Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-10-10 to 2018-10-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: human cornea model
Details on test animals or tissues and environmental conditions:
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) 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.

The EpiOcular™ human cell construct (MatTek Corporation) is used in this assay. This three-dimensional human cornea model allows the identification of a test item's potential to induce eye irritation or serious eye damage by assessing cell viability after treatment. The model is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells. Test materials can be applied topically to the model so that also water insoluble materials may be tested. Prior to use, each plate (6, 12, and 24-well) and its cover will be uniquely identified with a permanent marker by a plate number and/or test article number.
The cytotoxicity of the test article (and thus the ocular irritation potential) is evaluated by the relative viability of the treated tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test article-treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion).
Supplier: MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, Bratislava, Slovakia
Lot No.: 27073
Expiry date: 11 October 2018

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 μL (or a sufficient amount to cover uniformly the entire tissue surface)

Duration of treatment / exposure:
The plates with the treated tissue units were incubated for the exposure time of 30 ± 2 minutes at standard culture conditions (37±2°C in an incubator with 5 % CO2, ≥95% humidified atmosphere).
Duration of post- treatment incubation (in vitro):
120 ± 15 minutes
Number of animals or in vitro replicates:
2
Details on study design:

- RhCE tissue construct used:
EpiOcular™ human cell construct (MatTek Corporation), Lot No.: 27073

- Duration and temperature of:
exposure: 30 ± 2 minutes at standard culture conditions (37±2°C in an incubator with 5 % CO2, ≥95% humidified atmosphere)
post-exposure immersion: 12 ± 2 minutes immersion incubation (Post-Soak) at room temperature
post-exposure incubation: 120 ± 15 minutes at standard culture conditions
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm (± 10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752)
- Description of the method used to quantify MTT formazan :
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 μL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±2°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere. Each insert was removed from the 24-well plate after 3 hours ± 15 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 24-well plate containing 2 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) and were stored overnight at 4-10°C in the dark.
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (120 rpm) for approximately 3 hours at room temperature, protect from light. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken. Following the formazan extraction, 200 μL sample(s) from each tube (preferably 2×200 μL if possible) was placed into the wells of a 96-well plate (labelled appropriately) and Absorbance / Optical Density of the samples was determined in a 96-well plate spectrophotometer at the wavelength of 570 nm.
- Description of evaluation criteria used: The test chemical is identified as requiring 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 less than or equal (≤) to 60% of the negative control
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392-492-1722) using the fifteen proficiency chemicals according to OECD Test Guideline No. 492.
- Acceptable variability between tissue replicates for positive and negative controls :
The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
The acceptable percentage viability for positive control (mean of two tissues) is:
- 30 minute exposure: below 50% of control viability
- 6 hours exposure: below 50% of control viability
- Acceptable variability between tissue replicates for the test chemical
The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colourant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results
Irritation parameter:
other: % viability
Run / experiment:
1, mean of two replicates
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

OD values and viability percentages of the controls:

Controls

Optical Density (OD)

Viability (%)

Δ%

Negative Control:
Sterile deionized water

1

2.150

92

15

2

2.505

108

mean

2.328

100

 

Positive Control:
Methyl acetate

1

0.370

16

5

2

0.476

20

mean

0.423

18

 

 

OD values and viability percentages of the test item:

Test Item

Optical Density (OD)

Viability (%)

Δ%

Test item

1

2.123

91

3

2

2.188

94

mean

2.155

93

 

standard deviation (SD)

1.97

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation/corrosion potential under the applied testing conditions and is considered as not requiring classification and labelling according to UN GHS (UN GHS No Category).
Executive summary:

A study according to OECD Guideline 492 was conducted to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro. Before treatment the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item (50 μL/units) and incubated for 30 ± 2 minutes at standard culture conditions (37±2°C in an incubator with 5±1% CO2, ≥95% humidified atmosphere). Exposure of test material was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 12 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test items treated tissues were incubated for 120 ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh assay medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±2°C in an incubator with 5±1% CO2 protected from light, ≥95% humidified atmosphere. The formazan precipitated was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated 30 ± 2 minutes. The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 93 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to the eye. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.