Registration Dossier

SKIN
Not irritating, rabbit, OECD 404, EU Method B.4, Pooles (2009)
EYES
Not irritating, rabbit, OECD 405, EU Method B.5, Pooles (2009)

Reference

Endpoint:

skin irritation / corrosion, other

Remarks:

in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-3 to 2009-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Hillcrest, Belton, Loughborough, UK
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.45 to 2.61 kg
- Housing: Individually housed in suspended cages
- Diet: 2030 Teklad Global Rabbit diet (Harlan Teklad, Blackthorn, Bicester, Oxon, UK) available ad libitum.
- Water: Mains drinking water available ad libitum.
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): A minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour cycle

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST SUBSTANCE
- Amount(s) applied : 0.5 g of test substance sufficiently moistened in 0.5 mL of distilled water to achieve a paste.

Duration of treatment / exposure:

Animal were exposed for 4 hours

Observation period:

Animals were observed for 72 hours for skin reactions

Number of animals:

3 animals in total (1 animal used in the initial test, with a further 2 animals in the main test.)

Details on study design:

TEST SITE
- Area of exposure: The test substance was introduced a 2.5 cm x 2.5 cm cotton gauze patch
- Type of wrap if used: The trunk of each rabbit was wrapped in an elasticated corset

REMOVAL OF TEST SUBSTANCE
- Washing : Any residual test substance was removed by gentle swabbing with cotton wool soaked in distilled water
- Time after start of exposure: 4 hours post administration of test substance

SCORING SYSTEM: Draize JH (1959) “Dermal Toxicity” In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Associations of Food and Drug Officials of the United States, Austin, Texas p.46-59

Irritation parameter:

overall irritation score

Basis:

mean

Remarks:

of three animals

Time point:

other: 24, 48 and 72 hours post dosing

Score:

0

Max. score:

8

Reversibility:

other: Not applicable, no effects were noted during the study.

Irritant / corrosive response data:

The test substance produced a primary irritation index of 0.0.

Other effects:

Not reported

Measurement of pH

The pH of the test substance was determined prior to commencement of the study and found to be as follows:

Preparation	pH Measurement
Preparation	immediately	after 10 minutes
10 % w/w aqueous preparation of the test substance	8.1	8.3

Table 1: Individual Skin Reactions

Skin Reaction	Observation Time (Following Patch Removal)	Individual Scores – Rabbit Number and Sex			Total
Skin Reaction	Observation Time (Following Patch Removal)	68639 Male	68640 Male	68655 Male	Total
Erythema/Eschar Formation	Immediately	0	0	0	(0)
	1 hour	0	0	0	(0)
	24 hours	0	0	0	0
	48 hours	0	0	0	(0)
	72 hours	0	0*	0*	0
Oedema Formation	Immediately	0	0	0	(0)
	1 hour	0	0	0	(0)
	24 hours	0	0	0	0
	48 hours	0	0	0	(0)
	72 hours	0	0*	0*	0
Sum of 24 and 72-hour Readings (S): 0
Primary Irritation Index (S/6): 0/6 = 0.0
Classification: Non-Irritant

() = total values not included in calculating the primary irritation index

Table 2: Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
Rabbit Number and Sex	Day 0	Day 3	Bodyweight Change (kg)
68639 Male	2.45	2.54	0.09
68640 Male	2.61	2.65	0.04
68655 Male	2.52	2.64	0.12

Interpretation of results:

not classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the study the test substance was determined to be not classified for skin corrosion and irritation.

Executive summary:

The skin corrosion and irritation potential of the test substance was determined in an in vivo study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 404 and EU Method B.4.

During the study, 0.5 g of test substance, sufficiently moistened in 0.5 mL of distilled water to achieve a paste, was applied to the shaved trunk of three male rabbits. The test substance was held in place under a semiocclusive dressing. Four hours following topical administration, any residual test substance was removed by gentle swabbing with cotton wool soaked in distilled water. Skin reaction to treatment was assessed over a period of 72 hours according to the Draize scale.

None of the animals died during the study and all animals gained weight. No erythema or oedema was observed following treatment with the test substance. Therefore, under the conditions of the study the test substance was determined to be not classified for skin corrosion and irritation.

Endpoint conclusion:

no adverse effect observed (not irritating)

Reference

Endpoint:

eye irritation, other

Remarks:

in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-09 to 2009-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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
- Source: Harlan Laboratories U.K. Limited, Hillcrest, Belton, Loughborough, UK
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.21 to 2.77 kg
- Housing: Animals were housed individually in suspended cages
- Diet: Free access to 2030 Teklad Global Rabbit diet (Harlan Teklad, Blackthorn, Bicester, Oxon, UK)
- Water: Free access to mains drinking water
- Acclimation period: A minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): At least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour cycle.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST SUBSTANCE
- Amount(s) applied : 100 mg

Duration of treatment / exposure:

Up to 72 hours

Observation period (in vivo):

Animals were assessed up to 72 hours after exposure (assessments were made at 1, 24, 48 and 72 hours post treatment).

