Registration Dossier

Diss Factsheets

Administrative data

Description of key information

- Episkin test for skin irritation (OECD 439): 72% cell viability: Non irritant

- Episkin test for skin corrosion (OECD 431): 58% cell viability: Non corrosive

- ICET (Isolated Chicken Eye Test) test (OECD 438): No prediction can be made (Neither NC nor Corrosive (UN GHS Category 1))

- EpiocularTM(OECD 492): 2 % cell viability, Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2019 - 08 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Batch No.: 1808A-RO-C-I1-FD-Chg#3
- Storage Conditions: 2-8°C
Test system:
human skin model
Remarks:
EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN SNC Lyon, France
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EpiSkin Model has been validated for irritation testing in an international trial. The EpiSkin method is a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purposes of distinguishing between skin irritating and no-skin irritating test substances.
Vehicle:
unchanged (no vehicle)
Remarks:
epidermal surface was first moistened with 5 µL deionised water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number: 19-EKIN-019
- Expiry date: 13 May 2019
- Date of initiation of testing: 08 May 2019

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated over night at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

EXPOSURE (DAY 0)
- Test Item: The epidermal surface was first moistened with 5 µL deionised water (prepared by MILLIPORE Synergy UV HF ASTM 1: F8JA80461C in Toxi-Coop ZRT) in order to improve further contact between the test item and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
- Positive and negative control: A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Triplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL PBS 1x solution, once. rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source
- Observable damage in the tissue due to washing: none

POST-INCUBATION (DAY 0-2)
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, >=95% humidified atmosphere.

MTT TEST (DAY 2)
After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5±1 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION (DAY 2)
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for approximately four hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY MEASUREMENTS (DAY 2)
Following the formazan extraction, 2×200 µL sample from each tube 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 570 nm (±10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 mg of the test item.

POSITIVE AND NEGATIVE CONTROL
- Amounts applied: A volume of 10 µL positive or negative control.
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 ± 1h
Number of replicates:
Three replicates were used for the test item and controls, respectively.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
72
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean corrected value
Other effects / acceptance of results:
OTHER EFFECTS:
- Possible direct MTT reduction with test item: As the test item has an intrinsic colour (brown), the check-method for possible direct MTT reduction with test item was not completed for the following reason: The color of the MTT solution was close to black after it was mixed with test item. 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 5.329 %. As the NSMTT were below 50 % 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. This step prevents the false viability, which can possibly be caused by the stained surface of the tissues by the test item. 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 0.040. The Non Specific Colourliving % (NSCliving %) was calculated as 4.8 %. As the NSCliving is below 5%, true MTT metabolic conversion for colour interference is not undertaken.

ACCPETANCE OF RESULTS
- Acceptance criteria met for negative control: Mean OD value 0.834 and standard deviation value (SD) for the % viability 0.43 (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be = or < 18)
- Acceptance criteria met for positive control: 20% mean viability range standard deviation value (SD) for the % viability 7.27 (The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be = or < 18.)
- For test chemicals, the standard deviation value (SD) of the % viability should be = or < 18 : 6.57 % SD

OD values and viability percentages of the controls:

 Control  Optical Density (OD)     Viability (%)

 Negative Control            

1X PBS

 1  0.0831  100
 2  0.0833   100
 3  0.0838   100
 mean  0.0834   100
 standard deviation (SD)     0.43

Positive Control

SDS (5% aq.)             

