Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-07-22 to 2002-10-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(1996)
Deviations:
yes
Remarks:
(the test item was insoluble in several solvents so that the protocol was adopted to the publication Th. Maurer and R. Hess (1989), Fd Chem. Toxic. Vol. 27, No. 12, p. 807-811).
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
yes
Remarks:
(the test item was insoluble in several solvents so that the protocol was adopted to the publication Th. Maurer and R. Hess (1989), Fd Chem. Toxic. Vol. 27, No. 12, p. 807-811).
GLP compliance:
yes
Remarks:
statement
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid, fine scales

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, Gartenstrasse 27, D-33178 Borchen
- Age at study initiation: Approximatly 5 to 7 weeks
- Weight at study initiation: 318 g to 401g
- Housing: Until Day 0: Makrolon cages type III (23 cm x 39 cm bottom area, 18 cm height) with wire mesh lids, single caging. From Day 0: group caging in plastic containers (46 cm x 105 cm x 36 cm), partly shaded, 6 (control group) or 11 (test substance group) animals per container.
- Diet: Altromin Maintenance Diet No. 3122, rich in crude fiber, ad libitum, offered in stainless steel containers.
- Water: Tap water offered in Makrolon bottles with stainless steel canules, ad libitum.
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22.0 °C
- Humidity: Step 1: Average of 59.9 %
Step 2: Average of 49.3 %
- Air changes: 12 per hour
- Photoperiod: Artificial light from 6.00 a.m. to 6.00 p.m.



Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
Concentration of test material and vehicle used at induction: treatment: 0.1 mL FCA intradermal and subsequent epicutanous exposure of 500 mg test item at the site of injection. Concentration was 50 % in white petrolatum for the epicutanous induction exposures.
Control: white petrolatum
Concentration of test material and vehicle used for each challenge: 50 % in white petrolatum for the epicutanous challenge exposure.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
Concentration of test material and vehicle used at induction: treatment: 0.1 mL FCA intradermal and subsequent epicutanous exposure of 500 mg test item at the site of injection. Concentration was 50 % in white petrolatum for the epicutanous induction exposures.
Control: white petrolatum
Concentration of test material and vehicle used for each challenge: 50 % in white petrolatum for the epicutanous challenge exposure.
No. of animals per dose:
Number of animals in test group: 20 (10 males, 10 females)
Number of animals in negative control group: 10 ( 5 males, 5 females)
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE INTRADERMAL
- No. of exposures: 1, intradermal injections of FCA (to enhance a possible sensitisation) and immediately afterwards epicutaneous application of the test substance to the sites of the intradermal injections.
- Test groups: 3
- Control group: 1
- Site: 2 cm x 4 cm in the interscapular region.
- Frequency of applications: 1 x
- Duration: 24 hours
- Concentrations: 500 mg test item

INDUCTION EXPOSURE EPICUTANOUS (day 8)
- No. of exposures: 1
- Concentrations: test item 50 % (w/w) in white petrolatum
- Epicutaneous application of the test substance to the sites of the intradermal injections.

Day 8: The highest technically feasible test item concentration of 50 % in white petrolatum did not cause markable skin irritations in the preliminary test in the animals. According to B. Magnusson and A.M. Kligman, all animals of both groups were therefore pretreated with a formulation of n-dodecylsulfate, sodium salt (E. Merck, D-64271 Darmstadt, Art. No. 13760), 10 % (w/w) in white petrolatum, one day before the epicutaneous induction exposure on Day 8. About 0.5 g per animal were gently applied with a plastic spatula onto the appropriate area to give a slight local hyperaemia. The area was left uncovered. About 0.5 g of test item or about 0.4 g to 0.5 g of white petrolatum were applied to each animal. The exposure time was 48 hours.

B. CHALLENGE EXPOSURE (day 23)
- No. of exposures: 1
- Test groups: 3
- Control group: 1
- Site: 2 x 4 cm epicutaneous application of the test substance to the left flanks and application of the vehicle to the right flanks of all animals.
- Concentrations: test item 50 % (w/w) in white petrolatum
- Challenge exposure: 24 hours
- Evaluation: 24 and 48 hours after the end of the challenge

OTHER:
Skin reactions Score:
0 = no reactions
1 = very slight erythema
2 = well defined erythema
3 =severe erythema and/or oedema
Positive control substance(s):
not required

Results and discussion

Positive control results:
Historical control data confirmed the validity of the test system. 7/10 animals (70 %) represented the net rate of animals sensitised by "HEXYL CINNAMIC ALDEHYDE". The results prove the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the available data on sensitisation the test item is not subjected to classification and labelling according to Regulation 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).
Executive summary:

The aim of the study was to investigate the sensitising potential of the test item using a traditional sensitising test with guinea pig. The study was performed according to the OECD 406, adopted in 1992. 10 females and 5 females were used as treatment and negative control group respecitvely. For the first induction the test item was administered epicutaneously following the injection of Freund's complete adjuvant. Before the second induction exposure, the animals were pretreated with n-dodecylsulfate, sodium salt (10 % in white petrolatum, w/w). The second induction occured epicutanously at day 8 using a test item concentrations of 50 % (w/w) in white petrolatum. At day 23 the epicutanous challenge was performed again with 50 % (w/w) in white petrolatum. Test sites were covered with occlusive dressings. The results of skin examinations were decisive for the grading of the potential of sensitisation. No positive skin reactions were observed after 24 hours and 48 hours therefore the test item is considered to be "not sensitising" to skin.