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

Endpoint summary

Description of key information

Skin irritation (in vivo): non-irritating (OECD TG 404)
Eye irritation (in vivo): non-irritating (OECD TG 405)
Respiratory irritation: not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: 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:

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

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.

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

Irritation parameter:

iris score

Basis:

mean

Remarks:

for all animals

Time point:

other: 24 h, 48 h, 72 h

Score:

0

Max. score:

2

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.

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

The irritation potential of Nebulone was derived for skin, eye and respiratory tract.

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 the 1h and 24h, observation. 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. None of the erythema/oedema Draize scores exceeded the value of 2.

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 Draize score of 0.33.

For respiratory irritation mostly human data are used for the assessment because no suitable in vitro or in vivo tests are available that can identify respiratory irritation (REACH guidance R.7.2.3). There are no human data such as indicated in R7.2.3 of the ECHA guidance that indicate respiratory reactions of the substance e. g. from consumer experience or occupational exposure. In view of the absence of skin and eye irritation potential, irritation of the respiratory tract is also not indicated. The substance also has a low vapour pressure (0.36 Pa), which minimises the exposure via the inhalation route.

Justification for selection of skin irritation / corrosion endpoint:
The one in vivo study available is adequate to cover the skin irritation and corrosion potential.

Justification for selection of eye irritation endpoint:
The one in vivo study available is adequate to cover the eye irritation and corrosion potential.

Justification for classification or non-classification

Nebulone does not have to be classified as irritating to the skin and eyes in accordance with the criteria outlined in Annex I of 67/548/EEC and Annex VI of 1272/2008/EC.

Nebulone does not need to be classified for respiratory tract irritation because it is not an irritating substance according to the criteria outlined in Annex I of 67/548/EEC (DSD) and Annex VI of 1272/2008 (CLP).