[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Endpoint summary

Description of key information

In vitro skin corrosion: Reconstructed human epidermis (RHE) testing was carried out according to OECD guideline 431 and EU Method B.40 (In vitro skin corrosion: Transcutaneous electrical resistance test). The test item was considered to be non-corrosive to the skin. 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.

The skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model was tested according to OECD Guideline 439 (In vitro skin irritation: Reconstructed Human Epidermis test method) and EU Method B.46. 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).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation / corrosion, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

THis study was conducted between 05 October 2016 and 07 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

EC No. 440/2008 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: epithelial, derived from human skin, and formed into a stratified, cornified epithelium

Cell source:

other: Not specified as study used an EpiDerm™ Reconstructed Human Epidermis Model Kit

Source strain:

not specified

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recognised in vitro test for corrosivity

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN PREPARATION
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 04 October 2016
EpiDermTM Tissues (0.63cm2) lot number : 23361
Assay Medium lot number : 091916TMA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:37”C
- Temperature of post-treatment incubation (if applicable): Room temperature
REMOVAL OF TEST MATERIAL AND CONTROLS
- 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 item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: None
- Filter bandwidth: Not reported

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
- Concentration (if solution): as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Sterile distilled water
- Concentration (if solution): not applicable
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0N

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 Minute Exposure

Value:

ca. 77

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive to skin

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 Minute Exposure

Value:

ca. 25.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive to skin

Other effects / acceptance of results:

Quality Criteria
The mean OD562 for the negative control treated tissues was 1.623 for the 3 Minute exposure period and 1.750 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 5.7% 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.

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

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

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 Minute	100*	5.7	77.0
60 Minute	100*	5.7	25.8

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

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corosive 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.

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 L samples were transferred to the appropriate wells of a pre-labeled 96 well plate.  The optical density (OD) was measured at 562 nm (OD562).

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*	5.7	77.0
60 Minute	100*	5.7	25.8

*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.

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 02 November 2016 and 07 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: (μ(5-amino-1,3,3-trimethylcyclohexylamine-N,N')hexafluorodiboron
Batch: AEF0009100
Purity: Not supplied
Physical state/Appearance: Clear colorless liquid
Expiry Date: 29 August 2017
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

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.
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.

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 01 November 2016
EpiSkinTM Tissues (0.38cm2) lot number : 16-EKIN-044
Maintenance Medium lot number : 16-MAIN3-074
Assay Medium lot number : 16-ESSC-047

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL,
- Concentration (if solution): as supplied:

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): as a 5% w/v aqueous solution

Duration of treatment / exposure:

15 Minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Details on study design:

Study Design
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 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 or 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 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. 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 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 was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Data Evaluation
Quantitative MTT Assessment (Percentage Tissue Viability)
For the test item 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 item / 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 toTable 1 below.

Irritation / corrosion parameter:

other: other: relative mean viability

Run / experiment:

15 Minute exposure/42h observation

Value:

> 88.3 - < 115.5

Negative controls validity:

valid

Remarks:

Set to 100%

Positive controls validity:

valid

Remarks:

11.1

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 individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given below.  The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given below.

The relative mean viability of the test item treated tissues was 106.1% after a 15‑Minute exposure period and 42‑Hour post‑exposure incubation period.It was considered unnecessary to perform IL-1alpha- analysis as the results of the MTT test were unequivocal.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 11.1% relative to the negative control treated tissues and the standard deviation value of the viability was 2.7%.  The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.819 and the standard deviation value of the viability was 5.7%.  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 15.4%.  The test item acceptance criterion was therefore satisfied.

Table: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562of tissues	Mean OD562of triplicate tissues	±SDof OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.796	0.819	0.046	97.2	100*	5.7
	0.788			96.2
	0.872			106.5
Positive Control Item	0.116	0.091	0.022	14.2	11.1	2.7
	0.082			10.0
	0.076			9.3
Test Item	0.723	0.869	0.127	88.3	106.1	15.4
	0.946			115.5
	0.938			114.5

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 cannot be determined).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed 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 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‑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 562 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 106.1% 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 can not be determined).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted on 03 November 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:

26 July 20013

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:

EC NO. 440/2008

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: local abattoir as a by-product from freshly slaughtered animals
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): Adult cattle - 12 to 60 monthe old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes 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
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival at the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: none reported.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item as supplied or control items were applied to the appropriate corneas

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3

Details on study design:

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 65 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 with opacity values close to the median value of all corneas were 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 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.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled 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

Value:

ca. 45.7

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:

DEVIATIONS FROM STUDY PLAN
The following deviation from the Study Plan occurred:

The positive control group had an overall IVIS of 53.8. This was marginally higher than the criteria range set for an acceptable test. However, as the score was only marginally exceeded, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant.
This deviation was considered to have not affected the integrity or validity of the study.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was above the range of 29.6 to 52.0. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.
The negative control gave opacity of ≤2.9 and permeability ≤0.103. The negative control acceptance criteria were therefore satisfied.

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given below.

