Registration Dossier

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:
weight of evidence
Study period:
2018-08-30 - 2018-09-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
A temperature higher than 25°C was registered on 26 October 2017. The maximum value measured was 26°C. A relative humidity higher than 70% was registered on 06 November 2017. The maximum value measured was 92%.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
A temperature higher than 25°C was registered on 26 October 2017. The maximum value measured was 26°C. A relative humidity higher than 70% was registered on 06 November 2017. The maximum value measured was 92%.
GLP compliance:
yes (incl. certificate)
Remarks:
Groupe Interministeriel des Produits Chimiques
Type of study:
Buehler test
Justification for non-LLNA method:
The Buehler assay is considered more suitable for the test item.

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Specific details on test material used for the study:
For the purpose of the study, the test item was used freshly prepared in corn oil (Sigma, Batch No. MKCC0462) for the topical applications. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The test material was diluted at 1.5%, 7.5% and 15% in corn oil.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: albino guinea pigs of Dunkin-Hartley strain, supplied by Charles-River (69210 Saint-Germain-Nuelles, France)
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified]
- Microbiological status of animals, when known:
- Age at study initiation: 4,5 or 6 weeks old
- Weight at study initiation:
- Housing: groups of 5 at the maximum in polycarbonate containers
- Diet (e.g. ad libitum): ad libitum, tap-water from public distribution system)
- Water (e.g. ad libitum): ad libitum, ENVIGO, 2040C
- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22° C ± 3° C
- Humidity (%): from 30 % to 70 %,
- Air changes (per hr): at least 10 cycles per hour
- Photoperiod (hrs dark / hrs light): circadian cycle (12 hrs day / 12 hrs darkness).

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
100% of corn oil, 0.5 mL
Day(s)/duration:
3 topical applications at D0, D7 and D13
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
1.5 % test item in corn oil
Day(s)/duration:
3 topical applications at D0, D7 and D13
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
7.5% test item in corn oil
Day(s)/duration:
3 topical applications at D0, D7 and D13
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
15% of test item in corn oil
Day(s)/duration:
3 topical applications at D0, D7 and D13
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
100% corn oil in 0.5ml
Day(s)/duration:
3 topical applications at D0, D7 and D13
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
100% HCA in 0.5ml
Day(s)/duration:
3 topical applications at D0, D7 and D13
Adequacy of induction:
highest technically applicable concentration used
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
100% corn oil, 0.5 mL
Day(s)/duration:
14 days, D27 and D35
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
1.5% test item in corn oil
Day(s)/duration:
14 days, D27 and D35
Adequacy of challenge:
highest non-irritant concentration
No.:
#3
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
7.5% test item in corn oil
Day(s)/duration:
14 days, D27 and D35
Adequacy of challenge:
highest non-irritant concentration
No.:
#4
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
15% test item in corn oil
Day(s)/duration:
14 days, D27 and D35
Adequacy of challenge:
highest non-irritant concentration
No.:
#5
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
100% corn oil in 0.5 ml
Day(s)/duration:
14 days, D27 and D35
Adequacy of challenge:
highest non-irritant concentration
No.:
#6
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50% HCA
Day(s)/duration:
14 days, D27 and D35
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Test item: 20
Solvent control item: 10
Negative control (corn oil): 10
Positive control (HCA): 10
Details on study design:
MAIN STUDY

A. INDUCTION EXPOSURE
After shearing of the scapular zone, the 3 local applications were performed on D0, D7 and D13 for 6 hours under occlusive dressing (25mm x 50mm non woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M).
- No. of exposures: 3 topical applications at D0, D7 and D13
- Exposure period: 6 hours
- Test groups: 20 guinea pigs (10 male and 10 female). Total of 3 test groups.
- Control group: 10 guinea pigs (5 male and 5 female)
- Solvent control item: 10 guinea pigs (5 male and 5 female)
- Postivie control group: 10 guinea pigs (5 male and 5 female)
- Duration: 13-days follwed by 13-day rest phase
- Concentrations: 1.5%, 7.5%, 15% test item diluted in corn oil.

