L-Glutamic acid, N-coco acyl derivs., compds. with triethanolamine (1:1)

Endpoint summary

Description of key information

Skin corrosion in vitro, Varga-Kanizsai (2017)

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Skin Irritation in vitro, Varga-Kanizsai (2017)

Under the conditions of this study, the test material is not irritating in the in vitro skin irritation test.

Eye irritation in vitro, Varga-Kanizsai (2018)

Based on this in vitro eye irritation assay in the isolated chicken eyes 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.

To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections). Microscopic evaluation showed very slight to severe (in one case) erosion of the epithelium in 6/6 cases. Moderate vacuolation of the corneal epithelium in 1/6 sections was also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 1. Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion classification of UN GHS eye irritant Category 1.

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:

08 December 2016 to 09 December 2016

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)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity testing in an international trial 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)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM) (Manufacturer: SkinEthic, France)
- Tissue batch number(s): 16-EKIN-049
- Expiry date: 12 December 2016
- Use of this model is recommended by OECD 431.
- Quality control: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories.
-Storage: The kits were found to be in good order at reception. They were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

INDICATOR FOR POTENTIAL FALSE VIABILITY
-Check for direct MTT reduction with the test material: Approximately 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 materials which do not react with MTT turn yellow, test materials reacting with MTT turn blue or purple.
After three hours 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.
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.

MAIN STUDY
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 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 (22.9-24.2°C) covered with the plate lids.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the incubation time (4 hours), all test material treated tissues or 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 TEST AND FORMAZAN EXTRACTION (DAY 0)
-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 and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±15 minutes), protected from light.
-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.

NUMBER OF REPLICATE TISSUES: 2

CELL VIABILITY MEASUREMENTS (DAY 1)
-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 instrument was verified and calibrated.

CALCULATIONS
The data calculation using two replicates is shown below. Results were 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 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 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 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 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 % a nd ≤ 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 material treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

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


INTERPRETATION OF THE TEST RESULTS
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)
Otherwise:
-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.
- Sub Category 1A: If corrosive after 3 min exposure
- Sub Category 1B: If not corrosive after 3 min exposure and corrosive after 1 hour exposure
- Sub Category 1C: If not corrosive after 1 hour exposure and corrosive after 4 hours exposure
-Non corrosive: If not corrosive after 4 hours exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg with 100 µL physiological saline to ensure good contact with the epidermis

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 0.9 % (w/v)

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

4 hours (±10 min)

Duration of post-treatment incubation (if applicable):

3 hours (±15 minutes) with MTT

Number of replicates:

The test was performed in duplicate.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

86.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ADDITIONAL CONTROLS
As the test material was coloured, two additional test material-treated tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.011, Non Specific Colour % (NSCliving %) was calculated as 1.3 %. This is below the threshold of 5 %, therefore correction due to colouring potential was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The mean OD value for the test material treated skin samples showed 86.7 % relative 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 (0.836).
-The two positive control treated tissues showed 0.8 % viability demonstrating the proper performance of the assay.
- The difference of viability between the two test material-treated tissue samples in the MTT assay was 16.7 %.
- The difference of viability between the two negative control tissue samples in the MTT assay was 15.0 %.
-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.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Substance	Run	Optical density		Viability (% RV)
		Measured	Blank corrected
Negative control	1	0.819	0.773	92.5
	2	0.944	0.898	107.5
	Mean	-	0.836	100.0
Positive control	1	0.052	0.006	0.8
	2	0.053	0.007	0.8
	Mean	-	0.007	0.8
Test material	1	0.710	0.664	79.5
	2	0.831	0.785	94.0
	Mean	-	0.725	86.7

 

Mean blank value was 0.046.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Executive summary:

The potential of the test material to cause skin corrosion was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis under GLP conditions using a human skin model assay.

Disks of EPISKIN™ (SM) were treated with the test material in duplicate and incubated for 4 hours at room temperature. Exposure of the 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, also performed in duplicate. 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 86.7 % 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.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2017 to 18 February 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)

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:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, 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:

Version 1.8 (February 2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

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)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (SM)
- Tissue batch number(s): 17-EKIN-007
- Expiry Date: 20 February 2017
- Source: SkinEthic, France
- The model 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. 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.

