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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:
28 Apr - 29 May 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The analytical purity of the test substance was not specified.

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:
adopted in 1992
Deviations:
yes
Remarks:
analytical purity of test substance not specified
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
adopted in 1996
Deviations:
yes
Remarks:
analytical purity of test substance not specified
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006. In accordance with the same regulation, the data was included to avoid unnecessary testing.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: approx. 5 weeks
- Weight at study initiation: 381 g (mean value)
- Housing: groups of 5 animals in metal cages with wire mesh floors
- Diet: free access to standard guinea pig diet (LC 23-B, pellet diameter 4 mm; Hope farms, Woerden, The Netherlands), including ascorbic acid (1600 mg/kg); in addition, hay was provided once a week (B.M.I., Helmond, The Netherlands)
- Water: tap water, diluted with decalcified water, ad libitum (by automatic drinking system: ITL, Bergen, The Netherlands)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
50%
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
Day 8 / 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:
corn oil
Concentration / amount:
20%
Day(s)/duration:
Day 22 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 females for the test group; 5 for the control group
Details on study design:
RANGE FINDING TESTS: Prior to the start of the main study the intradermal and epidermal irritancy of the test substance were investigated for selection of suitable test substance concentrations for induction and challenge of the main study. The selection was based on the absence of toxicity. For the Induction phase the highest possible concentration that produced moderate irritation should be determined (including slight necrosis with diameter < 3 mm for intradermal application. The challenge concentration should be the maximum non-irritant concentration. Selection of this concentration depended on a number of factors and exact criteria did not always apply. The test system, procedures and techniques were identical to those used in the main study; Animals were between 5 and 9 weeks old; therefore, body weights could exceed 500 g. Body weights were determined prior to treatment.
Concentrations usd for preliminary range finding test:
Intradermal: 100%, 50%, 20%, 10%
Epidermal: 100%, 50%, 20%, 10%

Intradermal injections: A series of four test substance concentrations were used; the highest concentration being the maximum concentration that could technically be injected. 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.

Epidermal application: A series of four test substance concentrations was used; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2 x 3 cm) mounted on medical tape, held in place with Micropore tape and subsequently Coban elastic bandage. The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours the dressing was removed and the skin cleaned of residual test substance. The treated skin areas were assessed for irritation 24 and 48 hours after exposure

Based on the results of the preliminary study a 50% dilution of the test substance in corn oil was used for intradermal induction, and the undiluted substance was used for the epidermal induction exposure. A 20% test substance concentration was selected for the challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Intradermal induction on Day 1 and epidermal induction at Day 8 (for 48 h)
- Induction test groups:
Intradermal:
The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Difeo, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 50% concentration in corn oil
C) A 1:1 w/w mixture of the undiluted test substance and Freunds' Complete Adjuvant
Epidermal:
The scapular area between the injection sites was clipped and subsequently treated with 0.5 ml of undiluted test substance using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Caban elastic bandage. 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 .
- Induction control animals:
The control animals were treated for induction as described for the experimental animals, except that, instead of the test substance, the vehicle was administered.
- Site: Three pairs of intradermal injections into the scapular region of both sides; one of each pair injected on each side of the midline and from cranial to caudal)
- Concentrations: Intradermal: 50%; Epidermal: 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24 h under occlusive dressing
- Test groups: 20% test substance
- Control group: 20% test substance
- Site: On one of the flanks (previously clipped)
- Concentrations: 20% in corn oil
- Evaluation (hr after challenge): 48 and 72 h (24 and 48 h after removal of the dressing)
Challenge controls:
none
Positive control substance(s):
yes
Remarks:
alpha-hexyl cinnamic aldehyde, tech. 85%

Results and discussion

Positive control results:
The skin reactions in the experimental animals observed in response to the 10% and 5% positive control substance concentration in the challenge phase of an independent control experiment were considered indicative of sensitisation, based on the absence of any response in the respective control animals. The positive control substance exposure lead to a sensitisation rate of 100% to both the 10% and the 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 sensitisation studies in a Maximisation type of test.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
20%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
20%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
20%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
20%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Group:
positive control
Dose level:
5 and 10%
Remarks on result:
positive indication of skin sensitisation
Remarks:
100% sensitisation rate

Any other information on results incl. tables

Induction readings:

Animal number

Intradermal injection (reading at day 3)

Epidermal exposure (reading at day 10)

 

A

B

C

100%

Control

Erythema

Edema

31

E2

NA

E3

0

0

32

N2

NA

E4

0

0

33

E4

NA

E4

0

0

34

E2

NA

E3

0

0

35

E3

E1

E2

0

0

Experimental

36

E2

E1

E2

3

2

37

E3

NA

E3

3

2

38

E3

E2

E4a

4

3

39

E2

E1

E3a

3

1

40

E3

E1

E4a

4n

1

41

E3

E2

E3

4

1

42

E4

E3

E4

4

3

43

E4

E4

E4

4

3

44

E4

E3

E4a

4

3

45

E3

E2

E3a

4

1

A: 1:1 mixture of Freuds´complete adjuvant with water

B: Test substance (50%) in corn oil

C: 1:1 mixture of the undiluted test substance with Freuds´complete adjuvant

a: Moderate erythema visible approx. 2 cm caudally of the injection sites

n: Signs of necrosis

 

Skin effects intradermal injections:

NA: No abnormalities

E(.): Erythema (grade)

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

Challenge readings

Animal No.

Day 24

Day 25

Comments

20%

Vehicle

20%

Vehicle

Control

31

0

0

0

0

 

32

0

0

0

0

 

33

0

0

0

0

 

34

0

0

0

0

 

35

0

0

0

0

 

Experimental

36

0

0

0

0

Not sensitized

37

0

0

0

0

Not sensitized

38

0

0

0

0

Not sensitized

39

0

0

0

0

Not sensitized

40

0

0

0

0

Not sensitized

41

0a

0a

0

0

Not sensitized

42

0

0

0

0

Not sensitized

43

0

0

0

0

Not sensitized

44

0

0a

0

0

Not sensitized

45

0

0

0

0

Not sensitized

 a: Skin reactions grade 1 noted at the edges of the application area are considered non-specific, possibly provoked by the edges of the patches.

No deaths occurred. No significant differences in the gain of body weight were observed between treatment and control group.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified