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Description of key information

OECD 406 Guinea Pig Maximasation Test

OECD 429 Local Lymph Node Assay

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

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Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 16 to July 28 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: From sponsor; CIBA Inc.
- Batch No.of test material: 512051
- Expiration date of the lot/batch: December 2008
- Purity test date: certificate of analysis dated June 16, 2008
- pH: 7.15
- Colour: colourless
- Form: liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

The test item was considered as 100% for the study
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 20-22 g
- Housing: The animals were individually housed in suspended solid floor polyprpylene cages furnished with softwood woodflakes
- Diet: ad libitum
- Water:ad libitum
- Acclimation period: a minimum of 5 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
undiluted, 50 or 25%
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:

A preliminary screening was performed in one mouse:
Daily treatment with 25 ul undiluted test item, on dorsal surface of ear for 3 days
Observations on days: 1, 2, 3, 4 , 5 and 6
No signs of systemic toxicity were noted.
Dose levels were selected based on this

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph node assay
- Criteria used to consider a positive response:
Local reactions:
Increase in ear thickness:
<10% Non-irritant
10-30% slightly irritant
>30% Irritant

Stimulation index (SI) determination:
The proliferation response of lymph node cells was expressed as the numbver of lymphocytes per lymph node and as the ration of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (SI)

When usingt the pooled approach, the SI was calculated according to the following formula:

SI = cell count of treated group / cell count of control group

The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater that 1.4 compared to control values (SI > 1.4)

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated daily by application of 25 ul of the appropriate cincentration of test item (0, 25, 50 or 100%) on the dorsal surface of the ear for three consecutive days.
The test item formulation was administered using an automatic micropipette and sprea over the odrsal surface of the ear using the tip of the pipette.
Key result
Parameter:
SI
Value:
1.73
Test group / Remarks:
25% test item
Remarks on result:
other:
Remarks:
calculated by pooled approach
Key result
Parameter:
SI
Value:
2.76
Test group / Remarks:
50% test item
Remarks on result:
other:
Remarks:
calculated by pooled approach
Key result
Parameter:
SI
Value:
3.17
Test group / Remarks:
100% test item
Remarks on result:
other:
Remarks:
calculated by pooled approach
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
A stimulation index more than 1.4 was recorded for all three concentrations (25, 50 and 100%)

CLINICAL OBSERVATIONS:
No mortality and no signs of systemic toxicity were noted in the test and control animals during the test

BODY WEIGHTS
Bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the control animals

A slight dryness was registred at D6 in thre treated groups at 50% and 100%.

A moderate to important increase in ear thickness (+16.5%, +29.4%, +41.7%) and in ear weight (+18%, +42.5%, +19.5%) was recorded respectively at the concentrations of 25%, 50% and 100%. The test item must therefore be considered as slightly irritant at 25% and 50% and irritant at 100%.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In view of these results, under these experimental conditions, the itest item, Dermosoft DGMC.., must be classified in category 1B "skin sensitisation" according to the Globally Harmonized System (COM (2007)355 final). The signal word "warning" and the hazard statement H317 "may cause an allergic reaction" are required.
Accoridng to tests on the ear thickness after exposure, the test item must also be considered as slightly irritant at 25% and 50% and irritant at 100%.
Executive summary:

The test was performed to access the skin sensitisation potential of the test item, Dermosoft DGCM.., in the CBA/J mouse strain following topical applications to the dorsal surface of the ear. The test was performed according to OECD guideline 429.

Three groups of each four animals, were treated for three consecutive days with 50 ul (25 ul per ear) with the undilited test item and the test item as a solution in acetone(olive oil (4:1) at concentrations of 25% and 50% (v/v). A further group of four animals was treated with acetone/olive oil (4:1) alone.

No mortality and no signs of systemic toxicity were noted in the test and the control animals during the test.

A slight dryness was registred at D6 in thre treated groups at 50% and 100%. A moderate to important increase in ear thickness (+16.5%, +29.4%, +41.7%) and in ear weight (+18%, +42.5%, +19.5%) was recorded respectively at the concentrations of 25%, 50% and 100%. The test item must therefore be considered as slightly irritant at 25% and 50% and irritant at 100%.

The SI calculated by pooled approach was 1.71, 2.76 and 3.17, for the treated groups at 25%, 50% and 100%, respectively.