KIT RECEPTION AND STORAGE
- The pH of the agar medium used for transport was checked by checking the colour of the medium: orange colour = good and yellow or violet colour = not acceptable.
- The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C): white colour = good and grey or black colour = not acceptable.
- The kits were found to be in good order at reception.
- The EPISKIN™ (SM) kit was kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (22.8 to 26.7 °C).
- Temperature of post-treatment incubation: 37 °C

CHECK-METHOD FOR DIRECT MTT REDUCTION
- Approximately 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 (± 5 minutes) protected from light, and then any colour change was recorded: Test materials which do not react with MTT turn yellow and Test materials reacting with MTT turn 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 FOR COLOURING POTENTIAL OF THE 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 extracting solution (e.g. acidified 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.
- Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. This tissue 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.

APPLICATION/ TREATMENT
- 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.
- Test Material (Day 0): 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 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 (22.8 to 26.7 °C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The test material stuck on surface of the epidermis so additional rinsing was used. The test material was totally washed off from the skin. 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 (± 1 h) at 37 °C in an incubator with 5 % CO2 in a > 95 % humidified atmosphere.

MTT TEST (Day 2)
- After the 42 hours incubation, all EPISKIN™ (SM) units (except for two 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 at 37 °C in an incubator with 5 % CO2 in a >95 % RH % protected from light.
- 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.
- Cell viability measurements (Day 2): Following the formazan extraction, 2 × 200 μL samples 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: 240 72800)) at the required wavelength on each day before use.

NUMBER OF REPLICATE TISSUES: 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.


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 %) = (mean ODCTV / mean ODNC) × 100

ODCTV: test substance treated viable tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSCliving % is ≤ 5 % then the normal calculation mode is used.
If NSC % is > 5 % a nd ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as follows.

TODTT = [ODTT – mean ODCTV]

ODTT: test substance treated viable tissue (incubated with MTT)
ODCTV: test substance 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 substance must be considered as incompatible with the test.

- Data calculation for substance having both MTT interacting potential and colouring potential
For test substances detected as able to stain the tissues and interfere with MTT also require a third set of controls before calculation of the “true” viability %.

Non Specific Colour % with killed tissues (NSCkilled%) = (mean ODCTK / mean ODNC) × 100

ODCTK: test material treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

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


INTERPRETATION OF TEST RESULTS
- In case duplicates are spread over different categories, the test will be repeated.
- IIn the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is ≤ 50 % of the mean viability of the negative controls.
In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is ≤ 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
-The test material considered to be non-irritant to skin (No Category), if the mean relative viability of three individual tissues after 15 minutes exposure to the test material and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg (with 10 µL distilled water to improve contact with the eye)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 5 % (w/v)

Duration of treatment / exposure:

15 minutes (± 0.5 minutes)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

75.5

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 no colour change (yellow colour) was observed after three hours of incubation of the test materials in MTT working solution, thus the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary.
- The false estimation of viability could be excluded.
- As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues were 0.024, Non Specific Colour % was calculated as 2.7 %. This value was below 5 %, therefore additional data calculation was not necessary.

VIABILITY RESULTS
- The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The OD values for the test material treated skin samples showed 75.5 % relative viability compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.

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 (0.897). Standard deviation of the viability results for negative control samples was 3.3.
- The positive control treated tissues showed 4.3 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.5.
- The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 6.9.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
- All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Treatment	Optical Density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative control: PBS	1	0.931	0.884	98.6
	2	0.922	0.875	97.6
	3	0.978	0.931	103.8
	Mean	-	0.897	100.0
Positive control: 5 % (w/v) SDS solution	1	0.086	0.039	4.4
	2	0.089	0.042	4.7
	3	0.081	0.034	3.8
	Mean	-	0.039	4.3
Test material	1	0.748	0.701	78.2
	2	0.770	0.723	80.6
	3	0.654	0.607	67.7
	Mean	-	0.677	75.5

Notes: Mean blank value was 0.047. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not irritating in the in vitro skin irritation test.

Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions.

The in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay.

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 % RH %. 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 RH % 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 75.5 % 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.

Under the conditions of this study, the test material is not irritating in the in vitro skin irritation test.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

no guideline followed

Principles of method if other than guideline:

The primary dermal irritation of the test material was assessed in male New Zealand White rabbits. On the day before application of the test material, the hair on the left and right trunk of rabbits was shaved with an electric clipper.
On the day of application, about 0.3 mL of the test material was applied with an adhesive plaster for patch test under occlusive conditions on the clipped area, followed by attachment of a body cover.
At 24 hours after occlusive patch application, the body cover and the adhesive plaster for patch test were removed. About 30 minutes later, the degree of irritation by the test material was evaluated according to the Draize criteria. Then, the same evaluation was performed after 48 and 72 hours.

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: Animals were housed one animal per cage.
- Diet: Fed with 150 g/day of solid feed (RC-4, Oriental Yeast Co., Ltd.).
- Water: Ad libitum
- Acclimation period: Acclimated but the period was not specified.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3 °C
- Humidity: 55 ± 15 %

Type of coverage:

occlusive

Preparation of test site:

shaved

Remarks:

Shaved with an electric clipper

Vehicle:

other: LFC

Controls:

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.3 mL

VEHICLE
- Amount(s) applied: The sample solutions were prepared in LFC.

POSITIVE CONTROL
- Amount(s) applied: 0.3 mL

Duration of treatment / exposure:

72 h

Observation period:

72 h

Number of animals:

Four males per group.

Details on study design:

TEST SITE
- Area of exposure: On the day before application of the test material, the hair on the left and right trunk of rabbits was shaved with an electric clipper. On the day of application, about 0.3 mL of the test material was applied with an adhesive plaster for patch test
- Type of wrap if used: Under occlusive conditions on the clipped area, followed by attachment of a body cover.

OBSERVATION TIME POINTS
At 24 hours after occlusive patch application, the body cover and the adhesive plaster for patch test were removed. About 30 minutes later, the degree of irritation by the test material was evaluated according to the Draize criteria. Then, the same evaluation was performed after 48 and 72 hours.

SCORING SYSTEM:
- Method of calculation: According to the Draize criteria.

Irritation parameter:

erythema score

Remarks on result:

other: Not reported

Irritation parameter:

edema score

Remarks on result:

other: Not reported

Irritant / corrosive response data:

At 24 hours after application, erythema was noted. One week after application, animals recovered.
No oedema was noted.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study the classification of the test material could not be determined.

Executive summary:

The primary skin irritation potential of the test material was assessed in male New Zealand White rabbits.

At 24 hours after application, erythema was noted.  One week after application, animals recovered.

Under the conditions of the study the classification of the test material could not be determined.

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

For the justification for read-across, please refer to the read-across assessment framework report that is attached to Section 13.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Remarks on result:

other: Not reported

Irritation parameter:

edema score

Remarks on result:

other: Not reported

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study the classification of the test material could not be determined.

Executive summary:

The primary skin irritation potential of the test material was assessed in male New Zealand White rabbits.

At 24 hours after application, erythema was noted.  One week after application, animals recovered.

Under the conditions of the study the classification of the test material could not be determined.

Reference 5

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test material was administered to male New Zealand white rabbits as an aqueous solution to clipped skin via an adhesive patch. A torso cover was then fitted. The irritation was assessed at 24, 48 and 72 hours following application.

GLP compliance:

not specified

Specific details on test material used for the study:

Substance supplied as a 30 % solution.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: Each rabbit was kept in a separate cage.
- Diet: Each rabbit received 150 g of solid food (RC-4, Oriental Yeast Co., Ltd.) per day.
- Water: Ad libitum
- Acclimation period: Yes, but duration not specified.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 °C
- Humidity (%): 55 ± 15 %

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Concentration: the 30 % test material solution was dissolved in distilled water for injection to prepare a 5 % (w/v) solution.

Duration of treatment / exposure:

The test material was not removed during the study.

Observation period:

72 hours following application.

Number of animals:

Four

Details on study design:

TEST SITE
- Type of wrap if used: The test material was applied to an adhesive patch (Torii Pharmaceutical Co., Ltd.), and the patch was put on the shorn area. A torso cover was then fitted to the animal.

REMOVAL OF TEST SUBSTANCE
- Washing: The test material was not removed.

OBSERVATION TIME POINTS
- The cover and the patch were removed 24 hours following application, and the level of irritation was assessed 30 minutes later. The same procedure was carried out 48 and 72 hours following application.

SCORING SYSTEM:
- Method of calculation: Draize scale

Irritation parameter:

erythema score

Remarks on result:

not determinable

Irritation parameter:

edema score

Remarks on result:

not determinable

Irritant / corrosive response data:

Mild erythema was observed 24 hours following application. The symptoms gradually subsided.

Table 1: Erythema and oedema scores.

24 hours		48 hours		78 hours
Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
6	1	5	0	2	0
1.8		1.3		0.5

 

Draize Scale for Skin Reactions

Erythema and Eschar Formation	Score
Very slight erythema (barely perceptible)	1
Well defined erythema	2
Moderate to severe erythema	3
Severe erythema (beet redness) to slight eschar formation (injuries in depth)	4
Oedema Formation
Very slight oedema (barely perceptible)	1
Slight oedema (edges of area well defined by definite raising)	2
Moderate oedema (raised approx. 1 mm)	3
Severe oedema (raised more than 1 mm, and extending beyond area of exposure)	4

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study the test material was determined to be a slight irritant on the skin of rabbits.

Executive summary:

The skin irritation potential of the test material was investigated in a Modified Draize method study using rabbits. The 30 % test material solution was dissolved in distilled water for injection to prepare a 5 % (w/v) solution.

Mild erythema was observed 24 hours following application. The symptoms gradually subsided.

Under the conditions of this study the test material was determined to be a slight irritant on the skin of rabbits.

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:

09 December 2016

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:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- The test material was applied as supplied, no formulation was required.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Characteristics of donor animals: Approximately 7 weeks old
- Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue: Heads were collected by a slaughter house technician and heads transported to the testing laboratory 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). The heads were received at the testing laboratory and processed within approximately 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

Three test material treated eyes, three positive control treated eyes and one negative control and one parafilm control treated eye were examined.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Selection: 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.
- Preparation: 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
- 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 avoid too much pressure on the eye by the clamp. 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 after being 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 the physiological 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, 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.
- 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 corneal thickness changes (0.0 %) were observed in the eyes. 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:
- Three test material treated eyes, three positive control treated eyes and one negative control and one parafilm control treated eye were examined

NEGATIVE CONTROL USED
- 30 µL of physiological saline (Salsol solution, 0.9 % (w/v) NaCl)

POSITIVE CONTROL USED
- 30 mg of powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME
- 30 mg for 10 seconds
- 30 mg of test material was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material. Due to the physical nature of the test material, it was spread onto Parafilm, and was then applied so the test material was in contact with the cornea for the treatment. An additional parafilm control was placed onto one eye directly without using physiological saline.

REMOVAL OF TEST SUBSTANCE
- 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 20 mL saline was performed at each time point when the test material or control material remaining on the cornea was observed.

OBSERVATION PERIOD
- 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal thickness and corneal opacity were measured at all time points.
- Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.
- Haag-Streit BP 900 slit-lamp microscope was used for the measurements.
- Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test material to the cornea were recorded. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- Histopathology: The corneas were subject to processing to slides and histopathology examination. Histopathology was performed on all eyes. At least one slide per cornea was produced and examined (1 slide for each control eye and two slides for each test material-treated eye. The retinas were embedded in paraffin wax, sections were cut at 4 - 6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine. Histopathology evaluation was performed by light microscopy.
The stained slides were evaluated with respect to general quality of the morphology of the epithelium, stroma and endothelium (presence of necrosis), visibility and integrity of basement membrane, Bowman's membrane and Descemet's membrane.

SCORING SYSTEM:
- 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(30 min to 240 min) + SECOmax(30 min to 240 min) + TECOmax(30 min to 240 min)) / 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(30 min) + SEFR(30 min) + TEFR(30 min)) / 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

ICE CLASSIFICATION
- Corneal Thickness:
Mean Corneal Swelling 0 to 5 %: ICE Class I
Mean Corneal Swelling >5 to 12 %: ICE Class II
Mean Corneal Swelling >12 to 18 % ( >75 min after treatment ): ICE Class II
Mean Corneal Swelling >12 to 18 %( ≤75 min after treatment ): ICE Class III
Mean Corneal Swelling >18 to 26 %: ICE Class III
Mean Corneal Swelling >26 to 32 % ( >75 min after treatment): ICE Class III
Mean Corneal Swelling >26 to 32 % ( ≤75 min after treatment ): ICE Class IV
Mean Corneal Swelling >32 %: ICE Class IV

