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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:
key study
Study period:
From 2010-06-22 to 2010-08-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
other: Draft study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
as the OECD 429 guideline states, the LLNA test is known to cause false positive with surfactants

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Expiration date of the lot/batch: 11 February 2011
- Storage conditions of test material: at room temperature, protected from light and under nitrogen gas in a dry and well-ventilated room.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: on the first day of treatment, the animals of the main test were 1-2 months old
- Weight at study initiation: a mean body weight ± standard deviation of 349 ± 17 g for the males and 345 ± 15 g for the females
- Housing: housed individually in polycarbonate cages with stainless steel lid (43 cm x 22 cm x 20 cm). Autoclaved sawdust were placed in each cage
- Diet (e.g. ad libitum): free access to 106 pelleted diet
- Water (e.g. ad libitum): water (filtered with a 0.22 µm filter)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 220+/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: From: 22 June 2010 To: 07 August 2010.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: intradermal: corn oil / topical: acetone
Concentration / amount:
Induction phase:
Concentration for intradermal injection: 5% *
Concentration for topical application: 50% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 0.1%**
** highest non-irritant concentration based on preliminary assays
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: intradermal: corn oil / topical: acetone
Concentration / amount:
Induction phase:
Concentration for intradermal injection: 5% *
Concentration for topical application: 50% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 0.1%**
** highest non-irritant concentration based on preliminary assays
No. of animals per dose:
Number and sex:
- preliminary test: 14 animals (6 males and 8 females),
- main test: 30 animals (15 males and 15 females = for vehicle group: 5 males and 5 females + for treated group: 10 males and 10 females)
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: topical induction lasted 48h
- Site: induction: interscapular region
- Frequency of applications: once intradermal, once topical

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 4 weeks between injection (induction) and challenge
- Exposure period: 24h
- Site: right flank (test item) and left flank (vehicle)
- Evaluation (hr after challenge): 24, 48, 72 after removal of dressing
Positive control substance(s):
yes
Remarks:
mercaptobenzothiazole (within a 6-month interval of the test)

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: 1st reading, 24 or 48h (combined) after challenge
Group:
negative control
Dose level:
0.1%
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: other: 1st reading, 24 or 48h (combined) after challenge. Group: negative control. Dose level: 0.1%. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
0.1%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Dryness of skin, 10/10 animals. Reaction is irritation and not sensitisation.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0.1%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Dryness of skin, 10/10 animals. Reaction is irritation and not sensitisation..
Reading:
other: 1st reading, 24 or 48h (combined) after challenge
Group:
test group
Dose level:
0.1%
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
Dryness of skin at 48h, 5/20 animals. Reaction is irritation and not sensitisation.
Remarks on result:
other: see Remark
Remarks:
Reading: other: 1st reading, 24 or 48h (combined) after challenge. Group: test group. Dose level: 0.1%. No with. + reactions: 17.0. Total no. in groups: 20.0. Clinical observations: Dryness of skin at 48h, 5/20 animals. Reaction is irritation and not sensitisation..
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
0.1%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
Dryness of skin in 15/20 animals. Its severity masked the skin reading in 5/20 animals. Reaction is irritation and not sensitisation.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 72.0. Group: test group. Dose level: 0.1%. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: Dryness of skin in 15/20 animals. Its severity masked the skin reading in 5/20 animals. Reaction is irritation and not sensitisation..

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under these experimental conditions, the test itemdid not induce delayed contact hypersensitivity in guinea pigs.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and Commission Regulation (EC) (No. 440/2008, B.6, 30 May 2008) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

 Thirty guinea-pigs were allocated to 2 groups: a control group of 5 males and 5 females and a treated group of 10 males and 10 females.

 On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

.            Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups),

.            test item at the concentration of 5% in corn oil (treated group) or vehicle alone (control group),

.            test item at the concentration of 5% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v)

in a mixture FCA/0.9% NaCl (50/50, v/v) (control group).

 

On day 8, the treated group animals received a topical application of the test item at the concentration of 50% (w/w) in acetone to the same test site, which was then covered by an occlusive dressing for 48 hours. The control group animals received an application of the vehicle under the same experimental conditions.

 

On day 29, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 0.1% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions.

Cutaneous reactions were evaluated in each animal 24, 48 and 72 hours after removal of the dressing.

 

Each animal was observed at least once a day for mortality and clinical signs during the treatment and observation periods. Body weight was recordedon the day of allocation into the groups, on day 1 and day 25.

On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination. Skin samples were taken each flank (challenge application sites) of all animals. No histological examination was performed.

 

No unscheduled mortality and no clinical signs were recorded in any animals. After the challenge application of the test item, a moderate erythema was noted on the right flank (test item application) of 1/10 animals at the 72-hour reading. A discrete erythema was observed on the right flank of 6/10, 5/10 and 0/10 animals of the control group at the 24, 48 and 72-hour readings, respectively. Dryness of the skin was also recorded in 6/10 animals. No cutaneous reactions were noted on the left flank (vehicle application) of the control animals during the whole challenge phase.

In the treated group, a moderate erythema was recorded on the right flank (test item application) of 4/20 and 1/20 animals at the 48 and 72-hour reading. A discrete erythema was noted on the right flank of 13/20, 11/20 and 3/20 animals at the 24, 48 and 72-hour readings, respectively. Dryness of the skin was recorded in 5/20 and 15/20 animals at the 48 and 72-hour readings, respectively. A severe dryness of the skin masked the evaluation of cutaneous reactions in 5/20 animals at the 72-hour reading. No cutaneous reactions were noted on the left flank (vehicle application) of the treated animals.

 

A higher incidence and severity in local reactions was noted on 20% of the animals of the treated group at the 48-hour reading. However, as the cutaneous reactions observed at the 24 and 72-hour readings in the animals of the treated group were of similar incidence and severity when compared to those recorded in the animals of the control group, they were attributed to the irritant properties of the test item but not to delayed contact hypersensitivity.

 

Therefore, under the experimental conditions of this study,the test item did not induce delayed contact hypersensitivity in guinea pigs.