fibroblasts cells (V79) was investigated after pulse treatment with 4,4’-MDA at concentrations up to 500 µg/mL without metabolic activation.The study was performed similar to OECD TG 487 guideline with the following deviations: cells were only treated in the absence of S9 mix, evaluation criteria were not clearly defined and no information on HCD is given. A clear concentration-related and statistically significant increase in micronucleated cells was observed after MDA treatment in the absence of any cytotoxic effects. The frequency of kinetochore positive and negative MN was determined and revealed a clear trend towards kinetochore negative micronuclei indicates that MN were formed due to a clastogenic event. 

 

Robbiano et al., Toxicology and Applied Pharmacology 161, 153-159, 1999:

The potential of 4,4’-MDA to induce MN was investigated in primary rat and human kidney cells. A few deviations to the current OECD TG 487 guideline were identified: the determination of cytotoxicity is not in line with the guideline requirements, no clear definition of evaluation criteria and no historical control data. The positive controls included in the assay confirmed the validity of the assay and thus the study is considered reliable and appropriate for the assessment of genotoxicity. The analysis of MN revealed no increased incidence of MN in primary rat and human kidney cells after test substance treatment.

 

 

Gene mutation in mammalian cells

 

Chromosomal aberrations (structural/numerical) in vivo

 

Shelby et al., Environmental and Molecular Mutagenesis 21, 160 – 179, 1993:

The study provides data on the induction of micronuclei in mouse bone marrow after triple exposure with 4,4’-MDA. A few deviations to the current OECD guideline 474 were identified, such as reduced number of cells evaluated for cytotoxicity and cytogenetic damage and the lack of information on historical control data. In addition, the maximum tolerable dose might have beenexceeded since the number of animals used was reduced due to animal death and in general, intraperitoneal injection of substances in this test is not recommended. No detailed rationale for the selection of dose levels is provided. Due to these shortcomings, the study does not allow for a final assessment.

Based on the results the authors concluded that 4,4’-MDA induced micronuclei, however, with regard to the classification criteria of the current OECD guideline 474, the outcome of the study should be concluded as non-mutagenic. A dose-related positive trend is observed, at the highest applied dose showing a statistically significant increase in micronuclei. However, an increase in cytotoxic effects is also observed at the highest dose applied, not only evident as animal death, but also as cytotoxicity to the bone marrow of more than 20% when compared to the control. Additionally, all values are clearly within the range of the negative controls of the performing laboratory for this trial. Therefore, the positive trend observed is considered to have no biological relevance. 

 

Morita T., Mutation Research 389, 3 -122, 1997:

This publictaion on the potential of 4,4’-MDA to induce micronuclei in the mouse after single or double treatment in three independent experiments showed major deviations to the current OECD guideline 474. Only 1000 cells per animal were analysed in peripheral blood for the occurrence of micronuclei instead of the current OECD guideline 474 requirement to analyse 4000 cells per animal. No control animals were available for the different time points analysed (24, 48 and 72 hours), there were only control animals analysed at the start of the treatment period. The publication does not provide any information about the proportion of mature/immature cells and no historical laboratory control data range is available for the discussion of the results. The intraperitoneal application is generally not recommended for this type of study. Due to these shortcomings, the study does not allow for a final assessment.
The outcome of the micronucleus study is considered as inconclusive by the authors of this publication. With regard to the current classification criteria of the OECD guideline 474, 4,4’-MDA should be considered as being non-mutagenic. A dose-related increase was observed in one single experiment at time point 48 hours and could not be confirmed by additional experiments. Although there were statistically significant increases in the number of micronucleated cells at time points 48 and 72 hours no clear confirmation of this result has been obtained by repeat experiments. Since all values were within the range of the negative controls for this trial, the single increased incidences in micronucleated cells are not considered as being biologically relevant. 

 

Suzuki et al., Mutation Research 583, 133–145, 2005:

The publication of Suzuki et al. (2005) reports data of a collaborative study trial of the micronucleus assay in rat peripheral blood and the liver after single treatment with 4,4’-MDA. There is no OECD guideline in place for the performance and evaluation of a micronucleus assay in the liver. With regard to the current OECD guideline 474, deviations were identified such as the exceedance of the maximum tolerable dose, evident in animal cell death. This led to a reduced number of animals for analysis (hepatocytes: 2-4 animals, peripheral blood: 3-4 animals). A single sampling time was applied for the peripheral blood analysis. For hepatocyte analysis there were no control values available for days 3 and 4. For both tissues 2000 cells per animal were evaluated instead of 4000 cells per animal as requested by the current OECD guideline 474. No information on historical control data is available, but robust control data have been shown in the study trial for both tissues. There was no biologically relevant increase in the number of micronucleated cells in the liver or in peripheral blood. Thus, the outcome corroborates the findings of the studies by Shelby et al. (1992) and Morita et al. (1997). Although the study has several deviations from the guideline and its reporting is somewhat limited, it is considered to provide relevant information to the weight of evidence and indicates that 4,4’-MDA does not to induce an increased number of micronucleated cells and, thus, mutations, in vivo in rat liver and peripheral blood after single treatment. 

