Registration Dossier

Skin

Non irritating, rabbit (male/female), Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US AFDO, Sachsse (1973)

Eye

Non irritating, rabbit, (male/female), Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US AFDO, Sachsse (1973)

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO)

Deviations:

no

GLP compliance:

no

Remarks:

Study conducted in 1973, prior to inception of GLP

Specific details on test material used for the study:

- Physical form of test material: Not reported; the report implies that it is a liquid.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2 to 3 kg
- Housing: Kept separately
- Diet: ad libitum
- Water: ad libitum

Type of coverage:

occlusive

Preparation of test site:

other: shaved and slightly scarified

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

The test material was applied to each site (intact and slightly scarified) in quantities of 0.5 mL.

Duration of treatment / exposure:

24 hours

Observation period:

72 hours

Number of animals:

Six rabbits (3 males/3 females)

Details on study design:

TEST SITE
- Pre-treatment: The rabbits were shaved on the whole back and flanks two days before treatment with an electric clipper. The shaven skin on the left side was slightly scarified immediately before treatment.
- Area of exposure: 2.5 x 2.5 cm gauze pads were soaked with the test material and applied to the skin.
- Type of wrap if used: The gauze patch was covered with a plastic film 5 x 5 cm, which was fixed to the body with adhesive tape.

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: The gauze patches were removed 24 hours after application.

SCORING SYSTEM
- Erythema and Eschar Formation
No erythema: 0
Very slight erythema (barely perceptible): 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4

- Oedema Formation
No oedema: 0
Very slight oedema (barely perceptible): 1
Slight oedema (edges of area well defined by definite raising: 2
Moderate oedema (raised approximately 1 mm): 3
Severe oedema (raised more than 1 mm and extending beyond area of exposure): 4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of 6 animals

Time point:

other: Mean of 24 and 72 hour time points

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Intact skin

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of 6 animals

Time point:

other: Mean of 24 and 72 hour time points

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Scarified skin

Irritation parameter:

edema score

Basis:

mean

Remarks:

of 6 animals

Time point:

other: Mean of 24 and 72 hour time points

Score:

0.17

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Intact skin

Irritation parameter:

edema score

Basis:

mean

Remarks:

of 6 animals

Time point:

other: Mean of 24 and 72 hour time points

Score:

0.25

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Scarified skin

Irritant / corrosive response data:

Signs of erythema were observed on both the intact and abraded skin of 4 animals at the 24 hour observation; all reactions were scored as 1. All signs were fully reversible by the 72 hour observation. The remaining two animals exhibited no signs of erythema.

Signs of oedema were observed on both the intact and abraded skin of 2 animals at the 24 hour observation; three of the reactions were scored as 1 and the scarified skin site of one rabbit was scored as a 2. All signs were fully reversible by the 72 hour observation. The remaining four animals exhibited no signs of oedema.

Table 1: Evaluation of Skin Reactions

Animal No. and Sex		Skin Reaction	24 hours after application		72 hours after application
Animal No. and Sex		Skin Reaction	Intact Skin	Scarified Skin	Intact Skin	Scarified Skin
1	M	Erythema	1	1	0	0
1	M	Oedema	1	2	0	0
2	M	Erythema	1	1	0	0
2	M	Oedema	1	1	0	0
3	M	Erythema	0	0	0	0
3	M	Odema	0	0	0	0
4	F	Erythema	0	0	0	0
4	F	Oedema	0	0	0	0
5	F	Erythema	1	1	0	0
5	F	Oedema	0	0	0	0
6	F	Erythema	1	1	0	0
6	F	Oedema	0	0	0	0

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material was determined not to be a skin irritant to rabbits.

Executive summary:

The irritation/corrosion potential of the test material was investigated following the patch-test technique described in the “Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics” (1959) of the US Association of Food and Drug Officials (AFDO).

Six rabbits (3 male/3 female) of the New Zealand White strain were shaved on the whole back and flanks two days prior to treatment; the left side was slightly scarified immediately before treatment. A gauze patch soaked with the 0.5 mL of the test material was immediately applied to the prepared skin (both shaved and scarified sites). The patch was covered with a plastic film which was fixed to the body with adhesive tape. The gauze patches were removed 24 hours after the application. The reaction of the skin was appraised upon removal and 72 hours after application.

Signs of erythema were observed on both the intact and abraded skin of 4 animals at the 24 hour observation; all reactions were scored as 1. All signs were fully reversible by the 72 hour observation. The remaining two animals exhibited no signs of erythema.

Signs of oedema were observed on both the intact and abraded skin of 2 animals at the 24 hour observation; three of the reactions were scored as 1 and the scarified skin site of one rabbit was scored as a 2. All signs were fully reversible by the 72 hour observation. The remaining four animals exhibited no signs of oedema.

Under the conditions of this study, the test material was determined not to be a skin irritant to rabbits.

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:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics (1959) of the US Association of Food and Drug Officials (AFDO).

Deviations:

no

GLP compliance:

no

Remarks:

Study conducted in 1973, prior to inception of GLP

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Housing: Kept separately
- Diet: ad libitum
- Water: ad libitum

Vehicle:

unchanged (no vehicle)

Controls:

other: The right eye remained untreated and served as a control.

Amount / concentration applied:

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

Duration of treatment / exposure:

The test material was introduced into the conjunctival sac of the left eye with a syringe. After application, the eyelids were held open for a few seconds.

Observation period (in vivo):

24 hours and 2, 3, 4 and 7 days post instillation.

Number of animals or in vitro replicates:

Six rabbits (3 male/3 female)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eyes of three rabbits were each rinsed with 10 mL lukewarm water.
- Time after start of exposure: About 30 seconds after treatment.

TOOL USED TO ASSESS SCORE: hand-slit lamp

SCORING SYSTEM:

CORNEA
Opacity: degree of density (area most dense is taken for reading)
0: No ulceration or opacity
1: Scattered or diffuse areas of opacity, details of iris clearly visible
2: Easily discernible translucent areas, details of iris slightly obscured
3: Opalescent areas, no details of iris visible, size of pupil barely discernible
4: Opaque cornea, iris not discernible through the opacity

IRIS
0: Normal
1: Folds above normal, congestion, swelling, circumcorneal injection (any of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive)
2: No reaction to light, haemorrhage, gross destruction (any or all of all these)

CONJUNCTIVAE
- Redness: (refers to the most severe effect of palpebral and bulbar conjunctivae excluding cornea and iris)
0: Blood vessels normal
1 Vessels definitely injected above normal
2: More diffuse, deeper crimson red, individual vessels not easily discernible
3: Diffuse beefy red

- Chemosis: lids and/or nictating membranes.
0: No swelling
1: Any swelling above normal (includes nictating membranes)
2: Obvious swelling with partial eversion of lids
3: Swelling with lids about half closed
4: Swelling with lids about half closed to completely closed

DISCHARGE
0: No discharge
1: Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)
2: Discharge with moistening of the lids and hairs just adjacent to lids
3: Discharge with moistening of the lids and hairs a considerable area around the eye

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: Mean of 24, 48 and 72 hour observations

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Eyes rinsed

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: Mean of 24, 48 and 72 hour observations

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other: Eyes not rinsed

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: Mean of 24, 48 and 72 hour observations

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: Eyes rinsed

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: Mean of 24, 48 and 72 hour observations

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Remarks on result:

other: Eyes not rinsed

Irritation parameter:

other: combined redness, chemosis and discharge scores

Basis:

mean

Remarks:

of 3 animals

Time point:

other: Mean of 24, 48 and 72 hour observations

Score:

1.33

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: Eyes rinsed. The redness, chemosis and discharge scores were reported as one combined figure; it was not possible to assign a value to each of the individual parameters.

Irritation parameter:

other: combined redness, chemosis and discharge scores

Basis:

mean

Remarks:

of 3 animals

Time point:

other: Mean of 24, 48 and 72 hour observations

Score:

1.33

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Eyes not rinsed. The redness, chemosis and discharge scores were reported as one combined figure; it was not possible to assign a value to each of the individual parameters.

Irritant / corrosive response data:

No corneal or iridial effects were observed in any rabbit at any time point.
Day 1 after application of the test material, five out of the six rabbits (3 with rinsed eyes and 2 with not rinsed eyes) demonstrated signs of eye irritation (redness, chemosis and discharge); one score was reported for each animal for the three parameters. The maximum combined score was 5.
On Day 2, the signs of irritation in four animals were completely resolved. The remaining animal still exhibiting signs of irritation (eyes not rinsed) received a combined score of 3 for redness, chemosis and discharge. These signs were completely resolved by 72 hours post installation of the test material.

