Registration Dossier

Administrative data

Description of key information

Skin sensitisation, Buehler assay, OECD 406, result: skin sensitiser Sub-category 1B above 15% up to 100% of test item concentration based on the specific concentration limit test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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
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.
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.
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).
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
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
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.
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

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

 

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.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-10-16 - 2017-12-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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.
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. Indeed, the preparation of the test item at 50% in corn oil (v/v) was a thick brown solution.
Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ENVIGO (Kreuzelweg 53, 5961 NM HORST The Netherlands)
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified]
- Microbiological status of animals, when known:
- Age at study initiation: 4 weeks
- Weight at study initiation:
- Housing: groups of 3 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).
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% of test item, 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:
unchanged (no vehicle)
Concentration / amount:
100% of solvent control, 0.5 mL
Day(s)/duration:
3 topical applications at D0, D7 and D13
Adequacy of induction:
highest technically applicable concentration used
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50% of test item, 0.5 mL
Day(s)/duration:
14 days, D27 and D35
Adequacy of challenge:
highest non-irritant concentration
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% vehicle control, 0.5 mL
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
Details on study design:
RANGE FINDING TESTS:
The induction phase consisted of 3 topical applications at 100% under occlusive dressing. Observatin perios 24 and 48 hours. The maximum non itrritant concentration was 50%.
Maximum Non Irritant Concentration (M.N.I.C.) determination:
This test was carried out with a reduced number of animals, for the purpose of determining the maximal non-irritating test item concentration to be used for the challenge phase. Furthermore, this test evaluates the irritant potential of the test item, and defines, if possible, a mild to moderate irritant concentration to be used for the topical induction phase. Two female and two male guinea pigs were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing (25 mm x 50 mm 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) for a period of 6 hours at 4 different concentrations: 100%, and diluted at 75%, 50% and 25% in corn oil.
The animals treated at the concentrations of 100%, and diluted at 75%, 50% and 25% received 0.5 mL of the corresponding preparation.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings and rinsing with corn oil. The skin reaction was observed and recorded according to the grades described hereafter.

MAIN STUDY
A. INDUCTION EXPOSURE
- 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)
- Control group: 10 guinea pigs (5 male and 5 female)
- Solvent control item: 10 guinea pigs (5 male and 5 female)
- Duration: 13-days follwed by 13-day rest phase
- Concentrations: 100%

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
- Control group: 10 guinea pigs
- Concentrations: 50% test item, 100% solvent control item
- Evaluation (hr after challenge): 24/48/72 hrs
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
Positive control results:
First challenge:
In the treated group with positive control, no cutaneous reaction was noted on the area challenged with alpha Hexylcinnamaldehyde at 50%.
Due to the absence of reaction in the positive control group, a rechallenge was performed under the same experimental conditions after a rest phase of 7 days.

Second challenge:
In the treated group with positive control, a slight to well defined erythema was noted in 70% (7/10), 30% (3/10) and 10% (1/10) of the animals, 24, 48 and 72 hours after the rechallenge phase, on the area challenged with alpha Hexylcinnamaldehyde at 50%. In the control group of positive control (associated with the treatment dose of 50%), a discrete erythema was noted in 60% (6/10), 20% (2/10) of the animals, 24 and 48 hours after the rechallenge phase, respectively.
No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with corn oil.
As irritation was observed in the control group animals, only reactions in the test group animals that exceed the most severe reactions seen in the control group animals were attributed to skin sensitization. Therefore, a sensitization reaction was noted in 30% (3/10) and 10% (1/10) of the animals 24 and 72 hours after the challenge phase, respectively, on the area challenged with the positive control at 50%.
These reactions were significant of a skin sensitising response. So the study must be validated.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
9
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:
50%
No. with + reactions:
7
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:
72
Group:
test group
Dose level:
50%
No. with + reactions:
1
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:
negative control
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
discrete erythema
Remarks on result:
no indication of skin sensitisation
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 cutaneous reaction
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
other: solvent control item
Dose level:
100%
No. with + reactions:
4
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:
other: solvnet control item
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reaction
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
discrete to moderate erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
discrete to moderate erythema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
discrete to moderate erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
discrete erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
discrete erythema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
discrete erythema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
other: solvent control item
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
discrete erythema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
other: solvent control item
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reaction
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no cutaneous reaction
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
discrete to moderate erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
discrete to moderate erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
50%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
discrete to moderate erythema
Remarks on result:
no indication of skin sensitisation

Induction phase

Induction phase Solvent control item:

No cutaneous reaction was noted during the induction phase.

- Induction phase Control:

No cutaneous reaction was noted during the induction phase.

- Induction phase Test item:

No cutaneous reaction was noted in the animals (20/20) 24 hours after the first induction.

Discrete erythema was observed in nineteen animals (19/20) 24 hours after the second induction.

Discrete erythema was observed in four animals (4/20) 24 hours after the second induction and moderate erythema was observed in sixteen animals (16/20) 24 hours after the third induction.

Induction phase Positive control:

No cutaneous reaction was noted in the animals (10/10) 24 hours after the first induction.

Discrete erythema was observed in eight animals (8/10) 24 hours after the second induction and in all animals (10/10) 24 hours after the third induction.

- Induction phase Control of positive control:

No cutaneous reaction was noted during the induction phase.

- Induction phase Rechallenge group from positive control:

No cutaneous reaction was noted during the induction phase.

