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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Allocation of animals 25 February 2020 End of experimental phase Last day of necropsy 06 May 2020

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 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

Justification for study design:

The purpose of this study was to provide information on toxicity in male and female rats after repeated dosing, as well as any possible effects of Lithium 4,5-dicyano-2-(trifluoromethyl) imidazolate on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus and parturition.

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:

solid

Test animals

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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on 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

Details on mating procedure:

Pairing was monogamous (one male to one female) with one exception due to the mortality of the first male selected for pairing on Day 2 of pairing period.
A vaginal smear was taken from all females from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred.

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). 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 (post coitum Day 29/30). 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

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.

Examinations

Parental animals: Observations and examinations:

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 - Parental animals
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.

Vaginal smears and oestrous cycle
Stock females
Oestrous cycle was monitored by vaginal smears in all stock females for 2 weeks before allocation in order to exclude from the study females with irregular cycle. These data are not tabulated in this report, but will be archived with all raw data.
Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification of mating including not less than 2 weeks before the pairing.
The vaginal smear data were examined to determine the following:
– anomalies of the oestrous cycle;
– pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken from all females, before despatch to necropsy with the exception of one control female sacrificed for humane reasons.

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

Oestrous cyclicity (parental animals):

Pairing was monogamous (one male to one female) with the exception indicated below.
A vaginal smear was taken from all females from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred.
Female animal no. X1370077, due to the mortality of the first male selected for pairing (no. X1370078 found dead on Day 2 of pairing period) and resulting not mated after the first day of pairing, was paired with a second male (no. X1370062).

Sperm parameters (parental animals):

The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed

Litter observations:

Parturition and gestation length
A parturition check was performed from Day 20 to Day 25 post coitum. Three females (1 control female, 1 of mid-dose group and 1 of high dose group) which did
not give birth after 25 days of post coitum period were sacrificed onDay 29 or 30 post coitum. These animals were found not pregnant at necropsy. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post partum). Seven high dose females lost their litters fromDay 1 toDay 3 post partum andwere sacrificed within 4 days from occurrence.

Pups identification, weight and observations
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4 and 13 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters. After culling, all pups were sacrificed with the dams on Day 14 post partum.

Culling and pups selection for blood collection (serum hormone) at necropsy
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. At least two pups (males or females, selected for culling) were sacrificed for blood collection. Two male pups were selected for dam no. X1370055 (Group 3) while two female pups were selected for dam nos. X1370027 (Group 2) and X1370057 (Group 3). One male pup only was selected for dam no. X1370031 (Group 2). No culling was performed for dam no. X1370047 (Group 3) due to a reduced size of the litter (less than 8). In addition, due to the high mortality observed in pups of Group 4 dams, no culling was performed in the high dose group.

Anogenital distance (AGD)
The anogenital distance (AGD) of each pup was measured on Day 1 post partum. The measure of AGD was normalized to the cube root of the pup’s body weight measured on Day 1 post partum. Data of AGD of high dose group are reported in Appendix 21 only. The evaluation of AGD data of high dose pups was not performed due to the limited number of pups available.

Nipple count
The presence of nipples/areolae in male pups was checked on Day 13 post partum (a day before despatch to necropsy).

Blood collection and thyroid hormone determination (T3, T4 and TSH)
On Day 4 post partum, as part of the necropsy procedure, blood samples (approximately 0.2 mL) were taken from 2 pups (1 male and 1 female, if possible). The exceptions are described in section 4.3.12. On Day 14 post partum, as part of the necropsy procedure, blood samples (approximately 0.5 mL) were taken from 2 pups (1 male and 1 female). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture). The order of collection was equalised between groups.

Postmortem examinations (parental animals):

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

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

Parental 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.
Females
Parental females were examined also for the following:
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant females were immersed in a 20% solution of ammonium
sulphide to reveal evidence of implantation.

Organ weights
Parental animals
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.
In addition, the testes and epididymides of the 5 males selected from control and high dose groups were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germcell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject: Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS- H stained sections were used to identify the spermatogenic stages.

