Reaction product of 2,2'-oxydiethanol and 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate and hexan-6-olide and trimethylhexa-1,6-diyl diisocyanate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jan - 26 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministère de l'Économie et des Finances, vry-sur-Seine Cedex, France

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

EC Number:

276-957-5

EC Name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Cas Number:

72869-86-4

Molecular formula:

C23H38N2O8

IUPAC Name:

Reaction mass of 7,7,9-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diylbismethacrylate and 7,9,9-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diylbismethacrylate

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Japan Health Science Foundation
- Suitability of cells: recommended test system in international guidelines
- Number of passages if applicable: 24 and 39
- Methods for maintenance in cell culture: Cell cultures were maintained in suspension cell culture flasks at 37 °C and 5% CO2.

MEDIA USED
- Type and identity of media including CO2 concentration: The culture medium used was the R10 medium: Roswell Park Memorial Institute medium (RPMI 1640) + 10% horse serum, heat inactivated (v/v),
50 IU/mL penicillin, 50 µg/mL streptomycin + 1 mM sodium pyruvate + 0.05% pluronic acid.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I
4 h treatment (with and without metabolic activation): 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250 and 500 µg/mL

Experiment I Repetition
4 h treatment (without metabolic activation): 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 µg/mL

Experiment II
24 h treatment (without metabolic activation): 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 µg/mL

Test item concentrations were initially selected based on the solubility in the solvent ethanol. In cases where the test item is cytotoxic, the highest concentration should aim to reach a maximum cytotoxicity (cytostasis) of 55 ± 5%. In the first experiment (4 h) without metabolic activation, only two of the eight tested concentrations met the acceptability criteria, therefore the test was not valid. Cytotoxicity (cytostasis) of more than 71% was observed at concentrations of ≥ 15.6 µg/mL, but only when tested without metabolic activation. Therefore the first experiment (without metabolic activation) was repeated at lower concentrations.

Vehicle / solvent:

- Vehicle used: ethanol (1.0% (v/v))

Controls

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine (-S9), 0.1 µg/mL in ethanol (4 h exposure) and 0.01 µg/mL in ethanol (24 h exposure)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 2 x 10E6 cells/tube in 8 mL cultivation medium

DURATION
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20-24 h; 24 h treatment: 44-48 h

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates in all experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the incubation period, the cell cultures were homogenised, centrifuged and underwent hypotonic treatment. Afterwards, the cells were fixed with methanol-acetic acid (3:1 ratio) and spread on glas slides for microscopic analysis.

NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cytostasis
- Any supplementary information relevant to cytotoxicity: please refer to “Any other informations on materials and methods”

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the micronucleolus test if:
- At least one of the test concentrations showed a statistically significant increase in the frequency of micronucleated cells when compared to the concurrent negative control
- A dose effect was observed in at least one experimental condition when evaluated with an appropriate trend test
- The results were outside the data distribution range of historic negative controls

A test substance was considered negative (not clastogenic or aneugenic) in the micronucleus test if:
- None of the test concentrations showed a statistically significant increase in the frequency of micronucleated cells when compared with the concurrent negative control
- There was no concentration-related increase in the frequency of micronucleated cells when evaluated with an appropriate trend test
- All results were inside the distribution of the historical negative control data

Statistics:

Chi square test (p < 0.05)

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA: please refer to Table 2 under “Any other informations on results incl. tables”

Any other information on results incl. tables

Table 1: Experimental results of the micronucleus test

	Mononuclear cells number	Micronucleated cells number	P-value	Interpretation
Experiment I: 4 h + S9 mix
Vehicle control	994	6
	995	5
CP 1.5 µg/mL	979	21	1.84E-08	significant
	964	36
250 µg/mL	996	4	4.90E-01	non significant
	996	4
125 µg/mL	995	5	8.27E-01	non significant
	995	5
62.5 µg/mL	997	3	4.90E-01	non significant
	995	5
31.25 µg/mL	993	7	8.27E-01	non significant
	997	3
Negative control	999	1
	995	5
Experiment I (Repetition): 4 h - S9 mix
Vehicle control	995	5
	997	3
MMC 0.2 µg/mL	814	186	1.50E-94	significant
	781	219
Col 0.1 µg/mL	879	121	1.61E-43	non significant
	917	83
250 µg/mL	996	4	1.00E+00	non significant
	996	4
125 µg/mL	998	2	7.96E-01	non significant
	995	5
62.5 µg/mL	996	4	1.00E+00	non significant
	996	4
31.25 µg/mL	998	2	7.96E-01	non significant
	995	5
Negative control	997	3
	995	5
Experiment II: 24 h - S9 mix
Vehicle control	994	6
	996	4
MMC 0.02 µg/mL	916	84	3.34E-28	significant
	940	60
Col 0.01 µg/mL	961	39	1.23E-11	non significant
	969	31
250 µg/mL	997	3	4.66E-01	non significant
	996	4
125 µg/mL	998	2	4.66E-01	non significant
	995	5
62.5 µg/mL	997	3	3.16E-01	non significant
	997	3
31.25 µg/mL	998	2	6.37E-01	non significant
	994	6
Negative control	995	5
	997	3
CP: Cyclophosphamide; MMC: Mitomycin C; Col: Colchicine

Table 2: Historical control data

	Without S9						With S9
	4 h treatment (n=30)			24 h treatment (n=29)			4 h treatment (n=30)
	Vehicle*	Mitomycin	Colchicine	Vehicle*	Mitomycin	Colchicine	Vehicle*	Cyclophosphamide
Concentration (µg/mL)		0.2	0.1		0.02	0.01		1.5
Minimum	5	261	91	5	93	43	5	58
Maximum	13	489	326	14	228	231	15	233
Mean	8	368	180	8	135	76	9	128
Standard deviation	2	70	55	2	28	42	3	40
Vehicle*: culture medium or ethanol; data were generated in 2015-2018

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in mouse lymphoma L5178 cells with and without metabolic activation.