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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
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)
In the invalid experiment 1, the post-incubation time was 60 min instead of 120 min. This was seen as uncritical, as the experiment as invalid due to a high standard devia-tion between the replicates and a repetition was performed.
GLP compliance:
yes (incl. QA statement)

Test material

Reference substance name:
Lubricating oils, used, distd.
EC Number:
EC Name:
Lubricating oils, used, distd.
Cas Number:
Lubricating oils, used, distd.
Test material form:

Test system

Duration of treatment / exposure:
28 minutes
Number of animals or in vitro replicates:
Details on study design:
The EpiOcularTMEye Irritation Test (EIT) predicts the acute eye hazard potential of chemicals by measurement of tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model. Within a testing strategy, the EpiOcularTMEIT can be used as a replacement of thein vivoDraize Eye Irritation Test.
It is utilized for the classification and labelling of chemicals concerning their eye hazard potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required.
The liquid testitem will be applied topically to a three-dimensional RhCE tissue construct in duplicate for an exposure timeof 30 ± 2 minutes.
Eye hazard materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt. The formazan is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison with untreated negative controls is used to predict the eye hazard potential.

Results and discussion

In vitro

Irritation parameter:
other: optical density
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Any other information on results incl. tables

The test item Lubricating oils, used, distd. / Distillato leggero was applied to a three-dimen-sional human cornea tissue model in duplicate for an exposure time of 28 minutes.

50 μL of the liquid test item was applied to two tissue replicates.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The form-azan production was evaluated by measuring the optical density (OD) of the resulting solu-tion.

Demineralised water was used as negative control and methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the ab-sorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.8, OD was 2.0. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 29.1% (< 50%).

The variation within tissue replicates of the controls and the test item was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 86.3%.

This value is above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Under the conditions of the test, the test item Lubricating oils, used, distd. / Distillato leggero is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.
According to CLP regulation, the substance should not be classified as eye irritant.

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