Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Endpoint summary

Description of key information

In vitro skin corrosion: The test item was classified as non-corrosive to the skin.

In vitro skin irritation:  The test item was classified as non-irritant.

In vitro eye irritaiton: The test item was considered not to be an ocular corrosive or severe irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 18 February 2014; Experimental Completion Date: 24 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Not applicable. EPISKIN Reconstructed Human Epidermis (RHE) Model used.

Strain:

other: Not applicable.

Details on test animals or test system and environmental conditions:

Details on test animals not applicable for in-vitro study.

EPISKIN Reconstructed Human Epidermis Model Kit supplied by SkinEthic Laboratories, Lyon, France

Type of coverage:

other: Not applicable.

Preparation of test site:

other: Not applicable.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

10 mg of the test item was then applied to the epidermal surface.

Duration of treatment / exposure:

Tissues were treated with the test item for an exposure period of 15 minutes.

Observation period:

42 hours post exposure incubation period.

Number of animals:

Not applicable.

Details on study design:

TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION:
The test item was used as supplied.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS, MTT AND ACIDIFIED ISOPROPANOL:
The negative control item, PBS, was used as supplied.
The positive control item, SDS, was prepared as a 5% w/v aqueous solution.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Negative Control:
Phosphate Buffered Saline Dulbecco’s (PBS) with Ca++ and Mg++

Positive Control:
Sodium Dodecyl Sulphate (SDS).

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.

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, each 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, 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.

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. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve further contact between the test item and the epidermis. 10 mg 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 centre). After 7 Minutes 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. The plate(s) 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 plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into 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.

Irritation / corrosion parameter:

other: other: Relative mean viability (%)

Value:

89.7

Remarks on result:

other:

Remarks:

Basis: mean (test item). Time point: 15-Minute exposure period and 42 hours post-exposure incubation period.. Remarks: Non-irritant. (migrated information)

Irritant / corrosive response data:

The relative mean viability ofthe test item treated tissues was 89.7% after a 15-Minute exposure period and 42 hours post-exposure incubation period.

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

Other effects:

Direct MTT Reduction:
The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

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

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

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.870	0.988	0.102	88.1	100*	10.3
	1.046			105.9
	1.047			106.0
Positive Control Item	0.043	0.046	0.004	4.4	4.7	0.4
	0.050			5.1
	0.045			4.6
Test Item	0.899	0.887	0.047	91.0	89.7	4.7
	0.926			93.7
	0.835			84.5

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

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was 4.7% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.4%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₆₂ for the negative control treated tissues was 0.988 and the standard deviation value of the percentage viability was 10.3%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 4.7%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

not classified

Remarks:

Migrated information (non-irritating) Criteria used for interpretation of results: EU

Conclusions:

The test item was classified as non-irritant.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 21 January 2014; Experimental Completion Date: 24 January 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method B.40bis

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Not applicable. EPISKIN Reconstructed Human Epidermis (RHE) Model used.

Strain:

other: Not applicable.

Details on test animals or test system and environmental conditions:

Details on test animals not applicable for in-vitro study.

EPISKIN ™ Reconstructed Human Epidermis Model Kit supplied by SkinEthic Laboratories, Lyon, France

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

20 mg of the test item was applied topically to the tissues.

Duration of treatment / exposure:

Tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes.

Observation period:

3 hours post exposure incubation period.

Number of animals:

Not applicable

Details on study design:

TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION:
The test item was ground before use.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS:
The negative control item was used as supplied.
The positive control item was used as supplied.

Negative Control: 0.9% w/v sodium chloride solution
Positive Control: Glacial acetic acid

PRE-TEST PROCEDURE
ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT:
MTT Dye Metabolizm, 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 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.

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, each test item is checked for the ability to directly reduce MTT according to the following procedure:

20 mg of the test item was added to 2.0 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 turned blue, the test item is presumed to have reduced the MTT and the determination of skin corrosion potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

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

2.0 mL of maintenance medium, warmed to approximately 37ºC, was pipetted into two wells of the first column of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12 well plate was used for each test item, control and time point. The tissues were incubated at 37°C, 5% CO2 in air overnight.
After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.

MAIN TEST:
APPLICATION OF TEST ITEM AND RINSING:
2.0 mL of assay medium, warmed to approximately 37°C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 20 mg of the test item was applied topically to the corresponding tissues and 100 µL of 0.9% w/v sodium chloride solution was added for wetting of the test item.

Duplicate tissues, treated with 50 µL of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µL of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.

At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.

2.0 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12-well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ± 5 minutes at 37°C, 5% C02 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 taken using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENT:
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 an Anthos 2001 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (240 minutes)

Value:

99.9

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (60 minutes)

Value:

91.4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (3 minutes)

Value:

101.1

Other effects / acceptance of results:

Direct MTT Reduction:
The MTT solution containing the test item turned orange. This was taken to indicate that the test item did not reduce MTT.

