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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:
7 April 1998- 8 May 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD TG 406

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
other: method described by Magnusson and Kligman, 'Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens'
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid study from 1989 is available, which has been conducted as guinea pig maximisation test.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
SOURCE OF TEST MATERIAL
- Purity: 94.0 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark.
Specific details on test material used for the study:
Batch: 6263001
Expiry date: 01 September 1999
Description: Off white solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
albino guinea pig (SPF-quality)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany.
- Females: Nulliparous and non-pregnant
- Age at study initiation: approx, 5 weeks old
- Weight at study initiation: Individual body weights did not exceed 500 grams.
- Housing: Group housing of 5 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system
- Diet: Free access to standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4mm (Hope farms, Woerden, The Netherlands).
- Water: Free access to tap-water, diluted with decalcified water.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
Air-conditioned room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%.
Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

ACCOMODATION
Group housing of 5 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands). The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
0.2% / 0.1ml
Day(s)/duration:
day 1 / 72 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, semiocclusive
Vehicle:
corn oil
Concentration / amount:
50% / 0.5ml
Day(s)/duration:
day 8 / 48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50% / 0.5ml
Day(s)/duration:
day 23
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50% / 0.5ml
Day(s)/duration:
day 24
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10
Details on study design:
RANGE FINDING TESTS:
-Induction (Intradermal and epidermal): The highest possible concentration that produced moderate irritation. Each of two animals received two different concentrations in duplicate (0.1 ml/site) in the clipped-scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.
-Challenge: The maximum non-irritant concentration. Two different concentrations were applied (0.5 ml each) per animal to the clipped flank, using Metalline patches and treated skin areas were assessed for irritation 24 and 48 hours after exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 10
- Exposure period: 10 days
- Test groups: 10
- Control group: 5
- Site: Scapular region
- Duration: Intradermal Injection (1-3 days), Epidermal exposure (8-10 days)

Day 1
The scapular region was clipped and three pairs of intradermal injections (0.1 ml/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection.
B) The test substance at a 0.2% concentration.
C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant.

Day 3
The dermal reactions caused by the intradermal injections were assessed for irritation.

Day 7
The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

Day 8
The 10% SDS treated area between the injection sites was treated with 0.5 ml of a 50% test substance concentration using a Metalline patch (2x3 cm)mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.

Day 10
The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance and the dermal reactions caused by the epidermal exposure were assessed for irritation.

B. CHALLENGE EXPOSURE
- No. of exposures: 10
- Day(s) of challenge: day 21
- Exposure period: 24 and 48hours
- Test groups: 10
- Control group: 5
- Site: Epidermal
- Evaluation (hr after challenge): Granding Irritation Reactions (according to guideline) after 24 and 48 hours

Day 21
One flank of all animals was clipped and treated by epidermal application of a 50% test substance concentration and the vehicle (0.5 ml each), using Metalline patches (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape and subsequently Coban elastic bandage.

Day 23 and 24
The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

C. OBERSVATIONS
- Mortality/Viability: twice daily
- Toxicity: at least once daily
- Body weights: Prior to start and at termination of the study.
- Irritation: Skin reactions were graded according to a numerical scoring systems (according to guideline). Furthermore, a description of all other (local) effects was recorded. Whenever necessary, the treated skin-areas were clipped at least 3 hours before the next skin reading to facilitate scoring.

Challenge controls:
vehicle (corn-oil)
Positive control substance(s):
no

Results and discussion

Positive control results:
Reliability check
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques. Females of the albino Dunkin Hartley guinea pig (from Charles River, Germany) were checked for the sensitivity to ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85%.

Test substance concentrations selected for this study were:
Intradermal induction: A 5% solution in water (Milli-U, w/w).
Epidermal induction: undiluted.
Challenge: a 10% and a 5% solution in water (w/w).

