Registration Dossier

Administrative data

Description of key information

Skin Corrosion/Irritation

Under the conditions of the study, the test material is non-corrosive to the skin.

Under the conditions of the study, the test material was considered to be non-irritating to the skin.

Eye Irritation

Under the conditions of this study, the test material was non-irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN™(SM) is a three-dimensional human skin model.
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)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM), supplied by SkinEthic, France.
- Tissue batch number: 16-EKIN-049
- Expiry date: 12 December 2016
- 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.
- The EPISKIN™(SM) kits 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.
- In each case, the pH of the agar medium used for transport was checked by checking the colour of the medium: orange colour = good or 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 or grey or black colour = not acceptable. The kits were found to be in good order at reception.

CHECK FOR DIRECT MTT REDUCTION
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: yellow or Test materials reacting with MTT: blue or purple. After three hours incubation, yellow colour of the mixture was detected; therefore additional controls were not used in the experiment.

CHECK TO DETECT 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 isopropanol. 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.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (22.9 to 24.2 °C)
- Temperature of post-treatment incubation: 37 °C (with MTT)

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 to 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/ 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.

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 the blank.

NUMBER OF REPLICATE TISSUES: 2

CALCULATIONS OF VIABILITY PERCENTAGES
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% = [(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 = (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 % = (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 % = (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 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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 20 mg (+ 100 µL of physiological saline to ensure good contact with the epidermis)

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

POSITIVE CONTROL
- Amount(s) applied: 50 µL
Duration of treatment / exposure:
4 hours ± 10 minutes
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:
99.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- OTHER EFFECTS: ADDITIONAL CONTROLS
- Colour interference with MTT: 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 0.006. Non Specific Colour % (NSCliving %) was calculated as 0.8 %. 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 a 99.3 % relative viability.

ACCEPTANCE OF RESULTS:
-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 5.4 %.
-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

Optical density (OD)

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

0.853

0.807

96.6

2

0.898

0.852

101.9

Mean

-

0.830

99.3

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 the study, the test material is non-corrosive to the skin.
Executive summary:

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.Bis using the reconstructed human epidermis model, under GLP conditions.

EPISKIN(SM) is designed to predict and classify the corrosive 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) (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 99.3 % 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 the study, the test material is non-corrosive to the skin.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
04 July 1988 to 12 September 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiences which do not affect the quality of the relevant results.
Reference:
Composition 0
GLP compliance:
no
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 16-19 weeks old
- Weight at study initiation: 2.92 - 3.12 kg
- Housing: Animals were accommodated individually in bracket cage in Animal Room No. 127
- Diet (e.g. ad libitum): 150 g/day of commercially available pellet diet was restricted fed
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature was set at 23 ± 3 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 10-15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day of lighting

Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
other: Pharmaceutical vaseline
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 g of Vaseline gel containing 5 or 15 % test material

POSITIVE CONTROL
- Amount(s) applied: 0.5 mL
- Concentration: 5 %
Duration of treatment / exposure:
24 hours
Observation period:
72 hours as well as one week after application of the test and positive control substances.
Number of animals:
6 animals
Details on study design:
DOSING:
- Before patching of the test material, both flanks of each rabbit was clipped using an animal hair trimmer.
- The following day a commercial patch with adhesive plaster containing 0.5g of the test material in Vaseline gel, Vaseline gel alone (vehicle group) and 0.5 mL of 5 % SLS solution was then applied to each occlusive patch. The patch was covered with a vest to prevent it from being dislodged. The application was kept on for 24 hours.

TEST GROUP COMPOSITION:
-Group 1: White Vaseline (vehicle)
-Group 2: Vaseline containing 5 % test material
-Group 3: Vaseline containing 15 % test material
-Group 4: Vaseline containing 5 % SLS
-Group 5: 5 % SLS Aqueous solution

OBSERVATIONS

-Skin Irritation was assessed according to the Draize evaluation method at 24, 48 and 72 hours, as well as one week after application of the test material and positive control:
- Erythema and Scab Formation
No erythema = 0
Very slight erythema = 1
Well defined erythema = 2
Moderate to severe erythema = 3
Deep red, severe erythema = 4
- Oedema Formation
No oedema = 0
Very slight oedema = 1
Well defined oedema (edge of area well defined) = 2
Moderate oedema (area raised approximately 1 mm) = 3
Severe oedema (area raised more than 1 mm and extending beyond the area of exposure) = 4

-Animals were observed twice daily.
-Body weights were measured on arrival, at substance application and at assessment.
-After study completion, necropsy was performed for all animals with macroscopic examination.

