Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 - 31 Aug 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:
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 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
human
Strain:
other: EpiOcular™, OCL-200-EIT,
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcular™ 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 EpiOcular™ 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 EpiOcular™ 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 EpiOcular™ 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. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular™ tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm². Analyses 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 µL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
28 min
Duration of post- treatment incubation (in vitro):
105 min
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used :
EpiOcular™ 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 ± 1 °C, 5 ± 1% CO2 and 80 - 100% relative humidity). After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and then incubated at standard culture conditions for 30 min. Afterwards, 50 μL of the controls and the neat test substance were applied and incubated for 28 min at 37.0 ± 1.0 °C. After exposure the tissue constructs were thoroughly rinsed with DPBS and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 105 min at standard culture conditions, 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. Therefore, a 24-well-plate was prepared with freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 min at standard culture conditions. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was sealed and stored in the refrigerator overnight. On the next day, the plate was shaken for 2 h at room temperature, protected from light. The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

- RhCE tissue construct used, including batch number :
Keratinocyte strain: 4F1188, Lot Number: 27002

- Doses of test chemical and control substances used :
50 μL of undiluted 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:
28 min at 37.0 ± 1.0 °C, 12 min immersion incubation at room temperature, 105 min at 37 ± 1.0 °C post-exposure incubation

- Number of tissue replicates used per test chemical and controls (positive control, negative control) : 2

- Wavelength used for quantifying MTT formazan : 570 nm

- Description of the method used to quantify MTT formazan : The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Anthos Reader 2010 Flexi microtiter plate photometer. Then the mean OD of the blank isopropanol (ODBlk) was calculated, followed by the subtraction of mean ODBlk of each value of the same experiment (corrected values).
The mean OD of the two replicates for each tissue the mean OD of the two relating tissues for controls and test substance were then calculated.

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 :

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: < 20%
d) values for negative control and for positive control within range of historical data

A test item is considered to be positive in the in vitro eye irritation test if the relative mean tissue viability of two individual tissues after exposure to the test item is ≤ 60% of the mean viability of the negative controls, thus requiring classification for eye irritation (Cat. 2) or serious eye damage (Cat.1).

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : negative control range: 1.253 - 2.437; positive control range: 12.4 - 57.2%

- Complete supporting information for the specific RhCE tissue construct used :
Tissue viability: 1.269 ± 0.039 (accepted range: 1.1 - 3.0)
Barrier function: ET-50: 24.36 min (accepted range: 12.2 - 37.5)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: mean viability (%)
Remarks:
out of 2 replicates
Run / experiment:
test substance
Value:
102.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: mean viability (%)
Remarks:
out of 2 replicates
Run / experiment:
positive control
Value:
33.2
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:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: not in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD = 2.1 > 0.8 and < 2.5 (historical range: 1.253 - 2.437)
- Acceptance criteria met for positive control: yes, % viability = 33.2% < 50% (historical range: 12.4 - 57.2%)
- Range of historical values if different from the ones specified in the test guideline: 1 - 3.3% < 20%

Any other information on results incl. tables

Table 1: Results of MTT assay (28 min exposure)

 

Tissue No.

OD570

Mean (OD570) - blank

Mean tissue viability (% of negative control)

Negative

control

1

2.156

2.137

100

2.193

2

2.084

2.067

2.125

Positive control

1

0.750

0.721

33.9

33.2

0.750

2

0.716

0.685

32.6

0.730

Test substance

1

2.174

2.143

102.0

102.4

2.188

2

2.184

2.164

102.9

2.219

  OD = optical density

Applicant's summary and conclusion

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified