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

Endpoint summary

Description of key information

Skin irritation (OECD 439, EPISKIN): not irritating

Eye irritation (OECD 437, BCOP): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 06 Feb 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 26 Jul 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 06 Jul 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Gyógyszerészeti és Egészségügyi Minőség- és Szervezetfejlesztési Intézet (National Institute for Quality- and Organizational Development in Healthcare and Medicines), Budapest, Hungary

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin (EPISKIN SNC, Lyon, France)
- Tissue batch number: 15-EKIN-005
- Expiry date: 9 Feb 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The test substance was washed from the skin surface with phosphate buffered saline (PBS).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: 96-well plate spectrometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the final product was assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate.
- Morphology: Histological examination was performed to demonstrate a human epidermis-like structure. A well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum was observed.
- Contamination: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: A single experiment was conducted.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 min exposure and 42 h post-incubation is less than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL

Duration of treatment / exposure:

15 ± 0.5 min at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

in triplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

15 minutes exposure

Value:

79

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item showed slightly reduced cell viability in comparison to the negative control (mean value: 79%). However, all obtained test item viability results were above 50% when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.
Positive and negative controls showed the expected cell viability values within acceptable limits.

Table 3. MTT assay after 15 min exposure.

	Negative control			Positive control			Test substance
Tissue sample	1	2	3	1	2	3	1	2	3
OD570	0.854	0.862	0.835	0.212	0.110	0.192	0.639	0.622	0.759
OD570 (mean values of replicates)	0.85			0.171			0.673
Viability (%)	100			20			79

Possible direct MTT reduction with test substance:

No colour change was observed after 3 h of incubation. The test substance did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.

Colouring potential of test substance:

The test substance showed no ability to become coloured in contact with water. The intrinsic colour of the test substance is light yellow and therefore considered to be not able to significantly stain the tissues and lead to a false estimate of the viability. Additional controls and data calculations were not necessary. A false estimation of the viability can be excluded.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

Based on the experimental findings and under the conditions of the test, the test substance has no skin irritating properties.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jan 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Bensheim, Germany
- Characteristics of donor animals: Animals were at least 9 month old.
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) supplemented with penicillin/streptomycin at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes. The corneae were directly used in the BCOP test on the same day.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularisation, pigmentation, opacity and scratches were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

POSITIVE CONTROL
- Amount applied: 0.75 mL

NEGATIVE CONTROL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAE:
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAE:
At the end of the 1-hour equilibration period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity > 7 was discarded.

TREATMENT METHOD:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. The anterior compartment received the test substance or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with saline.
- POST-EXPOSURE INCUBATION: 2 h in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (Electro Design, France).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Versamax Molecular Devices) at 490 nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 corneae

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.
The positive control (2-ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging.
Relative to the negative control, the test substance did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 0.

Table 2. Results after 10 min incubation time.

Test group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative control	1	0.33	0.060	0.054	1.90	1.15
	0		0.051		0.77
	0		0.052		0.78
Positive control	56.67*		0.648*		66.38	67.84
	55.67*		0.724*		66.52
	54.67*		1.063*		70.61
Test substance	-0.33*		0.006*		-0.43	0.00
	-0.33*		0.006*		-0.43
	-0.33*		0.009*		-0.20

*: corrected values

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

Based on the experimental findings and under the conditions of the test, the test substance has no eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation / corrosion

The skin irritation potential of the registered substance was determined by an in vitro skin irritation test using a human skin model according to OECD guideline 439 and in compliance with GLP (Ágh, 2015). A volume of 10 µL test substance was applied evenly to the epidermal surface of human skin tissues (EpiSkinTM) with a tissue size of 0.38 cm² for 15 min. After a 42 ± 1 h post-incubation period, the cytotoxic (irritancy) effect was assessed. Cell viability was measured by dehydrogenase conversion of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazoliumbromide (MTT), present in cell mitochondria, into a blue formazan salt, that is quantitatively measured after extraction from tissues. Phosphate buffered saline (PBS) was used as negative control and 5% aq. sodium dodecylsulfate (SDS) solution as positive control. The relative mean tissue viability obtained after 15 min treatment with the test substance compared to the negative control tissues was 79%. Since the mean relative tissue viability for the test substance was above 50%, the test substance is considered to be non-irritant. The optical density of the negative control was well within the required acceptability ranges. The positive control revealed a mean cell viability of 20% after 15 min exposure thus ensuring the validity of the test system. Based on the results, the test substance was not irritating to the skin under the conditions of the test.

Eye irritation / serious eye damage 

The eye irritation potential of the registered substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD guideline 437 and in compliance with GLP (Roth, 2015). After a first opacity measurement of the fresh bovine corneae, the neat test substance was applied directly to the epithelial surface of three cattle corneae for 10 min at 32 ± 1 °C. After the incubation phase the test substance was rinsed from the corneae and the corneae were incubated for another 120 min at 32 ± 1 °C. Afterwards, opacity was measured a second time. In addition, the permeability of the corneae was determined by measuring spectrophotometrically the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Based on the results of the opacity and permeability measurement of the test substance an in vitro irritation score (IVIS) of 0.00 was calculated. The mean IVIS of the negative control was 1.15 and of the positive control (2-ethoxyethanol) was 67.84. All values of the negative and positive controls were within the acceptance criteria of the OECD guideline 437. Therefore, the test system was acceptable. Based on the results, the test substance was not irritating to the eyes under the conditions of the test.

Justification for classification or non-classification

The available data on skin and eye irritation of the test substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.