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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 report, compareble to OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
see principles
Principles of method if other than guideline:
This method, inspired by a method published by Magnusson and Kligman, is used to evaluate, according to a maximised protocol, the cutaneous, allergenic or sensitizing potential of a test article in the albino guinea pig by epicutaneous applications under an occlusive patch and using Freund's complete adjuvant administered by the intradermal route.
This method differs from that of Magnusson and Kligman in four essential characteristics :
a) The test article is applied as supplied for induction and is not mixed with Freund's adjuvant, this avoids any possible incompatibility.
b) It is not applied by the intradermal route, but by the epicutaneous route under an occlusive patch, i.e. by the route of administration recommended for man. Moreover, this protocol is applicable to test articles which have liquid, semi-liquid, paste, pulverised or solid presentations.
c) Each study animal is used as its own control, in order to eliminate risks of interpretation errors associated with differences in individual reactions.
d) The results of the positive and doubtful macroscopic observations are systematically completed by histopathological examination of the application area in order to :
- improve the accuracy of the study by facilitating the determination of the origin (allergic or orthoergic) of the macroscopic lesions observed ;
- obtain documents which may be reexamined, compared and archived.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Testing according to OECD 406 has been performed in 1990; back then LLNA has not been an official OECD method.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: Echantillon ref. 423683
Identification for the study : 07672 BO 002
Presentation white fine powder
Volumic mass = 0.8048 g/ml, considered as 0.80 g/ml for the study
Conditions of measurement : weighing of 1 ml of the test article in a callipered plastic tube on a Mettler AE 200 balance (d = 0.1 mg) - T = 19 °C.
Packaging : plastic container
Quantity and date received : about 250 g on 22 February 1990
Storage : at room temperature, in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Reason for the choice of species : the guinea-pig is recognized as the animal species most sensitive for the evaluation of delayed hypersensitivity by the cutaneous route. This species has been tradition-ally used for this type of study and is chosen by the regulatory authorities.
- Supplier : Elevage Lebeau (78950 Gambais - France)
- Age : young adult.
- Weight at the beginning of treatment : preliminary study : 390 g to 395 g ; -main study: 330 g to 500 g (the weights are recorded in appendix).
- Number and sex: preliminary study : 2 males, 2 non-pregnant females; main study: 10 males, 10 non-pregnant females.
Two complementary guinea pigs (1 male and 1 female) were treated in a similar way. as replace-ments for any possible non-treatment related deaths.

Cages: housed by sex and in groups of 5 or 6 (or 2 in the preliminary studies) in polystyrene cages with perforated flooring (internal dimensions: 560 x 355 x 315 mm).
- Temperature: 20+/- 3°C
- Humidity 30 to 70 % R.H.
- Lighting artificial, 12 hours out of 24 (photoperiod = 7h30 - 19h30).

Diet: complete pelleted guinea-pig maintenance diet, ad libitum (U.A.R. formula 114 - U.A.R., Vil-lemoisson - 91360 Epinay S/Orge - France).
- Water filtered (5 pm) and softened mains drinking water, ad libitum (automatic watering). Bacterio-logical and chemical analysis twice a year.

- Acclimatisation period :
preliminary study : 13 days before the beginning of treatment; main study : 18 days before the beginning of treatment.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.40 g and 0.20 g per animal of the test article. The test article was applied once only.
Methods of administration : the 2 dose levels were applied to the same animals, to the•clipped skin of the dorsal region and under an patch for 48 hours, and according to the methods of application.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.40 g and 0.20 g per animal of the test article. The test article was applied once only.
Methods of administration : the 2 dose levels were applied to the same animals, to the•clipped skin of the dorsal region and under an patch for 48 hours, and according to the methods of application.
No. of animals per dose:
20
Details on study design:

