Registration Dossier

- Skin irritation: not irritating, male/female, New-Zealand White rabbit, EPA 81-5, Glaza 1992

- Eye irritation: not irritating, male/female, New-Zealand White rabbit, EPA-81-4, Glaza 1992

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Sep 1992 to 05 Sep 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-5 (Acute Dermal Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hra:(NZW)SPF strain

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: adult
- Weight at study initiation: 2694 - 3066 g
- Housing: animals were individually housed in screen-bottom stainless steel cages in temperature- and humidity-controlled quarters.
- Diet: measured amount of rabbit Diet
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 44 - 57
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

IN-LIFE DATES: From: 02 Sep 1992 To: 05 Sep 1992

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 g

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 animals/sex

Details on study design:

TEST SITE
- Area of exposure: On day before treatment, the back and/or flanks of each animal were clipped free of hair to obtain an unblemished skin site. The test material was applied to the intact skin on each animal's back.
- Coverage: approximate exposure area of 6.25 cm2
- Type of wrap if used: 2.5 cm X 2.5 cm gauze patch secured with paper tape, loosely overwrapped with Saran Wrap and secured with tape.

REMOVAL OF TEST SUBSTANCE
- Washing: using tap water and disposable paper towels. The test material was removed from the test sites as thoroughly as possible irritating the skin.
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
- At 4, 24, 48 and 72 hours.
- Animals were weighed just before test material administration. Approximately 30 minutes after removal of the test material, the degree of erythema and oedema at each test site was read according to the Draize technique (recorded as the 4-hour score). The untreated skin of each animal was used for comparison.

SCORING SYSTEM:
- Method of calculation: method of Draize. See Table 1 in 'Any other information on materials and methods incl. tables'.

TERMINATION
- At termination the experimental phase, all animals were designated to be euthanised and discarded.

STATISTICAL ANALYSIS
- No statistical analyses were required by the protocol.

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #3, #4, #5, #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal: #1, #2, #3, #4, #5, #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Application of the test substance to the skin of rabbits under 4-hour semi occluded conditions resulted in no dermal irritation. The average of the 4-, 24-, 48-, and 72-hour scores is 0.0 (considered to be non-irritating).

Table 2: Individual irritation scores

Scores observed after	4 hours	24 hours	48 hours	72 hours
Erythema	0, 0, 0, 0, 0, 0	0, 0, 0, 0, 0, 0	0, 0, 0, 0, 0, 0	0, 0, 0, 0, 0, 0
Oedema	0, 0, 0, 0, 0, 0	0, 0, 0, 0, 0, 0	0, 0, 0, 0, 0, 0	0, 0, 0, 0, 0, 0

Table 3 Mean value irritation scores

Animal	mean 24-72 hrs
Animal	erythema	oedema
1	0	0
2	0	0
3	0	0
4	0	0
5	0	0
6	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

In this EPA 81-5 study, the test substance has been found not to be skin irritating to rabbits.

Executive summary:

The objective this study was to assess the primary dermal irritation potential of the test substance in vivo, according to the EPA OPP 81-5 test guideline following GLP principles. An amount of 0.5 g unchanged test material was applied under semi-occlusive conditions for 4 hours to the clipped backs of 3 male and 3 female albino rabbits (Hra: New-Zeeland White), covering a surface area of 6.25 cm2. After 4 hours the test item was removed using tap water. Scoring was performed at 4, 24, 48 and 72 hours and after 3 days of application following the Draize system.

No signs of irritation, either erythema or oedema reactions, were observed. The 24/48/72 mean averages for these parameters were 0 for all animals.

