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Description of key information

> Skin Irritation

Read-across to structurally similar substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8)

Following exposure to erbium gadolinium yttrium zirconium oxide, the mean relative viability was 86.4 % compared to the negative control value. This is above the threshold of 50 %, therefore the test material was considered being non-irritant to skin.

> Eye Irritation

Appl (2020)

Under the conditions of the study the test material was not classified according to EU criteria.

Orovecz (2019)

Under the conditions of the study 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.

Read-across to structurally similar substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8)

Based on the in vitro eye irritation assays in isolated chicken eyes, the test material was not classified as a severe irritant and not classified as non-irritant. 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 erosion of the corneal epithelium in 4/6 cases, and slight intensity in 2/6 sections. Slight necrosis of the top epithelial layer was seen in 1/6 cases. 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, erbium gadolinium yttrium zirconium oxide was classified as Category 2B under GHS.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2016 to 13 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test material in the solvent/vehicle: Not applicable, the test material was applied in its original form, no formulation was required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, the test material was applied in its original form, no formulation was required.

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified (adult)
Source strain:
other: not applicable
Justification for test system used:
The EPISKIN™(SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439). Therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model
- Manufacturer: SkinEthic, France
- Batch No.: 16-EKIN-019
- Expiry Date: 2016-05-16
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been 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.
- EPISKIN (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). 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.

PRE-INCUBATION
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight (at least 18 hours) at 37 °C in an incubator with 5 % CO2, in a >95 % humidified atmosphere.

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

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Disks of EPISKIN (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature (24.8 - 25.8 °C). 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 protected from light.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with Phosphate Buffered Saline (PBS) to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37 °C in an incubator with 5 % CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
MTT was diluted in PBS at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution was stored in refrigerator (2-8 °C) protected from light. It was diluted with pre-warmed (37 °C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Plate reader
- Wavelength: 570 nm
- After the 42 hours incubation, all EPISKIN (SM) units (except of 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 (± 5 min) at 37 °C in an incubator with 5 % CO2 protected from light.

FORMAZAN EXTRACTION
- Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04 N HCl (1.8 mL of 12 N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.
- 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.
- A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENTS
- Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The 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, Serial Number: 0920-14, Date of calibration: 02 September 2014, calibration is valid until September 2016) at the required wavelength on each day before use.

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test material is considered to be non-irritant to skin if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than 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 applied: 20 mg (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)

NEGATIVE CONTROL (PBS)
- Amount applied: 50 µL

POSITIVE CONTROL (5% SDS)
- Amount applied: 50 µL
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours ± 1 hour
Number of replicates:
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 tissues were used for the non-specific OD evaluation.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
relative mean tissue viability compared to the negative control tissues
Value:
86.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
relative viability of each test material sample: 77.7, 80.3, 101.2%
Other effects / acceptance of results:
As no colour change (yellow colour) was observed after three hours of incubation of the test materials in MTT working solution, 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 was 0.027. Non Specific Colour % was calculated as 3.2 %. This value was below 5 %, therefore additional data calculation was not necessary.

Validity results:
- The mean OD value of the three negative control tissues was in the recommended range (0.836). Standard deviation of the viability results for negative control samples was 4.9.
- The positive control treated tissues showed 7.4 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.3.
- The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 12.9.
- 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.
Interpretation of results:
other: Not classified according to EU criteria
Conclusions:
Under the conditions of the test the mean relative viability was 86.4 % compared to the negative control value. This is above the threshold of 50 %, therefore the test material was considered non-irritant to skin.
Executive summary:

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

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. 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 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 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 86.4 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

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

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across to structurally similar substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8). The only difference between the substance being registered and the read-across substance is that the substance to be registered is missing the gadolinium oxide content (about 1.4 %). The absence of this compound at such a low % is considered highly unlikely to affect its properties.
Reason / purpose for cross-reference:
read-across source
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
relative mean tissue viability compared to the negative control tissues
Value:
86.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
relative viability of each test material sample: 77.7, 80.3, 101.2%
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 October 2019 to 29 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 14 week old (young adult) males.
- Weight at study initiation: 3730 g - 3825 g.
- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: At least 36 days. Only animals in acceptable health condition were used for the test. The veterinarian certified health status. Both eyes of each animal provisionally selected for testing were examined prior to starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 23.0 °C.
- Humidity (%): 42 – 64 %.
- Air changes (per hr): 15 - 20 air exchanges/hour.
- Photoperiod (hrs dark / hrs light): Light 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:
unchanged (no vehicle)
Controls:
other: The untreated right eye served as control.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: A single amount of 0.1 g of the test material was administered to the left eye of each animal.
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
Three animals
Details on study design:
IDENTIFICATION OF pH
- The pH of the test material was measured prior to the start of treatment and it was found to be 6.25, so the test material is permitted for use in animal studies.

PRE-STUDY EXAMINATION
- Three male animals in acceptable health condition were selected for the test. Care was taken to select only those animals that had a normal eye condition.

ADMINISTRATION
- Initially only one rabbit was treated with test material. All scores were zero at 24 hours after the application, therefore two additional rabbits were treated after the 48-hour observation of the first rabbit. No clinical signs were observed in any animals during the observation period; the experiment was terminated after 72 hours observation of the second and third rabbits.
- The test material was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for at least one second in order to prevent loss of the material. The untreated contralateral eye served as the control.
- The eyes were examined at approximately 1, 24, 48, 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill-health during the study were recorded. At the end of the observation period, the animal was sacrificed by intravenous sodium pentobarbital. Death was verified by checking pupil and corneal reflex and the absence of respiration and pulse.
- All rabbits were examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health were recorded.
- Analgesic and Anaesthetic Treatment: Sixty minutes (60 ± 10 min) prior to test material application, a systemic opiate analgesic was administered by subcutaneous injection (SC) under direct Veterinary supervision. Repeat injections were given on the first and second days as appropriate to maintain an adequate level of analgesia. Five minutes (5 ± 1.5 min) prior to test material application, a topical ocular anaesthetic was applied to each eye (including the control eye to ensure direct comparison of any ocular observations).
Eight hours (8 to 9 hr) after test material application, a systemic opiate analgesic and a nonsteroidal anti-inflammatory drug (NSAID) were administered by subcutaneous injection under direct Veterinary supervision. The systemic opiate analgesic was again injected ~12 hours after the post-treatment analgesic.
Systemic Opiate Analgesic: Bupredine Multidose (0.3 mg/mL buprenorphine)
Topical Ocular Anaesthetic: Benoxi (4 mg/mL oxybuprocaine - hydrochloride)
Nonsteroidal Anti - inflammatory Drug: Melovem®(5 mg/mL meloxicam)

REMOVAL OF TEST SUBSTANCE
- Washing: Solid test material was observed in the treated eye of the test animals at the 1-hour observation point. As such the treated eye was rinsed with physiological saline solution.
- Time after start of exposure: At the 1-hour observation point.

SCORING SYSTEM
- Individual reactions of the animals were recorded at each observation time. The nature, severity and duration of all lesions observed were described.
- The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (09 October 2017).
Scoring and Assessment of Local Reaction
1. Conjunctivae
A. Redness (Palpebral and bulbar)
0 Normal
1 Some blood vessels hyperaemic (injected)
2 Diffuse, crimson colour, individual vessels not easily discernible
3 Diffuse beefy red.

B. Chemosis
0 Normal
1 Some swelling above normal (includes nictating membrane)
2  Obvious swelling with partial eversion of lids
3 Swelling with lids about half closed
4 Swelling with lids more than half closed.

C. Discharge
0 No discharge
1 Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)
2 Discharge with moistening of the lids and hairs just adjacent to lids
3 Discharge with moistening of the lids and hairs, on considerable area around the eye.

2. Iris
0 Normal
1 Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia: or injection: iris reactive to light (a sluggish reaction is considered to be an effect)
2 Haemorrhage, gross destruction, or no reaction to light.

3. Cornea
E. Opacity - Degree of Density (Area most dense taken for reading)
0 No ulceration or opacity
1 Scattered or diffuse areas of opacity (other than slight dulling of normal lustre): details of iris clearly visible
2 Easily discernible translucent area: details of iris slightly obscured
3 Nacrous area: no details of iris visible: size of pupil barely discernible
4 Opaque cornea: iris not discernible through the opacity.

F. Area of Cornea Involved
1 One quarter (or less), but not zero
2 Greater than one quarter, but less than half
3 Greater than half, but less than three quarters
4 Greater than three quarters, up to whole area.

Any Other Lesions in the Eye (e.g. pannus, staining, anterior chamber changes): Text description, or 0 if absent.

BODY WEIGHT
- Individual body weight was recorded on the day of treatment and at the end of observation period of each animal.
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: No effects observed.
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Examination of Ocular Reactions
No Initial Pain Reaction/Pain reaction (IPR/PR) was observed.
 
Animal 1 (No. 2091) Clinical Observation
At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Remaining test material was noted in the eye sac.
At 24, 48 and 72 hours after the application, no clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed.
 
Animal 2 (No: 2094) Clinical Observation
At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Remaining test material was noted in the eye sac.
At 24, 48 and 72 hours after the application, no clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed.
 
Animal 3 (No: 2199) Clinical Observation
At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Remaining test material was noted in the eye sac.
At 24 hours after the application, conjunctival redness (score 1) and chemosis (score 1) were noted in the rabbit.
At 48 and 72 hours after the application, no clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed.

As no clinical signs were observed, the experiment was terminated after 72 hours observation of the second and third rabbits. During the experiment, the control eye of each animal was symptom-free. Solid test material was observed in the treated eye of the test animals at the 1-hour observation point. As such, the treated eye of all animals was rinsed with physiological saline solution at the 1-hour observation point in line with the OECD guidance. The general state and behaviour of animals were normal throughout the study period.
Other effects:
MORTALITY
There was no mortality observed during the study.
 
BODY WEIGHTS
The body weight of the animals was considered to be within the normal range of variability.

CLINICAL OBSERVATIONS
There were no clinical signs observed that could be related to treatment.
Interpretation of results:
other: Not classified according to EU criteria.
Conclusions:
Under the conditions of the study the test material is not classified as irritant according to EU criteria.
Executive summary:

The in vivo eye irritation potential of the test material was assessed in rabbits according to OECD Test Guideline 405 and in compliance with GLP.

Three rabbits were treated with the test material. Prior to test material application, the animals were treated with analgesic and anaesthetic as per the regulatory guideline. The test material was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 g of the test material was administered to the left eye of each animal. The eyes were examined at 1, 24, 48 and 72 hours after application.

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed. At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the three rabbits. Remaining test material was noted in the left eye sac of all animals. As such the treated eye was rinsed with physiological saline solution at the 1 - hour observation point.

At 24 hours after application, conjunctival redness (score 1) and chemosis (score 1) were noted in one rabbit. No clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed in other animals. At 48 and 72 hours after the application, no clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed in any animals. During the experiment, the control eye of each animal was symptom-free. No mortality occurred during the study. The general state and behaviour of animals were normal throughout the study period. The bodyweights of all rabbits were considered to be within the normal range of variability. As no clinical signs were observed, the experiment was terminated after 72 hours observation of the second and third rabbits.

Test material, applied to rabbit eye mucosa, caused conjunctival effects at 1 hour and 24 hours after the treatment, which were fully reversible within a maximum of 48 hours. There were no effects on cornea and iris observed during the study.

Under the conditions of the study the test material was not classified according to EU criteria.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2018
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:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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.
- Storage, temperature and transport conditions of ocular tissue: Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature.
- Time interval prior to initiating testing: Heads were transported at the earliest convenience. The heads were received at Citoxlab Hungary Ltd. and processed within 2 hours of collection.
- Indication of any existing defects or lesions in ocular tissue samples: After 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).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: The test material was applied in its original form. 30 mg of the test material was applied onto the entire surface of the cornea.

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

Duration of treatment / exposure:
Ten seconds
Duration of post- treatment incubation (in vitro):
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.

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 physiological saline, three eyes with the test material and another three eyes with powdered Imidazole.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 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.
The eyeball 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 to 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 minute and the zero time. No changes in thickness (0.0 %) were observed in any eye. 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.

NEGATIVE CONTROL USED
The negative control eye was treated with 30 μL of physiological saline (Salsol solution, 0.9 % (w/v) NaCl) stored at room temperature.

POSITIVE CONTROL USED
Positive control eyes were treated with 30 mg powdered imidazole stored at room temperature.

SUBSIDIARY MATERIAL
Fluorescein Retention Test using fluorescein, 10 % (w/v) solution. This material was mixed with physiological saline solution to achieve the final concentration of 2 % (w/v). The final solution was stored at room temperature.

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. 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.

OBSERVATION AND ASSESSMENT OF CORNEAL EFFECTS
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ± 5 minutes were considered acceptable.
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 Bern 900 slit-lamp microscope was used for the measurements.

TEST MATERIAL REMOVAL
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with at least 3 x 20 mL saline was performed at each time point when the test material and positive control material remaining on the cornea was observed.

EVALUATION
Corneal swelling was calculated according to the following formulae:

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

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

Where:
CS = Cornea swelling
CT = Cornea thickness
FECS (at time t) = First eye cornea swelling at a given time-point
SECS (at time t) = Second eye cornea swelling at a given time-point
TECS (at time t) = Third eye cornea swelling at a given time-point

Small negative numbers for swelling (0 to -5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

Cornea opacity was calculated according to the following formulae:

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

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

Where:
CO at time t = Cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t = 0 = Baseline cornea opacity
ΔCO at time t = Difference between cornea opacity at t time and cornea opacity baseline
FECO = First eye cornea opacity
SECO = Second eye cornea opacity
TECO = Third eye cornea opacity
max (30min to 240min) = Maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

Fluorescein retention was calculated according to the following formulae:

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

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

Where:
FR at time t = Fluorescein retention at 30 minutes after the post-treatment rinse
FR at t = 0 = Baseline fluorescein retention
ΔFR at time t = Difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = First eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = Second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = Third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean values at up to 75 minutes and 240 minutes.
Value:
>= 4.3 - <= 5.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
0.83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

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

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

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments.

The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification.

Test Material

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

4.3 % 

I

Mean maximum corneal swelling at up to 240 min

5.9 % 

II

Mean maximum corneal opacity change

1.00 

II

Mean fluorescein retention change

0.83 

II

Other Observations 

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

Overall ICE Class 

3 x II

Positive Control

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

12.3 % 

III

Mean maximum corneal swelling at up to 240 min

27.3 % 

III

Mean maximum corneal opacity change

4.00 

IV

Mean fluorescein retention change

3.00 

IV

Other Observations

 

Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post - treatment rinse.

Overall ICE Class 

1 x III 2 x IV

The positive control imidazole was classified as severely irritating, UN GHS Classification: Category 1.

Negative Control

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0 %

I

Mean maximum corneal swelling at up to 240 min

0.0 %

I

Mean maximum corneal opacity change

0.00

I

Mean fluorescein retention change

0.00

I

Other Observations 

None

Overall ICE Class 

3 x I 

The negative control physiological saline was classified as non-irritating, UN GHS Classification: No Category.

