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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:
21 Jul - 05 Sept 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
adopted 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
adopted March 2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), November 2000, including the most recent partial revisions
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): AF-654
- Physical state: white powder
- Lot/batch No.: #CE-201
- Expiration date of the lot/batch: 01 Jan 2005
- Storage condition of test material: room temperature in the dark

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 Deutschland, Kisslegg, Germany
- Age at study initiation: approximately 4 weeks old
- Weight at study initiation: control group (mean): 360 ± 15 g; test group (mean): 344 ± 13 g
- Housing: group housing of maximally 5 animals per labelled cage containing purified sawdust as bedding material
- Diet: standard guinea pig diet, including ascorbic acid (1000 mg/kg) (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin, Germany), ad libitum and pressed hay twice a week
- Water: tap water, ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 22.7
- Humidity (%): 45 - 88
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Induction: intradermal 5%, epicutaneous 50%
Challenge: epicutaneous 50%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: intradermal 5%, epicutaneous 50%
Challenge: epicutaneous 50%
No. of animals per dose:
5 (controls), 10 (in test group)
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted, the selection of concentrations was based on the following criteria:
- the concentrations are well-tolerated systemically
- for induction exposure: the highest possible concentration that produced mild to moderate irritation (grades 2-3)
- for challenge exposure: the maximum not-irritant exposure
Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting concentration for each route. The starting and subsequent concentrations were taken from the series: 100, 50, 20, 10, 5, 2, 1% and if needed further lower concentrations using the same steps. The system and procedures were identical to those used for the main study.

Intradermal injections: A series of 4 test substance concentrations (10, 5, 2 and 1%) was 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 injection.

Epicutaneous application: A series of 4 test substance concentrations (50, 20, 10 and 5%) 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. The animals receiving intradermal injections were treated with the lowest concentrations and two additional animals with the highest concentrations. After 24 hours the skin was cleared of residual test substance using water and assessment for irritation was done 24 and 48 hours after exposure.

Based on the results of the preliminary studies, the test substance concentration selected for the intradermal induction in the main study was a 5% concentration. The intradermal injection of a 10% concentration was difficult and the reliability of the injected volume could not be established. For the epicutaneous induction exposure in the main study a 50% concentration was selected. No irritation reactions were observed to the highest test substance concentration epidermally tested. Therefore, 10% SDS was applied 24 hours before epicutaneous induction in the main study to induce mild inflammation.
A 50% test substance concentration was selected for the challenge phase.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (w/w) Freunds' Complete Adjuvant (FCA)/water
Injection 2: test substance at a 5% concentration in corn oil
Injection 3: 1:1 mixture (w/w) of FCA and the test substance at 10% concentration

Epicutaneous: 50% test substance in corn oil

- Control group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (w/w) FCA/water
Injection 2: corn oil
Injection 3: 1:1 mixture (w/w) of FCA and corn oil

Epicutaneous: corn oil

- Site: scapular region (intradermal and epicutaneous)
- Frequency of applications: every 7 Days
- Duration: Day 0 - 8
- Concentrations: intradermal 5%, epicutaneous 50%

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day(s) of challenge: Day 22
- Exposure period: 24 hours
- Test groups: test substance and vehicle
- Control group: test substance and vehicle
- Site: flank
- Concentration: 50%
- Evaluation (hr after challenge): 48 and 72 h

OTHER:
- On Day 7 10% SDS was applied to the topical application site to provoke a mild inflammatory reaction.
- Observations for mortality were recorded twice daily, toxicity at least once daily and body weights were measured at start and termination of the study. Skin reactions were graded according to Draize Scoring System.
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamicaldehyde (85%)

Results and discussion

Positive control results:
The positive control was used at regular intervals for a reliability check of the study protocol. Alpha-hexylcinnamicaldehyde (induction intradermal: 20% (in water), induction epicutaneous: undiluted, epicutaneous challenge: 20% (in water)) resulted in 60% positive sensitisation reactions in guinea pigs tested (NOTOX Project 383254).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction: 0%; challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
erythema was observed at intradermal and epicutaneous induction sites
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: induction: 0%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: erythema was observed at intradermal and epicutaneous induction sites.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
induction: 5%; challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythema was observed at intradermal and epicutaneous induction sites
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: induction: 5%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: erythema was observed at intradermal and epicutaneous induction sites.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
induction: 0%; challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
erythema was observed at intradermal and epicutaneous induction sites
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: induction: 0%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: erythema was observed at intradermal and epicutaneous induction sites.
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction: 5%; challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythema was observed at intradermal and epicutaneous induction sites
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: induction: 5%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: erythema was observed at intradermal and epicutaneous induction sites.

Any other information on results incl. tables

Table 1: Skin effects caused by the intradermal injections and epidermal exposure during the induction phase

Animal

Intradermal Injection (Day 3)

Epidermal exposure (Day 10)

A

B

C

D

Erythema (grade)

Erythema (grade)

Erythema (grade)

Erythema (grade)

Oedema (grade)

Control 1

3

2

2

1

0

Control 2

2

2

2

0

0

Control 3

3

1

2

0

0

Control 4

3

2

2

1

0

Control 5

3

1

1

1

0

Experimental 1

3

3

3

2

0

Experimental 2

3

3

3

1

0

Experimental 3

3

2

3

1

0

Experimental 4

3

3

3

2

0

Experimental 5

3

3

3

1

0

Experimental 6

2

3

3

2

0

Experimental 7

3

3

2

2

0

Experimental 8

3

3

3

2

0

Experimental 9

3

3

3

0

0

Experimental 10

3

3

2

1

0

A: 1:1 mixture of FCA and water for injection

B: A 5% test substance concentration (Experimental); vehicle (Control)

C: 1:1 Mixture of FCA and a 10% concentration (Experimental) or vehicle (Control)

D: A 50% test substance concentration (Experimental); vehicle (Control)

Erythema was noted at intradermal and epicutaneous induction sites. The skin reactions observed in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment. No skin reactions were evident after the challenge exposure in the experimental and control animals.

No mortality occurred and no symptoms of systemic toxicity were noted in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
DSD: not classified
CLP: not classified