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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TKA 40135 (CGI 819)

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Age at study initiation: 4 to 5 weeks
- Weight at study initiation: 325-378 g
- Housing: groups of five in suspended metal cages with wire mesh floors
- Diet (e.g. ad libitum): guinea-pig diet FD2 enriched with vitamin C, ad libitum; hay was additionally given weekly
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 29 °C
- Humidity (%): 38 - 64 %
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: acetone and acetone in Alembicol D
Concentration / amount:
Induction, intradermal injection: 1% w/v in 5% acetone in Alembicol D
Induction, topical application: 70% w/v in acetone
Challenge, topical application: 70 and 35% w/v in acetone
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: acetone and acetone in Alembicol D
Concentration / amount:
Induction, intradermal injection: 1% w/v in 5% acetone in Alembicol D
Induction, topical application: 70% w/v in acetone
Challenge, topical application: 70 and 35% w/v in acetone
No. of animals per dose:
Control animals were 5/group
Test animals were 10/group
Details on study design:
As the Sponsor advised that the test substance is light sensitive, the test formulations were prepared under safelight and the formulation containers wrapped in aluminium foil. Aluminium foil was incorporated in the dressings to minimise photoinduced degradation ofthe test material. The test substance was prepared prior to each application on the day of dosing in 5% acetone in Alembicol D" (product of coconut oil) and acetone alone.

RANGE FINDING TESTS
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.
Animals for these investigations were pre-treated with an intradermal injection of Freund's complete adjuvant, 1:1 with water for irrigation, approximately two weeks prior to the start of the preliminary investigations. The concentrations of TKA 40135 (CGI 819) used for the main study were selected based on the results of these preliminary investigations.

MAIN STUDY
A. INDUCTION EXPOSURE
Induction intradermal injections
The control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injection.
A 40 x 60 mm area of dorsal skin on the scapular region of each test animal was clipped free of hair. Three pairs of intradermal injections were made into a 20 x 40 mm area within the clipped area:
- Freund's complete adjuvant diluted 1:1 with water
- Test material 1%, w/v in 5% acetone in Alembicol D
- Test material 1% w/v in a 1:1 mixture of Freund's complete adjuvant and 5% acetone in Alembicol D

Induction topical application
The control animals were treated similarly to the test animals with the exception that the test substance was omitted from the topical application.
Six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.5 ml per site of 10% w/w sodium lauryl
sulphate in petrolatum. After 24 hours, a 20 x 40 mm patch saturated with ca. 0.4 mL of 70% test material in acetone (w/v) was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape. This in turn was firmly secured by elastic adhesive bandage wound round the torso of the animal and fixed with impervious plastic adhesive tape. The dressing was left in place for 48 hours.

B. CHALLENGE EXPOSURE
The control and test animals were challenged topically two weeks after the topical induction application; the test concentrations were 70 and 35% w/v in acetone. Hair was removed by clipping and then shaving from an area on the left flank of each animal. A 20 x 20 mm patch was saturated with approximately 0.2 mL of 70% test material in acetone and applied to an anterior site on the flank. The test material at a concentration of 35% in acetone was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours and the dressing was secured as described for the topical induction.

EXAMINATIONS
The dermal reactions were assessed using the following numerical system:
Erythema and eschar formation:
No erythema 0
Slight erythema 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Edema formation:
No edema 0
Slight edema 1
Well-defined edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 mm) 3
Severe edema (raised more than 1 mm and extending beyond the area of exposure) 4

INTERPRETATION OF RESULTS
Dermal reactions in the test animals elicited by the challenge application were compared with the findings simultaneously obtained in the control animals.
A test animal was considered to show positive evidence of delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals of the control group.
If the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result for that test animal was classified as inconclusive.
A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than the maximum reaction seen in animals of the control group.

Moreover, all animals were observed daily for signs of ill health or toxicity, and body weight gain was assessed.
Positive control substance(s):
yes
Remarks:
The sensitivity of the guinea-pig strain used is checked periodically at the testing facilities with known sensitisers hexyl cinnamic aldehyde (HCA), Benzocaine and mercaptobenzothiazole (MBT).

Results and discussion

In vivo (non-LLNA)

Results
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
70% test material in acetone
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
No signs of ill health or toxicity were recorded; body weight gain was as expected.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 70% test material in acetone. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: No signs of ill health or toxicity were recorded; body weight gain was as expected. .

Any other information on results incl. tables

Dermal reactions seen following the induction injections included necrosis at sites receiving Freund's Complete Adjuvant in test and control animals, slight irritation in test animals at sites receiving the test material (% w/v in 5% acetone in Alembicol D), and slight irritation in control animals receiving 5% acetone in Alembicol D.

Induction by topical application resulted in slight erythema in test animals treated with the test material (70% w/v in acetone) as well as slight erythema in the control animals.

After challenge, the dermal reactions seen for three of the ten test animals were more marked than those seen for the controls and, therefore, these animals gave positive responses. A further four animals gave inconclusive responses and the remaining three animals gave negative responses.

Applicant's summary and conclusion