Comparative evaluation of three methylene dianiline isomers in the bacterial reverse mutation assay,the in vitro gene mutation test,and the in vitro chromosomal aberration test

Abstract

4,4^′-MDA is classified as a genotoxic carcinogen based on numerous in vitro and animal data. The consequential assumption that a safe threshold does not exist is not only applied to 4,4^′-MDA but also to its structural isomers and impurities 2,2^′- and 2,4^′-MDA in the absence of substance-specific data. This constitutes a problem in human risk assessments for all three substances as the inherent risks of 2,2^′- and 2,4^′-MDA and their contribution as impurities to that of 4,4^′-MDA are essentially unknown. A comparative in vitro genotoxicity dataset consisting of the bacterial reverse mutation (Ames) test and the chromosomal aberration test in human lymphocytes (both performed according to the current OECD Guidelines) was generated for all three isomers. Furthermore, an in vitro gene mutation test in Chinese hamster ovary (CHO) cells (HPRT locus assay) was conducted with 2,4^′-MDA. The results indicate differences regarding the genotoxic mechanism and potential, respectively, between the three structures and suggest that the no-threshold assumption for 4,4^′-MDA may not be appropriate for 2,2^′- and 2,4^′-MDA.

Keywords

Methylenedianiline in vitro genotoxicity reverse bacterial mutation (Ames) test chromosomal aberration test HPRT locus assay risk assessment

Introduction

4,4^′-methylenedianiline (4,4^′-MDA; see Figure 1 for structure) is a high production volume commodity chemical used mainly as a precursor to the production of 4,4^′-methylene diphenyl diisocyanate (MDI), but has other industrial applications in epoxy resins and adhesives. It is produced as technical-grade MDA which is a mixture of 4,4^′-MDA (ca. 60% w/w), smaller quantities of the isomers 2,4^′-methylenedianiline (2,4^′-MDA) and 2,2^′-methylenedianiline (2,2^′-MDA; see Figure 1 for structures), and other polynuclear amines. Pure 4,4^′-MDA (≥98% w/w) which can be recovered from the technical product by distillation still contains traces (up to 2% w/w in total) of 2,2^′-MDA and 2,4^′-MDA (Hansen et al., 2001). In the European Union (EU), 4,4^′-MDA has a harmonized classification as Muta. 2 (substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans, H341) and Carc. 1B (presumed to have carcinogenic potential for humans, H350) according to Annex VI of Regulation (EC) No 1272/2008 (CLP). This is primarily based upon administration of 4,4^′-MDA dihydrochloride in the drinking water that caused tumors in the liver and the thyroid of rats and mice (Lamb et al., 1986; NTP, 1983) and is also supported by genotoxicity data. The liver is also the main target of specific organ toxicity of 4,4^′-MDA after single and repeated exposure, which indicates that liver activation, as a non-genotoxic process, plays a crucial part in the formation of tumors. Furthermore, there is a possibility that the observed thyroid tumors are not formed via a specific genotoxic mode of action but are rather secondary to induction of Uridine 5^′-diphospho-glucuronosyltransferase (UGT) in the liver, which leads to increased clearance of thyroxin and, by interruption of a negative feedback loop via the pituitary, thyroid-stimulating hormone (TSH)-mediated thyroid hyperplasia. However, in the absence of mechanistic data, the relevance of genotoxicity in the formation of the tumors cannot be conclusively evaluated. Therefore, 4,4^′-MDA is currently regarded as a genotoxic carcinogen without a safe threshold in a conservative approach by regulators (BAuA and AGS, 2010). Because of a lack of substance-specific data, this assumption is also adopted for 2,2^′-MDA and 2,4^′-MDA.

While the genotoxicity data base for 4,4^′-MDA is vast, available studies are mostly dated, of mixed reliability and quality, and none were conducted under current OECD guidelines. In the bacterial reverse mutation assay (Ames test) 4,4^′-MDA was found to be mutagenic in Salmonella typhimurium (S. typhimurium) strains TA 98 (frame shift mutations) and TA 100 (base pair substitutions) in the presence of a metabolic activation system in various studies (McCarthy et al., 1982; Messerly et al., 1987; Morgott et al., 1982; Oesch, 1977; Rannug et al., 1984; Rao et al., 1982; Tanaka et al., 1985; Zeiger et al., 1988). Induction of clastogenic chromosomal damage has been observed in V79 cells but not in primary rat or human kidney cells treated with 4,4^′-MDA (Robbiano et al., 1999; Zhong et al., 2001). A number of authors have suggested a potential for 4,4^′-MDA to cause DNA damage based on positive results in indicator tests in mammalian cell cultures (Comet assay, Sister Chromatid Exchange (SCE) assay, Unscheduled DNA Synthesis (UDS) test), but many of these studies have severe deficiencies, and the results are in many cases not sufficiently well reported for a final assessment (Gulati et al., 1989; Kenyon et al., 2004; Krueger and Chappelle, 2018; Martelli et al., 2002; Mori et al., 1988; Shaddock et al., 1989; Swenberg, 1981).

