Hexamethylene diisocyanate, oligomers, reaction products with 4-Hydroxybutyl acrylate

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

4-{[(6-{2,4,6-trioxo-3,5-bis[6-({[4-(prop-2-enoyloxy)butoxy]carbonyl}amino)hexyl]-1,3,5-triazinan-1-yl}hexyl)carbamoyl]oxy}butyl prop-2-enoate

EC Number:

929-915-1

IUPAC Name:

4-{[(6-{2,4,6-trioxo-3,5-bis[6-({[4-(prop-2-enoyloxy)butoxy]carbonyl}amino)hexyl]-1,3,5-triazinan-1-yl}hexyl)carbamoyl]oxy}butyl prop-2-enoate

Test material form:

solid

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:

33, 100, 333, 1000, 2500, 5000µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to 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:

other: sterility control

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene, 4-nitroquinoline-N-oxide

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:

slight cytotoxicity observed at 5000µg/plate with S9 mix in the SPT and PIT

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight cytotoxicity observed at 5000µg/plate in the PIT with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight cytotoxicity observed at 5000µg/plate in the PIT with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

Any other information on results incl. tables

Standard Plate Test

E.coli WP2 uvrA

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	23.3	6.1	20.7	5.9
33	24.7	5.9	24.3	7.5
100	19.0	5.3	26.3	5.5
333	25.0	10.0	27.7	2.1
1000	25.0	10.5	21.3	11.8
2500	19.3	3.2	21.3	3.1
5000	16.3	4.0	17.7	3.8
5.0 (4 -NQO)	1450.7	53.2	-	-
60 (2 -AA)	-	-	109.7	12.9

TA 1535

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	10.0	2.6	9.0	2.0
33	8.0	2.0	7.3	2.1
100	8.3	0.6	8.7	1.5
333	12.0	3.6	7.7	1.5
1000	8.0	2.6	7.7	2.3
2500	9.0	1.0	7.7	1.5
5000	8.3	2.1	4.7	0.6
5.0 (MNNG)	3479.0	378.9	-	-
2.5 (2 -AA)	-	-	232.0	9.8

TA 100

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	91.3	19.0	82.3	0.6
33	108.3	24.4	91.7	7.1
100	88.0	11.1	95.7	3.8
333	88.7	17.2	112.0	11.8
1000	114.0	20.7	101.3	11.4
2500	82.7	8.0	83.0	5.0
5000	82.0	10.1	60.3	6.7
5.0 (MNNG)	4008.7	386.0	-	-
2.5 (2 -AA)	-	-	2567.7	19.9

TA 1537

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	8.0	2.62	7.0	2.0
33	7.3	3.2	11.0	1.0
100	5.7	3.1	9.3	2.5
333	7.0	2.0	7.0	1.0
1000	6.7	3.1	9.7	4.0
2500	9.7	3.5	7.7	3.1
5000	6.3	3.1	9.0	2.6
100 (AAC)	1219.3	211.6	-	-
2.5 (2 -AA)	-	-	218.7	8.4

TA 98

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	16.0	2.6	25.0	2.6
33	15.0	4.4	19.7	2.9
100	18.7	2.9	29.3	8.3
333	20.3	2.1	22.7	4.0
1000	21.7	6.4	27.7	7.5
2500	16.7	1.5	25.7	2.1
5000	16.7	1.5	22.0	2.0
10 (NOPD)	539.3	32.7	-	-
2.5 (2 -AA)	-	-	2113.3	90.0

Preincubation Test

TA 1535

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	9.7	2.1	9.0	0.0
33	6.3	3.5	6.7	0.6
100	8.3	1.5	10.0	1.7
333	11.7	2.3	8.3	1.2
1000	9.7	3.2	7.0	2.6
2500	10.7	2.1	7.3	2.5
5000	6.7	1.5	4.7	0.6
5.0 (MNNG)	5509.3	382.9	-	-
2.5 (2 -AA)	-	-	332.0	16.5

TA 100

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	104.0	13.9	110.0	5.6
33	109 -3	5.5	105.7	10.8
100	106.3	13.4	105.7	13.9
333	101.0	15.9	104.3	7.4
1000	106.7	7.2	103.0	9.2
2500	102.0	15.6	105.7	21.8
5000	102.3	2.5	96.7	8.1
5.0 (MNNG)	4232.7	364.5	-	-
2.5 (2 -AA)	-	-	2966.0	49.9

TA 1537

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	8.0	1.0	9.0	1.0
33	8.3	1.5	8.0	4.0
100	10.0	3 .6	9.7	0.6
333	7.0	4.0	7.3	2.3
1000	8.3	3.5	9.0	2.0
2500	7.0	2.0	9.3	3.2
5000	9.3	4.5	5.3	4.0
100 (AAC)	943.3	172.1	-	-
2.5 (2 -AA)	-	-	257.7	18.5

TA 98

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	27.0	10.4	28.3	4.0
33	18.3	5.8	28.0	14.7
100	27.0	5.2	25.7	2.1
333	27.0	9.2	28.7	2.3
1000	21.3	5.1	27.0	6.2
2500	26.7	1.2	31.7	8.5
5000	25.0	4.0	29.3	6.0
10 (NOPD)	608.3	52.7	-	-
2.5 (2 -AA)	-	-	2473.0	14.1

E.coli WP2 uvrA

Dose [µg/plate]	Without S9 [Revertants/plate]		With S9 [Revertants/plate]
	Mean	Standard Deviation	Mean	Standard Deviation
DMSO	22.3	4.5	22.0	2.0
33	20.0	0.0	20.3	11.0
100	23.7	5.5	21.7	11.6
333	21.0	9 .0	24.0	6.6
1000	16.0	6.0	29.0	1.7
2500	16.0	4.6	23.0	5.6
5000	25.0	4.0	14.0	5.2
5.0 (4 -NQO)	746.3	69.6	-	-
60 (2 -AA)	-	-	94.3	11.2

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 (TA 1535, TA 100, TA 1537, TA 98) and Escherichia coli WP2 uvrA, in a reverse mutation assay up to the limit concentration of 5000 μg/plate. The standard plate test (SPT) and preincubation test (PIT) were performed both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 1000 μg/plate onward in the standard plate test and from about 2500 μg/plate in the preincubation test with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 μg/plate. A relevant increase in the number of his+ or trp+ revertants 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.