Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 July 2001 to 04 December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
There was a reliable in vivo skin sensitisation test available so performing a LLNA was not necessary.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Quest International; 55897/02380, Lot SR33405
- Expiration date of the lot/batch: Indicated as used within the expiry date
- Purity: Minimum 98% RPA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark


In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall, Newchurch, Burton-on-Trent, Staffs, UK.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Young adults
- Weight at study initiation: 253-337g
- Housing: 5 per cage in cages suitable for animals of this strain and weight range expected during the course of the study.
- Diet: FD1 diet supplied by Special Diets Services, Essex, UK ad libitum
- Water: Mains water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature:18±3°C
- Humidity: 30-70%
- Air changes (per hr): A minimum of 15 changes/hour
- Photoperiod (hrs dark / hrs light): Artificial, giving 12 hours light, 12 hours dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: Acetone/olive oil (1:9)
Concentration / amount:
3% w/v preparation in acetone/olive oil
Day(s)/duration:
7 days
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: Acetone/olive oil (1:9)
Concentration / amount:
75% w/v preparation in acetone/olive oil
Day(s)/duration:
48 hrs
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: 1:9 acetone/olive oil
Concentration / amount:
75%
Day(s)/duration:
24 hrs
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: 1:9 acetone/olive oil
Concentration / amount:
25%
Day(s)/duration:
24 hrs
Adequacy of challenge:
highest non-irritant concentration
No.:
#3
Route:
epicutaneous, occlusive
Vehicle:
other: 1:9 acetone/olive oil
Concentration / amount:
7.5%
Day(s)/duration:
24 hrs
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Test group - 20 animals
Control group - 10 animals
Details on study design:
RANGE FINDING TESTS:
The dose levels for each of the three stages of the main study were determined by a sighting phase in which groups of 2 or 4 females guinea pigs were used and up to 4 dose levels were tested on each group of animals (Appendix A). The procedure was:

Induction phase:
Intradermal injection: 0.3, 1, 3 and 10% w/v preparations of the test substance in (1:9) acetone/olive oil were tested to determine the highest concentration that could be well tolerated locally and systemically. The dose level selected for intradermal induction in the main study was 3% w/v, as this level caused no more than mild irritation.

Topical application: a 75% w/v preparation of the test substance in (1:9) acetone/olive oil was tested to ensure that it did not cause greater than a mild/moderate irritation response in animals that had been injected with Freund’s Complete Adjuvant (FCA) at least 14 days previously. As there was no skin irritation, this dose level was selected for topical induction in the main study.

Challenge phase
Topical application: 75, 50, 25 and 10% w/v preparations of the test substance in (1:9) acetone/olive oil were tested to determine the highest concentration which did not produce irritation in animals that had been injected with FCA at least 14 days previously. There was no evidence of skin irritation at any concentration. The dose levels selected for challenge in the main study were 75, 25 and 7.5% w/v.

MAIN STUDY
A. INDUCTION EXPOSURE
An area approximately 5x5cm on the scapular region of each animal was clipped free of hair with veterinary clippers and a row of 3 injections (0.05-0.1mL each) was made on each side of the mid-line. The injections were:

(i) Top: FCA plus acetone/olive oil in the ratio 1:1
(ii) Middle: 3% w/v preparation of the test substance in acetone/olive oil
(iii) Bottom: a 3% preparation of the test substance in a 1:1 preparation of FCA plus acetone/olive oil

Control animals were treated the same as the test animals, except that they were treated with acetone/olive oil in place of test substance.

The injection sites were checked for any adverse effects approximately 1 day after dosing.

One week after intradermal injection, the scapular area was clipped again and treated with a topical application of the test substance as a 75% w/v preparation in acetone/olive oil. This preparation (200-300mg) was applied on filter paper (approximate size 4cm x 2cm and covering the intradermal sites) which was held in place by a piece of surgical tape. The tape was covered by a strip of adhesive bandage (20-30cm x 5cm) and secured by a piece of self-adhesive PVC tape. This occlusive dressing was kept in place for at least 48 hours.

Acetone/olive oil only was applied to the filter paper for control animals.

The application sites were checked immediately after removal of the dressings, approx. 1 day later, and an additional twice prior to challenge.