Table 1: Summary of Individual Scores

	Eyes rinsed									Eyes not rinsed
Days after Application	Rabbit 1 (F)			Rabbit 2 (F)			Rabbit 3 (F)			Rabbit 4 (M)			Rabbit 5 (M)			Rabbit 6 (M)
Days after Application	Cornea	Iris	Conjunct.	Cornea	Iris	Conjunct.	Cornea	Iris	Conjunct.	Cornea	Iris	Conjunct.	Cornea	Iris	Conjunct.	Cornea	Iris	Conjunct.
1	0	0	3	0	0	5	0	0	4	0	0	5	0	0	4	0	0	0
2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	6	0	0	0
3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
7	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Conjunct. = The combined score for redness, chemosis and discharge.

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material was determined not to be an eye irritant to rabbits.

Executive summary:

The potential of the test material to cause eye irritation or corrosion was investigated following the technique described in the “Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics” (1959) of the US Association of Food and Drug Officials (AFDO).

The test material (0.1 mL) was administered into the left eye of 6 New Zealand White rabbits (3 male/3 female). The right eye remained untreated and served as a control. The eyes of the female rabbits were rinsed approximately 30 seconds after administration with lukewarm water.

The reactions were appraised with a slit-lamp after 24 hours and after 2, 3, 4 and 7 days.

No corneal or iridial effects were observed in any rabbit at any time point. Day 1 after application of the test material, five out of the six rabbits (3 with rinsed eyes and 2 with not rinsed eyes) demonstrated signs of eye irritation (redness, chemosis and discharge). On day 2, the signs of irritation in four animals were completely resolved. One remaining animal still exhibited signs of irritation (eyes not rinsed); these signs were completely resolved by 72 hours post installation of the test material.

Under the conditions of this study, the test material was determined not to be an eye irritant to rabbits.

Endpoint conclusion:

no adverse effect observed (not irritating)

Endpoint conclusion:

no study available

Skin Irritation

The irritation/corrosion potential of the test material was investigated following the patch-test technique described in the “Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics” (1959) of the US Association of Food and Drug Officials (AFDO). The study was assigned a reliability score of 2 in accordance with the principles for assessing data quality set forth by Klimisch et al. (1997).

Six rabbits (3 male/3 female) of the New Zealand White strain were shaved on the whole back and flanks two days prior to treatment; the left side was slightly scarified immediately before treatment. A gauze patch soaked with the 0.5 mL of the test material was immediately applied to the prepared skin (both shaved and scarified sites). The patch was covered with a plastic film which was fixed to the body with adhesive tape. The gauze patches were removed 24 hours after the application. The reaction of the skin was appraised upon removal and 72 hours after application.

Signs of erythema were observed on both the intact and abraded skin of 4 animals at the 24 hour observation; all reactions were scored as 1. All signs were fully reversible by the 72 hour observation. The remaining two animals exhibited no signs of erythema.

Signs of oedema were observed on both the intact and abraded skin of 2 animals at the 24 hour observation; three of the reactions were scored as 1 and the scarified skin site of one rabbit was scored as a 2. All signs were fully reversible by the 72 hour observation. The remaining four animals exhibited no signs of oedema.

Under the conditions of this study, the test material was determined not to be a skin irritant to rabbits.

Eye Irritation

The potential of the test material to cause eye irritation or corrosion was investigated following the technique described in the “Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics” (1959) of the US Association of Food and Drug Officials (AFDO). The study was assigned a reliability score of 2 in accordance with the principles for assessing data quality set forth by Klimisch et al. (1997).

The test material (0.1 mL) was administered into the left eye of 6 New Zealand White rabbits (3 male/3 female). The right eye remained untreated and served as a control. The eyes of the female rabbits were rinsed approximately 30 seconds after administration with lukewarm water.

The reactions were appraised with a slit-lamp after 24 hours and after 2, 3, 4 and 7 days.

No corneal or iridial effects were observed in any rabbit at any time point. Day 1 after application of the test material, five out of the six rabbits (3 with rinsed eyes and 2 with not rinsed eyes) demonstrated signs of eye irritation (redness, chemosis and discharge). On day 2, the signs of irritation in four animals were completely resolved. One remaining animal still exhibited signs of irritation (eyes not rinsed); these signs were completely resolved by 72 hours post installation of the test material.

Under the conditions of this study, the test material was determined not to be an eye irritant to rabbits.

With regard to the available study data, 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 irritation/corrosion or eye irritation.

In accordance with Annex VI, Regulation (EC) No. 1272/2008, the substance requires classification with respect to skin irritation as Category 2 (H315: Causes skin irritation) and eye irritation as Category 2 (H319: Causes serious eye irritation).