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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 May 2019 - 08 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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:
2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model (10 February 2017).
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reference substance 001
Cas Number:
67-47-0
Molecular formula:
C6H6O3
impurity 1
Chemical structure
Reference substance name:
Fructose
EC Number:
200-333-3
EC Name:
Fructose
Cas Number:
57-48-7
Molecular formula:
C6H12O6
IUPAC Name:
D-fructose
impurity 2
Chemical structure
Reference substance name:
1-(furan-2-yl)-2-hydroxyethanone
Cas Number:
17678-19-2
Molecular formula:
C6H6O3
IUPAC Name:
1-(furan-2-yl)-2-hydroxyethanone
impurity 3
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
impurity 4
Chemical structure
Reference substance name:
Sodium formate
EC Number:
205-488-0
EC Name:
Sodium formate
Cas Number:
141-53-7
Molecular formula:
CH2O2.Na
IUPAC Name:
sodium formate
impurity 5
Chemical structure
Reference substance name:
Sodium nitrate
EC Number:
231-554-3
EC Name:
Sodium nitrate
Cas Number:
7631-99-4
Molecular formula:
HNO3.Na
IUPAC Name:
sodium nitrate
impurity 6
Chemical structure
Reference substance name:
Glucose
EC Number:
200-075-1
EC Name:
Glucose
Cas Number:
50-99-7
Molecular formula:
C6H12O6
IUPAC Name:
D-glucose
impurity 7
Chemical structure
Reference substance name:
Sodium acetate
EC Number:
204-823-8
EC Name:
Sodium acetate
Cas Number:
127-09-3
Molecular formula:
C2H4O2.Na
IUPAC Name:
sodium acetate
impurity 8
Chemical structure
Reference substance name:
2-furaldehyde
EC Number:
202-627-7
EC Name:
2-furaldehyde
Cas Number:
98-01-1
Molecular formula:
C5H4O2
IUPAC Name:
2-furaldehyde
impurity 9
Chemical structure
Reference substance name:
Sodium 4-oxovalerate
EC Number:
243-378-4
EC Name:
Sodium 4-oxovalerate
Cas Number:
19856-23-6
Molecular formula:
C5H8O3.Na
IUPAC Name:
sodium 4-oxopentanoate
impurity 10
Chemical structure
Reference substance name:
Formaldehyde
EC Number:
200-001-8
EC Name:
Formaldehyde
Cas Number:
50-00-0
Molecular formula:
CH2O
IUPAC Name:
formaldehyde
Test material form:
solid: crystalline
Details on test material:
freeze-dried material
Batch: 1808A-RO-C-I1-FD-Chg#3
Appearance Crystal / brown
Storage: Refrigerator (2 - 8 °C)
Expiring date: 31 December 2019
Specific details on test material used for the study:
- Batch No.: 1808A-RO-C-I1-FD-Chg#3
- Storage Conditions: 2-8 °C

Test animals / tissue source

Species:
other: The EpiOcular™ human cell construct (MatTek Corporation)
Details on test animals or tissues and environmental conditions:
DETAILS ON TEST SYSTEM
- RhCE tissue construct used: EpiOcular™ (OCL-200-EIT)
- Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia
- Lot No.: 30608
- Expiry date: 30 May 2019
- Storage: The EpiOcular™ (OCL-200-EIT) units were stored at refrigerator (2-8°C) until the preparation of tissues for treatment is started. The assay media supplied with the kits were stored at refrigerator (2-8°C).
The EpiOcular™ human cell construct (MatTek Corporation) is used in this assay. This three-dimensional human cornea model allows the identification of test items with the 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 item number.


JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
The cytotoxicity of the test item (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 item treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion).
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™ EIT 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, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: Approx. 50 mg

POSITIVE AND NEGATIVE CONTROLS
- Amount applied: 50 µL
Duration of treatment / exposure:
6 hours (± 15 min)
Duration of post- treatment incubation (in vitro):
25 ± 2 minute immersion incubation (Post-Soak)
18 hours ± 15 minutes (Post-treatment Incubation)
Number of animals or in vitro replicates:
Two replicates of the test item and the controls, respectively.
Details on study design:
DETAILS ON THE TEST PROCEDURE
- Preparation of EpiOcular™ Tissues for Treatment:
After the test kit arrival, the tissues were equilibrated to room temperature for about
15 minutes. The Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37±2°C in an incubator with 5±1% CO2, =95% humidified atmosphere for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours).

-Application:
* Pre-Treatment: After the overnight incubation, the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
* Test Item: Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. To avoid that test item is spilled into the medium under the tissue inserts, the tissue insert was removed from the medium, placed onto a sterile surface (e.g. the lid of a microtiter plate) and dosed by pouring the solid test article onto the tissue surface so that the surface of the tissue was completely covered by the test item. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.
* Positive and Negative Control: A volume of 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the EpiOcular™ tissues surface if necessary.
* Additional controls for MTT direct interacting chemicals: In addition to the normal procedure, 2 killed test item treated tissues and 2 killed negative control treated tissues were used for the MTT evaluation in one run.
* Additional controls for dyes and chemicals able to colour the tissue: In addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).
* Additional controls for non-specific colour in killed tissues: In addition to the normal procedure, two killed test item treated tissues were used for avoiding a possible double correction for colour interference (NSCkilled).

-Exposure:
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±2°C in an incubator with 5±1% CO2, >=95% humidified atmosphere).

- Rinsing:
After the incubation time the plastic films were removed and the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as follows: Three clean beakers (glass or plastic with minimal 150 mL capacity), containing a minimum of 100 mL each of Ca++Mg++ Free-DPBS were used per test item. Each test item utilizes a different set of three beakers. The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps (taking care of not to damage the tissues by the forceps). The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues was dipped into the first beaker of DPBS and was swirled in a circular motion in the liquid for approximately 2 seconds and after was lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container.
This process was performed two additional times in the first beaker. The tissue was rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).

-Post-soak and post-incubation:
After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- MTT Test After Post-incubation:
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.

- Formazan Extraction:
Inserts were 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 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm (between the plate cover and upper edge of the wells).
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (120 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.

- Cell Viability Measurements:
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 read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at the wavelength of 570 nm ((±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using isopropanol solutions as the blank (8×200 µL).

- Check-method to Detect the Colouring Potential of Test Item:
The test item has an intrinsic colour (brown). If the test item is not white, off white, almost white and/or colorless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability which can possibly be caused by the stained surface of the tissues by the test item.
* Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD reading was made following the same conditions as for the other tissues.
* Additional controls for non-specific colour in killed tissues:
The test item is a possible MTT-reducer and has an intrinsic colour (brown). To avoid a possible double correction [TODTT (MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed.
In addition to the normal procedure, two killed test item treated tissues were used to avoid a possible double correction for colour interference. In this additional control, the test item was applied on two killed tissue replicates, which undergo the entire testing procedure but were incubated with medium instead of MTT solution during the MTT incubation step.

- Assay acceptance criteria:
* 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 (liquids)
6 hours exposure: below 50% of control viability (solids)
* 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 colorant 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: Mean tissue viability (%)
Run / experiment:
1
Value:
12
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean tissue viability: 100%
Positive controls validity:
valid
Remarks:
Mean tissue viability: 11%
Other effects / acceptance of results:
OTHER EFFECTS
- Possible direct MTT reduction with test item:
The check-method for possible direct MTT reduction with test item was not completed for the following reason: The color of the MTT 0.3 mg/mL solution was close to black after it was mixed with test item during the check-method in the in vitro skin irritation study (Study Code: 944-439-4530). So the interaction of the test item with MTT solution could not be determined. Based on this information the additional controls were automatically used. This step prevents the false viability which is the possible interaction with MTT, and it occurs if the test item cannot be removed totally from the surface of the tissues. In this situation the direct interaction with MTT was defined based on the OD results of additional controls.
The non-specific MTT reduction (NSMTT) was determined to be 9.885 %. As the NSMTT were below 60 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.

