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

Endpoint summary

Description of key information

A combined screening for toxicity to reproduction with short-term repeated-dose toxicity study via the oral was conducted on the registered substance.

Dose selection was based on the outcome of 14-day Dose Range Finding study.

Based on the results of this study, the NOAEL for the general toxicity was 25 mg/kg/day for males and 10 mg/kg/day for females.

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 105 Sprague Dawley SD rats (45 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival, the weight range for each sex was determined and found to be 206 - 221 g for males and 179 - 201 g for females and the animals were temporarily identified within the cage by means of a coloured mark on the tail.
A health check was then performed by a veterinarian.
An acclimatisation period of 33 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C±2°Cand 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period,
birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. Nesting material was changed at least 2 times a week. Drinking water was supplied ad libitum to each cage via water bottles
A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels of 5, 10 and 25mg/kg/day were selected based on information from a preliminary non-GLP compliant study

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the preparations made on two occasions during the study (Day 1 and again towards the end of the study) were analysed to check the concentration.
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC. The software used for this activity was Empower®2 Build No. 2154. Results of the analyses were within the acceptability limits stated in ERBC SOPs for solutions (90-110% for concentration)

Duration of treatment / exposure:

Males: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy (Days 35 and 36). Males were treated for a total of 34 or 35 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). Non pregnant females (nos. X1370017 - Group 1, X1370043 - Group 3 and X1370073 - Group 4) and one female of the Group 4 that lost the litter (no. X1290079 - Group 4) were dosed up to the day before necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7 and 13 post partum.

Frequency of treatment:

animals were dosed once a day, 7 days a week
The dose volume was 10 mL/kg body weight

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

Each group comprised 10 male and 10 female rats.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected on the basis of the outcome of a 2-week preliminary study conducted on the test substance.

Positive control:

Not included

Observations and examinations performed and frequency:

Mortality
Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs
Before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (0.5-1 hour after treatment).

Clinical Observations (Functional Observation Battery Tests)
Once before commencement of treatment and at least once a week from the start of treatment until termination, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes.
Grip strength and sensory reactivity to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. For males, the tests were performed on Day 29 of the study and for females on Day 12 post partum.
Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order. For males, the test was performed on Day 29 of the study and for females on Day 12 post partum.

Body weight
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

Clinical pathology investigations
Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of the treatment period, by random selection from 5 males and 5 females (females with viable litters) of each main group. Further blood samples for haematology, clinical chemistry and coagulation were taken under identical conditions at the end of the recovery period.
Males: Blood samples for haematological investigations, biochemical tests and hormone determination were collected in condition of food deprivation under isoflurane anaesthesia from the retro-orbital sinus. Blood samples for coagulation test (food available) were collected at necropsy from the vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
Females: As a part of the sacrificial procedure, blood samples for all determinations were withdrawn fromthe abdominal vena cava in condition of food deprivation under isoflurane anaesthesia. The order of collection was equalised between groups.

Urinalysis (Only males)
Individual overnight urine samples were collected during the last week of treatment from the same male animals selected for the clinical pathology investigations and under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.

Blood collection and thyroid hormone determination (T3, T4 and TSH)
Blood collection was performed for hormone determination (0.8 mL) from all animals at termination under condition of food deprivation. No sample was taken from one control female animal sacrificed for humane reasons.

Sacrifice and pathology:

Euthanasia (All groups)
Animals that had completed the scheduled test period, were killed by exsanguination under isoflurane anaesthesia. One control female sacrificed for humane reason (no. X1370009) was killed under carbon dioxide asphyxiation.

Males
The males were killed after the mating of all females on Days 35 and 36 of the study.

Females
The females with live pups were killed on Day 14 post partum. The females which did not give birth 25 days after positive identification of mating or with total litter loss were killed shortly after (see section 4.3.10).

Necropsy
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Organ weights
From all animals completing the scheduled test period, the organs indicated in section 4.5.7 were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in section 4.5.7 were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are listed in section 4.5.7. After dehydration and embedding in paraffin wax, sections of the tissueswere cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In the first instance, the examination was restricted as detailed below:
i Tissues specified in section 4.5.7 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose main group killed at term.
ii Tissues specified in section 4.5.7 from all animals sacrificed or dying during the treatment period.
iii All abnormalities in all groups.

Since findings were observed in the bone marrow, liver and spleen in high dose animals, the examination was extended to:
-Bone marrow, liver and spleen in all females of Groups 2 and 3 and in the remaining female animals of control and high dose groups.

Statistics:

Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males
In high dose males, clinical signs generally observed only during the first week of treatment were limited to ataxia in three males and also decreased activity in one male only.
No treatment-related clinical signs were observed in low and mid-dose groups.