Results lead to a sensitisation rate of 100 per cent to both the 10% and 5% concentrations. From these results, it was concluded that the female guinea pig of the albino Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a substance in a Maximisation type of test.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
moderate irritation (intradermal), no irritation (epidermal)
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
moderate irritation (intradermal), no irritation (epidermal)
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no clinical sings
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no clinical signs
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
three animals showed scaliness
Remarks on result:
positive indication of skin sensitisation
Remarks:
score 2: 5 animals, score 1: 5 animals
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
all animals showed scaliness
Remarks on result:
positive indication of skin sensitisation
Remarks:
score 2: 8 animals, score 1: 2 animals)

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

Based on preliminary results, a 0.2% test substance concentration was selected for the intradermal induction in the Main Study, as it produced moderate irritation (erythema score 3). A 50% concentration was selected for the epidermal induction exposure.

No signs of irritation were observed to the highest test substance concentration tested in the preliminary irritation study. Therefore, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction. A 50% test substance concentration was selected for the challenge phase.

MAIN STUDY

A. Induction phase

The skin effects caused by the intradermal injection and epidermal exposure during the induction phase are given in Table 1.

The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

B. Challenge phase

No skin reactions were evident after the challenge exposure in the experimental and control animals (Table 2).

C. Observations

Toxicity / Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Body Weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Table 1. Induction Readings.

Animal
Number
Intradermal Injection
(DAY 3)
Epidermal Exposure
(DAY 10)
A B C 50%#
Control
1 E4 NA N1 0 0
2 E4 NA E2 0 0
3 E4 NA E4 1 0
4 E4 NA E2 0 0
5 E4 NA E4 0 0
Experimental
6 E1 E1 E1 0 0
7 E2 E1 E2 0 0
8 E2 E1 E2 0 0
9 E2 E2 E2 0 0
10 E3 E2 E3 0 0
11 E2 E3 E3 0 0
12 E2 E2 E2 0 0
13 E2 E2 E3 0 0
14 E2 E2 E3 0 0
15 E2 E3 E4 0 0

A. 1:1 mixture of FCA(Freunds' Complete Adjuvant) and water for Injection.

B. A 0.2% test substance concentration (Experimental); vehicle (Control).

C. 1:1 mixture of FCA and a 0.4% concentration (Experimental) or vehicle (Control).

#. Test substance concent ti (Experimental); vehicle (Control).

Skin effects intradermal injections:

NA No abnormalities

E(.) Erythema (grade)

N( .) Signs of necrosis (mm in diameter)

Table2. Challenge readings

   Challenge    
Animal
Number
DAY 23
Readings
DAY 24
Readings
Comments
50%# Vehicle 50%#  Vehicle   
Control
1 0 0 0 0  
2 0 0 0 0  
3 0 0 0 0  
4 0 0 0 0  
5 0 0 0 0  
Experimental
6 E1 E1 E1 0 not sensitised
7 E2 E1 E2 0 not sensitised
8 E2 E1 E2 0 not sensitised
9 E2 E2 E2 0 not sensitised
10 E3 E2 E3 0 not sensitised
11 E2 E3 E3 0 not sensitised
12 E2 E2 E2 0 not sensitised
13 E2 E2 E3 0 not sensitised
14 E2 E2 E3 0 not sensitised
15 E2 E3 E4 0 not sensitised

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
acc. to CLP Regulation
Conclusions:
Phytosphingosine was not sensitising to skin under the test conditions. Based on these results and according to the CLP regulation, phytosphingosine does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.
Executive summary:

Assessment for contact hypersensitivity to phytosphingosine in the albino guinea pig (maximisation test) was carried out according to OECD 406 'Skin Sensitisation' and in compliance with GLP.

Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 0.2% concentration and epidermally exposed to a 50% concentration. In addition, five control animals were similarly treated with the vehicle (corn oil) only (negative control). Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentrationl and the vehicle. No skin reactions were evident after the challenge exposure in the experimental and control animals.

No evidence was obtained that phytosphingosine had caused skin hypersensitivity in the guinea pig, since no respones were observed in the experimental animal in the challenge phase. This result indicates a sensitisation rate of 0 percent. In conclusion, phytosphingosine was considered not sensitsing to skin.

The study was considered reliable and adequate for hazard assessment of human health.