Registration Dossier

Skin Irritation: Non-irritating, OECD 439 and EU Method B.46, Warren 2012.

Skin Corrosion: Non-corrosive, OECD 431 and EU Method B.40, Warren 2012.

Eye Irritation: Irritant (Category 2), OECD 405 and EU Method B.5, Sanders 2013.

Endpoint conclusion:

no adverse effect observed (not irritating)

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 November 2012 to 25 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 - 20 weeks old
- Weight at study initiation: 2.48 or 2.58 kg
- Housing: individually housed in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

Vehicle:

unchanged (no vehicle)

Controls:

other: the left eye of each animal remained untreated and was used for control purposes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL, weighing approximately 59 mg (as measured by gently compacting the required volume into an adapted syringe)

Duration of treatment / exposure:

Single exposure

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): None
- Time after start of exposure: Not applicable

SCORING SYSTEM: Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49

TOOL USED TO ASSESS SCORE: standard opthalmoscope

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

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritant / corrosive response data:

Scattered or diffuse corneal opacity was noted in both treated eyes at the 24 and 48-Hour observations and in one treated eye at the 72-Hour observation.
lridial inflammation was noted in both treated eyes one hour after treatment and at the 24, 48 and 72-Hour observations.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment. Severe conjunctival irritation was noted in one treated eye with moderate conjunctival irritation noted in the other treated eye at the 24-Hour observation. Moderate conjunctival irritation was noted in both treated eyes at the 48 and 72-Hour observations. Moderate conjunctival irritation persisted in one treated eye with minimal conjunctival irritation noted in the other treated eye at the 7-Day observation. Minimal conjunctival irritation was noted in one treated eye at the 14-Day observation.
One treated eye appeared normal at the 14-Day observation and the other treated eye appeared normal at the 21-Day observation.

Other effects:

Both animals showed expected gain in bodyweight during the study.

Table 1: Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	72696 Male						72767 Male
Rabbit Number and Sex	IPR = 2						IPR = 2
Time After Treatment	1 hr	24 hr	48 hr	72 hr	7 days	14 days	1 hr	24 hr	48 hr	72 hr	7 days	14 days	21 days
CORNEA E = Degree of Opacity F = Area of Opacity	0 0	1 2	1 2	1 1	0 0	0 0	0 0	1 1	1 1	0 0	0 0	0 0	0 0
Score (E x F) x 5	0	10	10	5	0	0	0	5	5	0	0	0	0
IRIS D	1	1	1	1	0	0	1	1	1	1	0	0	0
Score (D x 5)	5	5	5	5	0	0	5	5	5	5	0	0	0
CONJUNCTIVAE A = Redness B = Chemosis C = Discharge	2 3 2	2 3 3	2 2 1	2 1 1	1 1 1	0 0 0	2 2 3	2 2 3	2 2 1	2 2 1	1 2 1	1 1 0	0 0 0
Score (A + B + C) x 2	14	16	10	8	6	0	14	14	10	10	8	4	0
Total Score	19	31	25	18	6	0	19	24	20	15	8	4	0

Key: hr = hour(s) IPR = initial pain reaction

Interpretation of results:

Category II

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, the test material was determined to be irritating to the eye based on iritis, conjunctival redness and chemosis.

Executive summary:

An in vivo study was performed under GLP conditions to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. The method was designed to be compatible with OECD Guideline 405 and EU Test Method B.5.

A single application of the test item to the non-irrigated eye of two rabbits produced scattered or diffuse corneal opacity, iridial inflammation and moderate to severe conjunctival irritation. One treated eye appeared normal at the 14-Day observation and the other treated eye appeared normal at the 21-Day observation.

Under the conditions of the test, the test material was determined to be irritating to the eye based on iritis, conjunctival redness and chemosis.

Endpoint conclusion:

adverse effect observed (irritating)

Endpoint conclusion:

no study available

Skin irritation:

Two key in vitro studies are available to address this endpoint. The studies were performed to GLP and in accordance with recognised guidelines and as such have been assigned a reliability score of 1 in line with the principles for assessing data quality defined in Klimisch (1997).

The first test was conducted to evaluate the corrosivity potential of the test material using the EPISKIN^TM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to be compatible with OECD Test Guideline 431 and EU Test Method B.40.

The EPISKIN^TM 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 materials.

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test material 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-labelled 96-well plate. The optical density (OD) was measured at 540 nm (OD540).

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

The relative mean viability of the test material treated tissues was:

240 minutes exposure 90.3%

60 minutes exposure 89.5%

3 minutes exposure 108.4%

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

The test material was considered to be Non-Corrosive to the skin.

A second test was conducted to evaluate the skin irritation potential of the test material using the EPISKIN^TM Reconstructed Human Epidermis (RHE) 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 material by means of the colourimetric 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 material treated tissues, relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This test was designed to be compatible with OECD Guideline 439 and EU Test Method B.46.

Triplicate tissues were treated with the test material 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-labelled 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-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

The relative mean viability of the test material treated tissues was 102.1% after the 15-Minute exposure period.

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

The test material was considered to be Non-Irritant (NI).

The in vivo skin irritation test was waived, as adequate in vitro data is already available. The in vitro data available, generated using a validated test method, is adequate for the purpose of classification and labelling and is adequately and reliably documented in the study report. Furthermore a reliable acute dermal toxicity study ("Experimental ALC20110128 EXP1: Acute Dermal Toxicity (Limit Test) in the Rat" Sanders 2013, Study No. 41301569) is to be used as supporting information as it sufficiently covers the key parameters for evaluation and classification of skin irritation in vivo. This study is to be used as supporting information only, since the study was performed using rats and the preferred species for skin irritation testing is the rabbit. Based on all available information further testing for skin irritation in vivo was deemed unnecessary.

