Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2012 - 17 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 442b with a small deviation in the weight of the test animals used. This deviation has no impact on the outcome of the study. The study is therefore reliable without restriction.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
yes
Remarks:
Test animals 20-21 g instead of 21-25 g.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqueous solution
Details on test material:
- Name of test material (as cited in study report): Zirconium acetate solution
- Substance type: Unknow
- Physical state: Liquid (colourless solution)
- Lot/batch No.: 12/228
- Expiration date of the lot/batch: August 2013
- Storage condition of test material: Room temperature in the dark
- Solution contains 40.7% of zirconium acetate anhydrous (22 Wt % expressed as ZrO2+HfO2)
- Purity on dry content basis is > 99%

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/JN
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 21-25 g
- Housing: Individually housed in Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm with nesting material.
- Diet (e.g. ad libitum): ad libitum4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Range-finding test:
5 %, 10%, 25%, 50%, 100% w/w

Main test:
25, 50, 100% w/w

Concentrations were prepared in vehicle with the test item as supplied. No correction factor was applied.
No. of animals per dose:
Range-finding test: 1

Main test: 6
Details on study design:
RANGE FINDING TESTS:
- Irritation: excessive ear thickness > 25% at 100 %

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: increases in cell proliferation of draining lymph nodes

TREATMENT PREPARATION AND ADMINISTRATION:
- 25 µL test item to dorsal ear surface days 1, 2, and 3
- Day 5; A solution of BrdU, at a concentration of 10 mg/mL in physiological saline (0.9% NaCl) was administered at the dose volume of 0.5 mL/animal; Intraperitoneal injection, using a 25 G needle and a plastic graded syringe of a suitable volume.
- Asphixiation by carbon dioxide 24 hr after injection of BrdU.
- Auricular lymph nodes excised, drained and pooled for each group, and 1 mL PBS added to pool.
A single cell suspension of lymph node cells (LNC) was prepared from each animal by gentle mechanical disaggregation and passage through a 70 µm nylon mesh.
The suspensions thus obtained were centrifuged and the supernatant resuspended in 20 mL of 2% BSA-PBS.
BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001), according to manufacturer instructions.
Briefly, 100 μL of the LNC suspension was added to the wells of a flat-bottom 96-well microplate in triplicate. After fixation and denaturation of the LNC, 100 μL of anti-BrdU antibody labelled with peroxidase was added to each well and allowed to react.
Subsequently the anti-BrdU antibody was removed by washing and 100 μL of the substrate solution was then added and allowed to produce chromogen.
The reaction was finally stopped by adding 25 μL of Stop solution (1 M H2SO4).
Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.

Results and discussion

Positive control results:
In the group treated with the positive control item, a stimulation index (SI) of 5.83 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: Vehicle: Not applicable Test item: main study No increases in cell proliferation of draining lymph nodes were observed at any of the concentrations investigated. 25%-1.02 50%-1.06 100%-0.72

Any other information on results incl. tables

Range finding test:

- No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any tested concentrations.

- The evaluation of visible reactions showed no erythema at any of the tested concentrations.

- The evaluation of ear thickness indicated that the reaction was acceptable (increase of less than 25% compared to Day 1) at the concentration of 100% (undiluted test item).

- The evaluation of ear punch weight indicated that the reaction was acceptable (increased less than 25% with respect to the negative control) at all the investigated concentrations.

Main study:

- No mortality

- Clinical Observations: No significant clinical signs were recorded in animals treated at any dose level.

- Body weights: weight decreases/reduced body weight gains observed in some animals from all groups (including controls). They were considered of low entity and/or incidental and thus not toxicologically relevant.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results obtained in this study indicate that zirconium acetate solution (40.7% as zirconium acetate, anhydrous) does not elicit any sensitisation response in mice following dermal exposure. According to EU CLP (Council Regulation (EC) No. 1272/2008 and subsequent revisions), the substance tested under these experimental conditions does not need to be classified.