The increase of UDS after treatment with 4,4^′-MDA observed in vitro were not confirmed in vivo in the liver of mice or rats (Mirsalis et al., 1989). Increases in DNA fragmentation were reported in the liver, stomach, kidney, bladder, lung, brain, and, to a lesser extent, the colon of mice, but only at doses that caused distinctive jaundice and thus likely exceeded the maximum tolerated dose (MTD) (Sasaki et al., 1999). The National Toxicology Program (NTP (1986)) reported structural chromosomal aberrations and increases in SCE in mouse bone marrow, but these results cannot be fully evaluated because of a lack of detail in reporting. Micronucleus tests in mice and rats gave positive results in the liver after repeated administration of 4,4^′-MDA over 14 or 28 days while no effects were observed in the bone marrow or the peripheral blood after single and up to three consecutive treatments (Hamada et al., 2015; Morita et al., 1997; Sanada et al., 2015; Shelby et al., 1993; Suzuki et al., 2005). Mutagenicity on erythropoietic cells was reported in a Pig-a/PIGRET assay in rats after repeated treatment with 4,4^′-MDA (Sanada et al., 2014). The reported genotoxic effects in the repeated-dose liver micronucleus test and the Pig-a/PIGRET assay are equivocal as they were accompanied by hepatotoxicity and changes in the hematopoietic activity, respectively, and did not exhibit a conclusive dose- or time-dependency. It is therefore questionable if these effects represent a specific genotoxic response.

Genotoxicity data for 2,2^′-MDA and 2,4^′-MDA—which only occur as impurities in 4,4^′-MDA—are restricted to an old Ames study assessing the mutagenic potential of various steric isomers of MDA in the presence of metabolic activation that showed that o,p’-MDA (2,4^′-MDA) was only mutagenic in TA 98 and o,o’-MDA (2,2^′-MDA) did not lead to mutagenicity in any of the tested strains (Ross et al., 1983).

The combined data clearly indicate that 4,4^′-MDA causes point mutations in bacteria with metabolic activation. Furthermore, the data suggest that 4,4^′-MDA is genotoxic in vivo in the liver (where it also causes severe hepatotoxicity after acute and repeated dosing), but the results are to some extent inconclusive as the contribution of confounding tissue toxicity is unclear. One aim of the present work was therefore to shed light on the mechanism of 4,4^′-MDA-induced genotoxicity by means of state-of-the-art, OECD Test Guideline- and GLP-compliant in vitro tests that cover point mutagenicity and clastogenicity, respectively. To that end, 4,4^′-MDA was tested in the Ames test (OECD Guideline 471; OECD (2020)) and in the chromosomal aberration test (OECD Guideline 473; OECD (2016a)) in human lymphocytes. As data on 2,2^′- and 2,4^′-MDA are extremely limited, a second aim of this paper was to generate comparative in vitro genotoxicity data for these two isomers against 4,4^′-MDA. For the related aromatic amine toluene diamine (TDA), which is used as an intermediate to produce toluene diisocyanate (TDI), genetic toxicity and carcinogenicity studies conclusively demonstrated differences between the 2,6-TDA and 2,4-TDA isomers (for structures, see Figure 2). Although both isomers elicit genotoxicity in vitro, only 2,4-TDA was carcinogenic in mice and rats and produced mutations in the transgenic rodent gene mutation assay, while 2,6-TDA was negative regarding all of these effects (Hayward et al., 1995; National Cancer Institute (NCI), 1980; Sui et al., 2012). Similar differences can be hypothesized for structural isomers of MDA, and therefore, 2,4^′- and 2,2^′-MDA were examined in the same two test systems as 4,4^′-MDA for the present study. Additionally, 2,4^′-MDA was assessed in the gene mutation test using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in Chinese hamster ovary (CHO) cells (OECD, 2016b).

Methods

Test substances

4,4^′-MDA (CASRN 101-77-9) with a purity of 98.0% and 2,4^′-MDA (CASRN 1208-52-2) with a purity of 98.9% were provided by BASF Polyurethanes GmbH (Lemförde, Germany). 2,2^′-MDA (CASRN 6582-52-1) with a purity of 99.6% was provided by Covestro Deutschland AG (Leverkusen, Germany).

General conduct of the genetic toxicity tests

All tests were carried out in compliance with Good Laboratory Practices (GLP). Dimethyl sulfoxide (DMSO, CASRN 67-68-5) was used as a vehicle for the test items in all tests. Phenobarbital/β-naphthoflavone-induced rat liver S9 prepared by the executing laboratory was used as a metabolic activation system, and all tests were carried out both with and without metabolic activation. Test concentrations for the main experiments were determined in preliminary bacteriotoxicity/cytotoxicity tests.

