Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Endpoint summary

Description of key information

Skin Corrosion

Under the conditions of the test, the test material was not corrosive to the skin.

Skin Irritation

Under the conditions of the test, the test material was not classified as irritating to skin.

Eye Irritation

- Orovecz (2018): Under the conditions of the test, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

- Spohr (2018): Under the conditions of this study, the test material does not possess an eye irritating potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2017 to 15 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40. bis.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EPISKIN™(SM) model

Source species:

other: EPISKIN™(SM) model

Cell type:

other: EPISKIN™(SM) three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

SOURCE
EPISKIN™(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-037, Expiry Date: 18 September 2017) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

No formulation was required.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM) model
- Tissue batch number(s): Batch No.: 17-EKIN-037
- Expiry date: 18 September 2017
- Date of initiation of testing: 14 September 2017

- Pre-incubation (Day [-1]): The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was 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°C in an incubator with 5% CO2 in a > 95% humidified atmosphere.

- Application (Day 0): The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test material was applied evenly to the epidermal surface of each of two test material treated skin units and each additional control skin units and then 100 μL physiological saline was added to the test material to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (23.6 - 25.2°C) covered with the plate lids.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.6 - 25.2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the incubation time (4 hours), all test material treated tissues and also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 13 September 2017) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced.
- Incubation time: The plate was incubated at 37°C in an incubator with 5% CO2 in a > 95% humidified atmosphere for 3 hours (± 15 minutes), protected from light.
- Spectrophotometer: The OD (optical density or absorbance) of the samples was measured using a plate reader
- Wavelength: 570 nm

- Formazan extraction (Day 0): At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved 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 containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~ 150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

- Cell viability measurements: Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately).
The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

- Indicator for potential false viability
Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test material is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

- Check-method for possible direct MTT reduction with test material:
20 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere for 3 hours and then any colour change was observed:
- Test material which do not react with MTT: Yellow
- Test material reacting with MTT: Blue or purple
After three hours of incubation, yellow colour of the mixture was detected; therefore additional controls were not used in the experiment.

- Check-method to detect the colouring potential of test material:
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol* (simulating a tissue humid environment). As the test material had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
* Note: Water is the environment during exposure, isopropanol is the extracting solution.

Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.


NUMBER OF REPLICATE TISSUES:
- In this assay, two replicates per test material were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates: Duplicate

CALCULATION OF VAIBILITY PERCENTAGES
The data calculation using two replicates is shown below. Results are calculated in a similar way when more replicates are used.

Blank: The mean of the 6 blank OD values was calculated

Negative control: Individual negative control OD values (NCraw) were corrected with the mean blank
OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
- The corrected mean OD of the 2 negative control values was also calculated: This corresponds to 100 % viability

Positive control: Individual positive control OD values (PCraw) were corrected with the mean blank
OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
- The corrected mean OD of the 2 positive control values was calculated
- The % viability for each positive control replicate was calculated relative to the mean negative control:
Positive Control1 % = (ODPC1 / mean ODNC) ×100
Positive Control2 % = (ODPC2 / mean ODNC) ×100
- The mean value of the 2 individual viability % for positive control was calculated:
Mean PC % = (PC1 % + PC2 % ) / 2

Test material: Individual test material OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
- The corrected mean OD of the 2 test material values was calculated
- The % viability for each test material replicate was calculated relative to the mean negative control:
Treated Tissue1 % = (ODTT1 / mean ODNC) ×100
Treated Tissue2 % = (ODTT2 / mean ODNC) ×100
- The mean value of the 2 individual viability % for test material was calculated:
Mean TT % = (TT1 % + TT2 %) / 2
- The variability for 2 disks was calculated as:
(Disk1-Disk2)/((Disk1+Disk2)/2) x 100 %

Data calculation for test items having MTT-interacting potential
Test materials that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability% as follows:
Non-specific MTT reduction calculation (NSMTT%):
NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD
ODKT: test material treated killed tissues OD
ODNC: negative control OD
If NSMTT% is ≤ 50 %, then true MTT metabolic conversion (TODTT) has to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test material treated viable tissues
– The % relative viability (RV%) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSMTT% is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

