Registration Dossier

SKIN
Not irritating, OECD 404, EU Method B.4, Pooles (2010)
EYE
Not irritating, OECD 405, EU Method B.5, Pooles (2010)

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 December 2009 to 6 December 2009

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 minor 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 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: 2.05 to 2.74 kg
- Housing: Individually housed in suspended cages
- Diet: 2030 Teklad Global Rabbit diet (Harlan Teklad, Blackthorn, Bicester, Oxon, UK) avilable 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:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

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

Duration of treatment / exposure:

Animals 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 under 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:

primary dermal irritation index (PDII)

Basis:

mean

Remarks:

of 3 animals

Time point:

other: 24 and 72 hours post dosing

Score:

0

Max. score:

8

Reversibility:

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

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by six to give the primary irritation index of the test substance.

The test material 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 test substance	7.9	7.9

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)	68706 Male	68707 Male	68708 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)
68706 Male	2.05	2.08	0.03
68707 Male	2.74	2.78	0.04
68708 Male	2.26	2.31	0.05

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance produced a primary irritation index of 0.0 and was classified as a non-irritant to rabbit skin. No corrosive effects were noted.

Executive summary:

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

During the study no signs of skin irritation were noted at any of the observation points. The test substance was therefore concluded to be a non-irritant.

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:

7 December 2009 to 17 December 2009

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 minor 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: 2.67 to 2.78 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, with a further two animals

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 and 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 this point 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. If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

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

Remarks on result:

other: No corneal effects observed

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

10

Reversibility:

other: not applicable

Remarks on result:

other: No iridial effects observed

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

4

Max. score:

20

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

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

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	7.9	7.9

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

Rabbit Number and Sex	68709 Male				68732 Male				68733 Male
Rabbit Number and Sex	IPR = 2				IPR = 3				IPR = 2
Time After Treatment	1 Hr	24 Hrs	48 Hrs	72 Hrs	1 Hr	24 Hrs	48 Hrs	72 Hrs	1 Hr	24 Hrs	48 Hrs	72 Hrs
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	6	0	0	10	4	0	0	10	4	0	0

IPR = Initial pain reaction

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
68709 Male	10	4	0	0
68732 Male	10	4	0	0
68733 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)
68643 Male	2.78	2.92	0.14
68649 Male	2.70	2.79	0.09
68667 Male	2.67	2.73	0.06

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Slight conjunctival redness and chemosis (score 1) was observed 24 hours following administration of the test substance; all treated eyes appeared normal 48 hours after administration. Under the conditions of the study the test material was determined to be not irritating to eyes.

Executive summary:

The eye irritation potential of the test substance was investigated 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.

No corneal or iridial effects was noted during the study. Moderate conjuctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24-hour observation. Under the conditions of the study the test material was determined to be not irritating to eyes.

Endpoint conclusion:

no adverse effect observed (not irritating)

Skin

In vitro testing concluded that the test substance was unlikely to be irritating to skin following exposure. An in vivo test was subsequently performed to confirm findings of the in vitro study.

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

During the study no signs of skin irritation were noted at any observation point. The test material was therefore concluded to be a non-irritant to the skin.

Eye

In vitro testing concluded that the test substance was unlikely to be an eye irritant. An in vivo test was subsequently performed to confirm findings of the in vitro study.

The eye irritation potential of the test substance was investigated 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.

No corneal or iridial effects were noted during the study. Moderate conjuctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24-hour observation. The test substance was therefore concluded to be not irritating to eyes.

Justification for selection of skin irritation / corrosion endpoint:
The key study was selected as such because it represents the only available in vivo study.

Justification for selection of eye irritation endpoint:
The key study was selected as such because it represents the only available 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 either skin or eye irritation.