- Verification of the absence of individual reaction: application of the test article at the maximum dose level or concentration recognized as non-irritant during the preliminary study.
- Induction period: during which :”preparatory” or “sensitizing” contacts are made between the organ-ism and allergen. This engages the allergic process without provoking any clinical manifestation of hypersensitivity. The following procedures are performed on the clipped skin of the 20 guinea-pigs :
-- 1 intradermal injection of Freund's complete adjuvant
-- 7 consecutive applications of the test article as supplied by the enclosed epicutaneous route for 48 or 72 hours at the site of the intradermal injection.
- Rest period: or incubation period during which possible cellular transformations continue which brings about a change in reactivity.
12 days without treatment.
-"Challenge" phase : corresponding to the contact between the organism and the sensitizing agent, which provokes clinical manifestation of hypersensitivity :
--application of the test article by the enclosed epicutaneous route for 48 hours to an area not previ-ously treated, at the dose concentration which did not provoke a pathological orthoergic reaction.
The sensitization reaction is evaluated by macroscopic examinations and in certain cases by histopa-thological examinations of the cutaneous lesions observed 6 and possibly 24 and 48 hours after re-moval of the challenge application occlusive patch, comparison is made with the 1st application in the same animal (corresponding to the verification of the absence of individual reaction) .


Experimental design for the evaluation of sensitizing potential:
Induction
a) Intradermal injection of Freund's complete adjuvant :
Injection area : upper clipped costal region, immediately behind the right scapulum.
- Time : Day 1
- Methods of administration : one intradermal injection with a 1 ml sterile syringe, of a.l. ml of Freund's adjuvant diluted to 50 %(v/v) in an isotonic injectable solution (0.9 %NaCl).
b) Topical application of the test article :
- Application area: to the site of the intradermal injection of Freund's adjuvant.
- Administration time: Days 1, 3, 5, 8, 10, 12 and 15.
- Dose level : 0.40 g per animal of the test article as supplied.
- Reason for the choice of the dose level : it is the maximum dose level which can be applied under the occlusive patch.
- Methods of application : to the skin clipped on Day 1, under the occlusive patch as previously described
- Length of exposure: 48 hours (Days 1, 3, 8. 10 and 15) or 72 hours (Days 5 and 12).

Rest period
The animals were not subjected to treatment between Days 17 to 29, i.e. for a period of 12 Days.

"Challenge" application
Application area : left lateral abdominal region, not previously treated.
- Administration time : Day 29
- Dose level : 0.40 g per animal of the test article as supplied, quantity corresponding to the maximum non-irritant dose level determined during the
preliminary study.
- Methods of application: to the clipped skin and under the occlusive
patch previously described.
This patch was held in position by means of an adhesive cotton band 4 cm
wide which was applied onto a Codex hydrophilic gauze which covered the
whole of the clipped area to avoid any possible irritation reactions due to
this band.
- Length of exposure 48 hours.
Challenge controls:
not reported
Positive control substance(s):
no

Results and discussion

Positive control results:
not applicable

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
6
Group:
test group
Dose level:
400 mg
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 6.0. Group: test group. Dose level: 400 mg. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
400 mg
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 400 mg. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
other: 3rd reading
Hours after challenge:
48
Group:
test group
Dose level:
400 mg
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 400 mg. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The substance showed no signs of skin sensitization within the tested Guinea pig.
Executive summary:

Evaluation of the cutaneous delayed hypersensitivity of beta-cyclodextrin was performed in the albino Dunkin-Hartley guinea-pig, according to a maximized protocol using one treated group of 20 animals of both sexes according to a guideline equivalent to the Magnusson Kligman maximation test in OECD Guideline 406. The 7 applications corresponding to the induction phase and the challenge application were conducted with the test article as supplied and at. the dose level of 0.40 g per guinea-pig (quantity corresponding to a volume of 0,5 ml) under an occlusive patch moistened with 0.5 ml of purified water. Macroscopic cutaneous examinations were performed according to the Draize scale to the challenge application site, 6, 24 and 48 hours after removal of the occlusive patches. Histopathological examinations of the skin were carried out in the6guinea-pigs showing doubtful reactions at 6 hours. Macroscopic and histopathological examinations did not reveal any lesion of "delayed hypersensitivity with cell mediation" type in the guinea-pigs. From the results obtained under the experimental conditions employed, the test article did not provoke any reaction of cutaneous sensitization.