Sodium bis(trifluoromethylsulfonyl)imide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

sodium trifluoro[(trifluoromethanesulfonylazanidyl)sulfonyl]methane

EC Number:

804-361-2

Cas Number:

91742-21-1

Molecular formula:

C2F6NO4S2.Na

IUPAC Name:

sodium trifluoro[(trifluoromethanesulfonylazanidyl)sulfonyl]methane

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Purity: 99.9 %

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

- Justification of the test method (e.g. ICE, EIT, RhCE) and considerations regarding applicability: The test system and the number of eyes used in the study are in compliance with the relevant OECD guideline.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg test material in 1 mL physiological saline
- Concentration (if solution):

Duration of treatment / exposure:

45 min

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180 and 240 minutes after the post-treatment rinse

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined in the experiment.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old and 2.5 kg) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Charles River Laboratories Hungary Kft. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received and processed within 2 hours of collection. After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes and Identification
The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
Each eye was located in chamber identified by a unique number within the Test Facility.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes were noted in the eyes in the experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined in the experiment.

NEGATIVE CONTROL USED
30 μL of physiological saline

POSITIVE CONTROL USED
30 mg powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME
30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.

REMOVAL OF TEST SUBSTANCE
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 3*20 mL saline was performed at additional time point when the positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface.
- Damage to epithelium based on fluorescein retention: The fluorescein retention determination the settings of the slit lamp microscope was the same as for opacity assessment, but the green light filter was used.
- Macroscopic morphological damage to the surface: Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator. Morphological findings at any observation point was recorded, their frequency and degree are taken into evaluation during classification.

SCORING SYSTEM:
- Mean corneal swelling (%) small negative numbers for swelling following application (0 to -5%) are counted as zero (scored as class I). Large negative numbers (>12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).
- Mean maximum opacity score: Corneal opacity is scored using the area of the cornea that is most densely opacified. Maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye
- Mean fluorescein retention score at 30 minutes post-treatment:
ΔFRattimet= FRattimet–FRatt=0
Mean ΔFR = (FEFR (30min) + SEFR(30min) + TEFR(30min))/ 3
where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and the baseline value
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus the baseline value
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus the baseline value
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus the baseline value

DECISION CRITERIA: GHS CLASSIFICATION

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

corneal swelling 

Value:

>= 12 - <= 40.3

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Value:

>= 4 - <= 4

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table of individual data Sodium bis(trifluoromethane)sulfonimide

Chamber number ↓	Corneal thickness (instrument units)															Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	C h a n g e	30	change at 30	75	change at 75	Max change up to 75	120	change at 120	180	change at 180	240	change at 240	Max change up to 240	0	30	75	120	180	240	Max ΔOpac	0	30	Δ Flu ret
11	58	58	0.0%	66	13.8%	69	19.0%	19.0%	73	25.9%	77	32.8%	81	39.7%	39.7%	0	4	4	4	4	4	4.0	0	3	3.0
12	60	60	0.0%	65	8.3%	70	16.7%	16.7%	74	23.3%	78	30.0%	84	40.0%	40.0%	0	4	4	4	4	4	4.0	0.5	3	2.5
13	58	58	0.0%	66	13.8%	70	20.7%	20.7%	72	24.1%	76	31.0%	82	41.4%	41.4%	0	4	4	4	4	4	4.0	0	3	3.0
Mean values:					12.0%		18.8%	18.8%		24.4%		31.3%		40.3%	40.3%							4.00			2.83

Note: There were no morphological effects or other observations seen.

Table of individual data Imidazole

Chamber number ↓	Corneal thickness (instrument units)															Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	C h a n g e	30	change at 30	75	change at 75	Max change up to 75	120	change at 120	180	change at 180	240	change at 240	Max change up to 240	0	30	75	120	180	240	Max ΔOpac	0	30	Δ Flu ret
14	60	60	0.0%	64	6.7%	67	11.7%	11.7%	71	18.3%	72	20.0%	79	31.7%	31.7%	0	4	4	4	4	4	4.0	0	3	3.0
15	58	58	0.0%	64	10.3%	68	17.2%	17.2%	72	24.1%	72	24.1%	78	34.5%	34.5%	0	4	4	4	4	4	4.0	0	3	3.0
16	57	57	0.0%	63	10.5%	67	17.5%	17.5%	70	22.8%	73	28.1%	76	33.3%	33.3%	0	4	4	4	4	4	4.0	0.5	3	2.5
Mean values:					9.2%		15.5%	15.5%		21.8%		24.1%		33.2%	33.2%							4.00			2.83

Note: Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

 

Table of individual data Physiological saline

Chamber number ↓	Corneal thickness (instrument units)															Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	C h a n g e	30	change at 30	75	change at 75	Max change up to 75	120	change at 120	180	change at 180	240	change at 240	Max change up to 240	0	30	75	120	180	240	Max ΔOpac	0	30	Δ Flu ret
17	60	60	0.0%	60	0.0%	60	0.0%	0.0%	60	0.0%	60	0.0%	60	0.0%	0.0%	0	0	0	0	0	0	0.00	0	0	0.00

Note: There were no morphological effects or other observations seen.

 

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on this in vitro eye irritation assay in isolated chicken eyes with Sodium bis(trifluoromethane)sulfonimide, the test item was severe irritant, UN GHS classification: Category 1.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The study was conducted and irritation effects of the test item were evaluated according to the OECD No. 438 guideline (2018).

After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 mg test item. The three positive control eyes were treated in a similar way with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the study was considered to be valid.

Severe cornea thickness change (mean = 40.3%) was observed during the four-hour observation period on test item treated eyes. Severe cornea opacity change was observed (severity 4 on all three eyes) during the four-hour observation period on test item treated eyes. Severe fluorescein retention change (severity 2.5 on one eye and severity 3 on two eyes) was observed on test item treated eyes during the observation period. No morphological effect was observed.

Based on this in vitro eye irritation assay in isolated chicken eyes with Sodium bis(trifluoromethane)sulfonimide, the test item was severe irritant, UN GHS classification: Category 1.