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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 712132 & 801102
- Expiration date of the lot/batch: Lot No 712132 Nov 2020; Lot No 801102 Dec 2020
- Purity test date: >98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility of the test substance in the solvent/vehicle: in acetone, alcohol and toluene

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Test system: Reconstructed Human Corneal Epithelium, small (area: 0.5 cm2)
Source: EPISKIN SNC, 4, rue Alexander Fleming , 69366 - Lyon Cedex 7, FRANCE

Test system

Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
- Test substance: 30 ± 2 mg test item moistened with 30 ± 2 µl 0.01M PBS pH = 7.4
- Negative control substance: 30 ± 2 µl 0.01M PBS pH = 7.4
- Positive control substance: 30 ± 2 µl methyl acetate
Duration of treatment / exposure:
4 ± 0.1 hr
Duration of post- treatment incubation (in vitro):
at room temperature for 30 ± 2 min
Number of animals or in vitro replicates:
2
Details on study design:
Test system preparation
After receipt of the SkinEthicTM HCE kit, all of the corneal epithelium units used in the study were pre-incubated in maintenance medium (provided by kit supplier) at 37 ± 2 oC, 5 ± 1 % CO2, ≥ 90% humidity for at least overnight before dosing.
Dose of test and control substances
Before application, the 2 corneal epithelium units for test substance, negative control substance and positive control substance were transferred to fresh pre-warmed maintenance medium, respectively. The conditioning incubation was made at 37 ± 2 oC, 5 ± 1 % CO2, ≥ 90% humidity for at least 30 min.
The application was conducted by gently spreading each substance on the epithelium without touching it to ensure the substance cover all the tissue surface, 2 units per substance. The incubation was made at 37 ± 2 oC, 5 ± 1 % CO2, ≥ 90% humidity for 4 ± 0.1 hr.
Removal of test chemicals and post-soak incubation
After treatment, each treated corneal epithelium unit was rinsed with 25 ml sterile 0.01M PBS pH 7.4 (approximately 2 ml per push) to remove the residue of test chemicals from the epithelium surface. After rinsing, remaining PBS was removed by energized reversal; the surface was swept with cotton-swabs carefully without damaging the epithelium when necessary.
The rinsed unit was transferred to 4 ml fresh pre-warmed maintenance medium and incubated at room temperature for 30 ± 2 min to remove the test chemicals inside the tissue.
At the end of incubation, each unit was removed from the maintenance medium. The medium was decanted off the tissue by turning over the insert. The bottom of the insert was dried carefully by gently taping on a dry absorbent paper and cotton swab. The unit was then transferred to 1 ml maintenance medium and incubated at 37 ± 2 oC, 5 ± 1 % CO2, ≥ 90% humidity for 18 ± 0.5 hr.

MTT assay
Each corneal epithelium unit was transferred into 300 µl MTT solution (1 mg/ml in maintenance medium protected from light) and incubated at 37 ± 2 oC, 5 ± 1 % CO2, ≥ 90% humidity for 3 hr ± 15 min.
After incubation, each unit was rinsed in 300 µl PBS to remove excess MTT solution or maintenance medium and then dried on absorbent paper. The unit was then transferred to 1.5 ml isopropanol, sealed and stored at room temperature for 2-3 hours with gentle shaking on a shaker (~ 120 rpm) protected from light.
The extraction solution was homogenized vigorously until a homogenous solution was generated. The empty inserts were removed. Two 200 µl of solution from each well was transferred into a 96-well plate. The absorbance (OD) at 570 nm was read using microplate reader. Isopropanol was used as blank.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: viability
Run / experiment:
1
Value:
98.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: viability
Run / experiment:
2
Value:
106.9
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the SkinEthicTM HCE EIT (for the solid’s protocol) Validated Reference Method (VRM) and associated with UN GHS classification system, the test item- dl-alpha-Tocopheryl Nicotinate, Lot No: 712132 is considered No Category.
Executive summary:

-  The OD mean value of the 2 corneal epithelium units of the negative control substance (0.01 M PBS pH = 7.4) is 1.627 at 4hr exposure; the OD mean value is> 1.0 and ≤ 2.5, i.e., meets the requirement of acceptance;

- The mean viability of the 2 corneal epithelium units of the positive control substance (methyl acetate) is 0.6% at 4hr exposure; the mean viability is20%, i.e., meets the requirement of acceptance;

The difference of viability between the 2 corneal epithelium unit replicates are less than 20%, i.e., meets the requirement of acceptance;

- The mean viability of the test item is 102.8% (i.e., > 50%) at 4hr exposure. Therefore, based on the SkinEthicTMHCE EIT (for the solid’s protocol) Validated Reference Method (VRM) and associated with UN GHS classification system, the test item is considered as No Category.