Bismuth oxide salicylate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

See below

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

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

Test material

Test material information

Constituent 1

Reference substance name:

Bismuth oxide salicylate

EC Number:

238-953-1

EC Name:

Bismuth oxide salicylate

Cas Number:

14882-18-9

Molecular formula:

C7H5BiO4

IUPAC Name:

2-hydroxy-4H-1,3,2-benzodioxabismin-4-one

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor’s identification: Bismuth Subsalicylate
· Date received: 04 March 2015
· Container: glass flask (n=1) · Form: powder
· Quantity: 181.40 g (content + container) · Colour: white
· Batch n°: 2015000575 · Storage : room temperature, darkness
· Production date: 16 January 2015 · Expiry date: 16 January 2020
· Content 57.8% of Bismuth and 38.7% of salicylates
It was identified under the code number: PH-15/0171.

In vivo test system

Test animals

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.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Induction 2

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

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Challenge 2

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

Results and discussion

Positive control results:

Positive Control with alpha-Hexylcinnamaldehyde Buehler Test – 3 applications: results trigger classification as Skin Sens. 1B under the CLP Regulation.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

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.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

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.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

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.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

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.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

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.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

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.

Results 7

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.

Results 8

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.

Results 9

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.

Results 10

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.

Results 11

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.

Results 12

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.