Registration Dossier

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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

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Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 March 2015 to 22 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
Environmental parameters:
A relative humidity lower than 30% was registered during 24 cumulative hours on 06 & 07 April
2015. The minimum value measured was 21%.
A temperature higher than 25°C was registered during some minutes on 16 April 2015. The maximum
value measured was 26°C
Temperatures lower than 19°C were registered during 24 cumulative hours on 07, 08 & 09 April 2015.
The minimum value measured was 17°C.
These deviations are considered as without impact on the conclusion of the study.
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
There is evidence that the presence of metal ions such as bismuth ions may cause a false positive result in the LLNA study (ICCVAM, 2009).

Reference: Revised Draft Assessment of the Validity of the LLNA for Mixtures, Metals, and Aqueous Solutions. Addendum No. 1 to the ICCVAM Report: The Murine Local Lymph Node Assay (LLNA): A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds (NIH Pub. No. 99-4494, March 2009). Available at: http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-app/BRDcomplete.pdf
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
The animals were housed in groups of 2 in polycarbonate containers. The flooring of the cages was
covered with dust-free wood shavings and the top fitted a stainless steel lid containing with a feeding
device and drinking device of 500 mL.
The containers are placed in an air-conditioned animal holding facility:
- Air recycling: at least 10 cycles per hour,
- Temperature: 22° C ± 3° C,
- Relative humidity: from 30 % to 70 %,
- Lighting: circadian cycle (12 hrs day / 12 hrs darkness).

Food and drink
The drinking water (tap water from public distribution system) and food (SAFE 106) were supplied freely.
Microbiological and chemical analyses of the water were carried out once every six months by the Eurofins IPL Atlantique (Bordeaux).

Thirty female albino guinea pigs of Dunkin-Hartley strain, supplied by HARLAN (Kreuzelweg 53, 5961 NM HORST The Netherlands) were 3 or 4 weeks old at the beginning of the main test.
Prior to the test, the animals were kept for a minimum acclimatization period of 5 days, under housing and diet conditions identical to those of the test.
Before the experimentation process, they were identified individually by marking with picric acid and by means of a numbered ring on the edge of one ear.
The animals were carefully shorn before each test item application:
- On the inter-scapular zone for the induction phase,
- On the dorso-lumbar zone for the challenge phase.
At least 3 hours before the first reading (challenge phase) they were shorn a second time in this dorsolumbar zone.
The animals were weighed at the beginning, before the second induction, during the first challenge and at the end of the study.

Animal welfare
The standard study plan related to this study was approved by the registered Ethics Committee No. 76.
The study was performed in accordance with the guidelines regarding the care and use of animals for experimental procedures:
- the European Communities Council Directive 2010/63/EU of 22 September 2010,
- the French Decree No. 2013-118 of 01 February 2013.
The animals were provided with suitable environmental enrichment (Tunnel).
The study was designed and was conducted to cause the minimum of suffering or distress to the animals, according the guidelines and to our internal animal welfare’s procedure. In particular, in case of suffering, the animal could be euthanized after decision of the Study Director.
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
Maximum Non Irritant Concentration (M.N.I.C.) determination: Bismuth subsalicylate diluted at 60%, 40%, 20% and 10% in liquid paraffin.
Induction: Bismuth subsalicylate diluted at 60% in liquid paraffin
Challenge: Bismuth subsalicylate diluted at 60% and 30% in liquid paraffin
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
Maximum Non Irritant Concentration (M.N.I.C.) determination: Bismuth subsalicylate diluted at 60%, 40%, 20% and 10% in liquid paraffin.
Induction: Bismuth subsalicylate diluted at 60% in liquid paraffin
Challenge: Bismuth subsalicylate diluted at 60% and 30% in liquid paraffin
No. of animals per dose:
10 controls
20 in test group
Details on study design:
Preliminary study
Maximum Non Irritant Concentration (M.N.I.C.) determination:
This test was carried out with a reduced number of animals, for the purpose of determining the maximal item concentrations without risk of an irritant effect during the challenge phase.
Furthermore, this test evaluates the irritant potential of the test item, and defines, if possible, a mild to moderate irritant concentration during topical induction phase.
Three guinea pigs were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-layer Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for a period of 6 hours at 4 different concentrations: diluted at 60%, 40%, 20% and 10% in liquid paraffin.
The animals treated at the concentrations of 60%, 40%, 20% and 10% received 0.5 mL of the corresponding preparation.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings. The skin reaction was observed and recorded according to the grades described hereafter.
Washing of the skin after removal of the dressing was done with liquid paraffin.

Main study
GROUP 1 (negative control) : 10 female guinea pigs identified n° C9550 to C9559
GROUP 2 (treated) : 20 female guinea pigs identified n° C9560 to C9579
Note: The results of the 3 last positive groups (Reference substance: a-Hexylcinnamaldehyde CAS n° 101-86-0 - Tests n°16-18) carried out as method sensibility.

Induction phase
After shearing of the scapular zone, the 3 local applications were performed on D0, D7 and D15 during 6 hours under occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-layer Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide
hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M).
The animals of the control group received 0.5 mL of liquid paraffin and the animals of the treated group received 0.5 mL of the test item diluted at 60% in liquid paraffin.

Rest phase
The animals of both groups were left untreated for 13 days.

Challenge phase
The experimental procedure of this phase was identical for both groups Group 1 (Negative control) and Group 2 (Treated) according to this experimentation: on the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing (25mm x 25mm gauze patches hydrophilic Codex of 8-layer Gazin® from Lohmann & Rauscher held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M), was performed during 6 hours:
- One area containing 0.5 mL of the test item diluted at 60% in liquid paraffin (MNIC = maximal non irritant concentration) on the top of left flank, one area containing 0.5 mL of liquid paraffin the bottom of left flank and one area containing 0.5 mL of the test item diluted at 30% in liquid paraffin on the
right flank.

Macroscopic examinations and evaluation of cutaneous reactions
A macroscopic evaluation of the cutaneous reactions (erythema and oedema) was conducted and all the local or systemic reactions were recorded as Group 1 (Negative control) and Group 2 (Treated):
· Approximately 24 hours after removal of the occlusive dressing, the cutaneous reactions were observed and graded according to the scales, given below.
· Approximately 24 hours later (i.e. 48 hours after removal of the occlusive dressing), a second observation was made.
Grading scale
0 ....... No visible change
1 ....... Discrete or patchy erythema
2 ....... Moderate and confluent erythema
3 ....... Intense erythema and swelling
All the animals with scores above or equal to 1 during the challenge phase, were considered positive.

Interpretation of reactions
The percentage of animals that showed a sensitivity contact potential is calculated 24 and 48 hours after the removal of the occlusive dressings.
A comparison of the intensities and persistence of reactions at the test item challenge sites in the test and control animals permits identification of sensitisation reactions.
If the test item at the maximum non-irritant concentration produces reactions in test group animals at the 24 or 48 -hour readings, these reactions were attributed to skin sensitisation. This pre-supposes that no similar reactions were observed in the test material challenge sites of any of the control group animals. If irritation was observed in the control group animals, only reactions in the test group animals that exceed the most severe reaction seen in the control group animals were attributed to skin sensitisation. The results were expressed in terms of incidence and severity of responses, ie:
Incidence Score: The number of test group animals showing skin reactions greater than the most severe reaction observed in the control group animals, expressed as a fraction of the total number of test group animals.
Severity Score: The sum of the values assigned to the skin responses at the test item challenge sites of the test and control group animals, at each evaluation, divided by the number of animals in that group.
Challenge controls:
Challenge phase: One area containing 0.5 mL of the test item diluted at 60% in liquid paraffin (MNIC = maximal non irritant concentration) on the top of left flank, one area containing 0.5 mL of liquid paraffin (control) the bottom of left flank and one area containing 0.5 mL of the test item diluted at 30% in liquid paraffin on the right flank.
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde
Positive control results:
Positive Control with alpha-Hexylcinnamaldehyde Buehler Test – 3 applications: results trigger classification as Skin Sens. 1B under the CLP Regulation.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 60%, Challenge: 60%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induction: 60%, Challenge: 60%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 60%, Challenge: 60%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induction: 60%, Challenge: 60%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 60%, Challenge: 30%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induction: 60%, Challenge: 30%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 60%, Challenge: 30%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induction: 60%, Challenge: 30%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction:60%, Challenge 0%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induction:60%, Challenge 0%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 60%, Challenge: 0%
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induction: 60%, Challenge: 0%. No with. + reactions: 0.0. Total no. in groups: 19.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%, Challenge: 60%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Induction: 0%, Challenge: 60%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%, Challenge 60%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Induction: 0%, Challenge 60%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%, Challenge: 30%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Induction: 0%, Challenge: 30%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%, Challenge: 30%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Induction: 0%, Challenge: 30%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%, Challenge: 0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Induction: 0%, Challenge: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%, Challenge: 0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Induction: 0%, Challenge: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Preliminary study

MNIC determination:

No cutaneous reaction and no sign of systemic toxicity were noted 24 and 48 hours after the occlusive dressing at the concentrations of 60%, 40%, 20% and 10%.

In view of these results, the concentration selected was 60% for the 3 inductions of the main study and the concentrations selected were 60% (MNIC) and 30% (1/2 MNIC) for the challenge phase.

Main study:

Induction phase

No cutaneous reaction was recorded during the induction phase in the treated group.

No cutaneous reaction was recorded during the induction phase in the control group.

Challenge phase:

In the treatment group (treatment dose of 60% and 30%), no macroscopic cutaneous reactions attributable to allergy were observed 24 and 48 hours after the removal of the occlusive dressing.

In the control group (associated with the treatment dose of 60% and 30%), no cutaneous intolerance reaction was observed during the examination following the removal of the occlusive dressing.

No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (vehicle).

Weight evolution

The weight gain of control animals (Group 1) and treated animals (Group 2) is presented in tables 4 and 5, respectively.

No abnormalities and no differences in the body weight between the control and the treated group were observed.

Mortality

One treated animal was found dead on day 0.

Clinical signs

No abnormal clinical signs related to the administration of the test item were observed.

Interpretation of results:
GHS criteria not met
Conclusions:
In view of these results, under these experimental conditions, the test item Bismuth Subsalicylate is not a skin sensitiser.
In accordance with the Regulation EC No 1272/2008, the test item does not have to be classified. No signal word or hazard statement is required.
Executive summary:

The aim of the study was to evaluate the possible allergenic activity of the test item Bismuth Subsalicylate after topical administration in guinea pigs.

The induction phase was conducted with three topical applications of the test item Bismuth Subsalicylate diluted at 60% in liquid paraffin to 20 guinea-pigs with occlusive dressing and a 13-day rest phase. The challenge phase, conducted under an occlusive dressing for 6 hours, consisted of a single topical application of the test item at 60% and 30% or an application of a negative control (liquid paraffin). The experimental protocol was established from the O.E.C.D. guideline n°406 dated July 17th, 1992 and the method B.6 of the Council regulation No. 440/2008. One treated animal was found dead on day 0. No other mortality and no clinical signs were noted during the study. As no reactions were noted in the surviving 19 test group animals, the mortality did not affect the interpretation or reliability of the result.

In the treatment group (treatment dose of 60% and 30%), no macroscopic cutaneous reactions attributable to allergy were observed 24 and 48 hours after the removal of the occlusive dressing.

In the control group (associated with the treatment dose of 60% and 30%), no cutaneous intolerance reaction was observed during the examination following the removal of the occlusive dressing.

No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (vehicle).

In conclusion, in view of these results, under these experimental conditions, the test item Bismuth Subsalicylate is not a skin sensitiser.

In accordance with the Regulation EC No 1272/2008, the test item does not have to be classified. No signal word or hazard statement is required.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
19 March 2010 to 23 April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study. Rated as Klimisch 2 because it is a read-across study.
Justification for type of information:
Please see read-across justfication attached below.
Reason / purpose:
read-across source
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)
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
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.
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.
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.

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

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.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, however, limited data is provided.
Justification for type of information:
Read-across is justified based on the structural similarity between methyl salicylate and the subsalicylate component of bismuth subsalicylate.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
male/female
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 10% and 25%
No. of animals per dose:
4
Details on study design:
no data
Parameter:
SI
Remarks on result:
other: SI was < 3
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: no data
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions reported in this study, methyl salicylate was considered not to be sensitising.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication conducted according to scientifically acceptable methods.
Justification for type of information:
Read-across is justified based on the structural similarity between salicylic acid and the subsalicylate component of bismuth subsalicylate.
Reason / purpose:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
Method: Mouse ear swelling test. The test animals are initially injected twice with FCA (Freund’s complete Adjuvant) into the abdomen. Animals are then topically dosed with test material in solvent or solvent alone (controls) applied to the center of the shaved region on the abdominal skin. Topical application to the abdomen was repeated for three additional consecutive days. Seven days after the final topical application to the abdomen, the test item in solution was applied to the left ear of each animal (test and control) and the solvent was applied to the right ear. At both 24 and 48 hours after this challenge, the thicknesses of both ears measured.
GLP compliance:
not specified
Type of study:
mouse ear swelling test
Species:
mouse
Strain:
CF-1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 6-8 weeks
- Weight at study initiation: no information
- Housing: 5 per cage
- Diet : ad libitum, Purina Rodent Laboratory Chow 5001
- Water :ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no information
- Humidity (%): no information
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light): no information


