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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
other: Magnusson and Kligman test
Justification for non-LLNA method:
The first-choice method according to REACH Annex VII §8.3, the Murine Local Lymph Node Assay, is known to give false positive results with hydrocarbon substances.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance/item: sulphur 80% wg
Common name (active ingredient): sulfur
Name to be used in the report: sulphur 80% wg
Chemical name (IUPAC): sulfur
CAS no.: 7704-34-9
Batch no.: SML/RD/F/S-911
Manufactured and supplied by: Sulphur Mills Limited, Mumbai, India
Purity to be stated in the report: Min 80.00% w/w
Density: before compaction: 0.82 g/ml
after compaction: 0.86 g/ml
Physical appearance: brown coloured granules
Storage conditions: Ambient (+18 to +36 °C)
Analysed purity: 78.79% w/w


In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Albino, NIH (Hartley)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sri Venkateshwara Enterprises; Subramanyanagar; Bangalore - 560 021
- Age at study initiation: 8 weeks
- Weight at study initiation: males 305-360 g and females 273-382 g
- Housing: individually in suspended polypropylene bottom mesh cages (size approx. L 410 x B 28 x H 140 mm) with stainless steel top grill
- Diet (e.g. ad libitum): guinea pig feed manufactured by Nav Maharashtra Chakan Oil Mills Ltd., Pune-30, Maharashtra, India ad libitum
- Water (e.g. ad libitum): deep bore-well water passed through activated charcoal filter and exposed to UV rays. Vitamin C was supplemented in drinking water ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 30-70%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: intradermal: saline or saline/FCA; epicutaneous: unchanged
Concentration / amount:
intradermal injection (induction): 1.0% w/v in normal saline
topical application (induction and challenge): 0.5 g test item as paste in de-ionised water (completely applied to induction site)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: intradermal: saline or saline/FCA; epicutaneous: unchanged
Concentration / amount:
intradermal injection (induction): 1.0% w/v in normal saline
topical application (induction and challenge): 0.5 g test item as paste in de-ionised water (completely applied to induction site)
No. of animals per dose:
control group: 5 males and 5 females
treatment group: 10 males and 10 females
Details on study design:
1. IRRITANCY TESTING
Intradermal irritancy test: sulfur at concentrations of 1%, 2.5% and 5% w/v in normal saline was injected intradermally (0.1 ml, in duplicate) into the clipped shoulder region of 2 guinea pigs. Erythema and oedema (grade 1) present at 24 hr and 48 hr at all sites. 1% w/v in saline was selected as the concentration for intradermal induction.

Topical irritancy test: sulfur at concentrations of 25%, 50% and 75% w/v in acetone was applied topically (0.1 ml, in duplicate) under patches (see below) to the clipped flanks of 4 guinea pigs. The only reaction observed was very slight erythema (grade 1) in 1 animal exposed to 75% sulfur. Given the absence of dermal responses seen, 0.5 g sulfur as a paste in de-ionised water was selected for topical induction and challenge.

2. INTRADERMAL INJECTION
The hair of all the animals in the control (G1) and treatment (G2) groups was removed from the shoulder region (at least an area of 2 x 4 cm) using an electric clipper, approximately 24 hours before administration.
Day 1. The animals were injected using sterile 1 ml tubercuIin syringe fitted with 26 gauge needle at the shoulder region with 0.1 ml each of 3 pairs of intradermal injections so that one injection of each pair on either side of the midline. Injections 1 and 2 were given near the head region 1 cm apart and injection 3 towards caudal part of the body.

CONTROL GROUP (G1);
1) Two 0.1 ml injections of 1:1 mixture (v/v) FCA in normal saline at site marked '1' (Solution 1).
2) Two 0 1 ml injections of the undiluted vehicle (normal saline) at site marked '2'
3) Two 0.1 ml injections of 1:1 mixture of vehicle and solution 1 at sites marked '3'
Injections 1 and 2 were given near to each other (1 cm apart) while Injection 3 was given slighty away from site 2 (about 2 cm apart).

TREATMENT GROUP (G2)
1) Two 0.1 ml injections of 1:1 mixture (vlv) of Freunds Complete Adjuvant (FCA) in normal saline at sites marked '1',
2) Two 0.1 ml injections of test item in vehicle (normal saline) at concentration of 1%w/v at sites marked '2',
3) Two 0.1 ml injections of test item in vehicle mixed 1:1 with 1:1 mixture (v/v) of FCA and normal saline at sites marked 3, such that the final concentration of test item injected is the same as that it site 2. Injections 1 and 2 were given near to each other (1 cm apart) while injection 3 was given slightly away from 2 (about 2 cm apart).