Number of animals or in vitro replicates:

1 animal was initially treated; a further two animals were treated thereafter

Details on study design:

APPLICATION OF TEST SUBSTANCE:
Initially a single rabbit was treated with 100 mg of test substance. The test substance was administered 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 test substance and then released. The left eye served as a control and remained untreated. Immediately after administration of the test substance an assessment of the initial pain reaction was made. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

SCORING SYSTEM:
Assessment of ocular damage/irritation was made 1, 24, 48 and 72 hours post dosing using the scoring system from Draize JH (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. Any other ocular effects were also noted at these time points along with any clinical signs of toxicity, if present. Individual bodyweights were recorded on day 0 and 3. The numerical values corresponding to each animal, tissue and observation time were recorded. These scores were then assessed using a modified version of the system described by Kay JH and Calandra JC (1962), J. Soc. Cosmet. Chem. 13, 281-289.

TOOL USED TO ASSESS SCORE: standard ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

80

Reversibility:

other: not applicable - no corneal effects were noted

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

10

Reversibility:

other: not applicable - no iridial effects were noted

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

1.33

Max. score:

20

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

No corneal or iridial effects were noted at observations made at 24, 48 and 72 hours. Conjunctival effects were limited to mild (score 1) conjunctival redness, and mild (score 1) conjunctival chemosis, at 24 hours, in all three animals. These effects had completely resolved by the 48 hour observation in all three animals.

Other effects:

Not reported

Measurement of pH

The pH of the test substance was determined prior to commencement of the study and found to be as follows:

Preparation	pH Measurement
Preparation	immediately	after 10 minutes
10 % w/w aqueous preparation of the test substance	8.1	8.3

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

Rabbit Number and Sex	68647 Male				68665 Male				68666 Male
Rabbit Number and Sex	IPR = 2				IPR = 2				IPR = 2
Time After Treatment (hrs)	1	24	48	72	1	24	48	72	1	24	48	72
Cornea
E = Degree of opacity	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0
Iris
D	0	0	0	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctivae
A = Redness	2	1	0	0	2	1	0	0	2	1	0	0
B = Chemosis	2	1	0	0	2	1	0	0	2	1	0	0
C = Discharge	1	0	0	0	1	0	0	0	1	0	0	0
Score (A + B + C) x 2	10	4	0	0	10	4	0	0	10	4	0	0
Total Score	10	4	0	0	10	4	0	0	10	4	0	0

IPR = Initial pain reaction

The test substance produced a maximum group mean score of 10.0

To calculate the maximum group mean score, encompassing all reactions, the following equations were used:

Cornea = (E x F) x 5

Where:

E = Degree of opacity

F = Area of Cornea Involved

Iris = (D x 5)

Conjunctivae = (A + B + C) x 2

Where:

A = Redness

B = Chemosis

C = Discharge

Table 2: Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
Rabbit Number and Sex	1 hour	24 hours	48 hours	72 hours
68647 Male	10	4	0	0
68665 Male	10	4	0	0
68666 Male	10	4	0	0
Group Total	30	12	0	0
Group Mean Score	10.0	4.0	0.0	0.0

Table 3:Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
Rabbit Number and Sex	Day 0	Day 3	Bodyweight Change (kg)
68647 Male	2.21	2.25	0.04
68665 Male	2.40	2.50	0.10
68666 Male	2.77	2.88	0.11

Interpretation of results:

not classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the study the test substance was determined to be "not classified" for eye irritation.

Executive summary:

The eye irritation potential of the test material was determined in vivo, in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 405 and EU Method B.5.

During the 100 mg of test substance was instilled into one eye of each of 3 rabbits, the other eye remained untreated and served as the control. Signs of ocular reaction were assessed and recorded 1, 24, 48 and 72 hours following administration using the Draize scale.

No corneal or iridial effects were noted at observations made at 24, 48 and 72 hours. Conjunctival effects were limited to mild (score 1) conjunctival redness, and mild (score 1) conjunctival chemosis, at 24 hours, in all three animals. These effects had completely resolved by the 48 hour observation in all three animals.

Therefore, under the conditions of the study the test substance was determined to be "not classified" for eye irritation.

Endpoint conclusion:

no adverse effect observed (not irritating)

Endpoint conclusion:

no study available

Skin

In the key study, the skin corrosion and irritation potential of the test substance was determined in vivo. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 404 and EU Method B.4.

During the study, 0.5 g of test substance, sufficiently moistened in 0.5 mL of distilled water to achieve a paste, was applied to the shaved trunk of three male rabbits. The test substance was held in place under a semiocclusive dressing. Four hours following topical administration, any residual test substance was removed by gentle swabbing with cotton wool soaked in distilled water. Skin reaction to treatment was assessed over a period of 72 hours according to the Draize scale.

None of the animals died during the study and all animals gained weight. No erythema or oedema was observed following treatment with the test substance. Therefore, under the conditions of the study the test substance was determined to be not classified for skin corrosion and irritation.

Two in vitro studies are available the findings of which corroborate the results of the key study. In the first in vitro study, the skin corrosion potential of the test substance was investigated under GLP conditions and in accordance with the standardised guideline OECD 431.

