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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 February 2001 - 16 March 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
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Official Journal of the European Union No. L 248, p. 206 - 211 of September 30, 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed in 2001, i.e. before LLNA was officially considered as a preferred testing method.

Test material

Constituent 1
Chemical structure
Reference substance name:
Reference substance 001
EC Number:
607-739-3
Cas Number:
2550-52-9
Molecular formula:
C16H30O
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Crl:(HA)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, D-97320 Sulzfeld
- Microbiological status of animals, when known: SPF
- Age at study initiation: approximately 18 days at the start of acclimatization
- Weight at study initiation: 271.2 ± 18.3 g (n = 30)
- Housing: The animals were housed under semi-barrier conditions up to 5 to a cage (Makroion® T ype 4)
- Diet (e.g. ad libitum): ALTROMIN 1322, standard diet for guinea pigs, ad libitum (ALTROMIN, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum (municipal supply), Makrolon® b ottles, changed daily
- Acclimation period: 13 days before administration (main study)
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 30 - 60 %%, with a short falling below to 20 %
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 19 February 2001 To: 16 March 2001 (main study)

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
5%
Day(s)/duration:
Day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate (1:1 v/v)
Concentration / amount:
25% (w/v), 0.5 mL per flank
Day(s)/duration:
7
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate (1:1 v/v)
Concentration / amount:
25%; 0.5 mL per flank
Day(s)/duration:
21
Adequacy of challenge:
not specified
No. of animals per dose:
20/dose group, 10/control group
Details on study design:
RANGE FINDING TESTS:
Intracutaneous tolerance test:
Twenty four hours after preparation of the animals 4 pairs of the following intracutaneous injections of 0.1 ml volume were given to both animals in the shoulder region so that one of each pair lies on each side of the midline in cranial to caudal sequence.
Injection 1: 5 % (w/v) solution of the test item in AO
Injection 2: 3 % (w/v) solution of the test item in AO
Injection 3: 2 % (w/v) solution of the test item in AO
Injection 4: 1 % (w/v) solution of the test item in AO

Epicutaneous tolerance test:
Twenty four hours after preparation of the animals two layers of filter paper (2 x 4 cm) were loaded with 0.5 ml of the test item formulations and applied to the test areas.
Area 1 (left flank): 25 % (w/v) solution of the test item in ED
Area 2 (left shoulder): 10 % (w/v) solution of the test item in ED
Area 3 (right shoulder): 5 % (w/v) solution of the test item in ED
Area 4 (right flank): 1 % (w/v) solution of the test item in ED
The papers were covered with film and held in contact with the skin by occlusive dressing (Lohmann GmbH & Co., Neuwied). After 24 hours the patches were removed and the administration areas were rinsed with ED.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 epicutaneous
- Exposure period: 48 hours for epicutaneous induction
- Test groups: 20 animals/group
- Control group: 10 animals/group
- Site:
Intradermal:
Three pairs of intracutaneous injections of 0.1 ml volume were given to each animal in the shoulder region so that one of each pair lay on each side of the midline in cranial to caudal sequence.
Dose group:
Injection 1: mixture of water for injections and Freund' s Complete Adjuvant (FCA) (1:1, v/v)
Injection 2: 5 % (w/v) solution of the test item in arachis oil
Injection 3: 5 % (w/v) Suspension of the test item in FCA/water (1:1, v/v)
Control group:
Injection 1: mixture of water and FCA (1:1, v/v)
Injection 2: arachis oil
Injection 3: 50 % mixture (w/v) of arachis oil in a mixture of water and FCA (1:1, v/v)

Epicutaneous:
Dose group:
Two layers of filter paper (2 x 4 cm) were loaded with 0.5 ml of the 25 % (w/v) solution of the test item in ED. One patch each was then placed on the skin of both sides of the body.
Control group:
Two layers of filter paper (2 x 4 cm) were soaked with 0.5 ml of ED. One patch each was then placed an the skin of both sides of the body.
In both groups the papers were covered with film and held in contact with the skin by occlusive dressing (Lohmann GmbH & Co., Neuwied). After 48 hours the patches were removed and the administration areas were rinsed with ED.

- Frequency of applications: 1 intradermal (3 injections), 1 epicutaneous
- Duration: 48 hours for epicutaneous application
- Concentrations: 5% in arachis oil for intradermal induction, 25% in ethanol/diethylphthalate (1:1 v/v)

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: 21
- Exposure period: 24 hours
- Test groups: 20 animals
- Control group: 10 animals
- Site: right flank
- Concentrations: 25% in ethanol/diethylphthalate (1:1 v/v)
- Evaluation (hr after challenge): 24 and 48 hours after patch removal
Challenge controls:
Strips of gauze patch were loaded with 0.5 ml of the 25 % (w/v) solution of the test item in ethanol/diethylphthalate (1:1 v/v) and applied to the right flank of all animals. Other strips of gauze patch were soaked with 0.5 ml of ethanol/diethylphthalate (1:1 v/v) and applied to the left flank of all animals.
Positive control substance(s):
yes
Remarks:
Benzocaine at the same experimental conditions.

Results and discussion

Positive control results:
The following dosing scheme was used:
Intracutaneous induction day 0: 2% benzocaine aqueous solution
Epicutaneous induction day 7: 25 % suspension in liquid paraffin
Challenge day 21: 10 % suspension in liquid paraffin
The epicutaneous administration of the 10 % suspension of Benzocaine in liquid paraffin caused a slight to moderate erythema (grade 1-2) in all animals of the dose group on day 23. In 9 of 10 animals these irritating signs were also observed on day 24.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% in ethanol/diethylphthalate (1:1 v/v)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% in ethanol/diethylphthalate (1:1 v/v)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% in ethanol/diethylphthalate (1:1)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% in ethanol/diethylphthalate (1:1 v/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10% benzocaine in liquid paraffin
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
The epicutaneous administration of the 10 % suspension of Benzocaine in liquid paraffin caused a slight to moderate erythema (grade 1-2) in all animals of the dose group on day 23.
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10% benzocaine in liquid paraffin
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Slight to moderate erythema was observed in 9 animals on day 24
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the study performed according to OECD guideline 406 (guinea pig maximization test), the test substance cyclohexadecanone was not sensitizing to skin.
Executive summary:

In a reliable study, performed according to OECD guideline 406 (guinea pig maximization test) the test substance cyclohexadecanone was not sensitizing following intradermal induction with 5% in arachis oil and epicutaneous induction with 25% in ethanol/diethylphthalate (1:1 v/v). No skin reactions were observed in any of 20 test animals following epicutaneous challenge with 25% test substance in ethanol/diethylphthalate (1:1 v/v). Positive control benzocaine produced satisfactory positive response (10/10 animals exibiting slight to moderate erythema following challenge with 10% in liquid paraffin). Based on the results of the study, cyclohexadecanone is not considered to be sensitizing to skin.