Registration Dossier

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:
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:
yes
Remarks:
Stability of test or controls was not determined by testing facility. Test and controls not characterized by testing facility (performed under non-GLP conditions by sponsor prior to initiation of study).
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Purity: 97.9%

Test animals / tissue source

Species:
other: In vitro EpiOcular™ tissues containing stratified human keratinocytes
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: Test was performed according to OECD Guideline 492 using EpiOcular™ Eye Irritation Test (EIT)
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: commercially available RhCE tissue constructs

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 mg
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used: After an overnight incubation for 16-24 hours, 6-well plates containing the EpiOcular™ tissues were removed from the incubator. Prior to test or control substance applications, each tissue surface was moistened with 20 μL of Ca++Mg++-free D-PBS and incubated at standard culture conditions for 30 minutes. After incubation, the tissues were tested in duplicate with 50 μL of the positive control or negative control, or approximately 50 mg of test substance and incubated at standard culture conditions for 6 hours. At the end of the 6-hour treatment time, the test substance, or control articles were removed by extensively rinsing the tissues followed by transference to 5 mL of Assay Medium, in a prelabeled 12 well plate for 25 minutes of immersion incubation (Post-Soak) at room temperature to remove any test substance absorbed into the tissue. At the end of the Post-Soak immersion, each insert was removed, the medium decanted off the tissue, the insert blotted on absorbent material, and transferred to the appropriate well of the prelabeled 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for 18 hours at Standard Culture Conditions (Posttreatment Incubation). At the end of the Post-treatment Incubation, the constructs were removed, gently blotted on absorbent material, and transferred to appropriate wells containing 300 μL of MTT solution. The trays were incubated for 180 minutes at standard culture conditions, after which they were transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in a like manner. The plates were sealed with parafilm and stored in the refrigerator (2-8ºC) until the last exposure time was harvested. To extract the MTT, the plates were then placed on an orbital plate shaker and shaken for 2-3 hours at room temperature. At the end of the extraction period, the liquid within the cell culture inserts was decanted into the well from which the cell culture insert was taken. The extract solution was mixed, and two aliquots of 200 μL were transferred to the appropriate wells of a 96-well plate. Two hundred microliters of isopropanol were added to the two wells designated as the blanks. The absorbance at 550 nm (OD550) of each well was measured with a Molecular Devices Vmax plate reader.

- RhCE tissue construct used, including batch number: stratified human keratinocytes
- Doses of test chemical and control substances used: 50 mg test substance; 50 µL of positive or negative control
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 6-hour exposure and 18-hour post treatment incubation at 37°C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: negative control of sterile deionized water (50 µL) for MTT and isopropanol blank for colorant control test
- Number of tissue replicates used per test chemical and controls: duplicate for test substance, positive and negative controls
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 550 nm
- Description of the method used to quantify MTT formazan: If the MTT solution colour turned blue/purple, the test substance was presumed to have reduced the MTT
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Based on OECD Guideline 492, if the test article-treated tissue viability is >60% relative to negative control-treated tissue viability, the test article is identified as not requiring classification and labelling according to UN GHS (No Category). If the test article-treated tissue viability is ≤60% relative to negative control-treated tissue viability, the test article is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or 2)
- Positive and negative control means and acceptance ranges based on historical data: no
- Acceptable variability between tissue replicates for positive and negative controls: yes; the corrected mean OD550 value of the negative control was >0.8 and <2.5, and the mean relative viability of the positive control was ≤50%
- Acceptable variability between tissue replicates for the test chemical: yes

Results and discussion

In vitro

Results
Irritation parameter:
other: % tissue viability
Value:
92.2
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; OD550 was 1.473
- Acceptance criteria met for positive control: yes; viability was 17.5%

The test substance was not observed to reduce MTT directly in the absence of viable cells; therefore, a killed control experiment was not performed. The test substance was not observed to be a colorant in isopropanol; therefore, a colorant control was not performed.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was predicted to be a non-irritant (mean viability was 92.2%).
Executive summary:

The EpiOcular™ Eye Irritation Test (EIT) was used to evaluate the ocular irritation potential of the test substance in the context of classification of ocular irritation according to the UN GHS classification system. The ocular irritation potential was evaluated based upon measuring the relative conversion of MTT (3-[4, 5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide)in the test substance-treated tissues after exposure to the test substance for 6 hours, followed by an 18 hour post-exposure expression period. Ocular irritation potential of the test article was predicted if the relative viability was less than or equal to 60%. If the relative viability was greater than 60%, the test article was predicted to not require classification or labelling for ocular irritation (GHS No Category). The protocol met the requirements of the OECD test guideline “Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labeling for eye irritation or serious eye damage: (TG 492).

The corrected mean OD550value for the negative control was 1.473, and the viability of the positive control, Methyl Acetate, was less than 50%; therefore, the assay results were considered valid. Based upon the results of this assay the test substance resulted in a relative viability of 92.2%, and is predicted to not require classification or labelling for ocular irritation (GHS No Category).