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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:
1994-10-31 to 1994-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 17 July, 1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
December 1992
Qualifier:
according to
Guideline:
other: "Allergic Contact Dermatitis in the Guinea-Pig: Identification of Contact Allergens"
Version / remarks:
Magnusson B. Kligman A.M., 1970 published by C.C. Thomas, Springfield, Illinois, USA.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT study conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force.
Moreover, no indication for skin sensitisation was observed in this study and thus, no dose response information is needed. As described in OECD guideline 406, the LLNA is able to detect reliably moderate to strong sensitisers. Because the test substance is unlikely to be a sensitiser, the GPMT was considered appropriate. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Ceramide III
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stable at room temperature in the dark
- Solubility and stability of the test substance in the vehicle: stable for at least 4 hours in propylene glycol
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test substance was prepared in propylene glycol (w/w) prior to each treatment. No adjustment was made for specific gravity of vehicle. The test substance was homogenised using a mechanical stirrer and electric blender.
- Final preparation of a solid: 1, 5, 10 and 25 % (w/w)

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Species/strain: albino guinea pigs / Himalayan strain, SPF-quality
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: 369 – 500 g (excluding positive control group)
- Housing: in pairs in labelled metal cages with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands)
- Diet (e.g. ad libitum): guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4 mm (Hope farms, Worden, The Netherlands), ad libitum. Hay (B.M.I., Helmond, The Netherlands) was provided once a week.
- Water (e.g. ad libitum): Tap water diluted with decalcified water, ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 °C
- Humidity (%): 50 %
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark
- Air changes per hour: 15

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
propylene glycol
Concentration / amount:
Intradermal induction: 5 % (w/w) of the test item, in propylene glycol
Dermal induction: 25 % (w/w) of the test item, in propylene glycol
Day(s)/duration:
8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
Challenge:10 %, 5 %, 2 % (w/w) of the test item, in propylene glycol
Day(s)/duration:
3
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Number of animals in the test group: 10
Number of animals in the negative control group: 5
Number of animals in the dose range finding study: 4
Details on study design:
RANGE FINDING TESTS:
Intradermal Injections (one animal):
A 1 % and 5 % (w/w) test item concentration in propylene glycol were injected into injection sites (0.1 mL each) in the right and left clipped shoulder regions, respectively. The resulting dermal reactions were assessed 24 and 48 hours later for erythema, necrosis and diameter of necrosis.
Epidermal Application (same animal as mentioned above):
A 25 % (w/w) test item concentration in propylene glycol (0.5 mL) was applied epidermally on a shaved flank, using a Scotchpak-non-woven patch (2.5 x 2.2 cm) on Micropore tape (both 3M, St. Paul, USA) and held in place by Coban elastic bandage (3M, St. Paul, USA). After 24 hours, the dressing was removed and residual test substance removed using a tissue moistened with tap water. The treated skin was assessed 24 and 48 hours later.
Epidermal Application (three animals):
Four concentrations of the test substance in propylene glycol (25 %, 10 %, 5 % and 1 % w/w, 0.05 mL each) were applied occlusively on a shaved flank of each of the three animals, using Square chambers (v.d. Bend, Brielle, The Netherlands), mounted on Micropore tape and held in place by Coban elastic bandage. After 24 hours, the dressing was removed and residual test substance removed using a tissue moistened with tap water. The treated sites were assessed for erythema and oedema 24 and 48 hours later.
MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal (day 1)
- No. of exposures: 3 pairs of intradermal injections of 0.1 mL
Test group:
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: a 5 % concentration of the test item in propylene glycol
Injection 3: a 0.5 % concentration of the test item formulated in a 1:1 mixture (v/v) FCA/water
Control group:
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: 100 % propylene glycol
Injection 3: a 50 % (v/v) formulation of propylene glycol in a 1:1 (v/v) mixture FCA/water
Topical Application (day 8)
The shoulder region of the same animals was shaved again one day before the treatments and rubbed with 10 % sodiumdodecylsulfate (SDS, Boom, Meppel, The Netherlands) in petrolatum using a spatula.
Test Group:
The test item was suspended in propylene glycol at a concentration of 25 %. A patch (2 x 4 cm) was fully loaded with 0.5 mL of the prepared test item, applied to the test area and held in contact by an occlusive dressing for 48 hours. After 48 hours, the dressing was removed and residual test substance removed using a tissue moistened with tap water.
Control Group:
SDS 10 % treatment as described above.
A patch was fully loaded with 0.5 mL of the vehicle propylene glycol and applied to the test area and held in contact by an occlusive dressing for 48 hours.
B. CHALLENGE EXPOSURE (day 22)
Test and Control Group:
All animals were treated epidermally with 0.05 mL of each of the following test substance concentrations 10 %, 5 % and 2 %, w/w in propylene glycol and with the vehicle on the clipped and shaved flank, using Square chambers attached to Micropore tape and secured with Coban elastic bandage. After 24 hours, the dressing was removed and residual test substance removed using a tissue moistened with tap water. The skin reaction was observed and recorded 24 and 48 hours after patch removal.
For further information, please see section "any other details on materials and methods incl. tables".
Challenge controls:
5 animals challenged in the same manner without induction
Positive control substance(s):
yes
Remarks:
α-hexyl cinnamic aldehyde, 85%, was dissolved in physiological saline (intradermal induction) or in distilled water (challenge). For epidermal induction, undiluted test substance was used.

