Registration Dossier

Administrative data

skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:

In vitro test system

Test system:
human skin model
Source species:
other: artificial skin model
Justification for test system used:
Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test item [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)]. The OECD Guideline 431 does not require the use of live animals or animal tissue for the assessment of skin corrosivity.
The test described in the OECD Guideline 431 allows the identification of corrosive chemical substances and mixtures. It further enables the identification of non-corrosive substances and mixtures when supported by a weight of evidence determination using other existing information (e.g., pH, structure-activity relationships, human and/or animal data). It does not normally provide adequate information on skin irritation, but provides an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures in accordance with UN GHS (1). It also allows a partial sub-categorisation of corrosives.
For a full evaluation of local skin effects after single dermal exposure, it is recommended to follow the sequential testing strategy as appended to Test Guideline 404 and provided in the Globally Harmonised System. This testing strategy includes the conduct of in vitro tests for skin corrosion (as described in the OECD guideline 431) and skin irritation before considering testing in live animals.
unchanged (no vehicle)
Details on test system:
The test item was applied topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction assay) below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers. Prior to testing the assay was validated, using the Epidermis Model EpiDermTM (MatTek), by demonstrating the technical proficiency as detailed in OECD guideline 431.
6.1 Human skin model
The following Reconstructed Human Epidermis Model was used:
EpiDerm™ (EPI-200-SCT, Lot no. 30846) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.
This human skin model used for this test complied with the following:
6.1.1 Basic procedure
EpiDerm tissues were conditioned by pre-incubation (1 hour) in Maintenance Medium1 for release of transport stress related compounds and debris in the incubator (37°C, 5% CO2, 95% relative humidity). After pre-incubation tissues were transferred to fresh Maintenance Medium and topically exposed with the test chemicals for 3 min and 1 h, respectively. Two tissues were used per treatment, negative and positive control and exposition time (12 tissues in total). After exposure tissues were rinsed, blotted and assay medium was replaced by MTT assay medium2 (final concentration: 1 mg MTT3 (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue)/mL medium). After 3-h incubation, tissues were washed with Dulbecco's phosphate buffered saline (D-PBS)4, blotted and the blue formazan salt was extracted with isopropanol5. The optical density of the formazan extract was determined spectrophotometrically at 540 nm and cell viability was calculated for each tissue as % of the mean of the negative control tissues. Skin corrosion potential of the test materials was classified according to the remaining cell viability obtained after 3 minutes or 1 hour exposure with the test chemical.
6.1.2 General model conditions
Human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells were present under a functional stratum corneum. The skin model also had a stromal component layer. The stratum corneum was multi-layered with the necessary lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic markers. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue. Passage of test chemicals around the stratum corneum would lead to poor modelling of the exposure to skin. The skin model was free of contamination with bacteria (including mycoplasma) or fungi.
6.1.3 Functional model conditions
The magnitude of viability was quantified by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue). In these cases the optical density (OD) of the extracted (solubilised) dye from the negative control tissue was at least 20-fold greater than the OD of the extraction solvent alone. The negative control tissue has been shown to be stable in culture (provide similar viability measurements) for the duration of the test exposure period. The stratum corneum has been shown to be sufficiently robust to resist the rapid penetration of certain cytotoxic marker chemicals (e.g. 1% Triton X-100). This property was estimated by the exposure time required to reduce cell viability by 50% (ET50) (for the EpiDerm™model this is >4 hours). The tissue employed has been shown to demonstrate reproducibility over time between laboratories. Moreover, it has been shown to be capable of predicting the corrosive potential of the reference chemicals when used in the testing protocol selected.
6.2 Test for interference of chemicals with the assay procedures
Optical properties of the test item or its chemical action on the MTT may interfere with the assay leading to a false estimate of viability, as the test item may prevent or reverse the colour generation as well as cause it. This may occur when a specific test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
Prior to the testing, the test item was evaluated for colour changes. Concurrent negative controls (sterile deionised water6) were run in parallel.
50 μL of the test item were mixed with 300 μL sterile deionised water and incubated in the dark at 37°C, 5% CO2 and 95% relative humidity for 60 minutes. No discolouration of the test item was noted. In addition, 50 μL of the test item were added to 2 mL isopropanol and incubated at room temperature for three hours. No discolouration of the test item was noted.
Furthermore, the test item was evaluated for the potential to interfere with the MTT assay reagent (e.g. reduction). A concurrent negative control (sterile deionised water) was run in parallel. 50 μL of the test item were added to 1 mL of the MTT medium (1 mg MTT/mL) and incubated at 37°C, 5% CO2, and 95% relative humidity for 60 minutes. Untreated MTT solution was used as control. A discoloration of the test item to orange with black precipitation was noted. Hence, due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues had to be performed.