Mean OD₅₆₂ values and viabilities for the negative control, positive control and test item are given in the following table:

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

Item	Exposure Period	Mean OD562 of individual tissues	Mean OD562 of duplicate tissues	Relative mean viability (%)
Negative Control Item	240 Minutes	0.878	0.927	100*
		0.976
Positive Control Item	240 Minutes	0.043	0.046	5.0
		0.048
Test Item	240 Minutes	0.860	0.926	99.9
		0.991
	60 Minutes	0.818	0.847	91.4
		0.875
	3 Minutes	0.869	0.937	101.1
		1.004

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

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was 5.0% relative to the negative control treated tissues following the 240-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₆₂ for the negative control treated tissues was 0.927. The negative control acceptance criterion was therefore satisfied.

 

Interpretation of results:

not classified

Remarks:

Migrated information (non-corrosive) Criteria used for interpretation of results: EU

Conclusions:

The test item was classified as 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 Starting Date: 12 March 2014; Experimental Completion Date: 12 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Eyes from adult cattle

Details on test animals or tissues and environmental conditions:

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:

other: test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.

Controls:

no

Amount / concentration applied:

0.75 mL of the test item preparation or control items were applied to the appropriate corneas.

Duration of treatment / exposure:

Exposure of 240 minutes.

Observation period (in vivo):

Not applicable.

Number of animals or in vitro replicates:

Not applicable.

Details on study design:

TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION:
For the purpose of this study the test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution.
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.

PREPARATION OF NEGATIVE AND POSITIVE CONTROLS:
The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.

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

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 (epithelial side uppermost) 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 (MEM) and plugged. The holders were incubated at 32 ± 1°C 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 MEM.

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 MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation 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°C for 240 minutes.

At the end of the exposure period the test item preparation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN:
Following the 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 (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1°C 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.

EVALUATION OF RESULTS:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement:
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.

Permeability Measurement:
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.

In Vitro Irritancy Score:
The following formula was used to determine the In Vitro irritancy score:

In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

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.

Visual Observation:
The condition ofthe cornea was visually assessed immediately after rinsing.

Irritation parameter:

in vitro irritation score

Value:

0.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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.

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

Treatment	Cornea Number	Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment - Pre-Treatment	Corrected Value		Corrected value
Negative Control	1	3	6	3		0.052
	5	3	3	0		0.015
	18	3	4	1		0.112
				1.3		0.06		2.2
Positive Control	2	3	70	67	65.7	1.091	1.031
	4	2	71	69	67.7	1.235	1.175
	6	2	72	70	68.7	1.231	1.171
					67.3		1.126	84.2
Test Item	3	1	2	1	0	0.061	0.001
	7	3	3	0	0	0.074	0.014
	9	3	4	1	0	0.093	0.033
					0		0.016	0.2

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant and does not require classification for eye corrosion/irritation according to the BCOP test method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Irritaiton/Corrosion

In vitro skin corrosion:

Introduction:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

The EPISKIN™ model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.

Methods:

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. 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 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 (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 of the test item treated tissues were:

240 minutes exposure: 99.9%

60 minutes exposure: 91.4%

3 minutes exposure: 101.1%

Quality criteria:

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

Conclusion:

The test item was classified as non-corrosive to the skin.

In vitro skin irritation:

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test 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. The concentration of the inflammatory mediator IL-1 α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.

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 ofthe 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 89.7% 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.

Eye Irritation:

Bovine Corneal Opacity and Permeability (BCOP) Assay:

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 bioitem 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 it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS 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 Category 1 or 2 (2A or 2B), i.e. they are referred to

as UN GHS No Category.

Method:

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution 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 an In Vitro Irritancy Score (IVIS).

Interpretation:

The test item is classified according to the prediction model below:

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	0.2
Negative Control	2.2
Positive Control	84.2

Conclusion:

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

Justification for selection of skin irritation / corrosion endpoint:

Two in vitro studies were conducted to assess skin corrosion and skin irritation. Both studies were conducted on the test substance according to internationally recognised OECD guidelines. The studies are considered to be of reliability 1.

Justification for selection of eye irritation endpoint:

An in vitro study (BCOP) was conducted to assess eye damage. The study was conducted on the test substance according to internationally recognised OECD guidelines. The studiy is considered to be of reliability 1.

Justification for classification or non-classification

Skin corrosion/irritation:

The in vitro skin corrosion study results classified the substance as as non-corrosive to the skin.

The in vitro skin irritaiton study results classified the substance as a non-irritant to the skin.

The substance is therefore not classified for skin corrosion or irritation according to the CLP regulation (Regulation (EC) No 1907/2006).

Eye irritation:

Based on the results of the BCOP assay, the substance does not require classification for serious eye damage or irritation.