2,2'-methylenediphenyl 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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Justification for type of information:

Hypothesis: Under physiological conditions the MDI substance readily polymerizes at the MDI/aqueous interface forming insoluble polyureas and/or reacts with extracellular biological nucleophiles to form MDI-adducts rendering the free NCO completely unavailable to react with DNA thereby negating DNA reactivity concerns. Further, the toxicokinetic and metabolic pathways do not indicate the formation of toxicologically relevant metabolites.

Justification: Toxicokinetic and hydrolysis mechanisms demonstrate that MDI rapidly polymerizes to polyurea that the MDI/aqueous interface. Hydrolysis is highly unfavored except when reaction is stabilized by aprotic solvents (DMSO). When appropriate solvents are used, all tested MDI substances are negative for mutagenicity and is supported by this mechanism.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

deviation: no analytical data on chemical composition of the test item were submitted by the sponsor; however, this deviation did not limit the assessment of the results.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

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)

Details on test material:

- Name of test material (as cited in study report): MDI 22
- Physical state: solid
- Purity: approx. 100 % (as indicated by the sponsor)
- Lot/batch No.: WRS 3792
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

Plate incorporation test (first test): 0, 50, 158, 500, 1581, 5000 µg/plate (without and with S9 mix)
Plate incorporation test (second test): 0, 0.5, 5, 16, 50 µg/plate (without and with S9 mix)
Independent preincubation test (first test): 0, 0.5, 1.6, 5, 16, 50, 158, 500 µg/plate (without and with S9 mix)
Independent preincubation test (second test): 0, 1.6, 5, 16, 50, 158, 500 µg/plate (only TA 100 and TA 102, without and with S9 mix)

Vehicle / solvent:

Solvents used: ethylene glycol dimethylether (EGDE) dried with a molecular sieve 0.3nm (test substance), deionized water (mitomycin C), DMSO (sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide, 2-aminoanthracene)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535), nitrofurantoin (TA 100), 4-nitro-1,2-phenylene diamine (TA 1537, TA 98), mitomycin C (TA 102, plate incorp. assay), cumene hydroperoxide (TA 100, preincub. trials), 2-aminoanthracene

Remarks:

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

Not specified.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: strong, strain-specific bacteriotoxic effect at 158 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay (plate incorporation test - first trial) with MDI 22 (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	6	102	5	18	201
50	6	78	2	12	138
158	3	71	0	9	99
500	0	19	0	1	0
1581	0	23	0	3	0
5000	0	50	0	4	0
Positive control	1033	304	24	54	725
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	8	172	8	25	322
50	3	126	8	25	309
158	4	111	0	20	241
500	0	54	0	3	0
1581	0	42	0	5	0
5000	0	56	0	3	0
Positive control	106	2892	229	2431	652

Table 2: Summary of results from the Salmonella mutagenicity assay (plate incorporation test - second trial) with MDI 22 (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	6	132	3	15	148
0.5	6	157	3	15	143
1.6	5	144	3	16	148
5	5	155	4	21	162
16	6	146	3	19	124
50	6	103	2	13	108
Positive control	859	310	30	58	809
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	9	200	5	22	230
0.5	9	213	3	23	296
1.6	10	199	5	23	229
5	9	218	5	27	231
16	10	231	7	26	222
50	10	168	4	20	251
Positive control	133	2909	225	1943	557

Table 3: Summary of results from the Salmonella mutagenicity assay (independent preincubation test - first trial) with MDI 22

(mean values of revertants per plate)

  

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	9	173	7	23	265
0.5	---	157	9	---	---
1.6	6	---	10	25	279
5	11	171	8	36	275
16	9	198	7	31	286
50	10	188	6	20	264
158	6	132	6	15	235
500	0	144	---	14	131
Positive control	852	619	30	82	408
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	9	283	9	39	437
0.5	---	---	9	---	---
1.6	10	286	10	41	436
5	15	302	9	40	410
16	13	297	11	36	332
50	8	268	9	31	355
158	9	235	7	34	373
500	6	93	---	15	308
Positive control	130	2332	279	2831	805

Table 4: Summary of results from the Salmonella mutagenicity assay (independent preincubation test - second trial) with MDI 22

(mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	---	144	---	---	250
1.6	---	158	---	---	222
5	---	155	---	---	226
16	---	137	---	---	209
50	---	142	---	---	210
158	---	133	---	---	186
500	---	79	---	---	67
Positive control	---	291	---	---	809
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	---	216	---	---	310
1.6	---	200	---	---	295
5	---	216	---	---	298
16	---	237	---	---	322
50	---	207	---	---	338
158	---	179	---	---	340
500	---	92	---	---	254
Positive control	---	2396	---	---	609

 

In the plate incorporation test doses of up to and including 50 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Higher doses had a strong, strain-specific bacteriotoxic effect, so that this range could only partly be used up to and including 5000 µg per plate for assessment purposes. Substance precipitation occurred at the dose of 5000 µg per plate. In the preincubation test doses of up to and including 158 µg per tube did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Higher doses had a strain-specific bacteriotoxic effect, so that doses up to and including 500 µg per tube could only partly be used for assessment purposes.

Evidence of mutagenic activity of MDl 22 was not seen up to the highest evaluable doses. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenie effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

In an Ames test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 MDI 22 (2,2'-MDI) revealed no mutagenic activity in the absence and in the presence of a metabolic activation system (OECD TG 471).