Dicerium trisulphide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 September 1996 to 13 January 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MAGNUSSON, B. and KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guinea-pig : Identification of contact allergens, Thomas, C.C., Springfield, Illinois, U.S.A.MAGNUSSON, B. and KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guinea-pig

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Guinea Pig Maximisation Test (OECD 406) was selected based on previous experience with several comparable compounds, which learned that the irritating potential of the test substance may confound the results obtained with LLNA tests. Additionally, insoluble inorganic forms are often not able to penetrate the skin. Furthermore, OECD Guideline n° 429 (LLNA) test was not validated in 1996.

Test material

Test material information

Constituent 1

Reference substance name:

Dicerium trisulphide

EC Number:

234-603-7

EC Name:

Dicerium trisulphide

Cas Number:

12014-93-6

Molecular formula:

Ce2S3

IUPAC Name:

dicerium trisulphide

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Cerium sulphide

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 311-360 g
- Housing: animals were housed in groups of five in suspended metal cages with wire mesh floors in Building R 17 Room 14.
- Diet: a vitamin C enriched guinea-pig diet FD2 was povided ad libitum. Hay was given weekly.
The batch of diet used for the study was not analysed for nutrients, possible contaminants or micro-organisms.
- Water: ad libitum.
Results of routine physical and chemical examination of drinking water, as conducted by the supplier, were made available to the testing laboratory.
- Acclimation period: 5 days

Each animal was identified by a unique ear tatto number. Each cage was identified by a coloured label displaying the study schedule number, animal numbers and the initials of the study Director and Home Office licensee.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 46-54
These environmental parameters were recorded daily.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (0700-1900 hours)

IN-LIFE DATES: 9 October 1996 - 15 November 1996

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: water for irrigation: product of coconut oil

Concentration / amount:

5 % w/v in water for irrigation

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

other: distilled water

Concentration / amount:

80 % in distilled water

Day(s)/duration:

7 days

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: Distilled water

Concentration / amount:

80 and 40 %

Day(s)/duration:

1 day

Adequacy of challenge:

other: 80 %: maximal practical concentration and did not give rise to irritanting effects 40 %: safe guard dose

No. of animals per dose:

- 6 for the range finding test
- 15 for the main study: 10 test animals and 5 control animals

Details on study design:

RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify, where possible, concentrations of the test substance that would produce irritation suitable for the induction phase of the main study, and a maximum non-irritant concentration by the topical route of administration for the challenge phase.
Animals for these investigations were pre-treated with an intradermal injection of Freund's complete adjuvant, 50:50 with water for irrigation (Ph.Eur.), approximately two weeks prior to the start of the preliminary investigations.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (one intradermal and one cutaneous).
- Type of epicutaneous induction: occlusive
- Sodium lauryl sulphate application: Yes, 0.5 ml at 10% (w/w) in petrolatum.
- Exposure period: 48 hours (cutaneous induction)
- Test groups: three pairs of intradermal injections were made using :
1/ Freunds complete adjuvant (FCA) diluted with an equal volume of water for irrigation (Ph.Eur.)
2/ The test substance at 5% w/v In water for irrigation
3/ The test substance at 5 %w/v in a 50 : 50 mixture of Freund's complete adjuvant and water for irrigation.
Six days later, the same area was pretreated by gentle rubbing with 0.5 ml per site of 10 % w/w sodium lauryl sulphate in petrolatum. 24 hours after, a 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of the test substance. The patch was placed on the skin of the test animals and covered by a lenght of impermeable plastic adhesive tape (50 mm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage (50 mm wisth "Elastoplast") wound round the torso of the animal and fixed with "sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.

- Control group: control group animals were treated in the same manner as the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.

- Site: a 40 x 60 mm area of dorsal skin on the scapular cregion of the guinea-pig was clipped free of hair with electric clippers. Intradermal injections and topical applications were made into a 20 x 40 mm area within the clipped area.
- Frequency of applications: once
- Duration: 8 days (total duration of the induction period)

- Concentrations: On day 1, intradermal injections (0.1 ml each) were performed in all animals as follows:
1/ Freunds complete adjuvant (FCA) diluted with an equal volume of water for irrigation (Ph.Eur.) for both groups.
2/ Test substance at 5% w/v in water for irrigation for the treated group (highest concentration that caused irritation but did not adversly affect the animals), and water for irrigation for the control group
3/ Test substance at 5% w/v in a 50 : 50 mixture of Freund's complete adjuvant and water for irrigation for the treated group, and only the 50 : 50 mixture of Freund's complete adjuvant and water for irrigation for the control group.

One week later, the animals of the treated group received a topical application of the test item at the concentration of 80% in in distilled water (pre-treatment with 10 % sodium lauryl sulphate in petrolatum was necessary as the substance was not irritant). The animals of the control group received an application of the vehicle under the same experimental conditions.