 

Hamada et al., Mutation Research 780-781, 2–17, 2015;

Sanada et al., Mutation Research 780-781, 31–35, 2015:

Additional supporting information on the potential of 4,4’-MDA to induce micronuclei is presented by Hamada et al. (2015) and Sanada et al. (2015), reporting the outcome of a collaborative study trial in liver and bone marrow after 14-day and 28-day repeated oral dosing of rats. A reduced number of cells was evaluated in the study trial (2000 cells instead of 4000 cells according to the current OECD guideline 474), however, the study trial is considered to provide reliable data for the assessment of the mutagenicity of 4,4’-MDA. A dose-dependent increase in micronucleated cells in the liver was observed after 14-day and 28-day repeated dose treatment. The level of micronucleated cells was comparable after both treatment schedules, suggesting that no further accumulation of micronucleated cells was apparent, but also elimination of cells due to increased hepatotoxicity observed after 28-day treatment. In bone marrow statistically significant increases in micronucleated cells were observed after 14-day treatment, however, the increase did not show dose-dependency. With regard to the historical data of the laboratory, the outcome was considered to be positive. After 28-day treatment no increase in micronucleated cells was observed.

 

Robbiano et al., Toxicology and Applied Pharmacology 161, 153-159, 1999:

The potential to induce micronuclei (MN) in male rats was investigated by Robbiano et al. (1999) with only minor deviations to OECD guideline 474. Rats were subjected to unilateral nephrectomy and i.v injection of folic acid followed by either single oral dosing or triple oral dosing with 4,4’-MDA. Kidney cells were prepared and the analysis of MN revealed no increased incidence of MN after test substance treatment. 

 

NTP, Study ID: 535562, 1986:

An analysis of chromosomal aberrations in mouse bone marrow was performed after intraperitoneal injection of 4,4’-MDA. The study shows a few deviations to the current OECD guideline 475, such as the lack of a second sampling time (> 24 hours), no information on toxicity were given, the evaluation criteria were not clearly defined and there was no information on historical data. Furthermore, the results are only reported in a tabular form, and they are inconsistent across the individual runs. A detailed discussion of this inconsistency would be needed for a final assessment. In addition, intraperitoneal injection is not recommended by the current OECD guideline. Only 50 metaphases per animal were evaluated for cytogenetic damage instead of 200 metaphases per animal, however, an increased number of animals (8) was evaluated.

Three independent experiments were performed. A clear negative outcome was observed in the first experiment. In contrast, statistically significant increases in chromosomal aberrations were found in experiments 2 and 3, whereas only in the third experiment a clear dose-dependency was observed. The study with few methodological deficiencies is insufficient for a final assessment, but indicates a clastogenic potential of 4,4’-MDA in vivo in mouse bone marrow as part of the weight of evidence.

 

 

 

Indicator tests (DNA/chromosomal damage) in vivo

 

Mirsalis et al., Environmental and Molecular Mutagenesis 14:155-164, 1989:

A study on unscheduled DNA synthesis (UDS) after 4,4’-MDA treatment in rats and mice was published by Mirsalis et al. (1989). The number of animals treated for determination of UDS is considered low (3 animals), however, the studies performed according to the OECD guideline 486 are considered reliable and provide supportive information about the induction of UDS in vivo. Single oral doses of 4,4’-MDA did not result in an increase in UDS in both rat and mice suggesting that no damage to DNA occurred after treatment with the substance.

 

Parodi et al., Carcinogenesis, 2, 1317-1326, 1981:

There is no guideline in place for the alkaline elution procedure performed and published by Parodi et al. (1981). After treatment with 4,4’-MDA a statistically significant increase in DNA fragmentation occurred 4 and 24 hours after treatment in the rat liver. When compared to the current guideline version of the Comet assay (OECD guideline 489), only one dose was applied by single intraperitoneal injection instead of at least duplicate administration. The applied dose was reported as the LD50, which clearly indicates that the MTD in this study was exceeded. Thus, the study is considered unreliable and is disregarded.

 

 

Sasaki et al., Mutation Research 444, 249–255, 1999:

In a comet assay published by Sasaki et al. (1999), the substance was administered to mice at a single dose of 250 mg/kg bw. The animals were sacrificed 3, 8 or 24 h after treatment. Stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow tissue were examined. The earliest time point where an onset of significantly increased DNA damages (indicated by migration distance) was observed was after 8 h in stomach and bladder. After 24 h statistically significant increases in migration distance were observed in stomach, liver, kidney, bladder, lung and brain. Slight and dose-related increases were also observed in the colon, however, they did not reveal any statistical significance. No effects were observed in the bone marrow. 

 

Robbiano et al., Toxicology and Applied Pharmacology 161, 153-159, 1999:

The DNA damaging properties of the substance were assessed in an in vivo comet assay in rats by Robbiano et al. (1999). Rats were treated by either single oral dosing or triple oral dosing with 4,4’-MDA. Kidney cells were prepared for subsequent comet analysis. A few minor deviations to OECD guideline 489 were identified, i.e. the number of cells scored was lower than specified, no clear evaluation criteria and no HCD are given in the publication. Comet analysis showed no relevant increases in tail length or moment in kidney cells.

 

Schuetze et al., Chem. Res.Toxicol., 9, 1103-1112, 1996:

The DNA-binding potency of 4,4’-MDA was investigated in rat liver. Two animals were treated once and liver DNA was isolated 24 hours after treatment. The calculated covalent binding index indicates the DNA-binding potency to be in the range of weak genotoxic substances.

 

Vock et al., Carcinogenesis, 17, (5), 1069-73, 1996:

Formation of DNA adducts with MDA in vivo was investigated using the 32P-postlabelling assay. After triple oral administration of MDA at up to 50 mg/kg to rats, dose-dependent formation of DNA adducts in the liver was observed.

 

Vock et al., Naunyn-Schmiedeberg’s Archives of Pharmacology, Vol. 353, No. 4 SUPPL., pp. R136. 1996:

To determine the overall DNA-binding potency of the substance, female Wistar rats were treated topically with radiolabelled 4,4’-MDI (4,4’-methylenediphenyldiisocyanate) and 4,4’-MDA. The amount of radioactivity covalently bound to DNA isolated from skin, liver and lung was determined after 24 or 48 hours. A low DNA binding potency was observed for MDI and MDA in the liver. No DNA adducts were detected in lung and epidermis. Chromatin protein adducts were observed in all tissues.

 

 

Information related to other investigations

 

 

Mori et al., Mut. Res., 204, 683-688; Cited in: European Union Risk Assessment Report 4,4’-methylenedianiline, Vol:9; 2001:

The publication of Mori et al. (1988) provides data on the Unscheduled DNA Synthesis (UDS) assay in rat hepatocytes (rat strain: ACI). The treatment of isolated rat hepatocytes with 4,4’-MDA up to a cytotoxic concentration range showed a clear and dose-related induction of UDS. The animals were not pretreated with a liver metabolism enzym inducer.

 

Shaddock et al., Environmental and Molecular Mutagenesis 13:281-288, 1989:

Shaddock et al. (1989) investigated the potential of 4,4’-MDA to induce UDS in isolated rat hepatocytes with and without pre-treatment of the animals (strain Sprague-Dawley) with Aroclor or phenobarbital. Without pre-treatment of the animals with the liver metabolizing enzyme inducers there was no induction of UDS, whereas including the pre-treatment of the animals with Aroclor or PB, slight, but statistically significant increases in UDS were observed. 

 

Martelli et al., Toxicology and Applied Pharmacology 182, 219–225, 2002:

DNA repair synthesis was investigated in vitro in primary human and rat hepatocytes in accordance with the OECD guideline 482 (deleted in 2014) with a minor deviation (lack of cytotoxicity data in human cells).

In rat hepatocytes an increase in DNA repair synthesis was observed, which showed a concentration-dependent increase in the lower concentration range and exceeded the laboratory specific threshold. Increased incidences for DNA repair synthesis were also observed in human hepatocytes, however, they did not fulfill the criteria for a positive outcome. Thus, a clear positive outcome for the induction of DNA repair synthesis can only be found in rat hepatocytes.

