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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
EpiOcular™ Cornea Epithelial Model
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 28, 2016 to December 02, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Phosphoric acid, mono- and bis (C16-20 branched and linear alkyl) esters
- Batch: CH 200529/001
- Composition: UVCB
- Appearance: Lightly yellow liquid

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test substance to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 µL
Duration of treatment / exposure:
30 minutes
Observation period (in vivo):
-
Duration of post- treatment incubation (in vitro):
After exposure the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.
Number of animals or in vitro replicates:
3
Details on study design:
The test consists of application of the test substance (50 µL) to the surface of the cornea epithelial construct for 30 minutes. After exposure the cornea epithelial construct is thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. After transfer to fresh medium for a 2 hours incubation period determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment (mean absorption measurement - optical density reading at 570 nm). Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance.

Results and discussion

In vitro

Results
Irritation parameter:
other: cell viability (%)
Run / experiment:
reduction of MTT
Value:
85
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test substance compared to the negative control tissues was 85%. Since the mean relative tissue viability for the test substance was above 60%, the test substance was considered to be non-irritant.
The positive control had a mean cell viability of 27% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was 1.5, which is within the acceptability range from > 0.8 to < 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 11%, indicating that the test system functioned properly. It was concluded that this test was valid and that the test substance was non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Applicant's summary and conclusion

Interpretation of results:
other: CLP criteria not met
Remarks:
not classified
Conclusions:
Under the study conditions, the test substance was considered to be non irritant to human eye (EpiOcular™ Cornea Epithelial Model).
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance according to OECD Guideline 492 (EpiOcular™ Cornea Epithelial Model), in compliance with GLP. The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. The test consisted of an application of the test substance (50 µL) to the surface of the cornea epithelial construct for 30 minutes. After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. After transfer to fresh medium for a 2 h incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment (mean absorption measurement - optical density reading at 570 nm).  Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test substance compared to the negative control tissues was 85%. Since the mean relative tissue viability for the test substance was above 60%, the test substance was considered to be non-irritant. The positive control had a mean cell viability of 27% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was 1.5, which is within the acceptability range from > 0.8 to < 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 11%, indicating that the test system functioned properly. It was concluded that this test was valid. Under the study conditions, the test substance was considered to be non irritant to human eye (Verbaan, 2017).