Registration Dossier

SKIN
Not irritating (rabbit); OECD 404 and EU method B.4
EYE
Not irritating (rabbit); OECD 405 and EU method B.5

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 February 2006 to 10 February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute 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: David Percival Ltd., Moston, Sandbach, Cheshire, UK
- Age at study initiation: 12 - 20 weeks
- Weight at study initiation: 2.0 - 3.5 kg
- Housing: individually housed in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study. 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.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
Any occasional deviations from the above targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 7 February 2006 To: 10 February 2006

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: moistened with distilled water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g moistened with 0.5 ml of distilled water
- Concentration (if solution): N/A

Duration of treatment / exposure:

4 h

Observation period:

72 hours

Number of animals:

3 male

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm cotton gauze patch secured with surgical adhesive tape
- % coverage: NDA
- Type of wrap if used: the trunk of each rabbit was wrapped in an elasticated corset

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after exposure the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: four hours after application

SCORING SYSTEM: Draize scheme

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

See table below for raw data.

Other effects:

None

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 material. The test material was classified according to the following scheme devised by Draize J H (1959) “Dermal Toxicity” In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the United States, Austin, Texas, p. 47:

Primary Irritation Index Classification of Irritancy

0 Non-irritant

>0 to 2 Mild irritant

>2 to 5 Moderate irritant

>5 to 8 Severe irritant

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test material is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.

Individual skin reactions

Skin Reaction	Observation Time	Individual Scores – Rabbit Number and Sex			Total
		24 Male	25 Male	26 Male
Erythema/Eschar Formation	1 Hour 24 Hours 48 Hours 72 Hours	0 0 0 0	0 0 0 0	0 0 0 0	(0) 0 (0) 0
Oedema Formation	1 Hour 24 Hours 48 Hours 72 Hours	0 0 0 0	0 0 0 0	0 0 0 0	(0) 0 (0) 0
Sum of 24 and 72-hour Readings (S): 0
Primary Irritation Index (S/6): 0
Classification: Non-irritant

( ) = Total values not used for calculation of primary irritation index

Interpretation of results:

other: not classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test material produced a primary irritation index of 0.0 and was classified as non-irritant to rabbit skin according to the Driaze classification scheme. No corrosive effects were noted.

Executive summary:

In a dermal irritation study (0656/0281), male New Zealand White rabbits were dermally exposed to 0.5 g of the test material moistened with distilled water for 4 hours to a 2.5 cm x 2.5 cm area of skin. Animals then were observed for 3 days. Irritation was scored by the method of the Draize classification scheme.

No effects were observed. In this study, the test material is not classified as a dermal 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:

13 February 2006 to 23 February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute 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: David Percival Ltd., Moston, Sandbach, Cheshire, UK
- Age at study initiation: 12 - 20 weeks
- Weight at study initiation: 2.0 - 3.5 kg
- Housing: individually housed in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study. 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.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
Any occasional deviations from the above targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 13 February 2006 To: 23 February 2006

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml (found to weigh approximately 23 mg)
- Concentration (if solution): N/A

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Duration of treatment / exposure:

The test substance was instilled in the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control. One single exposure, eye was not washed out after exposure. Immediately after administration of the test material, 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.

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation from 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.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard opthalmoscope.

Number of animals or in vitro replicates:

3 male

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): None
- Time after start of exposure: N/A

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: standard opthalmoscope

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

other: overall at 24, 48 and 72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

See table below for raw data.
Reversibility of any observed effect: Changes fully reversible within 1 day

Other effects:

None

The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae = (A + B + C) x 2

Score for iris = D x 5

Score for cornea = (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J H and Calandra J C (1962), J. Soc. Cosmet. Chem.,13, 281-289 was used to classify the ocular irritancy potential of the test material. 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 eye irritancy potential of the test material.

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

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	95 Male				24 Male				25 Male
Rabbit Number and Sex	IPR = 2				IPR = 2				IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA E = Degree of Opacity F = Area of Cornea Involved	0 0	0 0	0 0	0 0	0 0	0 0	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 B = Chemosis C = Discharge	1 0 1	0 0 0	0 0 0	0 0 0	0 0 0Re	0 0 0	0 0 0	0 0 0	0 0 0Re	0 0 0	0 0 0	0 0 0
Score (A + B + C) x 2	4	0	0	0	0	0	0	0	0	0	0	0
Total Score	4	0	0	0	0	0	0	0	0	0	0	0

IPR = Initial pain reaction

Re = White residual test material noted in the treated eye

Interpretation of results:

other: not classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test material produced a maximum group mean score of 1.3 and was classified as "practically non-irritant (Class 2 on a 1 to 8 scale)" to the rabbit eye according to a modified Kay and Calandra classification scheme.

Executive summary:

In a primary eye irritation study (0656/0282), ca. 23 mg of neat test material was instilled into the conjunctival sac of the right eye of a male New Zealand White rabbit. The eye was not washed after treatment. The animal was then observed for 72 hours. Irritation was scored by the method of the Draize scoring system.

In the three animals tested, the test material only produced minimal conjunctival irritation in one treated eye one hour after treatment. Two treated eyes appeared normal throughout the study and the remaining treated eye appeared normal at the 24-hour observation. In this study, the test material should not be classified.

Endpoint conclusion:

no adverse effect observed (not irritating)

Endpoint conclusion:

no study available

Skin irritation

The irritation potential of the test material to the rabbit was investigated in accordance with the standardised guidelines OECD 404 and EU Method B.4 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Three male New Zealand White rabbits were exposed to 0.5 g of the test material moistened with 0.5 mL of distilled water for 4 hours in a semi-occlusive fashion. Animals were then observed for 72 hours. Irritation was scored by the method of the Draize classification scheme.

No effects were observed, therefore under the conditions of this study, the test material is not considered to be a dermal irritant.

Eye irritation

The irritation potential of the test material to the rabbit was investigated in accordance with the standardised guidelines OECD 405 and EU Method B.5 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Three male New Zealand White rabbits were exposed to 0.1 mL of the neat test material instilled into the conjunctival sac of each right eye. The eyes were not washed after treatment and the animals were observed for 72 hours. Irritation was scored by the method of the Draize scoring system.

In the three animals tested, the test material only produced minimal conjunctival irritation in one treated eye one hour after treatment. Two treated eyes appeared normal throughout the study and the remaining treated eye appeared normal at the 24-hour observation.

Under the conditions of this study, the test material is not considered to be an ocular irritant.

Justification for selection of skin irritation / corrosion endpoint:
Only one study available.

Justification for selection of eye irritation endpoint:
Only one study available.

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.