Registration Dossier

Administrative data

Description of key information

One in vitro skin irritation (OECD 439) and one in vitro skin corrosion studies (OECD 431) were performed on the test item.

In both cases , results were negative. The test item is neither irritant and nor corrosive in these both in vitro studies.

The OECD 437 (BCOP) was performed on the test item and the conclusion is that does not required classification to UN GHS or EU CLP.

The test item is neither irritating and corrosive for skin and eye.

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: 19 September 2018 and Experimental completion date: 24 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
updated 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Source species:
human
Cell type:
other: adult human-derived keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Cell source:
other: not spacified
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.

Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Supplier : EpiSkin Laboratories, Lyon, France
Date received : 18 September 2018
EpiSkinTM Tissues (0.38cm2) lot number : 18-EKIN-038
Maintenance Medium lot number : 18-MAIN3-046
Assay Medium lot number : 18-ESSC-041

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: Triplicate

Test for Direct MTT Reduction
The test item is checked for the ability to directly reduce MTT according to the following procedure:

10 mg 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
10 mg 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-labeled 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.. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal 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-labeled 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 labeled 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-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.


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 is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570for the negative control treated tissues is³0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
Test Item: 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:
10 mg (26.3 mg/cm2)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicate
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 replicates
Run / experiment:
mean of 3 replicates
Value:
94.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

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

94.4% after a 15‑Minute exposure period and 42‑Hour post‑exposure incubation period.

It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was7.8% relative to the negative control treated tissues and the standard deviation value of the viability was6.2%. The positive control acceptance criteria were therefore satisfied.

The mean OD570for the negative control treated tissues was0.745and the standard deviation value of the viability was5.0%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was2.7%. The test item acceptance criterion was therefore satisfied.

Mean OD570Values 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

0.760

0.745

0.037

102.0

100*

5.0

0.773

103.8

0.703

94.4

Positive Control Item

0.111

0.058

0.046

14.9

7.8

6.2

0.029

3.9

0.033

4.4

Test Item

0.725

0.703

0.020

97.3

94.4

2.7

0.698

93.7

0.685

91.9

OD=Optical Densit

SD=       Standard deviatio

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

Interpretation of results:
GHS criteria not met
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 can not be determined).
Executive summary:

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.

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-labeled 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-labeled 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).

The relative mean viability of the test item treated tissues was 94.4% 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.

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 can not be determined).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 30 April 2018 and Experimental Completion Date: 31 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006, 18 December 2006, of the European Parliament and of the Council on the REACH.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
other: EpiDerm™ Reconstructed Human Epidermis Model
Source species:
human
Cell type:
other: Epithelial, derived from human skin and fromed into a stratified, cornified epithelium
Cell source:
other: not available
Source strain:
not specified
Details on animal used as source of test system:
Not applicable
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)
Remarks:
used as supplied
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received :11 September 2018
EpiDermTM Tissues (0.63cm2) lot number : 28652
Assay Medium lot number : 090618MSA
Upon receipt of the EpidermTM 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:
- Observable damage in the tissue due to washing:


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/L MTT solution
- Incubation time: 3 hours
- Wavelength: 570 nm
- Labtech LT-4500 microplate reader and LT-com analysis software.


NUMBER OF REPLICATE TISSUES: duplicate for test item, duplicate for positive and duplicate for negative control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: No MTT interference.



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

Negative Control
- The absolute OD570 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 OD570 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
- Potassium Hydroxide 8.0N solution is used as a 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%.

DECISION CRITERIA:
Viability Measured after Exposure Time Points Prediction to be considered according to
EU CLP Regulation (EC) No 1272/2008 UN GHS


STEP 1
< 50% after 3 min exposure Corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure Corrosive
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure Non-corrosive

STEP 2 for test items identified as corrosive in step 1
< 25% after 3 min exposure H314
Sub-category 1A

≥ 25% after 3 min exposure H314
Combination of sub-categories 1B-and-1C

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg
Duration of treatment / exposure:
60 minutes
3 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
duplicate for test item
duplicate for negative control
duplicate for positive control
Irritation / corrosion parameter:
other: relative mean viability
Run / experiment:
exposure period 3 minutes
Value:
108.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non corrosive to the skin
Irritation / corrosion parameter:
other: relative mean viability
Run / experiment:
exposure period 60 minutes
Value:
103.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive to the skin
Other effects / acceptance of results:
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 did not become colored.
This was taken to indicate the test item did not have the potential to cause color interference.


Test Item, Positive Control Item and Negative Control Item.

Exposure Period Percentage Viability
Negative Control Positive Control Test Item
3 minute 100* 4.4 108.8
60 minute 100* 3.4 103.2
*The mean viability of the negative control tissues is set at 100%



ACCEPTANCE OF RESULTS:
- The mean OD570 for the negative control treated tissues was 2.273 for the 3 Minute exposure period and 2.214 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 0.075% 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.