B. CHALLENGE EXPOSURE
- No. of exposures: 2 challenges at D27 and D35
- Day(s) of challenge: 14th day
- Exposure period: 6 hours
- Test groups: 20 guinea pigs (10 male and 10 female). Total of 3 test groups.
- Control group: 10 guinea pigs (5 male and 5 female)
- Solvent control item: 10 guinea pigs (5 male and 5 female)
- Postivie control group: 10 guinea pigs (5 male and 5 female)
Concentrations:
- Test groups: 1.5%, 7.5%, 15% test item diluted in corn oil.
- Control group: 100% corn oil
- Solvent control item: HCA (50% in corn oil)
- Postivie control group: HCA (50% in corn oil
- Evaluation (hr after challenge): 24/48/72 hrs

C. RECHALLENGE EXPOSURE
Due to the absence of reaction in the positive control group and doubtful reactions in treated groups, a rechallenge was performed under the same experimental conditions after a rest phase of 7 days.



Challenge controls:
The challenge phase of animals induced with the solvent control item conducted under occlusive dressing for 6 hours, consisted of a single topical application of the solvent control item at 100% and of a negative control (corn oil).
Positive control substance(s):
yes
Remarks:
alpha Hexylcinnamaldehyde, 10 animals

Results and discussion

Positive control results:
First challenge:
Due to the absence of reaction in the positive control group and doubtful reactions in treated groups, a rechallenge was performed under the same experimental conditions after a rest phase of 7 days.

Second challenge:
In the treated group 6 with the positive control, a discrete to intense erythema was noted in 80% (8/10), 60% (6/10) and 40% (4/10) of the animals, 24, 48 and 72 hours after the challenge phase, respectively, on the area challenged with HCA at 50%. No cutaneous reaction was recorded in animals for control group after the challenge phase, on the area challenged with HCAe at 50%.

In the treated group 2 (treatment 1.5%), no macroscopic cutaneous reactions attributable to allergy were observed 24, 48 and 72 hours following the removal of the occlusive dressing.In the naïve control group 1bis (associated with the treatment dose of 1.5%), no cutaneous intolerance reactions were observed.

In the treated group 3 (treatment 7.5%), a discrete to intense erythema was recorded in 10% (2/20), 15% (3/20), 10% (2/20) of the animals, 24, 48 and 72 hours after the challenge phase, respectively.
In the naïve control group 1bis (associated with the treatment dose of 7.5%), no cutaneous intolerance reactions were observed. It must be noted that only one animal (No. C4567) (1/20) showed a positive response during the 1st challenge and during the 2nd challenge. Only these reactions may be indicative of sensitisation. The other reactions should not be considered as indicative of sensitisation.

In the treated group 4 (treatment 15%), no macroscopic cutaneous reactions attributable to allergy were observed 24, 48 and 72 hours following the removal of the occlusive dressing. In the naïve control group 1bis (associated with the treatment dose of 15%), no cutaneous intolerance reactions were observed.

The test item is not considered to be a skin sensitizer when it was tested at 1.5%, 7.5% and 15% in corn oil.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1.5%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
discrete to moderate erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
1.5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
discrete to moderate erythema
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
1.5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
discrete to moderate erythema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
7.5%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
discrete erythema, moderate and confluent erythema, intense erythema and swelling
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
7.5%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Intense erythema and swelling
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
7.5%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
discrete erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
15%
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
Discrete or patchy erythema; Moderate and confluent erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
15%
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
discrete to moderate erythema; Moderate and confluent erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
discrete erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
72
Group:
positive control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
1.5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No cutaneous reaction
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
1.5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no cutaneous reaction
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
1.5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
7.5%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Moderate and confluent erythema; Intense erythema and swelling
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
7.5%
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
discrete to moderate erythema; Moderate and confluent erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
7.5%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Discrete or patchy erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
15%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
15%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
15%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Discrete or patchy erythema; Moderate and confluent erythema; Intense erythema and swelling
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Discrete or patchy erythema; Moderate and confluent erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
positive control
Dose level:
50%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Discrete or patchy erythema; Moderate and confluent erythema
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Weight evolution

No abnormalities and no differences in the body weight between the control and the treated group were observed.

Mortality

No mortality was registered during the main test.

Clinical signs

No abnormal clinical signs related to the administration of the test item were observed.

