Registration Dossier

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: GLP study performed according to the Test Guideline B.6 of the EEC Directive 84/449/EEC and OECD Guideline for testing of chemicals n°406

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
A c. 700 g sample of the test substance was received from the sponsor on
September 19, 1988. It consisted of a fine, white powder, designated:
“AF—366”.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: albino (Bor:DHPW)
Sex:
male/female
Details on test animals and environmental conditions:
Young, SPF bred, albino guinea pigs (Bor:DHPW) were obtained from
F. Winkelmann, Institute for the Breeding of Laboratory Animals GmbH and
Co. KG, Borchen, F.R. Gerniany. During an acclimatization period of 8 days,
and throughout the test period, the animals were kept individually under
conventional conditions in suspended, stainless steel cages, fitted with
wire mesh floor and front in an animal room conditioned with respect to
temperature (21 ± 2°C), relative humidity (at least 40%), ventilation (at
least 10 air changes/hour) and lighting (12 hours light/12 hours dark
cycle). The animals were fed a pelleted, natural ingredient diet (Hope
Fans, Woerden, the Netherlands) and received unfluoridated tap water ad
libitum. Preliminary tests (to establish the concentrations of the test
substance to be used in the main study) were conducted on animals which had
already served as controls in a previous sensitization test, or with
animals never used before. The main study was carried Out with 15 males
(body weight 323—410 g) and 15 females (body weight 283—332 g) without
visible skin abnormalities. The animals ware received on February 14, 1989.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: maize oil and vaseline
Concentration / amount:
induction treatment by intradermal injections of
Freund’s Complete Adjuvant (FCA), a 10% dilution (w/w) of the test
substance in maize oil, and a 10% dilution (w/w) of the test
substance in a mixture (1:1) of FCA and maize oil
Intradermal induction: 10% (w/w)
Epicutaneous induction: 50%(w/w)
Challenge: 25 and 50% (w/w)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: maize oil and vaseline
Concentration / amount:
induction treatment by intradermal injections of
Freund’s Complete Adjuvant (FCA), a 10% dilution (w/w) of the test
substance in maize oil, and a 10% dilution (w/w) of the test
substance in a mixture (1:1) of FCA and maize oil
Intradermal induction: 10% (w/w)
Epicutaneous induction: 50%(w/w)
Challenge: 25 and 50% (w/w)
No. of animals per dose:
20 test animals and 10 controls
Details on study design:
The experiment was conducted essentially according to the guinea pig
maximization test as described by B. Magnusson and A.M. Kligman (1969 and
1970), and according to:
— EEC—Directive 84/449, Annex V, Part B: Methods for the determination of
toxicity, B.6. Acute toxicity, skin sensitization, dated September 1984,
— the QECD Guidelines for Testing of Chemicals, nr. 406, adopted 12 May
1981.
The study consisted of an induction treatment, followed by a resting period
of 14 days, which preceded the challenge treatment.
Preliminary observations were made to establish the concentrations of the
test substance to be used for intradermal injection and for topical
application in the main study.
RANGE FINDING TESTS:
Prior to the start of the main study, the intradermal and epidermal irritancy of AF-366 was investigated to select test substance concentrations suitable for the induction and challenge phase of the main study. The selection was based on the absence of toxicity and on the following criteria for each route and/or study phase:

Induction (intradermal and epidermal): The highest possible concentration that produced slight to moderate irritation.

Challenge: The maximum non-irritant concentration.

The test system, procedures and techniques were identical to those used during the main study, unless otherwise specified.

Intradermal injections:
0.1 mL of a 1, 5 and 10% (w/w) dilution of the test substance in maize oil was applied to each of 3 animals by intradermal injection in the clipped scapular (shoulder) area. The highest concentration was the maximum concentration that could technically be injected, as higher concentrations solidified in the syringe. The injection sites were assessed for irritation 24 hours after treatment. Slight erythema with or without edema was observed for the 1, 5 and 10% solutions.

Epidermal application:
A series of 3 test substance concentrations (10, 20 and 50%) was used. The 50% solution was the maximum concentration that could technically be applied, as higher concentrations formed very dry and tough pastes. Both flanks of each of 3 animals were clipped free from hair with electric clippers. Subsequently, cups (Finn chamber large, Epitest Ltd., Oy, Finland) were loaded with the various test solutions. The cups were placed on separate areas of the clipped skin ((the scapular area in the main study) and covered with a piece of hypoallergenic paper bandage (Leukopor) that was secured by an elastic adhesive bandage (Tensoplast 7.5 cm wide) wound around the torso of the animal. The dressing was left in place for 24 hours. After removal of the dressing and 24 hours later, the animals were examined for signs of skin irritation. The 10 and 25% solutions did not cause skin irritation, while the 50% solution induced very slight erythema that had cleared within one day. A 25 and 50% solution were selected for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, intradermal and epicutaneous
- Exposure period: single injection (intradermal) and 48 hours (epicutaneous)
- Test groups:
Intradermal, day 1 (3 pairs of injections, 0.1 mL/site):
Injection 1: FCA
Injection 2: 10% (w/w) test substance in maize oil
Injection 3: 10% (w/w) test substance in maize oil in a 1:1 mixture with FCA
24 h after intradermal injection (day 2), the skin reactions were assessed.

Adjuvant treatment, day 7: Six days after the intradermal injections, the dorsal skin in the scapular region of all treated animals was closely shaved again and subsequently treated with a 10% dilution of sodium lauryl sulfate in vaseline (open application) to induce skin irritation.

