Reaction mass of 4,4'-methylenediphenyl diisocyanate and o-(p-isocyanatobenzyl)phenyl isocyanate / methylene diphenyl diisocyanate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptible publication report

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Cited as Directive 84/449/EEC, B.14

GLP compliance:

yes

Remarks:

with deviations of no analysis of test substance and no QA inspections.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenediphenyl diisocyanate

EC Number:

202-966-0

EC Name:

4,4'-methylenediphenyl diisocyanate

Cas Number:

101-68-8

Molecular formula:

C15H10N2O2

IUPAC Name:

1,1'-methylenebis(4-isocyanatobenzene)

Constituent 2

Reference substance name:

o-(p-isocyanatobenzyl)phenyl isocyanate

EC Number:

227-534-9

EC Name:

o-(p-isocyanatobenzyl)phenyl isocyanate

Cas Number:

5873-54-1

Molecular formula:

C15H10N2O2

IUPAC Name:

1-isocyanato-2-(4-isocyanatobenzyl)benzene

Constituent 3

Reference substance name:

2,2'-methylenediphenyl diisocyanate

EC Number:

219-799-4

EC Name:

2,2'-methylenediphenyl diisocyanate

Cas Number:

2536-05-2

Molecular formula:

C15H10N2O2

IUPAC Name:

1,1'-methylenebis(2-isocyanatobenzene)

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium LT2 mutants TA1535, TA 100, TA 1537, TA 98

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0-1200 µg 4,4'-MDI (dissolved in EGDE) /plate

Vehicle / solvent:

Ethyleneglycol dimethylether (EDGE)

Details on test system and experimental conditions:

Ames test.

TEST DESIGN: For the mutant count, at least 3 plates were used for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained at least three plates per strain. In experiments without S9 mix, buffer was used as replacement. The amount of solvent for the test substance and for the controls was 0.1 ml/plate.

SOLVENT : Ethylene glycol diethylether
CONCENTRATIONS TESTED: 0, 8, 40, 150, 200, 300, 600, 1000, 1200, 2400 µg/plate
METABOLIC ACTIVATION: S9 mix containing 30% S9 fraction
METABOLIC ACTIVATION SYSTEM: S9 fraction was obtained from Aroclor 1254 (500 mg/kg in corn oil, single intraperitoneal injection, 5 days prior to sacrifice) induced male Spraque-Dawley rats (200-300g).

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 200 µg/plate and more

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solutions of 4,4-MDI in EGDE, showed consistent negative response in all strains tested, with and without metabolic activation.

Isomers of monomeric MDI as well as polymeric MDI, dissolved in EGDE, behaved in a similar manner to 4,4'-MDI. Increasing the water content in a solution that contained isomers of monomeric MDI had no pronounced influence on its stability either (Table 3). It can therefore be concluded that solutions of MDI in EGDE can be stored for a few hours before use.

HPLC analysis
The stability of solutions of 4,4'-MDI in DMSO and in EGDE with varying amounts of water was additionally analyzed by HPLC. The advantage of this method is that the concentration of MDI and of the possible degradation products can be monitored and quantified, if suitable reference substances are available.

Signals that relate to the reaction product of 4,4'-MDI and dibutylamine, indicating the presence of 4,4'-MDI, as well as to 4,4'-MDA, indicating the presence of one of the possible degradation products of 4,4'-MDI, can be identified in the chromatogram. Their reference substances are readily available. This is not the case with the different ureas of MDI, which are not easily accessible. As the location of their signals has already been described in the literature, they were identified by analogy.

Table 4 shows the influence of the two solvents as well as the effect of their water content on the stability of solutions of 4,4'-MDI. Within 30 min 2.13 mM (532 mg) of MDI, dissolved in relatively dry DMSO (0.04%, 2.2 mM of water) were almost completely degraded to a number of reaction products such as ureas, carbon dioxide, and as a minor fraction, 4,4'-diphenylmethanediamine (4,4'-MDA). After 45 min no more MDI could be detected. 4,4'-MDA, with a final concentration of 3% in DMSO, could not be found at all if EGDE was the solvent. This indicates, that the mode of degradation of 4,4'-MDI in EGDE is different to that in DMSO. 4,4'-MDI (2.12 mM (531 mg)) dissolved in 100 ml of EGDE, which is a concentration comparable to that in DMSO, and a nearly 3-fold increased water content 6.11 mM (0.11%), was relatively stable. Of the original 4,4'-MDI, 95.3% was detectable after 30 min and 87.6% after 4 h. The influence of increased amounts of water on the stability of 4,4'-MDI was monitored in a supplementary experiment. In nearly equimolar solutions (4.03 mM 4,4'-MDI and 3.89 mM water) 99.1% of the MDI was still present after a period of 4 h. Raising the water content to 26.11 mM, which brings the MDI:water ratio to approx. 1:6.5, led to a solution still containing 78.9% of the MDI after 4 h.

These findings can only be explained by the fact that the degradation of MDI is considerably accelerated in the presence of DMSO and may be complete in less than an hour. On the other hand, the presence of EGDE does not influence the stability of solutions of MDI tremendously. Even after 4 h and in an excess of water, most of the 4,4'-MDI is still available.

Remarks on result:

other: other: Salmonella typhimurium LT2 mutants TA1535, TA 100, TA 1537, TA 98

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results with 4,4-MDI and metabolic activation (30% S9 fraction), solvent EGDE.

ug/plate	TA 100	TA 98
0	108	27
8	-	26
40	-	28
150	119	-
200	-	23b
300	100b	-
600	97b
1000	-	16bp
1200	157p	-
2400	130p	-
AA 3	484*	716*

*Mutagenic effect b= background growth reduced p= precipitation AA= 2-aminoanthracene

Table 1.Results with the mixture of isomers of monomeric MDI and metabolic activation (30% S9 fraction), solvent EGDE.

ug/plate	TA 100	TA 98
0	155	35
8	143	40
40	162	43
75	-	-
150	-	-
200	130	35
300	-	-
750	-	-
900	-	-
1000	119 bp	27 bp
5000	p	bp
AA 3	1304*	653*

*Mutagenic effect b= background growth reduced p= precipitation AA= 2-aminoanthracene

Table 3: Shelf-life of solutions of MDI in EGDE: influence of the water content of EGDE and IR-spectroscopic determination of the relative NCO content as a function of time.

.	Type of MDI/solvent
	4,4'-MDI			Monomeric MDI isomers		Polymeric MDI
	EGDE	EGDE	EGDE	EGDE	EGDE	EGDE	EGDE
Weight of MDI in 100 ml of solvent	100 mg	500 mg	500 mg	500 mg	500 mg	100 mg	500 mg
mM MDI	0.40	2.00	2.00	2.00	2.00	0.40	2.00
Water content of EGDE	0.02%	0.02%	0.23%	0.04%	0.27%	0.02%	0.02%
mM water	1.11	1.11	12.78	2.22	15.00	1.11	1.11
.	.	.	.	.	.	.	.
Start	100%	100%	100%	100%	100%	100%	100%
15 min	99.6%	99.7%	99.6%	99.3%	99.4%	100%	99.6%
30 min	99.3%	99.6%	99.5%	99.2%	99.0%	99.8%	100%
45 min	99.3%	99.3%	99.6%	99.2%	98.9%	99.6%	99.7%
1 h	99.1%	99.3%	99.2%	99.0%	98.7%	100%	99.7%
4 h	98.1%	98.5%	97.3%	98.5%	95.9%	100%	99.7%

Table 4. Shelf-life solutions of 4,4'-MDI in DMSO and in EGDE: HPLC determination of residual free MDI and one of its reaction products as a function of time

.	Solvent
	DMSO		EGDE		EGDE	EGDE
Weight of MDI in 100 ml of solvent	532 mg		531 mg		1007 mg	1007 mg
mM 4,4'-MDI	2.13		2.12		4.03	4.03
Water content of solvent	0.04%		0.11%		0.07%	0.47%
mM water	2.22		6.11		3.89	26.11
.	Analyzed products
	MDI	MDA	MDI	MDA	MDI	MDI
Start	86.5%	0.2%	100%	ND*	100%	100%
15 min	22.1%	8.6%	98.0%	ND*	-	-
30 min	1.0%	4.5%	95.3%	ND*	100%	96.6%
45 min	ND*	3.4%	95.3%	ND*	-	-
1 h	ND*	3.0%	92.3%	ND*	99.1%	93.3%
4 h	ND*	3.0%	87.6%	ND*	99.1%	78.9%

*ND, not detectable, e.g. 0.05 mg/100 ml solvent.

Applicant's summary and conclusion

Conclusions:

The data in this study and that of Seel et al 1999 led to the conclusion in the EU Risk Assessment (2005) that overall, tests assessing the mutagenic potential of MDI in vitro and in vivo provide no convincing evidence of mutagenic and genotoxic activity.

One aspect that appears to have been resolved, relates to the positive Ames tests when using DMSO as a solvent for MDI. In these circumstances MDI was shown to be rapidly converted to the known mutagen MDA thus giving falsely positive results. (According to a report from industry, the same situation appears to hold when acetone was used as a solvent, but the data substantiating this are not available.) When an appropriate vehicle (EGDE) was used, MDI was shown not to be mutagenic by itself or in the presence of a biotransformation system.