 1  0.136  16
 2  0.136  16
 3  0.241  29
 mean  0.171  20
    standard deviation (SD)  7.27

OD values and viability percentages of the test item (including corrected values):

 Test Item  Optical Density (OD)     TODTT  Viability (%)  realtive Viability (%)
5-(Hydroxymethyl)
furfural
            
 1  0.668  0.623  80  75
 2  0.581  0.536  70  64
 3  0.682  0.637  82  76
 mean  0.643  0.599  77  72
 standard deviation (SD)        6.57  6.57

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

 Additional controls  Optical Density (OD)   
 

Negative control killed tissues:

1x PBS

1

2

3

mean

0.061

0.095

0.066

0.074 

 

Test item treated killed tissues:

5-(Hydroxymethyl)furfural

1

2

3

mean

0.145

0.099

0.111

0.118

OD values and NSC % of additional control:

 Additional colour control  Optical Density (OD)     Non Specific Colour % (NSCliving %)

 5-(Hydroxymethyl)furfural

(test item treated tissues without MTT

incubation)

1

2

mean

0.037

0.043

0.040 

4.8 

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item 5-(Hydroxymethyl)furfural is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Executive summary:

EpiSkinTMSM test of 5-(Hydroxymethyl)furfural has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439,28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2,=95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

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

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50 % of the negative control.As the NSClivingis below 5%, true MTT metabolic conversion for colour interference is not undertaken.

In thisin vitroskin irritation test using the EPISKIN model, the test item 5-(Hydroxymethyl)furfural did not show significantly reduced cell viability in comparison to the negative control (mean corrected value for possible MTT reduction: 72 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected OD and cell viability values within acceptable limits and standard deviation of all calculated viability values (positive and negative control, test item) was below 18. The experiment was considered to be valid.

The results obtained from thisin vitroskin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item 5-(Hydroxymethyl)furfural is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2019 - 08 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: INVITTOX Protocol No. 118; “EPISKIN(TM) Skin Corrosivity Test” (ECVAM Database Service on Alternative Methods to Animal Experimentation)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Batch No.: 1808A-RO-C-I1-FD-Chg#3
- Storage Conditions: 2-8°C
Test system:
human skin model
Remarks:
EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France, three-dimensional human epidermis model.
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Remarks:
100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 19-EKIN-020
- Expiry date: 20 May 2019
- Date of initiating testing: 15 May 2019

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37 °C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37±1 °C in an incubator with 5±1% CO2 in a = 95 % humidified atmosphere.

APPLICATION / EXPOSURE
- Test Item: Approximately 20 mg test item was applied evenly to the epidermal surface of the two test skin units/exposure times respectively. Subsequently, 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

- Positive and negative control: A volume of 50 µL positive control (glacial acetic acid) or negative control (NaCl 9 g/L) was applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly the complete epidermal 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 epidermis units were incubated for the exposure time of 4 hours (±10 min) at room temperature (18-28 °C).

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Duplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
After the incubation time the EpiSkinTMSM units were rinsed thoroughly with approximately 25 mL PBS 1x solution to remove the test item from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

MTT TEST
To terminate the exposure with the test item, the EpiSkinTMSM units were rinsed with PBS. Thereafter, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±1 °C in an incubator with 5±1 % CO2 protected from light, =95% humidified atmosphere.

FORMAZAN EXTRACTION
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated overnight at room temperature protected from light for formazan extraction. At the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 µL sample from each tube 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 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µL).

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin (SubCat.1A) if mean tissue viability is < 35 % after 3 min exposure
- The test substance is considered to be corrosive to skin (subCat.1B or 1C) if Mean tissue viability is = 35 % after 3 min exposure and < 35 % after 1 hour exposure OR Mean tissue viability is = 35 % after 1 hour exposure and < 35 % after 4 hours exposure
- The test substance is considered to be non-corrosive to skin if mean tissue viability is = 35 % after 4 hours exposure


Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: approx. 20 mg

VEHICLE
- no, but 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact
with the epidermis.

NEGATIVE AND POSITIVE CONTROL
- Amount applied: 50µL each
Duration of treatment / exposure:
4h ±10 min
Duration of post-treatment incubation (if applicable):
no post-treatment incubation
Number of replicates:
Two replicates were used for the test item and control(s) respectively.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
58
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The mean OD value of the two negative control tissues was 0.671
Positive controls validity:
valid
Remarks:
difference of viability between the two tissue replicates: 2.3%
Other effects / acceptance of results:
OTHER EFFECTS
- Visible damage in test system: not reported

- 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 16.429 %. As the NSMTT were below 50 % 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 0.016. The Non Specific Colourliving % (NSCliving %) was calculated as 2.40 %.

