Indeno[4,5-d]-1,3-dioxin, 4,4a,5,6,7,8,9,9b-octahydro-7,7,8,9,9-pentamethyl-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 23 April 2003 and 20 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with OECD TG 405 and under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

446-220-4

EC Name:

-

Cas Number:

365411-50-3

Molecular formula:

C16 H26 O2

IUPAC Name:

7,7,8,9,9-pentamethyl-2H,4H,4aH,5H,6H,7H,8H,9H,9bH-indeno[4,5-d][1,3]dioxine

Test material form:

other: Solid block and liquefied before use

Details on test material:

Sponsor's identification: 2-EYED MUSK
Description: solid block
Date received: 05 February 2003
Storage conditions: room temperature in the dark

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Three New Zealand White rabbits were supplied by David Percival Ltd, Moston, Sandbach, Cheshire, UK. At the start of the study the animals were in the weight range of 2.0 to 3.5 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended metal cages. Free access to mains drinking water and food (Certified Rabbit Diet (Code 5322) supplied by International Product Supplies Limited, Wellingborough, Northants, UK) was allowed throughout the study. 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 temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70 % respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

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.

Test system

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 mL of the molten test material

Duration of treatment / exposure:

4 hours

Observation period:

14 days

Number of animals:

3

Details on study design:

On the day before the test each rabbit was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.

One rabbit was initially treated. Three suitable sites were selected on the back of the rabbit. At each test site a quantity of 0.5 mL of the molten test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset and the animal was returned to its cage for the duration of the exposure period.

One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after application. Any residual test material was removed by gentle swabbing with cotton wool soaked in 74 % Industrial Methylated Spirits or distilled water.

After consideration of the skin reactions produced in the first animal, two additional animals were treated with 0.5 mL of test material. One patch was applied to the back of each rabbit, and was allowed to remain in contact with the skin for a period of four hours.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

erythema score

Basis:

mean

Remarks:

Animal 1

Time point:

other: 24 h, 48 h , 72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: All treated skin sites appeared normal at the 14-day observation.

Results 2

Irritation parameter:

erythema score

Basis:

mean

Remarks:

Animal 2

Time point:

other: 24 h, 48 h, 72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: All treated skin sites appeared normal at the 14-day observation.

Results 3

Irritation parameter:

erythema score

Basis:

mean

Remarks:

Animal 3

Time point:

other: 24 h, 48 h, 72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: All treated skin sites appeared normal at the 14-day observation.

Results 4

Irritation parameter:

edema score

Basis:

mean

Remarks:

Animal 1

Time point:

other: 24 h, 48 h, 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: All treated skin sites appeared normal at the 14-day observation.

Results 5

Irritation parameter:

edema score

Basis:

mean

Remarks:

Animal 2

Time point:

other: 24 h, 48 h, 72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: All treated skin sites appeared normal at the 14-day observation.

Results 6

Irritation parameter:

edema score

Basis:

mean

Remarks:

Animal 3

Time point:

other: 24 h, 48 h, 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: All treated skin sites appeared normal at the 14-day observation.

Irritant / corrosive response data:

4-Hour Exposure Period
The individual scores for erythema/eschar and oedema are given in Table 1.

Well-defined erythema was noted at all treated skin sites one hour after patch removal and at the 24-hour observation, with very slight or well-defined erythema at all treated skin sites at the 48 and 72-hour observations.
Very slight or slight oedema was noted at all treated skin sites one hour after patch removal and at the 24-hour observation with very slight oedema noted at all treated skin sites at the 48 and 72-hour observations.
Loss of skin elasticity was noted at one treated skin site at the 48-hour observation and at all treated skin sites at the 72-hour observation. Crust formation was noted at one treated skin site and severe desquamation was noted at two treated skin sites at the 7-day observation.
All treated skin sites appeared normal at the 14-day observation.

1-Hour Exposure Period
The individual scores for erythema/eschar and oedema are given in Appendix 1 (attachment 1)
Very slight erythema was noted one hour after patch removal with well-defined erythema noted at the 24, 48 and 72-hour observations.
Very slight oedema was noted one hour after patch removal and at the 24, 48 and 72-hour observations.
Loss of skin elasticity was noted at the 48 and 72-hour observations and crust formation was noted at the 7 -day observation.
The treated skin site appeared normal at the 14-day observation.

3-Minute Exposure Period
The individual scores for erythemaleschar and oedema are given in Appendix 1.
No evidence of skin irritation was noted during the study.

Any other information on results incl. tables

Table 1                       Individual Skin Reactions Following 4-hour Exposure

Skin Reaction	Observation Time	Individual Scores – Rabbit Number and Sex			Total
		62 Male	97 Male	98 Male
ErythemalEscharFormation	1 Hour	2	2	2	(6)
	24 hours	2	2	2	6
	48 Hours	1	1	1	(4)
	72 Hours	1Le	1Le	1Le	4
	7 Days	0Cf	0D	0D	(0)
	14 Days	0	0	0	(0)
Oedema Formation	1 Hour	1	2	1	(4)
	24 hours	1	2	1	4
	48 Hours	1	1	1	(3)
	72 Hours	1	1	1	3
	7 Days	0	0	0	(0)
	14 Days	0	0	0	(0)
Sum of24 and 72-hour Readings (S)        :          17
Primary Irritation Index (S/6)                   :          17/6 = 2.8
Classification                                        :          MODERATE IRRITANT

 

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

Le = Loss of skin elasticity

Cf = Crust formation

D = Severe desquamation

 Interpretation of Results

Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize Scheme

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.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: According to the Draize classification scheme. The test material did not meet the criteria for classification as irritant or corrosive according to the EU labelling regulations Commission Directive 2001/59/EC.

Conclusions:

The irritancy potential of the test material to the skin of rabbits was assessed according to OECD Guideline 404. The test material was determined to be not irritating but did produce a primary irritation index of 2.8. No corrosive effects were noted.

Executive summary:

The skin irritation of Nebulone was performed according to OECD TG 404. A single 4-hour, semi-occluded application of the molten test material to the intact skin of three New Zealand white rabbits produced well-defined erythema and very slight to slight oedema especially at 1 and 24 hours observation time. Loss of skin elasticity, crust formation and severe desquamation were also noted. All treated skin sites appeared normal at the 14-day observation. The 3-minute and 1-hour semi-occluded applications of the test material to the intact skin of one rabbit produced no corrosive effects.The test material produced a primary irritation index of 2.8. None of the erythema/oedema scores exceeded the value of 2.