Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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 (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
The test item was homogeneous by visual inspection.
Storage conditions: Room temperature
Batch No.: VFH-2016-08

Test animals / tissue source

Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

To assess the ability of the test material to directly reduce MTT a pretest was performed.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: yes (tissue incubations for positive and negative controls included)
Amount / concentration applied:
50 μL
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
Two tissue samples were used per group.
Details on study design:
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.

Results and discussion

In vitro

Results
Irritation parameter:
other: viability
Remarks:
[%]
Run / experiment:
mean
Value:
73.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. T. Thus for the test substance the final mean viability is given after KC correction.

Any other information on results incl. tables

Methyl acetate was used as positive control and deionised water as negative control and both gave the results to satisfy acceptance criteria.

Methyl acetate decreased the mean viability to 23.2 % of control cultures.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the EpiOcular test (OECD 492, GLP), the substance was found to be non irritating to eyes.
Executive summary:

The objective was to assess the eye irritating potential of test substance. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

However, in the current case the results derived with EpiOcular alone were sufficient for a final assessment. Therefore, further testing in BCOP was waived.

A GLP-conform guideline study was conducted.

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50μL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular™ eye irritation assay: The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced. The final relative mean viability of the tissues treated with the test substance was 73.4%. Potential compound residues remained on the tissues treated with the test substance after the washing procedure (viable tissues, only). Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria described in chapter 3.10, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.