Registration Dossier

Administrative data

Description of key information

Skin irritation / corrosion: Harlan (2013): Not irritating

Eye irritation: Harlan (2013, in vivo): Not irritating

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:
4 September 2012 - 15 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: in vitro Reconstructed Human Epidermis (RHE) Model
Details on test animals or test system and environmental conditions:
The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes 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 a 13-Day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN Model Kit
Supplier: SkinEthic Laboratories, Nice, France
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro concurrent positive and negative controls were used
Amount / concentration applied:
10 mg
Duration of treatment / exposure:
A treatment period of 15 minutes was followed by a post-exposure incubation period of 42 hours.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
MATERIAL PREPARATION
-Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control.
-Sodium Dodecyl Sulphate (SDS) 5 % w/v aqueous dilution was used as the positive control.
-A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
-A 0.04 N concentration of hydrochloric acid in isopropanol was prepared when required.

PRE-TEST
Assessment of Direct Test Material Reduction of MTT

MTT dye metabolism, cell viability assay:
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
The test material was checked for the ability to directly reduce MTT according to the following procedure:
10 mg of the test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5 % CO₂ in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test material turns blue, it is presumed to have reduced the MTT.
The test material was shown not to directly reduce MTT.

PRE-INCUBATION (Day 0: Tissue Arrival):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37 °C, 5 % CO₂ in air overnight.

MAIN TEST
APPLICATION OF TEST MATERIAL AND RINSING (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test material for an exposure period of 15 minutes. It was applied topically to the corresponding tissues, ensuring uniform covering. Approximately 10 mg of the test material was applied to the epidermis surface (which had been previously moistened with 5 µL of sterile distilled water to improve the contact between the solid test material and the epidermis). Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5 % w/v served as the positive controls. To ensure satisfactory contact with the positive control material, the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7 minutes of contact time, the SDS solution was re-spread with a pipette tip to maintain the distribution for the remainder of the contact period. The plate(s) were kept in a biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test material. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5 % CO₂ in air for 42 hours.

MTT LOADING/FORMAZAN EXTRACTION (Day 3)
Following the 42 hour post-exposure incubation period, each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination.

2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5 % CO₂ in air. At the end of the 3 hour incubation period, each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (Day 6)
At the end of the formazan extraction period, each tube was mixed thoroughly on a vortex mixer to produce an homogenous coloured solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

INTERPRETATION OF RESULTS
-Quantitative MTT Assessment (percentage tissue viability)
For the test material, the relative mean tissue viabilities obtained after the 15 minute exposure period followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD540 of test material / mean OD540 of negative control) x 100

Classification of irritation potential is based upon relative mean tissue viability according to the following:

Criteria for in vitro interpretation Classification
Relative Mean Tissue Viability is ≤50 % Irritant (I) R38
Relative Mean Tissue Viability is >50 % Non-Irritant (NI)

The results were evaluated according to EU labelling regulations Commission Directive 2001/59/EC for classification and labelling of dangerous items.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

-Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40 % relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18 %.

-Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ≥0.6, and the standard deviation value of the percentage viability is ≤18 %.

-Test Material:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18 %.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test material (mean)
Value:
116.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control (mean)
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control (mean)
Value:
7.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative and positive controls are given in Table 1.
The relative mean viability of the test material treated tissues was 116.5 % after a 15 minute exposure period.

Direct MTT Reduction

An assessment found that the test material was not able to directly reduce MTT.

Quality Criteria

- The relative mean tissue viability for the positive control treated tissues was 7.8 % relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.3 %. The positive control acceptance criterion was therefore satisfied.

- The mean OD540 for the negative control treated tissues was 0.753 and the standard deviation value of the percentage viability was 4.8 %. The negative control acceptance criterion was therefore satisfied.

- The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 13.1 %. The test material acceptance criterion was therefore satisfied.

 

Table 1: Mean OD540 Values and Percentage Viabilities for the Negative and Positive Controls and the Test Material

Material

OD540 of Tissues

Mean OD540 of Triplicate Tissues

± SD of OD540

Relative Individual Tissue Viability (%)

Relative Mean Viability (%)

± SD of Relative Mean Viability (%)

Negative Control

0.773

 

0.753

 

0.036

102.7

 

100*

 

4.8

0.711

94.4

0.774

102.8

Positive Control

0.070

 

0.058

 

0.010

9.3

 

7.8

 

1.3

0.054

7.2

0.051

6.8

Test

Material

0.977

 

0.877

 

0.099

129.7

 

116.5

 

