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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:
From 1992-07-28 to 1992-08-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study in accordance with recognised test guideline but: Lot/batch No.: not stated Expiration date of the lot/batch: not stated NOTE: study deemed acceptable because spectral data for test item are available, covering approximately before and after the test period - see section 1.4 Analytical Information.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
no
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:
This study was carried out conducted in accordance with OECD 406 and in compliance with OECD GLP before 10 May 2017.

Test material

Constituent 1
Specific details on test material used for the study:
- Substance type: Organic
- Physical state: Fine, off-white powder
- Analytical purity: Approximately 100%
- Storage condition of test material: ambient temperature, in the original container

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac Limited, Bicester, Oxfordshire, England
- Age at study initiation: six to eight weeks
- Weight at study initiation: all animals were within the bodyweight range 338 - 438 g
- Housing: stainless steel cages with grid floors and tops (Type GP7/TRI0 from Modular Systems and Development Company Limited, London, England). The grid floors ensured rapid removal of waste material to undertrays which were cleaned out as necessary. No more than five animals of the same sex were assigned to each cage. The cages were suspended in mobile stainless steel racks.
- Diet (e.g. ad libitum): commercially-available pelleted guinea-pig diet (Guinea-pig F.D.l., from Special Diets Services Limited, Witham, Essex, England) was fed without restriction. The diet contained no added antibiotic or other chemotherapeutic or prophylactic treatment. A regular supplement of autoclaved hay was also provided.
- Water (e.g. ad libitum): free access to tap water taken from the public supply
- Acclimation period: at least six days but not more than sixteen days was allowed between arrival at the laboratory and first administration of the test material.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18°C (range 15°-23°C)
- Humidity (%): 55% R.H. (range 40%-70% R.H.)
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): lighting cycle of 12 hours artificial light per day; there was no source of natural light

IN-LIFE DATES: From: 28 July 2009 To: 28 August 1992

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal
Vehicle:
propylene glycol
Concentration / amount:
First induction: 3% w/v test item in propylene glycol and 3% w/v test item in propylene glycol in FCA
Second induction: 50% w/v test item in propylene glycol
Day(s)/duration:
First induction: Day 1; Second induction: Day 8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
50% w/v test item in propylene glycol and 10% w/v test item in propylene glycol
Day(s)/duration:
Day 22/ 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20
Details on study design:
RANGE FINDING TESTS:
Phase 1 - Intradermal induction
Four naive guinea-pigs received intradermal injections (0.1 mL) into the skin overlying the scapulae. Six injections were administered to each guinea-pig; three concentrations of test material in the selected vehicle and three in an emulsion of FCA. Two guinea-pigs received the maximum practicable concentration in each medium and two dilutions. The other two animals received three lower concentrations in each medium.
Reactions to treatment were assessed approximately 24 and 48 hours and 7 days after injection.

Phase 2 - Topical induction administration
Two guinea-pigs were subjected to a single intradermal injection of 0.1 mL FCA at least five days before topical application to simulate the FCA insult that the main study animals receive before topical induction. The hair was removed from both flanks of the two animals. Topical application of 0.4 ml of the maximum practicable concentration of the test material and three lower concentrations in the selected induction vehicle was administered to the four test sites on each guinea-pig. Each test formulation was applied to a 2 x 2 cm absorbent patch (Whatman No.3 filter paper) which was applied to the skin and covered by an occlusive dressing for 48 hours.
Reactions to treatment were assessed 24 and 48 hours and 7 days after removal of the dressings.

Phase 3 - Topical challenge administration
Three guinea-pigs received a single intradermal injection of 0.1 mL FCA at least twenty days before topical application to simulate the FCA insult that the main study animals receive before challenge application. The hair was removed from both flanks of the three animals. Topical application of 0.03 mL of four concentrations of test material in the selected challenge vehicle was administered to the four test sites on each guinea-pig. Each test formulation was applied to a 1 cm diameter absorbent patch which was applied to the skin and covered by an dressing for 24 hours.
Reactions to treatment were assessed 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
- Induction procedures: The induction procedures were primary induction by intradermal injection on Day 1 and secondary induction by occluded topical application on Day 8. Dermal responses to primary and secondary induction were assessed approximately 24 hours and 48 hours after injection or removal of the occlusive dressings
- Constitution of study groups: the test group consisted of twenty animals and the control group ten animals both, evenly divided by sex. The control animals (Group 1) were treated identically to the test animals (Group 2) during the induction and challenge procedures, except that during induction the test material was replaced by vehicle.
- Primary induction: three pairs of injections (0.1 mL) were made deep into the dermis, such that on either side of the dorsal median line there were three injection sites in a row parallel to the spinal column. All injection sites lay near the periphery of a dermal test site 4 cm x 2 cm long, overlying the scapulae. The anterior and middle sites were positioned close together and distant from the posterior sites.
- Secondary induction: On Day 7, the clipped dorsa of all animals were subject to inunction with 10% w/v sodium lauryl sulphate in petrolatum. This was intended to enhance dermal absorption of formulations administered on the following day. On Day 8, the dermal site overlying the scapulae were treated by topical application of 0.6 mL of a test material formulation to test animals, while controls received 0.6 mL of the vehicle. Each dose was applied to a 4 x 2.5 cm absorbent patch which was applied to the skin and covered by an occlusive dressing for 48 hours. The application site was wiped with a paper tissue moistened with the vehicle immediately after removal of the bandage.

B. CHALLENGE EXPOSURE
Both flanks of all animals were clipped on Day 21 to expose areas (5 x 5 cm) on either side of the trunk. On Day 22 these areas were wet shaven to reveal a 5 x 5 cm area on the left flank and a 10 x 5 cm area on the right flank. Approximately one hour later the left site was treated by topical application of 0.03 ml of the vehicle while the right side received 0.03 ml of the maximum non-irritant concentration (as determined by Phase 3 of the primary skin irritation screen) to one site and a dilution to a second site. The doses were applied to 1 cm diameter absorbent patches (AI-test) and covered by an occlusive dressing for 24 hours. The test site was wiped with a paper tissue moistened with vehicle immediately after removal of the bandage.
The challenge sites were examined approximately 24 and 48 hours after removal of the occlusive dressings.
Challenge controls:
The control animals (Group 1) were treated identically to the test procedures, except that during induction the test material was replaced by vehicle.
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:
24
Group:
negative control
Dose level:
50% w/v in propylene glycol
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% w/v in propylene glycol
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50% w/v in propylene glycol
No. with + reactions:
6
Total no. in group:
18
Clinical observations:
slight erythema or more marked reaction
Remarks on result:
positive indication of skin sensitisation
Remarks:
Two animals humanely killed on Day 22
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50% w/v in propylene glycol
No. with + reactions:
0
Total no. in group:
18
Remarks on result:
no indication of skin sensitisation
Remarks:
Two animals humanely killed on Day 22
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10% w/v in propylene glycol
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10% w/v in propylene glycol
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10% w/v in propylene glycol
No. with + reactions:
1
Total no. in group:
18
Clinical observations:
slight erythema
Remarks on result:
no indication of skin sensitisation
Remarks:
Two animals humanely killed on Day 22
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10% w/v in propylene glycol
No. with + reactions:
0
Total no. in group:
18
Remarks on result:
no indication of skin sensitisation
Remarks:
Two animals humanely killed on Day 22
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
propylene glycol only
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
propylene glycol only
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
propylene glycol only
No. with + reactions:
0
Total no. in group:
18
Remarks on result:
no indication of skin sensitisation
Remarks:
Two animals humanely killed on Day 22
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
propylene glycol only
No. with + reactions:
0
Total no. in group:
18
Remarks on result:
no indication of skin sensitisation
Remarks:
Two animals humanely killed on Day 22
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

General health and bodyweight:

The main study animals generally remained in overt good health and achieved anticipated bodyweight gains. Two male test animals showed ulceration of their induction sites and were humanely killed on the day of challenge application (Day 22). Necropsy examination revealed areas of excoriation up to 30 x 30 mm in size and enlarged axillary lymph nodes: one animal also showed occasional pale areas on the median and left lobe of the liver. The damage was considered to be a spontaneous physical effect, probably resulting from scratching of the site of the animal, and not primary irritation following second induction.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
It was concluded that, under the conditions of this study and the criteria of the EEC, repeated administration of test item caused delayed contact hypersensitivity in guinea-pigs.
Executive summary:

The potential of test item, to cause delayed contact hypersensitivity in guinea-pigs was assessed by the Magnusson-Kligman Maximisation Test based on OECD 406. The closely-clipped dorsa of ten male and ten female Dunkin-Hartley guinea-pigs were subject to intradermal injections of Freunds Complete Adjuvant, 3% w/v test item in propylene glycol and 3% w/v test item in propylene glycol in the adjuvant on Day 1. Seven days later the same area of skin was treated by topical application of 50% w/v test item in propylene glycol and the test site was covered by an occlusive dressing for 48 hours. The same induction procedures were carried out on a contemporaneous control group of five male and five female animals, except that the test material was replaced by vehicle in all doses. On Day 22, all surviving animals were challenged by occluded application of propylene glycol to the left flank and 50% and 10% w/v test item in propylene glycol to two sites on the right flank. The occlusive dressings were removed on the following day and the condition of the test sites was assessed approximately 24 and 48 hours later. Dermal signs of reaction to treatment following intradermal injection of 3% w/v test item in either propylene glycol or propylene glycol in FCA were slight or moderate erythema, eschar formation and pallor. Dermal signs following occluded topical application of 50% w/v test item in propylene glycol were exfoliation and occasional barely perceptible or slight erythema. Two male test animals showing excoriation of the induction site were killed on Day 22. A significant dermal response (slight erythema or a more marked reaction) was observed in six test and no control animals following challenge application of 50% w/v test item in propylene glycol. A single significant response was observed amongst test animals following challenge application of 10% w/v test item in propylene glycol. No significant response was observed following challenge application of propylene glycol alone. It was concluded that, under the conditions of this study and the criteria of the EEC, repeated administration of test item caused delayed contact hypersensitivity in guinea-pigs.