- Data calculation for test material having colouring potential:
For test materials detected as able to stain the tissues the non-specific OD was evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % as detailed below:
Non Specific Colour % (NSCliving %):
NSCliving % = (mean ODCTV / mean ODNC)×100
ODCTV: test material treated viable tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode was used.
If NSC living % is > 5 % and ≤ 5 0 %, then additional correction (TODTT) has to be undertaken as follows:

TODTT = [ODTV - ODCTV]
ODTT: test material treated viable tissue (incubated with MTT)
ODCTV: test material treated viable tissue (not incubated with MTT)
– The % relative viability (RV% %) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSC living % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

- Data calculation for test materials having both MTT-interacting and colouring potential:
For test materials detected as able to both stain the tissues and interfere with MTT may also require a third set of controls before calculation of the “true” viability %.
Non Specific Colour % with killed tissues (NSCkilled %):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test materisl treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
In that case the true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: test material treated viable tissues (incubated with MTT)
ODKT: test material treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test material treated viable tissues (not incubated with MTT)
ODCTK: test material treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

PREDICTION MODEL / DECISION CRITERIA
The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016).
The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).
For 2 disks:
If both disks have mean viability of ≥ 35% = Non Corrosive
If both disks have mean viability of < 35 = Corrosive (at the corresponding incubation period)

For more than 2 disks:
If the mean value is ≥ 35% and the variability is less than 50% = Non Corrosive
If the mean value is < 35% and the variability is less than 50% = Corrosive

Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg test material

NEGATIVE CONTROL
- Amount applied: 50 µL physiological saline

POSITIVE CONTROL
- Amount(s) applied: 50 µL glacial acetic acid

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (23.6 - 25.2°C) covered with the plate lids.

Number of replicates:

The test material was applied to two skin units and each additional skin unit.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of two runs

Value:

104.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ADDITIONAL CONTROLS
As the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.007, Non Specific Colour% (NSCliving%) was calculated as 0.9%. This is below the threshold of 5% therefore correction due to colouring potential was not necessary.
As no colour change was observed after three hours of incubation of the test material in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

Validity of the Test

After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the two negative control tissues was in the recommended range (0.788).

The two positive control treated tissues showed 0.6% viability demonstrating the proper performance of the assay.

The OD data of one replicate tissue at the positive control in this study was lower (0.001) than the minimum OD of the historical control range (0.005). This fact has no impact on the results or integrity of the study since the positive control material showed severe effect.

The difference of viability between the two test material-treated tissue samples in the MTT assay was 6.1%.

The difference of viability between the two negative control tissue samples in the MTT assay was 1.5%.

The mean OD value of the blank samples (acidified isopropanol) was 0.046. All these parameters were within acceptable limits and therefore the study was considered to be valid.

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the test the test material was not corrosive to the skin.

Executive summary:

The corrosivity of the test material was evaluated according to the standardised giudlines OECD 431.

During the study, disks of EPISKIN™ (SM) (two units) were treated with the test material and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units /control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test material. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test material is considered to be corrosive to skin.

Following exposure with the test material, the mean cell viability was 104.2% compared to the negative control. This is above the threshold of 35%, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test with the test material, the results indicate that the test material is non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 September 2017 to 15 September 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ (SM) reconstituted human epidermis model.

Cell source:

other: EPISKIN™ (SM) reconstituted human epidermis model.

Source strain:

other: Not applicable

Justification for test system used:

The EPISKIN™ (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

No formulation was required.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (SM) reconstituted human epidermis model.
- Tissue batch number(s): 17-EKIN-037
- Expiry date: 18 September 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At room temperature (24.9 - 25.7°C).

NUMBER OF REPLICATE WELLS
In this assay, three replicates were used for the test material. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation.