ACCEPTANCE OF RESULTS
All validity criteria were within acceptable limits and therefore the study can be considered as valid.
- The mean OD value of the two negative control tissues was 0.671.
- The positive control result showed 3 % viability.
- The difference of viability between the two tissue replicates:
* Negative control: 2.6 %
* Positive control: 2.3 %
* Test item: 14.3 %
* Color control (NSCliving): 0.8 %

All validity criteria were within acceptable limits and therefore the study can be considered as valid.


OD values and cell viability percentages of the positive and negative control:

 Controls  Optical Density  (OD)   Viability (%)

Delta%

 Negative Control:      

NaCl (9g/l saline)

 1  0.662  99  2.6   
 2  0.68  101
 mean  0.671  100  -
 Positive Control:
Glacial acetic acid      
 1  0.026  4  2.3   
 2  0.011  2
 mean  0.019  3  -

OD values and viability percentages of the test item (including corrected values):

 Test item  Optical Density  (OD)   TODTT  Viability (%)  Relative Viability (%)

Delta%

 5-(Hydroxymethyl)
furfural

 1  0.454  0.343  68  51  14.3   
 2  0.549  0.439  82  65
 mean  0.501  0.391  75  58
   standard deviation (SD)

 

 

 10.09

 10.09

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

 Additional controls  Optical Density (OD)   
 

Negative control killed tissues:

NaCl (9 g/L saline)

1

2

mean

0.025

0.030

0.027 

 

Test item treated killed tissues:

5-(Hydroxymethyl)furfural

1

2

mean

0.115

0.161

0.138

OD values and NSCliving % of additional control:

 Additional colour control  Optical Density (OD)     Non Specific Colour % (NSCliving %) Delta % 

 5-(Hydroxymethyl)furfural

(test item treated tissues without MTT

incubation)

1

2

mean

0.013

0.019

0.016 

2.4 

 0.8

Remark:

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

TODTT:true MTT metabolic conversion

Interpretation of results:
other: Non-corrosive
Conclusions:
The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item 5-(Hydroxymethyl)furfural can be classified as Non-corrosive to skin.
Executive summary:

EpiSkinTMSM test of the test item 5-(Hydroxymethyl)furfural has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 431,29 July 2016.

Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours (±10 min) at room temperature. Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2in a = 95 % humidified atmosphere and protected from light. The formazan

precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively.

The test item has an intrinsic colour (brown), therefore two additional test item treated tissues were used for the non-specific OD 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. However, the NSCliving% was 2.40 % (below 5 %), so the (NSCkilled) was not determined and used during the calculation of true MTT metabolic conversion.

For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 58 % (corrected values) at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits.

All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from thisin vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item 5-(Hydroxymethyl)furfural can be classified as Non-corrosive to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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
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)
Specific details on test material used for the study:
- Batch No.: 1808A-RO-C-I1-FD-Chg#3
- Storage Conditions: 2-8 °C
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.
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.

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.

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%

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

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 May 2019 - 08 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2018
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
2010
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 1808A-RO-C-I1-FD-Chg#3
Storage: Refrigerator (2-8 °C)
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3 ºC to 20.5 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.03 g
POSITIVE CONTROL
- Amount(s) applied: 0.03 g
NEGATIVE CONTROL:
- Amount(s) applied: 30 µL
Duration of treatment / exposure:
10 Seconds
Duration of post- treatment incubation (in vitro):
240 Min
Number of animals or in vitro replicates:
3 (treatments and positive control group)
1 (negative control)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

PREPARATION OF EYES
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EYES EXAMINATION AND ACLIMATIZATION TIME
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t = 0) for each individual eye. The cornea thickness of the eyes should not change by more than ± 5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0% to 2%) were observed in the eyes. This finding is considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES
Three (treatment and positive control groups)
One (negative control)

NEGATIVE CONTROL USED
saline solution.

