Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): LCE10003
- Substance type:
- Physical state: White solid
- Purity test date: 100%
- Lot/batch No.: 2237JG
- Expiration date of the lot/batch: 2011-12-31
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature (range of 20 ± 5 °C, provided
by Harlan Laboratories Ltd.), light protected.

In vivo test system

Test animals

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

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Concentration / amount:
1 Intradermal Induction
The last test item concentration of 1% in PEG 300 was applicable and caused a
discrete/patchy erythema additionally with blanching. This concentration was selected for the
main intradermal induction.
2 Epidermal Induction
The 50% concentration of test item used for the epidermal induction was the highest technically
applicable concentration during the epidermal pretest and caused discrete/patchy erythema
suitable for the epidermal induction.
3 Epidermal Challenge
The 15% concentration of test item used for the challenge was the highest non-irritant
concentration tested during the epidermal pretest.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Concentration / amount:
1 Intradermal Induction
The last test item concentration of 1% in PEG 300 was applicable and caused a
discrete/patchy erythema additionally with blanching. This concentration was selected for the
main intradermal induction.
2 Epidermal Induction
The 50% concentration of test item used for the epidermal induction was the highest technically
applicable concentration during the epidermal pretest and caused discrete/patchy erythema
suitable for the epidermal induction.
3 Epidermal Challenge
The 15% concentration of test item used for the challenge was the highest non-irritant
concentration tested during the epidermal pretest.
No. of animals per dose:
The animals were distributed as follows:
- Intradermal Pretest : 1
- Epidermal Pretest : 2
- Control Group : 5
- Test Group : 10
Details on study design:
RANGE FINDING TESTS:
The test item concentrations described below were selected during a preliminary solubility
- Intradermal Injections
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete
Adjuvant/physiological saline were made into the shaved neck of one guinea pig (no. 219). Six
days later intradermal injections (0.1 mL/site) were made into the clipped flank of the same
guinea pig at concentrations of A = 10%, B = 5% and C = 1% of the test item in PEG 300.
Dermal reactions were assessed 24 hours later.
Based on the results, the test item concentration of 1% was selected for intradermal induction in
the main test.
-Epidermal Applications
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete
Adjuvant/physiological saline were made into the shaved neck of two guinea pigs (nos. 220 -
221). Six days later, four patches of filter paper (3 x 3 cm) were saturated with the test item at
D = 50%, E = 25%, F = 15% and G = 10% in PEG 300 and applied to the shaved flanks of the
same guinea pigs. The amount or volume of test item preparation applied was approximately
0.2 g at 50% and approximately 0.2 mL at 10%, 15% and 25%. The occlusive dressings were left
in place for 24 hours.
Based on the results the test item concentration selected for epidermal induction and challenge in
the main test was 50% and 15%, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE : Intradermal Injection 0.1mL/site
- No. of exposures: 1
- Dayof induction: 1
- Test groups:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test item at 1% in PEG 300.
3) The test item at 1% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
- Control group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) PEG 300.
3) 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
- Site: An area of dorsal skin from the scapular region
- Duration: 7 days

B.INDUCTION EXPOSURE N°2 : topical application 0.3g (patch)
No. of exposures: 1
- Day(s) of challenge: 8
- Test groups: patch of filter paper was saturated with the test item at 50% in PEG 300
- Control group: treated as described above with PEG 300 only 0.3mL
- Site: scapular area
- Concentrations: 50% in PEG 300
- Duration: 48h
- Evaluation (hr after challenge): 24 and 48 hours after removal of the bandage

C. CHALLENGE EXPOSURE : topical application 0.2 mL (patch)
- No. of exposures: 1
- Day(s) of challenge: 22
- Test groups and control: patch of filter paper was saturated with the test item at 15% in PEG 300
- Site: scapular area
- Concentrations: 15% in PEG 300
- Duration: 24h
- Evaluation (hr after challenge): 24 and 48 hours after removal of the bandage
Positive control substance(s):
yes
Remarks:
ALPHA-HEXYLCINNAMALDEHYDE