PRE-INCUBATION (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was 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°C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

APPLICATION
(Day 0)
Test Material
As the test material was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 20 mg of the test material was applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

Negative and positive controls
50 μL of negative control (PBS) or positive control (5 % (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (24.9 - 25.7°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
After the 15 minutes incubation time, the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
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, in a > 95% humidified atmosphere.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 12 September 2017) was stored in refrigerator (2 - 8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Measured using a plate reader.
- Wavelength: 570 nm

MTT test (Day 2)
After the 42 hours incubation, all EPISKIN™ (SM) units (except the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKIN™ (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere.

Formazan extraction (Day 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved 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 containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

FUNCTIONAL MODEL CONDITIONS
Cell viability measurements (Day 2)
Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.

NUMBER OF REPLICATE TISSUES: Triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
INDICATOR FOR POTENTIAL FALSE VIABILITY
- Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test material is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test material interference with the viability measurement.
Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

- Check-method for possible direct MTT reduction with test material
10 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
- Test materials which do not react with MTT: Yellow
- Test materials reacting with MTT: Blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test material did not react with MTT and therefore the use of additional controls was not necessary.

- Check-method to detect the colouring potential of test-materials
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol* (simulating a tissue humid environment). As the test material had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non-specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
* Water is the environment during exposure, isopropanol is the extracting solution.

Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step:
MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

CALCULATIONS OF VIABILITY PERCENTAGES
Data calculation for normal test materials

Blank: The mean of the six blank OD values was calculated
Negative control: Individual negative control OD values (NCraw) were corrected with the mean blank
OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The mean corrected OD of the 3 negative control samples were calculated: this value corresponds to 100 % viability

Positive control: Individual positive control OD values (PCraw) were corrected with the mean blank
OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The mean corrected OD of the 3 positive control samples were calculated
– The % viability for each positive control replicate was calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual relative viability % for positive control was calculated:
Mean PC %= (%PC1 +%PC2 +%PC3) / 3

Test material:
– Individual test material OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The mean corrected OD of the 3 test material samples were calculated
– The % viability for each test material replicate was calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual relative viability % for test material was calculated:
Mean TT %= (%TT1 +%TT2 +%TT3) / 3

- Data calculation for test materials having MTT-interacting potential
Test materials that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability % as detailed below:
Non-specific MTT reduction calculation (NSMTT%):
NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control killed tissues OD
ODKT: test material treated killed tissues OD
ODNC: negative control OD
If NSMTT% is ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test material treated viable tissues
The % relative viability (% RV) for each test material replicate is calculated relative
to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3
If NSMTT% is > 30 % relative to the negative control, then additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

- Data calculation for test materials having colouring potential
For test materials detected as able to stain the tissues the non-specific OD due to the residual chemical colour (unrelated to mitochondrial activity) is evaluated and subtracted before calculation of the “true” viability % as detailed below:
Non Specific Colour % with viable tissues (NSCliving %):
NSC living % = (mean ODCTV / mean ODNC)×100
ODCTV: test material treated viable tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode is used.

If NSC % is > 5 % and ≤ 30 %, then true MTT metabolic conversion (TODTT) is undertaken as follows.
TODTT = [ODTT – mean ODCTV]
ODTT: test material treated viable tissue (incubated with MTT)
ODCTV: test material treated viable tissue (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated:
Mean Relative Viability % = (% RV 1 +%RV 2 +%RV 3) / 3
If NSC living % is > 30 % relative to the negative control, additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

- Data calculation for substance having both MTT interacting potential and colouring potential
For test materials detected as able to stain the tissues and interfere with MTT is also require a third set of controls before calculation of the “true” viability %.
Non Specific Colour % with killed tissues (NSCkilled%):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test material treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
In that case the true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: test material treated viable tissues (incubated with MTT)
ODKT: test material treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test material treated viable tissues (not incubated with MTT)
ODCTK: test material treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

PREDICTION MODEL / DECISION CRITERIA
- The test material is considered to be irritating to skin if the viability after 15 minutes exposure is less than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: As the test material was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 20 mg of the test material was applied evenly to the epidermal surface.


