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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Study initiation date: 29 september, 2000 - Experimental observations completed on 8 December, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
other: Annex IV C to 96/54/EC Commission directive
Qualifier:
according to guideline
Guideline:
other: 91/507/EC Commission directive
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
Heparin, sodium salt
Cas Number:
9041-08-1
IUPAC Name:
Heparin, sodium salt
Details on test material:
- Name of test material (as cited in study report): Heparin sodique
- Physical state: white amorphic powder
- Purity: 100%
- Lot/batch No.: 0015865
- Storage condition of test material: at room temperature (10 to 30 °C) in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D hall Ltd, Burton
- Age: Based on information from the supplier the guinea pigs were approximately 6 to 8 weeks old on Day 1.
- Body Weight: Animals in the main study were in a body weight range from 454 to 558g prior to dosing on Day 1.
- Housing: guinea pigs were accomodated in suspended polypropylene cages (six per battery) with open tops, solid floors and stainless steel mesh front panels (providing minimum dimensions of 61x81x25 cm). Each cage held up to 5 animals and was relined with a whitewood bedding, Grade 10 Woodflakes from Datesand Ltd, Brooklands, three times each week. Each batch of bedding had been analysed for specific constituents.
- Diet : SQC FD1 (pelleted) diet from Special Diets Services Ltd freely available
- Water (e.g. ad libitum): via water bottles
- Acclimation period: up to 22 days
No contaminant were expected to be present in diet or water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 40-70%
- Air changes (per hr): 14 air changes/h
- the rooms were illuminated by fluorescent strip-lights for twelve hours daily.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: vaseline
Concentration / amount:
Intradermal injection: 10% m/v Heparin sodique in purified water and/or adjuvant
Topical induction: 50% m/m Heparin sodique in vaseline
Challenge application: 20 and 40% m/m Heparin sodique in vaseline
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
Intradermal injection: 10% m/v Heparin sodique in purified water and/or adjuvant
Topical induction: 50% m/m Heparin sodique in vaseline
Challenge application: 20 and 40% m/m Heparin sodique in vaseline
No. of animals per dose:
10 treated animals and 5 controls
Details on study design:
MAIN STUDY
INDUCTION PHASE-intradermal injection
The dorsum overlaying the scapulae of each guinea pig was clipped on the day before treatment commenced. The area was confirmed to be free from injury or irritation. On Day 1 three paired intradermal injections (approximately 0.1 ml per site) were placed in single rows parrallel to and on either side of the dorsal mid-line of each guinea pig such that the anterior and posterior injection sites marked the corners of an approximate 20 x 40 mm area. The middle injection sites were positioned close to the anterior sites. The concentration of test article selected after the first screen was administered as follows:
Anterior: Test group: FCA / Control group: FCA
Middle: Test group:Heparin sodique, 10% m/v in purified water / Control group: purified water
Posterior: Test group:Heparin sodique, 10% m/v in FCA / Control group: 50% v/v purified water in FCA
Irritation or other dermal changes at the injection site were recorded by group on Day 2.

INDUCTION PHASE- topical application
On Day 7 the areas of dorsum denuded for the first phase of induction were clipped. Since the formulation selected was non-irritating in the screening test, the denunded areas were subject to application of 10% m/m sodium lauryl sulphate in petrolatum on Day 7. A dose of approximately 0.4 ml was rubbed into the dorsum using a gloved finger. This procedure is intended to produce a mild irritation of the skin to be treated on the following day. On Day 8 each dorsum was shaved. Approximately three hours later the area of skin including the intradermal injection sites was subject to application of a 25 x 50 mm lint pad, loaded with approximately 2 ml of 50% mm Heparin sodique in vaseline (test animals) or vaseline alone (control group). Occlusion of the treated skin was effected by successive layers of Blenderm and elasticated bandage. The dressings remained in place for approximately 48 hours. The treated areas of skin were washed with water. Irritation or other dermal changes at sites of occluded topical application were recorded by group on Day 11.

