Cordierite (Mg2[Al4O3(SiO3)5])

Endpoint summary

Description of key information

The skin corrosion study was conducted on the registered substance according to OECD Testing Guideline 431 and gave a relative mean tissue viability calculated as a percentage of the negative control of 96.6% after the 60-minute exposure and 94.8% after the 3 minute exposure. The test substance is not classified as a skin corrosive according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The skin irritation study was conducted on the registered substance according to OECD Testing Guideline 431 and gave a relative mean tissue viability calculated as a percentage of the negative control of 100.5% after the 15-minute exposure followed by the 42-hour post-exposure. The test substance is not classified as a skin irritant according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The eye irritation study was conducted on the registered substance according to OECD Testing Guideline 431 and gave an IVIS ≤ 3 when tested under a Bovine Corneal Opacity and Permeability (BCOP) Assay implying that it is not categorized as irritant to the eye. The test substance is not classified as an eye irritant according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

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:

From 26 August 2015 to 31 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 439. The study was conducted on the registered substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit

Strain:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit

Details on test animals or test system and environmental conditions:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 25 August 2015
EpiSkinTM Tissues (0.38cm2) lot number : 15-EKIN-034
Maintenance Medium lot number : 15-MAIN3-034
Assay Medium lot number : 15-ESSC-034

Type of coverage:

other: The test item was applied topically to the corresponding tissues ensuring uniform covering.

Controls:

other: 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.

Amount / concentration applied:

Amount(s) applied (volume or weight with unit): 10 µL

Duration of treatment / exposure:

Exposure: 15 minutes
Post-exposure incubation: 42 hours

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

The negative control item, DPBS, was used as supplied.
The positive control item, SDS, was prepared as a 5% w/v aqueous solution.

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 contact between the test item and the epidermis. 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 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:

100.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minute exposure. Reversibility: other: not applicable. (migrated information)

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	1.280	1.245	0.061	102.8	100*	4.9
	0.281			102.9
	0.174			94.3
Positive Control Item	0.080	0.087	0.008	6.4	7.0	0.7
	0.084			6.7
	0.096			7.7
Test Item	1.272	1.251	0.130	102.2	100.5	10.5
	0.112			89.3
	0.369			110.0

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The Relative mean tissue viability calculated as a percentage of the negative control was 100.5% after the 15-minute exposure followed by the 42-hour post-exposure. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The in vitro skin irritation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 439. 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 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 562 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 tissue viability calculated as a percentage of the negative control was 100.5 after the 15-minute exposure followed by the 42-hour post-exposure. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Reference 2

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From to 5 August 2015 to 7 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 431. The study was conducted on the registered substance.

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 of Commission Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiDerm™ Reconstructed Human Epidermis Model Kit

Strain:

other: EpiDerm™ Reconstructed Human Epidermis Model Kit

Details on test animals or test system and environmental conditions:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 04 August 2015
EpiDermTM Tissues (0.5cm2) lot number : 21683
Assay Medium lot number : 073015SLC

Type of coverage:

other: The test item was applied topically to the corresponding tissues ensuring uniform covering

Vehicle:

unchanged (no vehicle)

Controls:

other: negative and positive controls

Amount / concentration applied:

Amount(s) applied (volume or weight with unit): 25 mg

Duration of treatment / exposure:

Exposure: 3 minutes / 60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

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

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading.
Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well.
The two plates were placed into the incubator until required.
After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 μL of sterile water was added for wetting of the test item. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.
When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37°C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

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

Irritation parameter:

other: Relative mean tissue viability

Basis:

mean

Time point:

other: 60 minute exposure

Score:

96.6

Reversibility:

other: not applicable

Irritation parameter:

other: Relative mean tissue viability

Basis:

mean

Time point:

other: 3 minute exposure

Score:

94.8

Reversibility:

other: not applicable

Irritant / corrosive response data:

Irritation response data is calculated based on the optical density of MTT at 562 nm. Viable cells can reduce the yellow tetrazolium salt to a blue formazan dye by mitochondrial succinate dehydrogenase. The quantity of viable cells depends on the irritation potential of the test item.