NEGATIVE CONTROL
- Amount applied: 50 µL


POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

93.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Additional Controls

As no colour change was observed after three hours of incubation of the test material in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

As the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.011, Non Specific Colour % was calculated as 1.7%. This value was below 5%, therefore additional data calculation was not necessary.

 

Viability Results

The mean OD values for the test material treated skin samples showed 93.7% relative viability compared to the negative control.

 

Validity of the Test

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues was in the recommended range (0.671). Standard deviation of the viability results for negative control samples was 1.2%.

The positive control treated tissues showed 5.3% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.2%.

The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 12.2%.

The mean OD value of the blank samples (acidified isopropanol) was 0.048.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of the test, the test material was not classified as irritating to skin.

Executive summary:

The irritation potential of the test material was evaluated according to the standardised guidelines OECD 439 and EU Method B.46.

During ths study, disks of EPISKIN™ (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units/ control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test material. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test material is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 93.7% compared to the negative control. This is above the threshold of 50%, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test, the results indicate that the test material is non-irritant to skin, UN GHS Classification: No Category (since the test material is known not to be corrosive).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The solubility of the test material in physiological saline was tested prior to the experiment (30 mg test material in 1 mL physiological saline). The test material did not dissolve in physiological saline.

Species:

chicken

Strain:

other: Ross 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box).
- Time interval prior to initiating testing: The heads were received at Citoxlab Hungary Ltd. and processed within 2 hours of collection.
- Indication of any existing defects or lesions in ocular tissue samples: 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 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of the test material was applied onto the entire surface of the cornea.

Duration of treatment / exposure:

10 seconds.

Duration of post- treatment incubation (in vitro):

Observations were made at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

Three eyes were exposed to the test material.

Details on study design:

PREPARATION OF ISOLATED EYES
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, 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.

EQUILIBRATION AND BASELINE RECORDINGS
- Eyes examination and acclimatisation 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 too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and then placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly that the physiological saline 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, 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, acclimatisation started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5°C) during the acclimatisation and treatment periods.

- The baseline assessments
At the end of the acclimatisation 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% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in the experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
One negative control, three posititve control and three test material-treated.

NEGATIVE CONTROL USED
Negative control eye was treated with 30 μL of physiological saline.

POSITIVE CONTROL USED
Positive control eyes were treated with 30 mg powdered imidazole.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. 30 mg of the test material was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material, taking care not to damage or touch the cornea. An exposure period of 10 seconds from the end of the application was used.

OBSERVATION PERIOD
- Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ± 5 minutes were considered acceptable.

REMOVAL OF TEST MATERIAL
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible. Additional gentle rinsing with at least 20 mL saline was performed at each time point when the test material or positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and corneal opacity were measured at all time points
- Damage to epithelium based on fluorescein retention: Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Haag-Streit Bern 900 slit-lamp microscope was used for the measurements

EVALUATION
Corneal swelling was calculated according to the following formulae:

CS at time t = ((CT at time t –CT at t=0) / (CT at t=0)) x 100

Mean CS at time t = (FECS(at time t)+ SECS(at time t) + TECS(at time t)) / 3

Where:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point

Small negative numbers for swelling (0 to -5%) following application are evaluated as class I. Large negative numbers (>12% below control) 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 (class II).

Cornea opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at t=0

Mean ΔCOmax = (FECOmax(30min to 240min)+ SECOmax(30min to 240min) + TECOmax(30min to 240min)) / 3

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

Fluorescein retention was calculated according to the following formulae:

ΔFR at time t = FR at time t – FR at t=0

Mean ΔFR = (FEFR (30min) + SEFR(30min) + TEFR(30min)) / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention


DECISION CRITERIA:
ICE classification criteria for corneal thickness:
Mean Corneal Swelling (%) ICE Class
0 to 5 I
>5 to 12 II
>12 to 18 ( >75 min after treatment ) II
>12 to 18 ( <75 min after treatment ) III
>18 to 26 III
>26 to 32 ( >75 min after treatment ) III
>26 to 32 ( <75 min after treatment ) IV
>32 IV

