Pentanedioic acid, compd. with (1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol (1:1)

Skin sensitisation

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:

29 December 1997 and 01 February 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Guinea pig maximisation test has been used.

Test material

Test material information

Constituent 1

Reference substance name:

Abacavir Hemisulphate

IUPAC Name:

Abacavir Hemisulphate

Constituent 2

Reference substance name:

188062-50-2

Cas Number:

188062-50-2

IUPAC Name:

188062-50-2

Details on test material:

- Name of test material (as cited in study report): 1592U89 Hemisulphate
- Molecular formula (if other than submission substance): Please see Attachment 1.
- Molecular weight (if other than submission substance): Please see Attachment 1.
- Structural formula attached as image file (if other than submission substance): Please see Attachment 1.
- Description: off-white granular solid
- Batch number: R1136/156/1
- Date received: 24 November 1997
- Storage conditions: room temperature, in the dark, over silica gel

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: David Hall Limited, Newchurch, Burton-on-Trent, Staffordshire, UK.
- Age at study initiation: Eight to twelve weeks old.
- Weight at study initiation: 329 to 444g
- Housing: Housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes.
- Diet: ad libitum: Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK.
- Water: ad libitum: Mains drinking water.
- Acclimation period: Ten days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22°C
- Humidity (%): 32 to 56%
- Air changes (per hr): Approximately 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (light cycle 06.00 to 18.00) and 12 hours darkness.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: Please see "Details on study design (Traditional tests)" for details.

Induction 2

Concentration / amount:

Test Material treated animals
Intradermal induction: 10% w/v formulation of the test material in arachis oil BP
10% w/v in a mixture of Freund's Complete Adjuvant plus distilled water (1:1)
Topical induction: 50% w/w in arachis oil BP
Challange exposure: 50% w/w in arachis oil BP and an application of 25% w/w in arachis oil BP to confirm the substance as a non-irritant.

Control animals
Intradermal induction: Freund's Complete Adjuvant plus distilled water in the ratio 1:1
arachis oil BP
a 50% formulation of arachis oil BP in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Topical induction: arachis oil BP
Challange exposure: 50% w/w in arachis oil BP and an application of 25% w/w in arachis oil BP to confirm the substance as a non-irritant.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Please see "Details on study design (Traditional tests)" for details.

Challenge 2

Concentration / amount:

Test Material treated animals
Intradermal induction: 10% w/v formulation of the test material in arachis oil BP
10% w/v in a mixture of Freund's Complete Adjuvant plus distilled water (1:1)
Topical induction: 50% w/w in arachis oil BP
Challange exposure: 50% w/w in arachis oil BP and an application of 25% w/w in arachis oil BP to confirm the substance as a non-irritant.

Control animals
Intradermal induction: Freund's Complete Adjuvant plus distilled water in the ratio 1:1
arachis oil BP
a 50% formulation of arachis oil BP in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Topical induction: arachis oil BP
Challange exposure: 50% w/w in arachis oil BP and an application of 25% w/w in arachis oil BP to confirm the substance as a non-irritant.

No. of animals per dose:

Thirty animals in total as follows:
Twenty animals each dosed with test material concentrations specified in the above section.
Ten control animals each dosed with the concentrations specified in the above section.

Details on study design:

RANGE FINDING TESTS:
Selection of Concentrations for Main Study (Sighting Tests)
The concentration of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:

Selection of Concentration for Intradermal Induction
Four concentrations of test material were investigated (1 %, 5%, 10% and 25% w/v in arachis oil BP). A total of three guinea pigs were treated. Each guinea pig received four 0.1 mL intradermal injections of only one concentration of test material. The highest of these concentrations that could be intradermally injected and that did not cause local necrosis, ulceration or systemic toxicity was selected for the intradermal induction stage of the main study.

Selection of Concentration for Topical Induction
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant ten days earlier) were treated with four preparations of the test material (5%, 10%,25% and 50% w/w in arachis oil BP).
The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure, was selected for the topical induction stage of the main study.

Selection of Concentration for Topical Challenge
Four preparations of the test material (5%, 10%, 25% and 50% w/w in arachis oil BP) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The highest non-irritant concentration of the test material, plus one lower concentration, were selected for the topical challenge stage of the main study.

MAIN STUDY
A group of thirty guinea pigs was used for the main study, twenty test and ten control. The bodyweight of each animal was recorded at the start and end of the study.

A. INDUCTION EXPOSURE
INTRADERMAL INDUCTION
Induction of the Test Animals: Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 mL each) was made on each side of the mid-line. The injections were:

i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
ii) a 10% w/v formulation of the test material in arachis oil BP
iii) a 10% w/v formulation of 1592U89 in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water

Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (injection site ii) was evaluated.

TOPICAL INDUCTION
One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation.
A filter paper patch (WHATMAN No.4: approximate size 40 mm x 20 mm), loaded with the test material formulation (50% w/w in arachis oil BP) as a thick, even layer was applied to the prepared skin and held in place with a strip of surgical adhesive tape (BLENDERM: 3M Health Care; approximate size 50 mm x 30 mm) covered with an overlapping length of aluminium foil.
The patch and foil were further secured with a strip of elastic adhesive bandage (ELASTOPLAST: Smith & Nephew Limited; approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.

Erythematous and oedematous reactions were quantified one and twenty-four hours following removal of the patches.

Any other dermal reactions were noted.

B. CHALLENGE EXPOSURE
Shortly before treatment on Day 21, an area, approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.
A square filter paper patch (WHATMAN No.4: approximate size 20 mm x 20 mm), loaded with a thick, even layer of test material at the maximum non-irritant concentration (50% w/w in arachis oil BP) was applied to the shorn right flank of each animal and held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). To ensure that a non-irritant concentration was used at challenge, the test material at a concentration of 25% w/w in arachis oil BP was similarly applied to a separate skin site on the shorn left flank. The patches were occluded with an overlapping length of aluminium foil and secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each an imal.
After 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded. The challenge sites were swabbed with cotton wool soaked in diethyl ether in order to remove residual test material. The position of the treatment sites was identified by using a black indelible marker-pen.
Approximately 21 hours after removal of the patches the flanks were clipped using veterinary clippers, to remove regrown hair.

Evaluation of Skin Reactions
Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified.
Any other dermal reactions were also noted.

Challenge controls:

Induction of the Control Animals: Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:

i) Freund's Complete Adjuvant plus distilled water in the ratio 1: 1
ii) arachis oil BP
iii) a 50% formulation of arachis oil BP in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.

Approximately 24 and 48 hours after intradermal injection the degree of erythema at the vehicle injection sites (injection site ii) was evaluated.
The topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals.

Positive control substance(s):

yes

Remarks:

Neomycin Sulphate, 2-Mercaptobenzothiazole and 2,4-Dinitrochlorobenzene.

Results and discussion

Positive control results:

Neomycin Sulphate: 60% incidence of sensitisation.
2-Mercaptobenzothiazole: 70% - 90% incidence of sensitisation.
2,4-Dinitrochlorobenzene: 100% incidence of sensitisation.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50% and 25%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No dermal reactions were noted at the challenge sites of the test animals.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% and 25%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No dermal reactions were noted at the challenge sites of the test animals..

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50% and 25%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No dermal reactions were noted at the challenge sites of the test animals.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% and 25%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No dermal reactions were noted at the challenge sites of the test animals..

Any other information on results incl. tables

Intradermal and Topical Sighting Tests

Based on these results, the following concentrations of the test material were selected for the main study:

Intradermal Induction: 10% w/v in arachis oil BP

Topical Induction: 50% w/w in arachis oil BP

Topical Challenge: 50% and 25% w/w in arachis oil BP

Main Study

Skin Reactions Observed After Intradermal Induction

Very slight, well-defined or moderate to severe erythema was noted at the intradermal injection sites of all test group animals at the 24 and 48-hour observations.

Very slight to well-defined erythema was noted at the injection sites of all control group animals at the 24-hour observation and at the injection sites of eight control group animals at the 48-hour observation.

Skin Reactions Observed After Topical Induction

Well-defined erythema was noted at the topical induction sites of all test group animals at the l-hour observation. Very slight oedema was noted at the induction sites of six test group animals and slight oedema at the induction sites of fourteen test group animals at the l-hour observation. Bleeding was noted at the injection sites of seventeen test group animals at the l-hour observation.

Very slight erythema was noted at the induction sites of ten test group animals and well-defined erythema at the induction sites of eight test group animals at the 24-hour observation. Very slight oedema was noted at the induction sites of nine test group animals with slight oedema at the induction sites of three test group animals at the 24-hour observation. Small superficial scattered scabs or a hardened dark brown/black coloured scab were noted at the induction sites of seven test group animals at the 24-hour observation and prevented the accurate evaluation of erythema and oedema at the induction sites of two test group animals at this time.

No skin reactions were noted at the treatment sites of the control group animals at the one and 24-hour observations. Bleeding was noted at the injection site of one control group animal at the l-hour observation.

Skin Reactions Observed After Topical Challenge

50% w/w in Arachis Oil BP

No dermal reactions were noted at the challenge sites of the test and control group animals at the 24 and 48-hour observations.

25% w/w in Arachis Oil BP

No dermal reactions were noted at the challenge sites of the test and control group animals at the 24 and 48-hour observations.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material produced a 0% (0/20) sensitisation rate and was classified as a NON-SENSITISER to guinea pig skin.

Executive summary:

A study was performed to assess the contact sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 17 July 1992) and Method B6 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Twenty test and ten control animals were used for the main study. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

Intradermal Induction: 10% w/v in arachis oil BP

Topical Induction: 50% w/w in arachis oil BP

Topical Challenge: 50% and 25% w/w in arachis oil BP

The test material produced a 0% (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin.