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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 21 May 2003 and 29 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with OECD TG 405 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 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye 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 / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues 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

Vehicle:

unchanged (no vehicle)

Controls:

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

Amount / concentration applied:

0. 1 mL of the test material

Duration of treatment / exposure:

Up to 1 hour

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

Initially, a single rabbit was treated. A volume of 0. 1 mL of the test material was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made according to a six point scale.

After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2, (from Draize J H ( 1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

for all animals

Time point:

other: 24 h, 48 h, 72 h

Score:

0

Max. score:

4

Results 2

Irritation parameter:

iris score

Basis:

mean

Remarks:

for all animals

Time point:

other: 24 h, 48 h, 72 h

Score:

0

Max. score:

2

Results 3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

for all animals

Time point:

other: 24 h, 48 h, 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: All treated eyes appeared normal at the 48-hour observation.

Results 4

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

for all animals

Time point:

other: 24 h, 48 h, 72 h

Score:

0.11

Max. score:

4

Reversibility:

fully reversible within: All treated eyes appeared normal at the 48-hour observation.

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in table 1 and 2.
No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation in all treated eyes at the 24-hour observation.

All treated eyes appeared normal at the 48-hour observation.

Any other information on results incl. tables

Interpretation of Results

The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae =         (A + B + C) x 2
Score for iris                  =         D x 5
Score for cornea           =         (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J H and Calandra J C ( 1962), J. Soc. Cosmet. Chem. 13, 28 1- 289 (see Appendix 3) was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test material.

If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

Table1               Individual Scores and Individual Total Scoresfor Ocular Irritation

Rabbit Number and Sex	147 Female				168 Male				143 Female
	IPR= 2				IPR = 2				IPR = 1
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	1	0	0	2	1	0	0	2	1	0	0
B = Chemosis	1	0	0	0	1	0	0	0	2	1	0	0
C = Discharge	1	1	0	0	1	0	0	0	1	1	0	0
Score (A + B + C) x 2	8	4	0	0	8	2	0	0	10	6	0	0
Total Score	8	4	0	0	8	2	0	0	10	6	0	0

IPR= Initial pain reaction

Table 2               Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
147 Female	8	4	0	0
168 Male	8	2	0	0
143 Female	10	6	0	0
Group Total	26	12	0	0
Group Mean Score	8.7	4.0	0.0	0.0

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: other: EU labelling regulations Commission Directive 200 1/59/EC.

Conclusions:

The irritancy potential of the test material to the rabbit eye was assessed according to OECD Guideline 405. The test material did not meet the criteria for classification as irritant according to EU labelling regulations Commission Directive 2001/59/EC but did produce a maximum group mean score of 8.7 and was classified as a MINIMAL IRRITANT (CLASS 3 ON A 1 TO 8 SCALE) to the rabbit eye according to a modified Kay and Calandra classification system.

Executive summary:

The eye irritation potential of Nebulone was tested using the OECD TG 405. The substance was heated to produce a liquid. A single application of the test material of 0.1 ml into the non-irrigated eye of three New Zealand rabbits produced moderate conjunctival irritation. All treated eyes appeared normal at the 48-hour observation. The test material produced a maximum mean score of 0.33.