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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:
2008-05-12 to 2008-06-12
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test performed in 2008.
LLNA method (OECD 442 B) was adopted in 2010.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): LCA08002
- Physical state: White powder
- Analytical purity: 100%
- Lot/batch No.: 1549 JG
- Expiration date of the lot/batch: 2009-04-10
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/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: 226g to 270g
- Housing: 2 or 3 animals/cage: polycarbonate containers
- Diet (e.g. ad libitum): C17.50 guinea pig breeder (SDS)
- Water (e.g. ad libitum): tap water from public distribution system
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 22°C
- Humidity (%): 44% to 61%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Remarks:
and liquid parrafin for topical application
Concentration / amount:
Intradermal induction: 25%
Topical induction: 60%
Topical Challenge: 30%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Remarks:
and liquid parrafin for topical application
Concentration / amount:
Intradermal induction: 25%
Topical induction: 60%
Topical Challenge: 30%
No. of animals per dose:
10 animals
Details on study design:
1-Preliminary studies:
1-1 Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC)
This test was conduced for the purpose of defining a MNNC of the test item which, on intradermic injection during the induction phase, does not risk causing too great lesion (non-necrotizing concentration), should be well-tolerated systemically and should be the highest to cause mild to moderate skin irritation.
Two animals received on both sides of the spine, a volume of 0.1mL of the test item, at 6 concentrations (25%; 12.5%; 6.25%; 3.125%; 1.56%; 0.78%) in olive oil.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours later and 48 hours later if necessary.

1-2 Dertermination 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-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: diluted at 60%, 30%, 15% and 7.5% in liquid paraffin.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing

1-3 Dertermination 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 wihtou risk of an irritant effect during the challenge phase.
Three guinea pig 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) of the main study.
During the challenge phase, the animals were treated with the test item placed onto the select treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations; diluted at 60%, 30%, 15%, 7.5% in liquid paraffin.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing

2-Main study
Group1: Negative control: 5 females
Group 2: treated: 10 females

2-1 induction phase:
2-1-1 intradermal induction
After shearing scapular zone, three pairs of intradermal injections of 0.1mL 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 follow:
Group1:
- 2 ID: Freund's Complete Adjuvant diluted at 50% in isotonic sodium chloride
- 2ID: Olive Oil
- 2 ID: a mixture with equal volume v/v: Freund's Complete Adjuvant at 50% and Olive oil
Group 2:
- 2 ID: Freund's Complete Adjuvant diluted at 50% in isotonic sodium chloride
- 2ID: test item at 25%
- 2 ID: a mixture with equal volume v/v: Freund's Complete Adjuvant at 50% and the test item at 50%

2-1-2 Topical induction:
Day6:
The scapular zone of all animals in each group, shorn beforehand, was brushed with a solution of SDS at 10% in thick vaseline, in order to create a local irritation.
Day 7:
A topical application under occlusive dressing for 48hours was performed on the injection sites of each animals.
Group 1: 0.5mL of liquid paraffin
Group 2: 0.5mL of the test item at 60% in liquid paraffin

2-2 rest phase:
The animals both groups were left for 10 days.

2-3 challenge phase:
Day 20
The experimental procedure of this phase was identicalfor both groups submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing was performed during 24 hours; 1 sample cup containing the test item at 60% (MNIC) and at 30% (1/2 MNIC)

Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:
3 tests : 2008-02-12 ; 2007-10-16 ; 2007-01-16.
In conclusion, in view of the results, under these experimental conditions, the substance alpha-Hexylcinnamaldehyde: must be classified R43 "may cause sensitization by skin contact" in accordance with the criteria for the classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. This substance must be characterised by the symbol "Xi" and the warning label"irritant".
In accordance with the GHS (COM(2007)355 final), the test item must be classified category 1 "skin sensitisation". The signal word "Warning" and hasard statement H317 "May cause an allergic skin reaction" are required.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
60%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 60%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no reaction.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 30%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no reaction.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
60%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 60%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no reaction.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 30%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no reaction.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
100 % of animals sensitized
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
100 % of animals sensitized
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
60%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reaction
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 60%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no reaction.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reaction
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 30%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no reaction.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
60%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reaction
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 60%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no reaction.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reaction
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 30%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no reaction.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
90 % of animals sensitized
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
70 % of animals sensitized
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
In view of these results, under these experimental conditions, the test item LCA08002 must not be classified, 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. No Symbol and risk phrase are required.
In accordance with the GHS (COM(2007)355 final), the test item must not be classified in category 1. No signal word and hazard statement are required.
Executive summary:

The aim of the study was to evaluate the possible allergic activity of the test item LCA08002 after intradermal injection and topical administration in guinea pig.

After induction (intradermal injection at 25% and topical application 60%) of 10 guinea pigs of treated group with the test item LCA08002 and a 10 days rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted to a single topical application of the test item diluted at 60% and at 30% in liquid paraffin. The experimental protocol was established according to the OECD guideline n0406 dated July 17th, 1992 and the method B.6 of the E.E.C. n°96/54 dated July 30th,1996.

No macroscopic cutaneous reactions attibutable to allergy was recorded during the examination following the removal of the occlusive 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.

In conclusion, in view of these results, under these experimental conditions, the test item LCA08002 must not be classified, 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. No Symbol and risk phrase are required. In accordance with the GHS (COM(2007)355 final), the test item must not be classified in category 1. No signal word and hazard statement are required.