Under the conditions of this study, the test substance was considered not to be irritating to the skin of 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:

04 Sep 1992 to 07 Sep 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-4 (Acute Eye Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hra:(NZW)SPF

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: adult
- Weight at study initiation: 2745 – 3047 g
- Housing: individually housed in screen-bottom stainless steel cages in temperature- and humidity controlled quarters.
- Diet: a measured amount of Laboratory Rabbit Diet
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 44 ± 67

IN-LIFE DATES: From: 04 Sep 1992 To: 07 Sep 1992

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL:
- Amount applied: 0.04 g (0.01 mL weight equivalent) of the test material

Duration of treatment / exposure:

Single treatment

Observation period (in vivo):

1, 24, 48 and 72 hours

Number of animals or in vitro replicates:

6 males / 3 females

Details on study design:

ANIMAL SELECTION
Six male and three female acclimated rabbits were selected and maintained during the study in the same manner as for the acclimation period. Variations from the prescribed environmental condition existed and were considered to have had no effect on the study outcome.
The animals' eyes were examined on the day before test material administration using sodium fluorescein dye procedures. Only those animals with no ocular injury or irritation were used. Animals were identified by number and corresponding ear tag. The rabbits were divided into two groups consisting of six rabbits in Group 1 and three rabbits in Group 2.

TREATMENT
Each rabbit received 0.04 g (0.1 mL weight equivalent) of the undiluted test material placed into the everted lower lid of the right eye, with the left eye serving as the untreated control. The upper and lower lids were gently held together for 1 second to prevent loss of material and then released. The eyes of the Group 1 rabbits remained unflushed following instillation of the test material. After 24 hours, the eyes were flushed with lukewarm tap water. The eyes of the Group 2 animals were flushed in the same manner for 1 minute starting 30 seconds after test material instillation.

OBSERVATIONS
Animals were weighed just before test material administration. The treated eyes of both groups were observed for ocular irritation at 1, 24, 48, and 72 hours after treatment. Irritation was graded and scored according to the Draize technique (see Table 1 in 'Any other information on materials and methods incl. tables').

TERMINATION
At termination of the experimental phase, all animals were euthanised.

Irritation parameter:

cornea opacity score

Basis:

animal: #1 to #6

Remarks:

Group 1

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 to #6

Remarks:

Group 1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Remarks:

Group 1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal: #2, #4, #5, #6

Remarks:

Group 1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal: #3

Remarks:

Group 1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal: #1 to #6

Remarks:

Group 1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The test substance, when evaluated for its primary eye irritation potential in rabbits, produced only slight to moderate conjunctival irritation in eyes not receiving a washout until 24 hours after the treatment. In treated eyes receiving a washout approximately 30 seconds after instillation, the test material produced only slight conjunctival irritation. All treated eyes in both groups had returned to a normal appearance by 72 hours after treatment.

Table 2. Individual Eye irritation Scores Group 1

Animal Number	Sex	Cornea		Iris	Conjunctivae			Total score*
		A	B	C	D	E	F
1 hour
1	male	0	0	0	1	1	0	4.0
2	male	0	0	0	1	1	1c	6.0
3	male	0	0	0	1	0	0	2.0
4	male	0	0	0	2	1	0	6.0
5	female	0	0	0	2	1	1c	8.0
6	female	0	0	0	1	1	1c	6.0
						Mean		5.3
24 hours
1	male	0	0	0	0	0	0	0.0
2	male	0	0	0	0	0	0	2.0
3	male	0	0	0	1	0	0	4.0
4	male	0	0	0	2	0	0	2.0
5	female	0	0	0	1	0	0	2.0
6	female	0	0	0	1	0	0	2.0
						Mean		2.0
48 hours
1	male	0	0	0	0	0	0	0.0
2	male	0	0	0	0	0	0	0.0
3	male	0	0	0	1	0	0	2.0
4	male	0	0	0	0	0	0	0.0
5	female	0	0	0	0	0	0	0.0
6	female	0	0	0	0	0	0	0.0
						Mean		0.3
72 hours
1	male	0	0	0	0	0	0	0.0
2	male	0	0	0	0	0	0	0.0
3	male	0	0	0	0	0	0	0.0
4	male	0	0	0	0	0	0	0.0
5	female	0	0	0	0	0	0	0.0
6	female	0	0	0	0	0	0	0.0
		0	0	0	0	0	0	0.0

Cornea

A - Degree of opacity

B - Area of involvement

Iris

C - Degree of iridal irritation

Conjunctivae

D - Redness

E - Chemosis

F - Discharge

* Total score = (A x B x 5) + (C x 5) + [(D + E + F) x 2].

c = Clear discharge.

