Lithium trifluoromethanesulphonate

Endpoint summary

Description of key information

Skin irritation / corrosion: not corrosive (OECD No. 431, EU Method B.40 BIS, GLP compliant, K V1), not irritating (OECD No. 439, EU Method B.46, GLP compliant, K V1).

Eye irritation or serious eye damage: no prediction can be made (OECD No. 437, GLP compliant, K V1), potentially irritant or corrosive to the eye (OECD No. 492, GLP compliant).

Respiratory irritation: no specific study was available.

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:

From 31-August-2016 to 24-October-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Except for the Test Item characterisation which was conducted in an ISO 9000 environment.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- At room temperature container flushed with nitrogen, desiccated

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model EPI-200
- Tissue batch number(s): 24364 Kit M and L
- Source: MatTek Corporation, Ashland, MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out at 37.0 ± 1.0°C (actual range 35.6 - 37.3°C).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: 3 hours (37°C)
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570) = 1.496+/-0.034 [1.0 - 3.0]
- Barrier function: ET-50 = 6.52 hrs [4.77 - 8.72 hrs]
- Morphology: Functional stratum corneum, viable basal cell layer, and intermediate spinous and granular layers
- Contamination: No contamination (absence of bacteria, fungus and mycoplasma)

NUMBER OF REPLICATE TISSUES: 4 tissues per test item together with a negative control and positive control (2 tissues were used for a 3-minute exposure to Lithium Trifluoromethanesulfonate and two for a 1-hour exposure)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 35.6 to 48.6 mg of the solid test item with 25 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue.

NEGATIVE CONTROL
- Amount applied: 50 µl Milli-Q water

POSITIVE CONTROL
- Amount applied: 50 µl KOH
- Concentration: 8N

Duration of treatment / exposure:

3-minute and 1-hour exposures

Number of replicates:

4 tissues per test item together with a negative control and positive control (2 tissues were used for a 3-minute exposure to Lithium Trifluoromethanesulfonate and two for a 1-hour exposure)

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean values

Run / experiment:

3-minute application

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

11%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean values

Run / experiment:

1-hour application

Value:

63

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

11%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit >=0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range (3-minute treatment: 1.324-2.615 / 1-hour treatment: 1.361-2.352).
The mean relative tissue viability following the 3-minute or 1-hour exposure to the positive control was 11%.

In the range 20-100% viability, the Coefficient of Variation (cut-off value <= 30%) between tissue replicates was <= 10% for the negative control and the 3-minute treatment with the test item, indicating that the test system functioned properly. For the 1-hour treatment with the test item the coefficient of variation was 69%, but because the individual results of the mean relative tissue were 30% and 97%, both above 15%, this does not affect the study outcome.

Preliminary tests

Lithium Trifluoromethanesulfonate was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed it was concluded that Lithium Trifluoromethanesulfonate did not interfere with the MTT endpoint.

Main test

The mean absorption at 570 nm measured after treatment with Lithium Trifluoromethanesulfonate and controls are presented in the following Table. The individual OD570 measurements are presented in an other Table below.

Mean absorption in the in vitro skin corrosion test with Lithium Trifluoromethanesulfonate

	3 -minute application					1 -hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.526	1.586	1.556	+/-	0.042	1.770	1.588	1.679	+/-	0.129
Lithium Trifluoromethanesulfonate	1.440	1.372	1.406	+/-	0.048	0.502	1.626	1.064	+/-	0.795
Positive control	0.180	0.155	0.167	+/-	0.018	0.205	0.163	0.184	+/-	0.030

SD= Standard deviation

Duplicate exposures are indicated by A and B

In this table the values are corrected for background absorption (0.0420). Isopropanol was used to measure the background absorption.

Individual OD measurements at 570 nm

	3-minute application (OD570) AB		1-hour application (OD570) AB
Negative control
OD570 measurement 1	1.5361	1.6025	1.7973	1.6268
OD570 measurement 2	1.5801	1.6369	1.8098	1.6275
OD570 measurement 3	1.5880	1.6449	1.8278	1.6348
Lithium Trifluoromethanesulfonate
OD570 measurement 1	1.5015	1.4262	0.5481	1.6941
OD570 measurement 2	1.4688	1.4062	0.5456	1.6541
OD570 measurement 3	1.4750	1.4106	0.5380	1.6556
Positive control
OD570 measurement 1	0.2226	0.2013	0.2484	0.2051
OD570 measurement 2	0.2199	0.1948	0.2513	0.2053
OD570 measurement 3	0.2221	0.1934	0.2425	0.2051

OD = Optical density

Duplicate exposures are indicated by A and B.

The following Table shows the mean tissue viability obtained after 3-minute and 1-hour treatments with Lithium Trifluoromethanesulfonate compared to the negative control tissues.

	3-minute application Viability, percentage of control	1 -hour application Viability, percentage of control
Negative control	100 (98 and 102) / SD: 3.8	100 (105 and 95) / SD: 10.3
Lithium Trifluoromethanesulfonate	90 (93 and 88) / SD: 4.7	63 (30 and 97) / SD: 69.1
Positive control	11 (12 and 10) / SD: 13.9	11 (12 and 10) / SD: 20.6

SD= Standard deviation

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Lithium Trifluoromethanesulfonate compared to the negative control tissues was 90% and 63% respectively. Because the mean relative tissue viability for Lithium Trifluoromethanesulfonate was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, Lithium Trifluoromethanesulfonate is considered to be not corrosive.

Interpretation of results:

GHS criteria not met

Conclusions:

Lithium Trifluoromethanesulfonate is not corrosive in the in vitro skin corrosion test (OECD Guideline No. 431, adopted 29 July 2016) under the experimental conditions described in this report.
The substance is not classified as corrosive to skin according to GHS criteria.

Executive summary:

The assessment of the corrosive potential to skin of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using an in vitro skin corrosive test based on the guidelines described in: OECD No. 431 (adopted 29 July 2016) and EU Method B.40 BIS.

The test consisted of topical application of Lithium Trifluoromethanesulfonate (35.6 to 48.6 mg of the solid test item with 25 µl Milli-Q water) on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue was thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Lithium Trifluoromethanesulfonate compared to the negative control tissues was 90% and 63% respectively.

Because the mean relative tissue viability for Lithium Trifluoromethanesulfonate was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, Lithium Trifluoromethanesulfonate is considered to be non-corrosive in the performed experiment. The substance is not classified as corrosive to skin according to GHS criteria.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30-November-2016 to 17-February-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Amended by EC No. 640/2012 OJ No. L193, 20 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Except for the Test Item characterisation which was conducted in an ISO 9000 environment.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- At room temperature container flushed with nitrogen, desiccated

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): 17-EKIN-001
- Source: SkinEthic Laboratories, Lyon, France

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out at 37.0 ± 1.0°C (actual range 36.5 - 37.3 °C)
- Temperature of post-treatment incubation: 37.0 °C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (37°C)
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: IC50 = 1.8 mg/mL [1.5 - 3.0]
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: No contamination (absence of bacteria, fungus and mycoplasma)

NUMBER OF REPLICATE TISSUES: 3 tissues per test item together with negative and positive controls

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure and 42 hours post incubation is less than or equal to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure and 42 hours post incubation is greater than 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 14.9 to 15.8 mg of the solid test item with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue.

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean values

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

3.2%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 3.2% (<= 50%). The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 5% (<= 18%), indicating that the test system functioned properly.

The mean absorption at 570 nm measured after treatment with Lithium Trifluoromethanesulfonate and controls are presented in the following Table. The individual OD570 measurements are presented in an other Table below.

Mean absorption in the in vitro skin irritation test with Lithium Trifluoromethanesulfonate

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.002	0.971	0.917	0.963	+/-	0.043
Lithium Trifluoromethanesulfonate	0.729	0.745	0.723	0.732	+/-	0.012
Positive control	0.030	0.038	0.032	0.030	+/-	0.007

OD= optical density

SD= standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

Individual OD measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control
OD570 measurement 1	1.0533	1.1062	0.9533
OD570 measurement 2	1.0361	0.9211	0.9649
Lithium Trifluoromethanesulfonate
OD570 measurement 1	0.7764	0.7791	0.8263
OD570 measurement 2	0.7671	0.7966	0.7040
Positive control
OD570 measurement 1	0.0685	0.0870	0.0650
OD570 measurement 2	0.0765	0.0739	0.0663

OD= optical density

Triplicate exposures are indicated by A, B and C.

