Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Apr 2005 - 22 Aug 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Crl:SA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory Animal Breeders, 88353 Kisslegg, Germany
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 258 - 320 g
- Housing: animals were conventionally kept in type IV Makrolon® cages [4], in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. Low-dust wood shavings supplied by Rettenmaier & Sohne, GmbH & Co., Rosenberg, Germany were used as bedding. All animals used in this study were kept in the same animal room, one in which guinea pigs from other sensitization studies were also housed. Adequate separation, unambiguous cage markings and suitable organization of the work ensured that animals were not mixed up.
- Diet: PROVIMI KLIBA 3420 - Maintenance Diet for Guinea Pigs (PROVOMI KLIBA AG), ad libitum.
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): >= 10 times per hour
- Photoperiod (hrs dark / hrs light): 12/12 hrs

IN-LIFE DATES: From: 12 Apr 2005 To: 06 May 2005

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Concentration / amount:
Induction application: 5% (20 mg test item/animal: intradermal) and 50% ( 250 mg test item/animal: epicutaneous)
Challenge application: 50% (250 mg test item/animal: epicutaneous)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
Induction application: 5% (20 mg test item/animal: intradermal) and 50% ( 250 mg test item/animal: epicutaneous)
Challenge application: 50% (250 mg test item/animal: epicutaneous)
No. of animals per dose:
Test group: 20
Control group: 10
Details on study design:
RANGE FINDING TESTS:
- Intradermal induction: One guinea pig (animal No. 1) was given intradermal injections twice, in each case, with 0.1 ml of the following test item concentrations: 0%, 1%, 2.5% and 5%. The injection sites were evaluated after 24 and 48 hours:
- topical induction: Three different concentrations (12%, 25%, 50%) and the vehicle were tested on four guinea pigs. The patches moistened with 0.5 ml of the test item formulations or the vehicle were applied to each animal under occlusive conditions for 24 hours. At the end of the exposure period, the remaining test item or the vehicle were removed with physiological saline solution and twenty-one hours later the treated areas were shorn. The skin reactions were evaluated 48 hours and 72 hours after the start of the application. No reactions were observed in any of the animals.
- challenge: One week prior to the challenge, the challenge concentration was determined on 2 guinea pigs in the main study that were treated in the same manner as the control animals during the inductions. Four patches each loaded with 0.5 ml test item formulation (12%, 25%, 50%) or the vehicle were applied to each animal under occlusive conditions for 24 hours. At the end of the exposure period, the remaining test item or the vehicle were removed with physiological saline solution and twenty-one hours later the treated areas were shorn. The skin reactions were evaluated 48 hours and 72 hours after the start of the application. No reactions were observed in any of the animals.

MAIN STUDY
Twenty guinea pigs weighing 258 - 320 g were randomly selected for the test group and ten guinea pigs were selected to be weight matched controls for challenge. The exposure doses were based on the results of the range-finding studies.

A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: On day 1, at the intradermal induction test animals were treated with 5% test material. One week later, during the second induction expocure, the test animals were treated with 50% test material by topical occlusive application over the induction injection sites. Control animals were treated with 0.5 ml polyethylene glycol 400. At the end of the 48-hour exposure period, the remaining test item was removed with sterile physiological saline solution.
- Test groups: 20 female guinea pigs
- Control group: 10 female guines pigs
- Site: dorsal, neck region
- Frequency of applications: one intradermal application, one topical application.
- Concentrations:
Test group, intradermal induction:
1st Injection site: 0.1 mL cranial/bilateral complete Freund's adjuvant (DIFCO LAB.) diluted 1:1 with sterile physiological saline solution.
2nd Injection site: 0.1 mL medial/bilateral 5% test substance formulated in polyethylene glycol 400.
3rd Injection site: 0.1 mL caudal/bilateral 5% test substance formulated at equal parts in polyethylene glycol 400 and complete Freund's adjuvant.
Test group, topical induction: 50% test material as 0.5 mL.

Control group, intradermal induction:
1st Injection site: 0.1 mL cranial/bilateral complete Freund's adjuvant (DIFCO LAB.) diluted 1:1 with sterile physiological saline solution.
2nd Injection site: 0.1 mL medial/bilateral undiluted polyethylene glycol 400.
3rd Injection site: 0.1 mL caudal/bilateral equal parts of polyethylene glycol 400 and complete Freund's adjuvant.
Control group, topical induction: 0.5 ml polyethylene glycol 400.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: Three weeks after the intradermal induction application the test animals and control animals were challenged by hypoallergenic patch for 24 hours. At the end of the exposure period, the remaining test item was removed with physiological saline solution, and 21 hours later the skin of the animals was shorn in the zone of the challenge area.
- Test groups: 20 female guinea pigs
- Control group: 10 female guinea pigs
- Site: the right (caudal) flank of the animals.
- Concentrations: 50.0% test material in a volume of 0.5 mL (test group and control group) or 0.5 mL vehicle (control patch). A patch only loaded with the vehicle was placed on the right flank (cranial) as control patch.
- Evaluation (hr after challenge): 48 and 72 hrs after application of the test substance.
Challenge controls:
10
Positive control substance(s):
no

Results and discussion

Positive control results:
For the intradermal induction a 5% test item formulation was used, for the topical induction a 25% formulation and for the challenge a 12% formulation. After the challenge with the 12% test item formulation in the test item group 100% of the test animals exhibited dermal reactions in the challenge treatment. There was no reddening of the skin to be observed on control group animals.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
slight localized redness
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 10.0. Total no. in groups: 20.0. Clinical observations: slight localized redness.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
slight localized redness
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 50%. No with. + reactions: 10.0. Total no. in groups: 20.0. Clinical observations: slight localized redness.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects.

Any other information on results incl. tables

Following the first induction, the animals in the control group and the test group showed clear effects of irritation (encrustation) at the injection sites.

The challenge with the 50% test substance formulation led to skin effects (grade 1) in 10 of 20 animals in the test group (50%). No skin effects were observed in the animals of the control group. This indicates that the test substance exhibits a skin-sensitisation potential.

The appearance and behaviour of the test item group were not different from the control group. At the end of the study, the mean body weight of the treatment group animals was in the same range as that of the control group animals

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU