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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test

Test material

Constituent 1
Chemical structure
Reference substance name:
1-benzyl-3-carboxylatopyridinium sodium chloride
EC Number:
268-692-9
EC Name:
1-benzyl-3-carboxylatopyridinium sodium chloride
Cas Number:
68133-60-8
Molecular formula:
C13H12NO2.Cl.Na
IUPAC Name:
1-benzyl-3-carboxylatopyridinium sodium chloride
Details on test material:
- Name of test material (as cited in study report): LUGALVAN BPC dry
- Physical state: solid
- Analytical purity: 95.5 area-%
- Lot/batch No.: 467602
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CHARLES RIVER (F-69592 L’ARBRESLE)
- Age at study initiation: 4 weeks
- Weight at study initiation: 229 - 282 g
- Housing: housed in groups of 2
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): at least 10 cycles per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
70% in distilled water (induction); 50% in distilled water (challenge)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
70% in distilled water (induction); 50% in distilled water (challenge)
No. of animals per dose:
10 animals (controls), 20 animals (test group)
Details on study design:
Preliminary study
Maximum Non Irritant Concentration (M.N.I.C.) determination:
This test was carried out with a reduced animal’s number, for the purpose of determining the maximal item concentrations without risk of an irritant effect during the challenge phase.
Furthermore, this test evaluates the irritant potential of the test item, and defines, if possible, a mild to moderate irritant concentration during topical induction phase.
Three guinea pigs were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-layer Gazin® from Lohmann & Rauscher 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: diluted at 70% (w/w), 50% (w/w), 25% (w/w) and 10% (w/w) in distilled water.
The animals treated at the concentrations of 70% (w/w), 50% (w/w), 25% (w/w) and 10% (w/w) 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. The skin reaction was observed and recorded.

Main study
GROUP 1 (negative control) : 10 female guinea pigs identified n° C1399 to C1408
GROUP 2 (treated) : 20 female guinea pigs identified n° C1409 to C1428

Induction phase
After shearing of the scapular zone, the 3 local applications were performed at D0, D6 and D13 during 6 hours under occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-layer Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M).
The animals of the control group received 0.5 mL of distilled water and the animals of the treated group received 0.5 mL of the test item at 50% diluted in distilled water.
After patch removal, the treated areas were rinsed with distilled water (test vehicle).

Rest phase
The animals of both groups were left untreated for 13 days.

Challenge phase
The experimental procedure of this phase was identical for both groups Group 1 (Negative control) and Group 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-layer Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M), was performed during 6 hours:
- 1 area containing 0.5 mL of the test item at 50% (w/w) (MNIC = maximal non irritant concentration) on the left flank and one area containing 0.5 mL of the vehicle (distilled water) on the right flank.

Macroscopic examinations and evaluation of cutaneous reactions
A macroscopic evaluation of the cutaneous reactions (erythema and oedema) was conducted and all the local or systemic reactions were recorded as Group 1 (Negative control) and Group 2 (Treated):
• Approximately 24 hours after removal of the occlusive dressing, the cutaneous reactions were observed and graded according to the scales, given below.
• Approximately 24 hours later (i.e. 48 hours after removal of the occlusive dressing), a second observation were made.
Grading scales:
Erythema
0 No visible modification
1 Slight or patches of erythema
2 Moderate confluent erythema
3 Intense erythema

Oedema
0 No visible modification
1 Slight oedema
2 Moderate oedema
3 Severe oedema

All the animals with scores above or equal to 1 (erythema or oedema) during the challenge phase, were considered positive.
Challenge controls:
Peridodic positive controls (Reference substance: α-Hexylcinnamaldehyde CAS n° 101-86-0 - Tests n°12-14) were carried out as method sensibility.
Positive control substance(s):
yes
Remarks:
α-Hexylcinnamaldehyde

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Concentrations selected

Preliminary study:

- MNIC determination:

24 hours after removal of the occlusive dressings, slight erythema was noted in one animal (1/3) at the concentration of 70%. 24 and 48 hours after removal of the occlusive dressings, dryness was noted in all animals (3/3) at the concentration of 70%.

24 and 48 hours after removal of the occlusive dressings, no cutaneous reaction was noted at the concentration 50%, 25% and 10%.

In view of these results, the concentration selected was 70% for the 3 inductions of the main study and the concentration selected was 50% (MNIC) for the challenge phase.

Main study:

- Induction phase

The induction phase of the treated group was performed by topical application on D0, D6 and D13 with 0.5 mL of the test item diluted at 70% in distilled water.

No cutaneous reaction was recorded during the induction phase in the treated group.

The induction phase of the control group was performed by topical application on D0, D6 and D13 with 0.5 mL of distilled water.

No cutaneous reaction was recorded during the induction phase in the control group.

- Challenge phase:

The challenge phase was performed by topical application of 0.5 mL of the test item at 50% during 6 hours on the left flank of both groups. On the other side (right flank) 0.5 mL of the vehicle (distilled water) was also applied during 6 hours.

Interpretation of reactions

The percentage of animals that showed a sensitivity contact potential is calculated 24, 48 and 72 hours after the removal of the occlusive dressings.

A comparison of the intensities and persistence of reactions at the test item challenge sites in the test and control animals permits identification of sensitisation reactions.

If the test item at the maximum non-irritant concentration produces reactions in test group animals at the 24 48 or 72-hour readings, these reactions were attributed to skin sensitisation.

This pre-supposes that no similar reactions were observed in the test material challenge sites of any of the control group animals. If irritation was observed in the control group animals, only reactions in the test group animals that exceed the most severe reaction seen in the control group animals were attributed to skin sensitisation. The results were expressed in terms of incidence and severity of responses, ie:

Incidence Score: The number of test group animals showing skin reactions greater than the most severe reaction observed in the control group animals, expressed as a fraction of the total number of test group animals.

Severity Score: The sum of the values assigned to the skin responses at the test item challenge sites of the test and control group animals, at each evaluation, divided by the number of animals in that group.

Sensitising potential assessment

No macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of the occlusive dressing (challenge phase) from the animals of the treated group with the test item at 50%.

No cutaneous intolerance reaction was recorded in animals from the negative control group.

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.

CONCLUSION

In view of these results, under these experimental conditions, the test item LUGALVAN BPC dry shows no skinsensitisation potential.

Applicant's summary and conclusion