Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-12-11 to 2008-02-25
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:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
For some chemical structures it has been demonstrated that the guinea pig model was more relevant rather than the LLNA model due to the high probability to have false positive in LLNA tests (SI > 3 at irritating concentrations)
"Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the Guinea pig maximization test (GPMT)", Kreiling R et al., 2008

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid, pearl
- Analytical purity: 100%
- Lot/batch No.: T71225
- Expiration date of the lot/batch: 21 March 2009 (re-test)
- Storage condition of test material: at the room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
For the main study, 15 female albino guinea pigs of Dunkin-Hartley strain, supplied by Charles River (F-69592 L’Arbresle) weighed between 261 and 297 at the beginning of the test and were 4 weeks old.
Prior to the test, the animals were kept for a minimum acclimatisation 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 a tattoo placed on their ear.
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.
The environmental parameters of the main test were :
- Temperature : between 20°C and 23°C
- Relative humidity : between 33% and 67%
- Lighting time: 12 hours daily
The drinking water (tap water from public distribution system) and food were supplied freely. Microbiological verification and chemical analysis were conducted every six months by the Institut Européen de l’Environnement de Bordeaux (I.E.E.B.).

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Isotonic sodium chloride for the intradermal injection and distilled water for the topic application
Concentration / amount:
(1) Preliminary study
-determination by intrademal injection of the Maximal Non Necrotizing Concentration (MNNC): 0.0975 ; 0.195% ; 0.39% ; 0.78% ; 1.56% ; 3.125% ; 6.25% ; 25% and 50%in isotonic sodium chloride.
-determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): 6.25 ; 12.5 ; 25 and 50% in liquid paraffin.
-determination by topical application of the Maximal Non Irritant Concentration (MNIC): 6.25 ; 12.5; 25 and 50% in distilled water.
(2) Main study
intradermal induction with the product at 0.78% in isotonic sodium chloride
topical induction with the product at 50% in distilled water
challenge with the product at 25% and 50% in distilled water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Isotonic sodium chloride for the intradermal injection and distilled water for the topic application
Concentration / amount:
(1) Preliminary study
-determination by intrademal injection of the Maximal Non Necrotizing Concentration (MNNC): 0.0975 ; 0.195% ; 0.39% ; 0.78% ; 1.56% ; 3.125% ; 6.25% ; 25% and 50%in isotonic sodium chloride.
-determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): 6.25 ; 12.5 ; 25 and 50% in liquid paraffin.
-determination by topical application of the Maximal Non Irritant Concentration (MNIC): 6.25 ; 12.5; 25 and 50% in distilled water.
(2) Main study
intradermal induction with the product at 0.78% in isotonic sodium chloride
topical induction with the product at 50% in distilled water
challenge with the product at 25% and 50% in distilled water
No. of animals per dose:
5 animals for the preliminary study
10 animals for the treated group of the main study
5 animals for the control group of the main study

Details on study design:
(1) Preliminary study
- for the determination of the MNNC, two animals received increasing concentration of the substance by intradermal injection on both sides of the spine. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours later.
- for the determination of the pre-MNIC, the product was applied on the dorso-lumbar zone of 2 animals shorn beforhand, with occlusive dressing for 24 hours. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.
- for the determination of the MNIC, 3 animals were treated according to the same treatment as negative controls for the induction phase (ie 2 intradermic injection (ID) of Freund's Complete Adjuvant (FCA) diluted at 50% in isotonic sodium chloride + 2 ID of isotonic sodium chloride + 2 ID a mixture with equal volumes v/v FCA at 50% and isotonic sodium chloride.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the occlusive dressing.
(2) Main study
A- Induction
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 (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,
GROUP 2 (Treated):
• 2 ID: Freund’s Complete Adjuvant diluted by 50 % in isotonic sodium chloride,
• 2 ID: test item at 0.78%,
• 2 ID a test mixture in equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and the test item at 1.56%.
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 for 48 hours was performed 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 50%
B - Rest phase
The animals of both groups were left for 10 days.
C - Challenge phase
Day 21
The experimental procedure of this phase was identical for both groups GROUP 1 (Negative control) and GROUP 2 (Treated) 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 50% (MNIC) and at 25% (1/2 MNIC).
Challenge controls:
Negative controls : 5 animals

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 (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,


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 for 48 hours was performed on the injection sites of each animal.
GROUP 1 (Negative control): 0.5 ml of distilled water

Rest phase
The animals of control groups were left for 10 days.

Challenge phase
Day 21
The experimental procedure of this phase was identical for both groups GROUP 1 (Negative control) and GROUP 2 (Treated) 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 50% (MNIC) and at 25% (1/2 MNIC).
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:
positive control at 50% : 100% of animals sensitized after 24h
positive control at 50% : 90% of animals sensitized after 48h
positive control at 25% : between 90% and 100% of animals sensitized 24h
positive control at 25% : between 70% and 100% of animals sensitized 48h

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
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: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
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: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
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: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
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: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
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: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
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: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
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: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
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: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In view of these results, under these experimental conditions, the test substance 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 Globally Harmonized System (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 allergenic activity of the test substance after intradermic injection and topical administration in guinea pigs.

After induction (intradermic injection at 0.78% and topical application at 50%) of 10 Guinea Pigs of treated group with the test substance 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 50% and at 25% in distilled water. The experimental protocol was established according the O.E.C.D.guideline n°406 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 attributable 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 substance needs 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 Globally Harmonized System (COM(2007)355 final), the test item must not be classified in category 1. No signal word and hazard statement are required.