ICE classification criteria for corneal opacity:
Mean Maximum Opacity Score ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 4.0 IV

ICE classification criteria for mean fluorescein retention:
Mean Fluorescein Retention Score at 30 minutes post - treatment ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 3.0 IV

Assessment of the general in vitro eye irritancy and regulatory UN GHS classification:
The following table is used to identify the probably eye irritancy potential of test materials. In the case where the result indicates Non-irritant or Corrosive/Severely Irritating, then the test material can be classified. In all other cases the probable level of irritancy can be reported, but a regulatory in vivo rabbit eye irritation test is required for regulatory classification and labelling purposes.

UN GHS Classification Combinations of the three ICE Classes
No Category 3×I
2×I, 1×II
No prediction can be made Other combinations
Category 1 3×IV
2×IV, 1×III
2×IV, 1×II*
2×IV, 1×I*
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of epithelium (in at least 1 eye)
* Combinations of categories less likely to occur.

Irritation parameter:

percent corneal swelling

Remarks:

Up to 75 min

Run / experiment:

Mean

Value:

2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

10.9 %

Remarks on result:

no indication of irritation

Remarks:

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Irritation parameter:

percent corneal swelling

Remarks:

Up to 240 min

Run / experiment:

Mean

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

25.1 %

Remarks on result:

no indication of irritation

Remarks:

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Irritation parameter:

cornea opacity score

Remarks:

Mean maximum

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

4.0 %

Remarks on result:

no indication of irritation

Remarks:

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

3.0 %

Remarks on result:

no indication of irritation

Remarks:

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
- The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
No other morphological effect was observed in the study.

RESULTS:
Based on this in vitro eye irritation in the isolated chicken eyes test with the test material, the test maerial is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The physological saline gave no indication of irritation.
- Acceptance criteria met for positive control: The imdazole positive control was severly irritating.
- Range of historical values if different from the ones specified in the test guideline: The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the test, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Executive summary:

An in vitro eye irritation study of the test material was performed with isolated chicken’s eyes. The irritation effects of the test material were evaluated according to the standardised guideline OECD 438 (26 July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 mg test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in the experiment. Thus, the experiment was considered to be valid.

No significant corneal swelling (mean ≤ 5%) was observed during the four-hour observation period on test material treated eyes. No significant cornea opacity change (severity 0.5) was observed on three eyes. No significant fluorescein retention change (severity 0.5) was noted on all three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. All three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. Although there were no adverse corneal effects observed, no conclusion of in vivo significance could be established due to the adherence of the test material to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur.

Under the conditions of the test the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 2018 to 05 July 2018

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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- EpiOcular™ (purchased from MatTek Corporation (82105 Bratislava, Slovakia), Lot: 27056)
-The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organised basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL®, 10 mm Ø).
- EpiOcular™ tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate on Tuesday. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
- Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70 % isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2) overnight (17.5 hours).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: Approximately 50 mg of the test item were tested topically on duplicate EpiOcular™ tissues.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes immersion incubation (post-soak)
18 hours post-treatment incubation
180 minutes with MTT

Number of animals or in vitro replicates:

2

Details on study design:

ASSESSMENT OF DIRECT MTT REDUCTION BY THE TEST MATERIAL
- Test materials may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test material prior to conducting any assays with viable tissues. For this purpose approximately 50 mg of the test material were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test material was presumed to have reduced the MTT.
- Since the MTT solution colour did not turn blue/purple, the test material was not presumed to be a MTT reducer, and an additional test with freeze-killed tissues was not necessary.