Females
In two females of the high dose group, ataxia and decreased activity were generally observed during the first 8 days of treatment (during the premating period). In addition, in one animal only, tremors, kyphosis and brown staining on or/around the eyes were noted. During the post coitum and first days of the post partum periods (because all females of the high dose group lost their litters and were thus sacrificed), piloerection and/or convulsions and/or tremors were generally noted in the animals.
No clinical signs were observed in low and mid-dose groups during the treatment period.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of 5 cases of premature death, one male and two females from the high dose group, one male from the mid-dose group and one female from the control group occurred during the study.

Based on the clinical signs, macroscopic and microscopic observation performed in the early decedent animals, the factors contributory to the illness status and consequently the death of the control female were ascribed to difficulty in delivery. For the mid-dose male animal, based on the macroscopic and microscopic findings, the cause of death of this animal was misdosing.

Regarding the mortalities noted in the high dose animals, the pathological picture observed in the male animal did not allow to establish the cause of death, while for the two high dose females, the microscopic findings in the bone marrow, similar to those observed in high dose females sacrificed at term, associated with presence of dead foetuses in the uterus were considered the factor contributory to the cause of death and therefore treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No relevant differences in body weight and body weight gain were noted between control and treated males throughout the study.

In female animals, no differences of toxicological significance were noted before pairing, while from the end of the post coitum and during the post partum period, in the high dose group only, a slight decrease in body weight was noted, when compared to the control group.

Body weight gain in high dose females only, was generally reduced during the post coitum and post partum periods.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was unaffected by treatment in males during the study and in females during the premating period.
During the post coitum period, reduction in food consumption was generally noted in mid and high dose females.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Most of high dose females showed slight anaemia associated with mild reticulocytosis and leucocytosis. Anisocytosis and polychromasia were also recorded in three high dose females.
Platelets were increased in two high dose females.
These findings could be correlated with the findings seen at the histopathological examination.
Mid-dose females showed an increase of leucocytes involving mainly neutrophils, lymphocytes and monocytes.
No relevant differences between control and treated males were recorded.

Coagulation
Increase in prothrombin time was recorded in high dose females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Decreases of alanine aminotransferase, cholesterol, urea, phosphorus and potassium were seen in high dose females while mid-dose females showed decrease of urea only.
The magnitudes and directions of these changes were not indicative of an adverse liver impairment, therefore they were considered to be not adverse.
Changes recorded in treated males (chloride, sodium and bilirubin) were of minimal severity or not dose-related, therefore they were considered to be incidental.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Decreases of triiodothyronine (T3) and thyroxine (T4) were recorded in parental males of all treated groups, with the exception of T3 in animals dosed at 5 mg/kg/day with no clear dose-relation.
No increase of the thyroid stimulating hormone (TSH) was recorded in the same animals, with the exception of one male animal treated at 5 mg/kg/day, which showed a 3.5 fold increase of TSH.
Treated females showed a decrease of T4 only with no dose-relation.
Due to the absence of other related changes (TSH increase, histopathology findings and/or increased thyroid weight), the above findings were not considered to be adverse.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant changes were observed in urine analysis performed in males.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Motor activity and landing foot splay, performed at the end of the treatment period, did not reveal changes attributable to the test item. Decrease in grip strength was recorded at the end of treatment in mid- and high dose males.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase was observed in absolute and relative spleen weight of high dose treated females when compared to controls. Such organ weight variation in high dose females was considered treatment-related also on the basis of histopathological evaluation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted following gross pathology examination.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were present in bone marrow, spleen and liver of high dose treated females only.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

mortality

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

Critical effects observed:

no

Conclusions:

The NOAEL for the general toxicity was 25mg/kg/day for parental males and 10 mg/kg/day for parental females.

Executive summary:

The toxic effects on Sprague Dawley rats after repeated oral dosing with Lithium 4,5-dicyano-

2-(trifluoromethyl)imidazolate were investigated in a study conducted in accordance with the OECD TG 422.

The dose levels used were: 0, 5, 10 and 25mg/kg/day. The vehicle was softened water.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy performed for at least 4 weeks. Females were treated for 2 weeks prior to pairing and thereafter during pairing, gestation and lactation periods until Day 13. The non pregnant female and the female sacrificed for humane reasons or females with total litter loss were dosed up to the day before necropsy.

A total of 5 animals died or were sacrificed during the course of the study: 1 female from the control group, 1 male from the mid-dose group, 1 male and 2 females from the high dose group. Mortality in mid-dose male were not considered treatment-related. Regarding the high dose level, no signs were seen in the early male decedent that can clearly established the cause of death while the mortality observed in the females were considered treatment related.