In view of these results, under these experimental conditions, the itest item, Dermosoft DGMC.., must be classified in category 1B "skin sensitisation" according to the Globally Harmonized System (COM (2007)355 final). The signal word "warning" and the hazard statement H317 "may cause an allergic reaction" are required.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 8th to October 10th 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
LLNA study is presented in a nother endpoint
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: From sponsor; CIBA Inc.
- Batch No.of test material: 512051
- Expiration date of the lot/batch: December 2008
- Purity test date: certificate of analysis dated June 16, 2008
- pH: 7.15
- Colour: colourless
- Form: liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

The test item was considered as 100% for the study
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (F-69592 L'Arbresle)
- Females (if applicable) nulliparous and non-pregnant: not specified

- Age at study initiation: 4 weeks
- Weight at study initiation: 240-289 g
- Housing: Groups of two or three in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid. Each cage conains sawdust bedding which was changed at least 2 times a week. Each cage was installed in conventional air conditioned animal husbandry
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hr light): 12

Route:
intradermal
Vehicle:
olive oil
Concentration / amount:
3.125 %/ 0.2 mL (a pair of 0.1 mL each)
Day(s)/duration:
1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% / 0.5 mL
Day(s)/duration:
Given on day 7
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
6.25 and 3.125 %
Day(s)/duration:
On day 20 - 24 hours occlusion
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Group 1 (negative control): 5 animals
Group 2 (treated) : 11 animals
Details on study design:
RANGE FINDING TESTS:

Determination by intradermal injection of the Maximal Non Nocrotizing Concentration (MNNC)
- tested concentrations: 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100% in olive oil
- A macroscopic evaluation ofthe cutaneous reactions was conducted 24 hours later and 48 hours later if necessary

Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC)
- tested concentrations: 12.5, 25, 50 and 100% in liquid paraffin

Determination by topical application of the Maximal Non Irritant Concentration (MNIC)
- tested concentrations: 1.56, 3.125, 6.25, 12.5 in liquid paraffin

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and topical)
- Exposure period: 7 days
- Test groups: 11 animals
- Control group: 5 animals
- Site: scapular zone of animals
- Frequency of applications: 2 (Intradermal day 0 and topical day 7)
- Duration: The topical application was under occlusive dressing for 48 hours
- Concentrations: intradermal 3.125% and topical 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: 24 hours
- Test groups: 1
- Control group: 1
- Site: scapular zone of animals
- Concentrations: 3.125 and 6.25 %
- Evaluation (hr after challenge): 224, 58 and 72 hours

Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
6.25%
No. with + reactions:
11
Total no. in group:
11
Clinical observations:
None
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
3.125%
No. with + reactions:
9
Total no. in group:
11
Clinical observations:
None
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
6.25%
No. with + reactions:
4
Total no. in group:
11
Clinical observations:
None
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
3.125%
No. with + reactions:
2
Total no. in group:
11
Clinical observations:
None
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
6.25%
No. with + reactions:
1
Total no. in group:
11
Clinical observations:
None
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
6.25%
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
6.125%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
3.125%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
6.25%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Group:
positive control
Remarks on result:
not measured/tested
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The results obtained under these experimental conditions (OECD 406) enable to conlcude that the test item Dermosoft DGMC) is sensitising to skin according to the scales of interpretattion retained. In accordance with the Globally Harmonized System (COM(2007)355 final) the test item must be classified in category 1B “skin sensitisation”. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.
Executive summary:

The skin sensibilisating properties of test item, Dermosoft DGMC, was tested in accordance with OECD guideline 406. After induction (intradermic at 3.125% and topical application at 100%) of 11 guinea pigs of treated group with the test item and a 10 day rest phase, the challenge phase, under occlusive dressing for 24 hous, consisted to a single topical application of the test item diluted at 6.25% and 3.125% in liquid paraffin.

A slight to moderate erythema associated or not with a slight oedema was recorded, in 100% (11/11), 36% (4/11) and 9% (1/11) of the animals from group treated with 6.25%24, 48 and 72 hours, respectively, after the challenge phase.

A slight to moderate erythema associated or not with a slight oedema was recorded, in 82% (9/11) and 18% (2/11) of the animals from group treated with 3.125% 24 and 48 hours, respectively, after the challenge phase.

A slight to moderate erythema associated or not with a slight oedema was recorded, in 40% (2/5), 20% (1/5) and 20% (1/5) of the animals from control group to 6.25% treatment 24, 48 and 72 hours, respectively, after the challenge phase.

No cutaneous intolerance reaction was recorded in animals from the negative control group for the 3.125% treatment.

The results obtained under these experimental conditions (OECD 406) enable to conlcude that the test item Dermosoft DGMC) is sensitising to skin according to the scales of interpretattion retained. In accordance with the Globally Harmonized System (COM(2007)355 final) the test item must be classified in category 1B “skin sensitisation”. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Both the Guinea Pig Maximation Test and the Local Lymph Node Assay resulted in a positive response for skin sensitisation at a degree corresponding to a classification as Skin Sens.1B H317.