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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:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
;1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
OECD 406 test guideline was the original OECD guideline study for determining skin sensitization before the development of the LLNA. Study was conducted before the development of the LLNA.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SEC-BUTANOL
- Physical state: Colorless liquid
- Analytical purity: 99.87%
- Lot/batch No.: 9609P0515
- Expiration date of the lot/batch: October 1997
- Storage condition of test material: at room temperature and protected from light.

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: Centre d'Elevage Lebeau, 78950 Gambais, France
- Age at study initiation: approximately 3 months old
- Weight at study initiation: 335 ± 17 g for males and 332 ± 19 g for females
- Housing: individually housed in polycarbonate cages equipped with a polypropylene bottle.
- Diet (e.g. ad libitum): free access to "106 diet" (U.A.R., 91360 Villemoisson-sur-Orge, France).
- Water (e.g. ad libitum): Drinking water filtered by a F.G. Millipore membrane (0.22 micron) was provided ad libitum
- Acclimation period: at least 5 days


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


Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
Test substance was undiluted for challenge and cutaneous induction.
Test substance was at 5% (w/w) in paraffin oil for intradermal induction.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
Test substance was undiluted for challenge and cutaneous induction.
Test substance was at 5% (w/w) in paraffin oil for intradermal induction.
No. of animals per dose:
Control group: 5/sex
Test group: 10/sex
Details on study design:
RANGE FINDING TESTS:
Preliminary test:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.

By intradermal route:
24 hours before treatment, the dorsal region of the animals was clipped, the test substance was prepared in an appropriate vehicle, intradermal administrations of the test substance (0.1 ml) at different concentrations were performed in the dorsal region between the shoulders, cutaneous reactionswere evaluated approximately 24, 48 hours and six days after injection.

By cutaneous route:
24 hours before treatment, both flank regions of the animals were clipped, if necessary, the test substance was prepared in an appropriate vehicle, a quantity of 0.5 ml of the test substance undiluted was prepared on a dry gauze pad of approximately 4 square centimeters which was held in place by an occlusive dressing for 24 hours, cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings.

Criteria for selection of concentrations:
The following criteria were used:
the concentrations should be well-tolerated systemically and locally, intradermal injections should cause moderate irritant effect (no necrosis or ulceration of the skin), topical application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, topical application for the challenge should be the highest concentration which does not cause irritant effect.


MAIN STUDY: For all animals and before each treatment, the application sites were: clipped on days 1 to 7 (scapular area 4 cm x 2 cm), clipped and shaved on day 21 (each flank 2 cm x 2 cm).
A. INDUCTION EXPOSURE
FIRST INDUCTION WEEK, INTRADERMAL ROUTE
On day 1, six injections were made deep into the dermis of a clipped area in the dorsal region between the shoulders, using a needle (diameter: 0.50 x 16 mm) mounted on a 1 ml glass syringe.

Three injections of 0.1 ml were made into each side of this shoulder region, as follows:
Injection sites: Treated group:
Anterior 1: FCA diluted at 50% (v/v) with 0.9% NaCl
Middle 2: test substance at 5% (w/w) in paraffin oil
Posterior 3: mixture of 50/50 (w/v) of 1 and 2

Injection sites: Control group:
Anterior 1: FCA diluted at 50% (v/v) with 0.9% NaCl
Middle 2: vehicle
Posterior 3: mixture of 50/50 (w/v) of 1 and 2

FCA : Freund's complete adjuvant

SECOND INDUCTION WEEK, CUTANEOUS ROUTE
On day 7, the scapular area was clipped. As the test substance was shown to be non-irritant during the preliminary tests, the animals were treated with 0.5 ml of sodium lauryl sulfate (10% w/w) in vaseline in order to induce local irritation. On day 8, a topical application to the region of the intradermal injections was performed.

Control group:
application of 0.5 ml of the vehicle.
Treated group:
application of 0.5 ml of the test substance undiluted.

The test substance and the vehicle were prepared on a dry gauze pad (Coopetrative Pharmaceutique Fran¸caise, 77000 Melun, France), which was then applied to the dorsal region between the shoulders and held in place for 48 hours by means of an adhesive hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Me´dicales, 92240 Malakoff, France).
No residual test substance was observed after removal of the dressing.
Cutaneous reactions were recorded one hour after removal of the occlusive dressing.

B. CHALLENGE EXPOSURE
On day 22, the animals from both groups received an application of 0.5 ml of the test substance undiluted to the posterior right flank, and 0.5 ml of the vehicle to the posterior left flank. This application was performed using a 1 ml glass syringe (0.01 ml graduations, Record: Carrieri, 75005 Paris, France). The test substance and the vehicle were prepared on a dry gauze pad (Cooperative Pharmaceutique Francaise, 77000 Melun, France), then applied to a 4 square centimeter (2 cm x 2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France). No residual test substance was observed after removal of the dressing.

Challenge controls:
During the challenge period, the group was treated with the vehicle as well as with the test article to check the maximum subirritant concentration of the test article in adjuvant-treated animals.
Positive control substance(s):
yes
Remarks:
2,4-DINITRO CHLOROBENZENE

Study design: in vivo (LLNA)

Vehicle:
not specified
Details on study design:
Not applicable
Statistics:
Not applicable

Results and discussion

Positive control results:
The sensitivity of the guinea-pigs was checked with a positive sensitizer: 2,4-DINITRO CHLOROBENZENE (DNCB). During the induction period, the test substance was applied at 0.1% (w/w) (day 1) and 1% (w/w) (day 8). For the challenge application, the DNCB was applied to the right flank at a concentration of 0.5% (w/w).
Under the experimental conditions and according to the Magnusson and Kligman method, the test substance 2,4-DINITRO CHLOROBENZENE at a concentration of 0.5% (w/w) induced positive skin sensitization reactions in 50% of the guinea-pigs.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: undiluted. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: undiluted. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: undiluted. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: undiluted. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.5%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
1 very slight erythema; 1 well-defined erythema; 2 very slight erythema+dryness of the skin; 2 very slight erythema+dryness of the skin+crusts; 2 dryness of the skin; 1 very slight erythema+slight edema; 1 very slight erythema+slight edema+dryness of skin
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.5%. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: 1 very slight erythema; 1 well-defined erythema; 2 very slight erythema+dryness of the skin; 2 very slight erythema+dryness of the skin+crusts; 2 dryness of the skin; 1 very slight erythema+slight edema; 1 very slight erythema+slight edema+dryness of skin.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.5%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
1 well-defined erythema+dryness of the skin+crusts; 2 very slight erythema+dryness of the skin+crusts; 5 very slight erythema+dryness of the skin; 2 dryness of the skin
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.5%. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: 1 well-defined erythema+dryness of the skin+crusts; 2 very slight erythema+dryness of the skin+crusts; 5 very slight erythema+dryness of the skin; 2 dryness of the skin.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
positive control
Dose level:
0.5%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
3 very slight erythema+dryness of the skin+crusts; 1 very slight erythema+dryness of the skin; 6 dryness of the skin
Remarks on result:
other: see Remark
Remarks:
Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: positive control. Dose level: 0.5%. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: 3 very slight erythema+dryness of the skin+crusts; 1 very slight erythema+dryness of the skin; 6 dryness of the skin.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions attributable to thesensitization potential of the test substance SEC-BUTANOL (batch No. 9609P0515) were observed in guinea-pigs.