Registration Dossier

- Skin irritation: irritating, female, New-Zealand White rabbit, OECD 404, Robinson 1998

- Eye irritation: irritating, female, New-Zealand White rabbit, OECD 405, Robinson 1998

- Respiratory irritation: irritating, male/female, rat, OECD 403, Bruce 1985

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Aug 1996 to 12 Sep 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EPA OPP 81-5 (Acute Dermal Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

albino

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult
- Weight at study initiation: 2305 - 3020 g
- Housing: Individually in aluminium sheet cage
- Diet: STANRAB SQC, ad libitum
- Water: Mains water, ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): approximately 25 - 30
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 20 Aug 1996 To:12 Sep 1996

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Remarks:

Technical solution

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

23 days

Number of animals:

3 females

Details on study design:

APPLICATION OF TEST SUBSTANCE
Approximately 24 hours prior to application of the test substance, the hair was removed with a pair of veterinary clippers from an area approximately 7 cm x 13 cm on the left flank of each animal. The undiluted test substance (0.5 mL) was applied, using a sterile disposable polypropylene syringe, to the test site (approximate size 2.5 cm x 2.5 cm) on the left flank of each rabbit. The treated area was covered with a piece of 4-ply surgical gauze (approximate size 2.5 cm x 2.5 cm), which was secured by two strips of surgical tape (approximate size 1 cm x 8 cm). This was covered by a piece of impermeable rubber sheeting (approximate size 35 cm x 13 cm) wrapped once around the trunk of the animal and secured with adhesive impermeable polyethylene tape. The dressings were left in position for approximately four hours. After this time each dressing was carefully removed and discarded. The application site was gently cleansed free of any residual test substance using clean swabs of absorbent cotton wool soaked in 3 % teepol in water and was then dried gently with clean tissue paper.

IRRITATION RESPONSE
The animals were assessed daily for up to 23 days for any signs of skin irritation. Irritation was scored by the method of Draize, to assess the degree of erythema and oedema at the application sites approximately 30 - 60 minutes and 1, 2 and 3 days after removal of the dressings and then at intervals for up to 23 days. Any other signs of skin irritation were also noted.

BODY WEIGHTS
The body weight of each animal was recorded at the start of the study.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 2 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Very slight or well-defined erythema was seen in two animals for up to 7 days and in one animal for up to 17 days. Very slight or slight erythema was seen in all animals for up to 2 or 7 days. There were no additional signs of irritation in one animal. Additional signs seen in the remaining two animals included desquamation, thickening and development of new skin with the associated sparse hair growth. All signs of irritation had completely regressed by day 23 in both animals.

Other effects:

There were no signs of ill-health in any animal during the study.

Table 1: Individual and mean skin irritation scores of test substance according to the Draize scheme

Time	Erythema			Oedema
Animal number	#1	#2	#3	#1	#2	#3
after 30 - 60 minutes	1	1	0	1	1	0
after 1 day	1	1	1	1	1	1
after 2 day	1	1	1	1	0	1
after 3 days	1	1	1	1	0	2
mean score 24 - 72 h	1.0	1.0	1.0	1.0	0.3	1.3
after 4 days	2	1	1	1	0	2
after 7 days	0 d, n, t	0	1 d, t	0	0	0
after 10 days	-	-	1 d, t	-	-	0
after 11 days	0 d, n, t	-	-	0	-	-
after 14 days	0	-	1 d,	0	-	0
after 17 days	-	-	0 d, n, sp	-	-	0
after 21 days	-	-	0 n	-	-	0
after 23 days	-	-	0	-	-	0

d – desquamation; n – new skin; t – thickening; sp – sparse hair growth

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test substance is a moderate to severe irritant following a single four-hour application to rabbit skin.

Executive summary:

In a primary dermal irritation study in accordance with OECD 404 and in compliance with GLP, 3 young adult female New Zealand White albino rabbits were dermally exposed to 0.5 mL of test substance to an area (approximately 2.5 cm x 2.5 cm) of the shorn flank for 4 hours. The animals were assessed for up to 23 days for any signs of skin irritation. Irritation was scored by the method of Draize.

