Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-30 till 2010-04-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, 11 December 2009, Vers. 4.
Deviations:
no
Qualifier:
according to
Guideline:
other: "Commission Regulation (EC) No. 440/2008 B 46"
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EpiSkin and EpiDerm assays on the Skin Integrity Function Test (Altern Lab Anim. 2007 Dec; 35(6): 559-601).
Deviations:
no
Principles of method if other than guideline:
In the present study the test item bismuth nitrate pentahydrate was tested for its potential to induce skin irritation in a human skin model.
GLP compliance:
yes (incl. certificate)
Remarks:
signed by Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz (2009-03-30).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Bismuth nitrate, pentahydrate
- Molecular formula (if other than submission substance): Bi(NO3)3*5H2O
- Molecular weight (if other than submission substance):489.07 g/mol
- Physical state: solid, colourless crystals
- Storage condition of test material: Store substance at room temperature, in closed container and keep under N2 atmosphere.

Test animals

Species:
human
Details on test animals and environmental conditions:
not applicable

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable, since no animals were tested
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): About 15 mg of the test item were applied to each of triplicate tissues (≙ 39.47 mg/cm2).

VEHICLE
not used

NEGATIVE CONTROL
Deionised water (Lot no.230310) was used as the negative control.
- Amount(s) applied (volume or weight with unit): 15 µL were applied to each of triplicate tissues for 15 ± 1 minutes.

POSITIVE CONTROL
A 5% SLS (Sodium lauryl sulphate, lot no. 1353471 51508322 solution in deionised water, prepared freshly prior to the performance of the experiment, was used as positive control.
- Amount(s) applied (volume or weight with unit): 15 µL were applied to each of triplicate tissues for 15 ± 1 minutes.
Duration of treatment / exposure:
Tissues were placed into the incubator for 15 ± 1 minutes at 37 ± 1.5 °C
Observation period:
approximately 50 hours; After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 4 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.
Number of animals:
not applicable, since no animals were tested
Details on study design:
TEST SITE
not applicable

TEST SYSTEM
- EpiSkin Kit (Lot No.: 10-EKIN-011): Components Needed for the Assay: Sealed 12-well plate (Contains 12 inserts with EpiSkin tissues on agarose), 12-well plate (For MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide] viability assay), 1 bottle of Assay Medium (Basic medium for use in MTT assays) and 1 bottle of EpiSkin Maintenance Medium (Basic medium for incubations)
- MTT-Solution: 3 mg MTT Formazan salt were dissolved in 1 mL PBS. Before treatment of the tissues the MTT solution was diluted with assay medium to reach a final concentration of 0.3 mg/mL.

CELL CULTURE
EpiSkin kits are purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate. On day of receipt EpiSkin tissues were transferred to 12-well plates with maintenance medium.

EXPERIMENTAL PERFORMANCE
- Prewarming of EpiSkin Tissues: After 3 hours and 15 minutes incubation of the EpiSkin tissues, they were treated with the test item. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium.
- Treatment: The negative and positive control, and the test item were added into the insert atop the concerning EpiSkin triplicate tissues. The 12-well plates were placed into the incubator for 15 ± 1 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 ± 1 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.
- Time after start of exposure: 15 minutes

MTT ASSAY
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. After a nearly 3 hours incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for 2 hours 45 minutes while shaking (~120 rpm) at room temperature.
Per each tissue sample 2 x 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.
Some test chemicals may reduce MTT, which will result in a blue colour without any involvement of cellular mitochondrial dehydrogenase. Although in the present assay the test chemicals were rinsed off and the DMEM (Dulbecco's Modified Eagle Medium) medium beneath the tissues was replaced before contact with MTT medium, some amount of a test chemical may be released by the tissues into the MTT medium and directly reduce the MTT, which would be interpreted as "tissue viability". MTT reducing capability of the test item was tested as described in the field "Any other information on materials and methods - Test for Direct MTT Reduction”. No colour change could be observed in the present study.

SCORING SYSTEM:
- Evaluation of Results: The mean OD of the 3 negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/OD negative control]*100
For the test item and the positive control the mean relative viability +/- standard deviation of the 3 individual tissues are calculated and used for classification according to the EU classification (acc. to Directive 67/548/EEC and Regulation 1272/2008/EC).
- Acceptability of the Assay: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is > 0.8. An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is < 40%.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: After treatment with the test item the relative absorbance values were irrelevantly decreased to 82.4%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
Value:
ca. 82.4

In vivo

Other effects:
After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.8 for the 15 minutes treatment interval thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 27.4% thus ensuring the validity of the test system.
The standard deviations between the % variabilities of the test item, the positive and negative controls were below 8% (threshold of the "OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation": 18%), thus ensuring the validity of the study.

Any other information on results incl. tables

Results after treatment with dibismuth trioxide 

Dose group

Treatment Interval

Absorbance 570 nm
Tissue 1*

Absorbance 570 nm
Tissue 2*

Absorbance 570 nm
Tissue 3*

Mean Absorbance of 3 Tissues

Standard Deviation in %

Rel. Absorbance

[% of Negative Control]**

Negative Control

15 min

1.109

0.993

1.034

1.045

5.6

100.0

Positive Control

15 min

0.326

0.212

0.322

0.287

6.2

27.4

Test Item

15 min

0.866

0.937

0.779

0.861

7.6

82.4

*       Mean of 3 replicate wells after blank correction
**
      relative absorbance (rounded values) = 100 * (absorbance of test item/absorbance of negative control)

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In the present study the test item bismuth nitrate, pentahydrate was tested for its potential to induce skin irritation in a human skin model.
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is non irritant to skin.
Executive summary:

This in vitro study was performed to assess the irritation potential of Bismuth (III) trinitrate, pentahydrate by means of the Human Skin Model Test.

3 tissues (each) of the human skin model EpiSkin™ were treated with either the test item, the negative or the positive control for 15 minutes.

About 15 mg of the test item were applied to each tissue (39.47 mg/cm2),spread to match the tissue size.

15 µL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.

After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD (optical density) >0.6 till≤1.5 for the15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system.

After treatment with the test item Bismuth (III) trinitrate, pentahydrate the relative absorbance values were decreased to 82.4%. This value is well above the threshold for irritancy of 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Bismuth (III) trinitrate, pentahydrate is non irritant to skin and therefore, the test item should not be classified and labelled as skin irritant according to regulation (EC) No.: 1272/2008.