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

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 February 2014 -- 11 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: CEGAV, Argenvilliers, France
- Age at study initiation: 2 to 4 months old on the day of treatment
- Fasting period before study: no
- Housing: noryl cages
- Diet: 110 pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 25 March 2014 to 11 April 2014

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 g/flank

Duration of treatment / exposure:

3 min, 1 h, 4 h.

Observation period:

1, 24, 48 and 72 h after removal of the dressing; if relevant, daily until reversibility of reactions

Number of animals:

Three males

Details on study design:

TEST SITE
- Area of exposure: on the day before treatment, two or four areas of skin (3.5 cm x 5 cm) were closely clipped using electric clippers on the right and left anterior and/or posterior flanks of animals
- Coverage: 6 cm2
- Type of wrap if used: gauze pad held in place by a non-irritation semi-occlusive dressing and a restraining bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: using a dry cotton pad
- Time after start of exposure: at removal of each dressing (see Duration of exposure)

SCORING SYSTEM:

- Erythema and eschar formation:
0 no erythema
1 very slight erythema (barely perceptible)
2 well-defined erythema
3 moderate to severe erythema
4 severe erythema (beet redness) to slight eschar formation (injuries in depth)

- Edema formation:
0 no edema
1 very slight edema (barely perceptible)
2 slight edema (edges of area well-defined by definite raising)
3 moderate edema (raised approximately 1 millimeter)
4 severe edema (raised more than 1 millimeter and extending beyond area of exposure)

- Any other lesions were noted

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

no data

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

no data

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: day 2

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

no data

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: day 4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

no data

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item was very slightly irritant when applied topically to rabbits.

Therefore, the test item should not be classified as irritating to skin according to the criteria of CLP/GHS Regulation.

Executive summary:

The potential irritant properties of Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate was evaluated following dermal application on rabbits. This study was conducted in compliance with the OECD Guideline No. 404 and the principles of Good Laboratory Practice. The test item was first applied for periods of 3 minutes, 1 hour and 4 hours to a single male New Zealand White rabbit. After the 4-hour application, since the mean value from grading at 24, 48 and 72 hours after patch removal was < 2.3 for erythema and for edema, the test item was applied on the skin of two other animals for 4 hours. A quantity of 0.5 g/flank was used. The test item was placed on a gauze pad, moistened with drinking water treated by reverses osmosis, which was then applied to a skin area of approximately 6 cm2. The gauze pad was held in place by a non-irritation semi-occlusive dressing and a restraining bandage. After required period of contact with the skin, the dressing was removed. Each animal was observed once a day for mortality and clinical signs. For each exposure period, cutaneous reactions were evaluated approximately 1, 24, 48 and 72 hours after removal of the dressing and then daily until the reversibility of cutaneous reactions. The mean values of the scores for erythema and edema were calculated for each animal. Body weight was recorded on the day of treatment and at the end of the evaluation of cutaneous reactions. On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination.

No unscheduled deaths occurred during the study and no clinical signs were noted in any animals. The body weight of the animals was unaffected by the test item treatment. After a 4-hour exposure, no skin reaction was observed in 1/3 animal and a very slight erythema was noted in 2/3 animals on Day 1, persisting until Day 3 in one of them. Dryness of the skin was recorded in one animal from Day 4 until Day 10. Mean scores calculated for each animal over 24, 48 and 72 hours were as follows:

- erythema: 0.0, 0.0, 0.7; showing no significant inflammation,

- edema: 0.0, 0.0, 0.0; showing no significant inflammation.

Under the experimental conditions of this study, Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate was very slightly irritant when applied topically to rabbits.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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)

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

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

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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).

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation

In vitro study

The corrosive potential of Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate was evaluated in the Episkin^TMreconstructed skin model.The study design was based upon international guidelines (OECD Guideline No. 431 and Commission Regulation (EC) No. 440/2008, B.40bis). The study was conducted in compliance with CiToxLAB’s standard operating procedures and the principles of Good Laboratory Practice. A preliminary test was performed to detect the ability of the test item to interfere with cell viability measurements by directly reducing MTT.Following the preliminary test, the corrosive potential of the test item was tested in the main assay. The test item, the negative and positive controls were topically applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 4 hours; test item for 3 minutes, 1 hour and 4 hours.At the end of the designated incubation periods, each tissue was rinsed with D-PBS. The cell viability was then assessed by means of the MTT reduction assay.Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

During the preliminary test, the MTT solution containing the test item did not turn blue/purple when compared to the negative control. The test item was therefore presumed not to reduce directly MTT. As a result, no additional controls were performed on water-killed tissues in parallel to the main test (performed on viable tissues).All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.At the end of the MTT incubation period, any tissue discoloration was evaluated with the naked eye. A blue discoloration of the test item-treated tissues following the 3 minutes, 1 hour and 4 hours exposure periods was noted. This discoloration was representative of viable tissues.

The relative mean viabilities of the test item-treated tissues were:

- 162% for the 3 minutes exposure,

- 184% for the 1 hour exposure,

- 191% for the4 hours exposure.

As the mean viabilities were=35%, the results met the criteria for a non corrosive response. However, the high viability values seem to indicate an effect on the mitochondrial activity which could mask a possible cytotoxic effect.

Under the experimental conditions of this study, Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate, tested in its original form, is considered to be non corrosive to the skin.

In vivo study

The potential irritant properties of Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate was evaluated following dermal application on rabbits.This study was conducted in compliance with the OECD Guideline No. 404 and the principles of Good Laboratory Practice.The test item was first applied for periods of 3 minutes, 1 hour and 4 hours to a single male New Zealand White rabbit.After the 4-hour application, since the mean value from grading at 24, 48 and 72 hours after patch removal was < 2.3 for erythema and for edema, the test item was applied on the skin of two other animals for 4 hours.A quantity of 0.5 g/flank was used.The test item was placed on a gauze pad, moistened with drinking water treated by reverses osmosis, which was then applied to a skin area of approximately 6 cm². The gauze pad was held in place by a non-irritation semi-occlusive dressing and a restraining bandage. After required period of contact with the skin, the dressing was removed.Each animal was observed once a day for mortality and clinical signs. For each exposure period, cutaneous reactions were evaluated approximately 1, 24, 48 and 72 hours after removal of the dressing and then daily until the reversibility of cutaneous reactions. The mean values of the scores for erythema and edema were calculated for each animal. Body weight was recorded on the day of treatment and at the end of the evaluation of cutaneous reactions.On completion of the observation period, the animals were sacrificed then discarded without macroscopicpost-mortemexamination.

No unscheduled deaths occurred during the study and no clinical signs were noted in any animals.The body weight of the animals was unaffected by the test item treatment.After a 4-hour exposure, no skin reaction was observed in 1/3 animal and a very slight erythema was noted in 2/3 animals on Day 1, persisting until Day 3 in one of them. Dryness of the skin was recorded in one animal from Day 4 until Day 10.Mean scores calculated for each animal over 24, 48 and 72 hours were as follows:

- erythema: 0.0, 0.0, 0.7; showing no significant inflammation,

- edema: 0.0, 0.0, 0.0; showing no significant inflammation.

Under the experimental conditions of this study, Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate was very slightly irritant when applied topically to rabbits.

Eye irritation

In vitro study

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 at32°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 individualIn VitroIrritancy 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. 

Justification for classification or non-classification

Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate should not be classified as irritating to skin according to the criteria of CLP/GHS Regulations.

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