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Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-08-05 to 2002-09-19
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test performed in 2002.
LLNA method (OECD 442 B) was adopted in 2010.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
15 male albino guinea pigs of Dunkin-Hartley strain, supplied by Centre de Production Animale (F-
45160 Olivet) were exposed to the test product after a 5-day acclimatisation period. For the main study, the animals weighted between 275g and 342g at the beginning of the test.
The environmental parameters were:
- Temperature: between 19 °C and 23°C
- Relative humidity: between 47% and 60%
Route:
intradermal and epicutaneous
Vehicle:
other:
Concentration / amount:
1st induction:
- 2 intradermal injections of the product diluted at 20% in an ethanol/distilled water (20/80: v/v) solution.
- 2 intradermal injections of Freund’s Complete Adjuvant diluted at 50% in a physiological saline solution.
- 2 intradermal injections of a mixture with equal volumes - Freund’s Complete Adjuvant at 50% and the product diluted at 40% in a ethanol/distilled water (20/80: v/v) solution.
Weighing of animals.
2nd induction: topical application, on the same zone, with the product at 100%, 24 hours after brushing with 0.5 ml of a solution of Sodium lauryl sulfate at 10%.
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Challenge phase: topical application under occlusive dressing at the following concentrations : 100% and 50%.
No. of animals per dose:
Negative control: 5 mal guinea pigs identified n°C7276 to C7280
Treated: 10 male guinea pigs identified n°C7281 to C7290
Details on study design:
1) Preliminary studies:
- Maximum Non Necrotizing Concentration (M.N.N.C.) determination:
2 female guinea pigs identified n°C7259 to C7260 were used.
The test product was injected by intradermal route at the following concentrations: 25%, 12.5%, 6.25% and 3.125% diluted in distilled water, 12.5% and 6.25% diluted in paraffin oil and 20% and 10% diluted in ethanol/distilled water (20/80; v/v).

- Pre-Maximum Non Irritant Concentration (M.N.I.C.) determination:
2 female guinea pigs identified n°C7259 to C7260 were used.
The product was applied under an occlusive dressing during 24 hours, at the following concentrations pure and diluted 50%, 25% and 12.5% in distilled water.

- Maximum Non Irritant Concentration (M.N.I.C.) determination:
3 female guinea pigs identified n° C7265 to C7267 were used.
After induction by intradermal injection with ethanol/distilled water (20/80: v/v) solution and by topical application with ethanol/distilled water (20/80: v/v) solution and a 17-days rest phase, the challenge phase under occlusive dressing for 24 hours consists in a single topical application of the test product at the following concentrations: 100%, diluted 50%, 25% and 12.5% in ethanol/distilled water (20/80: v/v) solution.

2) Main study
GROUP 1 (negative control) : 5 male guinea pigs identified n° C7276 to C7280;
GROUP 2 (treated) : 10 male guinea pigs identified n° C7281 to C7290;

Note : The results of the 3 latest positive group (Reference substance : neomycin sulfate Test 6 and benzocaïne Test 4 and 5) carried out as method sensibility, were presented in appendix.

The environmental parameters were :
- Temperature : between 19 °C and 23°C
- Relative humidity : between 47% and 60%

* Chronological development:
a) Preliminary studies:
2002/08/01: Arrival of animals.
2002/08/05 to 2002/09/04: Preliminary tests : pre MNIC, MNNC, and MNIC determinations.

b) Main study:
2002/08/20: Arrival of animals.

Induction phase
2002/08/25: 1st induction:
- 2 intradermal injections of the product diluted at 20% in an ethanol/distilled water (20/80: v/v) solution.
- 2 intradermal injections of Freund’s Complete Adjuvant diluted at 50% in a physiological saline solution.
- 2 intradermal injections of a mixture with equal volumes - Freund’s Complete Adjuvant at 50% and the product diluted at 40% in a ethanol/distilled water (20/80: v/v) solution.

2002/08/25: Weighing of animals.

2002/08/30: 2nd induction: topical application, on the same zone, with the product at 100%, 24 hours after brushing with 0.5 ml of a solution of Sodium lauryl sulfate at 10%.
2002/09/01 to 2002/09/16: Rest phase: 15 days

2002/09/1:6 Challenge phase: topical application under occlusive dressing at the following concentrations : 100% and 50%.

2002/09/17: 24-hours reading time.
2002/09/18: 48-hours reading time and weighing.
Challenge controls:
Main study:
2002/08/20: Arrival of animals.