IN-LIFE DATES: no information
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
10 %
Concentration was selected on the basis of solubility.
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
10 %
Concentration was selected on the basis of solubility.
No. of animals per dose:
Test group: 10-15 animals
Control group: 5-10 animals
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Day(s) of exposure: 1, 2 and 3
- Test groups: 100 µl of test item in vehicle
- Control group: 100 µl of vehicle
- Site: abdominal skin
- Concentrations: 10 %

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 10
- Test groups: 20 µl of test item in vehicle and 20 µl of vehicle
- Control group: 20 µl of test item in vehicle and 20 µl of vehicle
- Site: left and right ear
- Concentrations: 10 %
- Evaluation (hr after challenge): 24 and 48
Positive control substance(s):
no
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Salicylic acid did not induce sensitisation in mice in the mouse Ear Swelling Test (MEST).
% swelling = [(test ear thickness) / (control ear thickness)] x 100 = 102%
No mice were sensitised.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results of this study indicated that salicylic acid is not a skin sensitiser.
Executive summary:

Salicylic acid was tested using the method of Mouse Ear Swelling Test (MEST). The aim of this study was to develop and optimise a protocol for the MEST. To evaluate the performance of this final MEST study design, a battery of 72 test materials including salicylic acid was evaluated. The substance was tested in CF-1 female mice, 6 to 8 weeks old. The results of this study indicated that salicylic acid is not a skin sensitiser.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Modified LLNA study
Justification for type of information:
Read-across is justified based on the structural similarity between salicylic acid and the subsalicylate component of bismuth subsalicylate.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Proliferation was evaluated by light microscopy
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CD-1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles river, France
- Age at study initiation: 6-8 weeks

No further information.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 10%, 20%
No. of animals per dose:
4
Statistics:
One-way analysis of variance; pairwise comparison with control by Dunnett's procedure; homogeneity of variance by Levene's test; normality of residuals by normal probability plot and Shapiro-Wlik's test.
Key result
Parameter:
other:
Remarks on result:
other: See below for results

Statistically significant T-lymphocyte proliferation in the paracortex was detected in the short, but not in the long protocol. The maximum T/C ratio was 1.74. No proliferation was detected in the cortex. Rare, very slight, paracortical hyperplasia was detected by both protocols. These results were comparable to those obtained for another irritant, sodium lauryl sulphate, in the same study. The authors considered the threshold for a positive result to be 1.75. the authors concluded that salicylic acid at a concentration of 20% is not sensitising.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The weak proliferative effects observed using the short protocol were below the threshold for a positive reaction in this assay. The read-across substance, salicylic acid, was considered not sensitising on the basis of the results of this study.
Executive summary:

In a variation on the mouse local lymph node assay, two protocols were used. In the short protocol, topical administration of 25 ul to both ears on Day 1, 2 & 3 was followed by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection and euthanasia on Day 5. In the long protocol, topical application of 50 ul to flank on Day 1 was followed by patch removal on Day 3, topical application of 25 ul to both ears on Days 6, 7, 8, BrdU i.p. injection & euthanasia on Day 9. The draining auricular lymph nodes were excised, weighed and fixed. Two successive sections were taken through mid-sagittal plane. One section was stained with haematoxylin and eosin for microscopic examination. The following section was processed for immunohistochemistry and labelled nuclei counting.

Statistically significant T-lymphocyte proliferation in the paracortex was detected in short, but not long, protocol. The maximum T/C ratio was 1.74. No proliferation was detected in the cortex. Rare very slight, paracortical hyperplasia was detected by both protocols.

These results were comparable to those obtained for another irritant, sodium lauryl sulphate, in the same study.