The following Table shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues.

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	4.5
Lithium Trifluoromethanesulfonate	76	1.2
Positive control	3.2	0.8

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 76%. Since the mean relative tissue viability for the test item was above 50%, the test item is considered to be non-irritant in the performed experiment.

Interpretation of results:

GHS criteria not met

Conclusions:

Lithium Trifluoromethanesulfonate is not irritant in the in vitro skin irritation test (OECD Guideline No. 439, adopted 28 July 2015) under the experimental conditions described in this report.
The substance is not classified as irritant to skin according to GHS criteria.

Executive summary:

The assessment of the irritant potential to skin of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using an in vitro skin irritation test based on the guidelines described in: OECD No. 439 (adopted 28 July 2015) and EU Method B.46 (Amended by EC No. 640/2012 OJ No. L193, 20 July 2012).

The test consisted of topical application of Lithium Trifluoromethanesulfonate (14.9 to 15.8 mg of the solid test item with 5 µl Milli-Q water) on the skin tissue for 15 minutes. After exposure the skin tissue was thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

The relative mean tissue viability obtained after the 15 -minute treatment with Lithium Trifluoromethanesulfonate compared to the negative control tissues was 76%.

Since the mean relative tissue viability for the test item was above 50%, Lithium Trifluoromethanesulfonate is considered to be non-irritant. The substance is not classified as irritant to skin according to GHS criteria.

Finally, Lithium Trifluoromethanesulfonate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations based on this test and the in vitro skin corrosion study (project 514504).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29-August-2016 to 21-October-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

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)

Version / remarks:

Adopted July 26, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- At room temperature container flushed with nitrogen, desiccated

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Characteristics of donor animals: Young cattle
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

A 20% (w/v) solution of Lithium Trifluoromethanesulfonate was prepared in physiological saline

Duration of treatment / exposure:

240 +/- 10 minutes at 32 +/- 1°C

Duration of post- treatment incubation (in vitro):

90 minutes incubation period with sodium fluorescein

Number of animals or in vitro replicates:

3 corneas per treatment group

Details on study design:

PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

CORNEA SELECTION AND OPACITY READING
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test item was introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea.
Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1°C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:

Opacity = [(I0/I) − 0.9894] / 0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

APPLICATION OF SODIUM FLUORESCEIN
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1°C.

PERMEABILITY DETERMINATIONS
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

DECISION CRITERIA:
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category), as described in the OECD Guideline No. 437, are given hereafter:
In vitro score range: UN GHS:
≤ 3 No Category
> 3 ; ≤ 55 No prediction can be made
> 55 Category 1

Irritation parameter:

in vitro irritation score

Remarks:

Mean values

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

-0.1

Positive controls validity:

valid

Remarks:

102.9

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 103 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Lithium Trifluoromethanesulfonate was tested in a 20% (w/v) solution.

The opacity, permeability and in vitro scores of the individual corneas are shown in the following Tables:

Individual opacity scores

Treatment	Opacity before treatment	Opacity after treatment	Final opacity^1	Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	3.9	4.2	0.3		-0.2
	2.3	2.3	0.0
	2.3	1.5	-0.8
Positive control	2.6	76.9	74.3	74.3	75.3
	2.9	62.5	59.6	59.6
	3.9	96.0	92.1	92.1
Test item	2.5	9.3	6.8	6.8	6.5
	2.9	10.6	7.7	7.7
	3.9	8.4	5.0	5.0

¹ Final Opacity = Opacity after treatment – Opacity before treatment.

² Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control³

³ In case the mean final opacity of the negative control is below zero, no correction will be made.

Permeability score individual values (uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean Final negative control
Negative control	1	0.004	0.004	0.004	0.004	0.004	-0.2
	1	0.001	0.012	0.002	0.005	0.005
	1	0.005	0.005	0.006	0.005	0.005
Positive control	1	1.028	1.042	1.010	1.027	1.027
	6	0.552	0.545	0.535	0.545	3.270
	1	1.257	1.250	1.257	1.255	1.255
Test item	1	0.023	0.012	0.022	0.019	0.019
	1	0.009	0.010	0.011	0.010	0.010
	1	0.079	0.072	0.080	0.077	0.077

Permeability score individual values (corrected)

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	Negative control corrected OD490 Average	Negative control corrected Final OD490	Average OD
Positive control	1	1.023	1.037	1.005	1.022	1.022	1.838
	6	0.547	0.540	0.533	0.540	3.241
	1	1.252	1.245	1.252	1.250	1.250
Test item	1	0.018	0.007	0.017	0.014	0.014	0.031
	1	0.004	0.005	0.006	0.005	0.005
	1	0.074	0.067	0.075	0.072	0.072

¹ OD490 values corrected for the mean final negative control permeability (0.005).