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of
ALPHA-HEXYLCINNAMALDEHYDE which is recommended by the OECD 406 Guidelines
and is known to have moderate skin sensitisation properties in the guinea pig strain. The results
from the most recent test run (Harlan Laboratories Study C67818, performed from 06-Oct-2009
to 13-Nov-2009)
The study was performed with 15 (10 test and 5 control) male albino Dunkin Hartley Guinea Pig,
HsdPoc: DH, SPF, delivered by Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / The
Netherlands, Postbus 6174, 5960 AD Horst / The Netherlands.
The raw data from this study are kept in a separate file at Harlan Laboratories Ltd. The test
described was performed under GLP-conditions with a final QA-check.
The intradermal induction of sensitization in the test group was performed in the nuchal region
with a 10% dilution of the test item in PEG 300 and in an emulsion of Freund's Complete
Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted
for 48 hours under occlusion with the test item at 10% in PEG 300 one week after the
intradermal induction. The animals of the control group were intradermally induced with PEG
300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.
Two weeks after epidermal induction the control and test animals were challenged by epidermal
application of the test item at 3% in PEG 300 and PEG 300 alone under occlusive dressing.
Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

Based on the above mentioned findings in an adjuvant sensitization test (M&K-test) in guinea
pigs and in accordance to Commission Directive 2001/59/EC, ALPHAHEXYLCINNAMALDEHYDE
has to be classified and labelled as a skin sensitizer.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
PEG 300
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
RAS
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: PEG 300. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: RAS.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
PEG 300
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
RAS
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: PEG 300. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: RAS.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
LCE10003 15% (challenge)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
RAS
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: LCE10003 15% (challenge). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: RAS.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
LCE10003 15% (challenge)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
RAS
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: LCE10003 15% (challenge). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: RAS.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
PEG 300
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
RAS
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: PEG 300. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: RAS.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
PEG 300
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
RAS
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: PEG 300. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: RAS.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
LCE10003 15% (challenge)
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
discrete or patchy erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: LCE10003 15% (challenge). No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: discrete or patchy erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
LCE10003 15% (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
RAS
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: LCE10003 15% (challenge). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: RAS.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE 3%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
discrete or patchy erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: ALPHA-HEXYLCINNAMALDEHYDE 3%. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: discrete or patchy erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE 3%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
discrete or patchy erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: ALPHA-HEXYLCINNAMALDEHYDE 3%. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: discrete or patchy erythema.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The challenge indicated that 2 out of 10 guinea pigs had been sensitized (20% of the test animals
were affected, which is below the borderline incidence of positive reactions described in the
Regulation (EC) N0. 1272/2008; for an adjuvant guinea pig test method a response of at least
30% of the animals is considered positive”). As the reaction faded at the 48-hour reading, the
results are indicative of irritation and not sensitization.
Based on the above mentioned findings in an adjuvant sensitisation test (M&K-test) in guinea
pigs and in accordance to Regulation (EC) No 1272/2008, LCE10003 does not have to be
classified and labeled as a skin sensitizer.
Executive summary:

In order to assess the cutaneous allergenic potential of LCE10003, the Maximization-Test was

performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance

with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6.

The intradermal induction of sensitisation in the test group was performed in the nuchal region

with a 1% dilution of the test item in PEG 300 and in an emulsion of Freund's Complete

Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted

for 48 hours under occlusion with the test item at 50% in PEG 300 one week after the

intradermal induction. The animals of the control group were intradermally induced with PEG

300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.

Two weeks after epidermal induction the test and control animals were challenged by epidermal

application of the test item at 15% in PEG 300 and PEG 300 alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

Results - Skin Reactions after the Challenge Procedure:

After 24 hours                     After 48 hours

Positive / Total                     Positive / Total

% Positive of Total                   % Positive of Total

Control Group

LCE10003, 15% in PEG 300              0 / 5                                           0 / 5

(left flank)                                              0 %                                             0 %

PEG 300 only                                      0 / 5                                          0 / 5

(right flank)                                            0 %                                             0 %

Test Group

LCE10003, 15% in PEG 300              2 / 10                                          0 / 10

(left flank)                                             20 %                                           0 %

PEG 300 only                                      0 / 10                                          0 / 10

(right flank)                                           0 %                                             0 %

No toxic signs were evident in the guinea pigs of the control or test group.

No intercurrent deaths occurred during the course of the study.

The challenge indicated that 2 out of 10 guinea pigs had been sensitized (20% of the test animals

were affected, which is below the borderline incidence of positive reactions described in the

Regulation (EC) N0. 1272/2008; for an adjuvant guinea pig test method a response of at least

30% of the animals is considered positive”). As the reaction faded at the 48-hour reading, the

results are indicative of irritation and not sensitization.

Based on the above mentioned findings in an adjuvant sensitisation test (M&K-test) in guinea

pigs and in accordance to Regulation (EC) No 1272/2008, LCE10003 does not have to be

classified and labeled as a skin sensitizer.