ASSESSMENT OF COLOURED OR STAINING MATERIALS
- Coloured test materials or test materials which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement, if the colourant binds to the tissue and is extracted together with MTT. Therefore, each test material has to be checked for its colouring properties.
- All other intrinsically coloured test materials (e.g. red, yellow, green colorants) have to be tested for their ability to absorb significantly light at the wavelength used for the MTT determination. 50 µL of the test material were added to 2 mL of isopropanol, incubated in a glass tube, and placed on an orbital plate shaker for 3 hours at room temperature. Two 200 µL aliquots of isopropanol solutions and of pure isopropanol were transferred to a 96-well plate and the absorbance is measured with a plate reader at the MTT measurement wavelength.
- Since the test material was not completely soluble in isopropanol, it was centrifuged and 200 µL aliquots of the supernatant were used for measurement. Since, after subtraction of the OD for isopropanol, the OD of the test material solution was > 0.08 (approximately 5 % of the mean viability of the negative control) the test material was considered as possibly interacting with the MTT measurement and an additional test on additional viable tissues (without MTT addition) had to be performed.
- Since the test material was non-coloured, additional tests had to be performed to assess, if it becomes coloured after contact with water or isopropanol. For this purpose, approximately 50 mg each of the test material were added either to 1.0 mL of water or to 2 mL isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 in air for one hour, the isopropanol mixture for 3 hours at room temperature.
- Although the Test Material did not dye water or isopropanol but formed an opaque suspension with an OD570 value of >0.08 (measured value was 0.140), additional tests with viable tissues had to be performed.

PERFORMANCE OF THE TEST
- After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca2+Mg2+free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- After the 30 minute Ca2+Mg2+free-DPBS pre-treatment, the test and control material were tested by applying approximately 50 mg (test material) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 6 hours.
- At the end of the 6 hours treatment time, the test material was removed by extensively rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature).
- Three clean beakers containing a minimum of 100 mL each of Ca2+Mg2+-free DPBS were used per test material. The test material utilised a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control materials were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts are 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 culture was then rinsed in the second and third beaker 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 (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
- Since it was not possible to remove the visible test material completely, this was noted in the study file. No further rinsing was done.
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test material or control 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 transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 (post-treatment incubation).

MTT ASSAY
- At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
- Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were extracted immediately (shaken for approximately 3 hours at room temperature). For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
- The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
- The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

COLOURANT CONTROLS FOR ASSESSMENT OF COLOURED OR STAINING TEST MATERIALS
- Since the test material showed to have or to develop relevant colour, which could interact with the MTT measurement, an additional test had to be performed to determine the amount of colour bound to and extracted from the tissues. For this purpose the coloured test material was applied to two additional tissues (= colorant controls (CC)), and were treated in the same way as previously described. In contrast to the normal viability test, no MTT assay was performed. The bound colour was extracted and the absorbance of the isopropanol extracts was measured identically as in the MTT assay for coloured test materials. The amount of extracted colour was subtracted from the results of the viability assay.

DATA EVALUATION
1) The mean OD value of the blank control wells (ODBlk) for each experiment was calculated.
2) The mean value of the two replicates for each tissue was calculated.
3) The mean ODBlk from each mean OD value of the same experiment was subtracted (blank corrected values).
4) The mean value of the two relating tissues for each control (negative control (NC) and positive control (PC) and test material (TI) was calculated (ODTI, ODNC, ODPC).
5) The mean OD value of the negative control corresponds to 100 % viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100 % Viability

- Calculations for Viability Tests only
The percent viability of each of the two relating tissues for each control and test material relative to the negative control (= 100 %) was calculated:

Viability (%) = 100. [(ODTI/ODPC/ODNC)/meanODNC]

The difference of the viability between duplicate tissues was calculated. If the difference is >20 % the test is considered as non-qualified.
The mean test material viability (TI viability) was calculated and the test material was classified according to the prediction model.

- Calculation for Viability plus Colorant Control (CC) Test
The OD values of the additional viable tissue experiment (without MTT addition; each two tissues and two replicates) were determined and blank corrected. The mean value of the two replicates for the negative control (NC) and test material (TI) was calculated test material: ODTI_CC; negative control: ODNC_CC). The mean OD of the two negative control tissues was calculated (mean ODNC_CC).