Very slight or well-defined erythema was seen in two animals for up to 7 days and in one animal for up to 17 days. Very slight or slight oedema was seen in all animals for up to 2 or 7 days. There were no additional signs of irritation in one animal. Additional signs seen in the remaining two animals included desquamation, thickening and development of new skin with associated sparse hair growth. All signs of irritation had completely regressed by day 23 in both animals. The test substance is a moderate to severe irritant following a single four-hour application to rabbit skin.

Endpoint conclusion:

adverse effect observed (irritating)

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Aug 1996 to 5 Sep 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EPA OPP 81-4 (Acute Eye Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult
- Weight at study initiation: 3021 - 3077 g
- Housing: Individually in aluminium sheet cage
- Diet: Diet (STANRAB SQC), ad libitum
- Water: Mains water, ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): approximately 25 - 30
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 21 Aug 1996 To: 5Sep 1996

Vehicle:

unchanged (no vehicle)

Remarks:

Technical solution

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL

Duration of treatment / exposure:

Single treatment

Observation period (in vivo):

8 days

Number of animals or in vitro replicates:

3 females

Details on study design:

PREPARATION AND SELECTION OF ANIMALS
The eyes of the animals were examined one day prior to test item administration. Animals with overt signs of ocular injury or irritation which may have interfered with the interpretation of the results were not used in the test.

INSTILLATION
Initially, the test substance (0.1 mL) was instilled into the conjunctival sac of the left eye of one rabbit by gently pulling the lower lid away from the eyeball to form a cup into which the test substance was dropped. The lids were then gently held together for 1 - 2 seconds after which the animal was released. The other eye was untreated (control eye). Immediately after the instillation of the test substance, an assessment of initial pain was made on a 6 point scale.

CAGE SIDE OBERVATIONS
When the eye irritation potential had been fully assessed in the first animal, the test eye of the remaining two animals was assessed. Animals were observed frequently on the day of dosing and daily throughout the remainder of the study.

OCULAR SCORING
The eyes were examined for up to 8 days, to assess the grade of ocular reaction. Irritation was scored by the method of Draize 1959, to assess the grade of ocular reaction approximately one hour and 1, 2 and 3 days after instillation and then at intervals for up to 8 days. In addition, as an aid in the assessment of corneal damage, fluorescein staining was used at all readings from 1 day after instillation. A modified form of the Kay and Calandra (1962) system was used to interpret and classify the numerical scores. This scoring table is derived fom the Draize scoring table, which is included in the "Any other information on materials and methods incl. tables" field. This table will be used to interpret the results from this study.

BODY WEIGHT
The animals were weighed prior to the start of the study.

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: #1, #3

Time point:

24/48/72 h

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 8 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal: #2, #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Instillation into the eye caused slight initial pain (class 2 on a 0 - 5 scale). No corneal or iridial effects were observed. Conjunctival effects included slight to moderate redness, slight chemosis and slight to moderate discharge. The area around the eye of two of the animals was stained brown or brown/red by the test material. Additional signs of irritation included mucoid and Harderian discharge and erythema and thickening of the eyelids. All effects had completely regressed by day 8.

Other effects:

MORTALITY
No deaths occurred.

CLINICAL SIGNS
No systemic signs of toxicity were noted during the study

BODY WEIGHT:
The body weights of the rabbits were considered to be within the normal range of variability.

Table 1: Eye irritation scores of the test substance according to the Draize scheme

Time	Cornea			Iris			Conjunctiva
							Redness			Chemosis
Animal number	#1	#2	#3	#1	#2	#3	#1	#2	#3	#1	#2	#3
after approx. 1 hour	0	0	0	0	0	0	1 m, st	1 m	1 m, st	1	1	1
after 24 hours	0	0	0	0	0	0	2 m, d, st	2 e	2 m, r, st	1	1	1
after 48 hours	0	0	0	0	0	0	2 m, st	2 e, d, r	2 d, r, st,	1	0	0
after 72 hours	0	0	0	0	0	0	1 st	1 e, d, r	1 d, r, st	0	0	0
mean scores 24 - 72h	0	0	0	0	0	0	1.7	1.7	1.7	0.7	0.3	0.3
after 4 days	0	0	0	0	0	0	1 st	1 d	1 d, st	0	0	0
after 7 days	0	0	0	0	0	0	0	1	0 st	0	0	0
after 8 days	-	0	-	-	0	-	-	0	-	-	0	-

d – Harderian discharge; m – mucoid discharge; st – staining of peri orbital area; e – eyelids thickened;

r – erythema of the upper/lower eyelid

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The test substance is a mild irritant to the rabbit eye.

