Bismuth tris(2-ethylhexanoate)

Endpoint summary

Description of key information

Skin corrosion/irritation: irritating (OECD 435 and OECD 439; GLP)

Eye irritation: causes serious eye damage (OECD 405; GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2018-04-26

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: -15°C to 40°C

Test system:

artificial membrane barrier model

Source species:

other: not specified

Cell type:

other: synthetic macromolecular bio-barrier

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

The CORROSITEX™ Assay is a standardized, quantitative in vitro test for skin corrosivity and has been validated by the ECVAM for testing acids, bases and their derivatives (ECVAM, 2000)*. The bio-barrier membrane is constructed to have physico-chemical properties similar to rat skin.

Reference:
- ECVAM (2000) ESAC statement on the application of the Corrositex® assay for skin corrosivity testing

Vehicle:

unchanged (no vehicle)

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes (lot no. CT120516; supplier: Invitro International; Irvine, CA 92614)
- Components: a synthetic macromolecular bio-barrier and a chemical detection system (CDS)
- Apparatus and preparation procedures: the preparation was completed at least 2 hours prior to running tests. The entire content of the BIOBARRIER diluent was added to the vial of BIOBARRIER matrix powder. The vial was heated to 68°C (± 1°C) in a water bath under smooth agitation. After complete dissolution (approx. 20 min.) the solution was allowed to sit for 5 min. to allow any air bubbles to rise to the surface. 200 µL of the BIOBARRIER were pipetted into each membrane disc. The BIOBARRIERS were set on the tray and kept in the cold (2 - 8°C) for at least two hours.

WAS THE COMPATIBILITY TEST PERFORMED: yes
In order to test whether the test system is suitable for the test item, 150 µl of the test substance was added to the Qualify test tube. The vial was shaken to allow dissolution of the test substance and let stand for one minute. If the colour or consistency of the CDS changes at the sample/testing fluid interface, the test material is qualified for the assay. If no reaction is observed within five minutes, the sample is not qualified for the CORROSITEX TM assay.

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes
This step established the category of cut-off times for the sample. 150 µl of the test substance was added to the tubes labelled Tube A and Tube B. The vial was shaken to allow dissolution of the test substance. In case a colour change was observed in either of the tubes and colour was matched to the corresponding colour charts on the CORROSITEX™ Testing Protocol Poster. Test materials having high acid/alkaline reserves are defined as Category 1 materials, while those with low acid/alkaline reserves are defined as Category 2 materials. If no colour change had been observed in either tube, CONFIRM reagent was added to Tube B. After shaking, the resulting colour was matched to the colour chart on the CORROSITEX™ Testing Protocol Poster. If the test item has a strong inherent colour or shows other characteristics impairing a clear categorization according to the colour chart, the pH value can be measured in tubes A and B and is used to confirm/determine the category of the test item, according to the Corrositex® Reference Manual (1995)*.

TEMPERATURE USED DURING TREATMENT: room temperature (17 - 25 °C)

METHOD OF DETECTION
- Chemical or electrochemical detection system: chemical detection system (CDS)

METHOD OF APPLICATION (CLASSIFICATION TEST):
The CDS vials were warmed to room temperature (17 - 25˚C) before using. Four vials were utilized for test item sample replicate testing. One vial was utilized for a positive control sample and another vial for a negative control. Lastly, one vial served as a CDS colour control. One BIOBARRIER disc was added on top of the first vial (discs were not longer in the vial than two minutes before adding the test samples).
500 µl of the test item was applied evenly on the top of the BIOBARRIER disc and starting time was recorded. This step was repeated for the remaining vials, staggering each start time by e.g. 10 seconds (but not longer than 2 minutes). The start time difference for each vial was subtracted from the final time to determine the net response time. As soon as a reaction had been observed, the time was recorded.

NTERPRETATION OF THE RESULTS:
For Category 1 substances, test chemicals were categorized as non-corrosive in case no colour change occurs after 240 minutes. For Category 2 substances, test chemicals were categorized as non-corrosive in case no colour change occurs after 60 minutes. The start time difference for each vial was subtracted from the final time to determine the net response time.
The time (in minutes) elapsed between application and barrier penetration for the test substance was recorded in tabular form as individual replicate data.
The mean time (± standard deviation) of the four sample replicates to activate the CDS was calculated and reported in tabular form. Using the table as shown in the field "Any other information on materials and methods incl. tables" below, the test item was categorised.

TEST ACCEPTANCE CRITERIA:
The test meets acceptance criteria if:
- test item qualifies in qualification test
- positive control activates CDS > 3 - 60 min.
- negative control activates CDS not before 60 min.
The exact breakthrough time of the positive control should be determined to demonstrate, that the response is in the acceptable historical range of breakthrough times for the positive control (mean ± 2 - 3 standard deviations).

*Reference:
- InVitro International (1995), Corrositex® Reference Manual

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 µL of the test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL of citric acid (10%) in aqua dest.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL of phosphoric acid (85 %)

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Test item: quadruplicates
Negative control: single measurement
Positive control: single measurement

Irritation / corrosion parameter:

penetration time (in minutes)

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Colour change or structural change was not observed up to 60 minutes (treatment period)

Other effects / acceptance of results:

QUALIFICATION TEST
The test substance was compatible with the CORROSITEX™ Assay, as assessed in the qualification step. The categorization step and the classification step could be performed.

CATEGORIZATION TEST
A direct colour change was not observed. CONFIRM reagent was added to tube B and the category was read from the CORROSITEX™ colour chart. The chemical has been categorized to timescale category 2.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: negative control did not activate the CDS before 60 minutes (> 60 minutes).
- Acceptance criteria met for positive control: positive control activated the CDS between 3 - 60 minutes (26.18 min).

Please also refer to the field "Any other information on results incl. tables" below.

Results of the test item bismuth tris (2-ethylhexanoate)

	CORROSITEX™ Time [min]	Colour Change	Consistency Change
Replicate 1	> 60	no	no
Replicate 2	> 60	no	no
Replicate 3	> 60	no	no
Replicate 4	> 60	no	no
Mean ± SD	> 60
Positive control	26.18	yes	no
Negative control	> 60	no	no

The mean time, required to activate the CDS was > 60min.

 Historical Data

Mean time [min] until colour change of positive control (Phosphoric Acid (85% (v/v))	Standard Deviation [min]	number of control values	Range of LCL - UCL [min]
21.4	13.3	35	0.0 – 48.0

LCL:      Lower control limit (95%, mean – 2*SD)

UCL:     Upper control limit (95%, mean + 2*SD)

Historical data were generated from 2010 - 2018

Interpretation of results:

GHS criteria not met

Conclusions:

According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the test item bismuth tris(2-ethylhexanoate) is not corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-25 to 2018-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2014-11-07

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: -15°C to 40°C

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25899

TEST FOR MTT INTERFERENCE
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- after incubation verification of the colour by the unaided eye.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item were mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- after incubation verification of the colour by the unaided eye

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponded 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 (%) = [(mean ODtest item / positive control) / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS.
The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1", if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category”, if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL (47 µL/cm²) of the test item
Prior to treatment, all EpiDerm™ tissues were gently blotted to remove moisture. The test item was applied undiluted. 30 µL (47 µL/cm²) of the test item were dispensed directly atop the EpiDerm™ tissue. The test item was gently spread to match size of the tissue using a bulb-headed Pasteur pipette. After the washing steps, a small amount of the test material could not be removed from the edge of the tissue surface

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 ± 1 minute

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

4.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

After the washing steps, a small amount of the test material could not be removed from the edge of the tissue surface.

Other effects / acceptance of results:

TEST FOR MTT INTERFERENCE
The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, the test item did not directly reduce MTT (NSMTT equalled 0%) and a functional test with freeze-killed tissues and the quantitative corrections were not necessary.

TEST FOR COLOUR INTERFERENCE
The mixture of 30 µL of the test item per 300 µl aqua dest. showed no colouring detectable by unaided eye-assessment. The mixture of 30 µL of the test item per 300 µl isopropanol showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in isopropanol was measured in the range of 570 ± 30 nm. No absorption was measured in the relevant range. Therefore, an additional test with viable tissues (without MTT addition) was not necessary (NSC equalled 0%).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.849).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.2 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2 % - 2.4 %).
- The absorbance values were not below historically established boundaries.

Please also refer to the field "An other information on results incl. tables" below.

Table1:  Result of the test item bismuth tris (2-ethylhexanoate)

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.824	1.879	1.802	0.090	0.106	0.099	0.130	0.124	0.128
	1.898	1.893	1.800	0.094	0.105	0.109	0.133	0.128	0.133
Mean Absolute OD570	1.849****			0.101			0.129
OD570(Blank Corrected)	1.781	1.836	1.759	0.047	0.063	0.056	0.087	0.080	0.085
	1.855	1.849	1.757	0.051	0.062	0.066	0.090	0.085	0.090
Mean OD570of the Duplicates (Blank Corrected)	1.818	1.843	1.758	0.049	0.063	0.061	0.088	0.083	0.087
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.806*			0.058			0.086
SD OD570	0.044			0.007			0.003
Relative Tissue Viability [%]	100.6	102.0	97.3	2.7	3.5	3.4	4.9	4.6	4.8
Mean Relative Tissue Viability [%]	100.0			3.2**			4.8
SD Tissue Viability [%]	2.4***			0.4***			0.2***
CV [% Viabilities]	2.4			12.9			3.4

 

*Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is ≤ 20%.

***Standard deviation (SD) obtained from the three concurrently tested tissues is≤ 18%

****Mean absolute OD₅₇₀of the negative control tissues is≥0.8 and≤2.8.

Table2:  Historical Data

	Mean Absolute OD570±30nmNC	MeanAbsoluteOD570±30nmPC	Mean Relative Viability [%] PC	SD Viability [%] NC, PC, TI
Mean	1.861	0.114	3.7	4.4
SD	0.247	0.033	1.5	4.1
Range of LCL – UCL	1.367 – 2.355	0.048 – 0.181	0.7 – 6.8	0.0 – 12.5
n	25	25	25	117

LCL:      Lower control limit (95%, mean – 2*SD)

UCL:      Upper control limit (95%, mean + 2*SD)

n:            number of control values

Historical data were generated in 2017.

 

Interpretation of results:

other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria

Conclusions:

The test item, bismuth tris(2-ethylhexanoate), is either corrosive or irritating to the skin. Since the RhE test methods covered by OECD TG 439 cannot resolve between UN GHS Categories 1 or 2, further information on skin corrosion is required to decide on its final classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2018-09-24 to 2018-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on 2018-04-26

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 12 – 13 weeks old
- Weight at study initiation: 2.3 kg
- Housing: individually housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm²
- Diet (ad libitum): autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Relative humidity: 55 ± 10%
- Air changes: at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml of the test item applied in the conjunctival sac of one eye. Untreated eye served as control.

Duration of treatment / exposure:

not applicable

Observation period (in vivo):

1, 24, 48 and 72 hours as well as 4 to 21 days after test item application

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

1 male rabbit

Details on study design:

PREPARATION OF THE ANIMALS
Within 24 hours before the test and immediately prior to the application both eyes of the animal were examined.
Approx. 15 hours before the application the eyes were examined with the aid of a fluorescein solution (Fluoreszein SE Thilo®). The eyes were rinsed with physiological saline 0.9 % NaCl after the examination. The animal did not show eye irritation, ocular defects, or pre-existing corneal injury.

INITIAL AND CONFIRMATORY TEST
The in vivo test was performed initially using one animal. The results of the initial test indicated that the test item is corrosive or severe irritant to the eye using the procedure described. Therefore, no additional animals were treated due to animal welfare reasons.

USE OF TOPICAL ANESTHETICS AND SYSTEMIC ANALGESICS
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Temgesic®) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia.
Approx. 5 minutes prior to the application of the test item, 1-2 drops of an ocular anaesthetic (Proparakaine-POS® hydrochloride ophthalmic 0.5% solution) were administered in both the treated and the control eye of the animal.
To prevent pain and distress after the application of the test item the animal was treated with doses of buprenorphine and meloxicam to provide a continued therapeutic level of systemic analgesia:
- After test substance application the test animal was dosed with 0.03 mg/kg bw of buprenorphine at the discretion of the veterinarian: 11 hours (day 0) post-application once; day 1 and day 2 twice; day 3 once.
- After test substance application the test animal was dosed with 0.5 mg/kg bw of meloxicam: 8 hours (day 0) up to day 5 and from day 7 up to day 21.
- Day 6, 20 and 21 no analgesic medication.

REMOVAL OF TEST SUBSTANCE
- Washing: treated eye was rinsed with physiological saline 0.9 % NaCl after examination with fluorescein solution
- Time after start of exposure: 1 hour after the application
The eyes were not rinsed to remove residues of the test item, foreign bodies or incrustation 24 hours after application.

SCORING SYSTEM
according to Draize scale

TOOL USED TO ASSESS SCORE: slit lamp biomicroscope / fluorescein
Approximately 15 hours before the application the eyes were examined with the aid of a fluorescein solution.
24 hours post-application and from then on daily until end of the observation period, the treated eye was examined with the aid of a fluorescein solution and a slit lamp biomicroscope. After the examination the eye was rinsed with physiological saline 0.9% NaCl.