ANALYTICAL METHOD
-Total irritation score of two phenomenon's was calculated for each animal according to the Draize grading scale, and the per-animal mean score for each group was computed. The irritation potential for rabbit skin of test material and positive control material was evaluated according to the following:
0 to 2: Non irritant or mild irritant
3 to 5: Moderate irritant
6 to 8: Severe irritant
Irritation parameter:
overall irritation score
Basis:
other: mean at 5 %
Time point:
24/48/72 h
Score:
0.067
Max. score:
0.2
Reversibility:
fully reversible within: 48h
Irritation parameter:
overall irritation score
Basis:
other: mean at 15 %
Time point:
24/48/72 h
Score:
0
Max. score:
0

TABLE 1: Assessment of Irritation

 

24 hours Score

48 hours Score

72 hours Score

1 week Score

Vaseline

0.3

0

0

0

5% Test material in vaseline

0.2

0

0

0

15% Test material in vaseline

0

0

0

0

Vaseline with 5% SLS

5.5

4.5

4.8

0.3

5% SLS aqueous solution

8.0

7.2

6.7

2.2

After 24 hours application, slight erythema was observed in 2 of 6 animals in the vaseline (vehicle) application group, and in 1 of 6 animals in the vaseline gel containing 5 % test material application group. However after 48 hours these changes recovered and thereafter no changes were observed. No abnormalities were observed after application in the vaseline gel containing 15 % test material.

In the positive control groups, the SLS vaseline ointment and aqueous solution groups, 24 hours after application moderate erythema was observed in the former, and sever erythema and oedema was observed in the latter. After 48 hours, even scab formation was observed although there was gradual recovery thereafter. After one week, both groups presented partial scabbing and very slight to slight erythema at areas where the scab had peeled.

 

No notable symptoms of clinical signs were observed during the study, no notable changes in bodyweight were observed and after the study no notable abnormalities were observed in the macroscopic organ examination.

Under the conditions of this study and based on the results above, the test material is considered to be not irritating to rabbit skin.

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of the study, the test material was considered to be non-irritating to rabbit skin. No abnormalities were observed after application in the 15 % test material group.
Executive summary:

The potential for the test material to cause skin irritation was predicted in an in vivo study using male New Zealand white rabbits. The study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies which do not affect the quality of the relevant results.

Vaseline gel containing 5 % and 15 % of the test material and vaseline (as a vehicle) were applied with occlusive patch to the rabbit skin for 24 hours. Animals were observed for 72 hours and then a week and the mean score per animal at each reading time was recorded. SLS vaseline ointment and aqueous solution groups were used as positive control groups.

Under the conditions of the study, the test material was considered to be non-irritating to rabbit skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2016 to 17 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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:
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. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 °C, below 70 RH%).
Species:
chicken
Strain:
other: COBB 500 and ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption.
- Characteristics of donor animals: Approximately 7 weeks old.
- Storage, temperature and transport conditions of ocular tissue: Heads were collected by a slaughterhouse technician and heads transported to the 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 2 hours of collection.
- indication of any existing defects or lesions in ocular tissue samples: Corneas were examined to ensure that the cornea was in good condition.
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):
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.
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.
Number of animals or in vitro replicates:
One eye was treated with the negative control, three eyes with the test material and another three eyes with positive control in each experiment.
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.

-Examiniation and acclimatisation: The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position. 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, acclimatization 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.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. No changes in thickness (0.0 %) were observed in the eyes in each 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 eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.

NEGATIVE CONTROL USED: 30 µL physiological saline (0.9 % (w/v) NaCl).

POSITIVE CONTROL USED: 30 mg imidazole.

APPLICATION DOSE AND EXPOSURE TIME: 30 mg (test material or positive control) / 30 µL (negative control) for 10 seconds.

TREATMENT: 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. In each experiment, 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.

REMOVAL OF TEST SUBSTANCE: 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 20 mL saline was performed after treatment and at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes were rinsed additional gentle rinsing at least 2 x 20 mL saline after treatment in each experiment and with 20 mL physiological saline solution each time point when the test item 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.
- 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 900slit lamp microsccope was used for measurements.
- Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- At the end of the procedure, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid, to be used for potential histopathology and stored at room temperature.

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] x100

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

Remark:
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

Remark:
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

Remark:
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
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 1 Mean (Up to 75 min)
Value:
1.1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes (2/3) and at 180 minutes (1/3) after the post-treatment rinse.
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 1 Mean (Up to 240 min)
Value:
2.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes (2/3) and at 180 minutes (1/3) after the post-treatment rinse.
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment 1
Value:
0.17
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes (2/3) and at 180 minutes (1/3) after the post-treatment rinse.
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment 1
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes (2/3) and at 180 minutes (1/3) after the post-treatment rinse.
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 2 Mean (Up to 75 min)
Value:
0.5
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment 2 Mean (Up to 240 min)
Value:
1.6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment 2
Value:
0.33
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment 2
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.
Other effects / acceptance of results:
The test material showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes the test material was non-irritant, UN GHS Classification: No Category.

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 range in experiment. This experiment was considered to be valid.

Positive control:
Experiment I
Mean maximum corneal swelling at up to 75 min: 10.3 %
Mean maximum corneal swelling at up to 240 min: 27.7 %
Mean maximum corneal opacity: 4.0
Mean fluorescein retention: 3.0
Other Observations: Imidazole was stuck on all cornea surfaces after the posttreatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Experiment II
Mean maximum corneal swelling at up to 75 min: 10.3 %
Mean maximum corneal swelling at up to 240 min: 26.1%
Mean maximum corneal opacity: 4.0
Mean fluorescein retention: 3.0
Other Observations: Imidazole was stuck on all cornea surfaces after the posttreatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Negative control:
Experiment I: 0.0 for all parameters assessed.
Experiment II: 0.0 for all parameters assessed.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study the test material was not irritating to the eye.
Executive summary:

The irritation effects of the test material were investigated in accordance with the standardised guidelines OECD 438 and EU Method B.48 using the isolated chicken eye assay under GLP conditions.

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 experiment. Thus, the experiment was considered to be valid.

Experiment I: 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 one eye. No fluorescein retention change was noted on three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes (2/3) and at 180 minutes (1/3) after the post-treatment rinse.

Experiment II: 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 two eyes. No fluorescein retention change was noted on three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Under the conditions of this study, the test material was non-irritant, UN GHS Classification: No Category.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
04 July 1988 to 12 September 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiences which do not affect the quality of the relevant results.
Reference:
Composition 0
GLP compliance:
no
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 14 to 20 weeks old
- Weight at study initiation: 2.77 to 3.27 kg
- Housing: each animal was accommodated individually in bracket cage in Animal Room No. 127
- Diet: During the preliminary study period, 150 g/day of commercially available pellet diet was restricted fed
- Water: tap water (ad libithum)
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: The room temperature was set at 23 ± 3 °C
- Humidity: humidity at 55 ± 15 %
- Air changes: 10 to 15 air changes per hour
- Photoperiod: 12 hours/day of lighting (lights on at 7 AM; lights out at 7 PM)
Vehicle:
other: Olive oil
Controls:
yes, concurrent no treatment
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL:
- Amount applied: 0.1 mL
- Concentration: 5 %
Duration of treatment / exposure:
1 to 2 seconds
Observation period (in vivo):
Observations were made at 24, 48 and 72 hours as well as 1 week after application.
Number of animals or in vitro replicates:
6 animals
Details on study design:
DOSING:
- Procedure: Before administration, the both eyes of rabbits were examined that there were no injuries or abnormalities.0.1 mL of the test material and SLS aqueous solution was placed in the conjunctival sac, and then held the upper and lower eyelids closed for 1 or 2 seconds. The left eye was left untreated and was used as a control for comparison in the observation.

OBSERVATIONS

ASSESSMENT OF IRRITATION:
-Conjunctival irritation was assessed according to the Draize evaluation method:
1) Cornea

A. Opacity – Degree of Density (area most dense taken for reading)
0: Transparent, no opacity
1: Scattered or diffuse area - details of iris clearly visible
2: Easily discernible translucent areas, details of iris slightly obscured
3: Opalescent areas, no details of iris visible, size of pupil barely discernible
4. Opaque, iris invisible

B. Area of Cornea Involved
1: 0-1/4
2: 1/4-1/2
3: 2/16-3/4
4: 3/4-1

2) Iris

A. 0: Normal
1: Folds above normal, congestion, swelling, circumcorneal injection (any one or all of these or combination of any thereof), iris still reacting to light
2: No reaction to light, haemorrhage; gross destruction (any one or all of these)

3) Conjunctivae

A. Redness (palpebral and bulbar conjunctiva)
0: Normal blood vessels
1: Vessels definitely injected above normal
2: More diffuse, deeper crimson red, individual vessels not easily discernible
3: Diffuse beefy red

B. Chemosis
0: No chemosis
1: Any swelling above normal (includes nictitating membrane)
2: Obvious swelling with partial eversion of the lids.
3: Swelling with lids about half closed
4: Swelling with lids about half closed to completely closed

C. Discharge
0: No discharge observed
1: Any amount different from normal
2: Discharge with moistening of the lids and hairs just adjacent to the lids
3: Discharge with moistening of the lids and considerable area around the eye

-Animals were observed twice daily for clinical signs
-Bodyweights were measured on arrival, on administration and at assessment.
-After study completion, full necropsy was performed for all animals and macroscopic examination was conducted.

