Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 - 27 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ GLP guideline study with minor deviations (no medium exchange before overnight incubation of the insert (Preparation of EpiOcularTM Tissues for Treatment), no demonstration of technical proficiency)

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:
28 July 2015
Deviations:
yes
Remarks:
/ GLP guideline study with minor deviations (no medium exchange before overnight incubation of the insert (Preparation of EpiOcularTM Tissues for Treatment), no demonstration of technical proficiency)
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-di-tert-butyl-4-hydroxybenzyl alcohol
EC Number:
201-815-6
EC Name:
3,5-di-tert-butyl-4-hydroxybenzyl alcohol
Cas Number:
88-26-6
Molecular formula:
C15H24O2
IUPAC Name:
2,6-di-tert-butyl-4-(hydroxymethyl)phenol
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dry conditions. Protect from heat and direct sunlight

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe. It was concluded that the EpiOcularTM
EIT is able to correctly identify chemicals not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.
In order to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant), further testing is required.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcularTM human cell construct (OCL-200-EIT, MatTek Corporation) is used in this assay. It is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells. All biological components of the EpiOcularTM tissue and the kit culture medium have been tested and are free of the presence of viruses, bacteria and mycoplasma.
Analysis for tissue functionality and quality was performed and 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):
25 ± 2 min (Post-exposure immersion incubation)
18 h ± 15 min (Post-treatment incubation)
Number of animals or in vitro replicates:
2 replicates
Details on study design:
- Details of the test procedure used
The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by the relative viability of the treated RhCE tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT in control and test item treated cultures.

- RhCE tissue construct used, including batch number
The EpiOcularTM human cell construct (OCL-200-EIT, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 23742)

- Doses of test chemical and control substances used
50 mg test item; 50 μL positive control (methyl acetate) and 50 μL negative control (sterile deionized water)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
exposure: 6 h ± 15 min at 37 ± 1 °C
post-exposure immersion: 25 ± 2 min at room temperature
post-exposure incubation: 18 h ± 15 min at 37 ± 1 °C

- Description of any modifications to the test procedure
The inserts were transferred aseptically into the 6-well plates and incubated at standard culture conditions directly overnight (37 ± 1°C in an incubator with 5 ± 1% CO2, 90 ± 10% humidified atmosphere) instead of firstly for 1 h, after which the medium should have been exchanged by fresh medium and incubated then overnight.

- Number of tissue replicates used per test chemical and controls
2 replicates each

- 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
Inserts were removed from the 24-well plate after the incubation time inn MTT solution (3 h ± 10 min), the bottom of the insert was blotted on absorbent material and transferred to a 6-well plate containing 2 mL isopropanol per well in a manner avoiding the isopropanol to flow into the insert. The plate was sealed with parafilm. To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for ~2 h at room temperature. 200 μL samples from each tube were placed into the wells of a 96-well plate and the absorbance /optical density was measured in a 96-well plate spectrophotometer to determine cell. viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item requires classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60% of the negative control.
The test item requires no classification and labelling according to UN GHS (No Category), if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
The results obtained for the respective negative and positive controls are in the range of the historical control data.

- Complete supporting information for the specific RhCE tissue construct used
A copy of the test kit quality control sheet and of the certificate of analysis provided by the manufacturer are included in the study report.

- Reference to historical data of the RhCE tissue construct
yes, no further information available

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
no information provided

- Positive and negative control means and acceptance ranges based on historical data
negative control mean OD: 2.237, range: 2.060 - 2.378
positive control mean OD: 0.229, range: 0.064 - 0.500

- Acceptable variability between tissue replicates for positive and negative controls
Acceptable variability between tissue replicates for positive and negative controls < 20%.

- Acceptable variability between tissue replicates for the test chemical
Acceptable variability between tissue replicates for the test chemical < 20%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: mean viability (%)
Run / experiment:
negative control
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: mean viability (%)
Run / experiment:
positive control
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: mean viability (%)
Run / experiment:
test item
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no information provided

DEMONSTRATION OF TECHNICAL PROFICIENCY: no information provided

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (2.115) of the two negative control tissues lies between 0.8 and 2.5.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (4%) lies below 50% of the control viability.

Any other information on results incl. tables

Table 1: MTT results of eye irritation study

Controls

 

Optical Density (OD)

Viability (%)

Δ%

Negative Control: Sterile deionized water

1

2.06

97

5

2

2.17

103

mean

2.115

100

-

Positive Control: Methyl acetate

1

0.064

3

1

2

0.086

4

mean

0.075

4

-

Test item

1

2.063

98

10

2

1.855

88

mean

1.959

93

-

standard deviation (SD)

6.95

-

 

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