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Diss Factsheets

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:
09 September 2002 - 11 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(1996)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Adequate data from a guinea pig maximisation test are available since 2002.

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)
EC Number:
250-774-0
EC Name:
Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)
Cas Number:
31714-55-3
Molecular formula:
C32H19Cl2CrN4O4
IUPAC Name:
[1-{[5-chloro-2-(hydroxy-kappaO)phenyl]diazenyl}-2-naphtholato(2-)-kappaO]{1-[(5-chloro-2-hydroxyphenyl)diazenyl]-2-naphtholato-kappaO}chromium
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: black powder
- Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 311-395 g
- Housing: Singly or in pairs in solid-floor polypropylene cages
- Diet: Certified Guinea Pig Diet, International Product Supplies Limited, UK (ad libitum)
- Water: tap water (ad libitum)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
- 25% for the intradermal induction
- 50% for the epidermal induction
Challenge
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
- 25% and 10% for the challenge
No. of animals per dose:
- Range finding tests: 7 (see below for details)
- Main study: Test animals: 10; Control: 5
Details on study design:
RANGE FINDING TESTS
- Selection of concentration for intradermal induction: Intradermal injections (0.1 mL/injection site) were made on the clipped shoulder of 3 guinea pigs, using concentrations of 5, 10 and 25%. The degree of erythema at the injection sites was assessed approx. 24, 48, 72 hours and 7 days after injection. Any evidence of systemic toxicity was also recorded.
- Selection of concentration for epidermal induction: Two guinea pigs (intradermally injected with FCA 8 days earlier) were treated with test substance concentrations of 50, 25, 10 and 5%. Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approx. 1, 24 and 48 hours after dressing removal.
- Selection of concentration for challenge:The test substance concentrations 50, 25, 10 and 5% were applied to the clipped flanks of 2 guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approx. 1, 24 and 48 hours after dressing removal. Next to the highest non-irritant concentration, also one lower concentration was selected for the main study.

MAIN STUDY
A. INDUCTION EXPOSURE (test animals)
- No. of exposures: 2

1) Intradermal injections:
- Shortly before treatment on Day 0 the hair was removed from an area approx. 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers.
- A row of 3 injections, each of 0.1 mL, was made on each side of the mid-line into a 20 mm x 40 mm area:
-- FCA (50% in distilled water)
-- 25% of the test substance in the vehicle
-- a 25% formulation of the test substance in a 1:1 preparation of FCA plus vehicle
- Readings: Approx. 24 and 48 hours after intradermal injection the degree of erythema at the test substance injection sites was evaluated

2) Topical application:
- On day 7 the same area on the shoulder region used previously for the intradermal injections was clipped again.
- A filter paper patch (Whatman No. 4; 40 x 20 mm) loaded with the formulation of the test substance in vehicle (at 50%) was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bondage wound in a double layer around the torso of each animal.
- This occlusive dressing was kept in place for 48 hours.
- Readings: The degree of erythema and oedema was evaluated 1 and 24 hours after patch removal. Any other reactions were also recorded.

INDUCTION (control animals)
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

B. CHALLENGE EXPOSURE (all animals)
- Shortly before treatment on Day 21, an area of approx. 50 x 70 mm on both flanks of each animal was clipped free of hair with veterinary clippers
- Square filter paper patches (Whatman No. 4; approx. 20 x 20 mm) loaded with the test substance in vehicle:
-- Were applied at 25% to the shorn right flank of each animal
-- Were applied at 10% to the shorn left flank of each animal
- The patches were held in place with a strip of surgical adhesive tape
- The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bondage wound in a double layer around the torso of each animal
- After 24 hours, the dressing was carefully removed and the challenge sites were swabbed with cotton wool soaked in diethyl ether to remove residual test substance.
- Prior to the 24 hours observation, the flanks were clipped again
- The degree of erythema and oedema was evaluated 24 and 48 hours after the removal of the patches
(48 and 72 hours after the start of the exposure)
Positive control substance(s):
yes
Remarks:
(a reliability check is carried out at regular intervals to check the sensitivity of the test system)

Results and discussion

Positive control results:
The latest reliability check was carried out in May/June 2002 using 2-Mercaptobenzothiazole. In this study, a sensitisation rate of 80% was found, showing that the test system is reliable.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
4
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
4
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
4
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
4
Group:
positive control
Dose level:
50% and 25%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
other: Results for latest reliability check (May/June 2002)

Any other information on results incl. tables

RANGE FINDING TESTS

- Selection of concentration for intradermal induction: There was no evidence of systemic toxicity. Black coloured staining prevented evaluation of erythema. Focal eschar was observed at 10 and at 25%, after 7 days, in 2 of the 3 animals. Based on the results, the highest tested concentration of 25% was choosen for the main study.

- Selection of concentration for epidermal induction: At all concentrations, in both animals, moderate and confluent erythema was observed after 1 hour. After 24 hours, discrete or patchy erythema was observed at 50 and 25% in both animals. No erythema was observed after 48 hours. No oedema was observed. Black coloured staining was observed. Based on the results, the highest tested concentration of 50% was choosen for the main study (max. attainable concentration suitable for topical application for a solid).

- Selection of concentration for challenge: At 50%, moderate and confluent erythema was observed after 1 hour in both animals and discrete or patchy erythema after 24 hours in 1 of the 2 animals. At 25%, moderate and confluent erythema was observed after 1 hour in both animals. At 10%, moderate and confluent erythema in an animal and discrete or patchy erythema in the other animal was observed after 1 hour. At 5%, discrete or patchy erythema was observed in both animals only after 1 hour. Oedema was only observed after 1 hour, at 50%.

As no erythema nor oedema was observed after 24 hours at 25% (while erythema was observed at 50%) this concentration, as well as additionally 10%, was choosen for the main study.

MAIN STUDY

- Readings (intradermal induction):

-- Black coloured staining, which prevented evaluation of erythema, was noted at the induction sites of all test group aninals

-- Discrete or patchy erythema was noted at the induction sites of all control group animals, only 24 hours after the injections, on both sides of the mid-line

- Readings (topical induction):

-- Black coloured staining, which prevented evaluation of erythema, was noted at the induction sites of all test group aninals

-- Moderate and confluent erythema was noted at the induction sites of all control group animals, 1 hour after patch removal

-- Discrete or patchy erythema was noted at the induction sites of 3 of the 5 control group animals, 24 hours after patch removal

-- Bleeding from the intradermal injection sites was noted in all test group animals and in 1 control group animal

-- No oedema was observed

- Readings (challenge):

-- Black coloured staining was noted at the challenge sites of all aninals. This did not affect evaluation of skin reactions.

-- On Day 21, one of the control animals was humanely killed. This did not affect the integrity of the study.

-- No skin reactions were noted in any of the 14 animals at the challenge sites

- Other information:

-- Body weights and body weight gain of experimental animals remained in the same range as controls over the study period

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
In a guinea pig maximisation test performed according to OECD 406, the substance is not considered a skin sensitiser.
Executive summary:

The skin sensitisation potential of the substance (a solid) was investigated by performing a guinea pig maximisation test in accordance with OECD 406 and according to GLP principles. Arachis oil was used as vehicle. Test substance concentrations selected for the main study were based on the results of a preliminary study. Reliable positive and negative controls were included.

A concentration of 25% was used for the intradermal induction, 50% for the epidermal induction (max. attainable concentration) and 25% and 10% for the challenge. As none of the test group animals showed sensitisation effects after challenge, the substance does not need to be classified as a skin sensitiser in accordance with the CLP Regulation.