In Vitro irritancy score

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	0.3	0.004	0.4
	0.0	0.005	0.0
	-0.8	0.005	-0.7
Positive control	74.3	1.022	89.6
	59.6	3.241	108.2
	92.1	1.250	110.9
Test item	6.8	0.014	7.1
	7.7	0.005	7.7
	5.0	0.072	6.1

¹ In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

² Positive control and test item are corrected for the negative control.

The individual in vitro irritancy scores for the negative controls ranged from -0.7 to 0.4. The individual positive control in vitro irritancy scores ranged from 89.6 to 110.9. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with Lithium Trifluoromethanesulfonate showed opacity values ranging from 5.0 to 7.7 and permeability values ranging from 0.005 to 0.072. The corneas were translucent after the 240 minutes of treatment with Lithium Trifluoromethanesulfonate. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 6.1 to 7.7 after 240 minutes of treatment with Lithium Trifluoromethanesulfonate.

Since Lithium Trifluoromethanesulfonate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Interpretation of results:

study cannot be used for classification

Conclusions:

Evaluation of the eye hazard potential of Lithium Trifluoromethanesulfonate using the Bovine Corneal Opacity and Permeability test (BCOP test, OECD Guideline No. 437).
The test item induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.0 after 240 minutes of treatment.
Since Lithium Trifluoromethanesulfonate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Executive summary:

The assessment of the eye hazard potential of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using the Bovine Corneal Opacity and Permeability test (BCOP test) based on the OECD Guideline No. 437 (adopted 26 July 2013).

The test consisted of topical application of Lithium Trifluoromethanesulfonate on the epithelium of the bovine cornea for 4 hours. The non-surfactant solid test item was applied as a 20% solution. After exposure the corneas were thoroughly rinsed. The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 103 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Lithium Trifluoromethanesulfonate induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.0 after 240 minutes of treatment. Since Lithium Trifluoromethanesulfonate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30-November-2016 to 14-February-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Except for the Test Item characterisation which was conducted in an ISO 9000 environment.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- At room temperature container flushed with nitrogen, desiccated

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23460 kit J, supplied by MatTek Corporation, Ashland MA, U.S.A.)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
The cells used to produce EpiOcular tissue are screened for potential biological contaminants. Tests performed for each of the potential biological contaminants listed (HIV-1 virus, Hepatitis B virus, Hepatitis C virus, Bacteria, yeast and other fungi) were performed according to standardised test methods. The product resulted in "no detection" for the potential biological contaminants previously listed.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

56.8 to 57.6 mg of the test item applied directly on top of the skin tissue

Duration of treatment / exposure:

6 hours ± 15 minutes at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes at 37°C

Number of animals or in vitro replicates:

2 tissues per test item together with a negative control and positive control

Irritation parameter:

other: Tissue viability (percentage of control)

Remarks:

Mean values

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

22%

Remarks on result:

other: Potentially irritant or corrosive

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 22% (< 50%). The absolute mean OD570 of the negative control tissues was within 0.8 and 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 13% (< 50%), indicating that the test system functioned properly. Acceptance criteria were met.

Preliminary tests

Lithium Trifluoromethanesulfonate was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed it was concluded that Lithium Trifluoromethanesulfonate did not interfere with the MTT endpoint.

Main test

The following Table shows the mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues.

Mean tissue viability in the EpiOcular™ test with Lithium Trifluoromethanesulfonate

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	13
Test item	2.4	0.02
Positive control	22	5.3

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 2.4%.

Since the mean relative tissue viability for the test item was below 60%,it is considered to be potentially irritant or corrosive to the eye.

Interpretation of results:

other: UN GHS Category 1 or 2

Conclusions:

The mean relative tissue viability for the test item was below 60% it is considered to be potentially irritant or corrosive to the eye.
Lithium Trifluoromethanesulfonate is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report.

