Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14.05.2012 - 21.08.2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study performed according to GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
3,3,5-trimethylcyclohexyl 2-hydroxybenzoate
Test material form:
other: clear colorless to pale yellow liquid
Details on test material:
Purity: 99.8 %

Test animals

other: seeded human epidermal keratinocytes
other: supplied by SkinEthic Laboratories, Lyon, France
Details on test animals or test system and environmental conditions:
The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes 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 a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Type of coverage:
other: EPISKIN model
Preparation of test site:
not specified
unchanged (no vehicle)
other: sodium dodecyl sulphate (SDS) 5% as positive control
Amount / concentration applied:
10 µL of the undiluted test item were applied to each of triplicate tissues.
For the positive and negative controls also each 10 µL were dosed per tissue.
Duration of treatment / exposure:
The negative and positive control, and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were placed into the incubator for 15 min at 37 ±1.5 °C, 5 ±0.5% CO2 .
Observation period:
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ±1.5 °C, 5 ±0.5% CO2 .
Number of animals:
not relevant, measurements were made in triplicates for negative control, positive control and test item.
Details on study design:
The MTT concentrate was thawed on the day of testing and diluted with the MTT diluent. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO 2 ) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol / 2 N HCl 49:1 (v/v)) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 72 hours in the refrigerator.
Per each tissue sample 2 x 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax ® Molecular Devices, 85737 Ismaning, Germany) with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.
Some test chemicals may reduce MTT, which will result in a blue colour without any involvement of cellular mitochondrial dehydrogenase. Although in the present assay the test chemicals were rinsed off and the medium beneath the tissues was replaced before contact with MTT medium, some amount of a test chemical may be released by the tissues into the MTT medium and directly reduce the MTT, which would be interpreted as "tissue viability". MTT reducing capability of the test item was tested as described in section “9.5 Test for Direct MTT Reduction”.

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: Relative absorbance (% of negative control)
Remarks on result:
Basis: other: absorbance of negative control. Time point: 6 days following incubation. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)

Any other information on results incl. tables

Quality criteria

The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability meets the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 till ≤ 1.5. The standard deviations in between tissues of the same treatment group should be ≤ 18%. An assay meets the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%. The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the present report (the acceptance limit of the IC 50 should be between 1.0 and 3.0 mg/mL after 18 hours treatment with SLS). The historical data (means, standard deviation, and ranges) of the positive control as well as for the negative control obtained with the EpiSkin™ model at Harlan CCR are mentioned in the report.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: EU
The test item was considered to be Non-Irritant (NI) in this human epidermis skin model test.
Executive summary:

The relative mean absorbance of the test item treated tissues was 108.9% (relative to negative control) after a 15-Minute exposure period. Thus, the test substance had no effect on viability and the substance is considered non-irritant.