OBSERVATIONS
- body weight: prior to the administration, 72 hours after administration and at the end of the observation period
- clinical observations: The eyes were comprehensively evaluated for the presence or absence of ocular lesions one hour and then 24, 48 and 72 hours after application. The eyes were further examined for signs of irritation throughout the observation period.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

not reversible

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

not reversible

Irritant / corrosive response data:

Animal # 1:
- conjunctival redness score 1 one hour post-application. 24 hours post-application a conjunctival redness score 2 was observed before the score decreased to 1 from 72 hours until 12 days post-application. No conjunctival redness was detected 13 days after installation of the test item. 14 to 21 days after dosing the animal showed conjunctival redness score 1. The effect was not fully reversible within the observation period of 21 days after application of the test material. The mean score of animal no. 1, following grading at 24, 48 and 72 h after installation of Bismuth tris (2-ethylhexanoate), was calculated to be 1.66.
- conjunctival chemosis score 3 one hour and score 2 24 hours post-application. From 72 hours to 11 days post-administration of the test material conjunctival chemosis was assessed with score 1. 12 days after test item installation score 2 was observed, before the score decreased again to 1 from 13 days post-application until end of the observation period. The effect was not fully reversible within the 21 days-observation period. The mean score of animal no. 1, following grading at 24, 48 and 72 h after installation of Bismuth tris (2-ethylhexanoate), was calculated to be 1.33.
- Iris lesion score 1 one hour post-application and score 2 24 hours post-application. From 72 hours until end of the observation period iris lesion score 1 was observed in the tested animal. The effect was not fully reversible within the observation period of 21 days after dosing. The mean score of animal no. 1, following grading at 24, 48 and 72 h after installation of Bismuth tris (2-ethylhexanoate), was calculated to be 1.33.
- Cornea opacity score 1 throughout the 21 days-observation period. The effect was not fully reversible within the test period. The mean score of animal no.1, following grading at 24, 48 and 72 h after installation of the test material, was calculated to be 1.00.

Local effects were observed in animal #1:
The observed local effects comprised slight to moderate hypersecretion, half eyelid closure and incline of the head.

Upon fluorescein examinations starting 24 hours post-application, the treated eyes of animal no. 1 showed corneal daily lesions (starting with approx. 100% of the area) until 12 days post-application. From 13 days to 19 days after administration of the test item no stainable lesions were recorded in the test animal, while from 20 days to 21 days post-application slightly visible changes in the cornea were apparent (approx. 10% of the area).

Other effects:

- clinical observations: neither mortality nor significant clinical signs of toxicity were observed.
- body weight: the body weight development was within the expected range.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The substance is serious eye damaging.
According to Annex I of Regulation (EC) 1272/2008 and subsequent adaptations, the test item Bismuth tris (2-ethylhexanoate) has to be classified as serious eye damaging (Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation:

The substance was observed to be either corrosive or irritating to the skin in a reliable in vitro skin irritation/corrosion study according to OECD 439. As the results of an OECD 439 test are not suitable to differentiate between skin categories 1 and 2, the substance was tested in an in vitro skin corrosion test.

Bismuth tris(2 -ethylhexanoate) was tested in an in vitro skin corrosion test according to OECD 435 and the substance was not observed to be corrosive to the skin.

Eye irritation:

Bismuth tris(2 -ethylhexanoate) was tested in two in vitro test systems evaluating eye damaging potential (OECD 437; GLP and OECD 492; GLP) and in an in vivo test (OECD 405).

The in vitro eye irritancy potential of Bismuth tris (2-ethylhexanoate) could not be assessed in the bovine corneal opacity and permeability assay, since the experiment showed residual of Bismuth tris (2-ethylhexanoate) on the corneas. The study could not be performed according to the guideline OECD 437.

With the OECD 492 study a prediction of the ocular irritation potential of Bismuth tris (2-ethylhexanoate) could not be made. In accordance with OECD 492 guideline further testing with other test methods will be required, because this test cannot resolve between UN GHS Categories 1 and 2.

Based on these results of both in-vitro tests further in-vivo testing was necessary for assessing the acute eye irritation/corrosion potential of the test substance. Bismuth tris (2-ethylhexanoate) produced irritant effects in the rabbit, which were not fully reversible within an observation period of 21 days.

Justification for classification or non-classification

Skin irritation:

Bismuth tris(2 -ethylhexanoate) does possess a skin irritation potential based on in vitro OECD 435 and 439 studies. The substance does require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations (Category 2; H315).

Eye irritation:

Bismuth tris(2 -ethylhexanoate) does possess serious eye damaging potential based on an in vivo OECD 405 study. The substance does require classification as serious eye damaging according to Regulation (EC) No 1272/2008 and its subsequent adaptations (Category 1; H318).