4-Decenal, 9-hydroxy-5,9-dimethyl-

Endpoint summary

Description of key information

Skin irritation/corrosion

- In vitro skin irritation (OECD 439, K, Rel.1):

The mean relative absorbance value decreased to 7.9% versus 5.3% in the positive control (5% SLS). This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Under the experimental conditions of this study and in view of the supporting skin corrosion study results, the test item is classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

 

- In vitro skin corrosion (OECD 431, K, Rel.1):

The relative mean viabilities of the test item treated tissues were as follows:

3 minutes exposure: 118.9%, versus 28.6% in the positive control (8.0 N Potassium hydroxide).

60 minutes exposure: 69.5%, versus 13.2% in the positive control (8.0 N Potassium hydroxide).

The test item is considered to be non-corrosive to skin, since the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater than 15%.

Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS. This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.

 

Eye irritation

- In vivo eye irritation (OECD 405, K, Rel.1):

Mean individual scores at 24, 48 and 72 h after exposure for the 2 animals were 1.0 / 1.0 for cornea score; 0.66/ 0.33 for iris score; 2.0 / 2.0 for conjunctivae score and 1.66 / 1.66 for chemosis score. Both treated eyes appeared normal at the 7-Day observation.

Under the test conditions, the test item was classified as Eye Irr. Category 2 (H319: Causes serious eye irritation) according to the Regulation (EC) No. 1272/2008 (CLP) and as Eye Irr. Category 2B (H320: Causes eye irritation) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). The signal word "Warning" is required.

 

- In vitro eye irritation (BCOP, OECD 437, SS, Rel.1):

Relative to the negative control, the test item caused a slight increase of the corneal opacity and permeability. The calculated mean IVIS was 7.03 (threshold for corrosivity/severe irritancy: IVIS ≥ 55.1). According to OECD 437, the test item is classified as not corrosive/not severe irritant to the eye.

According to the current study and under the experimental conditions reported, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

 

Respiratory irritation

No information.

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:

15-17 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 439 without deviations.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted July 22, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP Compliance certificate (signed on 11 April 2013)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-Kit (EpiDerm™ Tissues) from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The tissues (surface of 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm of diameter).
- Tissue batch number(s): 18320
- Delivery date: 14 May 2013 (at 4°C on medium-supplemented agarose gels in a 24-well plate)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at 37ºC for 35 minutes and at RT into the sterile hood for 25 minutes.
- Temperature of post-treatment incubation (if applicable): 37 +/- 1.5°C, 5 +/- 0.5 % CO2.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: not reported (diluted with the MTT diluent)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices microplate reader
- Wavelength: 570+/- 1 nm filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 1.705, 1.994 and 1.998 (historical control equal to 1.838 with a range of viabilities between 1.423 and 2.651 ).
- Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system or rather of the test kit.

MTT DIRECT INTERFERENCE
For correct interpretation of results, it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 30 µL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
> Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 tissues/dose group

PREDICTION MODEL / DECISION CRITERIA
- The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%)= [OD-test item / OD-mean of negative control] * 100

For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
- For the current test, an irritation potential of a test item according to EU classification H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control:
- mean tissue viability <= 50% : irritant (I), H315 (category 2)
- mean tissue viability > 50% : non-irritant (NI)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the undiluted test item was dispensed directly atop the EpiDerm(TM) tissue and spread to match the surface of the tissue.
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of DPBS
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of 5% SLS
- Concentration (if solution): 5% w/v

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 35 at 37 °C, 5% CO2 and 95% RH and then removed from the incubator and placed in sterile hood until the 60 minutues expired.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for approximately 22 hours. The inserts were then transfered to new 6-well plates for another 18 hrs post-incubation at 37 +/- 1.5°C, 5 +/- 0.5 % CO2.

Duration of post-treatment incubation (if applicable):

- At the end of the 42 h post-treatment incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in air.
- At the end of the formazan extraction period: At the end of the formazan extraction period, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Por each tissue, 3 x 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices) with a 570 +/- 1 nm filter. Mean values were calculated from the 3 wells per tissue.