Executive summary:

The assessment of the eye hazard potential of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using the EpiOcular™ test based on the OECD Guideline No. 492 (adopted 28 July 2015).

The test consisted of topical application of Lithium Trifluoromethanesulfonate on the Reconstructed Human EpiOcular™ Model for 6 hours. The solid test item was directly applied. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect

The positive control had a mean cell viability of 22% after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within 0.8 and 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 13%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 2.4%. Since the mean relative tissue viability for the test item was below or equal to 60% after 6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

Lithium Trifluoromethanesulfonate is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. According to the UN GHS Regulation, based on the results of this test, the test substance is classified in Category 1 or 2. The EpiOcular™ test based on the OECD Guideline No. 492 do not allow to classify according to the Regulation (EC) 1272/2008 (CLP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:

Two key studies were identified for skin irritation / corrosion.

The assessment of the corrosive potential to skin of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using an in vitro skin corrosive test based on the guidelines described in: OECD No. 431 (adopted 29 July 2016) and EU Method B.40 BIS.

The test consisted of topical application of Lithium Trifluoromethanesulfonate (35.6 to 48.6 mg of the solid test item with 25 µl Milli-Q water) on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue was thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Lithium Trifluoromethanesulfonate compared to the negative control tissues was 90% and 63% respectively. Because the mean relative tissue viability for Lithium Trifluoromethanesulfonate was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, Lithium Trifluoromethanesulfonate is considered to be non-corrosive. The substance is not classified as corrosive to skin according to GHS criteria.

The assessment of the irritant potential to skin of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using an in vitro skin irritation test based on the guidelines described in: OECD No. 439 (adopted 28 July 2015) and EU Method B.46 (Amended by EC No. 640/2012 OJ No. L193, 20 July 2012).

The test consisted of topical application of Lithium Trifluoromethanesulfonate (14.9 to 15.8 mg of the solid test item with 5 µl Milli-Q water) on the skin tissue for 15 minutes. After exposure the skin tissue was thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

The relative mean tissue viability obtained after the 15 -minute treatment with Lithium Trifluoromethanesulfonate compared to the negative control tissues was 76%. Since the mean relative tissue viability for the test item was above 50%, Lithium Trifluoromethanesulfonate is considered to be non-irritant. The substance is not classified as irritant to skin according to GHS criteria.

Eye irritation or serious eye damage:

Two key studies were identified for eye irritation.

The assessment of the eye hazard potential of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using the Bovine Corneal Opacity and Permeability test (BCOP test) based on the OECD Guideline No. 437 (adopted 26 July 2013).

The test consisted of topical application of Lithium Trifluoromethanesulfonate on the epithelium of the bovine cornea for 4 hours. The non-surfactant solid test item was applied as a 20% solution. After exposure the corneas were thoroughly rinsed. The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 103 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Lithium Trifluoromethanesulfonate induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.0 after 240 minutes of treatment. Since Lithium Trifluoromethanesulfonate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

The assessment of the eye hazard potential of Lithium Trifluoromethanesulfonate was carried out, under GLP compliance, using the EpiOcular™ test based on the OECD Guideline No. 492 (adopted 28 July 2015).

The test consisted of topical application of Lithium Trifluoromethanesulfonate on the Reconstructed Human EpiOcular™ Model for 6 hours. The solid test item was directly applied. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 22% after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within 0.8 and 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 13%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 2.4%. Since the mean relative tissue viability for the test item was below or equal to 60% after 6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

Lithium Trifluoromethanesulfonate is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report.

Justification for classification or non-classification

Lithium Trifluoromethanesulfonate is non-irritant in the in vitro skin irritation test under the experimental conditions described in the report and should not be classified for skin irritation according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations based on the in vitro irritation test and the in vitro skin corrosion study (No classification).

Lithium Trifluoromethanesulfonate cannot be classified based on the results of the BCOP test. However, based on the results of the EpiOcular™ test, the test substance requires classification for eye irritation or serious eye damage (UN GHS Category 1 or 2). Therefore, based on the low IVIS obtained with the BCOP test, Lithium trifluoromethanesulfonate is classified H319: Causes serious eye irritation (Category 2) according to the Regulation (EC) 1272/2008 (CLP).

No study are available for the respiratory irritation assessment.