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:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Principles of method if other than guideline:
The HCE model is currently involved in the eye irritation validation conducted by COLIPA following ECVAM guidelines. Furthermore, it is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). This model is recognized as the model of choice and scientifically relevant as documented by several publications (Cotovio et. al., Tox. in Vitro, 24, 2010, 523-537).
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: Assessment of ocular irritation potential of the test substance by determination of its cytotoxic effect on a human corneal epithelium (HCE) model (exposure 60 min./rt followed by 16 hours incubation at 37 °C, subsequently MTT).
Details on test animals or tissues and environmental conditions:
The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells from the cell line HCE reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: as negative control: Phosphate Buffered Saline (PBS, 30 µL)
Amount / concentration applied:
30 µl per insert
Duration of treatment / exposure:
60 min./room temperature
Observation period (in vivo):
post-exposure incubation: 16 hours (37 °C, 5 % CO2, maximum humidity)
Details on study design:
The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelia. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item.
Tests were performed in triplets. The test item was applied at a 100 % concentration, i.e. 30 µl per insert, for 60 min at room temperature. After the exposure period the inserts were washed carefully with PBS. After a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction was performed. Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item.

Positive control: 1H-1,2,4-Triazole-3-thiol (30 mg, plus 30 µl PBS for moistening)

Results and discussion

Any other information on results incl. tables

A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.

Sample No.

 Test item

 %Viability

 

1 -3

negative control PBS

100

4 -6

positive control

1H-1,2,4-Triazole-3-thiol

3.42

16 -18

test substance

101.88

The test item was characterized by having no significant impact on cell viability after test item exposure.

Applicant's summary and conclusion

Interpretation of results:
other: no irritant property
Executive summary:

An in vitro study for assessing ocular irritation was conducted on a human corneal epithelial (HCE) cell model. This model is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). Undiluted test item was applied topically to the reconstructed HCE tissue (30 µl per insert). After an exposure period of 60 minutes (room temperature), followed by a 16 hours post-treatment incubation period (37 °C, 5 % CO2, maximum humidity), the cell viability was 102 % as measured by a MTT conversion assay. Thus, it is concluded that the substance in not an irritant to the eye.