Number of replicates:

Triplicate tissues for test item, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure period and 42 h post-exposure incubation period

Value:

7.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- Direct MTT Reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

- Test item: The relative mean viability of the test item treated tissues was 7.9% after a 60 minutes exposure period and 42 hours post-exposure incubation period.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.899 and the standard deviation value of the percentage viability was 8.8%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 12.8%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 10.7%. The test item acceptance criterion was therefore satisfied.

Table 7.3.1/1: Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item after a 60-minute exposure

 

Item	OD570 of tissues	Mean OD570 of triplicate tissues	Relative individual tissue viability (%)	Relative mean viability (% of negative control)	± SD of Relative mean viability (%)
Negative Control Item	1.705	1.899	89.8	100.0	8.8
	1.994		105.0
	1.998		105.2
Positive Control Item	0.089	0.100	8.4	5.3	12.8
	0.098		8.5
	0.114		6.9
Test Item	0.159	0.151	4.7	7.9	10.7
	0.161		5.2
	0.132		6.0

SD = Standard deviation

OD = Optical density

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study and based on the result of the in vitro skin corrosion study, the test item is classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EpiDerm™ Tissues reconstructed human epidermis model. 

Triplicate tissues were treated with the test item for an exposure period of 60 minutes. Each 30 µL of the test item, the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue, spread to match the tissue size. 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. 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 triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

After treatment with the negative control, the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 1.0 ≤ 2.5 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. 

After treatment with the test item, the mean relative absorbance value decreased to 7.9% versus 5.3% in the positive control (5% SLS). This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

 

Under the experimental conditions of this study and in view of the supporting skin corrosion study results, the test item is classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 12 to 13, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD test Guideline No. 431 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted April 13, 2004

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP Compliance certificate (signed on 11 April 2013)

Species:

other: EpiDerm™ Tissues

Details on test animals or test system and environmental conditions:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-Kit (EpiDerm™ Tissues) from MatTek Corporation (Ashland, MA 01721, USA). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The tissues (surface of 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm of diameter).
- Tissue batch number(s): 18336
- Delivery date: 11 June 2013 (at 4°C on medium-supplemented agarose gels in a 24-well plate).

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL of the undiluted test item was applied to each set of duplicate tissues.

Duration of treatment / exposure:

Duplicate tissues were treated with the test item, the negative control (Deionised water) and positive control (8.0 N Potassium Hydroxide) for exposure periods of 3 and 60 minutes.

Observation period:

At the end of the formazan extraction period, the optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Versamax® Molecular Devices microplate reader.

Number of animals:

Duplicate tissues for test item, negative and positive controls

Details on study design:

APPLICATION OF TEST ITEM AND RINSING
- Duplicate tissues were treated with the test item, positive or negative control for each of two different exposure periods: 3 minutes and 1 hour.
After the pre-incubation of the tissues was completed (1 hour 25 minutes for the 1 hour exposure and 2 hours 19 minutes for the 3 minutes exposure) the medium in each well was replaced by 0.9 mL fresha ssay medium. The negative control (50 µL deionised water) was added to the surface of duplicates tissues. Subsequently, the remaining tissues were exposed to the test item and the positive control in the same manner. The 6-well plates were then placed into an incubator (37 +/- 1.5°C, 5 +/- 0.5% CO2).

At the end of the exposure period, the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing DPBS to remove any residual test material. Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT ASSAY
The MTT concentrate was prepared on the day of testing and diluted with the MTT diluents (1:4). The resulting MTT solution was stored in the dark at 4°C for later use on the same day (not until next day). Two 24-well plates were prepared before the end of the tissues pre-warming period. MTT solution (300 µL) was added to each well and the plates were kept in an incubator (37 +/- 1.5 °C, 5 +/- 0.5% CO2) until required.
Following rinsing, the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period, the MTT solution was aspirated from the wells and the wells were rinsed three times with DPBS. The inserts were transferred into new 24-well plates and immersed in extractant solution by gently pipetting 2 mL of isopropanol into each insert ensuring that the tissue was completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for approximately 24 hours without shaking.
After the extraction period, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. The 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Por each tissue, 3 x 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate. The OD was read in a microplate reader (Versamax® Molecular Devices) at 570 nm without reference filter. The mean values were calculated from the 3 wells per tissue.