The viability of the two relating tissues was calculated according to the following formula:

Test material cc Viability (%) = 100 x (ODcc / meanODNC)

The difference of the viability of the two tissues was calculated. If the difference is >20 %, the additional viable tissues (without MTT addition) test is considered as non-qualified.
The mean TestMaterialCC Viability for the additional viable tissues (without MTT addition) was calculated and subtracted from the test material viability (TI viability) to determine the CC corrected test material viability.

CC corrected test material viability = TI viability – mean TestMaterialCC Viability

The test material was classified regarding the additional viable tissues (without MTT addition) corrected viability according to the prediction model.


PREDICTION MODEL
- If the test material-treated tissue viability is > 60 % relative to the negative control-treated tissue viability, the test material is labelled non-irritant.
- If the test material-treated tissue viability is ≤ 60 % relative to negative control-treated tissue viability, the test material is labelled irritant.
- According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60 ± 5 %, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

ACCEPTABILITY OF THE ASSAY
The results are acceptable according to MatTek Protocol, if:
- The negative control OD is > 0.8 and < 2.5,
- The mean relative viability of the positive control is below 50 % of the negative control viability.
- The difference of viability between the two relating tissues of a single test material is < 20 % in the same run (for positive and negative control tissues and tissues of test materials). This applies also to the freeze-killed tissues (materials and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
The positive and negative control data shall fall within the historical control data.

Irritation parameter:

other: Mean relative absorbance value

Run / experiment:

Test material mean

Value:

87.769

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS
- Although the Test Material did not reduce MTT additional tests with viable tissues had to be performed based on the fact that the Test Material was not soluble in deionised water and isopropanol.
- Optical evaluation of the MTT-reducing capacity of the test material with MTT-reagent did not show blue/purple colour. Therefore, an additional test with freeze-killed tissues was not necessary.
- The mean relative absorbance value of the test material, corresponding to the cell viability, decreased to 87.769 % (threshold for irritancy: ≤ 60 %), consequently the test material was not irritant to eye.
- Concerning acceptance criteria:
The negative control OD is > 0.8 and < 2.5 (1.980 and 2.144).
The mean relative viability of the positive control is below 50 % of the negative control viability (20.33 %).
- The difference of viability between the two relating tissues of a single material is < 20 % (values between 0.02 and 9.46 %) in the same run (for positive and negative control tissues and tissues of single test materials). This applied also to the killed controls (materials and negative control) and the additional viable tissues (without MTT addition) which were calculated as percent values related to the viability of the relating negative control.

Table 1: Results of the Pre-Test (Coloured or Colour Changing Test Materials)

	OD570 Well 1	OD570 Well 2	Mean OD570[Well 1/2]	Mean OD570blank corrected	Evaluation Mean OD570 (blank corrected) > 0.08 *
Negative control A.Deion.	0.036	0.036	0.036
Test Material + A.Deion	0.247	0.106	0.176	0.140	yes
Negative control Isopropanol	0.037	0.037	0.037
Test Material + Isopropanol	0.039	0.042	0.041	0.004	no

* Mean OD570 (blank corrected) > 0.08: If yes, additional viable tissues are necessary in the main experiment.   

Table 2: Results After Treatment for 6 hours with the Test Material and the Controls

Test Group		Well 1 [OD570]	Well 2 [OD570]	Mean [OD570] (Well 1 and well 2)	Mean [OD570] blank corr. (Well 1 and well 2)	Mean [OD570] of T1 and T2	Tissue viabil.* [%]	Rel. Viabil. of T1 and T2**	Diff. of Viabil. between T1 and T2 [p.p.]	Mean Viability of test material after data correct. procedure [%] if VT/FK available
Blank		0.037	0.037	0.037						87.769***
Negative Control	1	1.980	2.091	2.035	1.999	2.040	100.0	98.0	4.04
	2	2.144	2.091	2.118	2.081			102.0
Positive Control	1	0.548	0.548	0.548	0.511	0.415	20.33	25.1	9.46
	2	0.357	0.354	0.355	0.318			15.6
Test Material	1	1.897	1.824	1.861	1.824	1.795	88.01	89.4	2.79
	2	1.820	1.788	1.804	1.767			86.6
Blank		0.037	0.037	0.037
Viable Tissues Negative Control	1	0.041	0.052	0.047	0.010	0.008	0.41	0.5	0.15
	2	0.043	0.044	0.044	0.007			0.3
Viable Tissues Test Material	1	0.043	0.041	0.042	0.005	0.005	0.25	0.3	0.02
	2	0.041	0.042	0.042	0.005			0.2