6.3 Administration of the test, negative and positive reference items
The test item was used as supplied.
50 μL of test item was applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. A minimum of 70 μL substance applied per cm2 is required by the guidelines. Two replicate tissues for each treatment (exposure periods) were employed. At the end of the exposure period, the test item was carefully washed from the skin surface with D-PBS. The positive control item was 8 N KOH7 and the negative control was sterile deionised water. 50 μL of negative and positive controls were used.
6.4 Additional test for interference (reduction) of the test item with MTT
The frozen tissues were stored in the freezer (-20 ± 5°C).
The test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDermTM tissues. Then the data were corrected as described in section 6.7.
6.5 Cell viability measurements
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) reduction, which had been shown to give accurate and reproducible results, was used to measure cell viability. Each skin sample was placed in an MTT assay solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% relative humidity) for 3 hours. The precipitated blue formazan product was extracted using the solvent isopropanol and the concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer (Tecan Sunrise Magellan Version 6.48).
Cell viability measurements were carried out for both exposure periods (1st period: 3 min; 2nd period: 60 min). The measurements were made for each of the two tissues in triplicate.
6.6 Barrier function and quality controls (QC) of the model
The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by the manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek corporation determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm™ lot. The ET50 must fall within a range established based on a historical database of results.
6.7 Data correction procedure
For the MTT reducing test item, calculations were corrected by values of correspondent additional controls.
True viability = Viability of treated tissue – Interference from test chemical
= OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)
If the interference by the test substance is greater than 30% of the negative control value, additional steps must be taken into account or the test substance may be considered incompatible with this test.
6.8 Assay acceptability criteria
Assay acceptance criterion 1: Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile deionised water) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The tissues treated with the negative control should not be below historically established boundaries.
The assay meets the acceptance criterion if the mean ODof the NC tissues is ≥ 0.8 and ≤ 2.8.
Assay acceptance criterion 2: Positive control
A 8 N KOH was used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay.
Tissues treated with the PC, should reflect the ability of the tissues to respond to a corrosive chemical under the conditions of the test method (viability after 1 hour exposure: < 15%).
Assay acceptance criterion 3: variability between tissue replicates
Associated and appropriate measures of variability between tissue replicates should not exceed 30% (in the range of 20 – 100% viability).
6.9 Interpretation of results
The OD values obtained for each test sample were used to calculate a percentage viability relative to the negative control, which is arbitrarily set at 100%. The cut-off percentage cell viability value distinguishing corrosive from non-corrosive test items or discriminating between different corrosive classes, e.g. subcategories 1A, 1B and 1C, or the statistical procedure(s) used to evaluate the results and identify corrosive materials, is clearly defined and documented, and shown to be appropriate. The criteria of corrosivity associated with the EpiDermTM model are as follows:
- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A9), if the viability after 3 minutes exposure is less than 50%;
- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;
- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Negative controls validity:
Positive controls validity:
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
The EpiDerm™ model was employed. The test item, the negative control sterile deionised water or the positive reference item 8 N KOH were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.
Two tissues were used for each treatment and concurrent control groups. Cell viability was determined by using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.
The test item was applied topically as supplied (liquid). Sterile deionised water was used as the negative control. 8 N KOH was used as the positive reference item. The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.
In comparison to the negative control, the mean viability of cells exposed to the test item was 28.1% after a 3-minute exposure period and 14.1% after a 1-hour exposure (corrected viability calculated for MTT reducing test items using freeze-killed control tissues). The 3-minute exposure value was less than the cut-off percentage cell viability value of 50%, distinguishing corrosive from non-corrosive test items. Hence, the test item was corrosive in this skin model and is predicted to be corrosive to human skin (UN GHS Category 1).
The mean optical density (OD) of the negative control of 2 tissues was 1.480 (3-minute exposure) or 1.578 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The mean viability of cells treated with the positive reference item 8 N KOH was 5.7% or 5.1% (3 minute or 1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure.
The difference of viability between the two tissue replicates (at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled.

Under the present test conditions the test item tested at two exposure periods of 3 minutes or 1 hour was cytotoxic and, hence, predicted to be corrosive to skin (UN GHS Category 1) in an experiment employing an artificial three-dimensional model of human skin.
Executive summary:

Under the present test conditions the test item tested at two exposure periods of 3 minutes or 1 hour was cytotoxic and, hence, predicted to be corrosive to skin (UN GHS Category 1) in an experiment employing an artificial three-dimensional model of human skin.