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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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 material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

944-336-4

Molecular formula:

C21H34N2O8, C22H36N2O8, C23H38N2O8 as well as polymer content

IUPAC Name:

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

Test material form:

liquid

In vitro test system

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

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.