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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:
The study was performed between 2002-04-17 and 2002-05-25. Report signed off 2002-12-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997)

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:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
443-510-2
EC Name:
-
Cas Number:
738587-10-5
Molecular formula:
C26H14N10Ni2O18S4 as free acid
IUPAC Name:
dinickel(2+) sodium 3-carboxy-5-[(1E)-2-(7-oxido-2,6-disulfonaphthalen-1-yl)diazen-1-yl]-1H-1,2,4-triazol-1-ide 3-carboxy-5-[(1E)-2-(7-oxido-2-sulfo-6-sulfonatonaphthalen-1-yl)diazen-1-yl]-1H-1,2,4-triazol-1-ide

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
21 Male and female albino Dunkin Hartley guinea pigs were supplied by two different reputable suppliers. The animals used in the intradermal and topical induction sighting studies were obtained from one supplier and those for the main study were obtained from a different supplier. This was not considered to affect the purpose or integrity of the study. Females were nulliparous and non-pregnant. After an acclimatisation period of at least 5 days, each animal was selected at random and given an unique number within the study which was written on a small area of clipped rump using a black indelible marker pen. At the start of the study the animals were in the weight range of 300 to 450g and were eight to twelve weeks old.

The animals were housed singly or in pairs in solid floor polypropylene cages furnished with woodflakes. Free access to mains tap water and food (Certified Guinea Pig diet) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to acheive limits of 17 to 23 deg. C and 30 to 70% respectively. Any occasional deviation from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12h continuous light and 12h darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Remarks:
BP
Concentration / amount:
Concentration of test material and vehicle used at induction:
Intradermal: 10% w/w in arachis oil
Topical: 75% w/w in arachis oil

Concentration of test material and vehicle used for each challenge:
Topical challenge:

a) 75% in arachis oil

b) 50% in arachis oil
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Remarks:
BP
Concentration / amount:
Concentration of test material and vehicle used at induction:
Intradermal: 10% w/w in arachis oil
Topical: 75% w/w in arachis oil

Concentration of test material and vehicle used for each challenge:
Topical challenge:

a) 75% in arachis oil

b) 50% in arachis oil
No. of animals per dose:
Number of animals in test group: 10
Number of animals in negative control group: 5
Details on study design:
SELECTION OF TEST CONCENTRATIONS
Intradermal induction - Intradermal injections of 0.1ml/injection site on the clipped shoulder of 2 guinea pigs using 5%, 10% and 25% w/w in arachis oil BP. The degree of erythema at the injection sites was assessed approximately 24, 48, 72h and 7 days after injection. 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.

Topical Induction - Two guinea pigs (intradermally injected with Freunds Complete Adjuvant 9 days earlier) were treated with 4 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 48h. The degree of erythema and oedema was evaluated approximately 1, 24 and 48h after dressing removal. The highest concentration producing only mild to moderate dermal irritatiom was selected for the topical induction stage of the main study.

Topical challenge - Four preparations of the test substance (75%, 50%, 25% and 10% w/w in arachis oil BP) were applied to the clipped flanks of 2 guinea pigs under occlusive dressings for an exposure period of 24h. These guinea pigs were not 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 eveluated approx. 1, 24 and 48h after dressing removal. The highest non-irritant concentration of the test substance and one lower concentration were selected for the topical challenge of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal
- No. of exposures: A row of 3 injections (0.1ml each) each side of the mid-line in the test area of a) Freunds Complete Adjuvant plus distilled water in the ratio 1:1 b) a 10% w/w formulation of the test material in arachis oil BP c) a 10% w/w formulation of the test material in a 1:1 preparation of Freunds Complete Adjuvant plus distilled water.
- Test Group: 10 guinea pigs
- Control group: 5 guinea pigs
- Site: Shoulder region shorn with clippers.

Topical
- No. of exposures: One topical application on the same shoulder region used for intradermal injections using a filter paper patch loaded with 75% w/w in arachis oil BP.
- Exposure period: the occlusive dressing was kept in place for 48h.
- Test Group: 10 guinea pigs
- Control group: 5 guinea pigs
- Site: Sholder region shorn with clippers
- Duration: The degree of erythema and oedema was quantified 1 and 24h after removal of the patch.

B. CHALLENGE EXPOSURE
- No. of exposures: On day 21 a patch loaded with the test substance was applied to the shorn flanks of each animal
- Exposure period: Patch was removed after 24h.
- Test Group: 10 guinea pigs
- Control group: 5 guinea pigs
- Site: Both flanks
- Concentrations: 75% and 50% w/w in arachis oil BP.
- Evaluation (h after challenge): 24 & 48h after challenge patch removal the degree of erythema and oedema was quantified using the scale for erythema from modified OECD test guideline 406 & Method B6 and the scale for oedema from Draize (1997).
Challenge controls:
5 Guinea Pigs

The intradermal induction was performed using an identical procedure to that used for the test animals except that the test material was omitted from the intradermal injections. Injection b) was the vehice alone, injection c) was a 50% formulation of the vehicle in a 1:1 preparation of Freunds Complete adjuvant plus ditilled water. Similarly the topical induction procedure was indentical to that used for the test animals except that the test material was omitted.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Red coloured staining noted at topical challenge site
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Red coloured staining noted at topical challenge site.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Red coloured staining noted at topical challenge site
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Red coloured staining noted at topical challenge site.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Red coloured staining noted at topical challenge site
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Red coloured staining noted at topical challenge site.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Red coloured staining noted at topical challenge site
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Red coloured staining noted at topical challenge site.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
75 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Red coloured staining noted at topical challenge site
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 75 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Red coloured staining noted at topical challenge site.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Red coloured staining noted at topical challenge site
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Red coloured staining noted at topical challenge site.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
75 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Red coloured staining noted at topical challenge site
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 75 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Red coloured staining noted at topical challenge site.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Red coloured staining noted at topical challenge site
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Red coloured staining noted at topical challenge site.

Any other information on results incl. tables

Preliminary sighting studies

Maximum concentration not causing irritating effects in preliminary test: 75 %


Main Study

Signs of irritation during induction:

Intradermal:Red-coloured staining, which prevented evaluation of skin reactions, was noted at the intradermal induction sites of all test animals. No skin reactions were noted at the intradermal induction sites of control group animals.

Topical: Red-coloured staining, which prevented evaluation of skin reactions, was noted at the topical induction sites of all
test groups. Discrete or patchy erythema was noted at the topical induction sites of control group animals.

Evidence of sensitisation of each challenge concentration:
Number of animals showing evidence of sensitisation at each challenge concxentration: 0/9

Other observations:
Red-coloured staining was noted at the challenge sites ofall test and control group animals during the study. This
did not affect evaluation of skin reactions.

One test group animal was killed for humane reasons due to ill health on day 9. The absence of this animal was
considered not to affect the purpose or integrity of the study.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information not classified Criteria used for interpretation of results: EU
Conclusions:
The test material produced a 0% (0/9) sensitisation rate and was classified as a non-sensitiser to guinea pig skin under the conditions of the test. The test material does not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC or Regulation (EC) No. 1272/2008.
Executive summary:

Introduction

The study was performed to assess the contact sensitisation potential of the test substance in the albino guinea pig. The method was designed to meet the requirements of the following:

OECD Guidelines for the Testing of Chemicals No. 406 'Skin Sensitisation' (adopted 17 July 1992)

Commission Directive 96/54/EC Method B6 Acute Toxicity (Skin Sensitisation)

Results and Discussion

The test substance produced a 0% (0/9) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC or Regulation (EC) No. 1272/2008.