Lithium difluoro(oxalato)borate(1-)

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021.12-2022.2

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

We conducted this non-LLNA experiment for other regions' registration purpose. And this test was conducted previously which would not justify conducting an additional LLNA due to animal welfare.

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:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

Albino, NIH

Sex:

male/female

Details on test animals and environmental conditions:

Species : Guinea Pigs

Strain : Albino, NIH (Dunkin Hartley)

Scientific name : Cavia porcellus

Source : KCC Biolabs
Tumakuru,
Karnataka

No. of animals : Vehicle Control Group: 10 (5 Males + 5 Females)
Treatment Group: 20 (10 Males + 10 Females)

Note: Positive control data from Study No. G23413 was considered for this study and included in the report. The sensitivity and reliability check of the experimental technique was established once in six months.
For pre-study:
1 Male + 1 Female for intradermal induction
1 Male + 1 Female for topical boosting
Age : 10 to 11 weeks at intra-dermal induction

Body weight range at treatment : Males: 327.1 to 437.8 g
Females: 325.7 to 415.4 g

Identification : Guinea pig accession number. Identification of individual animals was by cage card and body markings using turmeric solution / crystal violet solution. The temporary body marking during acclimatization period was done with crystal violet.

Acclimatization : After physical examination for good health and suitability for the experiment, the animals were acclimatized for 7 days before start of the treatment. Females were nulliparous and non-pregnant. Animals were observed once daily during acclimatization period for any abnormalities.


Animals were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (13.4 to 14.3 air changes/hour). Environment: temperature 21 to 23°C relative humidity 65 to 67%, with 12 hours light and
12 hours dark cycle. The maximum and minimum temperature and relative humidity in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated from dry and wet bulb temperature recordings.

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.1 % w/v test item in Milli-Q water. A quantity of 0.01 g of test item made up to 10 mL with Milli-Q water and vortexed.

Day(s)/duration:

21 days

Adequacy of induction:

not specified

No.:

#1

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.1% w/v of test item in Milli-Q

Day(s)/duration:

24 hour

Adequacy of challenge:

not specified

No. of animals per dose:

5 animals / sex in the vehicle control and 10 animals / sex in the test item treated group

Details on study design:

Two groups (5 animals / sex in the vehicle control and 10 animals / sex in the test item treated group) were sensitized with intra-dermal injection and topical application (boosting) and then challenged by topical application. Positive control (2- Mercaptobenzothiazole) data generated in-house under study number G23413 was considered for this study.
For treatment group (G2), 0.1 mL of 1:1 mixture(v/v) of Complete Freund’s Adjuvant (FCA) in Milli-Q water was injected intra-dermally at site 1, 0.1 % w/v test item in Milli-Q water was injected at site 2 and 0.1 % w/v test item in Milli-Q water mixed with 1:1 mixture (v/v) of FCA in Milli-Q water, such that the final concentration of test item injected was same as that in site 2, was injected at site 3 for the treatment group animals. Similarly, for the vehicle control group animals (G1), 1:1 mixture of FCA & Milli-Q water was injected intra-dermally at site 1, Milli-Q water at site 2 and 1:1 mixture of vehicle (Milli-Q water) and FCA -Milli-Q water(1:1 mixture) at site 3.
On day 8, 0.1% w/v of test item in Milli-Q water was transferred on to a
filter paper (size: 2 x 4 cm) and applied on to the clipped area of skin (site of intradermal injection) by an occlusive dressing for 48 hours. Similarly, 0.5 mL of Milli-Q water was used for vehicle control group animals.
On day 22, the vehicle control and treatment group animals were challenged by transfer of 0.1% w/v of test item in Milli-Q water on to a filter paper
(size: 2 x 4 cm) and applied onto the prepared area of skin to the left flank by an occlusive dressing for 24 hours.Similarly 0.5 mL of Milli-Q water was applied on to the prepared area of skin of the right flank. The skin reaction was evaluated at 24 and 48 hours, post challenge using Magnusson and Kligman Grading Scale.

