Dodecahydro-3,8,8,11a-tetramethyl-5H-3,5a-epoxynaphth[2,1-c]oxepin

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-MAY-1997 to 22-JUL-1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The in vivo guinea pig maximisation test was already available (performed in 1997) and reliable to evaluate the skin sensitization potential and the classification determination.

Test material

Test material information

Constituent 1

Reference substance name:

rel-(3S,5aR,7aR,11aR,11bS)-3,8,8,11a-tetramethyldodecahydro-5H-3,5aepoxynaphtho[2,1-c]oxepine

Molecular formula:

C18H30O2

IUPAC Name:

rel-(3S,5aR,7aR,11aR,11bS)-3,8,8,11a-tetramethyldodecahydro-5H-3,5aepoxynaphtho[2,1-c]oxepine

Constituent 2

Reference substance name:

rel-(3R,5aS,7aR,11aR,11bS)-3,8,8,11a-tetramethyldodecahydro-5H-3,5aepoxynaphtho[2,1-c]oxepine

Cas Number:

220432-47-3

Molecular formula:

C18H30O2

IUPAC Name:

rel-(3R,5aS,7aR,11aR,11bS)-3,8,8,11a-tetramethyldodecahydro-5H-3,5aepoxynaphtho[2,1-c]oxepine

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Lot No.: 007806
Purity: 98.9% (major peak)
Expiry date : 17-APR-1998

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF

Sex:

male

Details on test animals and environmental conditions:

Number of animals for main study / pretest : 15 males/3 males
Age at delivery : 4- 6 weeks
Age at beginning of acclimatization period : 5 -7 weeks
Body weight at pretest start : Pretest groups 339-440 g
Body weight at beginning of acclimatization period : Control and test group 352-428 g
Identification : By unique cage number and corresponding ear tags
Randomization : Randomly selected at time of delivery
Acclimatization : One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretests. Only animals without any visible signs of illness were used for the study.
Conditions : Air-conditioned with 10-15 air changes per hour and continuously monitored environment with a target range for room temperature of 22 ± 3 °C and for relative humidity between 40-70 % (values above 70 % during cleaning process possible). The animals were provided with a 12-hour light, 12-hour dark cycle. Music was played during the light period.
Accommodation : Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz)
Diet : Pelleted standard Nafag Ecosan 845 25W4, batch nos. 18/97 and 29/97 guinea pig breeding / maintenance diet ("Nafag", Nahr- und Futtermittel AG, CH-9202 Gossau), ad libitum. Results of analyses for contaminants are archived at RCC.
Water : Community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/1) via the drinking water was provided. Results of bacteriological, chemical and contaminant analyses are archived at RCC

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline and Com oil.

Concentration / amount:

Amount = 0.1 ml/site :
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 5 % with com oil.
3) The test article diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Day(s)/duration:

injection peformed on test day 1

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

Amount : 0.3 g diluted at 50% in corn oil

Day(s)/duration:

48 hours performed on test day 8

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

Amount : 0.2 g of the highest non-irritating concentration of 50 % test substance in corn oil vehicule

Day(s)/duration:

The dressings were left in place for 24 hours.

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Indutcion: 6 intradermal applications per animal
Challenge: 2 patches are put in place

Details on study design:

INDUCTION

- INTRADERMAL INJECTIONS/ PERFORMED ON TEST DAY 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
Test Group;
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 5 % with com oil.
3) The test article diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Com oil.
3) 1:1 (w/w) mixture of com oil in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- EPIDERMAL APPLICATIONS/ PERFORMED ON TEST DAY 8
On test day 7 and approximately 24 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) of the animals of the control and test group was clipped, shaved free of hair and the test area was pretreated with a 10 % dilution of Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10 % concentration of SLS enhances sensitization by provoking a mild inflammatory reaction (Magnusson and Kligman 1970).
On test day 8, a 2 x 4 cm patch of filter paper was saturated with the test article (50 % in com oil) and placed over the injection sites of the test animals. The volume of test article applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test article.
The guinea pigs of the control group were treated as described above with com oil only. The volume applied was approximately 0.3 ml.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

CHALLENGE/ PERFORMED ON TEST DAY 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application. The test and control guinea pigs were treated in the same way.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 50 % (left flank) and the vehicle only (com oil applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 g and for the vehicle 0.2 ml. The dressings were left in place for 24 hours.
Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated

Challenge controls:

See details in the study design field above

Positive control substance(s):

no

Results and discussion

Positive control results:

The positive control was performed with Alpha-hexylcinnamaldehyde from 06-01-1997 to 13-02-1997.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % test substance in corn oil vehicule

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % test substance in corn oil vehicule

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% corn oil

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% Corn oil

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

25% in polyethylene glycol

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

Strong sensitizer

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

25% in polyethylene glycol

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

strong sensitizer

Any other information on results incl. tables

No positive reactions were observed in the animals either when treated with corn oil alone or when treated with the test article at 50%in corn oil. As there were no deaths during the course of the treatment period no necropsies were performed. No symptoms of systemic toxicity were observed in the animals. The body weight of the animals was within the range of physiological variability known for guinea pigs of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study none of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 50 % in com oil. No skin reactions were observed in the control group.
Therefore, the test article AMBERKETAL applied at a concentration of 50 % in com oil is considered not to be a sensitizer when used under the described test conditions

Executive summary:

In order to assess the cutaneous allergenic potential of AMBERKETAL, the Maximization­ Test in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6 was carried out in 15 (10 test and 5 control) male Albino guinea pigs.

The intraderrnal induction of sensitization was carried out with a 5% dilution of the test article in com oil and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological · saline. The epicutaneous induction of sensitization was conducted under occlusion with the test article at 50%in com oil. Two weeks after the epicutaneous induction application the challenge was completed by epicutaneous application of the test article at 50%in com oil under occlusive dressing. The animals of the control group were induced with com oil and FCA/physiological saline and challenged similarly to those of the test group. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hoursafterremovalofthedressing.

No toxic symptoms were evident in the guinea pigs of the control or test group. Nodeaths occurred. In this study none of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 50 % in com oil. No skin reactions were observed in the control group.

Therefore, the test article AMBERKETAL applied at a concentration of 50%in com oil is considered not tobea sensitizer when used under the described test conditions