1,2-dimethyl-3-propyl imidazolium bis((trifluoromethyl)sulfonyl)amide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 MAY 2016 to 02 JUN 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 on 26 july 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GROUPE INTERMINISTERIEL DES PRODUITS CHIMIQUES, France

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dimethyl-3-propyl imidazolium bis((trifluoromethyl)sulfonyl)amide

EC Number:

805-807-9

Cas Number:

169051-76-7

Molecular formula:

C10H15F6N3O4S2

IUPAC Name:

1,2-dimethyl-3-propyl imidazolium bis((trifluoromethyl)sulfonyl)amide

Test material form:

liquid

Details on test material:

Appearance : colorless oil
Composition: a base stock containing (C, 28.64; H, 3.61; F, 27.18; N, 10.02; O, 15.26; S, 15.29)
Molecular formula : C10H15F6N3O4S2
Molecular Weight : 419.12 g/mol
Purity >99% (purity determined by MNR)
Homogeneity : homogeneous

Specific details on test material used for the study:

Batch: L16-0180
storage condition: at room temperature
expiration ( or re-test) date: 19 April 2030

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.
- Age: bovine cattle were up to 12 months old.
- Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
- Upon arrival of the eyes, selection and preparation was performed as soon as possible.
- The eyes were carefully examined for defects and any defective eyes were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

As the test item was a non-surfactant liquid, it was tested undiluted (i.e. in its original form).
750 µL (± 8 µL) was applied on each cornea using the closed-chamber method.

Duration of treatment / exposure:

As the test item was a non-surfactant liquid, a treatment time of 10 minutes (± 30 seconds) was used in the study.

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes)

Number of animals or in vitro replicates:

A single experiment was performed unsing three corneas for each treated series (test item, positive and negative controls).

Details on study design:

PREPARATION OF CORNEAS
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS.
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber.
Both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea. The holders were pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C).
At the end of the pre-incubation period, the medium from both chambers of each holder was replaced with fresh cMEM (previously heated to +32°C). Corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0. Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

ALLOCATION OF THE CORNEAS
The test item, the negative and the positive controls were tested on three corneas each. The corneas were distributed as follows:
. the median value of the OPT0 values of all pre-incubated corneas (with OPT0 ≤ 7) was calculated,
. three corneas with opacity values close to the median value were selected as negative control corneas,
. the remaining corneas were shared out between test item and positive control-treated series using a manual distribution procedure.

TREATMENT OF CORNEAS
The medium was removed of the anterior chamber of the corneal holder and 750 µl of the test item or control substances were applied onto the appropriate corneas.
The treatment time of each series of three corneas was carefully measured with a chronometer, starting from the beginning of treatment of the first cornea of each series. Then each further operation (rinsing, measurement, etc.) was carried out in the same order for the three corneas of each series.
After application of the items, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for the selected treatment time: 10 minutes (± 30 seconds).
At the end of the exposure period, the test substance and control substances were removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed four times with pre-warmed cMEM containing phenol red before a final rinse with pre-warmed cMEM without phenol red. The anterior chamber was refilled with fresh pre-warmed cMEM without phenol red. The front cover was replaced. The holders were then incubated horizontally (corneas placed vertically) for 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C). On completion of the 2-hour incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (+32°C (± 1°C)), the second opacity measurement (OPT2) was then performed.

OPACITY MEASUREMENTS
An OPKIT opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.
Just before the first opacity measurement (i.e. OPT0), the opacitometer was calibrated using specific calibrators. Values obtained for each calibrator were as follows:
. calibrator No. 1: set to 75,
. calibrator No. 2: from 145 to 155,
. calibrator No. 3: from 218 to 232.
Just before the second opacity measurement (i.e. OPT2), the opacitometer was calibrated using the calibrator No. 1 set to 75.
Just after each opacity measurement (OPT0 and OPT2), the calibration of the opacitometer was checked by using the calibrator No. 1. The obtained value was between 73 and 77.
Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.
For opacity measurement, care was taken to make sure that no air bubbles were present within the holders containing corneas (by ensuring that each compartment was filled to overflowing with heated cMEM) and each holder was wiped dry.

PERMEABILITY DETERMINATION
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).
At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).

MACROSCOPIC EXAMINATION
After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium.
Then corneas were fixed in 10% neutral buffered formalin at room temperature. Based on the obtained results, it was decided that histological examination was not necessary.

