Hexamethylene diisocyanate, oligomers, reaction products with Bis-(Trimethoxysilylpropyl)amine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-{6-[3,5-bis(6-isocyanatohexyl)-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-3,3-bis[3-(trimethoxysilyl)propyl]urea

EC Number:

918-105-3

IUPAC Name:

1-{6-[3,5-bis(6-isocyanatohexyl)-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-3,3-bis[3-(trimethoxysilyl)propyl]urea

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17/0241-1, 294-296
- Expiration date of the lot/batch: 2020-05-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stable
- Stability under test conditions: yes

Method

Target gene:

histidine and tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2500, 5000µg/plate (SPT)
0, 33, 100, 333, 1000, 2500, 5000µg/plate (PIT, TA1535, TA100, TA98, E.coli WP2 uvrA, TA 1537 with S9)
0, 10, 33, 100, 333, 1000, 2500 µg/plate (PIT, TA1537 without metabolic activation)

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72h at 37°C in the dark

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the SPT with metabolic activation cytotoxicity was observed at 5000µg/plate and in the PIT without metabolic activation from 2500 and with metabolic activation from 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the SPT cytotoxicity was observed from 5000µg/plate without metabolic activation and in the PIT from 2500 without and from 5000 µg/plate with metabloic activation.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the SPT cytotoxicity was observed from 1000µg/plate onwards and in the PIT from 33 µg/plate without and from 2500µg/plate onwards with metabolic activation.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicty was observed in the SPT at 5000µg/plate and in the PIT without metabolic activation from 333µg/plate onwards and with metabolic activation at 5000µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Test substance precipitation of the test substance was found from about 1000 μg/plate onward with and without S9 mix in the standard plate test and without S9 mix in the preincubation test and from 2500 μg/plate with S9 mix in the preincubation test.

Any other information on results incl. tables

Standard Plate Test

E.coli WP2

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	24.0	1.7	22.0	3.6
33	23.7	4.5	27.7	4.7
100	26.3	2.5	22.7	8.0
333	20.0	1.0	26.3	8.5
1000	21.7	1.2	24.3	5.1
2500	25.3	3.5	20.0	5.2
5000	19.7	7.5	21.7	2.1
5.0 (4 -NQO)	740.0	51.1	-	-
60 (2 -AA)	-	-	86.7	11.6

TA 1535

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	9.3	3.2	8.7	3.1
33	12.0	0.0	10.0	4.4
100	10.7	1.5	10.3	1.2
333	9.0	4.4	7.7	0.6
1000	9.3	0.6	10.0	2.6
2500	7.3	0.6	8.7	1.5
5000	4.0	1.7	7.7	3.1
5.0 (MNNG)	4514.0	374.4	-	-
2.5 (2 -AA)	-	-	209.3	18.9

TA 100

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	106.7	14.4	120.3	21.5
33	111.7	16.9	122.0	19.1
100	89.7	5.1	117.0	11.5
333	87.7	6.5	116.7	4.2
1000	95.7	16.2	122.0	10.5
2500	91.0	10.4	122.0	15.1
5000	66.3	4.0	95.0	10.4
5.0 (MNNG)	3176.7	420.9	-	-
2.5 (2 -AA)	-	-	1659.7	266.2

TA 1537

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	8.3	2.3	7.3	4.0
33	8.7	3.5	7.7	3.8
100	7.3	3.2	5.7	2.1
333	7.7	4.7	7.3	5.8
1000	4.7	3.1	4.3	1.5
2500	3.7	1.2	4.7	0.6
5000	0.7	0.6	5.3	0.6
100 (AAC)	763.3	36.9	-	-
2.5 (2 -AA)	-	-	85.3	9.0

TA 98

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	19.7	4.6	23.0	1.0
33	20.7	6.7	23.3	6.0
100	20.0	3.0	22.3	3.5
333	17.0	7.0	17.7	4.9
1000	17.3	3.1	25.3	10.2
2500	19.3	4.0	24.0	3.6
5000	9.7	0.6	12.7	5.1
10 (NOPD)	922.0	56.6	-	-
2.5 (2 -AA)	-	-	854.3	170.8

Preincubation Test

TA 1535

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	8.0	3.0	9.7	1.5
33	9.0	7.2	6.7	2.1
100	10.7	5.7	7.7	2.1
333	8.0	4.6	8.0	4.4
1000	10.3	2.5	7.7	2.9
2500	3.7	2.1	8.0	3.6
5000	4.3	2.5	3.7	2.1
5.0 (MNNG)	3529.7	196.4	-	-
2.5 (2 -AA)	-	-	193.0	10.5

TA 100

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	92.7	4.2	95.0	19.7
33	84.3	9.9	93.0	3.5
100	93.7	7.6	90.0	3.0
333	74.3	8.0	81.7	5.9
1000	81.0	18.0	86.0	12.5
2500	57.0	5.6	89.3	5.9
5000	33.0	19.9	60.3	9.0
5.0 (MNNG)	2659.0	259.1	-	-
2.5 (2 -AA)	-	-	1317.7	40.5

TA 1537

Dose [µg/plate]*	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	9.7	3.2	7.3	2.9
10 / 33	11.3	1.5	6.3	2.1
33 / 100	5.7	1.2	9.3	1.2
100 / 333	5.7	3.5	6.0	2.6
333 / 1000	3.3	2.5	6.3	1.5
1000 / 2500	3.3	1.5	4.3	2.9
2500 / 5000	1.0	1.0	3.3	1.5
100 (AAC)	702.7	75.8	-	-
2.5 (2 -AA)	-	-	59.7	10.4

* Test concentration without S9 on the left and with S9 on the right (-S9 / + S9)

TA 98

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	17.3	3.8	22.7	2.5
33	12.7	3.1	18.0	4.6
100	16.7	3.8	19.7	2.1
333	9.3	2.1	17.3	6.7
1000	11.3	3.2	18.7	4.9
2500	8.0	1.7	17.3	5.0
5000	5.7	2.5	7.3	1.2
10 (NOPD)	912.3	38.0	-	-
2.5 (2 -AA)	-	-	952.7	18.1

E.coli WP2 uvrA

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	19.7	6.1	20.0	1.0
33	19.0	6.0	23.0	4.0
100	22.3	4.2	18.7	7.6
333	19.3	1.5	18.7	4.0
1000	18.3	2.5	18.0	6.6
2500	22.0	4.6	17.3	4.0
5000	13.7	2.3	18.7	5.0
5.0 (4 -NQO)	369.0	32.7	-	-
60 (2 -AA)	-	-	77.0	13.5

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test concentrations were 33 μg - 5000 μg/plate (SPT) and 10 μg - 5000 μg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found depending on the strain and test conditions from about 1000 μg/plate onward. A bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.