Negative and positive controls (reference substances).
- Duplicate tissues, treated with 50 µL of deionised water served as negative controls.
- Duplicate tissues, treated with 50 µL of 8.0 N Potassium Hydroxide served as positive controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

118.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

69.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Assessment of Direct Test Item Reduction of MTT:
The optical evaluation of the MTT-reducing capacity of the test item after an 1 hour incubation with MTT-reagent did not show blue colour and thereby was not considered to be an MTT reducer.


Quantitative MTT Assessment (Percentage Tissue Viability):
The test item is considered to be non-corrosive to skin:
- since the viability after 3 minutes exposure is greater than 50% (118.9%)
- the viability after 1 hour exposure is greater than 15% (69.5%).


Quality Criteria:
After exposure to the negative control, the absorbance values met the required acceptability criterion of mean OD570 ≥ 0.8 for both treatment intervals thereby confirming the acceptable quality of the tissues (1.597 for 3-minute exposure and 1.438 for 60-minute exposure).

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (28.6%) and for the 1 hour exposure period (13.2%) thus confirming the validity of the test system and the specific batch of tissue models.

Table 7.3.1/1: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Dose group	Exposure period	OD570 of individual tissues	Mean OD562 of duplicate tissues	Relative mean viability (%)	Relative standard deviation (%)
Negative Control	3 minutes	1.522	1.597	100.0	6.7
		1.673
Positive Control	3 minutes	0.458	0.457	28.6	0.2
		0.457
Test Item	3 minutes	1.918	1.899	118.9	1.4
		1.880
Negative Control	60 minutes	1.527	1.438	100.0	8.8
		1.348
Positive Control	60 minutes	0.201	0.190	13.2	8.1
		0.179
Test Item	60 minutes	1.098	1.000	69.5	14.0
		0.901

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS.

Executive summary:

An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Tissues reconstructed human epidermis model. 

 

Duplicate tissues were treated with the test item for two exposure periods of 3- and 60-minute. Each 50 µL of the test item, the negative control (deionised water) or the positive control (8.0 N Potassium Hydroxide) were applied to each tissue, spread to match the tissue size. At the end of the exposure period, the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing DPBS to remove any residual test material, 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 formalin crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

After exposure to the negative control, the absorbance values met the required acceptability criterion of mean OD570 ≥ 0.8 for both treatment intervals thereby confirming the acceptable quality of the tissues (1.597 for 3-minute exposure and 1.438 for 60-minute exposure).

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (28.6%) and for the 1 hour exposure period (13.2%) thus confirming the validity of the test system and the specific batch of tissue models.

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

 

The relative mean viabilities of the test item treated tissues were as follows:

3 minutes exposure: 118.9%, versus 28.6% in the positive control (8.0 N Potassium hydroxide).

60 minutes exposure: 69.5%, versus 13.2% in the positive control (8.0 N Potassium hydroxide).

 

The test item is considered to be non-corrosive to skin:
- since the viability after 3 minutes exposure is greater than 50%.
- the viability after 1 hour exposure is greater than 15%.

 

Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS. This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

weight of evidence

Study period:

May 24, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD test Guideline No. 437 without any deviation.

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:

No. 1152/2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP Certificate (Signed on May 12, 2014)

Species:

other: Freshly isolated bovine cornea

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Eyes from at least 9 month old donor cattle were obtained from a local abattoir. Excess tissue was removed from the excised eye. The isolated eyes were transported to the laboratory Hanks' Balanced Salt Solution (HBSS) supplemented with streptomycin/penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL was applied on each cornea
- Concentration: Undiluted

Duration of treatment / exposure:

The undiluted test item was applied for 10 minutes at 32 ± 1 °C.