Treatment-related clinical sings (e.g. ataxia, decreased activity, tremors, kyphosis, staining, convulsions) were noted in males and females of the high dose group only.

Neurotoxicity assessment, motor activity and landing foot splay, performed at the end of the treatment period, did not reveal changes attributable to the test item. Decrease in grip strength was recorded in mid- and high dose males.

No differences of toxicological significance were noted throughout the study in the body weight and body weight gain of treated males. In females of the high dose group, a slight decrease in body weight and body weight gain was noted during the post coitum and post partum periods.

No effects on food consumption were observed in males. In females of the mid- and high dose group, food consumption was slightly reduced during the post coitum period.

Changes observed in some haematological and coagulation parameters (erythrocytes, haemoglobin, haematocrit, leucocytosis, prothrombin time) in females of the high dose group were considered treatment-related and correlated with the findings observed at the histopathological examination.

Changes in chloride and sodium in high dose males were considered to be not adverse.

Changes in alanine aminotransferase, cholesterol, urea, phosphorus and potassium observed in high dose females and in urea for mid-dose females were not indicative of liver impairment, therefore were considered to be not adverse.

No relevant changes were observed in urine analysis performed in males.

Decreases of T3 and T4 were recorded in parental males of all treated groups, with the exception of T3 in males dosed at 5 mg/kg/day with no clear dose-relation.

In parental females, a decrease of T4 was noted with no dose-relation.

Due to the absence of other related changes (TSH increase, histopathology findings and/or increased thyroid weight), the changes noted in thyroid hormones were not considered to be adverse.

At macroscopic observation, no treatment-related changes were noted.

At the microscopic examination, treatment-related changes were present in bone marrow, spleen and liver of high dose treated females.

 

Conclusion

Based on the results of the present study, the NOAEL for the general toxicity was 25mg/kg/day for parental males and 10 mg/kg/day for parental females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was GLP-compliant and conducted according to a relevant OECD Testing Guideline.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

14-day DRF Study
The toxicity of Lithium 4,5-dicyano-2-(trifluoromethyl)imidazolate, after daily oral administration (gavage) to rats, was investigated over a period of 2 consecutive weeks.
Three groups, each of 5 male and 5 female Sprague Dawley rats, received the test item at dose levels of 25, 50 and 75mg/kg/day. Due to mortality and severe signs of toxicity observed at dose levels of 50 and 75 mg/kg/day, the treatment of these groups was stopped and the surviving animals sacrificed. Animals treated at 25mg/kg/day were treated for 2 consecutive weeks. A fourth group of 5 male and 5 female rats received the vehicle alone (purified water) and acted as a control.

The following investigations were performed: clinical signs, body weight and food consumption, post mortem organ weights (only for animals of Groups 1 and 2) and macroscopic observations.

All males and 4 out of 5 female animals dosed at 75 mg/kg/day and all males and 3 out of 5 females dosed at 50mg/kg/day were found dead or sacrificed for humane reasons between Days 2 and 4 of the study. Convulsions, prone posture, ataxia, tremors, semi closed eyes and pale appearance were observed in these animals. Although macroscopic observations (foamy contents of trachea and/or brown staining of the ventral region and red staining of muzzle) did not clearly indicate the cause which led to poor health conditions and death, these moralities were considered to be treatment-related. Therefore, in agreement with the Sponsor, the surviving animals of these two treatment groups were sacrificed on Day 4 of the study without any further investigation.
No mortality occurred in the animals treated at 25 mg/kg/day.

Convulsions, tremors, hunched posture and decreased activity were occasionally observed during the observation period in the males dosed at 25mg/kg/day. Piloerection was also frequently observed.
No clinical signs were observed during the study in the females receiving 25 mg/kg/day.

No relevant changes in body weight were observed during the study in male and female animals treated at 25 mg/kg/day.

Food consumption in animals treated at 25 mg/kg/day was comparable to the control group.

No changes were observed in terminal body weight and absolute and relative organ weights of control and treated animals that completed the treatment (Groups 1 and 2).

Animals killed at termination (Group 2) did not show relevant macroscopic changes that could be treatment-related.

On the basis of the above results, it can be concluded that severe toxicity occurred in the animals administered with the test item at dose levels of 50 and 75mg/kg/day, while the dose level of 25mg/kg/day could be selected as the high dose level for a subsequent toxicity study in rats.

Combined screening for toxicity to reproduction with short-term repeated-dose toxicity study
The toxic effects on Sprague Dawley rats after repeated oral dosing with Lithium 4,5-dicyano- 2-(trifluoromethyl)imidazolate were investigated in a study conducted in accordance with the OECD TG 422.

