Registration Dossier

Skin irritation / corrosion: PDII = 3.5 (OECD 404) - irritating to skin.
Eye irritation: maximum total score = 59 (OECD 405) - irritating to eyes.

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/01/91 - 29/01/1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 Sanbach, Cheshire, UK
- Age at study initiation: twelve to sixteen weeks old
- Weight at study initiation: 2.20 - 2.50 kg
- Housing: individually housed in suspended metal cages
- Diet (e.g. ad libitum): ad libitum (Spillers Rabbit Diet, Dalgety Agriculture Ltd., Almondsbury, Bristol)
- Water (e.g. ad libitum): ad libitum (drinking water)
- Acclimation period: minimum acclimatisation period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): animal room maintained at 17-22°C
- Humidity (%): relative humidity of 40-61%
- Air changes (per hr): approximately 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):0.5 g of test material was introduced under a 2.5 cm x 2.5 cm gauze patch and placed in position on the shorn skin.

Duration of treatment / exposure:

Four hours

Observation period:

1, 24, 48 and 72 hours following removal of the patches
An additional observation was made on day 7

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: A quantity of 0.5 g of test material was introduced under a 2.5 cm x 2.5 cm gauze patch and placed in position on the shorn skin.
- Type of wrap: The patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm x 4.0 cm). To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset (TUBIGRIP) and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- 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

SCORING SYSTEM: according to the scale of Draize J.H. (1959) Association of Food and Drug Officials of the United States, Austin, Texas, "The appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics"

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 1, 24, 48, 72 hours

Score:

2

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 7 days

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: crust formation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 1, 24, 48, 72 hours

Score:

1 - 2

Max. score:

4

Remarks on result:

other: haemorrage of dermal capillaries, blanching, light brown discolouration of the epidermis

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 7 days

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: crust formation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 1, 24, 48, 72 hours

Score:

1 - 2

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 7 days

Max. score:

4

Remarks on result:

other: adverse reactions prevent evaluation, hardened light brown-coloured scab

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 1, 24, 48, 72 hours

Score:

1 - 2

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 7 days

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 1, 24, 48, 72 hours

Score:

1 - 2

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 7 days

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 1, 24, 48, 72 hours

Score:

1

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 7 days

Max. score:

4

Remarks on result:

other: adverse reactions prevent evaluation of oedema

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

other: Sum of 24 and 72 hour readings for animal #1, #2, and #3 (S), PDII = S/6

Time point:

other: 24 and 72 hours

Score:

3.5

Max. score:

8

Remarks on result:

other: Classification: moderate irritant

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

2

Max. score:

4

Remarks on result:

other: positive criterion

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

1.7 - 2

Max. score:

4

Remarks on result:

other: positive criterion

Irritant / corrosive response data:

Very slight to well-defined erythema was noted at all treated skin sites one hour after patch removal with well-defined erythema at the 24, 48 and 72-hour observations.
Very slight to slight oedema was noted at all treated skin sites one hour after patch removal and at the 24, 48 and 72-hour observations.

Other effects:

Other adverse reactions noted at one treated skin site at these times were haemorrhage of the dermal capillaries, blanching and light brown discolouration of the epidermis. Crust formation was noted at two treated skin sites at the 7-day observation. A hardened light brown-coloured scab, preventing accurate evaluation of erythema and oedema was noted at one treated skin site at the 7-day observation.

Individual reactions

Skin reaction	Observation time	Individual Scores Rabbit Number and Sex (Bodyweight Kg)
Skin reaction	Observation time	165 Male (2.20)	166 Female (2.27)	170 Female (2.50)	Total
Erythema/eschar formation	1 h	2	1 Hd	1	(4)
	24 h	2	2 HdBl	2	6
	48 h	2	2 BrBl	2	(6)
	72 h	2	2 BrBl	2	6
	7 days	0 Cf	0 Cf	?eSp	(0-4)
Oedema formation	1 h	2	1	1	(4)
	24 h	2	2	1	5
	48 h	2	2	1	(5)
	72 h	1	2	1	4
	7 days	0	0	?od	(0-4)
Sum of 24 and 72-Hour readings (S): 21
Primary Irritation Index (S/6): 21/6 = 3.5
Classification: Moderate irritant

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

Cf = crust formation

Hd = haemorrhage of dermal capillaries

?e = adverse reactions prevent evaluation of erythema

Sp = hardened light brown-coloured scab

Br = light brown discolouration of the peidermis

?od = adverse reactions prevent evaluation of oedema

Bl = blanching

Individual daily and individual mean scores for dermal irritation following 4 -hour exposure required for EEC labelling regulations

Skin reaction	Reading (h)	Individual Scores ¿ Rabbit Number and Sex (Bodyweight Kg)
Skin reaction	Reading (h)	165 Male (2.20)	166 Female (2.27)	170 Female (2.50)
Erythema/eschar formation	24	2	2	2
	48	2	2	2
	72	2	2	2
Total		6	6	6
Mean Score		2.0+	2.0+	2.0+
Oedema formation	24	2	2	1
	48	2	2	1
	72	1	2	1
Total		5	6	3
Mean Score		0.8	2.0+	1.0

+ = positive criterion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test material, DTBP, produced a primary irritation index of 3.5 and was classified as a MODERATE IRRITANT to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.
The test material produced positive criteria in 3/3 rabbits according to the EEC labelling regulations and was classified as IRRITANT to rabbit skin. The symbol "Xi" and risk phrase R 38 "IRRITATING TO SKIN" are therefore required.