3. INDUCTION (BOOSTING) TOPICAL APPL ICATION
On day 7, the intradermal injection site (approximately 2 x 4 cm area) was closely clipped and the test area was painted with 0.5 ml of 10% w/v sodium lauryl sulphate in liquid paraffin to produce local irritation (since the test item did not cause skin irritation during pre-study). Approximately 24 hours after clipping on day 8, 0.5 g of test item as paste in de-ionised water was transferred completely on to the filter paper (2 x 4 cm) and applied on to the skin (site of intradermal injection) and covered with cotton gauze (size 3 x 4 cm - 6 ply).
This was held in place for 48 hours by adhesive tape (for occlusion) and crepe bandage wound around torso over which a clean cotton cloth (many tailed bandage) was tied to anchor the crepe bandage.
Control group: Handled similar to treatment group animals but the control patch was applied (fully loaded filter paper with de-ionised water).

4. CHALLENGE
TOPICAL APPLICATION
The treatment group and vehicle central group animals were challenged on the 22nd day of the test (the day of intradermal injections to animals is reckoned as day '1' of the test), by topical application of test item to control and the treatment group animals (at its highest non-irritant dose as determined by the pre-study). One male and two female guinea pigs of the vehicle control group were not challenged and retained naive.
i) One day before the challenge application, on day 21, the hair on both the flanks (approximately 80 sq cm- 8 x 10 cm ) was closely clipped.
ii) On day 22. 0.5 g of test item as a paste in deionised water was transferred completely onto the filter paper (size: 2 x 4 cm) and applied on to the left flank of the skin and covered with cotton gauze (size 3 x 4 cm - 6 ply).
Similarly, filter paper strip (2 x 4 cm) fully leaded with de-ionised water was applied on to the right flank and covered by 3 x 4 cm cotton gauze of 6 ply. The patches were held in place with the help of adhesive tape and crepe bandage wound around torso over which a clean cotton cloth (many tailed bandage) was tied to anchor the crepe bandage.
These challenge patches were kept in contact with the skin for a period of 24 hours. On the 23rd day, the patches were removed and the area of skin where the test patch was applied was cleaned with normal saline swabs.
iii) 24 hours after the removal of patches (viz 48 hours after challenge application), the skin was examined for skin reactions, which was scored using Draize criteria.
Skin was observed again at 48 hours after the removal of the test patch or on day 25 (72 hours after the challenge application) in order to confirm the previous days observations. All the observations were recorded.
Positive control substance(s):
yes
Remarks:
historical data (2-mercaptobenzothiazole)

Results and discussion

Positive control results:
Challenge application: 8 out of 10 guinea pigs had score of 1 (discrete or patchy erythema) at 24 hours and 7 out of 10 guinea pigs had score of 1 (discrete or patchy erythema) at 48 hours post removal of the test patch.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no dermal responses present
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no dermal responses present.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no dermal responses present
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no dermal responses present.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.5 g
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no dermal responses present
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.5 g. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no dermal responses present.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.5 g
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no dermal responses present
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.5 g. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no dermal responses present.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
2-Mercaptobenzothiazole
No. with + reactions:
10
Total no. in group:
10

Any other information on results incl. tables

Intradermal injection

At 24 hours (post administration) observation period erythema (score value of 1 - very slight erythema {barely perceptible}} and edema (score value of 1 - very slight edema {barely perceptible}) were observed in all the animals at sites 1, 2 and 3 which persisted through 48 hours at sites 1 and 3. However at site 2 erythema was observed in 7 and edema was observed in 6 out of 20 animals at 48 hours observation.

Topical boosting

There were no skin reactions at 1 and 24 haws post removal of the test patch.

Challenge

There were no skin reactions at 24 and 48 hours post removal of the test patch.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Sulfur was not a sensitizer under the conditions of the test.
Executive summary:

The sensitization potential of sulfur was evaluated in a GLP compliant guinea pig maximization study following OECD guideline 406. Following an irritancy screen, 1% w/v in normal saline was selected for intradermal induction and 0.5 g sulfur (as a paste in de-ionised water, applied under occlusive conditions) selected for topical induction and challenge. 2-Mercaptobenzothiazole was used as a positive control substance, and dermal responses were evaluated in all groups using Draize criteria. No erythema or oedema was present in either the control or test groups 24 hr or 48 hr post- challenge, however a satisfactory response was obtained in the positive control group.

It was concluded that sulfur was not a sensitizer under the conditions of the test.