Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 October 2015 to 07 January 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:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

EC Number:

600-273-1

Cas Number:

102089-74-7

Molecular formula:

C13H19NO3

IUPAC Name:

Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Justification for test system used:

The EPISKIN-SM model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Details on test system:

Human Skin
EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.: 15-EKIN-040, Expiry Date: 12 October 2015) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin-SM Test Kits used in the present study) and are documented in Appendix 2.

Kit Contents
Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”
(Batch No.: 15 MAIN3 041; Exp. Date: 14 October 2015)
A flask of sterile “Assay Medium”
(Batch No.: 15 ESSC 041; Exp. Date: 14 October 2015)

Number of Replicate Wells
In this assay, two replicates per test item per time point were used. Two negative controls and two positive controls were also run in the assay. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.

Kit Reception
In each case, the pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKIN-SM kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- 20 mg of test item was applied evenly to the epidermal surface of each of two test units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (23.6-23.9°C) covered with the plate lids.

Number of replicates:

two test units

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: optical density

Run / experiment:

Mean

Value:

0.831

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

93.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (0.889).
The positive control treated tissues showed 0.9% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 15.9%.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this in vitro EPISKIN model test with N-Boc-D-phenylglycinol, the results indicate that the test item is not corrosive to the skin.