During the study 20 mg test substance was applied topically to the reconstituted epidermis ensuring uniform coverage. At the end of the 3, or 60, minute exposure period each tissue was rinsed with Dulbecco's Phosphate Buffered Saline (DPBS) before being blotted and transferred to a well containing 300 µL of 0.5 mg/mL MTT solution. The tissues were then incubated at 37 °C, 5 % CO₂in air for 3 hours. At the end of the 3 hour MTT incubation period, the tissue cultures were blotted and transferred to pre-labelled 24-well plates for MTT extraction. The plates were left to stand overnight at room temperature to extract the reduced MTT (formazan dye) out of the tissue (ensuring the plate was covered to protect from light). At the end of the extraction period, each tissue was pierced and the extracted solution thoroughly mixed. For each tissue, triplicate 200 µL samples were taken.

The mean OD540 values of the triplicate wells, representing each SkinEthic RHE tissue, were automatically calculated by the plate reader. The mean OD540 values of the duplicate tissues were manually calculated. Each value had been corrected for blanks by the plate reader. For the test material, corrosivity potential was predicted from the relative mean tissue viabilities

Under the conditions of the study, the mean optical density of the test substance at 540 nm following a 3 and a 60 minute exposure was1.205 and 1.278, respectively. The mean % viability following a 3 and a 60 minute exposure was 96.9 and 92.5, respectively. Subsequently, the test substance was concluded to be "non corrosive" to skin.

In the second in vitro study, the skin irritation potential of the test substance was investigated under GLP conditions and in accordance with the standardised guideline EU Method B.46.

During the study 10 mg test substance was applied topically to the reconstituted epidermis ensuring uniform coverage. At the end of the 15 minute exposure period each tissue was rinsed with a Phosphate Buffered Saline solution containing Ca²⁺and Mg²⁺before being incubated for approximately 42 hours at 37 °C in 5% CO₂air. 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 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was then measured at 540 nm.

Under the conditions of the study, the mean optical density of the test substance at 540 nm was0.842 while the mean % viability was 111.5. Subsequently, the test substance was concluded to be "not irritating" to skin.

Since all studies were conducted under GLP conditions and in accordance with standardised guidelines all three studies were assigned a reliability score of 1 in line with the criteria of Klimisch (1997).

Eyes

In the key study, the eye corrosion and irritation potential of the test substance was determined in vivo. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 405 and EU Method B.5.

During the study, 100 mg of test substance was instilled into one eye of each of 3 rabbits, the other eye remained untreated and served as the control. Signs of ocular reaction were assessed and recorded 1, 24, 48 and 72 hours following administration using the Draize scale.

No corneal or iridial effects were noted at observations made at 24, 48 and 72 hours. Conjunctival effects were limited to mild (score 1) conjunctival redness, and mild (score 1) conjunctival chemosis, at 24 hours, in all three animals. These effects had completely resolved by the 48 hour observation in all three animals.

Therefore, under the conditions of the study the test substance was determined to be "not classified" for eye irritation.

An in vitro study is available, the findings of which corroborate the results of the key study. In the in vitro study, the eye irritation potential of the test substance was determined under GLP conditions.

During the study Reconstituted Corneal Epithelium were exposed to 30 mg of test substance for a period of 10 minutes after which time the cultures were rinsed with Dulbecco's Phosphate Buffered Saline. After rinsing, the tissues (two per group) were transferred into a pre-labelled 24-well plate containing 300 µL of a 0.5 mg/mL MMT solution prepared in maintenance medium. The MMT loading plate was placed into an incubator for approximately three hours at 37 °C, 5 % CO2 in air. After incubation with MMT, the tissues were visually examined and the degree of MMT staining was evaluated. The inserts were blotted on absorbent paper to remove residual MMT and transferred to a pre-labelled 24-well plate containing 0.75 mL of isopropanol in each of a sufficient number of wells. An extra 0.75 mL of isopropanol was added onto each tissue and the plate sealed to prevent isopropanol evaporation. The plate was wrapped in aluminium foil and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissues.

At the end of the extraction period, each tissue was pierced and the extracted solution thoroughly mixed. For each tissue, triplicate 200 µL samples were taken. The optical density was measured (quantitative measurement of tissue viability) at 540 nm (OD540) using Anthos 2001 microplate reader.

Under the conditions of the study, the mean optical density of the test substance at 540 nm was 0.994 while the mean % viability was 98.4. Subsequently, the test substance was concluded to be "not irritating" to eyes.

The key study was conducted under GLP conditions and in accordance with standardised guidelines; it was therefore assigned a reliability score of 1 in line with the criteria of Klimisch (1997). The supporting study was conducted under GLP conditions and followed sound scientific principles, it was therefore assigned a reliability score of 2.

Justification for selection of skin irritation / corrosion endpoint:
The key study is an in vivo study. Two additional in vitro studies are available which support the findings of the in vivo study.

Justification for selection of eye irritation endpoint:
The key study is an in vivo study. An additional in vitro study is available which supports the findings of the in vivo study.

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to skin and eye irritation.