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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:
key study
Study period:
05/11/2014-29/11/2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
variations from target temperature and relative humidity; no impact on animal health or outcome of the study and interpretation of results
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The guinea pig maximisation test is considered the most relevant test method for substances such as zirconium dinitrate oxide because false positive or inconclusive results have been obtained with similar substances when tested in the local lymph node assay.

Test material

Constituent 1
Chemical structure
Reference substance name:
Zirconium dinitrate oxide
EC Number:
237-529-3
EC Name:
Zirconium dinitrate oxide
Cas Number:
13826-66-9
Molecular formula:
N2O7Zr
IUPAC Name:
zirconium dinitrate oxide
Test material form:
solid
Details on test material:
Name of test material (as cited in study report): zirconium dinitrate oxide

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
female
Details on test animals and environmental conditions:
- Source: LAB-ÁLL Bt. Budapest, 1174 Hunyadi u. 7., Hungary
- Justification of strain: The guinea pig is the standard species used for skin sensitisation studies.
- Body weight range at the beginning of the study: 353 – 367 g
- Age of animals at arrival: young adult, 4 weeks
- Acclimatization time: 7 days
- Cage type: animals were housed in macrolon cages size IV, with 5 animals/cage to allow socialization
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 23.2 °C
- Humidity (%): 24 - 61%
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: 1% w/v methylcellulose solution formulated in PBS
Concentration / amount:
0.25% w/v
Day(s)/duration:
day 1 of treatment
Route:
epicutaneous, occlusive
Vehicle:
other: 1% w/v methylcellulose solution formulation in PBS
Concentration / amount:
25% w/v
Day(s)/duration:
day 8 of treatment, 48 h of exposure
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: 1% w/v methylcellulose solution formulated in PBS
Concentration / amount:
25%
Day(s)/duration:
day 22 of treatment, 24 hours of exposure
No. of animals per dose:
Preliminary test: 14 female animals

Main test:
Intra-dermal induction exposure:
- control group: 5
- test group: 10
Dermal induction exposure:
- control group: 5
- test group: 10
Challenge exposure:
- control group: 5
- test group: 10
Details on study design:
RANGE FINDING TESTS:
Test item concentrations:
- intra-dermal injection: 0.1, 0.25, 0.5, 1, 2.5 and 5% (w/v)
- dermal application: 10, 25, 50 and 75% (w/v)
Time of observations for local effects:
- 1, 4, 24, 48 and 72 hours after the intra-dermal treatment
- 1, 24, 48 and 72 hours after the patch removal in case of dermal treatment
Exposure time:
- For the dermal treatment: 48 hours. During the 48 h exposure, the skin reaction was examined 1, 4 and 24 h after application of the test item by opening and rebinding the patch to the same place. Where the skin reaction was severe (i.e. 75% (w/v)), the bandage was removed 24 h after the application.
Test item application and observations:
- For the intra-dermal application, 0.1 mL per concentration was injected intra-dermally into the hair free skin of the flanks. One concentration was injected on the right side and another concentration on the left side of the animals. Each concentration was injected in duplicate. Two or three animals were used per concentration.
- For the dermal application, approximately 0.5 mL per concentration was applied onto the clipped and shaved skin of the animals. A closed patch exposure was performed by means of an occlusive bandage using similar treatment procedures as for the main study. One concentration was used on the right side and another concentration on the left side of the animals. One - three animals were used per concentration. The observation was started with one animal/concentration. Depending on the observation, the preliminary work was stopped or continued with involving new animals. Time of exposure was 48 or 24 hours. Where the skin reaction was severe (i.e. 75% (w/v)), the bandage was removed 24 h after the application.

MAIN STUDY
A. Intradermal induction exposure
- Control group: 5 animals
- Test group: 10 animals
Concentration of the test item:
- Control: 1% methylcellulose in PBS
- Test group: 0.25% (w/v) in 1% methylcellulose in PBS
Day of treatment: 1
Site of treatment: shoulder
Time of observations: 24 hours after the treatment

B. Dermal induction exposure
- Control group: 5 animals
- Test group: 10 animals
Concentration of the test item:
- Control: 1% methylcellulose in PBS
- Test group: 25% (w/v) in 1% methylcellulose in PBS
Day of treatment: 8
Site of treatment: shoulder
Duration of exposure: 48 hours
Time of observations: 1, 24, 48 and 72 hours after the patch removal

C. Challenge exposure
- Control group: 5 animals
- Test group: 10 animals
Concentration of the test item:
- Control: 25% (w/v) in 1% methylcellulose in PBS
- Test group: 25% (w/v) in 1% methylcellulose in PBS
Day of treatment: 22
Site of treatment: flank
Duration of exposure: 24 hours
Time of observations: 24 and 48 hours after the patch removal
Challenge controls:
As described in details on study design
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

Positive control results:
Challenge with reference item 2-mercaptobenzothiazole resulted in a positive response in test animals previously sensitised. The net response values at the 24 and 48 hour observations represented an incidence rate of 90% and 80% and net score values of 0.90 and 0.80 respectively. In the control animals no visible changes were found either at the 24 or 48 hour examinations following challenge with the reference item.
The dermal scores represented discrete erythema (score 1) developed on the skin of sensitised guinea pigs.
On the basis of the results of the reliability check study, the reference item 2-mercaptobenzothiazole was classified as a skin sensitizer. This demonstrated that the experimental procedure and the test system were appropriate.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% w/v in 1% w/v methylcellulose in PBS
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reactions were found
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% w/v in 1% w/v methylcellulose in PBS
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reactions were found
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% w/v in 1% w/v methylcellulose in PBS
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reactions were found
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% w/v in 1% w/v methylcellulose in PBS
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reactions were found
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50% w/v (2-mercaptobenzothiazole)
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
discrete erythema (score 1) on the skin of the animals.
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50% w/v (2-mercaptobenzothiazole)
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
discrete erythema (score 1) on the skin of the animals.
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

The Guinea Pig Maximisation Test (OECD guideline 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. Although zirconium dinitrate oxide is soluble in pure water (accompanied by a pH decrease), it is converted to insoluble forms in physiologically relevant test media such as the sweat on the skin.

Mortality/clinical observations/body weight:

No treatment related mortality was observed during the study.

There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.

Body weight was recorded at randomisation, then at least weekly, including day 25 prior to euthanasia. There were no notable differences between the test animals group and the control group.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the conditions of the present assay the test item zirconium dinitrate oxide was shown to have no sensitisation potential and therefore it is classified as a non-sensitiser, according to current EU-regulations.