Corneal Epithelium Condition

The corneas treated with the test item were cloudy 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.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized below:

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	3	3	3	0		0.017
	2	3	3	3	0		0.012
	3	3	3	4	1		0.025
					0.3*		0.018**		0.6
Positive Control	4	3	37	38	35	34.7	1.150	1.132
	5	2	35	35	33	32.7	1.302	1.284
	6	2	34	35	33	32.7	1.699	1.681
						33.3 ***		1.366***	53.8
Test Item	7	1	50	23	22	21.7	0.349	0.331
	8	5	73	54	49	48.7	0.695	0.677
	9	0	69	43	43	42.7	0.620	0.602
						37.7***		0.537***	45.7

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 prediction of eye irritation can be made

Executive summary:

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	45.7
Negative Control	0.6
Positive Control	53.8

Conclusion

No prediction of eye irritation can be made.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 08 August 2017 an d27 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 405 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

02 October 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: (μ(5-amino-1,3,3-trimethylcyclohexylamine-N,N')hexafluorodiboron
Batch: AEF0009100
Purity: 100% UVCB
Physical state/Appearance: clear colorless liquid
Expiry Date: 29 August 2017
Storage Conditions: room temperature in the dark

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or tissues and environmental conditions:

Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 3.67 to 4.05 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.1 mL of undiluted test material

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

3

Details on study design:

Study Design
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% proxymetacaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Table 1.
Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later.
After consideration of the ocular responses produced in the first treated animal, two additional animals were similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977) given in Table 2.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Additional observations were made on Days 7 and 14 to assess the reversibility of the ocular effects.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Data Evaluation
Interpretation According to Kay and Calandra Classification System
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae = (A + B + C) x 2
Score for iris = D x 5
Score for cornea = (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C. (1962) (Table 3) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.
If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.
The results were also interpreted according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The results were also interpreted according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 1 h

Score:

24

Max. score:

0

Reversibility:

not specified

Remarks on result:

probability of moderate irritation

Irritant / corrosive response data:

Ocular Reactions
Individual and group mean scores for ocular irritation are given in Tables 4 and 5
Scattered or diffuse corneal opacity was noted in two treated eyes one and 24 hours after treatment and persisted in one treated eye at the 48, 72-Hour and 7-Day observations.
Iridial inflammation was noted in all treated eyes one and 24 hours after treatment and persisted in one treated eye at the 48 and 72-Hour observations.
Moderate conjunctival irritation was noted in all treated eyes one and 24 hours after treatment. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation in one treated eye at the 48-Hour observation. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation in two treated eyes at the 72-Hour observation. Minimal conjunctival irritation was noted in two treated eyes at the 7-Day observation.
An area of petechial hemorrhage, approximately 5 mm x 5 mm, on the nictitating membrane was noted in one treated eye at the 72-Hour observation.
One treated eye appeared normal at the 7-Day observation and the remaining two treated eyes appeared normal at the 14-Day observation.

Body Weight
All animals showed expected gain in body weight during the study.

Table 4    Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	75746Female						75770Female					75778Female
	IPR= 0						IPR =+					IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	14 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	14 Days
CORNEA
E = Degree of Opacity	1	1	0	0	0	0	0	0	0	0	0	1	1	1	1	1	0
F = Area of Cornea Involved	2	1	0	0	0	0	0	0	0	0	0	1	1	2	2	2	0
Score (E x F) x 5	10	5	0	0	0	0	0	0	0	0	0	5	5	10	10	10	0
IRIS
D	1	1	0	0	0	0	1	1	0	0	0	1	1	1	1	0	0
Score (D x 5)	5	5	0	0	0	0	5	5	0	0	0	5	5	5	5	0	0
CONJUNCTIVAE
A = Redness	2	2	2	2	1	0	2	1	1	1	0	2	2	2	2	1	0
B = Chemosis	3	2	2	1	1	0	2	1	1	1	0	2	2	2	1	1	0
C = Discharge	2	2	1	0	0	0	3	2	1	1Pt	0	3	2	1	1	1	0
Score (A + B + C) x 2	14	12	10	6	4	0	14	8	6	6	0	14	12	10	8	6	0
Total Score	29	22	10	6	4	0	19	13	6	6	0	24	22	25	23	16	0

IPR=Initial pain reaction

Pt =       Area of petechial hemorrhage, approximately 5 mm x 5 mm, on nictitating membrane

+ =        Not recorded due to technician error

Table 5   Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	14 Days
75746Female	29	22	10	6	4	0
75770Female	19	13	6	6	0	-
75778Female	24	22	25	23	16	0
Group Total	72	57	41	35	20	0
Group Mean Score	24.0	19.0	13.7	11.7	6.7	0.0

-  considered to be zero for calculation of Group Mean Score

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

The test item produced a maximum group mean score of 24.0 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item meets the criteria for classification as irritant (Category 2A) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. The signal word “Warning” and Hazard Statement “H319: Causes serious eye irritation” are therefore required.
The test item also meets the criteria for classification as irritating to eyes (Category 2A) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of three rabbits produced scattered or diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Petechial hemorrhage of the nictitating membrane was also noted in one treated eye. Treated eyes appeared normal at the 7 or 14-Day observations.

Conclusion

The test item produced a maximum group mean score of24.0and was classified as amoderateirritant (Class5on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandraclassification system.

The test item meets the criteria for classification as irritant (Category 2A) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. The signal word “Warning” and Hazard Statement “H319: Causes serious eye irritation” are therefore required.

The test item also meets the criteria for classification as irritating to eyes (Category 2A)  according to theGlobally Harmonized Systemof Classification and Labelling of Chemicals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)