Bacterial reverse mutation assay (Ames test)

Ames tests were performed in accordance with OECD Guideline 471 (OECD, 2020). The rate of induced back mutations from histidine auxotrophy (his^-) to histidine prototrophy (his⁺) was determined in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Furthermore, the rate of induced back mutations from tryptophan auxotrophy (trp^-) to tryptophan independence (trp⁺) was determined in Escherichia coli (E. coli) strain WP2. Each experiment included sterility controls (plates treated with soft agar, S9 mix, buffer, vehicle, and the test substance but without the addition of tester strain), vehicle controls (plates treated with vehicle instead of test substance), and the following positive controls: in experiments with S9 mix for all strains, 2-aminoanthracene (2-AA, CASRN. 613-13-8), and in experiments without S9 mix, N-methyl-N^′-nitro-N-nitrosoguanidine (MNNG, CASRN. 70-25-7) for S. typhimurium TA 1535 and TA 100, 4-nitro-o-phenylenediamine (NOPD, CASRN. 99-56-9) for S. typhimurium TA 98, 9-aminoacridine (AAC, CASRN. 90-45-9) for S. typhimurium TA 1537, and 4-nitroquinoline-N-oxide (4-NQO, CASRN. 56-57-5) for E. coli WP2 uvrA. Substances were tested at concentrations of 33, 100, 333, 1000, 2500, and 5000 μg/plate according to the plate incorporation method (standard plate test) and in case of negative results both with and without metabolic activation, subsequently in a preincubation test. In case of a positive result in the standard plate test, the experiment was repeated under the same conditions for confirmation. Individual plate counts and the mean number of revertant colonies per plate (based on three test plates per dose) were determined for all dose groups.

Chromosome aberration test in human lymphocytes in vitro

The in vitro chromosome aberration tests were conducted according to OECD Guideline 473 (OECD, 2016a). Human lymphocytes were obtained from blood samples drawn from healthy non-smoking and unmedicated donors who had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes. In culture, the cells were stimulated for proliferation with phytohemagglutinin (PHA) for 48 h before the start of treatment. Exposures to the test substances were conducted as pulse treatments of 4 h (with and without metabolic activation) and continuous treatments of 22 h (without metabolic activation), respectively. In all cases, metaphases were prepared 22 h after the beginning of treatment. In each experimental group two parallel cultures were analyzed, and at least 150 metaphases per culture were evaluated for structural chromosomal aberrations. Each experimental group included concurrent vehicle controls and the following positive controls: in experiments without metabolic activation ethylmethane sulfonate (EMS, CASRN. 62-50-0), and with metabolic activation cyclophosphamide (CPA, CASRN. 50-18-0). A validated test script of “R,” a language and environment for statistical computing and graphics, was used to confirm statistical significance with the Fisher’s exact test (modified) (probability value (p-value) <.05) and to assess dose dependency in the rates of aberrant cells by means of linear regression, respectively. The number of aberrant cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value was less than .05.

In vitro gene mutation test in Chinese hamster ovary cells (HPRT locus assay)

The HPRT test was performed according to OECD Guideline 476 (OECD, 2016b). CHO cells were exposed to 2,4^′-MDA for 4 h with and without metabolic activation, respectively. Subsequently, cells were cultured for six to 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies with gene mutations at the HPRT locus of each test group were fixed with methanol, stained with Giemsa and counted. Cytotoxicity was determined by means of cloning efficiency (CE) and relative survival (RS). The absolute cloning efficiency (CE₂) in % of each test group was calculated by dividing the total number of colonies in the test group by the total number of seeded cells in the test group and multiplying the quotient by 100. The corrected mutant frequency (MF_corr.) was subsequently calculated by dividing the sum of the mutant colony counts within each test group normalized per every 10⁶ cells seeded by the CE₂ value. The RS in % was calculated by dividing the cloning efficiency of the test group by the cloning efficiency of the vehicle control (both adjusted by loss of cells during treatment) and multiplying the quotient by 100. Finally, the mutant frequency (total number of mutant colonies per 10⁶ cells) was divided by CE₂ and multiplied by 100 to get the corrected mutant frequency (MF_corr.). In each experimental group two parallel cultures were analyzed. Each experiment included vehicle controls (containing the vehicle at the same volume and concentration as used in the treated cultures) and the following positive controls: in experiments with S9 mix 7,12-dimethylbenz[a]anthracene (DMBA, CASRN. 57-97-6), and in experiments without S9 mix EMS (CASRN. 62-50-0). A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency, and the independent variable was the dose. The calculation was performed using the EXCEL function RGP. A pair-wise comparison of each test group with the control group was carried out using Fisher’s exact test with Bonferroni-Holm correction. The calculation was performed using the EXCEL function HYPGEOM.VERT.