13.1

0.875

116.2

0.779

103.5

*The mean viability of the control group is set at 100 %

SD = Standard deviation

Interpretation of results:
other: not classified according to EU criteria
Conclusions:
Under the conditions of this study, the test material was considered to be a non-irritant and requires no classification in accordance with EU criteria.
Executive summary:

The skin irritation potential of the test material was evaluated in vitro using the EPISKIN reconstructed human epidermis model in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period, each tissue was rinsed before being incubated for 42 hours, after which each tissue was taken for MTT loading. After MTT loading, a total biopsy of each epidermis was made and formazan crystals were extracted out of the MTT-loaded tissues. Duplicate tissues treated with Dulbecco’s Phosphate Buffered Saline with Ca++ and Mg++ served as the negative control and duplicate tissues treated with 5 % w/v aqueous Sodium Dodecyl Sulphate served as the positive control. The optical density of all treated tissues was measured at 540 nm.

The relative mean viability of the test material treated tissues was 116.5 % after the 15 minute exposure period.

Under the conditions of this study, the test material was considered to be a non-irritant and requires no classification in accordance with EU criteria.

Endpoint:
skin irritation: in vivo
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Section 1.4 of Annex XI of REACH, the in vivo skin irritation study (as required in Section 8.1.1 of Annex VIII) is not required as this endpoint has been adequately addressed with in vitro study data.
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:
11 February 2013 - 21 February 2013
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
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: 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
- Strain: New Zealand White
- Age at study initiation: 12 - 20 weeks
- Weight at study initiation: 2.42 or 2.44 kg
- Housing: The animals were individually housed in suspended cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): Free access to mains drinking water.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): At least 15 per hour.
- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give twelve hours continuous light (0600 to 1800) and twelve hours darkness.

IN-LIFE DATES: From: 11 February 2013 To: 21 February 2013
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye of each animal remained untreated and was used for control purposes.
Amount / concentration applied:
100 mg
Duration of treatment / exposure:
The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material, and then released.
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
2. Initially, one animal was treated; after consideration of the ocular response, a second animal was treated.
Details on study design:
Immediately after administration of the test material, an assessment of the initial pain reaction was made.

SCORING SYSTEM: Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the Draize numerical evaluation.
Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period. Any clinical signs of toxicity, if present, were also recorded.

Draize Scale for Scoring Ocular Irritation

1. CONJUNCTIVAE

Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris):
Vessels normal 0
Vessels definitely injected above normal 1
More diffuse, deeper crimson red, individual vessels not easily discernible 2
Diffuse beefy red 3

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

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

2. IRIS

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

3. CORNEA

Degree of Opacity (most dense area used):
No opacity 0
Scattered or diffuse areas, details of iris clearly visible 1
Easily discernible translucent areas, details of iris slightly obscured 2
Opalescent areas, no details of iris visible, size of pupil barely discernible 3
Opaque, iris not discernible through the opacity 4

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


TOOL USED TO ASSESS SCORE: Any additional ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.5
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
other: discharge score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Irritant / corrosive response data:
Individual scores are given in Table 2.
No corneal effects were noted during the study. Iridial inflammation (Grade 1) was noted in both treated eyes one hour after treatment.
Moderate conjunctival irritation (Grade 2 redness, Grade 2 chemosis and Grade 2 discharge) was noted in both treated eyes one hour after treatment. Minimal conjunctival irritation (Grade 1 redness and Grade 1 chemosis) was noted in both treated eyes at the 24 hour observation. Minimal conjunctival irritation (Grade 1 redness in both treated eyes and Grade 1 chemosis in one treated eye) was noted in both treated eyes at the 48 hour observation.
Both treated eyes appeared normal at the 72 hour observation and the study was therefore terminated.
Other effects:
BODYWEIGHT
Both animals showed expected gain in body weight during the study.

Table 2: Individual Scores for Ocular Irritation

Rabbit Number and Sex

72956 Male

72987 Male

IPR = 2

IPR = 2

Time After Treatment

1 hour

24 hours

48 hours

72 hours

1 hour

24 hours

48 hours

72 hours

Cornea

Degree

Area

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

Iris

1

0

0

0

1

0

0

0

Redness

2

1

1

0

2

1

1

0

Chemosis

2

1

0

0

2

1

1

0

Discharge

2

0

0

0

2

0

0

0

IPR = Initial pain reaction; a score of 2 indicates slight initial pain. The rabbit blinks and tries to open the eye but reflex closes it.

Interpretation of results:
other: not classified according to EU criteria
Conclusions:
Under the conditions of this study, the test material requires no classification in accordance with EU criteria.
Executive summary:

The irritancy potential of the test material was assessed in vivo in accordance with the standardised guidelines OECD 405 and EU Method B.5.

A single application of 100 mg of the test material was sequentially administered to the right eye of two New Zealand White rabbits. The treated eyes were not irrigated; the left eye remained untreated and served as the control. The animals were observed for ocular effects 1, 24, 48 and 72 hours after administration of the test material.

The individual mean scores for corneal opacity and iritis were 0.0 for both animals. The individual mean scores for conjunctival redness were 0.7 for both animals. The individual mean scores for chemosis were 0.3 and 0.7. Both treated eyes appeared normal at the 72 hour observation and the study was therefore terminated.

Under the conditions of this study, the test material requires no classification in accordance with EU criteria.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 October 2012 - 18 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of the study was to determine the eye irritation potential of the test material using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of MTT to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
GLP compliance:
yes (incl. QA statement)
Species:
other: in vitro Reconstructed Human Corneal Epithelium (HCE) Model
Details on test animals or tissues and environmental conditions:
The SkinEthic HCE model consists of transformed human corneal epithelial cells of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum free and chemically defined medium.

SKINETHIC HCE Model (0.5 cm²)
Supplier: SkinEthic Laboratories, Lyon, France

RECEIPT OF TISSUES
On arrival, the SkinEthic HCE tissues (Day 6 cultures) were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 300 µL of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37 °C, 5 % CO₂ in air.
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro concurrent positive and negative controls were used
Amount / concentration applied:
30 mg test item to 1 mL of 0.5 mg/mL MTT solution.
Duration of treatment / exposure:
A treatment period of 10 minutes was followed by a post-exposure incubation period of 3 hours.
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
MATERIAL PREPARATION
-Solution A (as supplied) was used as the negative control. The composition for 1 L was as follows: Na2HPO4 0.142 g/L; Glucose 1.802 g/L; HEPES 7.149 g/L; KCl 0.224 g/L and NaCl 7.597 g/L.
-Sodium Dodecyl Sulphate (SDS) 2 % w/v prepared in sterile water was used as the positive control.

PRE-TEST
Assessment of Direct Test Material Reduction of MTT:
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of MTT to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material present on or in the tissues. To identify this possible interference, the test material was checked for its ability to reduce MTT directly.
30 mg of the test material was added to 1 mL of a 0.5 mg/mL MTT solution and incubated at 37 °C, 5 % CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test material turned blue, it was presumed to have reduced the MTT.
The MTT solution containing the test material remained yellow which indicated that the test material did not directly reduce MTT.

PREPARATION OF TISSUES
Using sterile techniques, 1 mL of maintenance medium at room temperature was dispensed into the appropriate number of wells of 6-well plates designated ‘treatment plates’. Each well was labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test material, negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7 Day old tissues were transferred from the ‘arrival plates’ into the wells of the ‘treatment plates’ containing the maintenance medium.

MAIN TEST
Triplicate tissues were treated with 30 mg of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µL of solution A to serve as negative controls and triplicate tissues were treated with 30 µL of 2 % w/v SDS to serve as positive controls. The plates were incubated at 37 °C, 5 % CO2 in air during the exposure time.
At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µL of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37 °C, 5 % CO2 in air.
At the end of the incubation period, the inserts were rinsed twice with phosphate buffered saline and blotted on absorbent paper to remove residual MTT solution and transferred to a pre labelled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of isopropanol in each of a sufficient number of wells. An extra 0.75 mL of isopropanol was added onto each tissue and the plate sealed to prevent isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.
At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.

TISSUE HISTOLOGY
One tissue for each treatment group was retained for possible tissue histopathology.
The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre labelled 1.5 mL Eppendorf tube containing 1 mL of 10 % Formalin and stored at room temperature.

