Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 17 to 19, 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
study report
Report Date:

Materials and methods

Principles of method if other than guideline:
Evaluation of eye irritation on 3D human corneal epithelium in vitro reconstituted (SkinEthic HCE) through the investigation of cytotoxicity by MTT test.
The test method is comparable to OECD guideline 492 (2017).
GLP compliance:
the available study was originally run not for REACH purposes; however, it is scientifically valid and well documented.

Test material

Test material form:
solid: particulate/powder

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
- Justification of the test method: evaluation of cell survival after the exposure to the substance through MTT assay. MTT assay method is a colorimetric assay that allows to determinate the percentage of living cells within a cell culture. This assay is based on the ability the mitochondrial succinate dehydrogenase enzyme to metabolise the tetrazolium salt, giving a colored compound.

- Description of the cell system used: human corneal epithelium in vitro reconstituted. A reconstructed artificial human skin model comprising transformed human corneal epitheliaI cells, kept for 5 days in chemically defined medium on inert polycarbonate filters. It was purchased from SkinEthic.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg
Duration of treatment / exposure:
1 hour at room T.
Duration of post- treatment incubation (in vitro):
16 hours at 37 °C, 5 % CO2.
Number of animals or in vitro replicates:
3 replicates
Details on study design:
MTT assay
At the end of the exposure the MTT assay was performed to evaluate the cell survival on the skin units.
The MTT assay is simple, accurate and yields reproducible results. The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT (Sigma M-2128). This product is of yellowish colour in solution. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.
After 16-hour incubation of corneal epithelium, the units are incubated for 3 h in 300 μl/well in a 24 well plate of 1mg/ml MTT solution at 37 °C. The solution is then removed and replaced with 1500 μl/well of isopropanol, with further 2 h incubation at room temperature under medium speed shaking.
The absorbance at 570 nm is measured with a microplate reader (Tecan modello Sunrise remote). The absorbance values are corrected by subtracting the reading of the blanks, with the diluent only.
The results are expressed in terms of viability:
% of cell viability = [OD(570 nm ) test product / mean OD(570 nm) negative control] × 100

Acceptance criteria of method
for negative control (CN): the mean OD value of the 3 tissue has to be ≥ 0.7, the standard deviation has to be ≤ 18 %
for positive control (CP): the viability mean (expressed as % of the NC) has to be ≤ 50 %, the standard deviation has to be ≤ 18 %
for the sample : the standard deviation has to be ≤ 18 %

Expression of results
Mean cell viability is predictive of irritant ocular potential of the sample.
Criteria for in vitro interpretation Classification
Mean tissue viability ≤ 50 % classified
Mean tissue viability > 50 % not classified

Results and discussion

In vitro

Irritation parameter:
other: mean cell viability in %
Run / experiment:
average of 3 replicates
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Sample % mean cell viability (ds) after 1 h treatment and 16 h incubation Comment
test substance 100.7 (± 1.2) Not irritant
SLS 0.5 % 1.2 (± 0.7) Irritant
negative control (CN) 100.0 (± 2.8) Not irritant

Applicant's summary and conclusion

Interpretation of results:
other: not irritant according to the CLP Regulation (EC 1272/2008)
Not irritant to the eye under test conditions.
Executive summary:


In vitro assay using a reconstituted 3D human corneal epithelium. Test sample was applied on 3 replicates for 1 hour at room T. Then, the substance was removed and tissues were incubated for 16 h at 37 °C and 5 % CO2. After incubation, evaluation of survival was done using the MTT assay and measuring absorbance at 570 nm. Based on absorbance values, a % of cell viability was derived.

Phosphate buffer was used as negative control; 0.5 % solution in water of SLS was used as positive control. Controls were run in 3 replicates.


Negative and positive controls were both valid. The mean cell viability of test substance was 100.7 ± 1.2 %, thus above the threshold of 50 %. Accordingly, the substance was considered as not irritant to the eye.