Results

Ames test

4,4^′-MDA produced a relevant, reproducible, and dose-dependent increase in the number of his⁺ revertants exceeding a factor of two compared to the concurrent vehicle control in S. typhimurium strains TA 100 and TA 98 with S9 mix in the standard plate test (Table 1, Figure 3; for detailed results, see Supplemental Table S1 and Supplemental Figure S1). It has thus been interpreted as being mutagenic in the Ames test.

Table 1.

Summary of bacterial reverse mutation (Ames) standard plate test (SPT) results for 4,4^′-MDA.

Strain	Test group	Factor above vehicle control
	(μg/plate)	Without S9	With S9	With S9 repeat
TA 1535	33	0.8	1.1
	100	1.1	1.2
	333	1.1	0.9
	1000	0.8	1.1
	2500	0.7	1.0
	5000	0.9	0.6
	Positive control	497.3	14.0
TA 100	0.33			0.9
	1.0			1.0
	3.3			1.4
	10			1.8
	33	0.9	3.2	3.0
	100	1.0	4.8	4.6
	333	1.0	6.8
	1000	1.2	7.9
	2500	1.4	7.3
	5000	1.1	4.8
	Positive control	30.1	11.5	19.0
TA 1537	33	1.4	1.0
	100	0.9	1.1
	333	1.3	0.7
	1000	0.8	0.8
	2500	0.8	1.1
	5000	0.7	0.9
	Positive control	109.4	13.9
TA 98	0.33			1.2
	1.0			1.2
	3.3			1.5
	10			2.1
	33	1.3	2.4	2.5
	100	1.0	2.4	2.8
	333	1.0	4.1
	1000	1.2	4.3
	2500	1.2	4.0
	5000	1.2	2.9
	Positive control	40.8	35.9	69.7
E. Coli	33	1.2	0.9
	100	0.9	0.9
	333	1.0	1.3
	1000	1.0	1.0
	2500	0.7	0.7
	5000	0.4	0.5
	Positive control	27.5	3.0

For solvent and positive controls, see Supplemental Information.

Ames: bacterial reverse mutation; SPT: standard plate; MDA: methylene dianiline.

With 2,4^′-MDA and 2,2^′-MDA, no increases in the number of his⁺ or trp⁺ revertants were observed in any of the test strains in the standard plate test or the preincubation test, neither with nor without the addition of a metabolizing system (Table 2, Table 3, Figure 3; for detailed results, see Supplemental Table S2a/b, Supplemental Figure S2a/b, Supplemental Table S3a/b, and Supplemental Figure S3a/b). Thus, both substances were found to be not mutagenic in the Ames tests.

Table 2.

Summary of bacterial reverse mutation (Ames) standard plate (SPT) and preincubation (PIT) test results for 2,4^′-MDA.

Strain	Test group	Factor above vehicle control (SPT)		Factor above vehicle control (PIT)
	(μg/plate)	Without S9	With S9	Without S9	With S9
TA 1535	33	1.2	0.9	0.7	1.1
	100	1.2	0.8	1.3	0.8
	333	1.5	0.9	0.6	1.2
	1000	1.3	0.8	0.9	1.0
	2500	1.1	0.9	1.0	0.9
	5000	0.9	0.6	0.4	0.0
	Positive control	497.3	14.0	333.0	20.7
TA 100	33	1.0	1.1	1.0	1.2
	100	1.0	1.0	1.0	1.3
	333	1.0	1.3	1.1	1.4
	1000	1.0	1.6	1.2	1.8
	2500	0.9	1.4	1.0	1.8
	5000	1.1	1.1	0.7	0.0
	Positive control	30.1	11.5	43.1	17.7
TA 1537	33	1.2	0.9	2.2	1.1
	100	1.3	1.0	0.8	1.9
	333	1.6	1.3	1.6	0.8
	1000	1.2	0.8	1.8	0.8
	2500	0.5	1.0	0.5	1.1
	5000	0.4	0.2	0.0	0.0
	Positive control	109.4	13.9	176.2	22.8
TA 98	33	1.0	1.7	1.1	0.8
	100	1.0	1.1	1.2	1.0
	333	1.0	1.1	0.7	1.0
	1000	1.0	0.6	0.9	1.2
	2500	0.8	0.8	0.8	0.9
	5000	0.5	0.8	0.5	0.8
	Positive control	40.8	35.9	60.5	57.3
E. Coli	33	1.4	0.9	1.1	0.9
	100	0.8	0.7	0.9	0.9
	333	0.7	1.1	0.9	0.7
	1000	0.9	0.5	0.9	0.8
	2500	0.5	0.3	0.6	0.6
	5000	0.3	0.4	0.5	0.2
	Positive control	27.5	3.0	16.6	4.6

For solvent and positive controls, see Supplemental Information.