OBSERVATION PERIOD
240 min

POSITIVE CONTROL USED
0.03 g Imidazole (ground before use).

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea. The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test substance, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.

One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.



REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

The Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation. The Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.

- Indicate any deviation from test procedure in the Guideline

METHODS FOR MEASURED ENDPOINTS:
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.: The cornea thickness was measured, cornea opacity and morphological effect were observed at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.
- Corneal opacity:
- Damage to epithelium based on fluorescein retention:
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting:
- Macroscopic morphological damage to the surface:
- Others (e.g, histopathology):

SCORING SYSTEM:
- Mean corneal swelling (%) :
CS at time t= ((CT at time t – CT at t=0)/(CT at t=0))*100
Mean CS at time t = (FECS(at time t)+ SECS (at time t) + TECS (at time t))/3

CS = Cornea swelling
CT = Cornea thickness
FECS = First eye cornea swelling
SECS = Second eye cornea swelling
TECS = Third eye cornea swelling
Mean CS = The mean percentage of corneal swelling
at time t = Observation time at 30, 75, 120, 180 or 240 minutes after the post-treatment rinse
at t=0 = Reference value
For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (> -12 %) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect.

- Mean maximum opacity score :
Score:
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris are clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
Delta CO at time t = CO at time t – CO at t=0
Mean CO(at time t) = (FE DeltaCO (at time t)+ SE DeltaCO(at time t) + TE DeltaCO(at time t))/3

CO = Cornea opacity
DeltaCO = Difference between cornea opacity and cornea opacity reference value
FE DeltaCO = Difference between first eye cornea opacity and first eye cornea opacity reference value
SE DeltaCO = Difference between second eye cornea opacity and second eye cornea opacity reference value
TE DeltaCO = Difference between third eye cornea opacity and third eye cornea opacity reference value
Mean CO = The mean corneal opacity value
at time t = Observation time at 30, 75, 120, 180 or 240 minutes after the post-treatment rinse
at t=0 = Reference value

- Mean fluorescein retention score at 30 minutes post-treatment
Score:
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein

DeltaFR at time t = FR at time t – FR at t=0
Mean FR = (FE DeltaFR (at time t) + SE DeltaFR(at time t) + TE DeltaFR at time t))/3

FR = Fluorescein retention
DeltaFR = Difference between fluorescein retention and fluorescein retention reference value
FE DeltaFR = Difference between first eye fluorescein retention and first eye fluorescein retention reference value
SE DeltaFR = Difference between second eye fluorescein retention and second eye fluorescein retention reference value
TE DeltaFR = Difference between third eye fluorescein retention and third eye fluorescein retention reference value
Mean FR = The mean fluorescein retention value
at time t = Observation time at 30 minutes after the post-treatment rinse
at t=0 = Reference value

DECISION CRITERIA: the decision criteria as indicated in the TG was used.
Irritation parameter:
percent corneal swelling
Remarks:
75 min
Value:
12
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class II
Irritation parameter:
percent corneal swelling
Remarks:
240 min
Value:
13
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class II
Irritation parameter:
cornea opacity score
Value:
1.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class II
Irritation parameter:
fluorescein retention score
Value:
2.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class III
Other effects / acceptance of results:
In this ICET, 5-(Hydroxymethyl)furfural did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were twice II (based on the corneal swelling of 13% within 240 minutes and corneal opacity score of 1.3), once III (based on the fluorescein retention score of 2.3).

Positive and negative controls showed the expected results. The experiment was considered to be valid.
Interpretation of results:
other:
Remarks:
No prediction can be made
Conclusions:
According to the guideline OECD 438, 5-(Hydroxymethyl)furfural overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item 5-(Hydroxymethyl)furfural by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The Imidazole (positive control) was ground before use in the study. The test item and positive control applied in an amount of 0.03 g/eye the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study.