* Tissue viability = [(100 x meanOD of T1&T2test material/positive control/negative control) / meanOD of T1&T2negative control]

** Relative Tissue viability = [(100 x meanOD blank corrected test material/positive control/negative control) / meanOD of T1&T2negative control]

*** Corrected mean viability = Tissue viability test material - Tissue viability VT test material

Discussion

This in vitro study was performed to assess the eye irritation potential of the test material by means of the Human Cornea Model Test.

Although the test material did not reduce MTT additional tests with viable tissues had to be performed based on the fact that the test material was not soluble in deionised water and isopropanol.

About 50 mg of the test material and each 50 µL of the controls, respectively, were applied to each of duplicate EpiOcular^™ tissue for 6 hours.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 20.33 %, thus the validity of the test system is ensured.

The acceptance criteria were met.

Since the viability value of the test material exposed tissues was 87.769 % and hence did not decrease below 60 %, the test material is not considered to possess an eye irritating potential.

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study, the test material does not possess an eye irritating potential.

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guideline OECD 492, under GLP conditions. This in vitro study was performed to assess the eye irritation potential of the test material by means of the Human Cornea Model Test.

Although the Test Material did not reduce MTT additional tests with viable tissues had to be performed based on the fact that the Test Material was not soluble in deionised water and isopropanol.

Each 50 mg of the test material, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50 % viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20 % in the same run (for test material tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the test material. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60 % (determined value for the test material 87.769 %).

Under the conditions of this study, the test material does not possess an eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In Vitro Skin Corrosion

The corrosivity of the test material was evaluated according to the standardised giudlines OECD 431.

During the study, disks of EPISKIN™ (SM) (two units) were treated with the test material and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9 % (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units /control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test material. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35 % of the negative control, the test material is considered to be corrosive to skin.

Following exposure with the test material, the mean cell viability was 104.2 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test with the test material, the results indicate that the test material is non-corrosive to the skin.

In Vitro Skin Irritation

The irritation potential of the test material was evaluated according to the standardised guidelines OECD 439 and EU Method B.46.

During ths study, disks of EPISKIN™ (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2, in a > 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units/ control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test material. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test material is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 93.7 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test, the results indicate that the test material is non-irritant to skin, UN GHS Classification: No Category (since the test material is known not to be corrosive).

In Vitro Eye Irritation: Orovecz (2018)

An in vitro eye irritation study of the test material was performed with isolated chicken’s eyes. The irritation effects of the test material were evaluated according to the standardised guideline OECD 438 (26 July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 mg test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in the experiment. Thus, the experiment was considered to be valid.

No significant corneal swelling (mean ≤ 5 %) was observed during the four-hour observation period on test material treated eyes. No significant cornea opacity change (severity 0.5) was observed on three eyes. No significant fluorescein retention change (severity 0.5) was noted on all three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. All three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. Although there were no adverse corneal effects observed, no conclusion of in vivo significance could be established due to the adherence of the test material to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur.

Under the conditions of the test the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

In Vitro Eye Irritation: Spohr (2018)

The eye irritation potential of the test material was investigated in accordance with the standardised guideline OECD 492, under GLP conditions. This in vitro study was performed to assess the eye irritation potential of the test material by means of the Human Cornea Model Test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Although the Test Material did not reduce MTT additional tests with viable tissues had to be performed based on the fact that the Test Material was not soluble in deionised water and isopropanol.

Each 50 mg of the test material, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50 % viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20 % in the same run (for test material tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the test material. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60 % (determined value for the test material 87.769 %).

Under the conditions of this study, the test material does not possess an eye irritating potential.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin or eye irritation.