Ames: bacterial reverse mutation; SPT: standard plate; PIT: preincubation; MDA: methylene dianiline.

Table 3.

Summary of bacterial reverse mutation (Ames) standard plate (SPT) and preincubation (PIT) test results for 2,2^′-MDA.

Strain	Test group	Factor above vehicle control (SPT)		Factor above vehicle control (PIT)
	(μg/plate)	Without S9	With S9	Without S9	With S9
TA 1535	10			1.1	0.9
	33	1.0	0.9	1.0	0.8
	100	1.0	0.9	1.1	0.8
	333	1.1	1.1	0.7	0.8
	1000	0.9	0.7	0.8	0.7
	2500	0.5	0.7	0.3	0.3
	5000	0.3	0.3
	Positive control	560.5	29.8	339.3	12.5
TA 100	10			1.1	1.0
	33	1.0	1.1	1.0	0.8
	100	0.9	1.0	1.0	0.8
	333	1.0	1.0	0.9	0.8
	1000	1.0	1.1	0.8	0.6
	2500	0.7	0.7	0.6	0.6
	5000	0.2	0.3
	Positive control	35.9	29.6	28.4	12.0
TA 1537	10			0.8	0.7
	33	0.9	0.7	1.1	1.2
	100	1.5	1.1	1.1	1.0
	333	1.2	0.8	0.7	1.0
	1000	0.8	1.3	0.9	0.9
	2500	0.3	0.5	0.1	0.2
	5000	0.1	0.1
	Positive control	122.9	9.8	110.3	13.3
TA 98	10			0.8	0.7
	33	1.2	0.7	0.7	0.7
	100	0.9	0.8	1.0	0.8
	333	1.1	0.6	0.8	0.6
	1000	0.9	0.5	0.9	0.6
	2500	0.2	0.3	0.4	0.2
	5000	0.1	0.2
	Positive control	48.7	86.1	45.7	45.5
E.Coli	10			0.7	1.0
	33	1.0	1.0	1.0	1.0
	100	0.9	1.0	0.8	1.1
	333	0.7	0.8	0.7	0.9
	1000	0.5	0.6	0.6	0.8
	2500	0.2	0.4	0.3	0.5
	5000	0.1	0.2
	Positive control	48.4	7.5	21.5	9.5

For solvent and positive controls, see Supplemental Information.

Ames: bacterial reverse mutation; SPT: standard plate; PIT: preincubation; MDA: methylene dianiline.

Chromosomal aberration test

After a pulse treatment for 4 h, 4,4^′-MDA did not cause statistically significant or biologically relevant increases in the number of cells carrying structural chromosomal aberrations in the presence or absence of S9 mix (Table 4, Experiment 4.I; for detailed results, see Supplemental Table S4 and Supplemental Figure S4). In a first experiment with continuous treatment for 22 h without metabolic activation (Experiment 4.IIB), increases exceeding the laboratory’s historical control data range (0.0–1.9%) were observed at all evaluated test substance concentrations. The increases were, however, not dose dependent, and only one of the values (2.7% at 300 μg/mL) was statistically significant. Two additional experiments with continuous treatment for 22 h without metabolic activation did not confirm the findings of the first experiment (Experiments 4.IIC and 4.IID). Thus, 4,4^′-MDA was evaluated as non-clastogenic in the chromosome aberration test.

Table 4.

Summary of results of the chromosomal aberration test with 4,4^′-MDA.

Experiment	Preparation interval (h)	Exposure period (h)	Test group (μg/mL)	Aberrant cells without S9 (%)		Aberrant cells with S9 (%)
				Excl. gaps^a	Carrying exchanges	Excl. gaps^a	Carrying exchanges
4.I	22	4	Solvent control	1.3	0.0	1.3	0.0
			377	1.3	0.0	1.7	0.7
			661	0.7	0.0	1.7	0.3
			1156^d	2.3	0.0	3.0	0.3
			Positive control	11.0^b	3.3	8.3^b	0.7
4.IIB	22	22	Solvent control^c	1.2	0.0
			150^c	2.2	0.2
			300^c	2.7^b	0.0
			500^c	2.0	0.0
			800	n.a.	n.a.
			Positive control	13.0^b	2.3
4.IIC	22	22	Solvent control	1.0	0.3
			50	0.3	0.0
			150	0.7	0.0
			400	0.7	0.0
			500	n.a.	n.a.
			Positive control	21.3^b	6.3
4.IID	22	22	Solvent control^c	1.3	0.0
			100^c	1.5	0.2
			200^c	1.5	0.2
			300^c	1.7	0.0
			400	n.a.	n.a.
			Positive control	20.7^b	7.3

^aIncluding cells carrying exchanges.