The authors considered the threshold for a positive result in this assay to be 1.75. Under the conditions of the study, read-across substance, salicylic acid, at a concentration of 20% is not sensitising.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a key study, the sensitising potential of bismuth subsalicylate was evaluated in guinea pigs (Buehler Test, OECD 406). A Buehler Test was conducted because the results of the LLNA study with bismuth subsalicylate were considered to be unreliable (see explanation below). In the treatment group (treatment dose of 60% and 30%), no macroscopic cutaneous reactions attributable to allergy were observed 24 and 48 hours after the removal of the occlusive dressing. In the control group (associated with the treatment dose of 60% and 30%), no cutaneous intolerance reaction was observed during the examination following the removal of the occlusive dressing. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (vehicle). In view of these results, under these experimental conditions, the test item bismuth subsalicylate is not a skin sensitiser.

The five supporting studies are detailed below:

A study was conducted to determine the sensitising potential of bismuth subnitrate in the local lymph node assay in mice. The study was performed according to OECD 429. Treatment with bismuth subnitrate at concentrations of 10% or 25% did not reveal statistically 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 not classified as sensitising. Therefore, by read across, the bismuth component of bismuth subsalicylate is not considered to be sensitising.

Read across substance, salicylic acid, was tested using the method of mouse ear swelling test (MEST). The aim of this study was to develop and optimise a protocol for the MEST. To evaluate the performance of this final MEST study design, a battery of 72 test materials including salicylic acid was evaluated. The substance was tested in CF-1 female mice, 6 to 8 weeks old. The results of this study indicated that salicylic acid is not a skin sensitiser.

A further study was conducted with read across substance, salicylic acid. In a variation on the mouse local lymph node assay, two protocols were used. In the short protocol, topical administration of 25 ul to both ears on Day 1, 2 & 3 was followed by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection and euthanasia on Day 5. In the long protocol, topical application of 50 ul to flank on Day 1 was followed by patch removal on Day 3, topical application of 25 ul to both ears on Days 6, 7, 8, BrdU i.p injection and euthanasia on day 9. The weak proliferative effects observed using the short protocol were below the threshold for a positive reaction in this assay. The read-across substance, salicylic acid, was considered not sensitising on the basis of the results of this study.

In addition, read across substance, methyl salicylate, was shown to be not sensitising in a local lymph node assay. Therefore, by read across, the subsalicylate component of bismuth subsalicylate is not considered to be sensitising.

A study was conducted to determine the sensitising potential of bismuth subsalicylate in the local lymph node assay in mice. The study was performed according to OECD 429. Under the conditions of the assay bismuth subsalicylate, tested in a suitable vehicle, was shown to have sensitisation potential (sensitiser), however, the results of this study are considered to be unreliable for the following reasons:

The results of the Buehler test with the test substance and the results of sensitisation studies with closely related structural analogues provided negative results (see above).

There is evidence that the presence of metal ions such as bismuth ions may cause a false positive result in the LLNA study (ICCVAM, 2009).

In conclusion, bismuth subsalicylate is not classified as a skin sensitiser.

 

Revised Draft Assessment of the Validity of the LLNA for Mixtures, Metals, and Aqueous Solutions. Addendum No. 1 to the ICCVAM Report: The Murine Local Lymph Node Assay (LLNA): A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds (NIH Pub. No. 99-4494, March 2009). Available at: http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-app/BRDcomplete.pdf



Migrated from Short description of key information:
A Buehler test was conducted with bismuth subsalicylate. Under the experimental conditions, the test item Bismuth Subsalicylate was shown not to be a skin sensitiser.

Justification for selection of skin sensitisation endpoint:
Considered to be the highest quality, most reliable study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Not assessed. No evidence of respiratory sensitisation was noted in any of the studies conducted, and it is proposed that the substance is not a respiratory sensitiser.


Migrated from Short description of key information:
Not assessed. No evidence of respiratory sensitisation was noted in any of the studies conducted, and it is proposed that the substance is not a respiratory sensitiser.

Justification for selection of respiratory sensitisation endpoint:
Not assessed. No evidence of respiratory sensitisation was noted in any of the studies conducted, and it is proposed that the substance is not a respiratory sensitiser.

Justification for classification or non-classification

The above studies have been ranked either reliability 1 or reliability 2 according to the Klimisch et al system with the exception of the bismuth subsalicylate LLNA study, which is considered to be unreliable. The ranking of 1 was deemed appropriate for the bismuth subsaliyclate Buehler Test because the study was conducted to GLP and in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification for sensitisation under the CLP Regulation (EC No 1272/2008).