Cyclohexadecen-1-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-03-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted in 2004, when the GPMT was still an accepted method to evaluate the skin sensitisation potential of a substance.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-300-4

EC Name:

-

Cas Number:

88642-03-9

Molecular formula:

C16H28O

IUPAC Name:

(6E)-cyclohexadec-6-en-1-one; (7Z)-cyclohexadec-7-en-1-one; (8E)-cyclohexadec-8-en-1-one; (8Z)-cyclohexadec-8-en-1-one

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals: SPF
- Age at study initiation: adult
- Weight at study initiation: 330 - 408 g
- Housing: two or three animals per cage; polycarbonate (macrolone type IV, floor area 1800 cm2)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 3 °C
- Humidity: 55 +/- 15 %
- Air changes: 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

other: sunflower oil; ethanol/diethylphthalate 1:1

Concentration / amount:

1.25 % w/v (intradermal)
75 % w/v (epicutaneous)

Day(s)/duration:

0 and 7

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol/diethylphthale (1:1)

Concentration / amount:

50 % (w/v)

Day(s)/duration:

21

No. of animals per dose:

10 (test group)
5 (control group)

Details on study design:

RANGE FINDING TESTS: yes

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: intradermal injection (once); epicutanous 48 h
- Test groups: 1
- Control group: 1
- Frequency of applications: on day 0 and day 7

Induction
Day -1 - Shaving of the shoulder region
An area of dorsal skin 4 x 6 cm in the scapular region was clipped free of hair with an electric clipper.

Day 0 - Intradermal injection in the shoulder region
Three pairs of intradermal injections were given simultaneously into this area. Each dose was 0.1 mL. The first two pairs of injections were given close to each other and most cranially, whereas the third pair of injections was given towards the caudal part of the test area.
1st pair: Freund's complete adjuvant (FCA) mixed 1:1 with sterile distilled water
2nd pair: - Group 1: SY04/F31358 1.25 °A) (w/v) in sunflower oil
- Group 2: sunflower oil
3rd pair: - Group 1: Equal amounts of SY04/F31358 2.5 % (w/v) in sunflower oil and FCA/water in the ratio 1:1 (v/v)
- Group 2: Equal amounts of sunflower oil and FCA/ water in the ratio 1:1 (v/v)

Day 2 — Observation of skin reaction
Two days after the intradermal induction the injection sites possible skin irritations.

Day 6 - Shaving/ Observation of skin reactions
Six days after the intradermal induction, the same 4 x 6 cm scapular area was clipped free of hair and examined for possible skin reactions.

Day 7 — Dermal induction in the shoulder region
A paper patch (Whatman No. 3M, 2 x 4 cm) was saturated with an amount of 0.25 mL of the 75 % test item (group 1) or the vehicle (group 2). The patch was placed on the skin and covered with impermeable adhesive bandage (Blenderm 5 x 7 cm). This was firmly secured with tape (Gothaplast 5 cm width) wrapped round the trunk. The dressing was left in place for 48 hours.

Day 9 — Observation of skin reactions
After the patch removal the application sites were examined for skin reactions.



B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 21
- Exposure period: 24 h
- Test groups: 1
- Control group: 1
- Evaluation (hr after challenge): 24 and 48 h after removal of the patch

Challenge
Day 20 – Shaving
Hair was removed with an electric clipper and an electric razor from a 4 x 6 cm area from the left flank of the guinea pigs.

Day 21 - Topical application at the flank region
A paper patch (Whatman No. 3M, 2 x 2 cm) was saturated with an amount of 0.1 mL of the 50 % (w/v) test item and placed on the guinea pigs. The patches were covered with impermeable adhesive bandage (Blen erm 4 x 5 cm width) and secured with tape (Gothaplast 5 cm width) wrapped round the trunk. After 24 hours of exposure the patches and tape were removed.

Day 23 and 24 - Observation of skin reactions
Each challenge site was examined approx. 24 and 48 hours after the removal of the patch. About three hours before the 24-hour-reading the sites were clipped and shaved in order to facilitate the evaluation.

Positive control substance(s):

yes

Remarks:

At regular intervals the sensitivity and reliability of the experimental technique was assessed using alpha-HEXYLCINNAMALDEHYDE, TECH., 85 % (10% for intradermal induction and undiluted for topical induction and challenge).

Results and discussion

Positive control results:

The latest positive control study was performed from November 6th, 2002 to November 20th, 2002 in which 100 % of the animals responded positively.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % (w/v) at challenge

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % (w/v) at challenge

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Results 3

Key result

Reading:

other: 1st and 2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Results 4

Key result

Reading:

other: first and second reading

Hours after challenge:

48

Group:

positive control

Dose level:

100 % at challenge

Remarks on result:

positive indication of skin sensitisation

Remarks:

100 % of the tested animals responded positively (no absolute numbers are given in the report)

Any other information on results incl. tables

Table 1 Challenge responses and body weights of test item treated group

Animal number	24-hour-observation		48-h-observation		Body weight (g)
	Left anterior	Left posterior	Left anterior	Left posterior	Day 0	Day 24
1	0	0	0	0	330	442
2	0	0	0	0	408	510
3	0	0	0	0	406	510
4	0	0	0	0	340	450
5	0	0	0	0	390	475
6	0	0	0	0	383	502
7	0	0	0	0	343	510
8	0	0	0	0	379	496
9	0	0	0	0	337	470
10	0	0	0	0	402	496

Table 2 Challenge responses and body weights of vehicle control group

Animal number	24-hour-observation		48-h-observation		Body weight (g)
	Left anterior	Left posterior	Left anterior	Left posterior	Day 0	Day 24
11	0	0	0	0	380	466
12	0	0	0	0	390	485
13	0	0	0	0	364	465
14	0	0	0	0	375	437
15	0	0	0	0	384	497

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, the test item showed no dermal sensitising potential in guinea pigs.

Executive summary:

The dermal sensitizing potential of the test item was modified investigated according to one of the methods recommended in the OECD Guidelines o. 406, "Skin Sensitization", 1992 and the EEC Guideline "EEC 92/69 part 6B", 1996.

The delayed contact hypersensitivity test used was the Guinea Pig Maximization Test described by B. Magnusson and A. M. Kligman.

15 animals divided into a test group of 10 animals and a control group of 5 animals were included in the study. The study comprised an induction and a challenge phase. The animals in the test group were induced with the test item whereas the animals in the control group were induced with the vehicle only. The induction procedure included intradermal injections and a closed patch topical application one week apart. All animals were challenged by a closed patch topical application of the test item for 24 hours. The challenge procedure included a closed patch topical treatment of the test item an the flank 3 weeks after the intradermal injection. The skin reactions were evaluated 24 and 48 hours after the challenge application. A 1.25 % (w/v) test item concentration was used for the intradermal induction and a 75 % (w/v) test item concentration for the topical induction. A 50 % (w/v) test item concentration was used for the challenge application. Under the experimental conditions described in this final report no evidence of delayed contact hypersensitivity was seen after the treatment with the test item.