Observation period (in vivo):

- The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea. Corneal opacity was measured after exposure, after rinsing and after incubation of the corneae for two hours (t130).
- Permeability endpoint was measured after the final opacity measurement when the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% w/v Sodium Fluorescein solution in HBSS. Corneawere incubated again in a horizontal postion for 90 minutes in a water-bath at 32 +/- 1°C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

Duration of post- treatment incubation (in vitro):

After the incubation phase the test item, positive and the negative controls were rinsed from the cornea and incubated for another 120 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

Total: 9 corneas - 3 corneas/group for test item, negative and positive controls

Details on study design:

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in guideline that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned againstthe sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 +/- 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0) and recorded. Each corneae with a value of the basal opacity > 7 was discarded.
Sets of three corneae were used for treatment with the test item and the negative and positive controls.

DETAILS ON TEST PROCEDURE
The anterior compartment received the test item or negative or positive control at a volume of 0.75 mL on the surface of the corneae and was incubated at 32 +/- 1°C in the water-bath, while the corneae were in a horizontal position. The incubation time lasted ten minutes.

After the test item and control items were rinsed off from the application side with saline. The corneae was then incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). In the second step of the assay, permeability of the cornea was determined.

Irritation parameter:

in vitro irritation score

Value:

7.03

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

See the attached document for information on tables of results

Interpretation of results:

GHS criteria not met

Conclusions:

According to the current study and under the experimental conditions reported, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Executive summary:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, 0.75 mL of undiluted test item was applied to isolated bovine corneas for 10 minutes followed by an incubation period of two hours at 32 °C. Three corneas were used for undiluted test item, negative control (saline solution) and positive control (2-Ethoxyethanol). 

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item; the positive and the negative controls were applied to corneae and incubated for 10 minutes at 32 +/- 1°C. After the incubation phase the test item, positive and negative controls were rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 +/- 1°C in incubation medium, and opacity was measured a second time (t130). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 +/- 1°C.

With the negative control neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.44).

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 65.13) corresponding to a classification as corrosive/severe irritant to the eye (CLP/GHS Cat.1).

Relative to the negative control, the test item caused a slight increase of the corneal opacity and permeability. The calculated mean IVIS was 7.03 (threshold for corrosivity/severe irritancy: IVIS ≥ 55.1). According to OECD 437, the test item is classified as not corrosive/not severe irritant to the eye.

 

According to the current study and under the experimental conditions reported, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 August 2013 to 16 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 405 without deviations affecting the purpose or integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted 02 october 2012

Deviations:

yes

Remarks:

On occasions the relative humidity was outside the target range of 30 to 70%. This was considered not to have affected the purpose or integrity of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No. 440/2008

Deviations:

yes

Remarks:

On occasions the relative humidity was outside the target range of 30 to 70%. This was considered not to have affected the purpose or integrity of the study.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 10 July 2012 / signed on 30 November 2012)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK.
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.60-2.74 kg
- Housing: Animals were individually housed in suspended cages.
- Diet: Food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum.
- Water: Mains drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Humidity: 30-70 %
- Air changes: 15 changes/h
- Photoperiod: 12 h dark / 12 h light

JUSTIFICATION FOR ANIMALS
The rabbit is the preferred species of choice as historically used for irritation studies and is specified in the appropriate test guidelines. The number of animals used was the minimum required to achieve the objectives of the study. Testing was conducted in two animals and the response in those animals was such that exposure of a third animal would not affect classification of the test item, therefore, no further testing was needed.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

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.

Observation period (in vivo):

1, 24, 48 and 72 h following instillation of test material and until 7 days.

Number of animals or in vitro replicates:

2 males

Details on study design:

PRE-TREATMENT:
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.

PROCEDURE:
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% tetracaine 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.

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 h later. No further analgesia was required. After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.

REMOVAL OF TEST SUBSTANCE
- Washing: No

SCORING SYSTEM: Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C. (1962) 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.

However, the results were also interpreted according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures and the Draize score as described in the OECD guideline No. 405 were calculated.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

other: Maximum group mean score

Basis:

mean

Time point:

24/48/72 h

Score:

20

Max. score:

110

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of moderate irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.66

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

- No initial pain reaction was noted in any animal following instillation of the test item.
- Diffuse corneal opacity was noted in both treated eyes at the 24, 48 and 72-Hour observations.
- Iridial inflammation was noted in both treated eyes one and 24 hours after treatment and in one treated eye at the 48-Hour observation.
- Moderate conjunctival irritation was noted in both treated eyes one hour after treatment and at the 24 and 48-Hour observations with minimal conjunctival irritation noted at the 72-Hour observation.
- Both treated eyes appeared normal at the 7-Day observation

See Table 7.3.2/1 below.