The dose levels used were: 0, 5, 10 and 25mg/kg/day. The vehicle was softened water.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy performed for at least 4 weeks. Females were treated for 2 weeks prior to pairing and thereafter during pairing, gestation and lactation periods until Day 13. The non pregnant female and the female sacrificed for humane reasons or females with total litter loss were dosed up to the day before necropsy.

A total of 5 animals died or were sacrificed during the course of the study: 1 female from the control group, 1 male from the mid-dose group, 1 male and 2 females from the high dose group. Mortality in mid-dose male were not considered treatment-related. Regarding the high dose level, no signs were seen in the early male decedent that can clearly established the cause of death while the mortality observed in the females were considered treatment related.

Treatment-related clinical sings (e.g. ataxia, decreased activity, tremors, kyphosis, staining, convulsions) were noted in males and females of the high dose group only.

Neurotoxicity assessment, motor activity and landing foot splay, performed at the end of the treatment period, did not reveal changes attributable to the test item. Decrease in grip strength was recorded in mid- and high dose males.

No differences of toxicological significance were noted throughout the study in the body weight and body weight gain of treated males. In females of the high dose group, a slight decrease in body weight and body weight gain was noted during the post coitum and post partum periods.

No effects on food consumption were observed in males. In females of the mid- and high dose group, food consumption was slightly reduced during the post coitum period.

Changes observed in some haematological and coagulation parameters (erythrocytes, haemoglobin, haematocrit, leucocytosis, prothrombin time) in females of the high dose group were considered treatment-related and correlated with the findings observed at the histopathological examination.

Changes in chloride and sodium in high dose males were considered to be not adverse.

Changes in alanine aminotransferase, cholesterol, urea, phosphorus and potassium observed in high dose females and in urea for mid-dose females were not indicative of liver impairment, therefore were considered to be not adverse.

No relevant changes were observed in urine analysis performed in males.

Decreases of T3 and T4 were recorded in parental males of all treated groups, with the exception of T3 in males dosed at 5 mg/kg/day with no clear dose-relation.

In parental females, a decrease of T4 was noted with no dose-relation.

Due to the absence of other related changes (TSH increase, histopathology findings and/or increased thyroid weight), the changes noted in thyroid hormones were not considered to be adverse.

At macroscopic observation, no treatment-related changes were noted.

At the microscopic examination, treatment-related changes were present in bone marrow, spleen and liver of high dose treated females.

Based on the results of the present study, the NOAEL for the general toxicity was 25mg/kg/day for parental males and 10 mg/kg/day for parental females.

 

 

Consistent adverse effects were observed during the 14-day DRF and combined screening for toxicity to reproduction with short-term repeated-dose toxicity study conducted on the test item; i.e. convulsions, tremors, ataxia, decreased activity,... All animals dosed above 25 mg/kg bw/d were prematurely sacrificed during the 14-day DRF study, and treatment-related mortality was observed in two females treated at 25 mg/kg bw/d during the combined study.

In the 14-day DRF, these effects were more marked in male animals than in female animals at all dose-levels.

In the combined study, these effects appeared in male animals at the high dose, but were more marked in female animals at the high dose during gestation.

This supports the assumption that effects of Li and some of its derivatives are related to the total dose received per animal, instead of the dose per kilograms. As male rats are heavier than female rats, they actually receive a more important dose of the test item, hence the more marked effects observed during the 14-day DRF study, and before gestation during the combined study. During gestation, the bodyweight of female rats increase significantly, thus the actual dose received increases as well.

Kidney saturation of the elimination of the test item is believed to be involved in this mechanism, although available experimental data do not allow reaching a conclusion.

Justification for classification or non-classification

Treatment-related adverse effects were observed during short-term repeated-dose toxicity testing  (DRF study) at doses of 25, 50 and 75 mg/kg bw/d. The most significant effects are convulsions, tremors, and ataxia. In addition, mortality occurred at 50 and 75 mg/kg bw/d indicating that the Maximal Tolerated Dose was exceed.
At 25 mg/kg bw/d, in OECD 422, adverse effects – including morbidity – in pregnant  female animals is considered related to an increase in actual dose received, consecutive to an increase in bodyweight during gestation. Therefore, this does not reflect actual exposure of non-pregnant individuals, and these effects shall not be taken into account when considering the classification of the registered substance for effects following a repeated exposure.
Consequently, Lithium 4,5-dicyano 2-(trifluomethyl) imidazolate should not be classified for STOT- RE in accordance with Regulation (EC) No 1272/2008.