Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 - 12 Aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ No demonstration of technical proficiency

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
28 July 2015
Deviations:
yes
Remarks:
/ No demonstration of technical proficiency
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Test animals / tissue source

Species:
human
Strain:
other: Keratinocyte strain: 4F1188
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek Corporation, Ashland MA, U.S.A.). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture.
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: 50 µL
- Concentration (if solution): neat/ undiluted
Duration of treatment / exposure:
30 ± 2 minutes
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
2 replicates
Details on study design:
- Details of the test procedure used
EpiOcularTM cornea epithelial model was used. The test item was applied undiluted (50 μL) directly on top of the tissue for 30 ± 2 minutes at 37.0 ± 1.0 °C. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours 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.

- RhCE tissue construct used, including batch number
Keratinocyte strain: 4F1188, Lot Number: 23438

- Doses of test chemical and control substances used
50 µL of undiluted test chemical, 50 µL of MilliQ water, 50 µL of Methyl Acetate

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
30 ± 2 minutes at 37.0 ± 1.0 °C, 12 ± 2 minutes immersion incubation at room temperature, 120 ± 15 minutes at 37 °C post-exposure incubation

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

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
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 TECAN Infinite® M200 Pro Plate Reader.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable)

- 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 acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20.

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, requiring classification for eye irritation or serious eye damage.

- Acceptable variability between tissue replicates for positive and negative controls
The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20

- Acceptable variability between tissue replicates for the test chemical
The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20

Results and discussion

In vitro

Results
Irritation parameter:
other: mean viability (%)
Run / experiment:
mean value of test item
Value:
88
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: no information provided

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1: MTT assay (30 ± 2 min exposure)

Tissue No.

OD570*

Mean (OD570)

Standard Deviation

Mean tissue viability

(% of negative control)

Standard Deviation
[%]

Negative
Control

1

1.713

1.778

0.091

100

5.1

2

1.842

Positive

Control

1

0.589

0.589

0.001

33

0.6

2

0.588

Test
Item

1

1.580

1.572

0.010

88

0.03

2

1.565

OD = optical density

SD = Standard deviation

 

* The values represent the mean values of two OD570 measurements per well and are corrected for background absorption (0.041).

Isopropanol was used to measure the background absorption.

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