Lithium trifluoromethanesulphonate

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Lithium trifluoromethanesulphonate

EC Number:

251-528-5

EC Name:

Lithium trifluoromethanesulphonate

Cas Number:

33454-82-9

Molecular formula:

CHF3O3S.Li

IUPAC Name:

lithium trifluoromethanesulphonate

Test material form:

solid: particulate/powder

Details on test material:

See confidential details on test material

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 animals / tissue source

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.

Test system

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

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.