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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Principles of method if other than guideline:
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. This is currently being addressed outside the validation study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Reaction mass of 2,2'-iminodiethanol and 2-{[bis(2-hydroxyethyl)amino]methyl}-4-[2-(4-hydroxyphenyl)propan-2-yl]phenol and 2,2-bis(4-hydroxyphenyl)propane and 2-{[bis(2-hydroxyethyl)amino]methyl}-4-[2-(3-{[bis(2-hydroxyethyl)amino]methyl}-4-hydroxyphenyl)propan-2-yl]phenol
- Appearance: viscous, clear, yellowish liquid

Test animals / tissue source

Species:
other: Assessment of the ocular irritation potential of the test item 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: sodium dodecyl sulfate 0.3 % (SDS, 30 µL)

Results and discussion

In vitro

Results
Irritation parameter:
other: cell viability (%)
Run / experiment:
mean
Value:
ca. 0.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

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

SDS 0.3 %

22.64

13 -15

test substance

0.4

Based on the result the test item was characterized by a significant impact on cell viability.

 

Applicant's summary and conclusion

Interpretation of results:
other: irritant/corrosive to the eye
Executive summary:

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial (HCE) cell model. 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, 2010, 523 -537), and is routinely used by cosmetic and pharmaceutical companies. Undiluted test item (30 µL) was applied topically to the reconstructed HCE tissue. 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 0.4 % as measured by a MTT conversion assay. As the cut-off for a non-irritant to the eye is 50 % (a test substance is predicted to be an ocular irritant if the relative tissue viability (%) exposed to the test substance is < 50), the test substance requires classification for eye irritation/corrosion based on this assay.