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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 24, 1999 to September 24, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 406 and EU Method B.6, in compliance with GLP
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
other: Pirbright-White
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Gartenstr. 27, 0-33178 Borchen, SPF breeding colony.
- Body weight at start of study: 312-420 g
- Housing: In fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet: Ssniff Ms-H (V2233), ad libitum
- Water: Tap water in plastic bottles, ad libitum
- Acclimation period: At least 7 d
- Animal identification: Fur marking with KMn04 and cage numbering
- Randomization procedure: Randomization scheme 1999.0310

ENVIRONMENTAL CONDITIONS
- Temperature: 20±3°C
- Humidity: 50±20%
- Photoperiod: 12 h light/dark cycle

IN-LIFE DATES: From: To: August 24, 1999 to September 24, 1999
Route:
intradermal and epicutaneous
Vehicle:
other: deionized water and 50 % Freund's Adjuvants were used for the intradermal applications and deionized water for the dermal applications
Concentration / amount:
Induction:
Concentration for intradermal injection: 5%
Concentration for dermal application: 25%

Challenge:
Concentration for dermal application: 25%
Route:
epicutaneous, occlusive
Vehicle:
other: deionized water and 50 % Freund's Adjuvants were used for the intradermal applications and deionized water for the dermal applications
Concentration / amount:
Induction:
Concentration for intradermal injection: 5%
Concentration for dermal application: 25%

Challenge:
Concentration for dermal application: 25%
No. of animals per dose:
Number of animals in test group: 10
Number of animals in negative control group: 5
Details on study design:
Test groups
The following test groups were used for the study:

Test group Number of animals
Determination of the primary non-irritant concentration 3
Determination of the tolerance of the intradermal injections 2
Control group 5
Treatment group 10


Test procedure

The following preparations were used for the intradermal injections:

Control group
1.) 50% Freund's Complete Adjuvant (i.e., FCA) emulsion
Original Freund's Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionized water.
2.) Deionized water (i.e., vehicle)
3.) 50% Freund's Complete Adjuvant emulsion mixed with an equal volume of the vehicle

Treatment group
1.) 50% Freund's Complete Adjuvant emulsion
Original Freund's Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionized water.
2.) 5% test substance in deionized water
3.) 5% test substance in a 50% Freund's Complete Adjuvant emulsion

For the intradermal injections of the test substance in 50% Freund's adjuvant, test substance was dissolved in deionized water and then mixed with an equal volume of Freund's Original Adjuvant [percentages w/v].
For the dermal treatments, test substance was suspended in deionized water [percentages w/v].

Main test for the sensitizing properties

Chronological description of the test procedure indicating the day, at which procedure was carried out:

Study Day 0
The body weight of the animals was determined. The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.
Study Day 1
Intradermal induction treatment

Two intradermal injections per animal of the following preparations were applied. The injection sites were all within a dorsal area of 2 x 4 cm. The injection sites were left uncovered.

Treatment group:
Site Appl. vol. Conc Preparation
1 2 x 0.1 mL _ 50% Freund's Adjuvant
2 2 x 0.1 mL 5% Test substance in deionized water
3 2 x 0.1 mL 5% Test substance in 50% Freund's Adjuvant

Control group:

Site Appl. Vol. Preparation
1 2 x 0.1 mL 50% Freund's Adjuvant
2 2 x 0.1 mL Deionized water
3 2 x 0.1 mL Equal volume of deionized water and 50% Freund's Adjuvant

Study Day 2-7
The administration area was examined for local tolerance. Systemic toxic effects were recorded, when apparent.


Study Day 8
Dermal induction treatment
An amount of 0.5 mL of the test substance preparation (i.e., treatment group) or the vehicle (i.e., control group) was administered to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The administration area was then kept under an occlusive bandage covered with an impermeable film and an elastic bandage for 48 h.

Treatment group 25.0% test substance in deionized water
Control group Deionized water


Study Day 10
Occlusive bandage was removed, irritant effects were recorded, when apparent.

Study Day 11-21
No treatment of control or treatment group. Test animals were kept under observation.

Study Day 22
Dermal challenge treatment

One area of approximately 5 x 5 cm on the left flank was shaved mechanically. An amount of 0.5 mL of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 h under an occlusive bandage covered with an impermeable film and an elastic bandage.

Treatment and control group (left flank):
25.0% test substance in deionized water

Study Day 23
Occlusive bandage was removed. Any remnants of the test substance were carefully washed off with warm water.

Study Day 24
Examination of the skin approximately 24 h after removal of the patches.

Study Day 25

Examination of the skin approximately 48 h after removal of the patches. Body weight of the test animals was determined.
Positive control substance(s):
yes
Remarks:
(alpha-hexyl cinnamic aldehyde tested in another study)
Positive control results:
After the challenge treatment positive response was observed in 80% of the treated animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% dermal application
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% dermal application. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% dermal application
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% dermal application. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: control
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control. Dose level: 25% dermal application. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control. Dose level: 25% dermal application. No with. + reactions: 0.0. Total no. in groups: 5.0.

Determination of the primary non-irritant concentration

No signs of irritation occurred after administration of the different test concentrations. Based on these results, a concentration of 25%test substance in deionized water was chosen for the challenge at Day 22.

 

Determination of the tolerance of the intradermal injections

 

-The intradermal injections with the 5.0% preparation caused well-defined erythema and edema as well as encrustions.

-The intradermal injections with the 1.0% and 0.2%preparation caused well-defined up to slight erythema and edema.

-Based on this preliminary test, a 5% preparation was selected for the intradermal injections in the main test.

 

Main test for the sensitizing properties

 

Body weight gains and clinical signs

 

-The body weight gains of the animals were not impaired.

 

-The treated animals showed no clinical signs of intoxication throughout the study.

 

Intradermal induction treatment

 

-The intradermal injections with Freund's Adjuvant (with and without test substance) caused severe erythema and edema as well as indurations and encrustations. The administration sites treated with the test substance in deionized water showed well-defined erythema and edema and encrustions. Intradermal injections of the vehicle alone exhibited no signs of irritation.

-Due to these strong irritation reactions of the skin, 10% sodium dodecylsulfate was not administered at Day 7.

 

Dermal induction treatment

-After the removal of the patches at Day 10, severe erythema and edema, indurated and encrusted skin as well as necrosis were observed at the sites previously treated with Freund's Adjuvant. The administration sites treated with the test substance showed well-defined erythema.

 

-The administration sites treated with the vehicle alone showed no signs of irritation.

 

Dermal challenge treatment

-No skin reactions were observed in the control and the treatment group 24 and 48 h after removal of the occlusive bandage.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the study conditions, the test substance showed no evidence of sensitizing properties.
Executive summary:

A guinea pig maximization test was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406 and EU Method B.6, in compliance with GLP.

 

Based on the results of a preliminary study, 5 and 25% of test substance in deionized water were selected as intradermal and dermal induction doses. The highest non-irritating concentration used for challenge application was 25% test substance in deionized water.

 

None of the animals of the test group showed skin reactions 24 and 48 h after removing the dressings. The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexyl cinnamic aldehyde for the maximization test.

 

Under the study conditions, the test substance showed no evidence of sensitizing properties.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A guinea pig maximization test was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406 and EU Method B.6, in compliance with GLP. Based on the results of a preliminary study, 5 and 25% of test substance indeionized waterwere selected as intradermal and dermal induction doses. The highest non-irritating concentration used for challenge application was 25% test substance indeionized water. None of the animals of the test group showed skin reactions 24 and 48 h after removing the dressings. The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexyl cinnamic aldehyde for the maximization test. Under the study conditions, the test substance showed no evidence of sensitizing properties (Seeberger, 1999c).


Justification for selection of skin sensitisation endpoint:
Guideline-compliant study conducted according to GLP.

Justification for classification or non-classification