Registration Dossier

Administrative data

Description of key information

Skin Corrosion (in vitro)

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

Skin Irritation (in vitro)

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Eye Irritation (in vitro/ex vivo)

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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:
09 May 2017 to 08 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines
Vehicle:
water
Remarks:
Deionised
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 Kit
- Lot No.: 25817
- Delivery date: 06 June 2017.
- Date of initiation of testing: On day of receipt

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
A test material may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test material is present in the tissues when the MTT viability test is performed. Some non-coloured test materials may change into coloured test materials in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility should be performed (step 1).
- Step 1: 25 ± 2 mg of the test material was added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated (37 ± 1.5°C, 5 ± 0.5% CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test material is presumed to have the potential to stain the tissue. An additional test on viable tissues (without MTT addition) should be performed (step 2).
Since the test material did not dye water relevantly (yellow) when mixed with it, step 2 did not have to be performed.
- Step 3: All test materials (including those already evaluated in step 1 and step 2 should be further evaluated for their potential to interfere with MTT. To test if a material directly reduces MTT, 25 ± 2 mg of the test material was added to 1 mL of a MTT/DMEM solution (1 mg/mL) and was incubated (37 ± 1.5°C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test material reduces MTT and an additional test on freeze-killed tissues (step 4) must be performed.
Since the test material did not prove to be a MTT reducer, step 4 did not have to be performed.

PRE-WARMING OF EPIDERM TISSUES
18 to 19 hours before dosing, EpiDerm tissues were unpacked, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 μL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5°C, 5 ± 0.5% CO2) until use.

TREATMENT
Duplicate EpiDerm tissues were treated with the test material, positive control or negative control for exposure times of 3 ± 0.5 minutes and 60 ± 5 minutes. After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5°C, 5 ± 0.5% CO2).

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT ASSAY
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 μL) was added to each well and the plates were kept in an incubator (37 ± 1.5°C, 5 ± 0.5% CO2) until required. Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5°C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for nearly 20 hours without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour. 3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

NUMBER OF REPLICATE TISSUES: 2
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 ± 2 mg (39.7 mg/cm²) of test material, applied with 25 μL of deionised water

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
Duration of treatment / exposure:
3 ± 0.5 minutes and 60 ± 5 minutes
Duration of post-treatment incubation (if applicable):
3 hours with MTT
Number of replicates:
Duplicate plates were treated for the test material, positive control and negative control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
97
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
104.6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
MTT INTERFERENCE POTENTIAL
- The optical pre-experiment (colour interference pre-experiment) to investigate the test material’s colour change potential in water did not lead to a relevant change in colour.
- Optical evaluation of the MTT-reducing capacity of the test material after 1 hour incubation with MTT-reagent did not show blue colour.

MAIN TEST
The test material is considered to be non-corrosive to skin since the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater than 15%. Results can be seen in Table 1.

ACCEPTABILITY OF THE TEST
The acceptance criteria are met:
- the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.455 to 1.575)
- the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (3.2%)
- the Coefficient of Variation (CV) in the range 20 to 100% viability between tissue replicates is ≤ 30% (range: 0.2 to 3.5%)

Table 1: Results of main study after treatment with test material and positive control

Treatment

3 minute relative absorbance

(% of negative control)

60 minute relative absorbance

(% of negative control)

Test material

97.0

104.6

Positive control

22.6

3.2

Negative control

100.0

100.0

Interpretation of results:
other: Not corrosive in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.
Executive summary:

The corrosivity potential of the test material to skin was investigated in accordance with the standardised guidelines OECD 431 and EU Method B40 under GLP conditions, by means of the Human Skin Model Test with an EpiDerm™ tissue model.

The test material did not reduce MTT (pre-test for direct MTT reduction), and it did not dye deionised water significantly (yellow), when mixed it (pre-test for colour interference). Therefore, additional tests with freeze-killed or viable tissues (without MTT addition) to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to the test material, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. Afterwards, the test and the control materials were rinsed off the tissues, and a 3 hour incubation period (37 ± 1°C, 5 ± 0.5% CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for nearly 20 hours at room temperature.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (22.6%) and for the 1 hour exposure period (3.2%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test material the relative absorbance value decreased slightly to 97.0% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was not reduced (104.6%). Both values did not fall below the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 2017 to 28 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
other: UN GHS
Version / remarks:
published 2003, last (6th) revision 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ Kit, SkinEthic Laboratories (69007 Lyon, France)
- Tissue lot number(s): 17-EKIN-030
- The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
- Delivery date: 25 July 2017

TEST FOR MTT REDUCTION AND COLOUR INTERFERENCE
- Prior to the start of the test, the test material’s colour interference potential had to be evaluated. For this purpose the test material (10 ± 2 mg) was mixed with 90 µL of deionised water in a pre-experiment. The test material/water mixture was gently shaken for 15 minutes at room temperature. The colour of the test material/water mixture did not change during the incubation period compared with the colour of the pure test material. Therefore, the measurement of the OD of the test material in water at 570 nm was not required and consequently not performed.
- For correct interpretation of results it is necessary to assess the ability of the test material to directly reduce MTT. To test for this ability 10 ± 2 mg of the test material was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5°C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. If the MTT solution colour turns blue/purple, the test material is presumed to have reduced the MTT. If the test material has shown its ability to reduce MTT, it is necessary to perform a functional test with killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues.
Since the colour did not turn blue/purple, the test material was not considered to be a MTT reducer.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ± 1.5°C

NUMBER OF REPLICATE TISSUES: 3

TREATMENT
- Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 23 hours.
- The test material tissues were wetted with 5 µL of deionised water. The negative control, positive control and the test material were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 hours at 37 ± 1.5°C, 5 ± 0.5% CO2.
If the results obtained by means of the MTT assay are unclear or borderline, additionally the IL-1 α concentration in the medium after 42 hours incubation can be determined by order of the Sponsor. For this purpose, samples of all treatment groups were taken from the wells (It was not necessary to perform the immunoassay, therefore the taken samples were discarded after report finalisation).

MTT ASSAY
- A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5°C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for 2 hours and 35 min at room temperature with gentle agitation.
- Per tissue sample 2 x 200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax®) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

DATA EVALUATION
- The mean OD of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test material or the positive control the individual relative tissue viability is calculated according to the following formula: Relative viability (%) = (meanOD test material or positive control / meanOD negative control) x 100
- For the test material and the positive control, the mean relative viability ± rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
- For the current test, an irritation potential leading to H315 classification of EU according to regulation (EC) 1272/2008, and GHS category 2 according to UN GHS (published 2003, last (6th) revision 2015) is recommended if the mean relative tissue viability of three individual tissues is reduced to < 50 % of the negative control.

ACCEPTABILITY OF THE ASSAY
- The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 to ≤ 1.5.
- The rel. standard deviations between tissues of the same treatment group should be ≤ 18%.
- An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 ± 2 mg
- 5 µL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis.

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5%
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
(Mean Rel. Absorbance %)
Run / experiment:
mean
Value:
95.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- The optical pre-experiment (colour interference pre-experiment) to investigate the test material’s colour change potential in water did not lead to a change in colour. Optical evaluation of the MTT-reducing capacity of the test material after 3 hour incubation with MTT-reagent did not show blue colour.
- The mean relative absorbance value of the test material, corresponding to the cell viability, decreased to 95.6% (threshold for irritancy: ≤ 50%), consequently the test material was not irritant to skin.

ACCEPTANCE OF RESULTS
The acceptance criteria were met:
- the mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 0.659 to 0.694).
- the rel. standard deviations between tissues of the same treatment group was ≤ 18% (range: 2.8 to 11.9%).
- the mean relative tissue viability of the positive control was ≤ 40% (6.9%).
- the acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (2.1 mg/mL).
- the results for the positive and negative controls are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH

Table 1: Summary of results

Test group

Mean Absorbance of 3 Tissues

Relative Standard Deviation [%]

Mean Rel. Absorbance

[%]

Negative control

0.672

2.8

100.0

Positive control

0.047

11.9

6.9

Test material

0.642

9.3

95.6

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is a non-irritant in the in vitro skin irritation test.
Executive summary:

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

This in vitro study was performed to assess the irritation potential of Yttrium acetate by means of the Human Skin Model Test.

The test material did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test material, the negative control (PBS) or the positive control (5% sodium lauryl sulphate) for 15 minutes. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD0.6 to ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test material the mean relative absorbance value decreased to 95.6 %. This value is above the threshold for irritancy of ≤ 50 %. Therefore, the test material is not considered to possess an irritant potential.

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Characteristics of donor animals (e.g. age, sex, weight): at least 9 months old donor cattle
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: The corneae were directly used in the BCOP test on the same day.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularisation, pigmentation, opacity and scratches were discarded.
Vehicle:
other: Saline (0.9% NaCl in deionised water)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20 % w/v

VEHICLE
- Amount(s) applied: 0.75 mL
- Concentration (if solution): 0.9 %
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1°C for permeability determination.
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
- All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularisation, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
- Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
- For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1°C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test material and for the negative and positive controls, respectively.

NUMBER OF REPLICATES: 3

VEHICLE CONTROL USED: 0.9% NaCl (saline)

POSITIVE CONTROL USED: 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline)

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of 20% test material suspension in saline for 240 minutes.

TREATMENT METHOD:
- The anterior compartment received the test material suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1°C in the water-bath. The incubation time lasted 240 minutes.

REMOVAL OF TEST MATERIAL
- The test material or the control materials, respectively, were each rinsed off from the according application sides with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240) using an opacitometer [OP_KiT opacitometer (Electro Design, 63-Riom, France)].

POST-EXPOSURE INCUBATION:
- Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1°C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate.
- The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
- Opacity: The change of the opacity value of each treated cornea or of the positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea. The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: The corrected OD490 value of each cornea treated with positive control or test material is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
- IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test material:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test material is classified into the following Category according to OECD guideline 437:
IVIS ≤ 3: No category
IVIS > 3 to ≤ 55: No prediction can be made
IVIS > 55: Category 1

VALIDITY OF THE TEST
The test will be acceptable if:
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
1.09
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
- For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.18).
- The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.30) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
- The test material was tested as suspension. Relative to the negative control, the test material did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.09 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test material is not categorised.

Table 1: Summary of results after 240 minutes treatment.

Test group

Mean IVIS

Proposed Classification

Negative control

1.18

Not categorised

Positive control

98.30

Category 1

Test material

1.09

Not categorised

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

The potential of the test material to cause eye irritation or damage was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions, with a BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test material, the positive and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1°C. After the incubation phase, the test material as well as the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1°C.

For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.18). The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.30) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test material did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.09 (threshold for serious eye damage: IVIS > 55).

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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

Additional information

Skin Corrosion (in vitro)

The corrosivity potential of the test material to skin was investigated in accordance with the standardised guidelines OECD 431 and EU Method B40 under GLP conditions, by means of the Human Skin Model Test with an EpiDerm™ tissue model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material did not reduce MTT (pre-test for direct MTT reduction), and it did not dye deionised water significantly (yellow), when mixed it (pre-test for colour interference). Therefore, additional tests with freeze-killed or viable tissues (without MTT addition) to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to the test material, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. Afterwards, the test and the control materials were rinsed off the tissues, and a 3 hour incubation period (37 ± 1°C, 5 ± 0.5% CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for nearly 20 hours at room temperature.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (22.6%) and for the 1 hour exposure period (3.2%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test material the relative absorbance value decreased slightly to 97.0% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was not reduced (104.6%). Both values did not fall below the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Skin Irritation (in vitro)

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

This in vitro study was performed to assess the irritation potential of Yttrium acetate by means of the Human Skin Model Test.

The test material did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test material, the negative control (PBS) or the positive control (5% sodium lauryl sulphate) for 15 minutes. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD0.6 tO ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test material the mean relative absorbance value decreased to 95.6 %. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test material is not considered to possess an irritant potential.

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Eye Irritation (in vitro/ex vivo)

The potential of the test material to cause eye irritation or damage was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions, with a BCOP assay using fresh bovine corneae. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test material, the positive and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1°C. After the incubation phase, the test material as well as the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1°C.

For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.18). The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.30) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test material did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 1.09 (threshold for serious eye damage: IVIS > 55).

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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 or corrosion.