The test item produced a maximum group mean score of 20.0 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.

Regarding the draize score system:
- the corneal opacity is ≥ 1 on both animals,
-the iritis is ≤ 1 on both animals,
- the conjunctival redness is ≥ 2 on both animals,
- the conjunctival oedema (chemosis) is ≤ 2 on both animals.

Other effects:

Both animals showed expected gain in body weight during the study.

Table 7.3.2/1: Eye irritation response data for each animal at each observation time

 

Score at time point	Cornea opacity (/4)	Iritis (/2)	Conjunctivae
			Redness (/3)	Chemosis (/4)
1 h	0 / 0	1 / 1	2 / 2	3 / 3
24 h	1 / 1	1 / 1	2 / 2	2 / 2
48 h	1 / 1	1 / 0	2 / 2	2 / 2
72 h	1 / 1	0 / 0	2 / 2	1 / 1
D 7	0 / 0	0 / 0	0 / 0	0 / 0
Average 24, 48 and 72 h	1 / 1	0.66 / 0.33	2 / 2	1.66 / 1.66
Reversibility	7 days	72 hours	7 days	7 days

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Conclusions:

Under the test conditions, the test item was classified as Eye Irr. Category 2 (H319: Causes serious eye irritation) according to the Regulation (EC) No. 1272/2008 (CLP) and as Eye Irr. Category 2B (H320: Causes eye irritation) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Executive summary:

In an eye irritation study performed according to the OECD Guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test item was instilled into the right eye of 2 males New Zealand White strain rabbits. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test item, and then released. The left eye remained untreated and served as control. The eyes were examined and the changes were observed at 1, 24, 48, 72 h and 7 days after instillation of test item and graded according to the Draize method.

 

No initial pain reaction was noted in any animal following instillation of the test item. Diffuse corneal opacity was noted in both treated eyes at the 24, 48 and 72-Hour observations. Iridial inflammation was noted in both treated eyes one and 24 hours after treatment and in one treated eye at the 48-Hour observation. Moderate conjunctival irritation was noted in both treated eyes one hour after treatment and at the 24 and 48-Hour observations with minimal conjunctival irritation noted at the 72-Hour observation. Both treated eyes appeared normal at the 7-Day observation.

 

Mean individual scores at 24, 48 and 72 h after exposure for the 2 animals were 1.0 / 1.0 for cornea score; 0.66/ 0.33 for iris score; 2.0 / 2.0 for conjunctivae score and 1.66 / 1.66 for chemosis score.

 

Under the test conditions, the test item was classified as Eye Irr. Category 2 (H319: Causes serious eye irritation) according to the Regulation (EC) No. 1272/2008 (CLP) and as Eye Irr. Category 2B (H320: Causes eye irritation) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). The signal word "Warning" is required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

- Skin irritation:

One key study was identified (Harlan, 2013).

In this in vitro skin irritation study performed according to the OECD Guideline 439 and in compliance with GLP, the EpiDerm™ Tissues reconstructed human epidermis model was used.

Triplicate tissues were treated with the test item for an exposure period of 60 minutes. Each 30 µL of the test item, the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue, spread to match the tissue size. 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. 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 triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

After treatment with the negative control, the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 1.0 ≤ 2.5 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. 

 

After treatment with the test item, the mean relative absorbance value decreased to 7.9% versus 5.3% in the positive control (5% SLS). This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Under the experimental conditions of this study and in view of the supporting skin corrosion study results, the test item is classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

 

- Skin corrosion:

One key study was identified (Harlan, 2013).

In this in vitro skin corrosion study performed according to the OECD Guideline 431 and in compliance with GLP, the EpiDerm™ Tissues reconstructed human epidermis model was used.

