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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March 2010 to 23 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products, CPMP/SWP/2145/00, adopted March 1, 2001.
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
signed by Freie und Hansestadt Hamburg, Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz (2009-11-12)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Bismuth hydroxide nitrate oxide
- Molecular formula (if other than submission substance): Bi5O(OH)9(NO3)4
- Molecular weight (if other than submission substance): 1461.99 g/mol
- Substance type: pure substance
- Physical state: solid, white, cloddy powder

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NMRI mice supplied by Charles River Deutschland GmbH were used in this experiment.
- Age at study initiation: 8 weeks
- Weight at study initiation: 22 - 28 g
- Housing: The animals were kept singly in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. Granulated textured wood was used as bedding material for the cages. The cages were changed and cleaned once a week.
- Diet: ad libitum; commercial diet ssniff R/M-H V1534. Food residue was removed.
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Humidity: 55% ± 15% (maximum range)
- Photoperiod: 12/12 hours dark/light cycle

Study design: in vivo (LLNA)

Vehicle:
other: a mixture of acetone / olive oil (3+1, v/v)
Concentration:
The test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 μL/ear.
No. of animals per dose:
30 (5 groups of 6 female animals each)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The vehicle (a mixture of acetone/olive oil) .was selected on the basis of maximising the test concentrations and solubility whilst producing a solution/suspension suitable for application of the test item.
- Irritation: A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10, 25 and 50% of Bismuth hydroxide nitrate oxide in acetone/olive oil (3+1 v/v), w/w, were examined.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
At the start of the experiment the mice were weighed and assigned to each of the 5 groups by a randomisation program (block size n = 6).
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The so-called stimulation (or LLN-) indices to determine the sensitising potential, were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones. An index above 1.4 is considered positive.
- Other: Furthermore, the stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.

TREATMENT PREPARATION AND ADMINISTRATION:
The experimental schedule of the assay was as follows:
- Day 1: The weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer. Open application of 25 μL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3: The application procedure carried out on day 1 was repeated.
- Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis): Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer. Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance. Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers would have been determined according to the Nalimov test.
In addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

Results and discussion

Positive control results:
The positive control group caused the expected increases in lymph node cell count (statistically significant at p ≤ 0.01). Even though also the lymph node weight was increased in the positive control (indicating factor to be a substance with irritating properties) the no observed “acute skin reaction” indicates that the positive control group has sensitising properties. The values for the stimulation index of lymph node cell count was 1.94. Therefore, the study could be regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Treatment with Bismuth hydroxide nitrate oxide at concentrations of 10% or 25% did not reveal statistical significantly increased values for lymph node cell count (SI of 1.181 and 1.145, respectively). Treatment with a concentration of 50% revealed indeed a statistical significantly increased value (SI of 1.246), but all stimulation indices for the lymph node cell count were beneath the threshold value of 1.4. Hence, the test item is classified as not sensitising. The threshold level for the ear weight of 1.1 was not exceeded and the lymph node weights were not increased in a dose-related way, i.e. no irritating properties were noted. A table with more details is attached to this endpoint study record.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method. The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by a European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility (iv) faster results.

Any other information on results incl. tables

No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In conclusion, under the present test conditions, Bismuth hydroxide nitrate oxide at concentrations of 10%, 25% or 50% (w/w) in acetone/olive oil (3+1, v/v) did not reveal any sensitising properties in the local lymph node assay and therefore should not be classified and labelled according to regulation (EC) No.: 1272/2008
Executive summary:

The purpose of this study was to determine the sensitising potential of Bismuth hydroxide nitrate oxide in the local lymph node assay in mice. The study was performed according to OECD 429.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method. 3 concentrations of Bismuth hydroxide nitrate oxide (10%, 25% and 50%, w/w) suspended in acetone/olive oil (3+1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. In addition, a positive control group (30% solution (v/v) of α-hexyl cinnamic aldehyde in acetone/olive oil (3+1, v/v)) was employed.

Treatment with Bismuth hydroxide nitrate oxide at concentrations of 10% or 25% did not reveal statistical significantly increased values for lymph nod cell count. Treatment with a concentration of 50% revealed indeed a statistical significantly increased value, but all stimulation indices for the lymph nod cell count were beneath the threshold value of 1.4. Hence, the test item is classified as not sensitising.