Bismuth oxide salicylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 to 24 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

Product name: Bismuth subsalicylate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: human

Details on animal used as source of test system:

Human Skin
EPISKIN-SM (Source: SkinEthic, France, Batch No.:12-EKIN-008, Expiry date: 27 February 2012) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EPISKIN model has been validated for irritation and corrosivity testing in multiple laboratories and accepted by ECVAM and OECD, it is considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Quality Control
EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

Kit Contents
Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis

Medium: A flask of sterile “Maintenance Medium” for incubations. (Batch No.: 12-MAIN3-008; Exp. Date: 29 February 2012)

A flask of sterile “Assay Medium”.
(Batch No.: 12-ESSC-011; Exp. Date: 29 February 2012)

Number of Replicate Wells

In this assay 3 replicates for the test item and 3 negative controls + 3 positive controls were used. Furthermore one additional control for coloured substance was used.

Kit Reception Quality Check

The colour of the agar medium used for transport was checked for its pH:

- orange colour = good
- yellow or violet colour = not acceptable

The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C:

- the indicator changes from white to grey at 40°C The kit was found to be in good order at reception.

Storage

The EPISKIN-SM kit was kept in its packaging at 37°C and the assay and maintenance medium supplied with the kit was stored at 2-8°C until the initiation of the test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

First 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of the test item was applied evenly to the epidermal surface of each of the three test skin units.

Duration of treatment / exposure:

The plates with the test item treated and the negative and positive control treated epidermis were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (20-37°C).

Duration of post-treatment incubation (if applicable):

After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1x solution (0.9%) to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source care was taken to avoid the damage of epidermis.

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.

Number of replicates:

In this assay 3 replicates for the test item and 3 negative controls + 3 positive controls were used. Further more one additional control for coloured substance was used.

Test system

Number of animals:

Not applicable

Details on study design:

INDICATOR FOR POTENTIAL FALSE VIABILITY

Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test substance is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed below.

Check-method for possible direct MTT reduction with test substance

An amount of 10 mg test item was added to 2 mL MTT ready to use solution and mixed. The mixture was incubated for three hours at room temperature protected from light and then any colour change was assessed:

- Test substances which do not interact with MTT: yellow
- Test substances interacting with MTT: blue or purple

If the MTT solution colour becomes blue or purple, the test substance interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non specific reduction of the MTT (i.e. by using killed epidermis).

The test item showed no direct interaction with MTT.

Check-method to detect the colouring potential of test-substances

Prior to treatment, chemicals were evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
As the test item has an intrinsic colour, further evaluation to detect colouring potential was not necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test substance to stain the epidermis by using additional control tissues.

Additional control(s) for dyes and chemicals able to colour the tissue

In addition to the normal procedure, one additional chemical-treated tissue was used for the non specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. This is to mimic the amount of colour from the test item that may be present in the test disks. OD readings were made following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY

Procedures were performed under aseptic conditions (in laminar hood using sterile equipments).

Pre-incubation (Day [-1])

The “maintenance medium” was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2.

Application and rinsing (Day 0)

- First 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of the test item was applied evenly to the epidermal surface of each of the three test skin units.
- 20 µl PBS was added to each of the three negative control skin units
- 20 µl SDS was added to each of the three positive control skin units

- For additional control for staining effects of the test item, 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of the test item was applied evenly to the epidermal surface.

The plates with the test item treated and the negative and positive control treated epidermis were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (20-37°C).

After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1x solution (0.9%) to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source care was taken to avoid the damage of epidermis.

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.


MTT test after 42 hours incubation (day 2)

After the 42 hours incubation all EPISKIN-SM units except the one staining control were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The one additional control for coloured substances was transferred to wells filled with fresh assay medium. Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

Formazan extraction (Day 2)

At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).

The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (Day 2)

Following the formazan extraction, 2×200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at
540 nm using acidified isopropanol solution blank (6×200 µL).

Note: The validity of the microplate reader was verified with a standard verification plate daily before use. The standard plate was calibrated yearly by the manufacturer.

CALCULATIONS OF VIABILITY PERCENTAGES

Data calculation for normal test substances

Blank:

– The mean of the 6 blank OD values was calculated

Negative control:

– Individual negative control OD values (NCraw) were corrected with the mean blank
OD:

OD Negative Control (ODNC) = ODNCraw – ODblank mean

– The corrected mean OD of the 3 negative control values were calculated: this corresponds to 100% viability

Positive control:

– Individual positive control OD values (PCraw) were corrected with the mean blank
OD:

OD Positive Control (ODPC) = ODPCraw – ODblank mean

– The corrected mean OD of the 3 positive control values were calculated
– The % viability for each positive control replicate was calculated relative to the mean negative control:

% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100

– The mean value of the 3 individual viability % for positive control was calculated:

Mean PC % = (%PC1 +%PC2 +%PC3) / 3


Test substance:

– Individual test substance OD values (TTraw) were corrected with the mean blank
OD:

OD Treated Tissue (ODTT) = ODTTraw – ODblank mean

– The corrected mean OD of the 3 test substance values were calculated
– The % viability for each test substance replicate was calculated relative to the mean negative control:

% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100

– The mean value of the 3 individual viability % for test substance was calculated

Mean TT % = (%TT1 +%TT2 +%TT3) / 3

Data calculation for MTT-interacting substances

Test substances that interfere with MTT can produce non specific reduction of the MTT. It is necessary to evaluate the OD due to non specific reduction and to subtract it before calculations of viability %.

– Non specific MTT reduction calculation (NSMTT):

NSMTT = [(ODKT- ODKU) / ODNC] × 100
ODKU: untreated killed tissues OD
ODKT: test substance treated killed tissues OD
ODNC: negative control OD


If NSMTT is > 30% relative to the negative control: additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.
– True MTT metabolic conversion (TODTT) is undertaken if NSMTT is < 30%:

TODTT = [ODTT – (ODKT – ODKU)]
ODTT: test substance treated viable tissues

– The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:

% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2= [TODTT1 / mean ODNC] × 100
% RV 3 = [TODTT1 / mean ODNC] × 100

- The mean value of the 3 individual relative viability % for test substance is calculated

Mean Relative Viability % = (% RV 1 +% RV 2 +% RV 3) / 3

Data calculation for dyes and chemicals able to colour the tissue

For test substances detected as able to stain the tissues the non specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability %.

– Non Specific Colour % (NSC%):

NSC % = (ODCT / mean ODNC) × 100
ODCT: test substance treated tissue (not incubated with MTT) ODNC: negative control OD (incubated with MTT)

If NSC % is ≤ 5% then the normal calculation mode is used (see 3.7.1).
If NSC % is > 30% relative to the negative control, additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.

– True MTT metabolic conversion (TODTT) is undertaken if NSC % is > 5% and
≤ 30%

TODTT = [ODTV - ODCT]
ODTV: test substance treated tissue (incubated with MTT) ODCT: test substance treated tissue (not incubated with MTT)

– The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:

% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2= [TODTT1 / mean ODNC] × 100
% RV 3 = [TODTT1 / mean ODNC] × 100

– The mean value of the 3 individual relative viability % for test substance is calculated

Mean Relative Viability % = (% RV 1 +% RV 2 +% RV 3) / 3

VALIDITY OF THE TEST

The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.

The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability due the MTT interaction can be precluded.
- Colour interference with MTT: As the test item has an intrinsic colour, one additional chemical-treated tissue was used for the non specific OD evaluation. Optical density (measured at 540 nm) of this tissue was determined as 0.040, Non Specific Colour % was calculated as 6%. Therefore additional data calculation was necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified.

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.636. The positive control result showed a mean of 17% viability. Each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Any other information on results incl. tables

The results of the optical density (OD) measured at 540 nm of each replicate and the calculated % viability of the cells is presented below:

 

Substance	Optical Density (OD)		OD after adjustment*	Viability (% )
Negative Control: PBS	1 2 3	0.631 0.619 0.659		99 97 104
	mean	0.636		100
	standard deviation (SD)			3.61
Positive Control: SDS	1 2 3	0.103 0.091 0.125		16 14 20
	mean	0.106		17
	standard deviation (SD)			3.06
Test Item: Bismuth Subsalicylate	1 2 3	0.685 0.592 0.639	0.645 0.552 0.599	101 87 94
	mean		0.599	94
	standard deviation (SD)			7.00

*For the test item, the material had a residual colour which was expected to cause an OD of 0.074 in the final solutions. This was subtracted from the measured OD values .

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro skin irritation test in the EPISKIN model with Bismuth Subsalicylate the results indicated that the test item is non irritant.

Executive summary:

Disks of EPISKIN (three units / chemical) were treated with Bismuth Subsalicylate and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.

SDS 5% and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue, adjusted OD was calculated and the tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

As the test item has an intrinsic colour, one additional chemical-treated tissue was used for the non specific OD evaluation. Optical density (measured at 540 nm) of this tissue was determined as 0.040, and this value was subtracted from the measured OD value for the test item. Non Specific Colour % was calculated as 6%, therefore additional data calculation was necessary.

Following exposure with Bismuth Subsalicylate, the mean treated skin value was 94% and therefore non-irritant. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

In this in vitro skin irritation test in the EPISKIN model with Bismuth Subsalicylate the results indicated that the test item is non irritant.