Table 1: Results from 1stChallenge

Treatment

Time

(hours)

Concentration at Challenge
(%)

Cutaneous Reaction
(Grade)

% Positive responses ≥1

% of Sensitised Animals

0

1

2

3

Control
(Corn oil)

24

1.5

10

0

0

0

0

0

48

10

0

0

0

0

0

72

10

0

0

0

0

0

24

7.5

10

0

0

0

0

0

48

10

0

0

0

0

0

72

10

0

0

0

0

0

24

15

10

0

0

0

0

0

48

10

0

0

0

0

0

72

10

0

0

0

0

0

24

Vehicle

10

0

0

0

0

0

48

10

0

0

0

0

0

72

10

0

0

0

0

0

Test Material
1.5 %

24

7.5

18

1

1

0

10

10

48

20

0

0

0

0

0

72

20

0

0

0

0

0

24

Vehicle

10

3

7

0

50

50

48

20

0

0

0

0

0

72

20

0

0

0

0

0

Test Material
7.5 %

24

7.5

15

2

2

1

25

25

48

19

0

0

1

5

5

72

19

1

0

0

5

5

24

Vehicle

13

3

4

0

35

35

48

20

0

0

0

0

0

72

20

0

0

0

0

0

Test Material
15 %

24

15

14

2

4

0

30

30

48

17

2

1

0

15

15

72

20

0

0

0

0

0

24

Vehicle

13

4

3

0

35

35

48

19

1

0

0

5

5

72

20

0

0

0

0

0

Positive control
Naïve control group

24

50

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

24

0

8

1

1

0

20

48

10

0

0

0

0

72

10

0

0

0

0

Positive control
alpha hexylcinnamaldehyde (HCA)

24

50

8

0

2

0

20

20

48

10

0

0

0

0

0

72

10

0

0

0

0

0

24

0

5

1

4

0

50

50

48

9

1

0

0

10

10

72

10

0

0

0

0

0

 

Table 2: Results from 2ndChallenge

Treatment

Time
(hours)

Concentration at Challenge
(%)

Cutaneous Reaction
(Grade)

% Positive responses ≥1

% of Sensitised Animals

0

1

2

3

Control (1 bis)
(Corn oil)

24

1.5

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

24

7.5

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

24

15

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

24

Vehicle

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

Test Material
1.5 %

24

7.5

20

0

0

0

0

0

48

20

0

0

0

0

0

72

20

0

0

0

0

0

24

Vehicle

20

0

0

0

0

0

48

20

0

0

0

0

0

72

20

0

0

0

0

0

Test Material
7.5 %

24

7.5

18

0

1

0

10

10

48

18

1

2

0

15

15

72

18

2

0

0

10

10

24

Vehicle

20

0

0

0

0

0

48

20

0

0

0

0

0

72

20

0

0

0

0

0

Test Material
15 %

24

15

20

0

0

0

0

0

48

20

0

0

0

0

0

72

20

0

0

0

0

0

24

Vehicle

20

0

0

0

0

0

48

20

0

0

0

0

0

72

20

0

0

0

0

0

Positive control
Naïve control group

24

50

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

24

0

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

Positive control
alpha hexylcinnamaldehyde (HCA)

24

50

2

1

6

1

80

80

48

4

3

3

0

60

60

72

6

2

2

0

40

40

24

0

10

0

0

0

0

0

48

10

0

0

0

0

0

72

10

0

0

0

0

0

Control Group
(1 ter)

24

Vehicle

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

24

Vehicle

10

0

0

0

0

48

10

0

0

0

0

72

10

0

0

0

0

 

Applicant's summary and conclusion

Interpretation of results:
other: The test item is not considered to be a skin sensitizer.
Conclusions:
The test item is not considered to be a skin sensitizer when tested at 1.5%, 7.5%, and 15% in corn oil.
Executive summary:

The aim of the study was to evaluate the possible allergenic activity of the test item magnesium metaborate and solvent control item after topical administration in guinea pigs. The experimental protocol was established according to OECD Guideline 406 and in compliance with GLP criteria.

According the results of the pretests, the induction phase (3 topical applications at 100%) under occlusive dressing was conducted with the test item to 20 guinea pigs and with the solvent control item to 10 guinea pigs and a 13-day rest phase. The challenge phase of animals induced with the test item conducted under occlusive dressing for 6 hours, consisted of a single topical application of the test item diluted at 50% in corn oil and of a negative control (corn oil).

The challenge phase of animals induced with the solvent control item conducted under occlusive dressing for 6 hours, consisted of a single topical application of the solvent control item at 100% and of a negative control (corn oil).

