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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Jan 2016
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report
Report date:

Materials and methods

Principles of method if other than guideline:
Test system HCE: Assessment of the ocular irritation potential by determination of the cytotoxic effect of a test item on the human corneal epithelium model (exposure 60 min./rt followed by 16 hours incubation at 37 °C, subsequently MTT).

The HCE model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for severe eye damage/eye irritancy (e.g. Cotovio et. al., Tox. in Vitro, 24, 2010, 523-537), and is routinely used by cosmetic and pharmaceutical companies. It has been prevalidated (van Goethem et. al., Tox in Vitro 20, 2006, 1-17; Alépée et al., Tox in Vitro 27, 2013, 1476-1488) and has entered formal ECVAM validation in 2010. Although a high reproducibility was attested within the validation process, a further need for optimization was identified. A recently conducted multi-laboratory validation study again demonstrated that the assay in general is a promising tool that may be used in combination with others for a solid eye irritation risk assessment (Alépée et al., Toxicol in Vitro 31, 2016, 43–53).
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
EC Number:
EC Name:
Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
Cas Number:
Molecular formula:
tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
impurity 1
Reference substance name:
Unknown impurities, none of which are present at ≥1%
Molecular formula:
Unknown impurities, none of which are present at ≥1%
Unknown impurities, none of which are present at ≥1%
Test material form:

Test animals / tissue source

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 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

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg per insert
Duration of treatment / exposure:
60 min. (room temperature)
Duration of post- treatment incubation (in vitro):
16 hours (37°C, 5% CO2, maximum humidity)
Number of animals or in vitro replicates:
3 inserts
Details on study design:
The irritation potential of the test item is assessed by determination of its cytotoxic effect on a reconstructed human ocular epithelium. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item.
The test substance was applied unchanged, i.e. 30 mg per insert (plus 30 µL PBS to moisten and ensure good contact with the tissue surface) for 60 min at room temperature. After the exposure period the inserts were washed carefully with PBS. MTT reduction was performed after a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity). 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.

Tests were performed in triplets for test substance, positive and negative control.

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

Results and discussion

In vitro

Irritation parameter:
other: cell viability (%)
Run / experiment:
decision criteria: ocular irritant if cell viability
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:

Any other information on results incl. tables

Summary of results:

 Sample No.  Test item  %Viability (mean)
 1 -3

 Negative control (PBS)


 4 -6

 Positive control (SDS 0.3%)


 13 -15

 test substance


Applicant's summary and conclusion

No indication for an eye irritant potential was seen in an in vitro test (human coreneal epithelial model; cp. Cotovio et al., Toxicol. in Vitro 24, 523-537, 2010).
Executive summary:

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial cell model (HCE; Episkin, France). This model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol. in Vitro 24, 523 -537, 2010), and is routinely used by cosmetic and pharmaceutical companies.

In this study 30 mg of this test item was applied topically to the reconstructed HCE tissue (plus 30 µL PBS to moisten and ensure good contact with the skin). 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 103.5 %, as measured by a MTT conversion assay. Based on this assay no potential for eye irritation was concluded.