The acute dermal toxicity study (Sanders 2013, Harlan Laboratories LTD, study number: 41301569) submitted as part of this registration can be used to support the in vitro skin irritation studies. This study was performed under GLP conditions and in line with standardised guidelines OECD 402 and EU Method B3. A group of ten animals (five males and five females) was given a single, 24 hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg body weight. Mortality, clinical signs and body weight development were monitored during the study. Local dermal reaction was scored daily for 14 days after patch removal according to the Draize scale (1977). All animals were subjected to gross necropsy.

Under the conditions of the test no mortality or systemic signs of toxicity were observed. Animals displayed overall expected weight gain and no abnormalities were observed at necropsy. The observed local dermal reactions are shown below in Tables 1 and 2. There were no signs of dermal irritation noted at the test site of one male. Very slight erythema was noted at the test sites of four males (grade 1) which was reversible by observation day 8. Well-defined erythema (grade 1-2) and very slight edema (grade 1) were noted at the test sites of all females, where erythema was reversible by day 14 and edema by day 3. Other signs of dermal irritation noted at the test sites of females were crust formation, small superficial scattered scabs and scab lifting to reveal glossy skin. Based on the criteria for classification of skin irritation as defined in Annex I of Regulation 1272/2008 the test material should not be classified since the mean value for erythema or edema did not exceed 2.3 at grading taken at 24, 48 and 72 hours.

Table 2: Dermal Skin Irritation Reaction According to the Draize Scale in Male Rats

Dose Level (mg/kg)	Animal No.	Observation	Days After Exposure
Dose Level (mg/kg)	Animal No.	Observation	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0	Erythema	1	1	1	1	1	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-1	Erythema	1	1	1	1	1	1	1	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2	Erythema	1	1	1	1	1	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-3	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-4	Erythema	1	1	1	1	1	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0 = No reaction

Table 2: Dermal Skin Irritation Reaction According to the Draize Scale in Female Rats

Dose Level (mg/kg)	Animal No.	Observation	Days After Exposure
Dose Level (mg/kg)	Animal No.	Observation	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	2-0	Erythema	2	2	1	1	1	1	1	0	0	0	0	0	0	0
		Edema	1	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	Cf	Cf	Cf	0	0	0	0	0	0	0
	2-1	Erythema	2	2	1	1	1	1	1	0	0	0	0	0	0	0
		Edema	1	1	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	Cf, Sg	Cf, Ss	Cf, Ss	Ss	Ss	Ss	Ss	Ss	Ss	Ss
	2-2	Erythema	2	2	2	2	2	2	2	1	1	1	1	1	1	0
		Edema	1	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	Cf, Sg	Cf, Sg	Cf, Sg	Ss, Sg	Ss, Sg	Ss, Sg	Ss, Sg	Ss, Sg	Ss, Sg	Ss
	2-3	Erythema	2	2	2	2	1	1	1	1	1	1	1	1	1	0
		Edema	1	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	0-4	Erythema	2	1	1	1	1	1	1	0	0	0	0	0	0	0
		Edema	1	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0 = No reaction

Cf = Crust Formation

Ss = Small superficial scabs

Sg = Cabs lifting to reveal glossy skin

Eye irritation:

One key in vivo study and one supporting in vitro study are available to address this endpoint. The key study was performed to GLP and in accordance with recognised guidelines and as such has been assigned a reliability score of 1 in line with the principles for assessing data quality defined in Klimisch (1997).

The key in vivo study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. The method was designed to be compatible with OECD Guideline 405 and EU Test Method B.5.

A single application of the test item to the non-irrigated eye of two rabbits produced scattered or diffuse corneal opacity, iridial inflammation and moderate to severe conjunctival irritation. One treated eye appeared normal at the 14-Day observation and the other treated eye appeared normal at the 21-Day observation.

Under the conditions of the test, the test material was determined to be irritating to the eye based on iritis, conjunctival redness and chemosis.

The supporting in vitro study was performed to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

The experimental design of the study consists of a test for direct reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test item for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 2% w/v Sodium Dodecyl Sulphate (SDS) served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test item was classified according to the following criteria:

i) If the percentage relative mean tissue viability was ≥60% the test item was considered to be non-irritant (NI).

ii) If the percentage relative mean tissue viability was <60% the test item was considered to be an irritant (I).

The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 62.7%.

It was considered unnecessary to proceed with tissue histopathology.

The quality criterion required for acceptance of results in the test was satisfied.

According to the study plan followed, the test item was considered to be a Non-Irritant (NI).

Justification for selection of skin irritation / corrosion endpoint:

In vivo testing for this endpoint is scientifically unjustified as adequate in vitro data is available. The key studies were performed in vitro, in line with GLP and OECD guidelines. They were assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). As the in vitro data available was generated using validated test methods, is adequate for the purpose of classification and labelling and is adequately and reliably documented in the study reports, they were therefore considered suitable to be the key studies for this endpoint.

Justification for selection of eye irritation endpoint:

The key study was performed in vivo, in line with GLP and OECD guidelines. It was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). It was therefore considered suitable to be the key study for this endpoint.

Effects on eye irritation: irritating

Skin Irritation:

According to the criteria outlined in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification as a skin irritant.

Eye Irritation:

According to the criteria outlined in Regulation (EC) No. 1272/2008, the test material meets the criteria for classification as a Category 2 eye irritant with the resultant hazard phrase, H319: Causes serious eye irritation.