Registration Dossier

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Administrative data

Description of key information

Skin corrosion in vitro: Negative

Skin irritation in vitro: Negative

Eye irritation in vitro: Negative

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:
02 September 2016 to 03 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 bis
Version / remarks:
2880
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Tissues (0.63 cm2)
- Supplier: MatTek
- Tissue batch number(s): Lot no.: 23352
- Storage conditions of tissues: The sealed 24 well plate was stored in a refrigerator until use

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: 2

TEST FOR DIRECT MTT REDUCTION BY THE TEST MATERIAL
A test material may interfere with the MTT endpoint if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT. 50 μL of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
The test material may interfere with the MTT endpoint if it is coloured; the MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 50 μL of test material was added to 300 μL of sterile water, the solution was incubated in the dark at 37 °C, 5% CO2 for 60 minutes. A visual assessment of the colour was then made.

MAIN TEST
-Pre-Incubation
0.9 mL of pre-warmed assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3- and 60-minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.

- Application of Test Material and Rinsing
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3- and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required. After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-minute exposure period was returned to the incubator, while the other was being dosed for the 60-minute exposure. For the 60-minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 μL of the test material and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5 % CO2) for the 60-minute exposure period.
When dosing for the 60-minute exposure period was complete, the same procedure was repeated for the 3-minute exposure period. Asthe exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. The plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60-minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

-Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

-Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3 and 60-minute exposure periods, compared to the mean of the negative control tissues (n = 2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test material / mean OD562 of negative control) x 100

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
-Negative Control
The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
-Positive Control
An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is <15 %.
-Coefficient of Variation
In the range 20 and 100 % viability, the Coefficient of Variation between tissue replicates should be ≤ 30 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
Duration of treatment / exposure:
3 minutes and 60 minutes of exposure
Duration of post-treatment incubation (if applicable):
3 hours with MTT
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure time
Value:
97.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure time
Value:
99.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The MTT solution containing the test material did not become coloured. This was taken to indicate the test material did not reduce MTT.
The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

QUALITY CRITERIA
The mean OD562 for the negative control treated tissues was 1.727 for the 3-minute exposure period and 1.603 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 4.8 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

Table 1: Relative mean viabilities for each treatment group

Exposure Period

Percentage viability

Negative control

Positive control

Test material

3 minutes

100*

5.1

97.7

60 minutes

100*

4.8

99.3

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

Interpretation of results:
other: Not corrosive in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material was determined to be non-corrosive.
Executive summary:

The corrosivity potential of the test material was determined in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis using the EpiDerm™ Human Skin Model under GLP conditions.

Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes and negative and positive control groups were also treated for each exposure period. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period, each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

The percentage viability of the skin after exposure to the test material was 97.7 and 99.3 % after 3 and 6 minute exposures, respectively.

The quality criteria required for acceptance of results in the test were satisfied and the test was therefore considered to be valid.

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September 2016 to 03 October 2016
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)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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™ Reconstructed Human Epidermis Model Kit (EPISKIN™, 0.38 cm²)
- Tissue lot number: 16-EKIN-039
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 of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Delivery date: 27 September 2016
- Source: SkinEthic Laboratories, Lyon, France

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

NUMBER OF REPLICATE TISSUES: 3

TEST FOR DIRECT MTT REDUCTION
The test material may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test material is checked for the ability to directly reduce MTT according to the following procedure: 10 µL 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 or purple it is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 10 µL of test material was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.

PRE-INCUBATION
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert; the tissues were satisfactory, the temperature indicator colour was satisfactory and the agar medium colour was satisfactory.
On day 0, 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 STUDY
On 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. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm2) of the test material was applied to the epidermis surface. Triplicate tissues treated with 10 µL of Dulbecco’s Phosphate Buffered Saline (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 the Sodium Dodecyl Sulphate (SDS) solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After a 7 minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
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
On 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 plates. 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
At the end of the formazan extraction period on day 6, 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 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

DECISION CRITERIA (choose relevant statement)
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 OD562 of test material / mean OD562 of negative control) x 100
Classification of irritation potential is based upon relative mean tissue viability following the 15 minute exposure period followed by the 42 hour post exposure incubation period according to the following:
- Relative mean tissue viability is ≤ 50 %: Irritant
- Relative mean tissue viability is > 50 %: Non-irritant

QUALITY CRITERIA
-The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤ 40 % relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤ 18 %.
-The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18 %.
-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 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 µL (26.3 µL/cm2)

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % w/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
95.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
The solution containing the test material was a very pale green colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.

MAIN STUDY
The individual and mean OD562 values, standard deviations and tissue viabilities for the test material, negative control and positive control are shown in Table 1.
The relative mean viability of the test material treated tissues was 95.9 % after a 15 minute exposure period and 42 hour post-exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

QUALITY CRITERIA
-The relative mean tissue viability for the positive control treated tissues was 6.8 % relative to the negative control treated tissues and the standard deviation value of the viability was 3.5 %. The positive control acceptance criteria were therefore satisfied.
-The mean OD562 for the negative control treated tissues was 1.062 and the standard deviation value of the viability was 8.8 %. The negative control acceptance criteria were therefore satisfied.
-The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 12.8 %. The test material acceptance criterion was therefore satisfied.

Table 1: Mean OD562 Values and Viabilities for the Negative Control, Positive Control and Test Material

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control

0.985

1.062

0.094

92.7

100*

8.8

1.166

109.8

1.034

97.4

Positive Control

0.114

0.072

0.037

10.7

6.8

3.5

0.057

5.4

0.045

4.2

Test Material

1.084

1.018

0.136

102.1

95.9

12.8

0.861

81.1

1.108

104.3

OD = Optical density

SD = Standard deviation

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


Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material was determined to be a non-irritant.
Executive summary:

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

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test material by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. 

Interference checks were performed before the main study. The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.  The solution prepared containing the test material was a very pale green colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.

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 incubating for 42 hours. At the end of the post exposure incubation period each tissue was taken for MTT-loading. Following this a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density was measured at 562 nm.

The relative mean viability of the test material treated tissues was 95.9 % after the 15 minute exposure period and 42 hour post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of the study the test material was classified as non-irritant. 

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:
06 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: Cattle used were typically 12 to 60 months old.
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported to the test facility over ice packs in supplemented Hanks’ Balanced Salt Solution (HBSS).
- Time interval prior to initiating testing: Eyes were transported to the test facility on the same day of slaughter. The corneas were prepared immediately on arrival.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: Yes; eyes were placed in HBSS supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes after the treatment with the test material
90 minutes with Sodium fluorecein
Number of animals or in vitro replicates:
3 replicates for each condition (test material, negative control and positive control)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

TREATMENT METHOD
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.
At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The corneal opacity readings were taken using a calibrated opacitometer. The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: Following the final opacity measurement, medium was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes to measure permeability. After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader. The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

ACCEPTABILITY OF THE ASSAY
For an acceptable test the following control criteria should be achieved:
-The test was acceptable if the positive control produced an IVIS which fell within two standard deviations of the historical mean collated during 2014 for the testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 52.0.
-The test was acceptable if the negative control produced an IVIS which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤ 2.9 and for permeability ≤ 0.103.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
A summary of the results can be seen in Table 1. The corneas treated with the test material and negative control material were clear post treatment and post incubation. The corneas treated with the positive control material were cloudy post treatment and post incubation

ACCEPTANCE OF RESULTS
The positive control IVIS score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.9 and permeability ≤0.103. The negative control acceptance criteria were therefore satisfied.

Table 1: Summary of opacity, permeability and in vitro irritancy scores

Treatment

Mean Opacity

Mean Permeability

Mean In Vitro Irritancy Score

Negative control

0.3

0.008

0.5

Positive control

32.3

1.206

50.4

Test material

0.2

0.021

0.5

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material had an IVIS score of 0.5 and was therefore determined to require no classification.
Executive summary:

The potential of the test material to cause serious eye damage was determined in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) assay.