One negative control eye was treated with 30 µL saline solution.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

The Imidazole was stuck on the corneas’ surface in all eyes (three eyes) at 30 minutes after the post-treatment rinse. The Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.

In this ICET, 5-(Hydroxymethyl)furfural did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were twice II (based on the corneal swelling of 13% within 240 minutes and corneal opacity score of 1.3), once III (based on the fluorescein retention score of 2.3).

Positive and negative controls showed the expected results. The experiment was considered to be valid.

According to the guideline OECD 438, 5-(Hydroxymethyl)furfural overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

- A valid EpiSkinTM SM test of 5-(Hydroxymethyl)furfural was performed under GLP to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015. EPISKIN discs were exposed in triplicate to 5-(Hydroxymethyl)furfural for 15 min at room temperature. The EPISKIN discs treated with the test item did not show significantly reduced cell viability (72% viability compared to the negative controls). Therefore 5-(Hydroxymethyl)furfural is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified). The study is rated as key study and as Klimisch 1 "reliable without restriction".

 

Skin corrosion:

- A valid EpiSkinTM SM test of 5-(Hydroxymethyl)furfural was performed under GLP to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 431, 29 July 2016. EPISKIN discs were exposed in triplicate to 5-(Hydroxymethyl)furfural for 4 hours at room temperature. The EPISKIN discs treated with the test item did not show significantly reduced cell viability (58% viability compared to the negative controls). Therefore 5-(Hydroxymethyl)furfural is considered to be non-corrosive to skin and is therefore not classified (UN GHS No Category / CLP not classified). The study is rated as key study and as Klimisch 1 "reliable without restriction".

Eye Irritation:

- A valid assessment of ocular irritation of 5-(Hydroxymethyl)furfural In Vitro Using the Isolated Chicken Eye Test (ICET) was performed under GLP according the the OECD guideline Nr, 438, 25 June 2018 in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. 5 -(Hydroxymethyl)furfural did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were twice II (based on the corneal swelling of 13% within 240 minutes and corneal opacity score of 1.3), once III (based on the fluorescein retention score of 2.3). According to the guideline OECD 438, 5-(Hydroxymethyl)furfural overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized asNo prediction can be made. The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

 

- A valid study was performed under GLP to determine the acute ocular irritation potential of the test item 5-(Hydroxymethyl)furfural on three-dimensional RhCE tissue in the EpiOcularmodel in vitro, according to OECD guideline Nr. 492, 28 July 2015. EpiOcular(two units) were treated with (50 mg/units) test item and incubated for 6 hours (±15 min) at standard culture conditions (37±1°C) in an incubator with 5±1 % CO2, 90±10% humidified atmosphere). The test item 5-(Hydroxymethyl)furfural showed significantly reduced cell viability, </= 60%, in comparison to the negative control (mean relative viability: 2 %). The results obtained from this in vitro eye irritation test, using the EpiOcularmodel, 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. Therefore an additional study was performed. The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

Justification for classification or non-classification

For Skin Irritation:

According to the results obtained with the EpiSkinTM SM test (OECD 439) and EpiSkinTM SM test (OECD 431), 5-(Hydroxymethyl)furfural (CAS 67-47-0) is considered to be non-irritant and non-corrosive to skin and is therefore not classified (UN GHS No Category /CLP not classified).

For Eye Irritation:

No prediction could be made for 5-(Hydroxymethyl)furfural (CAS 67-47-0) using the ICET (OECD 438) according to the UN GHS classifications and the CLP classification system.

Therefore a second test was performed. According to the results obtained with the EpiocularTMtest (OECD 492) 5-(Hydroxymethyl)furfural (CAS 67-47-0) is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1).

Since the ICET test does not indicate that the test item is eye corrosive, 5-(Hydroxymethyl)furfural (CAS 67-47-0) is therefore classified as Irritant (UN GHS Category 2)