Calcul:

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

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

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

4.4

108.8

60 minute

100*

3.4

103.2

Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

MeanOD570of individual tissues

Mean OD570of duplicate tissues

Standard Deviation

Coefficient of Variation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

2.059

2.273

0.302

13.3

100*

2.486

60 Minutes

2.364

2.214

0.212

9.6

2.064

Positive Control

3 Minutes

0.098

0.099

0.001

na

4.4

0.099

60 Minutes

0.077

0.075

0.003

na

3.4

0.073

Test Item

3 Minutes

2.602

2.473

0.182

7.4

108.8

2.344

60 Minutes

2.283

2.285

0.002

0.1

103.2

2.286

 

OD=Optical density

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

na=        Not applicable

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

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


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

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.

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 of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 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).

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

         Percentage viability
 Exposure period  Negative control Positive control  Test item 
 3 minute  100*  4.4  108.8
  60 minute  100* 3.4  103.2 

*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.

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 September 2018 and Experimental completion date: 02 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
09 October 2017
Deviations:
yes
Remarks:
these deviations were considered to have not affected the integrity or validity of the study (see below)
GLP compliance:
yes (incl. certificate)
Species:
other: Eyes from adult cattle (typically 12 to 60 months old)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Obtained from a local abattoir as a by-product from freshky slaughtered animals.
- Number of animals: not applicable
- Characteristics of donor animals (e.g. age, sex, weight): Eyes from adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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 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: penicillin at 100 IU/mL and streptomycin at 100 µg/mL
Vehicle:
other: Sodium Chloride 0.9% w/v.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
For the purpose of this study the test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.
Duration of treatment / exposure:
240 minutes at 32°C.
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
90 minutes at 32°C.
Number of animals or in vitro replicates:
- Test item: triplicate
- Negative control: triplicate
-
Positive control: triplicate
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.
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.


NUMBER OF REPLICATES : negative control, positive control and test item were all preformed in triplicate

NEGATIVE CONTROL USED : The negative control item, sodium chloride 0.9% w/v, was used as supplied.

SOLVENT CONTROL USED (if applicable) : negative control and solvent control (vehicle) are the same

POSITIVE CONTROL USED: The positive control item, Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v.

APPLICATION DOSE AND EXPOSURE TIME : 0.75 Ml of test item preparation was applied (test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v).

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: yes, 90 minutes at 32°C (application of Sodium Fluorescein)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Cornea were rinsed 3 times with fresh complete EMEM contaning phenol red before a final rinse with complete EMEM without phenol red.
A post-treatment opacity reading was taken and each cornea was visually observed.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control) 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: 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.
The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

- Others : In Vitro Irritancy Score (IVIS): The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Visual Observations: The condition of the cornea was visually assessed post treatment.

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS UN GHS
≤ 3: No Category
>3; ≤ 55: No prediction can be made
> 55: Category 1


DECISION CRITERIA: Criteria for an Acceptable test:

For an acceptable test the following positive control criterion should be achieved:
- 20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2017 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 71.2 to 132.9.

For an acceptable test the following negative control criteria should be achieved:
- Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.44


Irritation parameter:
in vitro irritation score
Run / experiment:
test item
Value:
1.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No category. Nor requiring classification to UN GHS or EU CLP.
Irritation parameter:
in vitro irritation score
Run / experiment:
negative control
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
positive control
Value:
88.8
Vehicle controls validity:
valid
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. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.3 and permeability ≤0.44. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.





Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD492)

In VitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

5

4

0

 

0.000

 

 

2

4

3

0

 

0.000

 

 

3

4

4

0

 

0.000

 

 

 

 

 

0.0*

 

0.000¨

 

0.0

Positive
Control

4

4

74

70

70.0

1.007

1.007

 

5

4

68

64

64.0

1.495

1.495

 

6

2

79

77

77.0

1.191

1.191

 

 

 

 

 

70.3·

 

1.231·

88.8

Test Item

7

5

6

1

1.0

0.043

0.043

 

9

4

5

1

1.0

0.016

0.016

 

10

4

6

2

2.0

0.027

0.027

 

 

 

 

 

1.3·

 

0.029·

1.8


OD= Optical density         * = Mean of the post-treatment -pre‑treatment values          ¨= Mean permeability                           ·= Mean corrected value

Corneal Epithelium Condition Post Treatment

Treatment

Cornea Number

Observation
Post Treatment

Negative Control

1

Clear

2

Clear

3

Clear

Positive Control

4

Cloudy

5

Cloudy

6

Cloudy

Test Item

7

Clear

9

Clear

10

Clear

 

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 corneain 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 test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 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 anIn VitroIrritancy Score (IVIS). 

Data Interpretation

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

IVIS

UN GHS

≤ 3

No Category

>3; ≤ 55

No prediction can be made

> 55

Category 1

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score (IVIS)

Test Item

1.8

Negative Control

0.0

Positive Control

88.8

 

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 is neither irritating and corrosive for skin and eye and does not required classification to UN GHS or EU CLP.