DATA EVALUATION
The In Vitro Irritancy Score (IVIS) was determined from the opacity and permeability measurements, as described below.
Opacity :
The change in opacity value of each individual cornea treated with test item, negative or positive control was calculated by subtracting the initial base-line opacity measurement (OPT0) from the post-treatment opacity reading (OPT2).
The average change in opacity for the corneas treated with the negative control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item or positive control to obtain a corrected opacity value (cOPT). The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.
Permeability :
The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item or positive control was calculated by subtracting the average negative control cornea OD490 nm value from the original OD490 nm value of each cornea. The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.
In Vitro Irritancy Score calculation :
The following formula was used to determine the In Vitro Irritancy Score (IVIS):
IVIS = cOPT + (15 x cOD490 nm)
The IVIS was calculated for each test item and positive control cornea. The mean IVIS for each series of three corneas was calculated from the individual scores. When the cOPT or cOD490 nm values were negative, they are considered equal to 0.
Acceptance criteria :
For the validation of an experiment, the following criteria had to be fulfilled:
. the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
. the mean opacity of the negative control corneas should be < 1.8,
. the mean OD490 nm of the negative control corneas should be < 0.0269 (see § Study plan adherence).
Data interpretation and classification :
The IVIS cut-off values for identifying the test item as inducing serious eye dam age (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are the following:
IVIS UN GHS
If the test item induces an IVIS ≤ 3 No category
If the test item induces an 3 < IVIS ≤ 55 No prediction can be made
If the test item induces an IVIS > 55 Category 1
A single experiment composed of at least three corneas is sufficient for the test item since the resulting classification is unequivocal.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 replicate ; 10 minutes of exposure

Value:

0

Negative controls validity:

other: The mean OD490nm of the negative control corneas was found at 0.030 instead of 0.0269 as specified in the study plan. As a result and based on the obtained results, this deviation is considered not to have any impact on the conclusion of the study.

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
No notable opaque spots or irregularities were observed on negative control and test item-treated corneas.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.
ACCEPTANCE OF RESULTS:
With one exception (mean OD490 nm of the negative control corneas at 0.030 instead of below 0.0269), all acceptance criteria were met.

Any other information on results incl. tables

Results:

GROUP		OPACITY				PERMEABILITY		SCORE
Negative control    (0.9% NaCl)	Holder	OPT0	OPT2	OPT2 -OPT0		OD490nm
	11	2	2	0		0.036
	14	2	2	0		0.022
	3	2	1	-1		0.031
	Mean			0		0.030
	SD			1		0.007
ITEM	Holder	OPT0	OPT2	OPT2 -OPT0	cOPT	OD490nm	cOD490nm
	29	1	2	1	1	0.016	-0.014	1
	26	2	2	0	0	0.014	-0.016	0
	13	3	3	0	0	0.027	-0.003	0
	Mean				0.3		-0.011	0
	SD				0.6		0.0	0.6
Positive control (100% ethanol)	Holder	OPT0	OPT2	OPT2 -OPT0	cOPT	OD490nm	cOD490nm
	29	1	21	20	20	2.340	2.310	55
	26	1	10	9	9	1.732	1.702	35
	13	3	21	18	18	1.776	1.746	44
	Mean				15.7		1.920	44
	SD				5.9		0.339	10.1

OD: optical density

cOD: corrected optical density

cOPT : corrected corneal opacity

SD: standard deviation

OPT0 : corneal opacity before treatment

OPT2: corneal opacity after the two hours recovery period

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

As the test item 1-propyl-2,3-dimethylimidaziolium bis((trifluoromethanesulfonyl)imide induced a mean IVIS < 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, 1-Propyl-2,3-Dimethylimidazolium bis(trifluoromethanesulfonyl)imide, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.
The design of this study was based on the guideline OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.
A single experiment was performed using three corneas for each treated series (test item, positive and negative controls). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, applied undiluted, and the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C.
At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on test item-treated corneas. With one exception (mean OD490nm of the negative control), all acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0. As the test item induced a mean IVIS < 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye dam age (UN GHS No Category).

Under the experimental conditions of this study, the test item 1-Propyl-2,3-Dimethylimidazolium bis(trifluoromethanesulfonyl)imide was identified as a test item not requiring classification for eye irritation or serious eye damage (UN GHS No Category).