CHALLENGE PHASE
Both flanks of all guinea pigs were clipped on Day 21 and shaved to remove hair stubble on Day 22. Approximately two hours later a lint pad, approximately 25 x 25 mm, loaded with approximately 1 ml 40% m/m Heparin sodique in vaseline was applied to the left flank of each animal. A second concentration 20% m/m Heparin sodique in vaseline was similarly applied to the right flank. The pads were kept in place by successive layers of Blenderm and elasticated bandage. The dressings were removed approximately 24 hours after application and the treated areas of skin were washed with arachis oil. Challenge site locations were marked with indelible ink immediately after the washing procedure.
The challenge sites were reshaved approximetaly 20 hours after removal of the dressings. Dermal responses to challenge were assessed approximately 24 and 48 hours after removal of the dressings. Responses to challenge were recorded individually.
Positive control substance(s):
yes
Remarks:
The strain of guinea pigs selected for this study has been subject to regular (6 monthly) testing to confirm its susceptibility to a moderate skin sensitizer, 2-mercaptobenzothiazole

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
40%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 40%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 20%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
40%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Slight erythema observed for one animal and desquamation observed for 2 animals. Inconclusive result for one animal, negative for all 9 other animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 40%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Slight erythema observed for one animal and desquamation observed for 2 animals. Inconclusive result for one animal, negative for all 9 other animals..
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Slight erythema observed for one animal and desquamation observed for 3 animals. Inconclusive result for one animal, negative for all 9 other animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Slight erythema observed for one animal and desquamation observed for 3 animals. Inconclusive result for one animal, negative for all 9 other animals..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
40%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Desquamation observed for 1 animal.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Desquamation observed for 1 animal..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Slight erythema observed for one animal and desquamation observed for 1 animal.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 20%. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: Slight erythema observed for one animal and desquamation observed for 1 animal..
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
40%
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
Slight erythema observed for 2 animals and desquamation observed for 4 animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 40%. No with. + reactions: 2.0. Total no. in groups: 5.0. Clinical observations: Slight erythema observed for 2 animals and desquamation observed for 4 animals..
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
20%
No. with + reactions:
3
Total no. in group:
5
Clinical observations:
Slight erythema observed for 3 animals and desquamation observed for all 5 animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 20%. No with. + reactions: 3.0. Total no. in groups: 5.0. Clinical observations: Slight erythema observed for 3 animals and desquamation observed for all 5 animals..

Any other information on results incl. tables

One animal from the test group was found dead on day 2. No cause of death was established. No other clinical observations of ill heath or toxicity were noted during the study.

All guinea pigs gained weight over the course of the experimental phase.

Intradermal injection: Slight or well defined erythema was noted at the anterior and posterior injection (treated with Freund's Complete Adjuvant emulsion FCA) sites for both test and control animals. No erythema was observed at the middle injection site in test animals receiving 10% m/v heparin sodique in purified water nor at the midlle injection site in control animals receiving purified water alone.

Topical application: No more than slight erythema was apparent in test animals following application of 50% m/m Heparin sodique in vaseline. No erythema was apparent at the topical application sites in the control guinea pigs.

Challenge phase: Slight erythematous reactions were seen at one of both treatment sites in the control group for three of the animals. Within 48 hours of the challenge application, all control animals had areas of desmaquation - this is commonly observed following occlusive bandaging of the skin and was not considered to be indicative of the test article irritation or sensitisation. In the test group similar reactions were seen but at a lower incidence. None of the animals developped reactions that were clearly more marked than those noted in the control group. The persistence of the reaction at one site was considered indicative of possible contact sensitivity but the individual result was deemed to be equivocal.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results from the challenge procedure indicated that no animals gave a positive response indicative of delayed contact hypersensitivity, the response for one animal was inconclusive.