Lithium difluoro(oxalato)borate(1-)

Endpoint summary

Description of key information

Skin Corrosion: Category 1(OECD431/GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021.01

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Provided by sponsor, Batch No.:G200810043

- Purity, including information on contaminants, isomers, etc.:
99.80%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Refrigeration (+2 to +8°C)

Test system:

human skin model

Remarks:

EpiSkin

Source species:

other: a three dimensional Reconstructed Human Epidermis model

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured on a collagen matrix at the air liquid interface

Details on animal used as source of test system:

SkinEthic Laboratories
4, rue Alexander Fleming - 69366
Lyon, Cedex 07
France

The EpiSkin kit is produced by SkinEthic and sourced through a local supplier.

Justification for test system used:

The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.

One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics 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:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
at room temperature
- Temperature of post-treatment incubation (if applicable):
37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the respective incubation periods, at room temperature, the epidermis units were removed from the wells and thoroughly rinsed with PBS placed on an absorbent paper to remove excess PBS. The epidermis units were then placed in a 12-well plate filled with 2 mL pre-warmed assay medium to rinse.

After rinsing, each of these epidermal units was gently tapped on an absorbent paper to remove excess assay medium.


DYE BINDING METHOD
- Dye used in the dye-binding assay: [ MTT ]
- Spectrophotometer:
Microplate reader Flex Station
- Wavelength:
570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)

Mean tissue viability % is ≤ 50 % Category 2 or Category 1
Mean tissue viability % is > 50 % Non-Irritant

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg
- Concentration (if solution):

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL PBS
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL Sodium Dodecyl Sulfate
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

4 hour

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

17.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The mean relative tissue viability for the test item, Lithium difluoro(Oxalato)borate(1-) was 17.83 % and hence, it is predicted to be irritant under the experimental conditions described in this report.

Executive summary:

The test item, Lithium difluoro(Oxalato)borate(1-) was tested for its possible skin irritation potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application for 15 minutes.

 

The test item is a powder and after moistening with 5 µL of water on the top of the skin tissues at 10 mg/tissue and exposed for 15 minutes.  

 

Ten microliters (10 µL) of PBS and 10 µL of 5% aqueous SDS were used as the negative and positive controls, respectively.

 

After 4-hour post-incubation period, irritation potential of Lithium difluoro(Oxalato)borate(1-) was evaluated by assessing the reduction of cell viability after exposure to the test item. Cell viability was measured by determining mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.The relative mean tissue viability obtained after 15 minutes treatment with the test item was compared to the negative control tissues.

 

The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was 0.827. The positive control had a mean cell viability of 4.88 % after 15 minutes exposure, indicating that the test system functioned properly.

 

The study indicated that the test item as the percent viability was 17.83 %, in this In Vitro Skin Irritation Test using Reconstructed Human Epidermis under the conditions of testing employed.

 

 

 

 

 

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted: 14 June 2019 (OECD, 2019)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by sponsor, Batch No.:G200810043

- Purity, including information on contaminants, isomers, etc.: 99.80%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigeration (+2 to +8°C)

Test system:

human skin model

Remarks:

EpiSkin

Source species:

other: a three dimensional Reconstructed Human Epidermis model

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured on a collagen matrix at the air liquid interface

Justification for test system used:

The assessment of skin corrosivity has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.

One of the validated in vitro test methods adopted by the OECD TG 431 makes use of reconstructed human epidermis (RhE). The test is designed to predict and classify the skin corrosivity potential of a test item by assessment of its effect on a reconstituted human epidermis, based on the experience that corrosive substances show cytotoxic effects following short-term exposure of the stratum corneum of the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the respective incubation periods, at room temperature, the epidermis units were removed from the wells and thoroughly rinsed with PBS placed on an absorbent paper to remove excess PBS. The epidermis units were then placed in a 12-well plate filled with 2 mL pre-warmed assay medium to rinse.

After rinsing, each of these epidermal units was gently tapped on an absorbent paper to remove excess assay medium.


DYE BINDING METHOD
- Dye used in the dye-binding assay: [ MTT ]
- Spectrophotometer: Microplate reader Flex Station
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)

< 35 % after 3 min exposure Corrosive • Optional sub-category 1A*
≥ 35 % after 3 min exposure AND < 35 % after 60 min exposure OR ≥ 35 % after 60 min exposure AND < 35 % after 240 min exposure Corrosive • A combination of optional Sub-categories 1B and 1C
≥ 35 % after 240 min exposure Non-Corrosive

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg



NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): Sodium chloride (NaCl) 0.9% (w/v)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

Duration of treatment / exposure:

240 mins

Duration of post-treatment incubation (if applicable):

3 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

>= 35.12 - <= 39.46

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:

- Direct-MTT reduction: Since, the color of MTT solution did not turn to either blue or purple, it was concluded that, the test item did not interact with MTT.
- Colour interference with MTT: Similarly, the water control tested did not show any coloring potential nor did it reduce the MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: It is considered that the NC meets the acceptance, if the mean OD value of the 2 tissues is ≥ 0.6 and ≤ 1.5 for every exposure time.
- Acceptance criteria met for positive control: It is considered that the Positive Control (glacial Glacial acetic acid) meets the acceptance if the mean viability expressed as % of the NC, is ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: the difference of viability between the two tissue replicates should not exceed 30%

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions above, the test item Lithium difluoro(Oxalato)borate(1-) is skin corrosive by determining the tissue viability after the 240 minute exposure.

Executive summary:

 

In a in vitro skin corrosion study ( G20740) according to OECD 431 TG under GLP compliance, a three dimensional Reconstructed Human Epidermis model (Episkin) were exposed to 20 mg of  Lithium difluoro(Oxalato)borate(1-) for 3, 60 and 240 minutes. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the respective treatment periods with the test item was compared to the negative control tissues.

 

The relative mean tissue viability obtained after 3, 60 and 240 minutes treatment duration with the test item compared to the negative control tissues was 81.87, 103.61 and 35.12 %, respectively.The positive control had a mean cell viability of 6.70 % after 240 minutes exposure. The absolute mean OD570of the negative control tissues was within the laboratory historical control data range.

 

Since, the tissue viability is > 35% after the 240 minute exposure, the test item Lithium difluoro(Oxalato)borate(1-) is predicted to be non-corrosive under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021.01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Provided by sponsor, Batch No.:G200810043

- Purity, including information on contaminants, isomers, etc.:
99.80%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Refrigeration (+2 to +8°C)

Species:

cattle

Strain:

other: Cornea

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
Local abattoir (Slaughter house), Near Frazer
town, Bengaluru.

- Number of animals:
3

- Characteristics of donor animals (e.g. age, sex, weight):
Between 3 to 4 years

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The bovine eyes were procured from local abattoir and transported in a jar containing Hank’s Balanced Salt Solution (HBSS) and penicillinstreptomycin (100 IU/mL & 100 μg/mL) in an ice box. The experiment was initiated within 6 hours from the collection of eyes.

- Time interval prior to initiating testing:

- Indication of any existing defects or lesions in ocular tissue samples:
none

- Indication of any antibiotics used:
penicillin-streptomycin (100 IU/mL & 100 μg/mL).

- Selection and preparation of corneas:
Post pretest examination, all eyes were dissected with approximately 2-mm to
3-mm sclera with cornea from surrounding tissue and then placed in a
container with fresh HBSS (Hank’s Balanced Salt Solution), containing
penicillin-streptomycin (100 IU/mL & 100 μg/mL).

- Quality check of the isolated corneas:
Immediately after receiving the eyes in the lab, all eyes were observed for any
evidence of vascularization, pigmentation, opacity or scratches. All fourteen
collected eyes were found to be suitable for the experiment purpose.

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

positive control: 0.75 ml 20% w/v Imidazole
negative control: 0.75 ml undiluted distilled water
test group: 0.75 ml 20% w/v test material

Duration of treatment / exposure:

4 hour

Duration of post- treatment incubation (in vitro):

1 hour

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES
3
NEGATIVE CONTROL USED
Negative control (Distilled water)

POSITIVE CONTROL USED
positive control (Imidazole)

APPLICATION DOSE AND EXPOSURE TIME
20% w/v
4 hour

TREATMENT METHOD:
closed chamber

POST-INCUBATION PERIOD:
yes. 1 hour

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
the test substances from anterior chambers were removed and washed with EMEM (with phenol red) until test material was completely removed from the corneal surface. A final wash was made with EMEM without phenol red. Both the chambers were filled with fresh EMEM (without phenol red) and final opacity was measured.

- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)
After final opacity measurement, the EMEM (without phenol red) was removed from anterior chamber of each cornea holder and 1 mL of 5 mg/mL sodium fluorescein solution (in Dulbecco’s Phosphate-Buffered Saline) was added.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

171.616

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

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

Conclusions:

In this study, the negative control (Distilled water) & positive control (Imidazole) exhibited expected response as No category (IVIS score 0.825) &
Category I (IVIS score 103.584) respectively. The test item Lithium Difluoro
(Oxalato) Borate (1-) exhibited IVIS score of 171.616.

From the results, it can be concluded as the test item Lithium Difluoro
(Oxalato) Borate (1-) has severe ocular hazard potential or compound causing serious eye damage, with the BCOP test method.

Executive summary:

The evaluation of the ocular hazard potential of Lithium Difluoro (Oxalato)
Borate (1-) was carried out using the Bovine Corneal Opacity and Permeability
test. A 0.75 mL of 20% w/v in distilled water of the test item (Lithium Difluoro
(Oxalato) Borate (1-), a 0.75 mL of 20% w/v in distilled water of positive
control (Imidazole) and a 0.75 mL of negative control (Distilled water) was
applied to each cornea by means of closed chamber method. The control and
treated corneas were then subjected to the opacity and permeability
measurements.
In this study, the negative control (Distilled water) was classified as UN GHS
No Category (IVIS score 0.825) and the positive control (Imidazole) was
classified under Category 1 (IVIS score 103.584). The test item Lithium
Difluoro (Oxalato) Borate (1-) exhibited an IVIS score of 171.616 and was
classified under UN GHS “Category 1”.
From the results, it can be concluded as the test item Lithium Difluoro
(Oxalato) Borate (1-) has severe ocular hazard potential or compound causing
serious eye damage, with the BCOP test method.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

In a in vitro skin corrosion study ( G20740) according to OECD 431 TG under GLP compliance, a three dimensional Reconstructed Human Epidermis model (Episkin) were exposed to 20 mg of  Lithium difluoro(Oxalato)borate(1-) for 3, 60 and 240 minutes. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the respective treatment periods with the test item was compared to the negative control tissues.

 

The relative mean tissue viability obtained after 3, 60 and 240 minutes treatment duration with the test item compared to the negative control tissues was 81.87, 103.61 and 35.12 %, respectively.The positive control had a mean cell viability of 6.70 % after 240 minutes exposure. The absolute mean OD570of the negative control tissues was within the laboratory historical control data range.

 

Since, the tissue viability is > 35% after the 240 minute exposure, the test item Lithium difluoro(Oxalato)borate(1-) is predicted to be non-corrosive under the experimental conditions described in this report.

 

The test item, Lithium difluoro(Oxalato)borate(1-) was tested for its possible skin irritation potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application for 15 minutes.

 

The test item is a powder and after moistening with 5 µL of water on the top of the skin tissues at 10 mg/tissue and exposed for 15 minutes.  

 

Ten microliters (10 µL) of PBS and 10 µL of 5% aqueous SDS were used as the negative and positive controls, respectively.

 

After 4-hour post-incubation period, irritation potential of Lithium difluoro(Oxalato)borate(1-) was evaluated by assessing the reduction of cell viability after exposure to the test item. Cell viability was measured by determining mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.The relative mean tissue viability obtained after 15 minutes treatment with the test item was compared to the negative control tissues.

 

The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was 0.827. The positive control had a mean cell viability of 4.88 % after 15 minutes exposure, indicating that the test system functioned properly.

 

The study indicated that the test item as the percent viability was 17.83 %, in this In Vitro Skin Irritation Test using Reconstructed Human Epidermis under the conditions of testing employed.

 

 

Eye irritation:

 

The evaluation of the ocular hazard potential of Lithium Difluoro (Oxalato)
Borate (1-) was carried out using the Bovine Corneal Opacity and Permeability
test. A 0.75 mL of 20% w/v in distilled water of the test item (Lithium Difluoro
(Oxalato) Borate (1-), a 0.75 mL of 20% w/v in distilled water of positive
control (Imidazole) and a 0.75 mL of negative control (Distilled water) was
applied to each cornea by means of closed chamber method. The control and
treated corneas were then subjected to the opacity and permeability
measurements.
In this study, the negative control (Distilled water) was classified as UN GHS
No Category (IVIS score 0.825) and the positive control (Imidazole) was
classified under Category 1 (IVIS score 103.584). The test item Lithium
Difluoro (Oxalato) Borate (1-) exhibited an IVIS score of 171.616 and was
classified under UN GHS “Category 1”.
From the results, it can be concluded as the test item Lithium Difluoro
(Oxalato) Borate (1-) has severe ocular hazard potential or compound causing
serious eye damage, with the BCOP test method.

Justification for classification or non-classification

Based on the available information in the dossier, the substance Lithium difluoro(Oxalato)borate(1-) (CAS No.409071 -16 -5) needs to be classified  for skin corrosion Category 1and Eye Damage Category 1 when considering the criteria outlined in Annex I of 1272/2008/EC.