Executive summary:

A study was performed to asses the irritancy potential of the test material, DTBP, to the skin of the New Zealand White rabbit. The method used followed that described in the OECD Guidelines for testing of Chemicals (1981) No. 404 "Acute Dermal Irritation/Corrosion" referenced as Method B4 in Commission Directive 84/449/EEC /which constitutes Annex V of Council Directive 67/548/EEC).

The results may be used as basis for classification and labelling under Annex VI of Council Directive 67/467/EEC (as adapted to technical progress by Commission Directive 83/467/EEC).

A single 4 -hour, semi-occluded application of the test material to the intact skin of three rabbits produced well-defined erythema and very slight to slight oedema. Other skin reactions noted were haemorrhage of the dermal capillaries, blanching, light brown discolouration of the epidermis, crust formation and hardened light brown-coloured scabs.

The test material produced a primary irritation index of 3.5 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

The test material was also classified as irritant according to EEC labelling regulations. The symbol "Xi" and risk phrase R 38 "IRRITATING TO SKIN" are therefore required.

Endpoint conclusion:

adverse effect observed (irritating)

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07/02/1991-09/02/1991

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:

referenced as Method B5 in Commission Directive 84/449/EEC

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 Sanbach, Cheshire, UK
- Age at study initiation: twelve to sixteen weeks old
- Weight at study initiation: 2.90 kg
- Housing: individually housed in a suspended metal cage
- Diet (e.g. ad libitum): ad libitum (Spillers Rabbit Diet, Dalgety Agriculture Ltd., Almondsbury, Bristol)
- Water (e.g. ad libitum): ad libitum (drinking water)
- Acclimation period: minimum acclimatisation period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): animal room maintained at 17-20°C
- Humidity (%): relative humidity of 52-57%
- Air changes (per hr): approximately 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Vehicle:

unchanged (no vehicle)

Controls:

other: the left eye remained untreated and was used for control purposes

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 ml of the test material, which was found to weigh approximately 98 mg (as measured by gently compacting the required volume into and adapted syringe)

Duration of treatment / exposure:

one second: The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released.

Observation period (in vivo):

1 hour, 24 and 48 hours following treatment

Number of animals or in vitro replicates:

one

Details on study design:

SCORING SYSTEM: Assessment of ocular damage/irritation was made according to the numerical evaluation from Draize J. H. 1959, Association of Food and Drug Officials of the united States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics".

TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope

Irritation parameter:

cornea opacity score

Remarks:

E: degree of opacity

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

1 - 2

Max. score:

4

Reversibility:

not specified

Remarks:

animal killed after 48h

Irritation parameter:

cornea opacity score

Remarks:

F: area of opacity

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks:

animal killed after 48h

Irritation parameter:

cornea opacity score

Remarks:

(E x F) x 5

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

0 - 40

Max. score:

80

Irritation parameter:

iris score

Remarks:

D

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

1

Max. score:

2

Reversibility:

not specified

Remarks:

animal killed after 48h

Irritation parameter:

iris score

Remarks:

D x 5

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

5

Max. score:

10

Irritation parameter:

conjunctivae score

Remarks:

A: redness

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

2 - 3

Max. score:

3

Reversibility:

not specified

Remarks:

animal killed after 48h

Remarks on result:

other: white areas over nictitating membrane. Sloughing of conjunctival membrane

Irritation parameter:

conjunctivae score

Remarks:

B: chemosis

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

2

Max. score:

4

Reversibility:

not specified

Remarks:

animal killed after 48h

Irritation parameter:

conjunctivae score

Remarks:

C: discharge

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

2

Max. score:

3

Reversibility:

not specified

Remarks:

animal killed after 48h

Irritation parameter:

conjunctivae score

Remarks:

(A + B + C) x 2

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

12 - 14

Max. score:

20

Irritation parameter:

overall irritation score

Remarks:

Total score

Basis:

animal #1

Time point:

other: 1, 24, 48 hours

Score:

17 - 59

Max. score:

110

Irritant / corrosive response data:

A dulling of the normal luster of the corneal surface was noted in the treated eye one hour after treatment. Areas of diffuse corneal opacity were noted at the 24-hour observation with areas of grade 4 corneal opacity at the 48-hour observation.
Iridial inflammation was noted in the treated eye at the one, 24 and 48-hour observations.
Moderate conjunctival irritation was noted in the treated eye one hour after treatment and at 24 and 48-hour observations. White areas over the nictitating membrane were also noted at the 24 and 48-hour observations. Sloughing of the conjunctival membrane was noted in the treated eye at the 48-hour observation.
Due to severe ocular reactions the animal was killed for humane reasons immediately after the 48-hour observation in accordance with Home Office guidelines. No further animals were treated.

Individual scores and total scores for ocular irritation

Rabbit number and sex IPR3 85 Male (bodyweight Kg) (2.90)
Time after treatment	1 hour	24 hours	48 hours
CORNEA
E = degree of opacity	d	1	2
F = area of opacity	4	4	4
Score (E x F) x 5	0	20	40
IRIS
D	1	1	1
Score (D x 5)	5	5	5
CONJUNCTIVAE
A = redness	2	2 W	3 SlW
B = chemosis	2	2	2
2 = discharge	2	2	2
Score (A + B + C) x 2	12	12	14
Total score	17	37	59

IPR = initial pain reaction

Sl = sloughing of conjunctival membrane

d = dulling of the normal lustre of the corneal surface

W = white areas over nictitating membrane

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The test material, DTBP, produced a maximum total score of 59 and was considered to be at least a severe irritant (class 6 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and calandra classification system (based on one rabbit only).
The test material produced positive criteria in the treated rabbit according to the EEC labelling regulations and was considered to be irritant to the rabbit eye.

Executive summary:

A study was performed to assess the irritancy potential of the test material, DTBP, to the eye of the New Zealand White rabbit. The method used followed that described in the OECD Guidelines for testing of Chemicals (1987) No. 405 "Acute Eye Irritation/Corrosion" referenced as Method B5 in Commission Directive 84/449/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

The results may be used as a basis for classification and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 83/467/EEC).

A single application of the test material to the non-irrigated eye of one rabbit produced areas of grade 4 corneal opacity, iridial inflammation and moderate conjunctival irritation. Other adverse effects noted were a dulling of the normal lustre of the corneal surface, white areas over nictitating membrane and sloughing of the consjunctival membrane.

The test material, DTBP, produced a maximum total score of 59 and was considered to be at least a severe irritant (class 6 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and calandra classification system (based on one rabbit only).

The test material produced positive criteria in the treated rabbit according to the EEC labelling regulations and was considered to be irritant to the rabbit eye.

Endpoint conclusion:

adverse effect observed (irritating)

Endpoint conclusion:

no study available

Skin irritation / corrosion:

In the key study (Tuffnell, P.P. 1991) the test material produced a primary irritation index of 3.5 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted. In a supporting study (Mürmann, P. 1983) the primary irritation index was 3.88 which also gave a similar classification. In a third study (Mallory, V.T. 1985), an occlusive application was applied to shaved skin and a corrosive result due to the presence of necrosis was reported. Based on the results of the key study (Tuffnell, P.P. 1991) and supported by Mürmann, P. 1983, the test material produced positive criteria in 3/3 rabbits, and was classified as irritant according to EEC labelling regulations. The hazard phrase H315 "Causes Skin Irritation" is therefore required.

Test Method:

The method used followed that described in the OECD Guidelines for testing of Chemicals (1981) No. 404 "Acute Dermal Irritation/Corrosion" referenced as Method B4 in Commission Directive 84/449/EEC /which constitutes Annex V of Council Directive 67/548/EEC).

The results may be used as basis for classification and labelling under Annex VI of Council Directive 67/467/EEC (as adapted to technical progress by Commission Directive 83/467/EEC).

A single 4 -hour, semi-occluded application of the test material to the intact skin of three rabbits produced well-defined erythema and very slight to slight oedema. Other skin reactions noted were haemorrhage of the dermal capillaries, blanching, light brown discolouration of the epidermis, crust formation and hardened light brown-coloured scabs.

Eye irritation:

In the key study (Tuffnell, P.P. 1991) the test material produced a maximum total score of 59 and was considered to be at least a severe irritant (class 6 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system (based on one rabbit only).An assessment of reversibility was not possible in this study as the animal was killed on welfare grounds after 48 hours.In the supporting study (Mürmann, P. 1983) an overall irritation mean score of 38.5 was reported with findings that were not fully reversible within 21 days. An interpretation of moderately irritating with risk of serious damage to the eyes was recorded. Based on the results of these studies with positive criteria 2,4-di-tert-butylphenol should be classified as irritant to eyes. The hazard phrase H318 "Causes Serious Eye Damage" is therefore required.

Test Method:

The method used followed that described in the OECD Guidelines for testing of Chemicals (1987) No. 405 "Acute Eye Irritation/Corrosion" referenced as Method B5 in Commission Directive 84/449/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

The results may be used as a basis for classification and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 83/467/EEC).

Skin irritation / corrosion:

The test material produced positive criteria in 3/3 rabbits therefore in accordance with EU CLP (Regulation (EC) No. 1272/2008) 2,4-di-tert-butylphenol should be classified as irritant to skin. The hazard phrase H315 "Causes Skin Irritation" is therefore required.

Eye irritation:

The test material produced positive criteria in the treated rabbit therefore in accordance with EU CLP (Regulation (EC) No. 1272/2008) 2,4-di-tert-butylphenol should be classified as irritant to eyes. The hazard phrase H318 "Causes Serious Eye Damage" is therefore required.