Results and discussion

Positive control results:
The sensitisation rate after application of the positive control substance α-hexyl cinnamic aldehyde, 85 %, was 100 %, confirming the reliability of the test system.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
other: No information given. Thus, it is assumed that the results refer to the second reading.
Hours after challenge:
48
Group:
positive control
Dose level:
10 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
No information given.
Remarks on result:
positive indication of skin sensitisation
Reading:
other: No information given. Thus, it is assumed that the results refer to the second reading.
Hours after challenge:
48
Group:
positive control
Dose level:
5 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
No information given.
Remarks on result:
positive indication of skin sensitisation
Reading:
other: No information given. Thus, it is assumed that the results refer to the second reading.
Hours after challenge:
48
Group:
positive control
Dose level:
2 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
No information given.
Remarks on result:
positive indication of skin sensitisation
Reading:
other: No information given. Thus, it is assumed that the results refer to the second reading.
Hours after challenge:
48
Group:
positive control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No information given.
Remarks on result:
other: in the 0 % group, no signs of skin sensitisation were observed.

Any other information on results incl. tables

Preliminary Study

The 25 % test substance concentration was chosen as the highest useable concentration, as higher concentrations were not homogeneous. The intradermal injections with a 5 % and 1 % concentration resulted in slight to well-defined erythema and some necrosis. Therefore, a 5 % concentration was selected for the intradermal induction.

The epidermal application with 0.5 mL of a 25 % concentration resulted in slight erythema at both reading times. Therefore, the 25 % concentration was selected for the epidermal induction.

The epidermal application with a series of concentrations (25 %, 10 %, 5 % and 1 %) using Square chambers resulted in two animals with slight erythema in response to the 25 % concentration and in one animal with slight erythema in response to the 10 % concentration. No skin reactions were observed in response to the other concentrations. Therefore, the 10 % concentration was selected as the highest concentration for the challenge.

The selection of the test substance concentration for the main study was based on the results of the preliminary study and in accordance with Magnusson and Kligman (1969).

No signs of systemic toxicity were observed during the preliminary study. However, body weight loss was noted in one of the four animals.

Main Study – Induction

Two experimental animals showed slight erythema after the 48 hours occluded epidermal induction exposure. One control animal showed very small crusts in the treated skin area.

