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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-11-25 to 1997-01-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable with restrictions - the stability of the test material was missing in the study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
, 1992-07-17
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 1996-02-27
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
Dipotassium hexafluorozirconate
EC Number:
240-985-6
EC Name:
Dipotassium hexafluorozirconate
Cas Number:
16923-95-8
IUPAC Name:
dipotassium hexafluorozirconate(2-)
Details on test material:
- Name of test material (as cited in study report): POTASSIUM HEXAFLUOROZIRCONATE
- Physical state: white crystalline solid
- Storage condition of test material: ambient temperature (<25°C), stored under artificial light

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately eight to twelve weeks old
- Weight at study initiation: 347 to 415 g
- Housing: the animals were 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 tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 21°C
- Relative humidity: 41 to 59%
- Air exchanges: approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
Intradermal induction:
- 1% w/v in arachis oil BP
Topical Induction
- 75% w/w in arachis oil BP
Topical Challenge:
- 25% and 10% w/w in arachis oil BP
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Intradermal induction:
- 1% w/v in arachis oil BP
Topical Induction
- 75% w/w in arachis oil BP
Topical Challenge:
- 25% and 10% w/w in arachis oil BP
No. of animals per dose:
- Sighting test: 6 animals (2 animals for intradermal induction; 2 animals for topical induction; 2 animals for topical challenge)
- Main study: 10 test animals and 5 control animals
Details on study design:
RANGE FINDING TESTS:
The concentrations 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 procedure was as follows:
- Selection of concentration for intradermal induction:
Two concentrations of test material were investigated (1% and 5% w/v in arachis oil BP). A total of two guinea pigs were used, each guinea pig receiving four 0.1 mL injections of only one concentration of test material, the degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and 7 days after injection according to the Draize scale. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, 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 eight days earlier) were treated with four preparations of the test material (75%, 50%, 25% and 10% w/w in arachis oil BP). 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 approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.
- Selection of concentration for topical challenge:
Four preparations of the test material (75%, 50%, 25% and 10% w/w in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure 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 degree of erythema and oedema was evaluated approximately 1, 24, and 48 hours after dressing removal. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

Results:
Based on the results, the following concentrations were selected for the main study:
- Intradermal induction: 1% w/v in arachis oil BP
- Topical induction: 75% w/w in arachis oil BP
- Topical challenge: 25% and 10% w/w in arachis oil BP

Please also refer to "Attached background material" below for further information on the results of the sighting study.

MAIN STUDY
A. INDUCTION EXPOSURE
1) Induction of 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:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 1% w/v formulation of the test material in arachis oil BP
c) a 1% w/v formulation of the test material 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 (ie. injection site b) was evaluated according to the Draize scale.
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 (75% 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: 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: 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.
The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches using the Draize scale.
Any other reactions were also recorded.

2) 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:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) arachis oil BP
c) a 50% w/v formulation of arachis oil BP in Freund's Complete Adjuvant/distilled water 1:1.
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.

B. CHALLENGE EXPOSURE
Shortly before treatment on Day 21, an area of 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), saturated with the test material at the maximum non-irritant concentration (25% w/w in arachis oil BP) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape (BLENDERM. approximate size 40 mm X 50 mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 10% w/w in arachis oil BP was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm X 75 mm) wound in a double layer around the torso of each animal.
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 to remove residual material.
Prior to the 24-hour observation 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 oedma was quantified using the Draize scale.
Any other reactions were also recorded.

OBSERVATIONS:
The bodyweight of each animal was recorded at the start and end of the study.