^bAberration frequency statistically significantly increased compared to corresponding solvent controls (p < .05).

^cEvaluation of 300 metaphases per culture.

^dPrecipitation occurred at the end of the treatment; n.a.: not analyzed because of strong cytotoxicity. For solvent and positive controls, see Supplemental Information.

MDA: methylene dianiline.

2,4^′-MDA produced neither a statistically significant nor biologically relevant increase in the number of cells carrying structural chromosomal aberrations in any of the experiments, neither with nor without S9 mix (Table 5; for detailed results, see Supplemental Table S5 and Supplemental Figure S5). Although one of the values obtained after continuous treatment without metabolic activation (2.0% at 370 μg/mL) exceeded the historical solvent control data range (0.0–1.9%), it did not reach statistical significance, and dose-dependency was not observed. Therefore, 2,4^′-MDA was considered non-clastogenic in the chromosomal aberration test.

Table 5.

Summary of results of the chromosomal aberration test with 2,4^′-MDA.

Experiment	Preparation interval (h)	Exposure period (h)	Test group (μg/mL)	Aberrant cells without S9 (%)		Aberrant cells with S9 (%)
				Excl. gaps^a	Carrying exchanges	Excl. gaps^a	Carrying exchanges
5.I	22	4	Solvent control	0.0	0.0	0.7	0.0
			214	0.3	0.0	0.0	0.0
			374	0.0	0.0	0.7	0.0
			655^c	1.0	0.0	1.3	0.3
			Positive control	16.7^b	7.7	10.0^b	2.7
5.IIB	22	22	Solvent control	1.0	0.0
			165	1.3	0.0
			247	1.0	0.0
			370	2.0	0.0
			556	n.e.	n.e.
			Positive control	9.7^b	4.0

^aIncluding cells carrying exchanges.

^bAberration frequency statistically significantly increased compared to corresponding solvent controls (p < .05).

^cPrecipitation occurred at the end of the treatment. n.e.: could not be evaluated because of strong cytotoxicity. For solvent and positive controls, see Supplemental Information.

MDA: methylene dianiline.

A pulse treatment for 4 h with 2,2^′-MDA without S9 mix did not result in an increase in the number of cells carrying structural chromosomal aberrations (Table 6, Experiment 6.I; for detailed results, see Supplemental Table S6 and Supplemental Figure S6). In the presence of S9 mix, however, one statistically significant increase was observed after treatment with 121 μg/mL (2.0% aberrant cells, excluding gaps). This value was in the range of the laboratory’s historical solvent control data (0.0–3.1% aberrant cells, excluding gaps), and no dose-dependency was observed. However, in a confirmatory experiment (Experiment 6.III, 4 h exposure with S9 mix), one statistically significant increase (3.3% aberrant cells, excluding gaps) was observed after treatment with 647 μg/mL. Dose-dependency was confirmed via trend test. After continuous treatment for 22 h in the absence of S9 mix (Experiment 6.II), the value of 3.3% aberrant cells, excluding gaps, at 327 μg/mL exceeded the range of the laboratory’s historical solvent control data (0.0–1.9% aberrant cells, excluding gaps) but was neither statistically significant nor dose dependent. However, in a second run (Experiment 6.III), two statistically significant and dose-dependent increases (5.0 and 6.3% aberrant cells, excluding gaps) exceeding the historical solvent control data range were observed after continuous treatment with 187 and 327 μg/mL without S9 mix. In conclusion, 2,2^′-MDA was considered to be clastogenic in the chromosomal aberration test.

Table 6.

Summary of results of the chromosomal aberration test with 2,2^′-MDA.

Experiment	Preparation interval (h)	Exposure period (h)	Test group (μg/mL)	Aberrant cells without S9 (%)		Aberrant cells with S9 (%)
				Excl. gaps^a	Carrying exchanges	Excl. gaps^a	Carrying exchanges
6.I	22	4	Solvent control	1.7	0.0	0.3	0.0
			69	0.3	0.0
			121	0.7	0.0	2.0^b,d	0.0
			211	0.3^e	0.0	0.3	0.0
			370			1.7^e	0.0
			Positive control	7.0^b	1.3	8.0^b	2.0
6.II	22	22	Solvent control	1.7	0.3
			107	1.3	0.0
			187	1.7	0.0
			327	3.3^e	0.0
			Positive control	47.0^b,c	20.0
6.III	22	22	Solvent control	0.7	0.3
			107	2.0	0.0
			187	5.0^b	1.0
			327	6.3^b,e	0.0
			Positive control	19.3^b	4.7
6.III	22	4	Solvent control			1.0	0.0
			211			2.0	0.3
			370			2.0	0.0
			647			3.3^b,e	0.3
			Positive control			5.7^b	1.0

^aIncluding cells carrying exchanges.

^bAberration frequency statistically significantly increased compared to corresponding solvent controls (p < .05).

^cEvaluation of 50 metaphases per culture.

^dEvaluation of 300 metaphases per culture.

^ePrecipitation occurred at the end of the treatment. For solvent and positive controls, see Supplemental Information.

MDA: methylene dianiline.

Gene mutation (HPRT) test

Three independent experiments were carried out (Table 7, Experiments 7.I, 7.II, and 7.III; for detailed results, see Supplemental Table S7 and Supplemental Figure S7). As the highest evaluated concentrations in Experiment 7.I (300 μg/mL without S9 mix and 400 μg/mL with S9 mix) did not cause test substance precipitation or cytotoxicity (RS 10–20%), these experiments were repeated in Experiments 7.III (without S9 mix) and 7.II (with S9 mix), respectively. The highest evaluated concentrations in these additional experiments did cause test substance precipitation and thus fulfilled the requirements of OECD Guideline 476 (for chemicals that are not cytotoxic at concentrations below the lowest insoluble concentration, the highest concentration analyzed should produce turbidity or a visible precipitate). 2,4^′-MDA did not cause an increase in the mutant frequencies either with or without S9 mix in any of the three independent experiments. The substance was therefore evaluated as not mutagenic in the HPRT locus assay.

Table 7.

Summary of results of the in vitro gene mutation (HPRT) test with 2,2^′-MDA.

Experiment	Exposure period (h)	Test groups (μg/mL)	Genotoxicity MFcorr (per 10⁶ cells) corrected with CE₂	Cytotoxicity RS (% of control)	Cytotoxicity CE₂ (% of control)	Genotoxicity MFcorr (per 10⁶ cells) corrected with CE₂	Cytotoxicity *** RS (%)	Cytotoxicity *** CE₂ (%)
			Without S9			With S9
7.I	4	Vehicle control	3.79	100.0	100.0	0.72	100.0	100.0
		0.6				n.c.1	126.1	n.c.1
		1.9				n.c.1	131.6	n.c.1
		5.6				5.76^a	113.5	105.7
		16.7				0.36	79.0	98.2
		50.0				4.76^a	49.8	90.3
		51.4	0.94	92.2	109.7
		92.6	0.70	101.7	99.0
		166.7	0.31	93.1	111.0
		300	3.07	86.1	90.0
		400				5.73^a	106.2	100.0
		500^b	n.c.2	n.c.2	n.c.2
		700^b	n.c.2	n.c.2	n.c.2	n.c.2	0.0	n.c.2
		Positive control	122.7^a	76.7	85.2	160.3^a	97.2	80.3
7.II	4	Vehicle control				0.7	100.0	100.0
		8.8				n.c.1	171.3	n.c.1
		15.9				n.c.1	86.0	n.c.1
		28.6				n.c.1	99.8	n.c.1
		51.4				2.49	56.0	113.0
		92.6				2.02	35.6	121.8
		166.7				2.93	29.7	132.0
		300^b				0.86	23.0	122.5
		500^b				n.c.3	n.c.3	n.c.3
		700^b				n.c.3	n.c.3	n.c.3
		Positive control				26.0^a	98.8	111.3
7.III	4	Vehicle control	4.76	100.0	100.0
		200	n.c.1	69.5	n.c.1
		250	3.54	76.7	67.3
		300	2.31	59.6	73.5
		350	1.42	65.2	95.9
		400^b	0.00	28.3	93.2
		450^b	n.c.3	n.c.3	n.c.3
		500^b	n.c.3	n.c.3	n.c.3
		Positive control	179.2^a	94.8	58.8

^aMutant frequency statistically significantly (p < .05) increased compared to controls.

^bVisible precipitation at the end of the exposure period; n.c.: culture not continued (1 = only four analyzable concentrations required; 2 = strong cytotoxicity; 3 = only one concentration beyond solubility limit required). For solvent and positive controls, see Supplemental Information.

HPRT: hypoxanthine-guanine phosphoribosyl transferase; MDA: methylene dianiline.

Discussion

Comparative testing of 2,2^′-, 2,4^′-, and 4,4^′-MDA in the Ames test and the chromosomal aberration test in human lymphocytes shows that the position of the amino moieties on methylene dianilines influences their genotoxicity in vitro (Table 8).

Table 8.

Summary of in vitro genotoxicity test results for 2,2^′-MDA, 2,4^′-MDA, and 4,4^′-MDA.

