Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 May - 23 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted in 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate

EC Number:

500-734-6

EC Name:

Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate

Cas Number:

162492-01-5

Molecular formula:

C39 H60 N6 O15

IUPAC Name:

Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC, CCL-93
- Suitability of cells: The cells were chosen because of their stable karyotype and their low spontaneous induction rate of micronucleus formation under standardized culture conditions.
- Methods for maintenance in cell culture: Thawed cultures were set up in 75 cm² cell culture plastic flasks at 37°C in 5% CO2 atmosphere. 5x10E5 cells per flask were seeded in 15 mL of minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and subcultures were made every 3 - 4 days.

MEDIA USED
- Type and identity of media including CO2 concentration: MEM supplemented with 10% FBS, 100 U/100 µg/mL penicillin/streptomycin solution, 2 mM L-glutamine, 2.5 µg/mL amphotericin and 25 mM HEPES; 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/ beta-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 1500 and 2000 µg/mL
Experiment I without metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 µg/mL
Experiment I with metabolic activation: 25, 50, 100, 120, 140, 160, 180, 200, 225 and 250 µg/mL
Experiment II: without metabolic activation: 0.5, 1.0, 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 µg/mL

A concentration of 2000 µg/mL was used in the pre-experiment since this is the highest recommended dose according to the guideline used. The concentrations used in the main experiments were based on cytotoxicity of the test item observed in the pre-experiment.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: colchicin

Remarks:

- S9: ethylmethanesulphonate (1200 µg/mL in experiment I, 900 µg/mL in experiment II), colchicin (1.5 µg/mL in experiment I, 0.16 µg/mL in experiment II) + S9: cyclophosphamide (2.5 µg/mL in experiment I)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: approximately 50000 cells per 25 cm² cell culture flask

DURATION
- Preincubation period: approximately 48 h
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): cytocalasin B (final concentration: 1.5 µg/mL)

STAIN (for cytogenetic assays): acridine orange solution

NUMBER OF REPLICATIONS: Duplicate cultures for every concentration/ control (except for the pre-experiment).

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Cells were then fixed with methanol + glacial acetic acid (3+1) and resuspended gently. The suspension was dropped onto clean glass slides, heat dried and stained with acridine orange.

NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration (1000 binucleated cells per slide)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: clearly surrounded by a nuclear membrane, having an area of less than one-third that of the main nucleus, being located within the cytoplasm of the cell, not linked to the main nucleus via nucleoplasmic bridges; mononucleated and multinucleated cells and cells with more than six micronuclei were not considered

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index from 500 cells

Evaluation criteria:

A test substance was considered positive in the micronucleus test if:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
b) the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
c) any of the results are outside the distribution of the historical negative/solvent control data.
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system. A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

non-parametric chi²-test, p < 0.05

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2.5 µg/mL and above (experiment I, -S9); at 200 µg/mL and above (experiment I, +S9); at 4.0 µg/mL and above (experiment II, -S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was noted at concentrations of 125 µg/mL and above without metabolic activation and at concentrations of 250 µg/mL and above with metabolic activation.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: see tables 1 and 2

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see tables 1 and 2

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 3
- Negative historical control data: see table 3

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Any other information on results incl. tables

Table 1 Results of experiment I (4 h treatment with and without metabolic activation, 24 h fixation period)

Test item	Concentration [µg/mL]	Cytostasis [%]	Relative cell growth [%]	Micronucleated cells frequency [%]
	without metabolic activation
Negative control	0	6	94	0.95
Solvent control	0	0	100	1.35
Test substance	0.5	8	92	0.75
	1.0	16	84	1.25
	2.5	47	53	1.25
EMS	1200	27	73	4.90
Colchicine	1.5	22	78	2.60
	with metabolic activation
Negative control	0	13	87	1.10
Solvent control	0	0	100	1.30
Test substance	25	1	99	0.65
	50	9	91	0.80
	100	12	88	0.70
	200	57	43	0.55
CPA	2.5	49	51	4.25

Negative control = cell culture medium, solvent control = DMSO or ethanol 1% v/v in cell culture medium, positive controls = ethylmethanesulfonate (EMS; without metabolic activation), colchicine (without metabolic activation), cyclophosphamide (CPA; with metabolic activation)

Cytostasis [%] = 100 – Relative Cell Growth [%]

Relative Cell Growth [%] = 100 x ((CBPI(test conc) – 1) / (CBPI(control – 1))

CBPI = Cytokinesis Block Proliferation Indix = ((mononucleate cells x 1) + (binucleate cells x 2) + (multinucleate cells x 3) / total number of cells

 

Table 2 Results of experiment II (24 h treatment without metabolic activation)

Test item	Concentration [µg/mL]	Cytostasis [%]	Relative cell growth [%]	Micronucleated cells frequency [%]
	without metabolic activation
Negative control	0	0	122	1.05
Solvent control	0	0	100	1.10
Test substance	2.0	10	90	0.83
	4.0	40	60	1.30
	6.0	60	40	1.25
EMS	900	54	46	4.55
Colchicine	0.16	15	85	4.60

Negative control = cell culture medium, solvent control = DMSO or ethanol 1% v/v in cell culture medium, positive controls = ethylmethanesulfonate (EMS; without metabolic activation), colchicine (without metabolic activation)

Cytostasis [%] = 100 – Relative Cell Growth [%]

Relative Cell Growth [%] = 100 x ((CBPI(test conc) – 1) / (CBPI(control – 1))

CBPI = Cytokinesis Block Proliferation Indix = ((mononucleate cells x 1) + (binucleate cells x 2) + (multinucleate cells x 3) / total number of cells

 

Table 3 Historical control data

	Negative control		Solvent Control		Positive controls
	Metabolic activation
	without	with	without	with	without (EMS)	without (Colchicine)	with (CPA)
Mean	0.91	1.07	0.88	1.03	4.19	4.21	4.00
SD	0.29	0.38	0.22	0.46	1.42	2.63	1.59
RSD	31.67	35.27	25.30	44.51	33.91	62.33	39.62
Min	0.45	0.50	0.55	0.55	2.25	1.85	2.30
Max	1.50	1.75	1.40	1.83	7.40	14.20	9.85
LCL	0.34	0.31	0.43	0.11	1.35	0.00	0.00
UCL	1.49	1.82	1.32	1.95	7.03	9.47	7.18
n	39	22	11	6	39	39	22

Negative control = cell culture medium, solvent control = DMSO or ethanol 1% v/v in cell culture medium, positive controls = ethylmethanesulfonate (EMS; without metabolic activation), colchicine (without metabolic activation), cyclophosphamide (CPA; with metabolic activation)

Mean: mean number of micronucleated cells (%), SD: standard deviation, RSD: relative standard deviation (%), Min: minimum number of micronucleated cells, Max: maximum number of micronucleated cells, LCL: lower control limit (95%, mean-2SD), UCL: upper control limit (95%, mean+2SD), n: number of assays

Applicant's summary and conclusion

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in vitro.