Bismuth chloride oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Mar - 23 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

different evaluation of cellular proliferation (cell count measurement instead of incorporation of 3H-methyl thymidine)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

6 Juli 2012

Deviations:

yes

Remarks:

different evaluation of cellular proliferation (cell count measurement instead of incorporation of 3H-methyl thymidine)

Test guideline 3

Qualifier:

according to guideline

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. QA statement)

Remarks:

Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Freie und Hansestadt Hamburg

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bismuth hydroxide nitrate oxide

EC Number:

215-136-8

EC Name:

Bismuth hydroxide nitrate oxide

Cas Number:

1304-85-4

Molecular formula:

Bi5H9N4O22

IUPAC Name:

Bismuth hydroxide nitrate oxide

Test material form:

solid: particulate/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 ± 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:

10%, 25% and 50% w/w

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)

Results

Results 1

Key result

Parameter:

SI

Value:

1.181

Test group / Remarks:

Bismuth hydroxide nitrate oxide at concentrations of 10%

Results 2

Key result

Parameter:

SI

Value:

1.145

Test group / Remarks:

Bismuth hydroxide nitrate oxide at concentrations of 25%

Results 3

Key result

Parameter:

SI

Value:

1.246

Test group / Remarks:

Bismuth hydroxide nitrate oxide at concentration of 50%

Remarks on result:

other:

Remarks:

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.

Cellular proliferation data / Observations:

Treatment with the test item at concentrations of 10% or 25% did not reveal statistical significantly increased values for lymph node cell count. Treatment with a concentration of 50% revealed indeed a statistical significantly increased value, 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.
No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

Any other information on results incl. tables

See tables attached under 'background material'.

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 node cell count. Treatment with a concentration of 50% revealed indeed a statistical significantly increased value, 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.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified