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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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
Chemical structure
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
Chemical structure
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.