Substance	OECD 471 (bacterial reverse mutation test)	OECD 473 (chromosomal aberration test in human lymphocytes)	OECD 476 (HPRT test in Chinese hamster ovarian cells)
2,2^′-MDA	Negative	Positive	Not conducted
2,4^′-MDA	Negative	Negative	Negative
4,4^′-MDA	Positive	Negative	Not conducted

HPRT: hypoxanthine-guanine phosphoribosyl transferase; MDA: methylene dianiline.

The consistently reported mutagenicity of 4,4^′-MDA in S. typhimurium strains TA 98 and TA 100 in the presence of a metabolic activation system in historical studies was reproduced in the current Ames test. Neither 2,4^′-MDA nor 2,2^′-MDA were mutagenic in the bacterial reverse mutation test in the present experiments. This is partly inconsistent with older results published by Ross et al. (1983) who found that 2,4^′-MDA (but not 2,2^′-MDA) was mutagenic in TA 98 and TA 100 with S9 mix. However, it must be noted that Ross et al. (1983) did not report details on the numbers of revertants per plate or on potential precipitation or bacteriotoxicity of the test items, and their results must therefore be considered with reservations. The lack of mutagenicity of 2,4^′-MDA in the Ames test was confirmed in the HPRT test in mammalian cells.

The genetic toxicity of many primary aromatic amines occurs through their metabolic activation by oxygenases of the cytochrome P450 family, leading to the formation of a DNA-reactive nitrenium ion via an aryl hydroxylamine intermediate. This toxification pathway can also be assumed from in vitro experiments for 4,4^′-MDA, which is in line with the fact that this isomer is generally only positive in the Ames test in the presence of a metabolic activation system (Kajbaf et al., 1992). Metabolism data are not available for 2,2^′-MDA or 2,4^′-MDA, but potentially, the extent of N-hydroxylation could be isomer dependent, which has been shown for isomers of another primary aromatic amine, aminobiphenyl (Ioannides et al., 1989). Comparative metabolism studies with the three MDA isomers investigating potential differences in their metabolic activation and/or detoxification could provide a mechanistic rationale for their different responses in the Ames test.

Of the three isomers, only 2,2^′-MDA produced clastogenicity in the in vitro chromosomal aberration test. Interestingly, 2,2^′-MDA showed clastogenic effects both with and without S9 mix. This marks a notable difference to 4,4^′-MDA, which required metabolic activation to yield positive results in the vast majority of historical in vitro genotoxicity studies. Like for the Ames assay, investigations of the metabolic behavior of the three MDA isomers may shed light on the underlying mechanisms of the differences in the chromosomal aberration test results.

4,4^′-MDA is currently classified as a genotoxic carcinogen for which, by default, no safe dose is presumed. While there are only limited data on 2,2^′- and 2,4^′-MDA, the same assumption has been applied to the two structurally similar isomers. The comparative in vitro results presented here do indicate differences regarding (a) the genotoxic potential in general, and (b) the mechanism of genotoxicity. These findings suggest that a general no-threshold assumption for the risk assessment of 2,2^′- and 2,4^′-MDA may not be appropriate. In the case of 2,4^′-MDA, the lack of a positive genotoxicity response in any of the three tests (Ames, chromosomal aberration, and HPRT) may even indicate that this isomer, in contrast to 4,4^′-MDA, is not genotoxic at all. Further in vivo testing is required to confirm the variances between the three isomers observed in these screening assays. Ultimately, comparative carcinogenicity studies in rodents could shed light on the carcinogenic potential of 2,2^′- and 2,4^′-MDA as well as the relative carcinogenic potency of the three isomers. These experiments may refine risk assessments for 2,2^′- and 2,4^′-MDA.

Supplemental Material

Supplemental Material - Comparative evaluation of three methylene dianiline isomers in the bacterial reverse mutation assay, the in vitro gene mutation test, and the in vitro chromosomal aberration test

Supplemental Material for Comparative evaluation of three methylene dianiline isomers in the bacterial reverse mutation assay, the in vitro gene mutation test, and the in vitro chromosomal aberration test by Elif Unterberger-Henig in Toxicology and Industrial Health.

Footnotes

Acknowledgment

The author is grateful to Dr Anne H. Chappelle of the International Isocyanate Institute, Inc. for reviewing the manuscript.

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The author is employed by BASF SE, a producer of MDA and MDI.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Part of this work was funded by the International Isocyanate Institute, Inc. The Institute is funded by producers of MDI and TDI.

Ethical approval

The author confirms that Ethical Committee approval was sought where necessary and is acknowledged within the text of the submitted manuscript.

Disclaimer

The opinions expressed in this manuscript are those of the author, not necessarily of BASF SE, the International Isocyanate Institute, Inc., or its member companies.

ORCID iD

Elif Unterberger-Henig

Supplemental Material

Supplemental material for this article is available online.

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