Epicutaneous, day 8:
50% (w/w) test substance in vaseline
The occlusive dressing was kept in place for 48 h. The degree of erythema and edema was evaluated directly after cleaning the skin area (day 10) and 24 hours later (day 11).

- Control group:
Intradermal, day 1 (3 pairs of injections, 0.1 mL/site):
Injection 1: FCA
Injection 2: maize oil
Injection 3: maize oil in a 1:1 mixture with FCA

Adjuvant treatment, day 7: Six days after the intradermal injections, the dorsal skin in the scapular region of all control animals was closely shaved again and subsequently treated with a 10% dilution of sodium lauryl sulfate in vaseline (open application) to induce skin irritation.

Epicutaneous, day 8: vaseline

- Site: the scapular region
- Frequency of applications: once (intradermal on day 1 and epicutaneous on day 8)
- Duration: day 1 (intradermal), day 8-10 (epicutaneous)
- Concentrations: 10% (intradermal), 50% (epicutaneous)

B. CHALLENGE EXPOSURE
- No. of exposures: one (no rechallenge)
- Day(s) of challenge: 24
- Exposure period: 24 h
- Test groups: test substance in vaseline (left flank) and vaseline alone (right flank)
- Control group: test substance in vaseline (left flank) and vaseline alone (right flank)
- Site: on one flank of the animals
- Concentration: 25 and 50% (w/w)
- Evaluation (hr after challenge): 48 and 72 h

OTHER:
the intradermal injections No. 1 and 3 listed above were performed with FCA, where the OECD guideline 406 specifies the use of a 1:1 mixture with FCA and water/ physiological saline.
Challenge controls:
The topical challenge was carried out two weeks after the topical induction
as follows:
An area of 5 x 5 cm on the left and right flank of each test and control
animal was clipped free from hair. Subsequently, cups were loaded with a
25% or a 50% dilution (w/w) of the test substance in vaseline or with
vaseline alone. The cups with the 25% and 50% test dilution were placed
separately on the shaved area of the left flank of each test and control
animal. The cup with vaseline alone was placed on the shaved area of the
right flank of each test and control animal. The cups were covered with
Leukopor bandage, and held in place by Tensoplast for 24 hours. Skin
readings were made at 24 and 48 hours after removal of the cups.
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene (DNCB) in vaseline

Results and discussion

Positive control results:
A reliability check is carried out at regular intervals with DNCB to check the sensitivity of the test system and the reliability of the experimental methods as used by the test laboratory. No justification for the use of a different positive control substance from those recommended in OECD guideline 406 was given. In an independent study performed in June 1988, DNCB induced sensitisation in 100% (10/10) of the animals challenged with a 0.05% solution in vaseline. None of the control animals had a positive reaction. A 0.1% solution in maize oil was used for intradermal induction, and a 0.1% solution in vaseline for topical induction.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% dilution (w/w) of "AF-366" in vaseline
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
The challenge treatment with vaseline alone, did not induce skin reactions in any of the test animals or controls. The chalienge treatment with the 25% and 50% test dilution in vaseline, did not induce skin reactions in any of the test animals or controls
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% dilution (w/w) of "AF-366" in vaseline. No with. + reactions: 0.0. Total no. in groups: 19.0. Clinical observations: The challenge treatment with vaseline alone, did not induce skin reactions in any of the test animals or controls. The chalienge treatment with the 25% and 50% test dilution in vaseline, did not induce skin reactions in any of the test animals or controls.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% dilution (w/w) of "AF-366" in vaseline
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
The challenge treatment with vaseline alone, did not induce skin reactions in any of the test animals or controls. The chalienge treatment with the 25% and 50% test dilution in vaseline, did not induce skin reactions in any of the test animals or controls
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% dilution (w/w) of "AF-366" in vaseline. No with. + reactions: 0.0. Total no. in groups: 19.0. Clinical observations: The challenge treatment with vaseline alone, did not induce skin reactions in any of the test animals or controls. The chalienge treatment with the 25% and 50% test dilution in vaseline, did not induce skin reactions in any of the test animals or controls.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% dilution (w/w) of "AF-366" in vaseline
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
The challenge treatment with vaseline alone, did not induce skin reactions in any of the test animals or controls. The chalienge treatment with the 25% and 50% test dilution in vaseline, did not induce skin reactions in any of the test animals or controls
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% dilution (w/w) of "AF-366" in vaseline. No with. + reactions: 0.0. Total no. in groups: 19.0. Clinical observations: The challenge treatment with vaseline alone, did not induce skin reactions in any of the test animals or controls. The chalienge treatment with the 25% and 50% test dilution in vaseline, did not induce skin reactions in any of the test animals or controls.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% dilution (w/w) of "AF-366" in vaseline
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
The challenge treatment with vaseline alone, did not induce skin reactions in any of the test animals or controls. The chalienge treatment with the 25% and 50% test dilution in vaseline, did not induce skin reactions in any of the test animals or controls
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% dilution (w/w) of "AF-366" in vaseline. No with. + reactions: 0.0. Total no. in groups: 19.0. Clinical observations: The challenge treatment with vaseline alone, did not induce skin reactions in any of the test animals or controls. The chalienge treatment with the 25% and 50% test dilution in vaseline, did not induce skin reactions in any of the test animals or controls.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
On the basis of the resuits obtained under the conditions of this study, it
is concLuded that, according to the EEC-standards (as published in the
Official Journal of the European Communities, L257, Volume 26, 16 September
1983), the substance “AF-366” is not a sensitizer.