Registration Dossier

Administrative data

Description of key information

SKIN
Not irritating, in vitro skin irritation test in the EPISKIN Model, OECD 439, EU Method B.46, Hargitai (2015)
EYE
Not irritating, in vitro eye irritation test in isolated chicken eyes, OECD 438, EU Method B.48, Gönczöl (2015)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 March 2015 to 15 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Reference:
Composition 1
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: EPISKIN MODEL
Strain:
other: Reconstructed Human Epidermis
Details on test animals and environmental conditions:
TEST SYSTEM:
HUMAN SKIN
- EPISKIN-SM is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al. 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
- For killed epidermis, living epidermis units were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5 % CO₂, in a >95 % humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 28 November 2014. Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.

QUALITY CONTROL
- 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).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
- The EPISKIN-SM model has been validated for irritation testing in an international validation study [9] 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.

KIT CONTENTS
- Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm₂) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plate
- Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis
- Medium: Flask of sterile “Maintenance Medium”
- Flask of sterile “Assay Medium”

NUMBER OF REPLICATE WELLS
- In this assay, three replicates were used for the test material. Three negative controls and three positive controls were also run in the assay. As the test material was coloured, one additional test material-treated tissue was used for the non-specific OD evaluation. Furthermore, as the test material might have an MTT interacting potential, three additional test material-treated killed epidermis and three negative control treated killed epidermis were used in the study.

KIT RECEPTION
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- Orange colour = good
- Yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C):
- White colour = good
- Grey or black colour = not acceptable
The kits were found to be in good order at reception.

STORAGE
- The EPISKIN-SM kit was kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2 to 8 °C until the initiation of the test.
Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
other: Positive and negative controls were included in the experiment
Amount / concentration applied:
TEST MATERIAL
- No formulation was required; the test material was applied as supplied.
- As the test material was solid, 10 μL distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis, then 20 mg of test material were applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a curved flat spatula (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

CONTROLS
Positive and negative controls were included in the experiment. Furthermore, as the test material was coloured, one additional control tissue sample was used in the experiment for determination of the non-specific colour. Additional controls on killed epidermis (three test material treated and three negative control treated skin units) were also used in the study to determine the MTT interacting potential of the test material.
- Negative Control: Phosphate Buffered Saline (PBS), (outsourced)
- Positive Control: 5 % (w/v) Sodium Dodecyl Sulphate solution (SDS), prepared freshly in laboratory
- 50 μL of positive control (5 % (w/v) SDS solution) or negative control (PBS) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
Duration of treatment / exposure:
The disks were treated with test material and incubated for 15 minutes; the test material was then rinsed. 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.
Details on study design:
TEST SITE
- Area of exposure: Disks of EPISKIN (three units)

SCORING SYSTEM
- Optical Density (OD) measured, reported as percentage relative viability.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS)
- Time after start of exposure: The disks were treated with test material and incubated for 15 minutes; the test material was then rinsed with Phosphate Buffer Saline (PBS).

PERFORMANCE OF THE STUDY:
PRE-INCUBATION (DAY -1)
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 37 °C in an incubator with 5 % CO₂, in a >95 % humidified atmosphere.

APPLICATION AND RINSING (DAY 0)
- Test Material
As the test material was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis, then 20 mg of test material were applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a curved flat spatula (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
- Positive and negative control
50 μL of positive control (5 % (w/v) SDS solution) or negative control (PBS) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (24.4 to 25.5 °C).
The test material was showed to be an MTT-interacting substance, in addition to the normal procedure, three test material treated killed epidermis and three negative control treated killed epidermis was used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed in case of the killed tissues as in case of the living tissues.
After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37 °C in an incubator with 5 % CO₂.

MTT TEST (DAY 2)
After the 42 hours incubation, all EPISKIN-SM units (except of one colour control unit) 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 (DAY 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit).
The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

CELL VIABILITY MEASUREMENTS (DAY 2)
Following the formazan extraction, 2 × 200 μL 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 were measured using a plate reader at 570 ±30 nm and 690 nm (the test material has a violet colour, but this wavelength was considered to be suitable for this purpose based on the observed spectral results of the MTT checking test). The mean of 6 wells of acidified isopropanol solution (200 μL / well) was used as blank.
Irritation / corrosion parameter:
other:
Value:
73
Remarks on result:
other:
Remarks:
Basis: mean. Time point: Assessed after 42 hour incubation period. (migrated information)
Irritant / corrosive response data:
As the test material was coloured, one additional test material-treated tissue was used for the non-specific OD evaluation. The optical density (measured at 570 nm) of this tissue was 0.011, Non Specific Colour % was calculated as 1.6 %. This value was below 5 %, therefore additional data calculation was not necessary.
As colour change (blue) was observed after three hours of incubation of the test material in MTT working solution, thus the test material might interact with MTT (this fact could not be properly determined due to the interfering colour of the test material). Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on these observed (negative) OD value, the calculated NSMTT was considered to be insignificant, thus additional data calculation was not necessary.
The OD values for the test material treated skin samples showed 73.0 % relative viability.