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

Item	Exposure Time	Mean OD562	Percentage Viability
Negative Control	3 minutes	2.199	100
	60 minutes	2.195	100
Positive Control	3 minutes	0.088	4.0
	60 minutes	0.080	3.6
Test Item	3 minutes	2.085	94.8
	60 minutes	2.120	96.6

Interpretation of results:

not irritating

Remarks:

Migrated information

Conclusions:

The Relative mean tissue viability calculated as a percentage of the negative control was 96.6% after the 60-minute exposure and 94.8% after the 3 minute exposure. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The in vitro skin corrosion of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 431. 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. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

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

The Relative mean tissue viability calculated as a percentage of the negative control was 96.6% after the 60-minute exposure and 94.8% after the 3_minute exposure. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

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:

From 16 September 2015 to 18 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 437. The study was conducted on the registered substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The positive control In Vitro Irritancy Score fell slightly short of the expected historical values. This was thought not to have affected the purpose or integrity of the study.

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

yes

Remarks:

The positive control In Vitro Irritancy Score fell slightly short of the expected historical values. This was thought not to have affected the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine cornea

Strain:

other: bovine cornea

Details on test animals or tissues and environmental conditions:

Not applicable

Vehicle:

physiological saline

Controls:

other:

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20% w/v solution in 0.9% w/v sodium chloride solution.

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% w/v sodium chloride solution
- Lot/batch no. (if required): 3011424
- Purity: 0.9%

Duration of treatment / exposure:

Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

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 (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (Appendix 1). 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 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 ºC for 240 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.

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 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:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

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

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	2.5
Negative Control	2.8
Positive Control	65.1

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item produced an IVIS ≤ 3 when tested under a Bovine Corneal Opacity and Permeability (BCOP) Assay implying that it is not categorized as irritant to the eye. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The potential of the test item to be irritant to the eye was determined in accordance with the OECD Guideline for Testing of Chemicals 437. 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 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)

The BCOP produced an IVIS ≤ 3 implying that it is not categorized as an irritant to the eye. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

The skin corrosion potential of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 431. 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. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The skin irritation potential of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 439. 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 formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The Relative mean tissue viability calculated as a percentage of the negative control was 100.5 after the 15-minute exposure followed by the 42-hour post-exposure. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The eye irritation potential of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 437. 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 BCOP produced an IVIS ≤ 3 implying that it is not categorized as an irritant to the eye. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Justification for selection of skin irritation / corrosion endpoint:

Study was conducted on the registered substance according to OECD Testing Guideline 439. According to the Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 4.0), reconstructed human epidermis (RHE) tests are considered scientifically valid for the prediction of irritant (Category 2) and non-irritant (no category) substances for Annex VIII according to the rules laid down in Annex XI, and therefore additional in vivo testing is not considered as scientifically justified.

Justification for selection of eye irritation endpoint:

Study was conducted on the registered substance according to OECD Testing Guideline 437. In accordance with the Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 4.0), the Bovine Corneal Opacity and Permeability (BCOP) test method is recommended to identify substances that do not require classification for eye irritation or serious eye damage i.e. leading to non-classification under CLP, without further testing. A GLP compliant Bovine Corneal Opacity and Permeability (BCOP) test was performed on the substance according to OECD Guideline 437, and produced a clear negative answer as the IVIS was < 3, leading to a non-classification under CLP. Therefore further in vivo testing is not considered as scientifically justified.

Justification for classification or non-classification

The skin corrosion study was conducted on the registered substance according to OECD Testing Guideline 431 and gave a relative mean tissue viability calculated as a percentage of the negative control of 96.6% after the 60-minute exposure and 94.8% after the 3 minute exposure. The test substance is not classified as a skin corrosive according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The skin irritation study was conducted on the registered substance according to OECD Testing Guideline 431 and gave a relative mean tissue viability calculated as a percentage of the negative control of 100.5% after the 15-minute exposure followed by the 42-hour post-exposure. The test substance is not classified as a skin irritant according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The eye irritation study was conducted on the registered substance according to OECD Testing Guideline 431 and gave an IVIS ≤ 3 when tested under a Bovine Corneal Opacity and Permeability (BCOP) Assay implying that it is not categorized as irritant to the eye. The test substance is not classified as an eye irritant according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.