Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23- 25 Sep 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: 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) (2015)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, 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 MODEL (EpiOcular™ Kit)
- Source: MatTek Corporation, Ashland, USA
- Lot No.: 21572

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 basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. Irritant materials are identified by their ability to damage the underlying cell layers which is determined through a decrease in cell viability as determined by MTT reduction.

ADAPTATION TO CELL CULTURE CONDITIONS
1.0 mL assay medium (37 °C) was aliquoted into 6-well plates. The inserts with EpiOcular™ tissues were transferred aseptically into the plates and pre-incubated at standard culture conditions for 1 h. Afterwards, the medium was replaced by 1 mL fresh assay medium and the EpiOcular™ tissues were incubated at standard culture conditions overnight (18 h). After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca²+ Mg²+ free DPBS. The tissues were incubated at standard culture conditions for 30 min.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5
- Humidity (%): 95

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: The negative control was deionised water and methyl acetate was used as positive control.
Amount / concentration applied:
TEST MATERIAL
- Applied volume: 50 µL

POSITIVE SUBSTANCE
- Substance: methyl acetate
- Applied volume: 50 µL

NEGATIVE CONTROL
- Substance: deionised water
- Applied volume: 50 µL
Duration of treatment / exposure:
30 min
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
The test was performed in duplicate for each treatment and control group.
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the treatment time, the test substance was removed by extensively rinsing the tissues with Ca²+Mg²+ free DPBS in clean beakers.
- Post-treatment incubation period: 2 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, a volume of 300 µL MTT solution was added to each well for 3 h at standard culture conditions. After removal of the MTT solution, wells were rinsed three times with Ca²+Mg²+ free DPBS. Extraction of the formazan product was carried out in 2 mL isopropanol. At the end of the extraction period the optical density (OD) was measured.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: cell viability (%)
Basis:
other: mean values of 2 tissues
Time point:
other: 30 min
Score:
95.1
Reversibility:
other: not applicable
Remarks on result:
other: Test substance
Irritation parameter:
other: cell viability (%)
Basis:
other: mean values of 2 tissues
Time point:
other: 30 min
Score:
6.9
Reversibility:
other: not applicable
Remarks on result:
other: Positive control
Irritation parameter:
other: cell viability (%)
Basis:
other: mean value of 2 tissues
Time point:
other: 30 min
Score:
100
Reversibility:
other: not applicable
Remarks on result:
other: Negative control

Any other information on results incl. tables

Table 1. Results after 30 min incubation time

Test group

Absorbance*

Mean absorbance of 2 tissues*

Rel. absorbance (%)**

Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2

Rel. absorbance (% of negative control)**

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Negative control

1.568

1.653

1.611

97.4

102.6

5.3

100.0

Positive control

0.107

0.114

0.110

6.6

7.1

0.5

6.9

Test substance

1.459

1.606

1.533

90.6

99.7

9.1

95.1

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

The optical pre-experiment (colour interference pre-experiment) to investigate the test substance’s colour change potential in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour. Therefore, additional tests with viable or freeze-killed tissues were not performed.

All acceptance criteria were met.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the conducted test, the test substance did not exhibit irritating properties towards human-derived epidermal keratinocytes in the EpiOcular TM model.