Historical Control Data (Updated on 22 May 2019)

Negative Control: Physiological Saline

Observation 

Min. Value 

Max. Value 

Maximum corneal swelling at up to 75 min 

-3.2 % 

3.4 % 

Maximum corneal swelling at up to 240 min 

-4.8 % 

3.4 % 

Maximum corneal opacity change 

0.00 

0.50 

Fluorescein retention change 

0.00 

0.50 

Number of studies 

462

 

Positive Control: Imidazole

Observation 

Min. Value 

Max. Value 

Maximum corneal swelling at up to 75 min 

-6.6 % 

25.0 % 

Maximum corneal swelling at up to 240 min 

-15.9 % 

36.7 % 

Maximum corneal opacity change 

3.50 

4.00 

Fluorescein retention change 

2.00 

3.00 

Number of studies 

209

 

Summary Table for UN GHS Classification

Criteria for ‘No Category’ (All True)

 

3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:

False

 

No severe corneal morphological changes:

True

Test material was not stuck to the cornea at 240 minutes after the post-treatment rinse:

False

 

 

Criteria for ‘Category 1’ (One or More True)

 

2 or more endpoints classed as IV:

False

Corneal opacity = 3 at 30 min (in at least 2 eyes):

False

Corneal opacity = 4 at any time point (in at least 2 eyes):

False

Severe loosening of epithelium (in at least 1 eye):

False

Criteria for ‘No Prediction Can Be Made’ (One or Two True)

 

Based on the endpoints not classifiable for No Category, or for Category 1:

True

Particles of test material were stuck to the cornea and could not be washed off during the study:

True

 

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of the study the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.
Executive summary:

The in vitro eye irritation of the test material was assessed according to OECD Guideline 438 and in compliance with GLP using isolated chicken eyes.

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 exposure time, 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). 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 this experiment. Thus, the study was considered to be valid.

Slight cornea swelling (mean swelling 5.9 %) was observed during the four-hour observation period on test material treated eyes. Slight cornea opacity change (severity 1) was observed on any eyes. Slight fluorescein retention change (severity 1 on two eyes and severity 0.5 on one eye) was observed. Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

No severe corneal morphological changes were observed.

Under the conditions of the study 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.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

> Skin Irritation

Read-across to structurally similar substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8)

The skin irritation potential of the test material was investigated in vitro in a study which was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions. The study was conducted on the read-across substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8). The only difference between the substance being registered and the read-across substance is that the substance to be registered is missing the gadolinium oxide content (about 1.4 %). The absence of this compound at such a low % is considered highly unlikely to affect its properties.

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. 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 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 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 86.4 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

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

> Eye Irritation

Appl (2020)

The in vivo eye irritation potential of the test material was assessed in rabbits according to OECD Test Guideline 405 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Three rabbits were treated with the test material. Prior to test material application, the animals were treated with analgesic and anaesthetic as per the regulatory guideline. The test material was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 g of the test material was administered to the left eye of each animal. The eyes were examined at 1, 24, 48 and 72 hours after application.

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed. At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the three rabbits. Remaining test material was noted in the left eye sac of all animals. As such the treated eye was rinsed with physiological saline solution at the 1 - hour observation point.

At 24 hours after application, conjunctival redness (score 1) and chemosis (score 1) were noted in one rabbit. No clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed in other animals. At 48 and 72 hours after the application, no clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed in any animals. During the experiment, the control eye of each animal was symptom-free. No mortality occurred during the study. The general state and behaviour of animals were normal throughout the study period. The bodyweights of all rabbits were considered to be within the normal range of variability. As no clinical signs were observed, the experiment was terminated after 72 hours observation of the second and third rabbits.

Test material, applied to rabbit eye mucosa, caused conjunctival effects at 1 hour and 24 hours after the treatment, which were fully reversible within a maximum of 48 hours. There were no effects on cornea and iris observed during the study.

Under the conditions of the study the test material was not classified according to EU criteria.

Orovecz (2019)

The in vitro eye irritation of the test material was assessed according to OECD Guideline 438 and in compliance with GLP using isolated chicken eyes. 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 exposure time, 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). 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 this experiment. Thus, the study was considered to be valid.

Slight cornea swelling (mean swelling 5.9 %) was observed during the four-hour observation period on test material treated eyes. Slight cornea opacity change (severity 1) was observed on any eyes. Slight fluorescein retention change (severity 1 on two eyes and severity 0.5 on one eye) was observed. Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

No severe corneal morphological changes were observed.

Under the conditions of the study 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.

Justification for classification or non-classification

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