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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017 July to 2017 September
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 (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Supplier: Borica Co., Ltd.
- Lot number: 000577Z
- Expiration date of the lot/batch: 2018-Jul-06
- CAS number: 23519-77-9

Test animals / tissue source

Species:
human

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 microliter
- Concentration (if solution): neat

VEHICLE
- Not used
Duration of treatment / exposure:
30 minutes +/- 2 min
Duration of post- treatment incubation (in vitro):
12 +/- 2 min post-treatment immersion and 2 hours +/- 15 min post-treatment incubation.
Number of animals or in vitro replicates:
3 replicates per run.
Details on study design:
- Details of the test procedure used
Prior to the study, the required compatibility checks confirmed that the test item interfered with MTT and therefore additional freeze killed negative controls (NC-FK) and freeze killed test item controls (TA-FK) were included in the assay to determine the background level of interference (manifesting as altered viability) produced by the test item. The level of viability measured in the interference controls was subtracted from the standard test viability value to give an adjusted final viability. No water colouration was observed and therefore colorant control was not required.

After pre-wetting tissues with 20 microliter PBS (Sterile Dulbecco’s phosphate buffered saline) for 30 +/- 2 min, single topical application of 50 microliter of neat test item or of reference items to the surface of the EpiOcular™ models for 30 +/- 2 min, followed by a 12 +/- 2 min post-treatment immersion, and 2 hr +/- 15 min post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n = 3).


- RhCE tissue construct used, including batch number
EpiOcular™ EIT (OCL-200) Reconstructed Human Ocular Epithelium
Supplier: MatTek Corporation
Keratinocyte Strain: 4F1188
Lot number: 23799
Description of the RhCE tissue: The EpiOcular™ tissue model (OCL200-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.


- MTT test conditions
Spectrometer: BMG LabTech FluoStar Optima
Wavelength for O.D. measurements: 570 nm


- Positive and negative control means and acceptance ranges based on historical data
1. Mean OD570 of the negative control (treated with sterile water) tissues is > 0.8 and < 2.5.
2. Mean of the positive control relative percentage viability is below 50% of negative control viability after 30 +/- 2 minutes exposure.


- Acceptable variability between tissue replicates for positive, negative controls and test items
The SD between three tissues replicates should not exceed 18% in the same run (for negative and positive control tissues and tissues of test items).



Results and discussion

In vitro

Results
Irritation parameter:
other: Tissue viability
Run / experiment:
1 run, 3 replicates per run
Value:
ca. 15.27
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

Table 3. Mean and SD of viability measurements and of viability percentages after 30 +/- 2 min of application and 2 h +/- 15 min post-incubation of test and reference items.

 Consition  Mean of OD  SD of OD  mean of viability %  SD of viability (%)  CV %  Classification
 NC  1.657  0.016  100.00  0.938  0.938  Non-irritant
 PC  0.522  0.115  31.479   6.964  22.122  Irritant
 TA2  0.253  0.008  15.277  0.481

 3.150

 Irritant

Note:

NC: Negative control (sterile H2O)

PC: neat methyl acetate

TA2: Zirconium tetrapropanolate

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
A Test item is considered to be irritant to the eye (i.e. would require labelling as either GHS 1 or 2) if they eye model viability after exposure and post-treatment incubation is ≤ 60%.
A test item is considered as a non-irritant to the eye (i.e. would not require a warning label in the European chemical classification systems) if the eye model viability after exposure and post-treatment incubation is > 60%.
The current assay is not intended to differentiate between GHS class 1 and GHS class 2 or R36 and R41 (degree of stromal damage).
The percentage of viability obtained with test item Zirconium tetrapropanolate was 5.602%, therefore it is considered as Irritant to the eye.
Executive summary:

The eye irritation potential of Zirconium tetrapropanolate was assessed in vitro according to OECD Test Guideline 492 (Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labeling for eye irritation or serious eye damage).

After a 30 +/-2 minute exposure on the surface of EpiOcular™ reconstructed ocular epithelium and 2 h +/- 15 mins post-incubation time, the viability of the tissues was assessed and compared to a negative control. The percentage viability was 5.602% and therefore:

Zirconium tetrapropanolate

was classified as Irritant to the human eye.