Calcium tin trioxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been conducted according to standard testing guidelines, and is considered to be adequate, relevant and reliable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442B

Deviations:

no

Principles of method if other than guideline:

OECD 442B has been implemented in July 2010. Similar to the LLNA of OECD 429, the LLNA: BrdU-ELISA studies the induction phase of skin sensitization and provides quantitative data suitable for dose-response assessment. Furthermore, an ability to detect skin sensitizers without the necessity for using a radiolabel for DNA eliminates the potential for occupational exposure to radioactivity and waste disposal issues

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Calcium tin trioxide

EC Number:

234-585-0

EC Name:

Calcium tin trioxide

Cas Number:

12013-46-6

Molecular formula:

Ca.O3Sn

IUPAC Name:

calcium oxostannanebis(olate)

Details on test material:

- Name of test material (as cited in study report): Calcium Stannate
- Substance type: solid
- Physical state: wet powder, white odorless, inorganic
- Analytical purity: 98%
- Impurities (identity and concentrations): confidential details on test material
- Purity test date: July 9, 2010
- Lot/batch No.: 61341-100601M000000
- Expiration date of the lot/batch: not assessed
- Stability under test conditions: stable
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany (breeder); delivery from Harlan, Horst, Netherlands
- Age at study initiation: approx. 11 weeks
- Weight at study initiation: 21.8 ± 1.8 g at start of the main experiment
- Housing: single housed in Makrolon®-cages Type III, non-barriered conditions
- Diet: ad libitum (food of type "1324" from Altromin, Lage, Germany as pelleted diet - batch No. 1323; expiry date 2010-11-24)
- Water: ad libitum (normal tap water from municipal source - SWK AQUA GmbH, Krefeld, Germany)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 -25°C
- Humidity (%): 30 % - 70 %
- Air changes (per hr): 8 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours dimmed light, 12 hours dark

IN-LIFE DATES: From 2010-09-07 TO 2010-09-16

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

CAS No. 57-55-6, Carl Roth GmbH&Co KG; Lot 13894655, Expiry 2013-12-31

Concentration:

In a pilot experiment the following three concentrations of the test substance in vehicle were selected: 50% (w/w), 25% (w/w) and 10% (w/w).
As no excessive local irritation and no ear thickness increase was observed in the pilot experiment the same concentrations were selected for the main experiment.

No. of animals per dose:

Two animals per dose group were used in the pilot study.
Four animals per group were treated in the main study.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: acceptable up to 50 % (w/w) preparation in vehicle.
- Irritation: only slight local irritation in the highest concentration group but ear thickness values were unaffected
- Lymph node proliferation response: not tested

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Irritation: only slight local irritation in the positive control group and t50% concentration group; ear thickness values were unaffected.
- Name of test method: LLNA:BrdU-ELISA (ELISA kit from Roche Diagnostics GmbH, Mannheim, Germany - Lot 12015100, Expiry March 2012)
- Criteria used to consider a positive response: The test substance is regarded as positive when at least with one concentration the
SI is ≥ 1.6. The test is considered valid if the positive control causes a positive result, i.e. SI ≥ 1.6.


