Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-06-29 to 2005-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Complete guideline conform study report available. Study is performed under GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): OPGE022005
- Physical state: liquid
- Purity approx. 100%
- Lot/batch No.: HS180505
- Storage condition: light protected at about 20 °C

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: PEG 300
Concentration / amount:
Pretest
intradermal injection in one animal:
Day 1: 4 intradermal injections (0.1 mL/site) of a 1:1 (V/V) mixture of Freud's Complete Adjuvant/physiol. saline into the shaved neck
Day 7: intradermal injections (0.1mL/site) were made into the clipped flank of same animal at concentrations A= 100%, B= 75%, C= 50 % of test item. PEG 300 was used for dilutions. Based on the result of the pretest, 100% was selected for intradermal injection in the main study.

Epidermal application in two animals:
Day 1: 4 intradermal injections (0.1 mL/site) of a 1:1 (V/V) mixture of Freud's Complete Adjuvant/physiol. saline into the shaved neck of two guines pigs.
Day 7: both flanks of each animal were clipped and shaved just prior to application. Thereafter, 4 patches of filter paper (3x3 cm) were saturated (about 0.2mL)with the testitem at concetrations D= 100%, E= 75%, F= 50% and G= 25% in PEG 300 and applied to the shaved flanks. Patches were covered by a strip of aluminium foil and firmly secured by elastic plaster. Dressings were removed after an exposure period of 24 hours. Twenty one hours after removal of dressing the application site was depilated with an aproved depilation cream. Approximately 3 hours later (48 hours from the epidermal application) the skin reaction was observed for reactions and a second observation was performed 72 hours later.

Main study:
Based on the results obtained in the pretest studies the following concentrations were selected:
induction: 100%
challenge: 25 %
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: PEG 300
Concentration / amount:
Pretest
intradermal injection in one animal:
Day 1: 4 intradermal injections (0.1 mL/site) of a 1:1 (V/V) mixture of Freud's Complete Adjuvant/physiol. saline into the shaved neck
Day 7: intradermal injections (0.1mL/site) were made into the clipped flank of same animal at concentrations A= 100%, B= 75%, C= 50 % of test item. PEG 300 was used for dilutions. Based on the result of the pretest, 100% was selected for intradermal injection in the main study.

Epidermal application in two animals:
Day 1: 4 intradermal injections (0.1 mL/site) of a 1:1 (V/V) mixture of Freud's Complete Adjuvant/physiol. saline into the shaved neck of two guines pigs.
Day 7: both flanks of each animal were clipped and shaved just prior to application. Thereafter, 4 patches of filter paper (3x3 cm) were saturated (about 0.2mL)with the testitem at concetrations D= 100%, E= 75%, F= 50% and G= 25% in PEG 300 and applied to the shaved flanks. Patches were covered by a strip of aluminium foil and firmly secured by elastic plaster. Dressings were removed after an exposure period of 24 hours. Twenty one hours after removal of dressing the application site was depilated with an aproved depilation cream. Approximately 3 hours later (48 hours from the epidermal application) the skin reaction was observed for reactions and a second observation was performed 72 hours later.

Main study:
Based on the results obtained in the pretest studies the following concentrations were selected:
induction: 100%
challenge: 25 %
No. of animals per dose:
Pretest:
Intradermal incection: one animal
dermal application: two animals

