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Toxicological information

Skin sensitisation

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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:
January 2008 to March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study: the test was conducted according the O.E.CD. guideline no. 406 dated July 17, 1992 and the method B.6 oI the E.E.C no 96/54 dated July 3, 1996.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
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 (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Valid study from the year 2008 was available.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (F-69592 L' Arbresle)
- Age at study initiation: 4 weeks
- Weight at study initiation: between 228 and 275 g
- Housing: The animals were housed either in groups of 2 or 3 in polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted a stainless steel lid with a feeding device and drinking device of 500 ml.
- Diet: C17.50 guinea pig breeder (Special Diets Services)
- Water: tap water from public distribution system
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 °C
- Humidity (%): between 36% and 58%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
unchanged (no vehicle)
Concentration / amount:
see "details on study design"
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
see "details on study design"
No. of animals per dose:
MAIN STUDY: Group 1 (negative control): 5 female
Group 2 (treated): 10 female
Details on study design:
RANGE FINDING TESTS:
Preliminary studies:
1. Determination by intradermal injection of the Maximal Non Necrotizing Concentration (CMNN):
This test was conducted for the purpose of determing a MNNC of the test item.
Two animals received on both sides of the spine, a volume of 0.1 mL of the test item, at 6 concentrations: diluted at 50%,25%, 12.5%, 6.25%,3 .125% and 1.56% in isotonic sodium chloride. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours later and 48 hours later
if necessary.

2. Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC):
This test, which allowed to evaluate the irritant 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-Iumbar zone of two guinea pigs shom be forehand, with occlusive dressing for 24 hours, at 4 different concentrations: diluted at 80%, 40%, 20% and 10% in distilled water. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.

3. Determination by topical application ofthe 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 I (negative control) for the induction phase (i.e. isotonic sodium chloride and distilled water). During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occJusive dressing for aperiod of 24 hours at 4 different concentrations: diluted at 80%, 40%, 20% and 10% in distilled water.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the occlusive dressing.


MAIN STUDY
A. INDUCTION EXPOSURE

INTRADERMAL INDUCTION:
- No. of exposures: Group 1 (negative control): 5 female x 3 pairs of ID = 15 exposures
Group 1 (treated):10 female x 3 pairs of ID = 30 exposures
- Test groups:
GROUP 2 (Treated):
• 2 ID: Freund's Complete Adjuvant diluted by 50 % in isotonic sodium chloride,
• 2 ID: test item at 3.125%,
• 2 ID a test mixture in equal volumes v/v: - Freund's Complete Adjuvant at 50% and the test item at 6.25%.
- Control group:
GROUP 1 (Negative control):
• 2 ID: Freund's Complete Adjuvant diluted at 50 % in isotonic sodium chloride.
• 2 ID: isotonic sodium chloride
• 2 ID: a mixture with equal volumes v/v: Freund's Complete Adjuvant at 50% and isotonic sodium chloride

- Site: on the scapular zone in such a way as an injection on each pair is placed to either side of the spine
- Frequency of applications: three pairs of intradermal injections (ID)
- Concentrations: 0.1 mL

TOPICAL INDUCTION:
Day 6: The scapular zone of all the animals in each group, shom be forehand, was brushed wilh 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 for 48 hours was perfonned on the injection sites of each animal.
GROUP 1 (Negative control): 0.5 mL of distilled water
GROUP 2 (treated): 0.5 mL of the test item at 80%
After the removal ofthe pateh, the treated areas were rinsed with distiJled water.
Rest phase: 10 days

B. CHALLENGE EXPOSURE
- No. of exposures: 6
- Day(s) of challenge: Day 20
- Exposure period: 24 hours
- Test groups: GROUP 1 (Negative control) and GROUP 2 (treated)
- Site: on the previously shorn dorso-Iumbar zone, an application on either side of the spine
- Concentrations: - 1 sampIe cup containing the test item at 80% (MNIC) and at 40% (112 MNIC).
Challenge controls:
No use of a challenge (i.e. naive) control group.
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:
a-Hexyceinnamaldehyde (CAS# 101-86-0) is used in the laboratory in order to check the sensibility of the method since this substance is known to have mild to moderate skin sensitisation properties

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
80 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 80 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
80%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 80%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
40%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
40%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
80%
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: 80%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
80%
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: 80%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
40%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
40%
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: 40%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Sensitising potential assessment:

No macroscopic cutaneous reactions attributable to allergy were recorded during the examination following the removal of the occusive dressing (challenge phase) from the animals of the treated group with the test item. No cutaneous intolerance reaction was recorded in animals from the negative control group.

Weight evolution:

Not any abnormality was recorded in the body weight gain of both groups.

Mortality:

No mortality was registered during the main test.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In view of the results under the experimental conditions, the test item needs not to be classified.
Executive summary:

No macroscopic cutaneous reactions attributable to allergy were recorded after induction of Guinea Pigs with the test item.