Registration Dossier

Administrative data

Description of key information

The test item was classified as non-irritant. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 cannot be determined).

The test item was considered to be non-corrosive to the skin.

Eye irritiation- No category. Not requiring classification to UN GHS or EU CLP.

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:
Experimental start date 18 October 2017. Experimental completion date 30 October 2017
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:
yes
Remarks:
Due to a technical error in the initial run it was necessary to repeat the test in one further run. This deviation was considered to have not affected the integrity or validity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Due to a technical error in the initial run it was necessary to repeat the test in one further run. This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
Batch: 52611021
Purity: 95 – 100%
Physical state/Appearance: Clear yellow liquid
Expiry Date: 01 December 2018
Storage Conditions: Room temperature in the dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 24 October 2017
EpiSkinTM Tissues (0.38cm2) lot number: 17-EKIN-043
Maintenance Medium lot number: 17-MAIN3-045
Assay Medium lot number: 17-ESSC-042
Vehicle:
unchanged (no vehicle)
Details on test system:
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric 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/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item 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 by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item 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 item is checked for the ability to directly reduce MTT according to the following procedure:
10 μL of the test item 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 item turns blue/purple, the test item 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 Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
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 item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item 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 item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. 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 item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). 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 item). The plates were kept in the 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 item. 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 homogenize 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 ºC 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 EPISKINTM 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 a homogenous colored 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 570 nm (OD570) (without a reference filter) using the Labtech LT 4500 microplate reader and LT-com analysis software.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. The test item was used as supplied.
10 μL of DPBS served as the negative controls. The negative control item, DPBS, was used as supplied.
10 μL of SDS 5% w/v served as the positive controls. The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution and was used within 2 hours of preparation.
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:
93.3
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 item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 93.3% 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 10.5% relative to the negative control treated tissues and the standard deviation value of the viability was 7.8%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.819 and the standard deviation value of the viability was 1.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 8.2%. The test item acceptance criterion was therefore satisfied.

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD570of tissues

Mean OD570of triplicate tissues

±SDof OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.831

0.819

0.012

101.5

100*

1.5

0.820

100.1

0.807

98.5

Positive Control Item

0.040

0.086

0.064

4.9

10.5

7.8

0.058

7.1

0.159

19.4

Test Item

0.842

0.764

0.068

102.8

93.3

8.2

0.732

89.4

0.719

87.8

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:
other: EU CLP Not classified for Irritation.
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 cannot be determined).
Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM 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 item 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/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item 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. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading 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 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 93.3% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 cannot be determined).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 20 September 2017 Experimental completion date 22 September 2017
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008,
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
Batch: 52611021
Purity: 95 – 100%
Physical state/Appearance: Clear yellow liquid
Expiry Date: 12 January 2018
Storage Conditions: Room temperature in the dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 19 September 2017
EpiDermTM Tissues (0.63cm2) lot number : 25842
Assay Medium lot number : 091417ALA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.
Justification for test system used:
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Vehicle:
unchanged (no vehicle)
Details on test system:
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colorimetric 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 item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item 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 by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item 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 item was checked for the ability to directly reduce MTT according to the procedure below:
50 μL of the test item 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 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turns blue/purple relative to the control, the test item was presumed to have reduced the MTT.

Assessment of Color Interference with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 50 μL of test item was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then
made.

Main Test
Pre-Incubation
The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3-Minute 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 Item and Rinsing
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure 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 item 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. Because the 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. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item. 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. 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 570nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μL of the test item. The test item was used as supplied.
50 μL of sterile distilled water. The negative control item was used as supplied.
50 μL of 8.0 N Potassium Hydroxide. The positive control item was used as supplied.
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
92.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
108
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 item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item was a pale yellow color. This color was not considered capable of causing color interference.

Quality Criteria
The mean OD570 for the negative control treated tissues was 1.504 for the 3-Minute exposure period and 1.437 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 3.6% 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.

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue
Exposure
Period
Mean OD570 of
individual
tissues
Mean OD570 of
duplicate
tissues
Standard
Deviation
Coefficient of
Variation
(%)
Relative
Mean
Viability
(%)
Negative
Control
3 minutes 1.584 1.504 0.113 7.5 100*
1.424
60 minutes 1.389 1.437 0.067 4.7
1.484
Positive
Control
3 minutes 0.065 0.064 0.002 na 4.3
0.62
60 minutes 0.052 0.052 0.001 na 3.6
0.051
Test Item 3 minutes 1.42 1.389 0.045 3.2 92.4
1.357
60 minutes 1.39 1.552 0.228 14.7 108.8
1.713

Relative mean % tissue viability =(mean OD570 of test item/mean OD570 of negative control) x 100

Coefficient of variation =(standard deviation/ mean OD570 of duplicate tissues) x 100

OD = Optical density

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

na = Not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item 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 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated

tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period Percentage Viability
Negative Control Positive Control Test Item
3 minute 100* 4.3 92.4
60 minute 100* 3.6 108

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

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive 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:
Experimental start date: 20 October 2017. Experimental completion date: 20 October 2017.
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)
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
Batch: 52611021
Purity: 95-100%
Physical state/Appearance: clear yellow liquid
Expiry Date: 12 January 2018
Storage Conditions: room temperature in the dark
Species:
cattle
Details on test animals or tissues and environmental conditions:
Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the appropriate corneas.
For the purpose of this study the test item was used as supplied.
The negative control item, sodium chloride 0.9% w/v, was used as supplied.
The positive control item, ethanol, was used as supplied.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
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 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
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.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items 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 ºC 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.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber 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 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labelled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
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.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

6

5

6

0

 

0.003

 

 

2

4

6

7

3

 

0.012

 

 

3

7

7

5

0

 

0.024

 

 

 

 

 

 

1.0*

 

0.013~

 

1.2

Positive Control

4

3

34

33

30

29.0

0.816

0.803

 

5

3

37

32

29

28.0

0.920

0.907

 

6

6

38

31

25

24.0

0.312

0.299

 

 

 

 

 

 

27.0#

 

0.670#

37.0

Test Item

7

3

3

2

-1

0.0

0.032

0.019

 

8

4

9

4

0

0.0

0.049

0.036

 

9

6

9

7

1

0.0

0.045

0.032

 

 

 

 

 

 

0.0#

 

0.029#

0.4

OD= Optical density           * = Mean of the post-incubation -pre‑treatment values          ~= Mean permeability                     #= Mean corrected value

Interpretation of results:
GHS criteria not met
Conclusions:
No category. Not requiring classification to UN GHS or EU CLP.
Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items 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).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS

Classification

≤ 3

No category. Not requiring classification to UN GHS or EU CLP

> 3; ≤55

No prediction of eye irritation can be made

> 55

Category 1. UN GHS or EU CLP Causes serious eye damage

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

0.4

Negative Control

1.2

Positive Control

37.0

Conclusion

No category. Not requiring classification to UN GHS or EU CLP.

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

Additional information

Justification for classification or non-classification

The test item EC 500-215-4 is not classified for Skin or Eye irritation.

The test item is also not classified for Skin corrosion.

All studies conducted were concluded as non-hazardous.