ANALYTICAL METHOD
-For each animal, total irritation score for three indices was summed, the mean value per animal in each group was calculated, and irritation of rabbit ocular membranes by the test material and positive control were evaluated with reference to the following criteria:
-Irritation score of cornea = (Score A) x (Score B) x 5
-Irritation score of iris = (Score A) x 5
-Irritation score of conjunctiva = (Score A+B+C) x 2 :
0 to 5 = Non-irritant
5 to 15 = Mild irritant
15 to 30 = Irritant
30 to 60 = Moderate irritant
60 to 80 = Moderate to strong irritant
80 to 110 = Strong irritant
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0

Table 1: Assessment of Irritation: Results

TEST SAMPLE

 

24-hour Score

48-hour Score

72-hour Score

1 week Score

Olive oil

Right Eye

0

0

0

0

Left Eye

0

0

0

0

5 % test material

Right Eye

0

0

0

0

Left Eye

0

0

0

0

5 % positive control

Right Eye

21.0

11.0

4.7

0.3

Left Eye

0

0

0

0

Based on the results above, no abnormalities of any ocular membranes were observed in the test material group or the olive oil group. On the other hand, in the positive control group, 24 hours after application, slight opacity of the cornea, mild congestion of the iris, strong erythema and moderate edema of the conjunctiva, and discharge were observed, but these symptoms recovered gradually thereafter, and abnormalities were nearly unobservable after 1 week.

No notable symptoms of clinical signs were observed during the study period, no notable changes to bodyweights were observed and after study completion no notable abnormalities were observed in the major organs of any animals.

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of the study, the test material was determined to be non-irritating as no abnormalities of any ocular membranes was observed throughout the study.
Executive summary:

The potential for the test material to cause eye irritation was assessed in an in vivo irritation study in rabbits. The study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies which do not affect the quality of the relevant results. The study was given a reliability score of 2 in accordance with the criteria for assessing data quality as set forth by Klimisch (1997).

The test material was administered to male New Zealand white rabbits, in a single application. Rabbits were assessed over 72 hours for signs of eye irritation, clinical sign of toxicity, mortality and bodyweight gain. Following the test material administration, no abnormalities of any ocular membranes were observed in the test material treated group. The positive control substance group showed symptoms including opacity of the cornea, congestion of the iris, erythema and oedema.

Under the conditions of the study, the test material was determined to be non-irritating.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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 corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.Bis using the reconstructed human epidermis model, 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 potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.Bis using the reconstructed human epidermis model, under GLP conditions.

EPISKIN(SM) is designed to predict and classify the corrosive 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) (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 99.3 % 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 the study, the test material is non-corrosive to the skin.

Skin Irritation In Vivo (Fujimoto, 1988)

The potential for the test material to cause skin irritation was predicted in an in vivo study using male New Zealand white rabbits. The study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies which do not affect the quality of the relevant results.

Vaseline gel containing 5 % and 15 % of the test material and vaseline (as a vehicle) were applied with occlusive patch to the rabbit skin for 24 hours. Animals were observed for 72 hours and then a week and the mean score per animal at each reading time was recorded.SLS vaseline ointment and aqueous solution groups were used as positive control groups.

Under the conditions of the study, the test material was considered to be non-irritating to rabbit skin.

Eye Irritation In Vitro (Varga-Kanizsai, 2017)

The irritation effects of the test material were investigated in accordance with the standardised guidelines OECD 438 and EU Method B.48 using the isolated chicken eye assay 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 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 experiment. Thus, the experiment was considered to be valid.

Experiment I: 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 one eye. No fluorescein retention change was noted on three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes (2/3) and at 180 minutes (1/3) after the post-treatment rinse.

Experiment II: 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 two eyes. No fluorescein retention change was noted on three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Under the conditions of this study, the test material was non-irritant, UN GHS Classification: No Category.

Eye Irritation In Vivo (Fujinomoto, 1998)

The potential for the test material to cause eye irritation was assessed in anin vivoirritation study in rabbits. The study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies which do not affect the quality of the relevant results. The study was given a reliability score of 2 in accordance with the criteria for assessing data quality as set forth by Klimisch (1997).

The test material was administered to male New Zealand white rabbits, in a single application. Rabbits were assessed over 72 hours for signs of eye irritation, clinical sign of toxicity, mortality and bodyweight gain. Following the test material administration, no abnormalities of any ocular membranes were observed in the test material treated group. The positive control substance group showed symptoms including opacity of the cornea, congestion of the iris, erythema and oedema.

Under the conditions of the study, the test material was determined to be non-irritating.

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 irritation/corrosion or eye irritation.