Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From December 05, 2017 to April xx, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This in vitro risk assessment assay predicts the acute eye irritation potential of a chemical by measurement of its cytotoxic effect on the EpiOcular™ corneal epithelial model.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: White pastilles

Test animals / tissue source

Species:
other: EpiOcularTM tissue model (OCL-200-MatTek Corporation)
Strain:
other: Keratinocyte 4F1188
Details on test animals or tissues and environmental conditions:
Test system
The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

The EpiOcular TM eye irritation test (EIT) using the MatTek EpiOcularTM tissue model OCL-200, was validated by the European Union Reference Laboratory for Alternative to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular TM EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose, as an alternative to Draize Rabbit Eye Test with excellent correlation of in vivo to in vitro test results.

QC results for the specific lot of models received (Lot# 27002) were checked in-house for MatTek acceptance ranges with the following outcome:
- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 0.3 % Triton X-100) where ET50 is the time taken for 0.3 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS



Test system

Vehicle:
other: PBS (Sterile Dulbecco’s Phosphate Buffered Saline)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
20 µL of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) plus 50 mg of test substance.
Duration of treatment / exposure:
6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion
Duration of post- treatment incubation (in vitro):
18 h ± 15 minutes post-treatment incubation
Number of animals or in vitro replicates:
Three tissues per condition (n=3)


Results and discussion

In vitro

Results
Irritation parameter:
other: Percentage of viability (relative to negative control)
Value:
84.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Prior to the study, the required compatibility checks (as per SOP L0069) confirmed that the test substance did not interfere with MTT or solvent.

Applicant's summary and conclusion

Interpretation of results:
other: CLP criteria not met
Remarks:
non-irritant to eye
Conclusions:
Under the study conditions, the test substance was determined to be non-irritant to the eye.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide', using Reconstructed human cornea-like Epithelium (RhCE), according to OECD Guideline 492, in compliance with GLP. EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH. On Day 1, after pre-wetting tissues with 20 µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, the tissues in triplicate were exposed to single topical application of 50 mg neat test substance or 50 µL of reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation, prior to the MTT endpoint. On Day 2, MTT tests and measurement were performed 570 nm without reference filter. The viability of the tissues were assessed and compared to a negative control. The percentage viability obtained with the test substance was determined to be 84.2%,which is well above the threshold (i.e., >60%) indicating no irritation potential. Under the study conditions, the test substance was determined to be non-irritant to the eyes (XCellR8, 2019).