Main Study - Challenge

Control Group: No skin reactions were evident after the challenge exposure.

Experimental Group: No skin reactions were evident after the challenge exposure.

Toxicity symptoms / Mortality

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

Body Weights

The average body weight gain of the control animals was markedly lower than of the experimental animals. No reason can be given for the cause of the difference.

Data on skin reactions from the preliminary study are shown in Table 1a-c.

In the main study induction phase, two animals showed slight erythema after the 48 hours occluded epidermal induction exposure. One control animal showed very small crusts in the treated skin area. No skin reactions were evident after the challenge exposure.

Table 1a: Preliminary study: intradermal injection (one animal)

24 h reading

Concentration % (w/w)

Erythema score

Necrosis

Diameter (mm)

 

1 %

1

yes

2

 

5 %

1

yes

2

48 h reading

Concentration % (w/w)

Erythema score

Necrosis

Diameter (mm)

 

1 %

2

yes

2

 

5 %

2

yes

2

Table 1b: Preliminary study: epidermal application using scotchpak-non-woven patch (same animal as used for intradermal injection)

24 h reading after bandage removal

Concentration % (w/w)

Erythema score

Oedema score

 

25 %

1

0

48 h reading after bandage removal

Concentration % (w/w)

Erythema score

Oedema score

 

25 %

1

0

Table 1c: Preliminary study: epidermal application using square chambers (3 animals)

24 h reading after bandage removal

Concentration % (w/w)

Erythema score

Oedema score

 

25 %

1/3 animals: 1

0

 

10 %

0

0

 

5 %

0

0

 

1 %

0

0

48 h reading after bandage removal

Concentration % (w/w)

Erythema score

Oedema score

 

25 %

1/3 animals: 1

0

 

10 %

1/3 animals: 1

0

 

5 %

0

0

 

1 %

0

0

Intradermal induction: 5% (w/w) of the test item, in propylene glycol

Dermal induction: 25% (w/w) of the test item, in propylene glycol

Challenge:10%, 5%, 2% (w/w) of the test item, in propylene glycol

POSITIVE CONTROL

α-hexyl cinnamic aldehyde, 85 %, was dissolved in physiological saline (intradermal induction) or in distilled water (challenge). For epidermal induction, undiluted test substance was used. See Table 2 for results.

Table 2: Skin reactions to positive control (10 animals in the test group).

 

α-hexyl cinnamic aldehyde concentration

 

10 %

5 %

2 %

0 %

Animals with skin reactions (scores 1-4)

10

10

10

0

Control animals with skin reactions (scores 1 only)

5

3

0

0

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions used in this study, no positive skin reactions were observed in any of the animals after the challenge exposure. The test item is therefore considered not to be a dermal sensitizer.
Executive summary:

In this skin sensitization study according to OECD Guideline No. 406 (adopted 17 July 1992) and EEC Directive 92/69/EEC, Part B.6 (December 1992), it was evaluated whether Ceramide III induces skin hypersensitivity in guinea pigs after intradermal and epidermal exposure.

After assessment of the slightly irritating and the non-irritating test substance concentrations in a preliminary study, a main study was performed with the selected test substance concentrations. Ten animals were intradermally injected with a 5 % concentration and epidermally exposed to a 25 % concentration, while five control animals were treated similarly, but with the vehicle only. Immediately after epidermal exposure, the skin irritation was scored. Two weeks after the epidermal application all animals were challenged with test substance concentrations of 10 %, 5 % and 2 %, and the vehicle propylene glycol. The challenge reactions were assessed 24 and 48 hours after bandage removal.

The epidermal exposure to Ceramide III in the induction phase resulted in slight erythema in two experimental animals only. The epidermal exposure to Ceramide III in the challenge phase resulted in no positive reactions in response to any of the concentrations tested.

Under the conditions of this study, Ceramide III induced no skin sensitisation in guinea pigs and is therefore considered to be a non-sensitizer.