Positive control substance(s):

yes

Remarks:

(2- Mercaptobenzothiazole

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

8% w/v of test item in Milli-Q water

No. with + reactions:

6

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

8% w/v of test item in Milli-Q water

No. with + reactions:

6

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.1% w/v of test item in Milli-Q water

No. with + reactions:

0

Total no. in group:

20

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.1% w/v of test item in Milli-Q water

No. with + reactions:

0

Total no. in group:

20

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.1% w/v of test item in Milli-Q water

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.1% w/v of test item in Milli-Q water

No. with + reactions:

0

Total no. in group:

10

Interpretation of results:

GHS criteria not met

Conclusions:

The comparison of the skin reaction (at challenge) of the test item treated animals with those of the concurrent control and positive control group animals showed that the test item “LITHIUM DIFLUORO(OXALATO)BORATE(1-)” is considered as “Non-Sensitizer” to Guinea Pigs under the stated experimental conditions and it is “Unclassified” as per Globally Harmonized System of Classification and Labelling of Chemicals (GHS), 2021.

Executive summary:

The test item, LITHIUM DIFLUORO(OXALATO)BORATE (1-) was tested for its skin sensitization potential in Guinea Pigs using Magnusson and Kligman Test.

Two groups (5 animals / sex in the vehicle control and 10 animals / sex in the test item treated group) were sensitized with intra-dermal injection and topical application (boosting) and then challenged by topical application. Positive control (2- Mercaptobenzothiazole) data generated in-house under study number G23413 was considered for this study.

For treatment group (G2), 0.1 mL of 1:1 mixture(v/v) of Complete Freund’s Adjuvant (FCA) in

On day 8, 0.1% w/v of test item in Milli-Q water was transferred on to a
filter paper (size: 2 x 4 cm) and applied on to the clipped area of skin (site of intradermal injection) by an occlusive dressing for 48 hours. Similarly, 0.5 mL of Milli-Q water was used for vehicle control group animals.

On day 22, the vehicle control and treatment group animals were challenged by transfer of 0.1% w/v of test item in Milli-Q water on to a filter paper
(size: 2 x 4 cm) and applied onto the prepared area of skin to the left flank by an occlusive dressing for 24 hours.Similarly 0.5 mL of Milli-Q water was applied on to the prepared area of skin of the right flank. The skin reaction was evaluated at 24 and 48 hours, post challenge using Magnusson and Kligman Grading Scale.

There were no clinical signs, pre-terminal deaths or skin reactions at 24 and 48 hours, post challenge in both vehicle control and treatment group.

In the positive control (2- Mercaptobenzothiazole) study (Stu

The comparison of the skin reaction (at challenge) of the test item treated animals with those of the concurrent control and positive control group animals showed that the test item “LITHIUM DIFLUORO(OXALATO)BORATE (1-)” is considered as “Non-Sensitizer” to Guinea Pigs under the stated experimental conditions and it is “Unclassified” as per Globally Harmonized System of Classification and Labelling of Chemicals (GHS), 2021.

 

 

 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021.05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

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)

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

The cell line was exposed to the test item for 48 h, and subsequently luciferase induction was evaluated. The assay was run in 96 well plates, and test item was tested in parallel at 12 concentrations ranging from 0.98 μM to 2000 μM.

Additionally, each plate contained:
- Six wells with solvent control (cells receiving solvent only)
- One blank well with no cells added
- Five wells with the positive control, cinnamic aldehyde.

Each plate was tested in parallel in triplicate for analysis of luciferase induction and one additional replicate plate was used for cytotoxicity assessment. The full test in triplicate analysis was independently conducted three times.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

1 847.23 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The luminescence reading and the absorbance reading obtained were analyzed in an excel sheet provided by Givaudan. In trial I, the dose-response curve of test item was observed to be biphasic,thus the results of trial I was regarded as ambiguous.Test item showed statistically significant induction above the threshold of 1.5 or 50% at 1000 µM in trial II and III of the non-cytotoxic concentration over the solvent control in two out of three trials. Though there is a statistically significant induction was observed at 2000 µM in the three trials, not considered as it was a cytotoxic dose. Out of three trials, test item was positive at non-cytotoxic concentrations in two trials. Hence, test item was considered as positive. Under the same circumstances the positive control cinnamaldehyde showed statistically significant induction over the solvent control confirming the sensitivity of the assay procedure used.

Based on these results it is concluded that the test item, Lithium difluoro(Oxalato)borate(1-) is positive in the in vitro skin sensitisation assay by ARE-Nrf2 Luciferase test method (KeratinoSens™ assay).

