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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 October 05 to 29, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 406 without any deviation.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on September 22, 1999 / signed on January 18, 2000
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of study performance (1999), the LLNA was not the first choice method for skin sensitisation testing.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state: White solid (but some samples were received as liquid, due to stability in supercooled state)
- Storage condition of test material: At room temperature, protected from light and under nitrogen atmosphere


In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, 76410 Saint-Aubin-Ies-Elbeuf, France
- Age at study initiation: Approximately 3 months
- Weight at study initiation: Males: 344 ± 13 g; Females: 355 ± 12 g
- Housing: During the acclimatization period and throughout the study, the animals were housed individually in polycarbonate cages (48 cm x 27 cm x 20 cm)
- Diet: 106 pelleted diet (UAR, 91360 Villemoisson-sur-Orge, France), ad libitum
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 30-70 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: October 05, 1999 To: October 29, 1999

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
5% (w/w)
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50% (w/w)
Day(s)/duration:
Day 8 / 48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
25% (w/w)
Day(s)/duration:
Day 22 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 5 males + 5 females
Treated group: 10 males + 10 females
Details on study design:
RANGE FINDING TESTS: The choice of the concentrations was based on the results of a previous incomplete study on the same test substance (Study No. 18854 TSG), where the maximum injectable concentration was 10%, the concentration which induced irritation without necrosis was 5%, and topical applications of the substance at 50% concentration did not induce irritation during the preliminary assay (but did so after the challenge). The concentrations of the test substance retained for the present study were therefore 5% for intradermal injections (day 1), 50% for topical induction (day 8) and 25% for the topical challenge application (day 22).

MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: One
- Test groups: Intradermally injected with 3 injections (0.1 mL each) of FCA diluted at 50% (v/v) with 0.9% NaCl, test substance at 5% (w/w) in corn oil and test substance at 5% (w/w) in a mixture FCA /0.9% NaCl 50/50 (v/v) at anterior, middle and posterior sites, respectively on day 1.
- Control group: Intradermally injected with 3 injections (0.1 mL each) of FCA diluted at 50% (v/v) with 0.9% NaCl, corn oil and vehicle at 50% (w/w) in a mixture FCA /0.9% NaCl 50/50 (v/v) at anterior, middle and posterior sites, respectively on day 1.
- Site: Each side of the interscapular region

B. INDUCTION EXPOSURE: TOPICAL
- Day 7: As the test substance was shown to be non-irritant during the preliminary test, the animals were treated with 0.5 mL of sodium lauryl sulfate at the concentration of 10% (w/w) in vaseline, in order to induce local irritation.
- No. of exposures: One application, on Day 8
- Exposure period: 48 hours
- Test groups: On day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the test substance at the concentration of 50% (w/w) and was then applied to the interscapular region of the animals of the treated group, via occlusive dressing.
- Control group: On day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the vehicle alone and was then applied to the interscapular region of the animals of the treated group, via occlusive dressing.
- Frequency of applications: Single application

C. CHALLENGE EXPOSURE
- No. of exposures: One application
- Day(s) of challenge: Day 22
- Exposure period: 24 hours
- Test groups: The filter paper of a chamber (Finn Chamber®) was fully-loaded with the test substance at the concentration of 25% (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank.
- Evaluation (hr after challenge): 24 hrs and 48 hrs after removal of the challenge application

OTHERS:
Clinical examinations: The animals were observed at least once a day during the study in order to check for clinical signs and mortality.
Body weight: The animals were weighed individually on the day of allocation into the groups, on the first day of the study (day 1) and on the last day of the study (day 25).
Pathology: At the end of the study, all animals were killed by carbon dioxide asphyxiation. No necropsy was performed. No skin samples were taken.
Challenge controls:
None

Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole

Results and discussion

Positive control results:
The species and strain which were used showed a satisfactory sensitization response in 100% animals treated with Mercaptobenzothiazole (20 % w/w).

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% (w/w)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% (w/w)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No cutaneous reactions were observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20 % (w/w/)
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
dryness of the skin
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

No clinical signs and no mortality were observed during the study. The body weight gain of the treated animals was similar to that of the control animals. Only signs of irritation during induction were observed by intradermal injection on day 1.

 

After the challenge application, no cutaneous reactions were observed in the animals of the control and treated groups.

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
Under the test conditions, the test material did not induce delayed contact hypersensitivity in guinea-pigs. The test material is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and of the GHS.
Executive summary:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406/EU Method B.6 and in compliance with GLP, Dunkin-Hartley guinea pigs (10/sex) were induced with three pairs of intradermal injections (0.1 mL each) of:

1/ FCA diluted at 50% (v/v) with 0.9% NaCl,

2/ test substance at 5% (w/w) in corn oil [ highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically] maximal, and

3/ test substance at 5% (w/w) in a mixture FCA /0.9% NaCl 50/50 (v/v)

at anterior, middle and posterior sites of interscapular region, respectively on day 1. Similarly, control group (5/sex) was intradermally induced with 0.1 mL of FCA diluted at 50% (v/v) with 0.9% NaCl, corn oil and vehicle at 50% (w/w) in a mixture FCA /0.9% NaCl 50/50 (v/v). On day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation. On day 8, the test substance (50% (w/w - highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically) in corn oil) or the vehicle (control group) was applied to the same test site which was then covered by an occlusive dressing for 48 hours. On day 22, all animals of the treated and control groups were challenged by a cutaneous application of the test substance (25% (w/w, highest non-irritant concentration) in corn oil) to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 hours. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

 

No clinical signs and no deaths were noted during the study. After the challenge application, no cutaneous reactions were observed in the animals of the control and treated groups. The species and strain which were used showed a satisfactory sensitization response in 100% animals treated with Mercaptobenzothiazole.

 

Under the test conditions, the test material is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and of the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.