Duplicate tissues were treated with the test item for two exposure periods of 3- and 60-minute. Each 50 µL of the test item, the negative control (deionised water) or the positive control (8.0 N Potassium Hydroxide) were applied to each tissue, spread to match the tissue size. At the end of the exposure period, the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing DPBS to remove any residual test material, 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 formalin crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

After exposure to the negative control, the absorbance values met the required acceptability criterion of mean OD570 ≥ 0.8 for both treatment intervals thereby confirming the acceptable quality of the tissues (1.597 for 3-minute exposure and 1.438 for 60-minute exposure).

 

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (28.6%) and for the 1 hour exposure period (13.2%) thus confirming the validity of the test system and the specific batch of tissue models.

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

The relative mean viabilities of the test item treated tissues were as follows:

3 minutes exposure: 118.9%, versus 28.6% in the positive control (8.0 N Potassium hydroxide).

60 minutes exposure: 69.5%, versus 13.2% in the positive control (8.0 N Potassium hydroxide).

The test item is considered to be non-corrosive to skin:
- since the viability after 3 minutes exposure is greater than 50%.
- the viability after 1 hour exposure is greater than 15%.

 

Under the experimental conditions of this study, the test substance is not classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS. This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.

 

Eye irritation/corrosion

- In vivo eye irritation:

A key study was identified (Harlan, 2013, rel. 1, K).

In this eye irritation study performed according to the OECD Guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test item was instilled into the right eye of 2 males New Zealand White strain rabbits. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test item, and then released. The left eye remained untreated and served as control. The eyes were examined and the changes were observed at 1, 24, 48, 72 h and 7 days after instillation of test item and graded according to the Draize method.

No initial pain reaction was noted in any animal following instillation of the test item. Diffuse corneal opacity was noted in both treated eyes at the 24, 48 and 72-Hour observations. Iridial inflammation was noted in both treated eyes one and 24 hours after treatment and in one treated eye at the 48-Hour observation. Moderate conjunctival irritation was noted in both treated eyes one hour after treatment and at the 24 and 48-Hour observations with minimal conjunctival irritation noted at the 72-Hour observation. Both treated eyes appeared normal at the 7-Day observation.

 

Mean individual scores at 24, 48 and 72 h after exposure for the 2 animals were 1.0 / 1.0 for cornea score; 0.66/ 0.33 for iris score; 2.0 / 2.0 for conjunctivae score and 1.66 / 1.66 for chemosis score.

 

Under the test conditions, the test item was classified as Eye Irr. Category 2 (H319: Causes serious eye irritation) according to the Regulation (EC) No. 1272/2008 (CLP) and as Eye Irr. Category 2B (H320: Causes eye irritation) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). The signal word "Warning" is required.

 

- In vitro eye irritation:

A supporting in vitro study was identified (Harlan, BCOP, 2013, rel. 1, SS).

In this in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, 0.75 mL of undiluted test item was applied to isolated bovine corneas for 10 minutes followed by an incubation period of two hours at 32 °C. Three corneas were used for undiluted test item, negative control (saline solution) and positive control (2-Ethoxyethanol). 

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item; the positive and the negative controls were applied to corneae and incubated for 10 minutes at 32 +/- 1°C. After the incubation phase the test item, positive and negative controls were rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 +/- 1°C in incubation medium, and opacity was measured a second time (t130). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 +/- 1°C.

 

With the negative control neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.44).

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 65.13) corresponding to a classification as corrosive/severe irritant to the eye (CLP/GHS Cat.1).

Relative to the negative control, the test item caused a slight increase of the corneal opacity and permeability. The calculated mean IVIS was 7.03 (threshold for corrosivity/severe irritancy: IVIS ≥ 55.1). According to OECD 437, the test item is classified as not corrosive/not severe irritant to the eye.

 

According to the current study and under the experimental conditions reported, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 and to the GHS.

Self classification:

Skin irritation:

Based on the available data, the test item is classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and the GHS. 

 

Eye irritation:

Based on the available data, the test item was classified as Eye Irr. Category 2 (H319: Causes serious eye irritation) according to the Regulation (EC) No. 1272/2008 (CLP) and as Eye Irr. Category 2B (H320: Causes eye irritation) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). The signal word "Warning" is required.

 

Respiratory irritation:

No data was available regarding respiratory irritation.