TREATMENT PREPARATION AND ADMINISTRATION:
Day 1: the ear thickness of both ears, the weight of each animal and any clinical observation were recorded prior to the application of the test or control substances. 25 µl of the selected test substance concentration, reference substance or vehicle was applied to the dorsal side of each ear.
Day 2: any clinical observation was recorded; the application procedure carried out on day 1 was repeated.
Day 3: any clinical observation was recorded ; the application procedure carried out on day 1 was repeated.
Day 4: no treatment.
Day 5: 5-bromo-2-deoxyuridine (BrdU) was solved in 0.9 % saline solution in a concentration of 10 mg/mL and filter sterilised (0.22µm filter).
0.5 mL of the BrdU-solution was injected intraperitoneal to each mouse (= 5 mg/mouse).
Day 6: the thickness of both ears and the weight of each animal were recorded.
Approx. 24 hours after BrdU injection the animals were sacrificed and the draining auricular lymph nodes from each mouse ear were exercised. Both lymph nodes of one animal were pooled and further processed for each animal separately in phosphate buffered saline (PBS).
From each mouse a single-cell suspension of lymph node cells was prepared by gentle mechanical disaggregation by the use of a disposable plastic pestle to crush the lymph nodes followed by passage through a 74 µm nylon mesh. Cell counts with the negative control group revealed a mean cell number of 3.27 x 106 cells/ml. The target volume of the cell suspensions was adjusted by 1:10 dilution with PBS to the optimised volume for the determination of cellular proliferation (= 3.3 x 104 cells/100 µl). 100 µl of the lymph node cell suspension was added to the wells of a flat-bottom microplate in triplicate. The microplate was centrifuged at 300 g for 10 minutes to affix the cells to the bottom. The PBS was carefully removed by aspiration by pipetting and the cells were dried at approximately 60°C in an oven for 1 h. The dried cells were stored refrigerated overnight until cell proliferation measurement on the next day.
Determination of the cellular proliferation was performed by the measurement of BrdU content in the DNA of the lymphocytes. BrdU was measured colorimetric by ELISA using a commercial kit from Roche Diagnostics GmbH, Mannheim, Germany (Lot 12015100, Expiry March 2012) according to the procedure given in the instruction manual.
After fixation and denaturation anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody excess was removed by washing and the substrate solution was then added and allowed to produce chromogen. Absorbance at 360 nm with a reference wavelength of 485 nm was then measured.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The BrdU-labelling index was calculated for each mouse from the measured optical density (OD) taking into account the dual wave difference data and respective blank values. Results for each treatment group were expressed as the mean Stimulation Index SI. The SI was derived by dividing the mean BrdU labelling index per mouse within each test substance group and the PC group by the mean BrdU labelling index for the negative control group (vehicle group).

Results and discussion

Positive control results:

Only slight symptoms of local irritation at the ears of the positive control group and the highest test substance concentration group were observed.
The positive control showed a clear positive response (SI ≥ 1.6) indicating the adequate function of the test system.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

For each treatment group the mean Stimulation Index SI was calculated from the BrdU-labelling indices for each mouse: Negative control group (vehicle): SI = 1.00 Positive control group (HCA 25 %) SI = 1.84 Test group 10 % Calcium Stannate SI = 1.23 Test group 25 % Calcium Stannate SI = 1.19 Test group 50 % Calcium Stannate SI = 1.29

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not applicable (ELISA method)

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

From the test results it is concluded that the test substance Calcium stannate has no sensitising properties under the given experimental conditions.

Executive summary:

The test substance Calcium Stannatewas tested regarding its skin sensitisation potential. A Local Lymph Node Assay (LLNA) with CBA/Ca mice was performed and 5-bromo-deoxyuridine (BrdU) was used to measure lymphocyte proliferationaccording to OECD No. 442B method (Local Lymph Node Assay: BrdU-ELISA).Propylene glycol was used as vehicle to suspend the solid preparation. The maximum concentration which was feasible for application was 50 % (w/w) of the test substance in vehicle.The maximum compatible dose was determined in a pilot experiment.

In the main experiment the test substance was applied as 10 % (w/w), 25 % (w/w) and 50 % (w/w) preparation in vehicle.In addition a concurrent negative control group (NC) treated only with the vehicle and a positive control group (PC) treated with 25 % (v/v) hexyl cinnamic aldehyd (HCA) in acetone/olive oil (3+1) as reference substance was used. Four animals per group were treated.25 µl of the selected test substance preparation, reference substance, or vehicle have been applied to the dorsal side of each ear for three consecutive days. No treatment took place on day 4.On day 5 each mouse received BrdU-solution injected intraperitoneally (5 mg BrdU/mouse). The animals were sacrificed on day 6 and the draining auricular lymph node from each mouse ear was exercised. A single-cell suspension of the lymph node cells was prepared separately for each animal.

Determination of the cell proliferation was performed by measurement of BrdU content in the DNA of the lymphocytes. BrdU was quantified by ELISA using a commercial kit according to the procedure given in the instruction manual.The ear thickness of both ears and the weight of each animal was determined prior to the first application and at termination on day 6. Animals were observed for any visible clinical symptom of toxicity.  Body weight development of the animals was as expected and within normal ranges. Increases of the ear thickness following test substance application indicating strong irritation properties of the used test substance concentrations or symptoms of systemic toxicity were not observed. Only slight symptoms of local irritation at the ears of the highest test substance concentration group and the positive control group were observed.

For each treatment group the mean Stimulation Index SI was calculated from the BrdU-labelling indices for each mouse. The Stimulation Indices for Calcium Stannate were lower than 1.6 for all tested concentrations. The positive control showed a clear positive response (SI≥1.6) indicating the adequate function of the test system. From the test results it is concluded that the test substance Calcium stannate has no sensitising properties under the given experimental conditions.