At the request of sponsor, a positive control group treated with a reference allergen alpha Hexylcinnamaldehyde was selected.

In the treated group 2 (treatment dose of 1.5%), a discrete to moderate erythema was recorded in 10% (2/20) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted 48 and 72 hours after the challenge phase. A discrete to moderate erythema was recorded in 50% (10/20) of the animals, 24 hours after the challenge phase on the treated area with corn oil. No cutaneous reaction was noted 48 and 72 hours after the challenge phase. In the control group 1 (associated with the treatment dose of 1.5%), no cutaneous reaction was noted after the challenge phase.

In the treated group 3 (treatment dose of 7.5%), a discrete to intense erythema was recorded in 25% (5/20), 5% (1/20), 5% (1/20) of the animals, 24, 48 and 72 hours, respectively, after the challenge phase. A discrete to moderate erythema was recorded in 35% (7/20) of the animals, 24 hours after the challenge phase on the treated area with corn oil. No cutaneous reaction was noted 48 and 72 hours after the challenge phase. In the control group 1 (associated with the treatment dose of 7.5%), no cutaneous reaction was noted after the challenge phase.

In the treated group 4 (treatment dose of 15%), a discrete to moderate erythema was recorded in 30% (6//20), 15% (3/20) of the animals, 24 and 48 hours, respectively, after the challenge phase. No cutaneous reaction was noted 72 hours after the challenge phase. A discrete to moderate erythema was recorded in 35% (7/20), 5% (1/20) of the animals, 24 and 48 hours, respectively, after the challenge phase on the treated area with corn oil. No cutaneous reaction was noted 72 hours after the challenge phase. In the control group 1 (associated with the treatment dose of 15%), no cutaneous reaction was noted after the challenge phase.

In the treated group 6 with the positive control, moderate erythema was noted in 20% (2/10), of the animals, 24 hours after the challenge phase, on the area challenged with alpha Hexylcinnamaldehyde at 50%. No cutaneous reaction was noted 48 and 72 hours after the challenge phase. A discrete to moderate erythema was recorded in 50% (5/10), 10% (1/10) of the animals, 24 and 48 hours after the challenge phase on the treated area with corn oil. No cutaneous reaction was noted 72 hours after the challenge phase. No cutaneous reaction was recorded in animals for control group 5 after the challenge phase, on the area challenged with alpha Hexylcinnamaldehyde at 50%. A discrete to moderate erythema was recorded in 20% (2/10) of the animals, 24 hours after the challenge phase on the treated area with corn oil. No cutaneous reaction was noted 48 and 72 hours after the challenge phase.

Due to the absence of reaction in the positive control group and doubtful reactions in treated groups, a rechallenge was performed under the same experimental conditions after a rest phase of 7 days.

Second challenge:

In the treated group 6 with the positive control, a discrete to intense erythema was noted in 80% (8/10), 60% (6/10) and 40% (4/10) of the animals, 24, 48 and 72 hours after the challenge phase, respectively, on the area challenged with HCA at 50%. No cutaneous reaction was recorded in animals for control group after the challenge phase, on the area challenged with HCAe at 50%.

In the treated group 2 (treatment 1.5%), no macroscopic cutaneous reactions attributable to allergy were observed 24, 48 and 72 hours following the removal of the occlusive dressing.In the naïve control group 1bis (associated with the treatment dose of 1.5%), no cutaneous intolerance reactions were observed.

In the treated group 3 (treatment 7.5%), a discrete to intense erythema was recorded in 10% (2/20), 15% (3/20), 10% (2/20) of the animals, 24, 48 and 72 hours after the challenge phase, respectively.

In the naïve control group 1bis (associated with the treatment dose of 7.5%), no cutaneous intolerance reactions were observed. It must be noted that only one animal (No. C4567) (1/20) showed a positive response during the 1st challenge and during the 2nd challenge. Only these reactions may be indicative of sensitisation. The other reactions should not be considered as indicative of sensitisation.

In the treated group 4 (treatment 15%), no macroscopic cutaneous reactions attributable to allergy were observed 24, 48 and 72 hours following the removal of the occlusive dressing. In the naïve control group 1bis (associated with the treatment dose of 15%), no cutaneous intolerance reactions were observed.

The test item is not considered to be a skin sensitizer when it was tested at 1.5%, 7.5% and 15% in corn oil.