B. CHALLENGE EXPOSURE
- No. of exposures: One.Two weeks after the topical induction application by the test substance, control and test animals were challenged using a single topical application of Cerium sulphide (test substance).
- Day(s) of challenge: Day 22
- Exposure period: 24 hours
- Test group and Control group: the animals were challenged by a topical application of the test item at the concentrations of 80 and 40 % in distilled water
- Site: Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of Cerium sulphide at the tested concentrations and applied to anterior (80 %) and posterior (40 %) sites on the flank. 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".
- Concentrations: test substance at 80 and 40 % w/v in distilled water.
- Evaluation (hr after challenge): 24 and 48

OTHER:
- All animals were observed daily for signs of ill health or toxicity.
- The bodyweight of each guinea-pig on the main study was recorded on Day 1 (intradermal injections) and on the last day observations were made of dermal responses to the challenge applications.
- The dermal reactions resulting from intradermal injection and topical application on the preliminary study, and topical application at the challenge were assessed using a numerical system (scoring from 0 to 4). The approximate diameter (mm) of the dermal response at the intradermal injection sites was recorded in the preliminary study only to assist in the choice of concentrations for the main study. Any other lesion not covered by the scoring system was described.

Positive control substance(s):

yes

Remarks:

Hexyl cinnamic aldehyde (HCA), Benzocaine and 2-Mercaptobenzothiazole (MBT)

Results and discussion

Positive control results:

The sensitivity of the guinea-pig strain used is checked periodically at Huntingdon Life Sciences with known sensitisers Hexyl cinnamic aldehyde (HCA), Benzocaine and 2-Mercaptobenzothiazole (MBT). A summarising table of positive control data performed between 1995 and 1996 using the Magnusson and Kligman test method was provided with the study report.
The reference items induced positive skin sensitization reactions in at least 60% guinea pigs and thus, the sensitivity of the guinea-pigs strain from the same source was considered satisfactory.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

40 and 80% w/v in distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

red staining on all dose sites that did not interfere with scoring

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

40 and 80% w/v in distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

red staining on all dose sites that did not interfere with scoring

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

40 and 80 % (w/v) in distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

red staining on all dose sites that did not interfere with scoring

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

40% and 80 % w/v in distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

red staining on all dose sites that did not interfere with scoring

Results 5

Group:

positive control

Remarks on result:

other: Not available

Any other information on results incl. tables

- No signs of ill health or toxicity were recorded.

- Bodyweight increases of the test animals were not statistically different from those of the negative control animals.

- Dermal reactions:

1) Intradermal injections: Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals.

Slight irritation was seen in test animals at sites receiving Dicerium trisulphide at 5% w/v in water for irrigation and no irritation was observed in control animals receiving water for irrigation.

2) Topical application: Slight erythema was observed in test animals following topical application with Dicerium trisulphide at 80% w/v in distilled water.

Slight erythema was seen in the control guinea-pigs.

3) Challenge: There were no dermal reactions seen in any of the test or control animals, therfore all test animals gave negative responses. Red staining was noted on all dose sites, however this did not interfere with scoring.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions of the study, Dicerium trisulphide did not produce evidence of skin sensitisation (delayed hypersensitivity) in any of the ten test animals.

Executive summary:

The potential of the test substance Dicerium trisulphide to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to EU Method B.6 (Skin Sensitisation) guideline and in compliance with the principles of Good Laboratory Practice Regulations.

 

Method

Fifteen guinea pigs were allocated to two groups: a control group of five males and a treated group of ten males.

On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

• Freund's complete adjuvant (FCA) diluted to 50% (v/v) with water for irrigation (both groups),

• Test substance at the concentration of 5% (w/v) in water for irrigation (treated group) or vehicle alone (control group),

• Test substance at the concentration of 5% (w/v) in a mixture FCA/water for irrigation (50/50, v/v) (treated group) or vehicle at the concentration of 50% (w/v) in FCA (50/50, v/v) (control group).

On day 7, a topical application of sodium lauryl sulfate at 10% (w/w) in petrolatum was performed to the same area of the animals of both groups, in order to induce a local irritation.

On day 8, the animals of the treated group received a topical application of the test item at the concentration of 80% (w/v) in distilled water to the same test site under an occlusive dressing for 48 hours. The animals of the control group received an application ofthe vehicle under the same experimental conditions.

On day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentrations of 80 and 40% (w/v) in distilled water to the left flank (anterior and posterior sites) under an occlusive dressing for 24 hours. The vehicle was applied to the right flank under the same experimental conditions.

Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

 

Results

No deaths and no systemic clinical signs were noted during the study.

After the challenge application of the test substance, no dermal reaction was seen in any of the test or control animals, therefore all test animals gave negative responses. Red staining was noted on all dose sites, however this did not interfere with scoring.

 

Conclusion

Under the experimental conditions of this study and according to the maximization method of Magnusson and Kligman, the test substance Dicerium trisulphide did not induce delayed contact hypersensitivity in guinea pigs and therefore should not be classified according to the EU criteria.