 

 

Martelli et al., Toxicology and Applied Pharmacology 182, 219–225, 2002:

The potential to induce DNA damage in primary human and rat hepatocytes and thyreocytes was assessed by Comet analysis after pulse (4 hours) and continuous (20 hours) exposure to 4,4’-MDA. There is no guideline in place for the performance of a Comet assay in vitro. When comparing the protocol to the current OECD 489 for Comet analysis in vivo and relevant OECD guidelines for the determination of genotoxic/mutagenic potential of substances in vitro a few deviations and reporting deficiencies are apparent. No information on toxicity of4[IK1] ,4’-MDA in human cells for both treatment schedules and in rat for the pulse treatment is provided in the publication. Only 100 cells were evaluated for Comet instead of 150 cells as requested by the OECD guideline 489 and the determination of hedgehogs was not included in the current study. The evaluation criteria for classification of positive/negative outcome are not defined and there is no information on the historical control data range in the respective cell types. Despite the few methodological deficiencies the study is considered reliable, scientifically valid and appropriate for the assessment of DNA damage in vitro.

Statistically significant increases in tail length and moment was observed after 4 and 20 hours treatment with 4,4’-MDA in both rat and human hepatocytes. The values of all applied concentrations were statistically significantly increased when compared to the control values and showed a concentration dependent increase. At the two highest applied concentrations after pulse treatment and at the highest applied concentration after continuous treatment the values show a slight decrease suggesting the occurrence of cytotoxic effects. This hypothesis, however, is based on cytotoxicity data available only for rat hepatocytes at the 20-hour exposure interval, where a moderate toxicity was observed at the highest applied concentration. A comparison of the exposure times shows a higher degree of DNA damage present at 4 hours exposure and a species comparison reveals that rats are more sensitive to DNA damage than humans. In rat and human thyreocytes statistically significant increases in tail length and moment were observed after 4 and 20 hours exposure with 4,4’-MDA, similar to the findings obtained for hepatocytes. When comparing the values of the cell types examined, the findings suggest a higher sensitivity for DNA damage in thyreocytes. A study schedule with 4 hours treatment and Comet analysis 16 hours after treatment revealed that DNA repair occurred only partially, since still statistically significant values for tail length and moment were found in rat and human cells in both tissues.

The authors report the absence of any DNA damage in primary cultures of cells from kidney (rat, human), urinary bladder mucosa (rat, human) and brain (rat). 

 

Robbiano et al., Toxicology and Applied Pharmacology 161, 153-159, 1999:

The DNA damaging potential of 4,4’-MDA was assessed in an in vitro comet assay in primary rat and human kidney cells. No guideline is available for the performance of the assay in vitro. A few deviations to the current OECD guideline 489 for Comet in vivo testing were apparent: Only 100 cells were evaluated instead of 150 cells, the determination of hedgehogs was not included, no evaluation criteria are defined and no information on the historical control data range is provided. Despite these deviations, the study considered reliable, scientifically valid and appropriate for the assessment of DNA damage in vitro. Comet analysis showed no relevant increases in tail length or moment in rat and human kidney cells.

 

Gulati et al., Environ. Mol. Mutagen., 13, 133-193, 1989:

In a sister chromatid exchange assay in CHO similar to OECD TG 479 (deleted in 2014), the cells were treated with 4,4’-MDA in the absence and presence of metabolic activation. The deviations to guideline include the lack of cytotoxicity data and the lack of an independent experiments repeat with S9 mix. With and without S9 mix, only marginal increases of SCE frequencies were found; the maximum effect was ca. 1.3-fold with S9 mix and 1.4-fold without S9 mix when compared to the negative control. Under the conditions of the present test, the test substance seems to slightly induce sister chromosome exchanges in presence and absence of an exogenous metabolism system.

 

Kenyon et al., Toxicology 196, 65–75, 2004:

The dermal absorption of the radiolabelled substance was determined in rat and human ex vivo skin patches after 24 or 48 hours. DNA was isolated from human skin patches 24 h after treatment and DNA adducts were determined using the32P-postlabelling method. In addition, peroxidase activity was measured in homogenised human skin patches. No significant difference was seen between the percutaneous penetration of the substance through human or rat skin for three different treatment levels, however, there was a significant difference in penetration between the doses applied, i.e. the highest absorption through skin occurred at the highest applied dose. A dose-related and statistically significant increase in the adduct level was observed after MDA treatment of human skin and peroxidase activity was apparent.

 

Swenberg J., Short-term tests for chemical carcinogens.ISBN 3-540-90496-4, Springer-Verlag, Berlin, Heidelberg, New York, 1981:

DNA strand breaks were investigated in V79 Chinese hamster lung fibroblasts after 4,4’-MDA treatment by using the alkaline elution technique. The cells were exposed for 1, 2 and 4 hours in the presence of metabolic activation up to a toxic concentration of the substance. DNA fragmentation was observed at substance concentrations of 1.0 mM and above. 

 

 

Indicator tests (chromosomal damage) in vivo

 

Parodi et al., Mutation Research, 108 (1983) 225-238:

The study by Parodi et al., 1983, was disregarded.. No guideline is in place for this study type. When compared with the OECD 479 guideline for in vitro SCE analysis (deleted in 2014) and current OECD guidelines for cytogenetic damage in vivo there are a severe methodological deficiencies: Only 4 or 3 animals instead of 5 animals were evaluated, no information on toxicity is available, there are no clear evaluation criteria reported and no information on the historical control data range is given. Also, the number of metaphases investigated was extremely low. Furthermore, the application of the substance via intraperitoneal injection is not recommended by current cytogeneticguidelines . In conclusion, this study is considered insufficient for assessment.

 

National Toxicology Program, Study ID: 535562, 1986:

In the study by NTP (1986), single significant increases in SCE were observed in all four experiments performed. In addition to the statistical significances observed, a clear dose-dependency was found in 2 out of 4 experiments. Thus, the outcome was considered to be positive. Due to the limited reporting and several inconsistencies (results only given in tabular form, results inconsistent across individual experiments), the study is considered not assignable.

 

Goreka-Turska et al., Bromat.Chem. Toksykol. XVI, 1, 37-42, 1983:

Additional published data for SCE in bone marrow are available by Gorecka-Turska et al. (1983), showing slight statistically significant increases in the number of SCE per chromosome after treatment of mice. Since the publication is Polish no details on methods are available, the publication provides an English translation of the tabulated results only.

 

 

Mutagenicity tests in vivo

 

Sanada et al., Genes and Environment, Vol. 36, No, 4 pp. 179-185, 2014:

In the study by Sanada et al. (2014), the administration of a single dose of 4,4’-MDA did not lead to an increased mutation frequency in erythrocytes and reticulocytes 7, 14 and 28 days after dosing. Repeated dosing of 4,4’-MDA resulted in statistically significant increases in mutation frequency in erythrocytes after 14 and 28 days of dosing, with a higher frequency observed after 14 days. In reticulocytes a statistically significant increase in mutation frequency was observed after 14 of dosing, whereas the mutation frequency after 28 days of dosing was comparable to the control value. The in vivo gene mutation assay in erythrocytes (Pig-a assay) and reticulocytes (PIGRET assay) is able to detect mutations in genes being involved in the production of glycosylphosphatidylinositol anchor proteins on the cell surface. No guideline is available for this test system.The study has some limitations, e.g. the statistical significance of the findings is not clearly reported, and positive mutagenicity results were found mostly in the presence of hematoxicity, which may have been a confounding factor

 

Cell transformation in vitro

ICI Central Toxicology Laboratory, unpublished report, Report No. CTL/P/749, 1982:

Two substance batches of 4,4’-MDA were assessed for its potential to induce cellular transformation in vitro in baby hamster kidney fibroblasts (BHK21/C13) in the presence of metabolic activation in a scientifically robust study similar to EU method B.21. Up to cytotoxic concentration ranges no transformation of BHK21/C13 cells was observed and thus there is neither an indication that 4,4’-MDA acts as a genotoxic carcinogen not as a non-genotoxic carcinogen. 

 

 

In silico data

 

Klopman et al., Environmental Mutagenesis 7:625 -644, 1985:

In an in silico QSAR-approach the mutagenic potential of the substance was assessed in the Salmonella typhimurium tester strains TA 98 and TA 100 with metabolic activation (Aroclor induced rat liver) using the CASE software. A mutagenic potential was predicted for both tester strains.

Justification for classification or non-classification

The available information on in vitro and in vivo genotoxicity are reliable and suitable for classification according to Regulation (EC) No 1272/2008. The genotoxic potential found in in vitro genotoxicity studies was confirmed in in vivo studies in somatic cells. There is no evidence that the substance has potential to cause mutations to germ cells and there are no positive results from tests showing mutagenic effects in the germ cells of humans. Consequently, 4,4’-MDA is classified as mutagenic (UN GHS Category 2, H341: suspected of causing genetic defect) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.