2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

EC Number:

247-953-0

EC Name:

2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Cas Number:

26747-90-0

Molecular formula:

C18H12N4O4

IUPAC Name:

bis(3-isocyanato-4-methylphenyl)-1,3-diazetidine-2,4-dione

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2014-07-27

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:

0,10, 25, 50, 160, 500, 1600, 5000 µg/plate (without and with S9 mix)

Vehicle / solvent:

Solvents used: ethylene glycol dimethylether (EGDE) dried with a molecular sieve 0.3nm (test substance), phosphate buffer (sodium azide, mitomycin C, cumene hydroperoxide ), DMSO (2-nitrofluorene, 4-nitro-1,2-phenylene diamine, 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 100, TA 1535), 2-nitrofluorene (TA 98), 4-nitro-o-phenylenediamine (TA 1537), mitomycin C (TA 102, plate incorp.), cumene hyd (TA 102, preincub. trials), 2-aminoanthracene (all strains)

Remarks:

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

Details on test system and experimental conditions:

METHOD: Standard plate test and preincubation test

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 1537, TA 100 and TA 98 this increase should be about twice that of negative controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria 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

Test results 1

Key result

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay (plate incorporation test) with Metalink U (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)	15	140	9	19	178
10	16	142	8	20	196
25	15	142	10	20	172
50	16	129	9	24	189
160	14	139	11	16	169
500	12	134	9	18	204
1600	15	128	8	18	180
5000	11	128	7	18	177
Positive control	210	489	71	771	538
Dose (µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	17	143	16	42	257
10	15	142	15	43	254
25	16	148	15	45	251
50	17	140	15	49	259
160	17	146	15	42	261
500	15	155	15	37	251
1600	13	153	16	36	230
5000	13	140	12	43	232
Positive control	140	1087	183	1348	379

Table 2: Summary of results from the Salmonella mutagenicity assay (independent preincubation test) with Metalink U (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)	16	95	9	20	190
10	15	85	9	19	218
25	15	96	8	18	180
50	14	93	9	17	188
160	13	97	8	19	170
500	13	95	9	20	184
1600	12	114	6	18	181
5000	11	92	7	17	137
Positive control	703	805	87	919	537
Dose (µg per plate)	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	18	98	18	37	258
10	15	95	16	38	261
25	16	111	18	35	257
50	15	97	17	43	243
160	15	116	17	36	266
500	16	137	17	37	268
1600	14	126	18	36	255
5000	14	113	17	38	240
Positive control	90	988	209	679	540

 

Metalink U was initially investigated for point mutagenic effects using the Salmonella/microsome plate incorporation test. The test item, dissolved in EGDE dried with a molecular sieve, was administered in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Substance precipitation occurred at the dose of 5000 µg per plate.

Metalink U was investigated in an independent repeat using the preincubation modification of the Salmonella/microsome test. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Substance precipitation occurred at the dose of 5000 µg per plate.

Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well in the preincubation modification, under the experimental conditions applied.

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

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The mutagenic potential of Metalink U was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen. No biologically relevant increase in the mutant count, in comparison with the solvent controls, was observed. Based on this test, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.