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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:
August/September 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1986

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Guinea pig maximisation test
Deviations:
not applicable
Qualifier:
according to guideline
Guideline:
other: Guinea pig maximisation test
Version / remarks:
As described by B. Magnusson and A. M. Kligman (1970) in "Allergic Contact Dermatitis in the Guinea—Pig: Identification of Contact allergens, 11 published by C，C. Thomas, Springfield, Illinois, U.S.A.
Deviations:
not specified
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test was performed before adoption of LLNA or in vitro guidelines.

Test material

Constituent 1
Chemical structure
Reference substance name:
Diamminediisocyanatozinc
EC Number:
401-610-3
EC Name:
Diamminediisocyanatozinc
Cas Number:
122012-52-6
Molecular formula:
C2H6N4O2Zn
IUPAC Name:
copper(2+) bis(carbonylazanide) diamine
Test material form:
solid: particulate/powder
Remarks:
white powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
female albino guinea-pigs of the Hartley/Dunkin strain
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: not stated
- Weight at study initiation: 391 - 491 g, mean 433 - 435 g
- Housing: The guinea-pigs were housed in suspended cages with wire mesh floors.
- Diet (e.g. ad libitum): Vitamin C-enriched Guinea-Pig Diet F.D.I (Special Diets Services Limited) ad libitum. Hay was given once weekly.
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: The guinea-pigs selected for the study were all acclimated to the laboratory environment.
- Indication of any skin lesions: All animals were observed daily for signs of ill health or toxic signs, no skin leasions were noted

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 21°C
- Humidity (%): 30-70%
- Air changes (per hr): Air exchange was maintained at approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light in each 24 hour period.
- IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
A 4 x 6 cm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made simultaneously into this area.
Injectables were prepared as follows：
1. Freund's complete adjuvant* was diluted with an equal volume of water for irrigation.
2. Test item, 0.25% w/w in liquid paraffin.
3. Test item, 0.25% w/w in a 50 : 50 mixture of Freund's complete adjuvant and Liquid paraffin.
Day(s)/duration:
7 days until 2nd induction
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
One week after the injections, the same 4 x 6 cm interscapular area was clipped and shaved free of hair.
A 2 x 4 cm patch of Whatman No. 3 paper was saturated with the test item, 15% w/w in liquid paraffin. The patch was placed on the skin and covered by a length of impermeable plastic adhesive tape (5 cm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage ("Elastoplast" 5 cm width) wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape- The dressing was left in place for 48 hours.
Day(s)/duration:
48h exposure, in total 14 days until challenge
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
The test and control animals were challenged topically two weeks after the induction period using the test item, 5% and 2.5% w/w in liquid paraffin.
Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 2 x 2 cm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of the test item, 5% w/w in liquid paraffin and applied to an anterior site on the flank.
The test item, 2.5% w/w in liquid paraffin was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".
Day(s)/duration:
24h exposure, then reading 24, 48 and 72 hours after removal
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 females / group
Details on study design:
RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of the test item in liquid paraffin which was considered to be a suitable vehicle, was investigated to identify (a) irritant test substance concentrations suitable for the induction phase of the main study and (b) non-irritant concentrations by the topical route of administration for the challenge phase.
The following concentrations of the test item were selected:
Induction
Intradermal injection: 0.25% w/w in liquid paraffin.
Topical application: 15% w/w in liquid paraffin.
Challenge
5% and 2.5% w/w in liquid paraffin.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: once intradermal, 48 h topical exposure, for 7 resp 14 days
- Test groups: 1 group of 20 animals
- Control group: 1 group of 20 animals
- Site: scapular region
- Frequency of applications: each one
- Duration: once intradermal, 48 h topical exposure
- Concentrations: 0,25% intradermal, 15% topical

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 24h exposure, reading after 24, 48 and 72 hours after removal
- Exposure period: 24h
- Test groups: 1 group of 20 animals
- Control group: 1 group of 20 animals
- Site: anterior and posterior site on the flank.
- Concentrations: 2.5 and 5%
- Evaluation (hr after challenge): 24, 48 and 72 hours after removal og the test item
Positive control substance(s):
not required

Results and discussion

Positive control results:
n/a

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Slight to well-defined erythema, slight edema
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
2.5%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Slight to well-defined erythema, slight edema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Slight to well-defined erythema, slight edema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
2.5%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Slight to well-defined erythema, slight edema
Remarks on result:
positive indication of skin sensitisation
Reading:
other: 3rd
Hours after challenge:
72
Group:
test chemical
Dose level:
5%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
Slight to well-defined erythema, slight edema
Remarks on result:
positive indication of skin sensitisation
Reading:
other: 3rd
Hours after challenge:
72
Group:
test chemical
Dose level:
2.5%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
Slight to well-defined erythema, slight edema
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Freund's treated controls, anterior and posterior site
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
barely perceptible erythema
Remarks on result:
other: slight irritation noted
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Freund's treated controls, anterior and posterior site
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
barely perceptible erythema
Remarks on result:
other: slight irritation noted
Reading:
other: 3rd
Hours after challenge:
72
Group:
negative control
Dose level:
Freund's treated controls, anterior and posterior site
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
barely perceptible erythema
Group:
positive control
Remarks on result:
not measured/tested

Applicant's summary and conclusion

Conclusions:
The Study was performed scientifically reasonable according to the method by Magnusson and Kligman, was sufficiently documented, contains the relevant results as required from OECD TG 406 and the given results allow a classification according to the recent Regulation 1272/2008. So the available study is considered sufficiently reliable and to allow a proper classification. Despite the fact that the duration between first induction and challenge was shortened compared to the recent guideline, the response of the immune system is evident. After 24 and 48 hours (as indicated in the recent OECD TG 406), the response of 20/20 animal of the test group was obviously more severe than the control group treated with Freund's adjuvans. The intradermal induction was a 0.25% solution of the test item in paraffin oil.
According to Regulation 1272/2008, Table 3.4.3, Animal test results for sub-category 1A, a substance must be classified as such, if in the Guinea pig maximisation test the numer of animals are ≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose. Since the latter is the case here, the test item must be classified as skin sensitizer Cat. 1A.
Executive summary:

In a study acc. to the guinea-pig maximisation test described by B. Magnusson and A.M. Kligman (1970) in "Allergic Contact Dermatitis in the Guinea-Pig : Identification of contact allergens", published by C.C. Thomas, Springfield, Illinois, U.S.A, each 20 female Dunkin-Hartley guinea pigs were sham-exposed or exposed to 0.25% (intradermal 1st challenge), 15% (topical 2nd challenge) and 5% an 2.5% (topical challenge) of the test item in paraffin. After 24 and 48 h after removal of the challenge test item pad, 20/20 animals showed a clear response to the test item, while the control (Freund's adjuvans) only induced max. barely perceptible erythema. In consequence, the test item needs to be classified as skin sensitizer Cat. 1A. acc. Regulation 1272/2008.