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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-10-03 to 2012-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Experimental investigations according to GLP and OECD guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethan-1-ol
EC Number:
608-245-0
Cas Number:
28770-01-6
Molecular formula:
C8H17NO2
IUPAC Name:
2-[2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethan-1-ol
Test material form:
liquid: viscous

In vitro test system

Test system:
human skin model
Source species:
human
Details on animal used as source of test system:
TEST SYSTEM
- Source: SKINETHIC Laboratories; 4, rue Alexandre Fleming, 69007 – LYON, France.
- Batch No.: 12-EKIN-038
- Expiry date: 22 October 2012

The EpiSkinTMSM model has been validated for irritation testing in an international trial and is considered to be suitable for this study.

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 via the MTT cell viability test using the cytotoxic test compound sodium dodecyl sulphate (SDS).

KIT CONTENTS:
- Units: EpiSkin SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm² per unit) 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” for incubations. (Batch No.: 12-MAIN3-046; Exp. Date: 24 October 2012): A flask of sterile “Assay Medium” for use in MTT assays. (Batch No.: 12-ESSC-045; Exp. Date: 24 October 2012)

KIT RECEPTION QUALITY CHECK:
- The colour of the agar medium used for transport was checked for its pH:
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 indicator changes from white to grey at 40 °C). The kit was found to be in good order at reception.
- Storage: The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2 - 8 °C.
Vehicle:
unchanged (no vehicle)
Details on test system:
Performance of the Study

Pre-incubation (day [-1]):
The “maintenance medium” was pre-warmed to 37 °C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO2.

Application (day 0):
A volume of 20 μL test item was applied on the skin surface by using a suitable pipette. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
A volume of 20 μL positive control (SDS 5 % aq.) or negative control (1 x PBS) was applied to the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Exposure (day 0):
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

Rinsing (day 0):
After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).

Post-incubation (day 0 - 2):
After rinsing the units were placed into the plate wells filled with fresh pre-warmed “Maintenance Medium” (2 mL/well) below them and then incubated for 42 hours at 37 °C in an incubator with 5 % CO2.

MTT test after 42 hours incubation (day 2):
After the 42 hours incubation the EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours at 37 °C in an incubator with 5 % CO2 protected from light.

Formazan extraction (day 2):
At the end of incubation with MTT a formazan extraction step was undertaken:
A defined disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch. The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a Vortex Mixer to achieve a good contact of all of the material to the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (day 2):
Following the formazan extraction, 2 × 200 μL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD’s of each well were recorded using a 96-well plate spectrophotometer at a wavelength of 570 nm while using an acidified isopropanol solution blank (6 × 200 μL).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 20 µL/ EpiSkin SM unit
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
MTT test after 42 hours post-incubation
Number of replicates:
3 EpiSkin SM for test item incubation
3 EpiSkin SM for positive control
3 EpiSkin SM for negative control

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Basis: mean. Time point: 42 hours after 15 min exposure.
Value:
84
Other effects / acceptance of results:
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-irritating to skin.

Any other information on results incl. tables

Validity of the test

The mean OD value of the three negative control tissues was 0.648. The mean OD value obtained for the positive control was 0.119 and this result corresponds to 18 % viability when compared to the results obtained from the negative controls. The calculated standard deviation values (SD) for the % viability of the positive and negative control were below 18.

The SD of the % viability calculated for the test item was 24.62, which is out of acceptability range, however, all viability results were above 50 % when compared to the control and, thus, unequivocally confirm that the test item is not irritant to skin. Therefore, the study can be considered as valid.

Indicator for potential false viability

Possible direct MTT reduction with test substance:

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

Colouring potential of test substances:

The test item showed no ability to become coloured in contact with water, therefore, additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Incozol 3 is considered as non-irritant to skin and is, therefore, not classified.
Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item Incozol 3 on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50% when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item Incozol 3 did not show significantly reduced cell viability in comparison to the negative control. 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. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, Incozol 3 is considered as non-irritant to skin and is, therefore, not classified.