- Corneal Opacity:
Mean Maximum Opacity Score 0.0 - 0.5: ICE Class I
Mean Maximum Opacity Score 0.6 - 1.5: ICE Class II
Mean Maximum Opacity Score 1.6 - 2.5: ICE Class III
Mean Maximum Opacity Score 2.6 – 4.0: ICE Class IV

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

DECISION CRITERIA
- 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.
- No category = 3×I or 2×I, 1×II
- Category 1: 3×IV or 2×IV, 1×III or 2×IV, 1×II or 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).
- No prediction can be made: Other combinations

Irritation parameter:

percent corneal swelling

Run / experiment:

75 minutes

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

percent corneal swelling

Run / experiment:

240 minutes

Value:

7.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class IV

Other effects / acceptance of results:

TEST MATERIAL
- Results can be seen in Table 1.
- The overall ICE class was 1xII 1xI 1xIV, based on these results 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.

POSITIVE CONTROL
- The positive control (Imidazole) was classified as severely irritating.

NEGATIVE CONTROL
- The negative controls Physiological saline and parafilm were non-irritating.

HISTOPATHOLOGY
- Semi-quantitative microscopic evaluation was performed on the cornea in the ICET.
- The classification of histopathology findings was performed based on three publications, M.K. Prinsen et al./Toxicology in Vitro 25 (2011), 1475-1479), Elodie Cazelle et al./ Toxicology in Vitro 28 (2014), 657-666 and Atlas of histopathological lesions of Isolated Chicken Eyes, M.V.W. Wijnands and M.K. Prinsen, June 2015.
- The negative control Physiological saline (Salsol solution 0.9 %) and the Parafilm control covering cornea showed no abnormalities. Positive control, 30 mg powdered Imidazole associated with moderate epithelial erosion. No compromised basement membrane, Bowman’s and Descemet’s membrane, or as well as no stromal and endothelial changes were recorded.
- The test material produced from very slight to severe (in one case) erosion of the epithelium in 6/6 cases. Moderate vacuolation of the corneal epithelium in 1/6 sections was also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 1.

Table 1: Summary of Results

Observation	Test Material		Positive Control		Negative Controls (Physiological Saline and Parafilm)
	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.6 %	I	10.3 %	II	0.0 %	I
Mean maximum corneal swelling at up to 240 min	7.2 %	II	27.2 %	III	0.0 %	I
Mean maximum corneal opacity	0.50	I	4.00	IV	0.00	I
Mean fluorescein retention	3.00	IV	3.00	IV	0.00	I
Other Observations	None		Imidazole 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.		None
Overall ICE Class	1xII; 1xI; 1xIV		1xIII; 2xIV		3xI

Validity of the Test

- The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data. This experiment was considered to be valid.

Interpretation of results:

other: EU Criteria: Category 1, causes serious eye damage (H318).

Conclusions:

Based on this in vitro eye irritation assay in the isolated chicken eyes 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.
To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections). Microscopic evaluation showed very slight to severe (in one case) erosion of the epithelium in 6/6 cases. Moderate vacuolation of the corneal epithelium in 1/6 sections was also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 1. Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion classification of UN GHS eye irritant Category 1.

Executive summary:

An in vitro eye irritation study of the test material was performed in isolated chicken’s eyes. The irritation effects of the test material were evaluated according to the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions.

After the zero reference measurements, the eye was held in horizontal position and 30 mg of test material was applied onto the centre of the cornea as a layer on parafilm such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution) and the parafilm control eye was treated directly with parafilm without using 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 and one parafilm control treated eye were examined. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

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

Slight corneal swelling was observed during the four-hour observation period on test material treated eyes. No significant corneal opacity change (severity 0.5) was noted on all three eyes. Severe fluorescein retention change (severity 3) was noted on all three eyes. No other corneal effect was observed.

Based on this in vitro eye irritation assay in the isolated chicken eyes 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.