Executive summary:

In a primary eye irritation study following OECD TG 405 and in compliance with GLP, 0.1 mL of test substance was instilled into the conjunctival sac of the left eye of each of 3, young adult female, New Zealand White rabbits. Immediately after the instillation of the test substance, an assessment of initial pain was made. The eyes were examined for up to 8 days, to assess the grade of ocular reaction. Irritation was scored by the method of Draize 1959.

Instillation into the eye caused slight initial pain (class 2 on a 0 - 5 scale). No corneal or iridial effects were observed. Conjunctival effects included slight to moderate redness, slight chemosis and slight to moderate discharge. The area around the eye of two of the animals was stained brown or brown/red by the test material. Additional signs of irritation included mucoid and Harderian discharge and erythema and thickening of the eyelids. All effects had completely regressed by day 8.

The test substance is a mild irritant to the rabbit eye.

Endpoint conclusion:

adverse effect observed (irritating)

Endpoint conclusion:

adverse effect observed (irritating)

Skin irritation

In a primary dermal irritation study in accordance with OECD 404 and in compliance with GLP, 3 young adult female New Zealand White albino rabbits were dermally exposed to 0.5 mL of test substance to an area (approximately 2.5 cm x 2.5 cm) of the shorn flank for 4 hours. The animals were assessed for up to 23 days for any signs of skin irritation. Irritation was scored by the method of Draize, 1959.

Very slight or well-defined erythema was seen in two animals for up to 7 days and in one animal for up to 17 days. Very slight or slight oedema was seen in all animals for up to 2 or 7 days. There were no additional signs of irritation in one animal. Additional signs seen in the remaining two animals included desquamation, thickening and development of new skin with associated sparse hair growth. All signs of irritation had completely regressed by day 23 in both animals.

Eye irritation

In a primary eye irritation study following OECD TG 405 and in compliance with GLP, 0.1 mL of test substance was instilled into the conjunctival sac of the left eye of each of 3, young adult female, New Zealand White rabbits. Immediately after the instillation of the test substance, an assessment of initial pain was made. The eyes were examined for up to 8 days, to assess the grade of ocular reaction. Irritation was scored by the method of Draize 1959.

Instillation into the eye caused slight initial pain (class 2 on a 0 - 5 scale). No corneal or iridial effects were observed. Conjunctival effects included slight to moderate redness, slight chemosis and slight to moderate discharge. The area around the eye of two of the animals was stained brown or brown/red by the test material. Additional signs of irritation included mucoid and Harderian discharge and erythema and thickening of the eyelids. All effects had completely regressed by day 8.

The test substance was rated as a mild irritant to the rabbit eye.

Respiratory irritation

In an acute inhalation toxicity study in accordance with OECD TG 403 and in compliance with GLP, groups of young adult Sprague-Dawley CD rats (5/sex) were exposed by inhalation (whole body) for 4 hours, to aerosols of test substance, in distilled water, to average total aerosol concentrations of 0.16,1.13 and3.86 mg/L as determined gravimetrically. Specified tissues submitted for histological examination revealed exposure-related, subchronic inflammation, congestion, oedema and alveolar/ septal thickening in the lungs of all rats exposed to 1.1 and 3.9 mg/L. Three males and two females in the 0.16 mg/L group had subchronic inflammation and alveolar / septal thickening of the lungs.

It is concluded that the test substance causes respiratory irritation.

Based on the available information, the test substance is classified for Skin irritation, Category 2, H315: Causes skin irritation in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.

Based on the available information, the test substance is classified for Eye irritation Category 2, H319: Causes serious eye irritation in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.

Based on the available information, the test substance is classified for STOT-SE Cat. 3, H335: May cause respiratory irritation in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.