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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2014-Janauary 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Method B.6
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (6E)-2,6-dimethyl-10-methylenedodeca-2,6-diene and (6E)-3,7,11-trimethyldodeca-1,6,10-triene and (6E,10E)-2,6,10-trimethyldodeca-2,6,10-triene and (6E,10Z)-2,6,10-trimethyldodeca-2,6,10-triene
EC Number:
942-471-3
Cas Number:
1581740-29-5
Molecular formula:
C15H26
IUPAC Name:
Reaction mass of (6E)-2,6-dimethyl-10-methylenedodeca-2,6-diene and (6E)-3,7,11-trimethyldodeca-1,6,10-triene and (6E,10E)-2,6,10-trimethyldodeca-2,6,10-triene and (6E,10Z)-2,6,10-trimethyldodeca-2,6,10-triene
Test material form:
other: liquid
Details on test material:
Identification: Myralene
Physical State/Appearance: clear colorless liquid
Batch: RV-873-75-2
Purity: 99.98%
Date of manufacture: 9/15/2014

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Thirty albino guinea pigs of Dunkin-Hartley strain, supplied by HARLAN (Kreuzelweg 53, 5961 NM
HORST- The Netherlands) were 4 or 5 weeks old at the beginning of the main test.
Prior to the test, the animals were kept for a minimum acclimatization period of 5 days, under stabling
and nutritional conditions identical to those of the test.
Before the experimentation process, they were identified individually by marking with picric acid and
by means of a numbered ring on the edge of one ear.

The animals were housed individually or in groups of 2 in polycarbonate containers, the flooring of
which was covered with dust-free cuttings and the top fitted with a stainless steel lid with a feeding
device and drinking device of 500 mL.
The temperature and relative humidity of the main test were controlled to remain within target ranges
of 19 to 25°C and 30 to 70%, respectively.
The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time
switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.

The drinking water (tap water from public distribution system) and food (SAFE, 106) were supplied
freely. Microbiological and chemical analyses of the water were carried out once every six months by
Eurofins IPL Atlantique (Bordeaux).

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
1st Intradermal Induction:
Day 0
After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were
performed on the scapular zone in such a way as an injection on each pair is placed to either side of
the spine as follows:
GROUP 1 (Control):
· 2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
· 2 ID: olive oil
· 2 ID: a mixture with equal volumes v/v : Freund’s Complete Adjuvant at 50% and olive oil
GROUP 2 (Treated):
· 2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
· 2 ID: test item at 5% in olive oil,
· 2 ID: a test mixture in equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and the test item at 10% in olive oil

2nd Topical Induction:
Day 6
The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of
sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.
Day 7
A topical application under occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-
layer Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide
hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 hours was
performed on the injection sites of each animal.
GROUP 1 (Control): 0.5 mL of liquid paraffin
GROUP 2 (treated): 0.5 mL of the test item at 100%

Challenge phase
Day 20
The experimental procedure of this phase was identical for both groups GROUP 1 (Control) and
GROUP 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone,
an application, under occlusive dressing, was performed during 24 hours:
- 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing the test item
diluted at 20% (MNIC) and 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®)
containing the test item diluted at 10% (1/2 MNIC).
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
1st Intradermal Induction:
Day 0
After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were
performed on the scapular zone in such a way as an injection on each pair is placed to either side of
the spine as follows:
GROUP 1 (Control):
· 2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
· 2 ID: olive oil
· 2 ID: a mixture with equal volumes v/v : Freund’s Complete Adjuvant at 50% and olive oil
GROUP 2 (Treated):
· 2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
· 2 ID: test item at 5% in olive oil,
· 2 ID: a test mixture in equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and the test item at 10% in olive oil

2nd Topical Induction:
Day 6
The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of
sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.
Day 7
A topical application under occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-
layer Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide
hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 hours was
performed on the injection sites of each animal.
GROUP 1 (Control): 0.5 mL of liquid paraffin
GROUP 2 (treated): 0.5 mL of the test item at 100%

Challenge phase
Day 20
The experimental procedure of this phase was identical for both groups GROUP 1 (Control) and
GROUP 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone,
an application, under occlusive dressing, was performed during 24 hours:
- 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing the test item
diluted at 20% (MNIC) and 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®)
containing the test item diluted at 10% (1/2 MNIC).
No. of animals per dose:
GROUP 1 (negative control) : 10 female guinea pigs identified n° C7098 to C7107
GROUP 2 (treated) : 20 female guinea pigs identified n° C7108 to C7127;
Details on study design:
RANGE FINDING TESTS:

1 - Determination by intradermal injection of the Maximal Non Necrotizing Concentration
(MNNC)
This test was conducted for the purpose of defining a MNNC of the test item which, on intradermal
injection during the induction phase, does not risk causing too great a lesion (non-necrotizing
concentration), should be well-tolerated systemically and should be the highest to cause mild-tomoderate
skin irritation.
Two animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4
concentrations: 100% and diluted at 50%, 20% and 10% in olive oil in order to determine the MNNC.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after the injections.
Due to necrosis observed in all animals, the same animals received in the same experimental
conditions the test item at 3 concentrations: diluted at 5%, 2% and 1% in olive oil in view to determine
the MNNC.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after injection.

2 Determination by topical application of the Pre-Maximal Non Irritant Concentration
(Pre-MNIC)
This test, which allowed evaluating the irritancy potential of the test item, defined whether an
application of sodium lauryl sulfate would be needed during topical induction phase.
The test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with
occlusive dressing (gauze patches hydrophilic Codex of 8-layer Gazin® from Lohmann & Rauscher
held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from
3M and Blenderm™ from 3M) for 24 hours, at 4 different concentrations: 100% and diluted at 50%,
20% and 10% in liquid paraffin.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the
dressing.