Induction phase
2002/08/25: 1st induction:
- 2 intradermal injections of the product diluted at 20% in an ethanol/distilled water (20/80: v/v) solution.
- 2 intradermal injections of Freund’s Complete Adjuvant diluted at 50% in a physiological saline solution.
- 2 intradermal injections of a mixture with equal volumes - Freund’s Complete Adjuvant at 50% and the product diluted at 40% in a ethanol/distilled water (20/80: v/v) solution.

2002/08/25: Weighing of animals.

2002/08/30: 2nd induction: topical application, on the same zone, with the product at 100%, 24 hours after brushing with 0.5 ml of a solution of Sodium lauryl sulfate at 10%.
2002/09/01 to 2002/09/16: Rest phase: 15 days

2002/09/1:6 Challenge phase: topical application under occlusive dressing at the following concentrations : 100% and 50%.

2002/09/17: 24-hours reading time.
2002/09/18: 48-hours reading time and weighing.
Positive control substance(s):
yes
Remarks:
Neomycin sulfate Benzocaine
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Skin reaction
Score 1: 1
Score 2: 0
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Skin reaction
Score 1: 1
Score 2: 0
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
Skin reaction
Score 1: 3
Score 2: 0
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 6th test)
Score 1: 4
Score 2: 5
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 6th test)
Score 1: 2
Score 2: 5
Score 3: 0
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
12.5%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 4th test)
Score 1: 4
Score 2: 5
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
12.5%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 4th test)
Score 1: 2
Score 2: 5
Score 3: 0
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
12.5%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 5th test)
Score 1: 5
Score 2: 4
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
12.5%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 5th test)
Score 1: 3
Score 2: 5
Score 3: 0
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
6.25%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 5th test)
Score 1: 5
Score 2: 4
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
6.25%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 5th test)
Score 1: 3
Score 2: 5
Score 3: 0
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
75%
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 6th test)
Score 1: 2
Score 2: 4
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
75%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 6th test)
Score 1: 1
Score 2: 4
Score 3: 0
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
38%
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 6th test)
Score 1: 3
Score 2: 3
Score 3: 0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
38%
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Skin reaction (treated group 6th test)
Score 1: 2
Score 2: 4
Score 3: 0

Concentrations selected
Preliminary studies:
- MNNC determination :
No necrosis has been observed since the concentrations of 20% (dilution in ethanol/distilled water (20/80: v/v) solution), the first induction has been carried out by intradermal injection at the same concentration.
-Pre MNIC determination :
24 hours after the removal of the occlusive dressings, no macroscopic cutaneous reaction was recorded.
In view of these results, the concentrations selected were pure for the 2nd induction of the main study and the MNIC began at the concentration of 100%.
- MNIC determination :
24 hours after removal of the occlusive dressings, no cutaneous reaction was recorded.
In view of this result, the concentrations selected were 100% (MNIC) and 50% (1/2 MNIC) for the challenge phase.


Main study :
- induction phase :
The induction phase was performed by intradermal injection at D0 with the test product diluted at 20% in an ethanol/distilled water (20/80: v/v) solution and by topical application at D7 with the test product at 100%, after brushing with a solution of sodium lauryl sulfate.
- challenge phase :
The test product has been used pure and diluted at 50% in an ethanol/distilled water (20/80: v/v) solution (1/2 MNIC).


 


Sensitising potential assessment
No macroscopic cutaneous reactions attributable to allergy was recorded during the examinationfollowing the removal of the occlusive dressings (challenge phase) from the animals of the treated group with the test product.
No cutaneous intolerance reaction was recorded in animals from the negative control group.


 


Weight evolution
Not any abnormality was recorded in the weight growth of both groups.

Interpretation of results:
other: not classified
Conclusions:
In view of these results, under these experimental conditions, the product MADECASSOSIDE, in accordance with the criteria for classification, packaging and labelling of dangerous substances of the E.E.C. Directives 67/548 and 99/45, must not be classified.
Executive summary:

After induction (intradermic injection and topical application) of 10 animals (male) of treated group with the test product MADECASSOSIDE and a 15-days rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted to a single topical application of the test product at 100% and diluted at 50% in an ethanol/distiller water solution (20/80, v/v), according to the experimental
protocol established from 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 examinationfollowing the removal of the occlusive dressing (challenge phase) from the animals of the treated group with the test product.
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 product MADECASSOSIDE, must not be classified and in accordance with the criteria for classification, packaging and labelling of dangerous substances of the E.E.C. Directives 67/548 and 99/45.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

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