1H-Imidazole-4,5-dicarbonitrile, 2-(trifluoromethyl)-, lithium salt (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 March 2014 -- 20 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Test guideline 2

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

lithium(1+) 4,5-dicyano-2-(trifluoromethyl)-1H-imidazol-1-ide

EC Number:

691-963-1

Cas Number:

761441-54-7

Molecular formula:

C6 F3 N4. Li

IUPAC Name:

lithium(1+) 4,5-dicyano-2-(trifluoromethyl)-1H-imidazol-1-ide

Test material form:

other: white powder

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Species: bovine cattle.
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.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
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 at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swivelled in order to observe the fringe areas and any scratches directly under the light.
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 until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature.

The prepared corneas were stored and used within 24 hours.
(Pre)Incubation T°C: 32°C
Dates of experimental phase: from 19 March 2014 to 20 March 2014

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Duration of treatment / exposure:

Exposure period of 4 hours ± 5 minutes, followed by rinsing

Observation period (in vivo):

Opacity measurement:
- before treatment
- after rinsing

Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement

Number of animals or in vitro replicates:

Not applicable
Triplicate corneas for each tested substance (test item, negative control, positive control)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Rinsing: the anterior part of the eye was emptied and then rinsed with at least three times with pre-warmed cMEM

NEGATIVE CONTROL:
As the test item was tested in a vehicle, the negative control was replaced by a vehicle control.

POSITIVE CONTROL:
20% imidazole solution

SCORING SYSTEM/TOOL
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average vehicle control value from values in positive control and test item.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average vehicle control value from values in positive control and test item.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the experimental conditions of this study, the test item, was classified as a test item inducing serious eye damage (UN GHS Category 1).

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate. 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 study design was based on the guideline OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating proceduresand 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. Three corneas were used for each treated series (test item, positive control and vehicle control). Before the treatment, a first opacity measurement was performed using an opacitometer. The test item was applied at the concentrations of 20% (w/v) in the vehicle (NaCl 0.9%), in a single experiment using a treatment time of 4 hours and the closed-chamber method.

At the completion of the treatment period, the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed. A second opacity measurement was then performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluoresceine solution. The holders were then incubated vertically for 90 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.

Opacity, cornea thickening and fluoresceine fixation were observed on the corneas treated with the test item following treatment. The individual In Vitro Irritancy Score (IVIS) obtained for the three test item-treated corneas were: 40, 61 and 70. The mean IVIS was 57. On the basis of the individual scores, the results could be considered as equivocal since the first of the three corneas gave a discordant prediction from the mean of all three corneas and since this discordant result was >10 IVIS units from the cut-off threshold of 55. In this case a second experiment may be considered. However, since the mean IVIS was > 55, it was decided to not perform a second experiment and to directly classify this test item as UN GHS Category 1. 

 

Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate was classified as a test item inducing serious eye damage (UN GHS Category 1).