Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD test guideline 406)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Description: blue powder
- Expiration date of the batch: 01-Apr-1997
-Stability under storage conditions: stable

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 355-449 g
- Housing: in groups of 2 animals per labelled metal cage with wire-mesh floors
- Diet: standard guinea pig diet including ascorbic acid (1600 mg/kg bw; LC 23-B, Hope Farms, Woerden, The Netherlands), ad libitum. Hay (Broekman Institute, Someren, The Netherlands) was provided once a week.
- Water: tap water, diluted with decalcified water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C
- Humidity: 55 %
- Air changes: 15 changes/hour
- Photoperiod: 12 hours dark / 12 hours light

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
propylene glycol
Concentration / amount:
Induction: 5 % (intradermal), 50 % (epicutaneous)
Challenge: 50 %, 25 %, 10 % (epicutaneous)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
Induction: 5 % (intradermal), 50 % (epicutaneous)
Challenge: 50 %, 25 %, 10 % (epicutaneous)
No. of animals per dose:
Experimental group: 20 animals
Control group: 10 animals
Details on study design:
INDUCTION - EXPERIMENTAL ANIMALS

Intradermal injections:
On day 1 an area of the dorsal skin from the scapular region (approximately 4 x 6 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 2 x 4 cm area in the clipped region as follows:
A) Test item dissolved to 5 % (w/w) with propylene glycol.
B) Freunds' Complete Adjuvant, 50:50 with water for injection
C) Test item at twice the concentration used in (A), emulsified in a 50:50 mixture of Freunds' Complete Adjuvant.

On day 7, approximately 24 hours prior to the epidermal induction application, the scapular area (approximately 6 x 8 cm) was clipped and shaved free of hair and pretreated with 10 % sodium dodecylsulfate (SDS) in petrolatum. The SDS was massaged into the skin with a spatula without bandaging.

Epicutaneous applications:
Seven days after the intradermal injections, the scapular area (approximately 6 x 8 cm) was again treated. A Scotchpak-non-woven patch ( 2 x 4 cm) mounted on Micropore tape was applied with 0.5 mL of a 50 % (w/w) concentration of the test item in vaseline and placed between the injection sites of the test animals. The Micropore tape was firmly secured, wrapped around the trunk of the animal and secured with Coban elastic bandage. After 48 hours, the dressings and residual test item were removed using a moistened tissue. The epidermal application procedure described ensured intensive contact of the test item even if insoluble in the vehicle used. Reaction sites were assessed for erythema and oedema immediately after removal of the dressings.

INDUCTION - CONTROL ANIMALS

The guinea pigs of the control group were treated in a similar method as described above by the intradermal and epicutaneous inductions with omission of the test item. Reaction sites were assessed and summarised similarly as described above.

CHALLENGE - ALL ANIMALS

The experimental and control guinea pigs were challenged two weeks after the epicutaneous induction application. Hair was clipped and shaved from a 5 x 5 cm area on the left flank of each guinea pig. A volume of 0.05 mL of each of the following three test item concentrations and the vehicle were applied using Square chambers attached to Micropore tape:

a = 50 % (w/w) in vaseline
b = 25 % (w/w) in vaseline
c = 10 % (w/w) in vaseline
d = vaseline

The patches were placed on the shaved area, the Micropore tape firmly secured around the trunk of the animals and held in place by Coban elastic bandage. The dressings and residual test item were removed after approximately 24 hours, using a moistened tissue. The test sites were assessed for redness and swelling 24 and 48 hours after removal of the dressings, using a numerical grading system (modified from Kligman A.M., J. Invest. Dermatol. 47, 1966).

The test sites were re-shaved with an electric razor after the first reading.

All animals were sacrificed at the end of the test period by oxygen/carbon dioxide asphyxiation.

In addition to the skin reactions the following data were recorded:
Mortality/viability/toxicity: once daily
Body weights: prior to start and at termination of the study.


.
Positive control substance(s):
yes
Remarks:
Formaldehyde

Results and discussion

Positive control results:
A positive control experiment is carried out once a year as a sensitivity check of the test system. The most recent test was performed in June 1993. The test confirmed sensitivity.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50, 25, 10, 0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Remaining test item was dried into the skin after challenge in 1 animal.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50, 25, 10, 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Remaining test item was dried into the skin after challenge in 1 animal..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50, 25, 10, 0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Remaining test substance was dried into the skin after challenge in 2 animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50, 25, 10, 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Remaining test substance was dried into the skin after challenge in 2 animals..
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50, 25, 10, 0
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
The control site in 1 animal was stained blue. It was considered possible that some of test item from another square chamber had leaked into the square chamber containing vehicle alone.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50, 25, 10, 0. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: The control site in 1 animal was stained blue. It was considered possible that some of test item from another square chamber had leaked into the square chamber containing vehicle alone..
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50, 25, 10, 0
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
One animal showed red spots in response to the 50 % and one other animal showed red spots in response to the 10 % test item concentration.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50, 25, 10, 0. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: One animal showed red spots in response to the 50 % and one other animal showed red spots in response to the 10 % test item concentration..

Any other information on results incl. tables

Induction Phase:

None of the experimental animals showed erythema or oedema. All animals showed blue discolouration of the treated skin area after the 48 hours occluded epidermal induction exposure, which made skin reading difficult. Seven control animals showed well defined erythema and brown discolouration of the treated skin area after the epidermal exposure. The brown discolouration was possibly caused by the SDS treatment on day 7.

 

Challenge Phase:

- Control group:

No skin reactions were evident after the challenge exposure. All challenge treated skin sites of all animals showed a blue discolouration, which made skin reading difficult.

- Experimental group:

One animal showed red spots in response to the 50 % and one other animal showed red spots in response to the 10 % test item concentration. All challenge treated skin sites of all animals showed a blue discolouration.

 

No symptoms of systemic toxicity were observed in main study animals during the study period. There were no incidences of mortality. The mean body weight gain of experimental animals was slightly lower as compared to the respective control values.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information