Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 - 19 Sep 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to MatTek’s EpiOcular (ET50) protocol, equivalent to MatTek’s EpiOcular (EIT) protocol under current (2014) ECVAM validation and GLP. Study was perfomed before release of OECD 492 in July 2015.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
equivalent or similar 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:
Jul 2015
Deviations:
yes
Remarks:
study was performed before publication of OECD 492; i.e. there are deviations in application of the test and control substance and in tissue viability measurements
Principles of method if other than guideline:
The study was performed according to MatTek’s EpiOcular (ET50) protocol, equivalent to MatTek’s EpiOcular (EIT) protocol under current ECVAM validation.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
TEST EYE MODEL
- Source: MatTek Corporation, Ashland, USA
- Lot No.: 19185

TEST METHOD
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

ADAPTATION TO CELL CULTURE CONDITIONS
At least 1 hour before dosing, EpiOcular™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into cell culture plates containing the pre-warmed assay medium. Additional cell cultures plates containing an adequate volume of medium were prepared as holding plates. The holding plates were pre-warmed in an incubator until use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: The negative control was deionised water and 0.3% Triton X-100 solution was used as positive control.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 100 µL

POSITIVE CONTROL SUBSTANCE
- Positive control substance: 0.3% Triton X-100 solution in deionised water
- Amount applied: 100 µL

NEGATIVE CONTROL SUBSTANCE
- Negative control substance: deionised water
- Amount applied: 100 µL
Duration of treatment / exposure:
Test substance: 3, 30 and 60 min
Positive control: 15 and 45 min
Negative control: 60 min
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
The test was performed in duplicates for each treatment and control group.
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The tissues were gently rinsed with PBS to remove any residual test material.
- Time after start of exposure: 3, 30 and 60 min (test substance); 15 and 45 min (positive control); 60 min (negative control)
- Post-treatment incubation period: at least 10 minutes but not longer than 20 minutes

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed after the incubation period. Therefore, tissues were incubated in 300 µL freshly prepared MTT-reagent for 3 h at 37 ± 1.5 °C and 5 ± 0.5% CO2. After aspiration of the MTT reagent, tissues were rinsed three times with PBS. Extraction of the formazan product was carried out in 2 mL isopropanol for 18.5 h. The optical density was measured at 570 nm wave length in a microplate reader.

Results and discussion

Results of ex vivo / in vitro studyopen allclose all
Irritation parameter:
other: rel. absorbance (% of negative control) / mean value of 2 tissues
Run / experiment:
3 min exposure
Value:
112
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
other: rel. absorbance (% of negative control) / mean value of 2 tissues
Run / experiment:
30 min exposure
Value:
117.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
other: rel. absorbance (% of negative control) / mean value of 2 tissues
Run / experiment:
60 min exposure
Value:
94.8
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritant / corrosive response data:
The relative absorbance values of the test item, corresponding to the cell viability, did not or did only irrelevantly decrease (94.8%) compared with the result of the negative control, consequently the test item was classified as non irritant.
Due to the lack of cytotoxicity, an ET50 value could not be calculated.

Any other information on results incl. tables

Table 2. Results after treatment with the test substance.

 

Treatment interval (min)

Mean Absorbance of
2 tissues*

Rel. Absorbance
(% of negative control)

Negative control

60

1.1600

100.0

Positive control

15

0.6920

59.7

45

0.3262

28.1

Test substance

3

1.2988

112.0

30

1.3678

117.9

60

1.0992

94.8

* Mean of three replicate wells after blank correction

ET50 of the test item = could not be calculated since viability was not reduced below 50%

ET50 of the positive control = 24.2 min

Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent did not show blue colour.

Both acceptability criteria of the assay were met:

The corrected mean OD values of the two tissues exposed to the negative control were ≥ 0.8 (1.2024 and 1.1175).

The ET50-value of the positive control is determined during the exposure period ≤ 30 min (24.2 min). 

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the human cornea model test the test substance does not possess any eye irritating potential.