Executive summary:

The potential of the test item, Lithium difluoro(Oxalato)borate(1-) to cause skin sensitisation was evaluated in ARE-Nrf2 Luciferase Test (KeratinoSens™ Assay).      

 

KeratinoSens™ assay is an in vitro cell based assay in which the KeratinoSens™ cells are exposed to the test item for 48 hours and subsequently measured for luciferase activity using a luminometer.

 

The assay was run in 96 well plates, and test item was tested at
12 concentrations (ranging from 0.98 to 2000 μM) along with the solvent control, control blank and the positive control cinnamic aldehyde (ranging from 4 μM to 64 μM).  

 

Three white plates were tested in parallel for luciferase induction and one additional transparent plate was used for cytotoxicity assessment. The full test in triplicate analysis was independently conducted three times.

 

The cells were exposed to the test concentrations for 48 hours at 37±1°C in a carbondioxide incubator. After exposure, cells were lysed with passive lysis buffer, luciferase substrate was added and the luminescence activity at each concentration was integrated for 1500 ms (1.5 seconds).

 

The viability assay plates containing the cells were treated with treatment medium containing MTT solution for 4 hours, and then cells lysed with
10% SDS and incubated overnight. After overnight incubation, the absorbance was read at 600 nm using a photometer.

 

The luminescence reading and the absorbance reading obtained were analyzed in an excel sheet provided by Givaudan. In trial I, the dose-response curve of test item was observed to be biphasic,thus the results of trial I was regarded as ambiguous.Test item showed statistically significant induction above the threshold of 1.5 or 50% at 1000 µM in trial II and III of the non-cytotoxic concentration over the solvent control in two  out of three trials. Though there is a statistically significant induction was observed at 2000 µM in the three trials, not considered as it was a cytotoxic dose. Out of three trials, test item was positive at non-cytotoxic concentrations in two trials. Hence, test item was considered as positive. Under the same circumstances the positive control cinnamaldehyde showed statistically significant induction over the

 

Based on these results it is concluded that the test item, Lithium difluoro(Oxalato)borate(1-) is positive in the in vitro skin sensitisation assay by ARE-Nrf2 Luciferase test method (KeratinoSens™ assay).

 

Reference 3

Endpoint:

skin sensitisation: in chemico

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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

not specified

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)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25±2.5ºC. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by High Performance Liquid Chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model (Section 12) which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

1.82 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

The mean of cysteine and lysine peptide depletion by the positive control (cinnamaldehyde) was 52.31% which shows that the positive control is a sensitizer with high reactivity, according to cysteine 1:10/lysine 1:50 prediction model, in Direct Peptide Reactivity Assay (DPRA) confirming the sensitivity of the test.
Whereas the mean of cysteine and lysine peptide depletion by the test item at 100 mM concentration was 0.91% respectively, which shows that the test item has no or minimal reactivity at 100 mM concentration. Under the testing conditions, test item [Lithium Difluoro(Oxalato)Borate(1-)] was concluded as negative or non-sensitiser with no or minimal reactivity in Direct Peptide Reactivity Assay (DPRA).

Executive summary:

Skin sensitisation test by in chemico test method, Direct Peptide Reactivity Assay (DPRA) was carried out using the nucleophile containing synthetic cysteine and lysine peptides to evaluate the skin sensitisation potential of the test item, Lithium difluoro(oxalato)borate(1-) and to classify as skin sensitiser or non-skin sensitiser.

Solubility check was performed for the test item before test sample analysis. The test item, Lithium difluoro(oxalato)borate(1-), was found to be soluble in acetonitrile and formed a homogenous solution at 100 mM concentration.

On the day of test sample preparation, appropriate amount of cysteine and lysine peptide was weighed and dissolved in phosphate buffer and ammonium acetate buffer respectively. The stock concentration of cysteine peptide prepared was 0.501 mg/mL (0.667 mM) and lysine peptide was 0.518 mg/mL (0.667 mM). The preparations were done immediately before use.  

Test item at 100 mM and positive control solution at 100 mM concentration and used in the test sample preparation for analysis. Test item, positive control and reference control was added to cysteine and lysine peptides and incubated in dark at 25°C for 24±2 hours. Post incubation, the test samples were analysed using HPLC as per section 9 to measure the peptide depletion.

 

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available