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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:
weight of evidence
Study period:
13 Sep - 06 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
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)
Version / remarks:
adopted in 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Departement Of Health Of The Government of the United Kingdom

Test material

Constituent 1
Chemical structure
Reference substance name:
Dodecyl myristate
EC Number:
218-039-9
EC Name:
Dodecyl myristate
Cas Number:
2040-64-4
Molecular formula:
C26H52O2
IUPAC Name:
dodecyl tetradecanoate

Test animals / tissue source

Species:
human
Strain:
other: EpiOcularTM (OCL-200-MatTek)
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcularTM EIT is used as a replacement of the Draize Eye Irritation Test. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcularTM EIT is intended to differentiate substances that are “not eye irritant” from those that require labelling as either GHS category 1 or 2 for serious eye damage resp. eye irritation potential.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts. Analysis for tissue functionality and for potential contaminants was passed.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
Duration of treatment / exposure:
6 h ± 15 min
Duration of post- treatment incubation (in vitro):
18 h ± 15 min
Number of animals or in vitro replicates:
3 replicates
Details on study design:
- Details of the test procedure used :
EpiOcularTM tissue model was used. After the assessment and exclusion of direct MTT reduction and colouring potential of the test substance, the well plates were prepared and the tissues were pre-incubated in warm medium under standard culture conditions (37°C, 5% CO2 and ≥ 95% relative humidity). After overnight incubation, the tissues were pre-wetted with 20 μL PBS buffer and then incubated at standard culture conditions for 30 ± 2 min. Afterwards, 50 μL of the controls and 50 mg of the neat test substance were applied and incubated for 6 h ± 15 min, then removed by washing which was followed by 25 ± 2 min post treatment immersion and 18 h ± 15 min post treatment incubation, prior to determination of the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

- RhCE tissue construct used, including batch number :
EpiOcular TM (OCL-200), Keratinocyte strain: 4F1188, Lot Number: 27007

- Doses of test chemical and control substances used :
50 μg of neat test substance, 50 μL of demineralised water (negative control), 50 μL of methyl acetate (positive control)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
6 h ± 15 min treatment; 25 ± 2 min post treatment immersion and 18 h ± 15 min post treatment incubation

- Number of tissue replicates used per test chemical and controls (positive control, negative control) : 3

- Wavelength used for quantifying MTT formazan : 570 nm

- Description of the method used to quantify MTT formazan : The amount of extracted formazan per tissue was determined spectrophotometrically at 570 nm in duplicate with a spectrophotometer (BMG LabTech FluoStar Optima).

To calculate the relative absorbance, the following equation was used: % viability = (OD corrected of test substance or positive control/ OD corrected of mean negative control)*100

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :

Acceptance criteria
The in vitro eye irritation test is considered valid if it meets the following criteria:
a) mean OD570 of the negative control: > 0.8 and < 2.5
b) mean relative tissue viability of the positive control: < 50% relative to the negative control
c) variation within replicates (of negative/ positive control and test substance): SD ≤ 18%

Evaluation criteria
A test substance is considered to have a positive result in the in vitro eye irritation test if the relative mean tissue viability after exposure and post treatment incubation is ≤ 60%, thus requiring classification for eye irritation (Cat. 2) or serious eye damage (Cat.1).
A test substance is considered to be non-irritant to the eye if the relative mean tissue viability after exposure and post treatment incubation is > 60%.

- Complete supporting information for the specific RhCE tissue construct used :
Tissue viability: 1.331 ± 0.206 (accepted range: 1.1 - 3.0)
Barrier function: ET-50: 29.61 min (accepted range: 12.2 - 37.5)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: mean viability (%)
Run / experiment:
test substance
Value:
39.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
other: mean viability (%)
Run / experiment:
positive control
Value:
2.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: not in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD = 1.421 > 0.8 and < 2.5
- Acceptance criteria met for positive control: yes, % viability = 2.4% <50%
- Acceptance criteria for variation within replicates met: SD (OD) = 1.7 - 16.6% ≤ 18%

Any other information on results incl. tables

Table 1: Results of MTT assay (6 h ± 15 min exposure, 18 h ± 15 min post-incubation)

 

Tissue No.

OD570(blank corrected)

Mean (OD570)

Mean
(OD570± SD)

Mean viability
(% of NC) ± SD

Aliquot 1

Aliquot 2

Negative control (NC)

1

1.337

1.356

1.347

1.421 ± 0.068

100 ± 4.8

2

1.438

1.434

1.436

3

1.480

1.481

1.481

Positive control (PC)

1

0.061

0.065

0.063

0.035 ± 0.025

2.4 ± 1.7

2

0.018

0.020

0.019

3

0.026

0.017

0.022

Test substance

1

0.836

0.835

0.836

0.565 ± 0.235

39.7 ± 16.6

2

0.411

0.412

0.412

3

0.446

0.448

0.447

 

Applicant's summary and conclusion

Interpretation of results:
other: irritating potential (Eye Irrit. 2 or Eye Dam. 1 according to Regulation (EC) No. 1272/2008)
Conclusions:
Under the conditions of the test, the test substance was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue in the EpiOcular™ EIT prediction model. The result does not allow a final conclusion on the classification of the registered substance as irritant or corrosive and therefore further data are required. In a Bovine Corneal Opacity and Permeability Test (BCOP) capable of identifying substances that can induce serious eye damage and substances not requiring classification for eye irritation or serious eye damage, no prediction could be made for the registered substance. In conclusion, accounting for both in vitro investigations, the registered substance is considered to meet the criteria for classification as Eye Irrit. 2, H319, according to the CLP Regulation (EC) No. 1272/2008.