- Colouring potential of test item:
The test item has an intrinsic colour (brown). If the test item is not white, off withe, almost white and/or colorless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. Two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.022. The Non Specific Colourliving % (NSCliving %) was calculated as 1.06 %.
In order to avoid a possible double correction [TODTT (MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was determined. Two additional test item-treated killed tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.011. The Non Specific Colourkilled % (NSClkilled %) was calculated as 0.54 %.

ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: mean OD value 2.05
- Acceptance criteria met for positive control: 11 % viability at 6h exposure
- Difference of viability between the two tissue replicates: 0.1 to 4.6 %
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Any other information on results incl. tables

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below: 

OD values and viability percentages of the controls:

 Controls

 Optical Density (OD)   

 Viability (%)

 Delta%

       Negative Control

sterile deionized water

 1  2.084  102  3.4   
 2  2.015  98
 mean  2.05  100  

Positive Control

Methyl acetate       

 1  0.235  11  0.2   
 2  0.230  11
 mean  0.233  11

 

 OD values and viability percentages of the test item:

 Test item

 Optical Density (OD)   

 Viability (%)

 Delta%

5-(Hydroxymethyl)furfural

 1  0.256  12  1.1
 2  0.233  11
 mean  20.244  12  

OD values of additional controls for MTT-interacting test item:

 

Additional controls

    

Optical Density (OD)

 
 Delta%

Negative control treated

killed tissues:

Sterile deionized water

1

2

mean 

0.037 

0.039

0.038

0.1

Test item treated killed tissues:

5-(Hydroxymethyl)furfural

1

2

mean 

0.194

0.287

0.240 

 4.6

OD values and NSCliving % of additional control:

 Additional colour control  Optical Density (OD)     Non Specific Colour % (NSCliving %)  Delta %
 5-(Hydroxymethyl)furfural

1

2

mean

0.010

0.033

0.022

 1.06  1.1

Remark: Delta%: The difference of viability between the two relating tissues

OD values and NSCkilled % of additional control:

 Additional colour control  Optical Density (OD)     Non Specific Colour % (NSCkilled%)  Delta %
 5-(Hydroxymethyl)furfural

1

2

mean

0.009

0.013

0.011

 0.54  0.2

Remark: Delta%: The difference of viability between the two relating tissues

True tissue viability % (TTV%):

 Parameters  Percent value (%)

Mean[%Viability test item]

[%NSMTT]

[%NSCliving]

[%NSCkilled]

12 

9.885 

1.06

0.54

 MeanTTV %

2*

Remark:NSMTT: Non-Specific MTT reduction

NSCliving: Non-Specific Colour in living tissues

NSCkilled: Non-Specific Colour in killed tissues

TTV%: True tissue viability

*: rounding result, the originally number is 1.595%

Applicant's summary and conclusion

Interpretation of results:
other: Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1)
Conclusions:
The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item 5-(Hydroxymethyl)furfural indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2.
Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test item 5-(Hydroxymethyl)furfuralon three-dimensional RhCE tissue in the EpiOcular™ modelin 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 and incubated for 6 hours (± 15 min) 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 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 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% CO2protected from light, =95% humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (brown), therefore two additional test item treated tissues were used for the non-specific OD (Optical Density) evaluation (NSCliving).

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (brown). To avoid a possible double correction [TODTT*(MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed test item treated tissues were used to avoid a possible double correction for colour interference.

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 for 6 hours ± 15 minutes.

The test item 5-(Hydroxymethyl)furfural showed significantly reduced cell viability in comparison to the negative control (true tissue viability: 2 %). All obtained test item viability results were below 60% when compared to the viability values obtained from the negative control.

Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

The results obtained from thisin vitro eye irritation test, using the EpiOcular™ model, with the test item 5-(Hydroxymethyl)furfural indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2.

 

* TODTT: OD true MTT metabolic conversion for treated tissues