To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections). Microscopic evaluation showed very slight to severe (in one case) erosion of the epithelium in 6/6 cases. Moderate vacuolation of the corneal epithelium in 1/6 sections was also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 1. Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion classification of UN GHS eye irritant Category 1.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

no guideline followed

Principles of method if other than guideline:

The eye irritation potential of the test material was assessed in male New Zealand White rabbits. Before instillation, both eyes of rabbits were macroscopically examined for ocular symptoms and injury, and rabbits with no abnormalities were used. In instillation, 0.1 mL of the sample was placed into the conjunctival sac of both eyes, formed by pulling the lower lid from the eyeball. Then, the upper and lower eyelids were gently held together. The degree of irritation was evaluated at 24, 48, and 72 hours after instillation according to the Draize criteria.

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Housing: Animals were housed one animal per cage.
- Diet: 150 g/day of solid feed for rabbits/guinea pigs (RC-4, Oriental Yeast Co., Ltd.)
- Water: Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3 °C
- Humidity: 55 ± 15 %

Vehicle:

water

Remarks:

Distilled water for injection

Controls:

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 mL
- Concentration: 5 %

Duration of treatment / exposure:

The test material was not removed

Observation period (in vivo):

72 h

Number of animals or in vitro replicates:

Four animals

Details on study design:

Before instillation, both eyes of rabbits were macroscopically examined for ocular symptoms and injury, and rabbits with no abnormalities were used. In instillation, 0.1 mL of the sample was placed into the conjunctival sac of both eyes, formed by pulling the lower lid from the eyeball. Then, the upper and lower eyelids were gently held together.

SCORING SYSTEM: The degree of irritation was evaluated at 24, 48, and 72 hours after instillation according to the Draize criteria.

Irritation parameter:

cornea opacity score

Remarks on result:

other: Not reported

Irritation parameter:

iris score

Remarks on result:

other: Not reported

Irritation parameter:

conjunctivae score

Remarks on result:

other: Not reported

Irritation parameter:

chemosis score

Remarks on result:

other: Not reported

Irritant / corrosive response data:

At 24 hours after instillation, almost no irritation was noted in the test material group. The symptoms returned to normal by 72 hours after instillation.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study the classification of the test material could not be determined.

Executive summary:

The eye irritation potential of the test material was assessed in male New Zealand White rabbits.

At 24 hours after instillation, almost no irritation was noted in the test material group.  The symptoms returned to normal by 72 hours after instillation.

Under the conditions of the study the classification of the test material could not be determined.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

For the justification for read-across, please refer to the read-across assessment framework report that is attached to Section 13.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Remarks on result:

other: Not reported

Irritation parameter:

iris score

Remarks on result:

other: Not reported

Irritation parameter:

conjunctivae score

Remarks on result:

other: Not reported

Irritation parameter:

chemosis score

Remarks on result:

other: Not reported

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study the classification of the test material could not be determined.

Executive summary:

The eye irritation potential of the test material was assessed in male New Zealand White rabbits.

At 24 hours after instillation, almost no irritation was noted in the test material group.  The symptoms returned to normal by 72 hours after instillation.

Under the conditions of the study the classification of the test material could not be determined.

Reference 4

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

0.1 mL of the test solution was applied to the eyes of male New Zealand White rabbits. The level of irritation was assessed 24, 48 and 72 hours following application of the test solution, in accordance with the Draize scale.

GLP compliance:

not specified

Specific details on test material used for the study:

The test material was supplied as a 30 % solution.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Housing: Each rabbit was kept in a separate cage in the animal-keeping room.
- Diet: 150 g of solid food per day
- Water: Ad libitum
- Acclimation period: Yes for a specified period (not stated in protocol).

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3 °C
- Humidity: 55 ± 15 %.

Vehicle:

water

Remarks:

Distilled water for injection

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Concentration: the 30 % test material solution was dissolved in distilled water for injection to prepare a 5 % (w/v) solution. 0.1 mL of the test solution was applied to the eye.

Duration of treatment / exposure:

The test material was not removed during the test.

Observation period (in vivo):

72 hours. The level of irritation was assessed 24, 48 and 72 hours following application of the test solution

Number of animals or in vitro replicates:

4 male rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The test materia was not removed.

