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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 - 23 Jun 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
FREIE UND HANSESTADT HAMBURG, Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Test material

Constituent 1
Reference substance name:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol
EC Number:
EC Name:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol
Cas Number:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol

Test animals / tissue source

other: three-dimensional Reconstructed human Cornea-like Epithelium (RhCE) tissue
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: EpiOcular RhCE tissue construct (Mattek) consist of relevant human-derived cells and reconstruct a cornea-like epithelium three-dimensional tissue, which is composed of progressively stratified but not cornified cells. Multiple layers of viable, non-keratinized epithelial cells are present in the reconstructed cornea-like epithelium. The RhCE tissue construct has an epithelial surface in direct contact with air so as to allow for direct topical exposure of test chemicals in a fashion similar to how the corneal epithelium is exposed in vivo. The RhCE tissue construct forms a functional barrier with sufficient robustness to resist rapid penetration of cytotoxic benchmark substances, e.g., Triton X-100. The containment properties of the RhCE tissue construct prevent the passage of test chemical around the edge of the viable tissue, which could lead to poor modelling of corneal exposure. The RhCE tissue construct was free of contamination by bacteria, viruses, mycoplasma, and fungi. The test item was applied topically to three-dimensional Reconstructed human Cornea-like Epithelium (RhCE) tissue constructs and tissue viability was measured following exposure and a post-treatment incubation period.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied: 50 µL
- Concentration: 100% (undiluted)
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
duplicate tissues for each treatment
Details on study design:
- Details of the test procedure used: Two tissue replicates were used for each treatment, negative and positive control. Concurrent negative (NC) and positive controls (PC) were used to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. Prior to treatment the tissues were pre-wetted with 20 μL DPBS (without Mg2+ and Ca2+) and incubated at 37°C, 5% CO2 and 95% relative humidity for 30 ± 2 minutes (pre-treatment). After the 30 ± 2 minutes pretreatment each test item, negative control and positive control was tested by applying 50 μL topically on EpiOcular tissues. The tissues were incubated at 37°C, 5% CO2 and 95% relative humidity for 30 ± 2 minutes (exposure). At the end of treatment, test items were removed by extensively rinsing the tissues with DPBS (without Mg2+ and Ca2+) at room temperature. The rinsing was performed by dipping the tissues three times in containers pre-filled with fresh 100 mL DPBS (without Mg2+ and Ca2+). For each treatment different fresh DPBS (without Mg2+ and Ca2+) solutions were used. The rinsing was repeated twice, each with fresh DPBS (without Mg2+ and Ca2+). After rinsing, the tissues were immediately transferred in 5 mL pre-warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for 12 ± 2 minutes (post-soak). After the Post-Soak each insert is removed and remaining media was decanted, the inert is blotted on absorbent material and transferred to the appropriate well of the pre-labeled 6-well containing 1 mL of warm Assay Medium. The tissues were incubated at 37°C, 5% CO2 and 95% relative humidity for 120 ± 2 minutes (post-treatment). After the Post-treatment Incubation the MTT assay was performed. Therefore, tissues were transferred into wells of a 24-well plate containing 0.3 mL of MTT solution (1 mg/mL). The tissues were incubated at 37°C, 5% CO2 and 95% relative humidity for 180 ± 10 minutes. After incubation each insert was removed from the 24-well plate. The bottom of the insert blotted on absorbent material, and then the insert was transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol will flow into the insert on the tissue surface. The plates were sealed with parafilm and were either stored overnight at 2°C to 8 °C in the dark or immediately extracted on a plate shaker for 2 to 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert decanted into the well from which it was taken. The extract solution was mixed and transferred to a 96-well plate. The absorbance (Optical Density (OD) at 540 nm) was determined with a microplate reader. As the test item was supposed to reduce the MTT solution, two freeze-killed tissues per control and test item were used. All assay procedures were performed as for the viable tissue.
- RhCE tissue construct used, including batch number: OCL-200-EIT, MatTek In Vitro Life Science Laboratories, Bratislava II, Slovak Republic (Lot. No. 23715)
- Doses of test chemical and control substances used: 100% (undiluted)
- Duration and temperature of pre-treatment, exposure and post-exposure incubation periods: 30 ± 2 min (pre-treatment, exposure) and 120 ± 2 min (post-exposure) at 37 °C, 5% CO2 and 95% relative humidity (each treatment period)
- Number of tissue replicates used per test chemical and controls (positive control, negative control: duplicate tissues each
- Wavelength and band pass used for quantifying MTT formazan: 540 - 590 nm
- Description of the method used to quantify MTT formazan: MTT assay was used for quantifying tissue viability. Viable cells of the EpiOcular RhCE tissue construct reduce the vital dye MTT into a blue MTT formazan precipitate, which was then extracted from the tissue using isopropanol. The extracted MTT formazan was quantified using a standard absorbance (Optical Density (OD)) measurement procedure in the range between 540 and 590 nm
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The individual % of control OD540 values was averaged to calculate the mean percent of control. The relative tissue viability was determined against the negative control. If the test item-treated tissue viability is >60% relative to negative control-treated tissue viability, the test item is predicted to be non-irritant. If the test item-treated tissue viability is ≤60% relative to negative control-treated tissue viability, the test article is predicted to be an irritant.
- Positive and negative control means and acceptance ranges based on historical data: 1.885 OD (= 100% viability for negative control), 30.65% (positive control)
- Acceptable variability between tissue replicates for positive and negative controls: 1.0 - 2.9% (negative control), 0.4 - 5.6% (positive control)
- Acceptable variability between tissue replicates for the test chemical: the difference of viability between two replicate tissues is <20%

Results and discussion

In vitro

Irritation parameter:
other: % tissue viability
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
viability 100%
Positive controls validity:
viability 30.1
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Acceptance criteria met for negative control: the negative control OD is >0.8 and <2.5
- Acceptance criteria met for positive control: the mean relative viability of the positive control is below 50% of control viability
- Acceptance criteria met for test item: the difference of viability between two replicate tissues is <20%

Any other information on results incl. tables

Table 1: Summarized results of in vitro eye irritation


Test item

Negative control

Positive control

Test item (corrected reduction control)

Mean corrected OD (n=2 tissues)





Percent difference tissues 1 + 2 (%)





Viability compared to control (%)





Reduction corrected viability (%) of test item





Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
CLP: not classified