First challenge

As irritation was observed at 24 hours in the control group animals, only reactions in the test group animals that exceed the most severe reactions seen in the control group animals were attributed to skin sensitization. Therefore, a sensitization reaction was noted in 30% (6/20), 35% (7/20) 5% (1/20) of the animals 24, 48 and 72 hours after the challenge phase, on the area challenged with the test item at 50%, respectively.

Second challenge

No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with corn oil. As irritation was observed in the control group animals, only reactions in the test group animals that exceed the most severe reactions seen in the control group animals were attributed to skin sensitization. Therefore, a sensitization reaction was noted in 85% (17/20), 50% (10/20), 45% (9/20) of the animals 24,48 and 72 hours after the challenge phase, respectively, on the area challenged with the test item at 50%.

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.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item has to be classified in Sub-category 1B as a skin sensitizer.
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 first challenge in the group treated with test item (treatment dose of 50%), a discrete to moderate erythema was recorded in 45% (9/20), 35% (7/20) 5% (1/20) of the animals, 24, 48 and 72 hours after the challenge phase, respectively.

In the control group of test item (associated with the treatment dose of 50%), a discrete erythema was noted in 10% (1/10) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted 48 hours after the challenge phase.

No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with corn oil. In the treated group with solvent control item (treatment dose of 100%), a discrete erythema was recorded in 40% (4/10) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted 48 hours after the challenge phase.

In the treated group with positive control, no cutaneous reaction was noted on the area challenged with alpha Hexylcinnamaldehyde at 50%. Due to the absence of reaction in the positive control group, a rechallenge was performed under the same experimental conditions after a rest phase of 7 days.

In the second challenge in the group treated with test item (treatment dose of 50%), a discrete to moderate erythema was recorded in 90% (18/20) of the animals, 24, 48 and 72 hours after the challenge phase.

In the control group of test item (associated with the treatment dose of 50%), a discrete erythema was noted in 60% (6/10), 30% (3/10), 10% (1/10) of the animals, 24, 48 and 72 hours after the challenge phase, respectively. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with corn oil.

In the treated group with solvent control item (treatment dose of 100%), a discrete erythema was recorded in 10% (1/10) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted 48 hours after the challenge phase.

The reactions from the positive control were significant of a skin sensitising response and the study therefore is valid.

In conclusion, the test item has to be classified in Sub-category 1B as a skin sensitizer, in accordance with the Regulation EC No 1272/2008 on classification, labelling and packaging of substances and mixtures, because the criteria set in Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures of ≥ 15 % responding at > 20 % topical induction dose for the Buehler assay were met.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Skin sensitisation

The aim of the key study (Richeux, 2018) 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 first challenge in the group treated with test item (treatment dose of 50%), a discrete to moderate erythema was recorded in 45% (9/20), 35% (7/20) 5% (1/20) of the animals, 24, 48 and 72 hours after the challenge phase, respectively.

In the control group of test item (associated with the treatment dose of 50%), a discrete erythema was noted in 10% (1/10) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted 48 hours after the challenge phase.

No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with corn oil. In the treated group with solvent control item (treatment dose of 100%), a discrete erythema was recorded in 40% (4/10) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted 48 hours after the challenge phase.

In the treated group with positive control, no cutaneous reaction was noted on the area challenged with alpha Hexylcinnamaldehyde at 50%. Due to the absence of reaction in the positive control group, a rechallenge was performed under the same experimental conditions after a rest phase of 7 days.

In the second challenge in the group treated with test item (treatment dose of 50%), a discrete to moderate erythema was recorded in 90% (18/20) of the animals, 24, 48 and 72 hours after the challenge phase.

In the control group of test item (associated with the treatment dose of 50%), a discrete erythema was noted in 60% (6/10), 30% (3/10), 10% (1/10) of the animals, 24, 48 and 72 hours after the challenge phase, respectively. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with corn oil.

In the treated group with solvent control item (treatment dose of 100%), a discrete erythema was recorded in 10% (1/10) of the animals, 24 hours after the challenge phase. No cutaneous reaction was noted 48 hours after the challenge phase.

The reactions from the positive control were significant of a skin sensitising response and the study therefore is valid.

Second study to evaluate the specific concentration limit

The aim of the key study (Richeux, 2018)was to evaluate the possible allergenic activity of the test itemOS104242Mat three concnetrations (1.5%, 7.5% and 15%)after topical administration in guinea pigs.The experimental protocol was established according to OECD Guideline 406 and in compliance with GLP criteria. 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. A positive control group treated with a reference allergen alpha Hexylcinnamaldehyde was selected.

The induction phase (3 topical applications at Day 0, Day 7 and Day 13) under occlusive dressing was conducted with the test itemOS104242 diluted at 1.5%, 7.5% and 15% in corn oil to 3 different treated groups of 20 Guinea pigsand 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 1.5% or 7.5% or 15% in corn oil respectively. A negative control of 10 Guinea Pigs was induced by the vehicle (corn oil at Day 0, Day 7 and Day 13) and challenged with the test itemOS104242 diluted at 1.5%, 7.5% and 15% in corn oilin view to verify the absence of any primary cutaneous irritation effect.

 

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.

Justification for classification or non-classification

In conclusion, the test item doesn't need to be classified up to the use of 15% in product; however, the test item should be classified as Category 1B if used final concentration is above 15%.

.