Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 4th to 6th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

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 model is the biological model required by OECD TG 492.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcular TM model is manufactured by MatTek in vitro Life ScienceLaboratories (Mlynske Nivy 73, Bratislava 821 05 -Slovakia). A stratified squamous epithelium similar to the corneal epithelium is obtained from Normal, Human Epidermal Keratinocytes (NHEK), derived from Neonatal-foreskin issue, grown in a medium chemically defined without the addition of fetal serum, on specially prepared cell culture insert (9mm) at the air-liquid interface.The batch is certified for the absence of HIV, Hepatitis B, Hepatitis C ,fungi,bacteria and yeast. Media are sterile. The inserts are sent by courier at 4 ° C in 24-well plates containing an agarose gel nutritious. The plates are wrapped in a plastic container. Including time in transit, tissues shell life is 96h.
- NAME and BATCH: EpiOcularTM batch N° 23774
- STATE MANUFACTURER MatTek IVLSL
- Cell viability OD (540nm-570 nm) [range indicated by manufacturer 1.1 - 3.0]: 1.603 ± 0.058 (Accepted)
- Barrier Function: ET-50 [range indicated by manufacturer 12.2 - 37.5 min]: 15.3 min (Accepted)
- DATE OF ARRIVAL: 04.04.2017
- EXPIRATION DATE: including time in transit, tissues shell life is 96h
- PREPARATION OF CULTURE: immediately after the arrival in the laboratory the EpiOcularTM were equilibrated to room temperature for 15 minutes. Tissues were then removed from the agarose nutrient solution under a sterile air flow cabin. The inserts were rapidly placed in a 6-well plate previously filled with 1 ml of medium pre-warmed at 37°C. The wells were placed in incubator at 37 °C, 5% CO2 and saturated humidity for 1h. After the incubation period the medium was replaced with 1ml of fresh medium and tissues were incubated at 37 °C, 5% CO2 and saturated humidity overnight. The test item was applied the day after the arrival of the test system.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: positive control: methyl acetate; negative control: ultrapure water
Amount / concentration applied:
50 mg of test item and 50 µl of the controls were directly and uniformy topically applied.
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
Two replicates for test item, positive and negative controls.
Details on study design:
- Details of the test procedure used: EpiOcular TM model according to OECD492 (July 2015)

- RhCE tissue construct used, including batch number: a stratified squamous epithelium similar to the corneal epithelium is obtained from Normal, Human Epidermal Keratinocytes (NHEK), derived from Neonatal-foreskin tissue, grown in a medium chemically defined without the addition of fetal serum, on specially prepared cell culture insert (9mm) at the air-liquid interface (EpiOcular TM batch N° 23774).

- Doses of test chemical and control substances used: 50μl for negative and positive controls and 50 mg of test item. Before the application the tissue surface is pre-treated with 20 μl of Ca++ and Mg++ free Dulbecco’s phosphate buffered saline.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): the exposure was carried out for 6 hours at 37 °C, 5% CO2 and the post incubation was carried out for 18 hours always at 37 °C, 5% CO2.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Not required. No colour interference and direct MTT reduction for test item.

- Number of tissue replicates used per test chemical and controls: test item, positive and negative controls were evaluated in duplicate on viable tissues. For each tissue were made two technical replicates.

- List of instruments used for the test:
ANALYTICAL BALANCE XS204
CO2 INCUBATOR
LAMINAR FLOW CABIN
MICROPLATE AUTOREADER INFINITE M200
MICROPIPETTES
REFRIGERATOR FRL 360
NEX POWER 1000 SYSTEM

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): optical density measured at 570 nm with TECAN INFINITE M-200 spectrophotometer is used for quantifying MTT formazan.

- Description of the method used to quantify MTT formazan: the method allows to evaluate a toxic event and related cytotoxicity by a colorimetric assay. Mitochondrial dehydrogenase of viable cells cleaves the tetrazolium ring yielding blue/purple MTT crystals which are insoluble in aqueous solutions. MTT crystals are extracted and optical density is measured at 570 nm.

- Data Acquisition: the accuracy of the spectro-photometrical measurements are in the range between 0 and 2 OD: < +/-(1% +10 mOD). The Raw data of OD were directly recorded in the Microplate autoreader (TECAN INFINITE M-200), reported in a Excel file and further processed (Software Magellan Tracker 6.5).
For each tissue were made two technical replicates.
The mean of 8 Blank OD (2-propanol) was subtracted to every single OD value.
The Biological Replicate OD mean was calculated on two technical replicates.
The viabilty of each Biological Replicate was calculated dividing the respective OD mean for the optical density average of the two Negative Control (NC) Biological Replicates in accordance with the following formula:
% Viability= (OD biological replicate Positive Control or sample/mean OD of Negative Control biological replicates)*100
The viability % mean of the two tissues treated with the same substance was calculated.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: according to the United Nations Globally Harmonized System (UN GHS) of Classification and Labelling of Chemicals and EU CLP (EC) No 1272/2008 if the mean relative tissue viability (%) of treated tissues exposed to the test substance is > 60 % the test substance is identified as not requiring classification and labelling(NO Category). If the mean relative viability is ≤ 60 % the test substance is identified as potentially requiring classification and labelling as Category 1 (risk phrase H318: causes serious eye damage) or Category 2 (risk phrase H319: Causes serious eye irritation). No further discrimination within Category 1 and Category 2 is possible. This evaluation criteria are the same reported in section ' Interpretation of Results and Prediction Model' of OECD 492 (July 2015)

- Acceptable variability between tissue replicates for positive and negative controls: for positive and negative controls the difference of viability between the 2 tissues replicates must be < 20 %. Moreover for negative control mean OD value of 2 tissue must be > 0.8 and < 2.5 and for positive control the mean cell viability expressed as percentage compared to negative control must be < 50 %.

- Acceptable variability between tissue replicates for the test chemical: for test item the difference of viability between the 2 tissues replicates must be < 20 %. If the colorant control result and/or the direct reduction of MTT by the test item is> 50 % of the viable negative control, the product may be considered as not testable.

- Statistics: no specific statistical analysis has been carried out except the standard deviation calculations.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of two replicates
Value:
ca. 66.94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: negative control meets the acceptance if the mean OD negative control value of the 2 tissues is > 0,8 and < 2,5 and if the difference of viability between the 2 tissues replicates is < 20 %.
Values OD mean of two tissues for negative control: 1.7748 and 1.7888, Mean: 1.7818 inside range 0.8 - 2.5. Difference of viability between the 2 tissues replicates for negative control : 100.39 - 99.61 = 0.78 % < 20 % .
Based on these considerations the acceptance criteria for negative control were satisfied.

- Acceptance criteria met for positive control: positive control meets the acceptance criteria if mean cell viability expressed as percentage compared to negative control is < 50% and if the difference of viability between the 2 tissues replicates is < 20%.
Mean cell viability for positive control: 12.61 % < 50 %. Difference of viability between the 2 tissues replicates for positive control: 14.27 - 10.94 = 3.33 % < 20 % .
Based on these considerations the acceptance criteria for positive control were satisfied.

- Acceptance criteria for test item: for test item the difference of viability between the 2 tissues replicates must be < 20 %. Difference of viability between the 2 tissues replicates for positive control: 69.39 - 64.49 = 4.9 % < 20 % .
Based on these considerations the acceptance criteria for test item were satisfied.

Applicant's summary and conclusion

Interpretation of results:
other: not eye irritant according to the CLP Regulation (EC n.1272/2008)
Conclusions:
Non-eye irritant.
Executive summary:

The study was conducted in order to assess the eye irritation of the test item using the EpiOcularTMEye Irritation Test (EIT) according to OECD TG 492 (July 2015) specific for solid chemicals. Eye irritation was assessed at 6h exposure followed by product washing and a post incubation period of 18 h by using the MIT test method to quantify the residual cell viability. Based on the adopted prediction model and to MIT results (66.94 %) the test item was not irritant according to UN GHS and according to the CLP Regulation (EC n.1272/2008).