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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:
2000-04-18 until 2000-05-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japan MAFF guideline, 59 Noh San No. 4200, January 28,1985
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was performed before LLNA was formally validated and available as OECD guideline in 2002.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-(4-Diethylamino-2-hydroxybenzoyl )-benzoesaeurehexylester
- Physical state and appearance: Solid, crystalline with white-yellowish colour.
- Analytical purity: 99.35 g/100 g (by GC analysis)
- Lot/batch No.: R323/681
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, FRG.
- Age at study initiation: Young adult animals
- Weight at study initiation: 327 - 375 g
- Housing: 5 animals per cage. Stainless steel wire mesh cages with plastic-coated
grating. Minimum floor area: 2000 cm2.
- Diet: Kliba Labordiät (Kaninchen-Meerschweinchen-Haltungsdiät) ad libitum. Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water: Tap water ad libitum
- Acclimation period: 7 days before the beginning of the study in the laboratory

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
For the intradermal induction: 5% of test substance
For the epicutaneous induction: 25% of test substance
For the challenge: 25% of the test substance
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
For the intradermal induction: 5% of test substance
For the epicutaneous induction: 25% of test substance
For the challenge: 25% of the test substance
No. of animals per dose:
Main test:
Number of animals per control group: 5
Number of animals of the test group: 10
Details on study design:
RANGE FINDING TESTS:
The concentrations of the test substance suitable for use in the main experiment were determined in a pretest (intradermal and epicutaneous):

- Intradermal Pretest
Amount applied:
6 intradermal injections in groups of two per animal were applied to each animal.
A) front row 2 injections each of 0.1 mL Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCl-solution in a ratio of 1: 1
B) middle row. 2 injections each of 0.1 mL of a test substance formulation in an appropriate vehicle (olive oil) at the selected concentration (5%)
C) back row 2 injections each of 0.1 mL Freund's adjuvant /0.9% aqueous NaCl-solution (1: 1) with test substance at the selected concentration (5%)

Site of application: shoulder
No. of animals: 2 per test substance concentration
Reading: 24 h after the beginning of application

- Epicutaneous Pretest
Amount applied:
2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing 0.5 mL of the test substance formulation were applied to the skin of the flanks under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (5 x 5 cm from Pfaelzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.

Exposure period:
The test substance was applied 2 times for 24 hours within a period of 96 hours

Site of application: flank, on the same area respectively
Number of test animals: 5 per test concentration
Readings: 24 h and 48 h after the beginning of application
Concentrations applied: 10%, 25% in olive oil

Applicability:
- After injection of 5% test substance preparation in olive oil or in Freund's adjuvant / 0.9% aqueous NaCl-solution (1 :1) with a syringe the concentration was well-tolerated locally and systemically.
- The application of a 25% test substance preparation in olive oil did not cause any skin reaction (48 hours after beginning of application).

The following concentrations for induction and the challenge were selected on the basis of the pretest:
Intradermal induction: Test substance 5% in olive oil or 5% in Freund's adjuvant / 0.9% aqueous NaCl-solution (1:1)
Epicutaneous induction: Test substance 25% in olive oil
Challenge test substance: Test substance 25% in olive oil


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures and concentrations: 2 exposures:

1. Intradermal induction that included 6 injections to each animal of the test group as follows:
A) front row: 2 injections each of 0.1 mL Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCI-solution in a ratio of 1: 1
B) middle row: 2 injections each of 0.1 mL of 5% test substance in olive oil
C) back row: 2 injections each of 0.1 mL Freund's adjuvant /0.9% aqueous NaCl-solution (1: 1) with test substance- 5%.
Site of application: shoulder
Reading: 24 h after the beginning of application

2. Epicutaneous induction: 2 x 4 cm gauze containing 25 % test substance in olive oil (1 mL of the test substance formulation was applied to each animal) under occlusive dressing.
- Dressing: 2 x 4 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test substance formulation were applied to the skin of the shoulder under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 6 cm from Russka) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
- Exposure period: The epicutaneous induction was carried out one week after the intradermal induction. 14 days after the intradermal induction, the challenge exposure was performed.
- Test groups: One test group that contains 10 animals

- Control group: 2 control groups (5 animals per group). Each animal in these control groups got the same induction exposure (intradermal and epicutaneous) treatments as written above but without the test substance.
- Site: The site of the injections (intradermal induction) and the gauze patches (epicutaneous induction) were at the same area on the shoulder. For the exact location of the different injections see above.
- Frequency of applications and duration: One time 6 injections for each animal (intradermal induction) and one time application of the patches in the epicutaneous induction which lasted 48 hours.


B. CHALLENGE EXPOSURE
- No. of exposures: One time challenge exposure
- Day(s) of challenge: 14 days after the epicutaneous induction.
- Site of application:
test substance formulation: posterior right flank
vehicle: posterior left flank
- Application / amount applied:
- Dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. ) containing the test substance formulation were applied to the skin of the flank under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (5 x 5 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG. 0.5 mL l of the test substance formulation was applied to each animal.
- Concentrations: 25% of the test substance in olive oil (0.5 mL on 2x2 cm gauze patches-occlusive dressing)
- Exposure period: The challenge exposure lasted 24 hours
- Test groups: 1 test group
- Control group: One control test (control group 1) was treated with the test substance as the test group (in both groups each animal was also applied with olive oil as a vehicle control on the other side of the flank). Whereas the other control group (control group 2) received only olive oil ( no challenge exposure).
- Evaluation (hr after challenge): 24 and 48 hours after the removal of the patch.
Challenge controls:
Control group 1 which had induction exposure without the test substance but a challenge exposure like the test group, i.e. with the test substance. In case of a borderline results at the (first) challenge exposure the control group 2 (which had no exposure induction and no challenge induction) were kept for a potential second challenge.
Positive control substance(s):
yes
Remarks:
A positive control with a known sensitizer is performed in separate study ttwice a year with Alpha-Hexylcinnamaldehyde techn. 85%. The test system proved to be able to detect sensitizing compounds under the laboratory conditions chosen.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
This data refers to control group 1 (see below).
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: This data refers to control group 1 (see below)..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
This data refers to control group 1 (see below).
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: This data refers to control group 1 (see below)..

Applicant's summary and conclusion