B. CHALLENGE EXPOSURE
Two weeks after the topical induction, an area, aprprox 15cm x 5 cm, on both flanks of all the test and control animals, was clipped free of hair with a pair of clippers. An occlusive dressing was prepared which consisted of 3 pieces of filter paper (1cm x 2cm) stitched to a piece of rubber sheeting (12cm x 5cm).

Preparations of the test substance in (1:9) acetone/olive oil were applied to each of the pieces of filter paper. The dressing was placed on the shorn flank of the guinea pig so that the 75% w/v preparation was on the top left, the 25% w/v preparation was on the bottom left and the 7.5% w/v preparation was on the top right. It was then covered with a strip of adhesive bandage (25-40cm x 7.5cm) which was secured by a self-adhesive PVC tape.

After 24 hours, the dressings were carefully cut, using blunt-tipped scissors, removed and discarded. The position of the pieces of filter paper on the skin were identified using a black waterproof marker-pen.

Skin sites were examined approx. 1 and 2 days after removal of the dressings.

OTHER:
Clinical observations: General
Prior to the start of the study, all guinea pigs were examined to ensure that they were physically normal and behaved normally. Throughout the study, the animals were observed daily.

Clinical observations: Sensitisation response
Following the challenge application, erythematous reactions were quantified and recorded, using the 4-point scale shown below, approx. 1 and 2 days after removal of the dressing.

0-no reaction
1-scatttered mild redness
2-moderate and diffuse redness
3-intense redness and swelling

Body weights
The animals were weighed on the day before dosing and at the end of the study. Individual bodyweights are presented in Appendix C.

Positive control substance(s):
yes
Remarks:
Hexylcinnamaldhyde in corn oil. The reliability of the test system is assessed at approx. 6 monthly-intervals (Appendix B).

Results and discussion

Positive control results:
Following challenge of previously induced guinea pigs with the 75% w/v preparation, scattered mild redness was observed in 6 of the 10 animals. There was no erythematous response in any of the control animals. The net percentage was calculated to be 60% and, under the conditions of the, hexylcinnamaldehyde is classified as sensitising.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% w/v
No. with + reactions:
5
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75% w/v
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% w/v
No. with + reactions:
3
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% w/v
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
7.5% w/v
No. with + reactions:
3
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
7.5% w/v
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
75% w/v
No. with + reactions:
1
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
75% w/v
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% w/v
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% w/v
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
7.5% w/v
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
7.5% w/v
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
75% w/v
No. with + reactions:
6
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
75% w/v
No. with + reactions:
3
Total no. in group:
10

Any other information on results incl. tables

To classify the sensitisation response, the percentage of control animals were subtracted from the percentage of the test animals that responded.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In a skin sensitisation test in guinea pigs, musk ketone is not sensitising.
Executive summary:

In a dermal sensitization study (38808) with musk ketone (≥98%) in acetone/olive oil, young adult Dunkin-Hartley albino guinea pigs were tested in a maximisation test. For induction, 3% w/v musk ketone in acetone/olive oil (intradermal injections) and 75% w/v musk ketone in acetone/olive oil (topical application) was used. For challenge, 7.5, 25 and 75% w/v musk ketone in acetone/olive oil was used for topical application. The evaluation of skin reactions after challenge was carried out at 24 and 48 hrs. The positive control was hexylcinnamaldehyde.

The body weight of animals increased during the study and was not affected by the treatment. The positive control, hexylcinnamaldehyde, gave the appropriate response.

Following challenge of previously induced guinea pigs with the 75% w/v preparation of the test substance in acetone/olive oil, scattered mild redness was observed in 5 of the 20 animals and on 1 of the 10 control animals. The net percentage was calculated to be 15%. Following challenge of previously induced guinea pigs with the 25% w/v preparation of the test substance in acetone/olive oil, scattered mild redness was observed in 3 of the 20 animals. There was no erythematous response in any of the control animals. The net percentage was calculated to be 15%. Following challenge of previously induced guinea pigs with the 7.5% w/v preparation of the test substance in acetone/olive oil, scattered mild redness was observed in 3 of the 20 animals. There was no erythematous response in any of the control animals. The net percentage was calculated to be 15%.

Based on these results, the substance is not sensitising.