SCORING SYSTEM: Draize scale

OTHER:
- Before applying, the rabbits' eyes were macroscopically examined to select rabbits with eyes that were free from disease and injury.
- The test material was applied to the eye by pulling the lower eyelid into a cup-shape. The upper and lower eyelids were gently held together following application.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

not specified

Irritant / corrosive response data:

Slight redness in the conjunctivae was observed following application in half of the rabbits in the test material group. No other abnormal condition was observed.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the test material has been determined to be non irritating.

Executive summary:

The potential of the test material to cause irritation to the eye was investigated in a Modified Draize method test using New Zealand White male rabbits.

Before applying, the rabbits' eyes were macroscopically examined to select rabbits with eyes that were free from disease and injury. 0.1 mL of the test material solution was applied to the eye by pulling the lower eyelid into a cup-shape. The upper and lower eyelids were gently held together following application.

Slight redness in the conjunctivae was observed following application in half of the rabbits in the test material group. No other abnormal condition was observed.

Under the conditions of the study the test material has been determined to be non irritating.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion In Vitro: Varga-Kanizsai (2017)

The potential of the test material to cause skin corrosion was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis under GLP conditions using a human skin model assay. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Disks of EPISKIN™(SM) were treated with the test material in duplicate and incubated for 4 hours at room temperature. Exposure of the 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, also performed in duplicate. 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 86.7 % 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.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Skin Irritation In Vitro: Varga-Kanizsai (2017)

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay.

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 % RH %. 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 RH % 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 75.5 % 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.

Under the conditions of this study, the test material is not irritating in the in vitro skin irritation test.

Skin Irritation In Vivo: Hanzawa (1992)

The skin irritation potential of the test material was investigated in a Modified Draize method study using rabbits. The 30 % test material solution was dissolved in distilled water for injection to prepare a 5 % (w/v) solution.

Mild erythema was observed 24 hours following application. The symptoms gradually subsided.

Under the conditions of this study the test material was determined to be a slight irritant on the skin of rabbits.

Skin Irritation In Vivo - Read-Across Substance: Hanzawa (1991)

The primary skin irritation potential of the test material was assessed in male New Zealand White rabbits. The study was awarded a reliability score of 4 in accordance with the criteria set forth by Klimisch et al. (1997).

At 24 hours after application, erythema was noted.  One week after application, animals recovered.

Under the conditions of the study the classification of the test material could not be determined.

Eye Irritation In Vitro: Varga-Kanizsai (2018)

An in vitro eye irritation study of the test material was performed in isolated chicken’s eyes. The irritation effects of the test material were evaluated according to the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

After the zero reference measurements, the eye was held in horizontal position and 30 mg of test material was applied onto the centre of the cornea as a layer on parafilm such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution) and the parafilm control eye was treated directly with parafilm without using 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 and one parafilm control treated eye were examined. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

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

Slight corneal swelling was observed during the four-hour observation period on test material treated eyes. No significant corneal opacity change (severity 0.5) was noted on all three eyes. Severe fluorescein retention change (severity 3) was noted on all three eyes. No other corneal effect was observed.

Based on this in vitro eye irritation assay in the isolated chicken eyes 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.

To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections). Microscopic evaluation showed very slight to severe (in one case) erosion of the epithelium in 6/6 cases. Moderate vacuolation of the corneal epithelium in 1/6 sections was also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 1. Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion classification of UN GHS eye irritant Category 1.

Eye Irritation In Vivo: Hanzawa (1992)

The potential of the test material to cause irritation to the eye was investigated in a Modified Draize method test using New Zealand White male rabbits. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Before applying, the rabbits' eyes were macroscopically examined to select rabbits with eyes that were free from disease and injury. 0.1 mL of the test material solution was applied to the eye by pulling the lower eyelid into a cup-shape. The upper and lower eyelids were gently held together following application.

Slight redness in the conjunctivae was observed following application in half of the rabbits in the test material group. No other abnormal condition was observed.

Under the conditions of the study the test material has been determined to be non irritating.

Eye Irritation In Vivo - Read-Across Material: Hanzawa (1991)

The eye irritation potential of the test material was assessed in male New Zealand White rabbits.

At 24 hours after instillation, almost no irritation was noted in the test material group.  The symptoms returned to normal by 72 hours after instillation.

Under the conditions of the study the classification of the test material could not be determined.

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 corrosion or irritation.

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified as Category 1 with respect to eye irritation. Causes serious eye damage (H318).