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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:
28.06.1991 - 22.07.1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
May 1981
Reference: B. Magnusson and A. M. Kligman: "The identifieation of contact allergens by animal assay. The guinea pig maximization test" (I Invest. Dermatol., 52, 268-276, 1969) and "Allergie Contact Dermatitis in the Guinea Pig" (Thomas Publishers, Springfield, Illinois, 1970).
Deviations:
yes
Remarks:
no skin reaction after intradermal induction
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was done before LLNA as first-choice method for in vivo testing was set into force.

Test material

Constituent 1
Chemical structure
Reference substance name:
4-cyclohexyl-4-methylpentan-2-one
EC Number:
225-562-6
EC Name:
4-cyclohexyl-4-methylpentan-2-one
Cas Number:
4927-39-3
Molecular formula:
C12H22O
IUPAC Name:
4-cyclohexyl-4-methylpentan-2-one
Specific details on test material used for the study:
Test material is a formulation of 10% in white vaseline.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Pirbright white Bor:DHPW (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: male : 349 - 458 g, female : 305 - 387 g
- Housing: up to maximum 5 animals per cage (Macrolon type IV) with "LIGNOCEL 3/4 Fasern" bedding
- Diet: ad libitum, Ssniff-G pellets
- Water: ad libitum, drinking water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature: 22 +/- 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): artificial lighting (120 lux) from 7.00 a.m. - 7.00 p.m.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
peanut oil
Concentration / amount:
5%
Day(s)/duration:
Day 0/single injections
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
10%
Day(s)/duration:
Day 7/48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
peanut oil
Concentration / amount:
5%
Day(s)/duration:
Day 21/24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 (test group and control)
Details on study design:
RANGE FINDING TESTS:
Intradermal Injection:
- Solvent: water and Freund's complete adjuvant (FCA; Sigma, 8024 Deisenhofen)
- Concentration: 5% of test substance (= a factor 2 dilution of the original 10% formulation in white vaseline)
- Amount animals: 2
- Recordings: 48 h after treatment
- Result: no specific findings

Dermal Application:
- Concentration: 10, 7.5 and 5% of test substance (= undiluted 10% formulation in white vaseline, a factor 1.33 and factor 2 dilution of the original 10% formulation in white vaseline)
- Procedure: closed patch by means of an occlusive bandage
- Amount animals: 2 per concentration
- Recordings: 48 h post application
- Results: 10%: slight erythema; 7,5%: partly slight erythema; 5%: no skin irritation

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2;
1: 3 pairs of intradermal injections: symmetrically in 2 rows an either side of the spine
2: 7 days later: test article was spread in a thick layer which was firmly secured by an occlusive dressing
- Exposure period: 48 h (epicutaneous)
- Site:
1: 4 x 6 cm clipped shoulder region
2: 4 x 5 cm same area
- Test group:
Intradermal:
1 .0.1 mL FCA (diluted w/w in water)
2. 0.1 mL test article diluted in peanut oil (final concentration: 5%, a factor 2 dilution of the original 10% formulation in white vaseline)
3. 0.1 mL test article emulsified in FCA/water (final concentration: 5%, a factor 2 dilution of the original 10% formulation in white vaseline)
Epicutaneous: undiluted test substance (concentration: 10%)
- Control group:
Intradermal:
1 .0.1 mL FCA (diluted w/w in water)
2. 0.1 mL vehicle (undiluted)
3. 0.1 mL vehicle (emulsified w/w with FCA)
Epicutaneous: vehicle

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 14 days after second stage induction
- Site: 5 x 5 cm on each flank, sealed for 24 h under an occlusive dressing
- Concentrations: maximal non-irritating concentration to the left flank and the vehicle to the right flank
- Evaluation (hr after challenge): 24 and 48 hours


SCORING SYSTEM:
0: No reaction
1: Scattered mild redness
2: Moderate and diffuse redness
3: Intense redness and swelling
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene (extreme sensitizer) and benzocaine (moderate sensitizer)

Results and discussion

Positive control results:
The reaction to the positive contml substances 2,4-dinitrochlorobenzene (extreme sensitizer) and benzocaine (moderate sensitizer) is tested periodically. The last test with an acceptable level of response to each of these substances was performed in April, 1991.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction intradermal: 0%; Challenge: 5%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 5%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Induction intradermal: 5%; challenge: 5%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Induction intradermal: 5%; challenge: 5%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
other: CLP criteria not met
Conclusions:
No animal showed an allergic response, the test article is not sensitising.
Executive summary:

The aim of the current study was to assess the contact sensitizing potential of the test item, according to the OECD test guideline 406 with particular reference to the publications by B. Magnusson and A. M. Kligman (1969 and 1970).

 

The potential skin sensitizing properties were assessed in the guinea pig maximization test using 20 test and 20 control animals. Following induction exposure to the test article or the vehicle, the animals were subjected two weeks later to a challenge exposure with the test article. Allergic responses to the challenge procedure were evaluated 24 and 48 hours after the end of the exposure period.

In the range finding study no specific findings were observed after intradermal injection of 50% test material (actual concentration of test substance 5%). After dermal application of 100% (actual concentration 10% in white vaseline) of the test item slight erythema was observed, while a concentration of 50% (actual concentration 5%) did not show any signs of skin irritation. These were the tested concentrations in the main study (intradermal induction: 5%, epicutaneous induction: 10%; epicutaneous challenge: 5%). In the main study the sensitization rate at 24 h and 48 h after challenge was 0%. At day 17 p.a. one guinea pig of the test group died, however, this was due to a disease of the respiratory tract and independent of the treatment.

According to the CLP Regulation (EC) No 1272/2008 the sub-categories for a skin sensitiser are as follows:

Sub-category 1A: Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered. The obtained data from the maximsation animal test can be used in the classification for sub-category 1A (Table 3.4.3): >= 30% responding at =< 0.1% intradermal induction dose or >= 60% responding at > 0.1% to =< 1% intradermal induction dose

Sub-category 1B: Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered. The obtained data from the maximisation animal test can be used in the classification for sub-category 1B (Table 3.4.4): >= 30% to <60% responding at > 0.1% to =< 1% intradermal induction dose or >= 30% responding at > 1% intradermal induction dose

Since no animal showed an allergic response, the test article does not have to be classified as sensitising.