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Diss Factsheets

Administrative data

Description of key information

Dibismuth trioxide is not considered to be irritating to skin or to eyes. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
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:
guideline study
Qualifier:
according to guideline
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
Guideline:
other: "Commission Regulation (EC) No. 440/2008 B 46".
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EpiSkin and EpiDerm assays and 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 dibismuth trioxide was tested for its potential to induce skin irritation in a human skin model.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed by Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz (2009-03-30).
Specific details on test material used for the study:
EpiSkin
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
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:
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%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
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
Duration of post-treatment incubation (if applicable):
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 replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Value:
85.2
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:
After treatment with the test item dibismuth trioxide the relative absorbance values were irrelevantly decreased to 85.2%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
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 12% (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.

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

0.855

0.791

1.026

0.890

11.6

85.2

*       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.

Interpretation of results:
GHS criteria not met
Conclusions:
In the present study the test item dibismuth trioxide 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 Dibismuth trioxide by means of the Human Skin Model Test.

3 tissues 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 Dibismuth trioxide the relative absorbance values were decreased to 85.2%. This value is well above the threshold for irritancy of50%. 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 Dibismuth trioxide is non irritant to skin and therefore, the test item should not be classified and labeled as skin irritant according to regulation (EC) No.: 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2010-06-08 till 2010-06-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / The Netherlands
- Age at study initiation: 17 weeks
- Weight at study initiation: 2530 - 3435 g
- Housing: Individually in stainless steel cages.
- Diet: ad libitum; Pelleted standard Kliba Nafag 3418 rodent maintenance diet (batch no. 05/10 provided by Provimi Kliba AG, Switzerland)
- Water: ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod: 12 hours dark/light cycle
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount applied (volume or weight with unit): The test item was applied at 0.1 g/animal, the dose specified in the test guidelines for a solid test item.

VEHICLE
Not applicable, test item was applied undiluted as it was delivered.
Duration of treatment / exposure:
72 hours
Observation period (in vivo):
at approximately 1, 24, 48, 72 hours after administration
Number of animals or in vitro replicates:
3 males
Details on study design:
A single animal was treated first. As neither a corrosive effect nor a severe irritant effect was observed after the 1- and 24-hour examinations, the test was completed using the two remaining animals.

REMOVAL OF TEST SUBSTANCE
Substance was not removed.

SCORING SYSTEM: The eye reactions were assessed according to the numerical scoring system listed in the Commission Regulation (EC) No 440/2008, B.5. Scleral reddening and ocular discharge were also assessed.
The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, separately.

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, 4153 Reinach / Switzerland).
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
of animal #1
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
for animal #2
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
for animal #3
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
of animal #1
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
for animal #2
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
of animal #3
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
of animal #1
Time point:
other: 72 hours after instillation
Score:
0.33
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
of animal #2
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
of animal #3
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of animal #1
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of animal #2
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
of animal #3
Time point:
other: 72 hours after instillation
Score:
0
Max. score:
0
Irritant / corrosive response data:
Slight reddening of the conjunctivae and slight reddening of the sclerae were noted in all 3 males 1 hour after instillation. The slight reddening of the conjunctivae persisted in one animal up to the 24-hour reading. Slight ocular discharge was present in one animal 1 hour after instillation.
No abnormal findings were observed in the treated eyes of any animals 24 or 48 hours after treatment.
No staining produced by the test item was observed in the treated eyes.
No corrosion of the cornea was observed at any of the reading times.
Other effects:
No intercurrent deaths occurred during the course of the study. No clinical signs were recorded throughout the entire observation period. The body weight of the animals was within the range commonly recorded for this strain and age.
Green test item remnants were observed in the treated eyes of all animals at the 1-hour reading.
Interpretation of results:
GHS criteria not met
Conclusions:
Based upon the referred classification criteria (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), dibismuth trioxide does not have to be classified with respect to eye irritation in rabbits.
Executive summary:

The primary eye irritation potential of dibismuth trioxide was investigated according to OECD test guideline 405. The test item was applied by instillation of 0.1 g into the left eye of each of 3 young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris light reflex were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.33, 0.00 and 0.00 for reddening and 0.00 for all animals for chemosis. The instillation of dibismuth trioxide into the eye resulted in mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, as well as ocular discharge. These effects were reversible and were no longer evident 24 or 48 hours after treatment. No abnormal findings were observed in the cornea or for the iris light reflex of any animals at any of the examinations. No corrosion was observed at any of the measuring intervals. No staining of the treated eyes by the test item was observed and no clinical signs were observed. Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

The results of an available in vitro skin irritation study (human skin model) with dibismuth trioxide indicate that this substance is not irritating to skin.

Results of an available in vivo eye irritation study (rabbits) with dibismuth trioxide indicate that the substance is not irritating to eyes.


Justification for selection of skin irritation / corrosion endpoint:
Only one study is available.

Justification for selection of eye irritation endpoint:
Only one study is available.

Justification for classification or non-classification

Results of an available in vitro skin irritation study and an available in vivo eye irritation study with dibismuth trioxide indicate that the substance is not irritating to skin or eyes.

Based these experimental results, dibismuth trioxide requires no classification neither as irritating to skin nor to eyes in accordance to regulation (EC) No.: 1272/2008.