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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-20 - 2018-05-04 (experimantal phase)
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
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:
OECD Guideline for the Testing of Chemicals No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage, 09 Oct 2017
Deviations:
no
Principles of method if other than guideline:
EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary, 22 July 2015
EpiOcular™ Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model, 29 June 2015
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid
EC Number:
604-610-3
Cas Number:
147858-26-2
IUPAC Name:
Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 °C; in closed packaging

Test animals / tissue source

Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
Justification for the Selection of the Test System
This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.
Justification for the Selection of the Test Method
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). Therefore, it can be used for regulatory purposes as an initial step in the bottom-up approach or as one of the last steps in a top-down approach to test eye irritation/corrosion potential. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye to formazan conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The test was carried out with the EpiOcular™ reconstructed human cornea-like epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

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):
Approximately 50 mg (83.3 mg/cm2) of the test item were applied directly atop the EpiOcular™ tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue.
Duration of treatment / exposure:
6 ± 0.25 h
Duration of post- treatment incubation (in vitro):
18 ± 0.25 h
Number of animals or in vitro replicates:
duplicates
Details on study design:
- Details of the test procedure used
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37  2 °C, 5.0% CO2 / 95% air. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37  2 °C, 5.0% CO2 / 95% air for 16 - 24 h.
After the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37  2 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. While the test item was applied, the tissue inserts were placed on a sterile surface. After dosing, the inserts were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37  2 °C, 5.0% CO2 / 95% air. At the end of the exposure period the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper.
- RhCE tissue construct used, including batch number
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), Lot No. 27039, keratinocyte strain 4F1188
- Doses of test chemical and control substances used
1. Negative Control 50 µL Aqua dest.
2. Positive Control 50 µL methyl acetate
3. Test Item 50 mg
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
After rinsing the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37  2°C, 5.0% CO2 / 95% air.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
None, information gathered from pre-tests
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) duplicates
- 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
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h  15 min at 37  2 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out immediately by shaking on an orbital plate shaker for 2 - 3 h at room temperature. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Data Analysis
Ocular irritation / damaging potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with Aqua dest. The test item is considered to be irritant / damaging to the eye but it cannot be differentiated between UN GHS “Category 1” or “Category 2”, if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant in accordance with UN GHS “No Category” if relative tissue viability is higher than 60%.

Table Prediction Model
Mean tissue viability
(% negative control) Prediction I / NI
 60 % Irritant (I): UN GHS “Category 1” or “Category 2”
> 60 % Non-Irritant (NI): UN GHS “No Category”

Test Acceptance Criteria
The test meets acceptance criteria if:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
See below
- Positive and negative control means and acceptance ranges based on historical data
See below
- Acceptable variability between tissue replicates for positive and negative controls: < 20%.
- Acceptable variability between tissue replicates for the test chemical: < 20%.

Results and discussion

In vitro

Results
Irritation parameter:
other: tissue viability [%]
Run / experiment:
mean
Value:
2.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
mean tissue viability 24.9%
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not stated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The study was conducted under GLP according to OECD guideline 492 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro. In this study under the given conditions the test item showed eye irritant or potentially damaging effects. The test item is classified as “eye irritant / damaging“ in accordance with UN GHS “Category 1” or “Category 2.” Although the test as such was not initially developed to serve as standalone test in case of a positive result, because not discrimination between Category 1 or 2 can be made, it is considered that this test suffices. The substance was determined to be a skin irritant, but not corrosive to the skin, so the classification as eye damage Cat. 1 is unlikely. Further, based on the current state of art, all available in vitro eye irritation / corrosion tests can not distinguish between Cat. 1 and 2, so a second in vitro test may not give further relevant information. A definitive conclusion can only be derived from an in vivo test, which is not required for this tonnage band and so can be omitted for animal welfare. So, the substance will be, based on all available information, classified as eye irr. Cat. 2.
Executive summary:

In the present study according to OECD TG 492 under GLP the eye irritating / damaging potential of 'Decanoic acid, 3-[[6-deoxy-2-O-(6-deoxy-a-L-manno-pyranosyl)-a-L-mannopyranosil-1-(carboxymethyl)octyl ester, mixture with 1-(carboxymethyl)octyl 3-[(6-deoxy-a-L-mannopyranosyl)oxy]decanoate' was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcularÔ, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6 h exposure period and 18 h post-treatment period and compared to those of the concurrent negative controls.

The mixture of 50 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 50 mg test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (2.2%).

Conclusion: In this study under the given conditions the test item showed eye irritating or potentially damaging effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.