Table 3. Individual Eye irritation Scores Group 2

Animal Number	Sex	Cornea		Iris	Conjunctivae			Total score
		A	B	C	D	E	F
1 hour
1 t	female	0	0	0	1	1	1c	6.0
2 t	female	0	0	0	1	1	1c	6.0
3 t	female	0	0	0	1	1	0	4.0
						mean		5.3
24 hour
1 t	female	0	0	0	1	0	0	2.0
2 t	female	0	0	0	1	0	0	2.0
3 t	female	0	0	0	1	0	0	2.0
						mean		2.0
48 hours
1 t	female	0	0	0	0	0	0	0.0
2 t	female	0	0	0	1	0	0	2.0
3 t	female	0	0	0	0	0	0	0.0
						mean		0.7

Cornea:

A - Degree of opacity

B - Area of involvement

Iris:

C - Degree of iridal irritation

Conjunctivae:

D - Redness

E - Chemosis

F - Discharge

* Total score = (A x B x 5) + (C x 5) + [(D + E + F) x 2].

c = Clear discharge

t = No pain response after test material instillation.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this study, it was concluded that test substance was considered to be not irritating to the eyes of rabbits.

Executive summary:

In a primary eye irritation study (EPA 81-4) performed following GLP principles, 0.1 mL of the undiluted test substance was instilled into the everted lower lid of the right eyes of nine rabbits, six with treated eyes unwashed and flushed after 24 hours, while three animals were flushed in the same manner for 1 minute starting 30 seconds after test material instillation. The left eye remained untreated and served as a control. Observations were made 1 hour, 24 hours, 48 hours and 72 hours after instillation of the test substance. Ocular irritation was evaluated by the Draize scale method.

The mean scores, calculated from the 24, 48 and 72 hour readings, for the 6 tested animals with eyes washed at 24 hours were 0 for cornea, 0 for iritis, 0.4 for conjunctiva redness, and 0 for oedema of conjunctiva. The reactions were fully reversible within the observation period. The mean scores calculated from the 24, 48 and 72 hour readings for each of the 3 tested animals with eyes washed at 30 seconds after treatment were also below the threshold values for all effects.

Under the conditions of this study, the test substance was considered to be not irritating to the eyes of rabbits.

Endpoint conclusion:

no adverse effect observed (not irritating)

Endpoint conclusion:

no study available

Skin irritation in rabbits, Glaza 1992

The objective this study was to assess the primary dermal irritation potential of the test substance in vivo, according to the EPA OPP 81-5 test guideline following GLP principles. An amount of 0.5 g unchanged test material was applied under semi-occlusive conditions for 4 hours to the clipped backs of 3 male and 3 female albino rabbits (Hra: New-Zeeland White), covering a surface area of 6.25 cm2. After 4 hours the test item was removed using tap water. Scoring was performed at 4, 24, 48 and 72 hours and after 3 days of application following the Draize system.

No signs of irritation, either erythema or oedema reactions, were observed. The 24/48/72 mean averages for these parameters were 0 for all animals.

Under the conditions of this study, the test substance was considered not to be irritating to the skin of rabbits.

Eye irritation in rabbits, Glaza 1992

In a primary eye irritation study (EPA 81-4) performed following GLP principles, 0.1 mL of the undiluted test substance was instilled into the everted lower lid of the right eyes of nine rabbits, six with treated eyes unwashed and flushed after 24 hours, while three animals were flushed in the same manner for 1 minute starting 30 seconds after test material instillation. The left eye remained untreated and served as a control. Observations were made 1 hour, 24 hours, 48 hours and 72 hours after instillation of the test substance. Ocular irritation was evaluated by the Draize scale method.

The mean scores, calculated from the 24, 48 and 72 hour readings, for the 6 tested animals with eyes washed at 24 hours were 0 for cornea, 0 for iritis, 0.4 for conjunctiva redness, and 0 for oedema of conjunctiva. The reactions were fully reversible within the observation period. The mean scores calculated from the 24, 48 and 72 hour readings for each of the 3 tested animals with eyes washed at 30 seconds after treatment were also below the threshold values for all effects.

Under the conditions of this study, the test substance was considered to be not irritating to the eyes of rabbits.

Based on the available information, classification for skin and eye irritation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.