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

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative Control: Phosphate buffered saline

1

0.713

0.668

93.8

2

0.795

0.749

105.2

3

0.765

0.720

101.1

mean

 

0.712

100.0

Postive Control: 5 % (w/v) SDS solution

1

0.077

0.032

4.5

2

0.092

0.046

6.5

3

0.093

0.047

6.6

mean

 -

0.042

5.9

Test Material

1

0.443

0.398

55.8

2

0.604

0.559

78.5

3

0.650

0.604

84.8

mean

0.520

73.0

Notes:

1. Mean blank value was 0.045.

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

3. Based on the absorbance values measured at 690 nm, no significant absorbance due to possible test material remaining on the Episkin units could be detected compared to the negative control samples (the test material appeared to stain permanently the epidermis and matrix, but this fact did not interfere with the formazan extraction and absorbance reading, and was considered not to adversely affect the results or intergrity of the study).
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Following exposure with the test material, the mean cell viability was 73.0 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.
Executive summary:

The in vitro irritation test of the test material was investigated in accordance with the standardised guidelines OECD Guideline 439 (In Vitro Skin Irritation) and EU Method B.46 (In Vitro Skin Irritation: reconstructed human epidermis model test) under GLP conditions. The study was awarded a reliability score of 1 in accordance with the principles for assessing data quality set forth by Klimisch et. al. (1997).

Disks of EPISKIN (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. PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). An additional disk was used to provide an estimate of colour contribution from the test material. Following the exposure with the test material, the mean cell viability was compared to the negative control.

Under the conditions study, the test material was determined to be not irritating to the skin.

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:
16 February 2015 to 07 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: Chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to laboratory at earliest convenience.
- After collection, 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). Heads were processed within approximately 2 hours of collection.

SELECTION AND PREPARATION OF EYES FOR THE TEST

EYES SELECTION
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline.
The fluorescein- treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head; to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

PREPARATION OF THE EYES
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EYES EXAMINATION AND ACCLIMATISATION TIME
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, 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.

IDENTIFICATION
The eyes were identified by chamber number, marked on the door of the chamber.

BASE LINE ASSESSMENTS
At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye.
The cornea thickness of the eyes should not change by more than 5 % between the -45 min and the zero time. No changes in thickness (0.0 %) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Vehicle:
physiological saline
Controls:
other: Positive and Negative control eyes
Amount / concentration applied:
TEST MATERIAL
- The test material was applied in powdered form in an amount of 30 mg
Duration of treatment / exposure:
- Exposure period: 10 seconds
Observation period (in vivo):
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.
Number of animals or in vitro replicates:
Three test material-treated eyes, three positive control-treated eyes and one negative control-treated eye were examined.
Details on study design:
REMOVAL OF TEST SUBSTANCE
The time of application was observed, 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 at ambient temperature, taking care not to damage the cornea but attempting to remove as much of the residual the test material as possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the test material or control material remaining on the cornea was observed.

SCORING SYSTEM
ICE Classification System

TOOL USED TO ASSESS SCORE
Haag-Streit Bern 900 slit-lamp microscope
Irritation parameter:
other: Corneal swelling (value as a percentage)
Basis:
mean
Time point:
other: at up to 75 mins
Score:
0.5
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Corneal swelling (value as a percentage)
Basis:
mean
Time point:
other: at up to 240 mins
Score:
0.5
Remarks on result:
other: ICE Class I
Irritation parameter:
other: Corneal opacity (value as a percentage)
Basis:
mean
Score:
1
Remarks on result:
other: ICE Class II
Irritation parameter:
other: Fluorescein retention (value as a percentage)
Basis:
mean
Score:
1
Remarks on result:
other: ICE Class II
Irritant / corrosive response data:
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. Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Table 1: Eye Irritation Results of the Test Material

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.5%

I

Mean maximum corneal swelling at up to 240 min

0.5%

I

Mean maximum corneal opacity

1.00%

II

Mean fluorescein retention

1.00%

II

Other Observations

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

Overall ICE Class

1 x I 2 x II

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant.
Executive summary:

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

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

No significant corneal swelling was observed during the four hour observation period.

Corneal opacity change (severity 1) and fluorescein retention change (severity 0.5 or 1 or 1.5) was noted on all test material treated eyes; particles of test material were stuck to the cornea and could not be washed off during the study. The effects were clearly greater that a negative effect, but not sufficient to classify as severe. The particles stuck to the cornea could potentially result in mechanical damage in vivo.

Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN

An in vitro skin irritation test of the test material was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test material was evaluated according to the OECD No. 439 guideline and in under GLP conditions. The study was awarded a reliability score of 1 in accordance with the principles for assessing data quality set forth by Klimisch et al. (1997).

Disks of EPISKIN (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). An additional disk was used to provide an estimate of colour contribution from the test material. Furthermore, three additional test material treated and three negative control treated killed epidermis units were used to determine the MTT interacting potential of the test material. For each treated tissue, the viability was expressed as a percentage 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 73.0 % 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 model test, the results indicate that the test material is non-irritant to skin.

EYE

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

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

No significant corneal swelling was observed during the four hour observation period. Corneal opacity change (severity 1) and fluorescein retention change (severity 0.5 or 1 or 1.5) was noted on all test material treated eyes; particles of test material were stuck to the cornea and could not be washed off during the study. The effects were clearly greater that a negative effect, but not sufficient to classify as severe. The particles stuck to the cornea could potentially result in mechanical damage in vivo.

Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant.


Justification for selection of skin irritation / corrosion endpoint:
Only one study available.

Justification for selection of eye irritation endpoint:
Only one study available.

Justification for classification or non-classification

SKIN

Based on the in vitro skin irritation test in the EPISKIN Model, and in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material is non-irritant.

EYE

Based on the in vitro eye irritation in isolated chicken eyes test, and in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material is not classified.