Main test
control group I: 5 animals
treatment group: 10 animals
control group II: 5 animals
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:
A study was performed with alpha-Hexylcinnamaldehyde in 15 (10 test and 5 control) male albino DunkinHartley guinea pigs.
Induction intradermal with 10% diluted alpha-Hexylcinnamaldehyde in PEG 300. One week after intradermal induction, epidermal induction with the test item at 10 % in PEG 300 for 48h under occlusion followed.
Two weeks after epidermal induction control and test animals were challenged by epidermal application of test item at 1% and 0.1% in PEG 300 and PEG 300 alone under occlusive dressing.
Two weeks after the first challenge a second challenge was performed in the same way.
Results: No toxic signs were evident in the guinea pigs of the control or test group. 4/10 (at 24h reading) and 1/10 (at 48h reading) test animals showed dicrete/patchy erythema after the first challenge treatment with alpha-Hexylcinnamaldehyde at 1% (w/w) in PEG 300. No skin effect were observed in the test group at 0.1%. No skin effect was observed in the control group treated in the same conditions during the challenge procedure.
7/10 animals (at 24 h reading) and 4/10 animals (at the 48 h reading) showed discrete/patchy erythema after the second challenge treatment with alpha-Hexylcinnamaldehyde at 1% (w/w) in PEG 300. One test animal was noted with the same reaction at the 24- and 48-hour reading when treated with the test item at 0.1%. Based on the above mentioned findings alpha-Hexylcinnamaldehyde at 1% is considered to be a sensitizer.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group I, Ieft flank
Dose level:
25 % in PEG 300
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group I, Ieft flank. Dose level: 25 % in PEG 300. No with. + reactions: 5.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
48
Group:
other: Control group I, left flank
Dose level:
25 % in PEG 300
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: Control group I, left flank. Dose level: 25 % in PEG 300. No with. + reactions: 5.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group I, right flank
Dose level:
PEG 300 only
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group I, right flank. Dose level: PEG 300 only. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
48
Group:
other: Control group I, right flank
Dose level:
PED 300 only
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: Control group I, right flank. Dose level: PED 300 only. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 % in PEG 300
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 % in PEG 300. No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
25 % in PEG 300
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 % in PEG 300. No with. + reactions: 9.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
PEG 300 only
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: PEG 300 only. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
PEG 300 only
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: PEG 300 only. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
24
Group:
other: Control Group II, right cranial flank
Dose level:
test item 15 % in PEG 300
No. with + reactions:
1
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: other: Control Group II, right cranial flank. Dose level: test item 15 % in PEG 300. No with. + reactions: 1.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group II, right cranial flank
Dose level:
test item 15 % in PEG 300
No. with + reactions:
2
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group II, right cranial flank. Dose level: test item 15 % in PEG 300. No with. + reactions: 2.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
24
Group:
other: Control group II, right caudal flank
Dose level:
test item 10 % in PEG 300
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: other: Control group II, right caudal flank. Dose level: test item 10 % in PEG 300. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group II, right caudal flank
Dose level:
test item 10 % in PEG 300
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group II, right caudal flank. Dose level: test item 10 % in PEG 300. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
24
Group:
other: test group, right cranial flank
Dose level:
test item 15 % in PEG 300
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: other: test group, right cranial flank. Dose level: test item 15 % in PEG 300. No with. + reactions: 4.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test group, right cranial flank
Dose level:
test item 15 % in PEG 300
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test group, right cranial flank. Dose level: test item 15 % in PEG 300. No with. + reactions: 5.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
24
Group:
other: test group, right caudal flank
Dose level:
test item 10 % in PEG 300
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: other: test group, right caudal flank. Dose level: test item 10 % in PEG 300. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test group, right caudal flank
Dose level:
test item 10 % in PEG 300
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test group, right caudal flank. Dose level: test item 10 % in PEG 300. No with. + reactions: 0.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the above mentioned findings in an adjuvant sensitization test in guinea pigs, the test item is considered not to be a sensitizer.
Executive summary:

In order to asses the coutaneous allergic potential of the test item a Magnusson & Kligman study was performed in 20 guinea pigs according to OECD 406. Based on the results of two separate pretests, for intradermal induction the undiluted test item and an emulsion of Freund's Adjuvannt (FCA/ physiol. saline) was used. For the epidermal induction the undiluted test item was used. The animals of the control group I were intradermally induced with PEG 300 and FCA/ physiol. saline and epidermally induced with PEG 300 under occlusion.

Two weeks after the induction period the challenge was perfomed by dermal application of the test item at 25 % (w/w) in PEG 300 and PEG 300 alone under occlusive dressing. Two weeks after the first challenge a second challenge was perfomed in the same way as previous challenge using the same test group and an additional control group II, a naive skin site and the test item concentrations of 15% and 10 % (w/w) in PEG 300. Coutaneous reactions were evaluated at approximately 24 and 48 hours after removal of the dressing.

Results:

No death occured and no toxic signs were evident in all groups. At the 24 h-reading all (10/10) and at the 48 h-reading nine of ten animals showed discrete /patchy erythema after first challenge treatment with the test item at 25 % (w/w) in PEG 300. The same reaction was noted in all animals of the control I group. No skin effect was observed in the test item and control I group when treated with PEG 300 only. Four (at the 24h-reading) and five (at the 48 h-reading) out of 10 test animals showed again discrete/patchy erythema after a second challenge treatment with the test item at 15 % (w/w) in PEG 300. The same reaction was seen in one (at 24h-reading) and two (at 48 h-reading) out of 5 animals of control group II treated with the test item at 15 % (w/w) in PEG 300. No skin effect was observed in the test and control group when treated with the test item at 10 % (w/w) in PEG 300.