Challenge controls:
5 male Dunkin-Hartley guinea pigs were used as a vehicle control group.
During challenge they received 25% and 10% w/w of the test material in arachis oil BP
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole (10% in arachis oil BP for intradermal induction; 50% in acetone:PEG 400 (70:30) for topical induction; 50% and 25% in acetone:PEG 400 (70:30) for challenge)

Results and discussion

Positive control results:
90% (9/10) of the animals responded positively.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% w/w of test material in arachis oil BP
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Very slight erythema was noted at the challenge sites of two test group animals. Residual test material around treatment site was noted in two test group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% w/w of test material in arachis oil BP. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: Very slight erythema was noted at the challenge sites of two test group animals. Residual test material around treatment site was noted in two test group animals..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% w/w of test material in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% w/w of test material in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10% w/w of test material in arachis oil BP
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Very slight erythema was noted at the challenge sites of two test group animals. Residual test material around treatment site was noted in one test group animal.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% w/w of test material in arachis oil BP. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: Very slight erythema was noted at the challenge sites of two test group animals. Residual test material around treatment site was noted in one test group animal..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10% w/w of test material in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Desquamation was noted in one test group animal.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% w/w of test material in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Desquamation was noted in one test group animal..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% w/w of test material in arachis oil BP
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Residual test material around treatment site was noted in one control group animal.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% w/w of test material in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Residual test material around treatment site was noted in one control group animal..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% w/w of test material in arachis oil BP
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% w/w of test material in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10% w/w of test material in arachis oil BP
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10% w/w of test material in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10% w/w of test material in arachis oil BP
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10% w/w of test material in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 5.0.
Group:
positive control
Dose level:
50% and 25% of 2-Mercaptobenzothiazole in acetone:PEG 400 (70:30)
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
other: Group: positive control. Dose level: 50% and 25% of 2-Mercaptobenzothiazole in acetone:PEG 400 (70:30). No with. + reactions: 9.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Main study:

Skin reactions observed after intradermal induction:

Well-defined erythema was noted at the intradermal induction sites of all test group animals at the 24 -hour observations with very slight to moderate to severe erythema at the 48 -hour observation.

Very slight erythema was noted at the intradermal induction sites of all control group animals at the 24 and 48 -hour observations.

Skin reactions observed after topical induction:

Well-defined erythema and very slight to slight oedema were noted at the induction sites of nine test group animals at the 1 - hour observation. Very slight to well-defined erythema and incidents of very slight oedema were noted at the induction sites of eight test group animals at the 24 -hour observation. Other reactions noted in test group animals were bleeding from induction site, crust formation, superficial cracking of the epidermis, small superficial scattered scabs and a hardened light brown coloured scab.

Skin reactions prevented the accurate evaluation of erythema and oedema at the induction site of one test group animal at the 1 and 24 -hour observations.

No skin reactions were noted at the treatment sites of control group animals at the 1 and 24 -hour observations.

Skin reactions observed after topical challenge:

- 25% w/w in arachis oil BP: positive skin responses (very slight erythema - grade 1) were noted at the challenge sites of two test group animals at the 24 -hour observation. No skin reactions were noted at the challenge sites of test group animals at the 48 -hour observation. Residual test material was noted at the challenge sites of two test group animals at the 24 -hour observation.

No skin reactions were noted at the challenge sites of control group animals at the 24 and 48 -hour observations. Residual test material was noted at the challenge site of one control group animal at the 24 -hour observation.

- 10% w/w in archis oil BP: positive skin reactions (very slight erythema - grade1) were noted at the challenge sites of two test group animals at the 24 -hour observation. Desquamation was noted at the challenge site of one test group animal at the 48 -hour observation. Residual test material was noted at the challenge site of one test group animals at the 24 -hour observation.

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

Please refer to "Attached background material" below for further information on the results of the topical challenge.

Observations

- Body weight: bodyweight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material is not considered to be a skin sensitiser. The test material produced a 20% (2/10) sensitisation rate.
According to 67/548/EC and subsequent regulations,dipotassium hexafluorozirconate is not classified as a skin sensitiser.
According to the EC Regulation No. 1272/2008 and subsequent regulations, dipotassium hexafluorozirconate is not classified as a skin sensitiser.