3. Determination by topical application of the Maximal Non Irritant Concentration (MNIC)
This test was carried out for the purpose of determining the MNIC of the test item without risk of an
irritant effect during the challenge phase.
Three guinea pigs were treated according to the same treatment as animals from GROUP 1 (negative
control) for the induction phase (i.e. olive oil and liquid paraffin
During the challenge phase, the animals were treated with the test item placed onto the selected
treatment sites and covered with an occlusive dressing (gauze patches hydrophilic Codex of 8-layer
Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide
hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for a period of 24
hours at 4 different concentrations: diluted at 20%, 10%, 5% and 2% in liquid paraffin.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of
the occlusive dressing.

MAIN STUDY


GROUP 1 (negative control) : 10 female guinea pigs identified n° C7098 to C7107
GROUP 2 (treated) : 20 female guinea pigs identified n° C7108 to C7127
Note: The results of the 3 last positive control groups (Reference substance:
alpha-Hexylcinnamaldehyde Tests 26-28) carried out in order to assess the sensitivity of the strain of
guinea pig used at these laboratories to a known sensitiser are presented in Appendix 2.
1 - Induction phase
1st Intradermal Induction:
Day 0
After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were
performed on the scapular zone in such a way as an injection on each pair is placed to either side of
the spine as follows:
GROUP 1 (Control):
· 2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
· 2 ID: olive oil
· 2 ID: a mixture with equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and olive oil
GROUP 2 (Treated):
· 2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
· 2 ID: test item at 5% in olive oil,
· 2 ID: a test mixture in equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and the test item at 10% in olive oil

Day 6
The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of
sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.
Day 7
A topical application under occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-
layer Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide
hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 hours was
performed on the injection sites of each animal.
GROUP 1 (Control): 0.5 mL of liquid paraffin
GROUP 2 (treated): 0.5 mL of the test item at 100%
Day 9
Occlusive dressing removal.
2. - Rest phase
The animals of both groups were left for 10 days



B. CHALLENGE EXPOSURE

Challenge phase
Day 20
The experimental procedure of this phase was identical for both groups GROUP 1 (Control) and
GROUP 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone,
an application, under occlusive dressing, was performed during 24 hours:
- 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing the test item
diluted at 20% (MNIC) and 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®)
containing the test item diluted at 10% (1/2 MNIC).
Day 21
Occlusive dressing removal.
Day 22
Macroscopic evaluation of the cutaneous reaction.
Day 23
Macroscopic evaluation of the cutaneous reaction.
Day 24
Macroscopic evaluation of the cutaneous reaction.
Challenge controls:
yes
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:
The results of the 3 last positive control groups (Reference substance:
alpha-Hexylcinnamaldehyde Tests 26-28) carried out in order to assess the sensitivity of the strain of
guinea pig used at these laboratories to a known sensitiser are attached in background materials.

the reference substance
a-Hexylcinnamaldehyde has to be classified R 43 “may cause sensitization by skin contact” in
accordance with the criteria for classification, packaging and labelling of dangerous substances and
preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. This substance has to be
characterised by the symbol “Xi” and the warning label “Irritant”.
In accordance with the Regulation (EC) No. 1272/2008 on classification, labelling and packaging of
substances and mixtures, the reference substance has to be classified in category 1 “Skin sensitisation”
sub-category 1B. The signal word “Warning” and hazard statement H317 “May cause an allergic
skin reaction” are required.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20%
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20%. No with. + reactions: 9.0. Total no. in groups: 20.0.
Reading:
other: Third reading
Hours after challenge:
72
Group:
test chemical
Dose level:
20%
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
other: Reading: other: Third reading. . Hours after challenge: 72.0. Group: test group. Dose level: 20%. No with. + reactions: 6.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
other: third reading
Hours after challenge:
72
Group:
test chemical
Dose level:
10%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: other: third reading. . Hours after challenge: 72.0. Group: test group. Dose level: 10%. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
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.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
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.
Reading:
other: third reading
Hours after challenge:
72
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: third reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
n view of these results, under these experimental conditions, the test item Myralene has to be
classified R43 “May cause sensitisation by skin contact”, in accordance with the criteria for
classification, packaging and labelling of dangerous substances and preparations of the E.E.C.
Directives 67/548, 2001/59 and 99/45. This item has to be characterised by the symbol “Xi” and the
warning label “Irritant”.
In accordance with the Regulation (CE) No. 1272/2008, the test item has to be classified in category 1,
Sub-category 1B. The signal word “Warning” and hazard statement H317 “May cause an allergic
skin reaction” are required
Executive summary:

The aim of the study was to evaluate the possible allergenic activity of the test item after intradermal

and topical administration in guinea pigs.

After induction (intradermic injection at 5% and topical application at 100%) of 20 Guinea Pigs of

treated group with the test item Myralene and a 10-day rest phase, the challenge phase, under

occlusive dressing for 24 hours, consisted to a single topical application of the test item diluted at 20%

and at 10% in liquid paraffin. The experimental protocol was established according to the OECD

guideline No. 406 dated July 17th, 1992 and the test method B.6 of the council regulation No.

440/2008.

In the treated group (treatment dose of 20%) a discrete to well-defined erythema was recorded in 45% (9/20) and 30% (6/20) of the animals, at the 48 and 72 hour readings. No skin reaction was noted at the 24 hour reading.

In the treated group (treatment dose of 10%) a discrete erythema was recorded in 5% (1/20) of the animals, at the 48 and 72 hour readings. No skin reaction was noted in the 24 hour reading.

In accordance with the Regulation (CE) No. 1272/2008, the test item has to be classified in category 1,

Sub-category 1B. The signal word “Warning” and hazard statement H317 “May cause an allergic

skin reaction” are required.