In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability. The undiluted test material was applied to the corneas for 10 minutes followed by an incubation period of 120 minutes. Negative and positive controls, sodium chloride and ethanol respectively, were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS scores for the test, negative and positive control materials were 0.5, 0.5 and 50.4, respectively. The corneas treated with the test material and negative control material were clear post treatment and post incubation. The corneas treated with the positive control material were cloudy post treatment and post incubation

The positive and negative controls met the acceptability criteria of the study and the so the test is considered to be valid.

Under the conditions of this study, the test material had an IVIS score of 0.5 and was therefore determined to require no classification.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosion

The corrosivity potential of the test material was determined in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis using the EpiDerm™ Human Skin Model under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes and negative and positive control groups were also treated for each exposure period. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period, each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

The percentage viability of the skin after exposure to the test material was 97.7 and 99.3 % after 3 and 6 minute exposures, respectively.

The quality criteria required for acceptance of results in the test were satisfied and the test was therefore considered to be valid.

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

Skin Irritation

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

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test material by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. 

Interference checks were performed before the main study. The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.  The solution prepared containing the test material was a very pale green colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.

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 incubating for 42 hours. At the end of the post exposure incubation period each tissue was taken for MTT-loading. Following this a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density was measured at 562 nm.

The relative mean viability of the test material treated tissues was 95.9 % after the 15 minute exposure period and 42 hour post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of the study the test material was classified as non-irritant. 

Eye Irritation

- Eye Damage (ex vivo)

In the key study, the potential of the test material to cause serious eye damage was determined in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using the Bovine Corneal Opacity and Permeability (BCOP) assay. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability. The undiluted test material was applied to the corneas for 10 minutes followed by an incubation period of 120 minutes. Negative and positive controls, sodium chloride and ethanol respectively, were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS scores for the test, negative and positive control materials were 0.5, 0.5 and 50.4, respectively. The corneas treated with the test material and negative control material were clear post treatment and post incubation. The corneas treated with the positive control material were cloudy post treatment and post incubation

The positive and negative controls met the acceptability criteria of the study and the so the test is considered to be valid.

Under the conditions of this study, the test material had an IVIS score of 0.5 and was therefore determined to require no classification.

- Eye irritation (in vivo)

In the first supporting study, the potential of the test material to cause eye irritation was investigated using methodology similar to that outlined in the standardised guideline OECD 405. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Three albino rabbits were treated with 0.1 mL of test material at a concentration of 5.0 % in ‘Carnation’ mineral oil. The test material was instilled into the conjunctival sacs of the test animals. The test material was allowed to fall on the everted lower lid of each rabbit, the upper and lower lids were then gently held together for one second before releasing to prevent the loss of the test material. The eyes were observed after 1 hour and every 24 hours over a 7 day period. A 2 % instillation of fluorescein was made on each animal at least once during the course of the experimental period. Macroscopic readings were facilitated with microscopic readings by slit lamp examinations.

All three animals received a score of 1 for hyperaemia at the 24 hour observation point. This had fully resolved by the following day in all three animals.

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

In the second supporting study, the potential of the test material to cause eye irritation was investigated using methodology similar to that outlined in the standardised guideline OECD 405. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Three albino rabbits were treated with 0.1 mL of test material at a concentration of 2.0 % in ‘Carnation’ mineral oil. The test material was instilled into the conjunctival sacs of the test animals. The test material was allowed to fall on the everted lower lid of each rabbit, the upper and lower lids were then gently held together for one second before releasing to prevent the loss of the test material. The eyes were observed after 1 hour and every 24 hours over a 7 day period. A 2 % instillation of fluorescein was made on each animal at least once during the course of the experimental period. Macroscopic readings were facilitated with microscopic readings by slit lamp examinations.

All three animals received a score of 1 for hyperaemia at the 1 hour observation points. This had fully resolved by the 24 hour examination in all three animals.

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

Justification for classification or non-classification

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