INTERPRETATION OF RESULTS
The mean OD540 values of the duplicate tissues were calculated. Each of these OD540 values had already been corrected for blanks by the microplate reader.
The relative mean tissue viability (percentage of the negative control) was calculated as follows:
Relative mean tissue viability (%) = (mean OD540 of the test material / mean OD540 of the negative control) x 100

The mean tissue viability for the test material was compared to the negative control and classified according to the following:

Relative Mean Tissue Viability Prediction
(%of negative control)

Tissue viability <60 Irritant (I)
Tissue viability ≥60 Non-Irritant (NI)

ASSAY ACCEPTANCE CRITERION
The results of the assay are considered acceptable if the following assay acceptance criterion was achieved:
- Positive Control: The assay meets the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is <60% relative to the negative control treated tissues.
Irritation parameter:
other: OD540
Run / experiment:
Test material (mean)
Value:
0.799
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: OD540
Run / experiment:
Negative control (mean)
Value:
0.859
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: OD540
Run / experiment:
Positive control (mean)
Value:
0.034
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Relative mean viability
Run / experiment:
Test material
Value:
93
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Relative mean viability
Run / experiment:
Negative control
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Relative mean viability
Run / experiment:
Positive control
Value:
4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The individual and mean OD540 values and relative mean tissue viabilities for the test material, negative and positive controls are given in Table 1.
The relative mean viability of the test material treated tissues after a 10 minute exposure was 93.0 %. The test material was therefore considered to be a non-irritant.
It was considered unnecessary to proceed with tissue histopathology. The quality criterion required for the acceptance of results in the test was satisfied.

Table 1: Assessment of Eye Irritation Potential - Viability of HCE Tissues

Material

OD540 of Individual Tissues

Mean OD540

Relative Mean Viability (%)

Negative Control

0.921

0.859

100*

0.796

Positive Control

0.038

0.034

4.0

0.029

Test Material

0.835

0.799

93.0

0.762

*The mean viability of the negative control is set at 100 %

Interpretation of results:
other: not classified according to EU criteria
Conclusions:
Under the conditions of this study, the test material was considered to be a Non-Irritant (NI) and therefore requires no classification in accordance with EU criteria.
Executive summary:

The eye irritation potential of the test material was investigated in vitro using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. The MTT assay, a colourimetric method of determining cell viability, is based on reduction of MTT to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

Triplicate SkinEthic tissues were treated with 30 mg of the test material for 10 minutes. Triplicate tissues treated with 30 µL of Solution A served as the negative control and triplicate tissues treated with 30 µL of 2 % w/v Sodium Dodecyl Sulphate (SDS) served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed and two tissues per group taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading, the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The relative mean viability of the test material treated tissues after a 10 minute exposure period was 93.0 %.

Under the conditions of this study, the test material was considered to be a Non-Irritant (NI) and therefore requires no classification in accordance with EU criteria.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion

The skin irritation potential of the test material was evaluated in vitro using the EPISKIN reconstructed human epidermis model in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997).

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period, each tissue was rinsed before being incubated for 42 hours, after which each tissue was taken for MTT loading. After MTT loading, a total biopsy of each epidermis was made and formazan crystals were extracted out of the MTT-loaded tissues. Duplicate tissues treated with Dulbecco’s Phosphate Buffered Saline with Ca++ and Mg++ served as the negative control and duplicate tissues treated with 5 % w/v aqueous Sodium Dodecyl Sulphate served as the positive control. The optical density of all treated tissues was measured at 540 nm.

The relative mean viability of the test material treated tissues was 116.5 % after the 15 minute exposure period.

Under the conditions of this study, the test material was considered to be a non-irritant and requires no classification in accordance with EU criteria.

Eye Irritation

An in vivo study was conducted accordance with the standardised guidelines OECD 405 and EU Method B.5, under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria detailed by Klimisch (1997).

A single application of 100 mg of the test material was sequentially administered to the right eye of two New Zealand White rabbits. The treated eyes were not irrigated; the left eye remained untreated and served as the control. The animals were observed for ocular effects 1, 24, 48 and 72 hours after administration of the test material.

The individual mean scores for corneal opacity and iritis were 0.0 for both animals. The individual mean scores for conjunctival redness were 0.7 for both animals. The individual mean scores for chemosis were 0.3 and 0.7. Both treated eyes appeared normal at the 72 hour observation and the study was therefore terminated.

Under the conditions of this study, the test material requires no classification in accordance with EU criteria.

An in vitro study was conducted using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. The MTT assay, a colourimetric method of determining cell viability, is based on reduction of MTT to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

Triplicate SkinEthic tissues were treated with 30 mg of the test material for 10 minutes. Triplicate tissues treated with 30 µL of Solution A served as the negative control and triplicate tissues treated with 30 µL of 2 % w/v Sodium Dodecyl Sulphate (SDS) served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed and two tissues per group taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading, the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The relative mean viability of the test material treated tissues after a 10 minute exposure period was 93.0 %.

Under the conditions of this study, the test material was considered to be a Non-Irritant (NI) and therefore requires no classification in accordance with EU criteria.

Justification for classification or non-classification

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