Postmortem examinations (offspring):

Euthanasia (All groups)
Pups
Pups that had completed the scheduled test period (Day 4 or Day 14 post partum) and those sacrificed for humane reason (dam sacrificed) were euthanised by intraperitoneal injection of Sodium Thiopenthal. Pups selected for blood collection for hormone determination were killed on the day of blood sampling (see section 4.4.4).

Necropsy
Pups
All pups found dead in the cage or those sacrificed for humane reasons (dam sacrificed) were examined for external and internal abnormalities.
All culled pups sacrificed on Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

Nipples retention at Day 14 post partum
No nipples were retained since no nipples were found at Day 13 post partum.

Organ weights
Pups at Day 14 post partum
Thyroids were weighed from one male and one female pup selected for blood collection for hormones determination and preserved in 10% neutral buffered formalin. The thyroid weights were determined after fixation.

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.

Reproductive indices:

Group mean values, where possible, were calculated for all parameters. Data from non pregnant females and from females with total litter loss, were excluded from group mean calculations as considered appropriate by the Study Director.
The following reproductive indices were calculated:
Males
Copulatory Index (%) = No. of males with confirmed mating/No. of males cohabitatedx 100
Fertility Index (%) = No. of males which induced pregnancy/No. of males cohabitated x 100

Females
Copulatory Index (%) =No. of females with confirmed mating/No. of females cohabitated x 100
Fertility Index (%) =No. of pregnant females/No. of females cohabitated x 100

Offspring viability indices:

Pre- implantation loss was calculated as a percentage from the formula:
(No. of corpora lutea – no. of implantation) / No. of corpora lutea x 100

Pre-natal loss was calculated as a percentage from the formula:
(No. of visible implantations – Live litter size at birth)/ No. of visible implantations x 100

Post natal loss at Day 0 post partum was calculated as a percentage from the formula:
Total litter size – Live litter size/Total Litter size X 100

Post natal loss at Day 4 post partum (before culling) was calculated as a percentage from the formula:
(Live litter size at birth - live litter size at Day 4 (before culling)/Live litter size at birth X 100

Post natal loss at Day 13 post partum (after culling) was calculated as a percentage from the formula:
(Live litter size on Day 4 (after culling) – Live litter size on Day 13 )/Live litter size on Day 4 (after culling) X 100

Anogenital distance in pups was presented as normalized to the cube root of body weight collectedon Day 1 post partum.
Sex ratios was calculated at birth, on Day 4 and on Day 14 post partum and will be presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

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.

Other effects:

no effects observed

Description (incidence and severity):

Regular layering in the germinal epithelium was noted in the seminiferous tubules of the observed high dose males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle was similar between the treated groups and control animals.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive parameters were similar between the treated groups and control animals.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

gross pathology

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs in pups, did not reveal any treatment related effects.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Litter data did not show any treatment related effects in low and mid-dose groups. An increased incidence of pup loss at birth was recorded in high dose females. All dams of the high dose group lost their litters within Day 3 post partum.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No treatment-related effects were observed in anogenital distance measured in male and female pups of the low and mid-dose groups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were found in male pups at Day 13 of lactation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A slight decrease in pups thyroid absolute weight was noted in the mid-dose group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings in deceased pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.

Details on results (F1)

At Day 4 post partum, T3 and T4 were decreased in pups from all groups, with the exception of T4 in female pups of group receiving 5 mg/kg/day with no dose-relation.
At Day 14 post partum, T4 was decreased in male and female pups of groups treated at 5 and 10 mg/kg/day with dose-relation. In addition, T3 showed a decrease in males of group treated at 10 mg/kg/day.
Since no other related changes were recorded (TSH increase and/or increased thyroid weight), the above findings were not considered to be adverse.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

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.
The NOAEL for reproductive toxicity for parental males and females was 25 mg/kg/day.
The NOAEL for pup developmental toxicity was 10 mg/kg/day.

Executive summary:

The toxic effects on Sprague Dawley rats after repeated oral dosing with Lithium 4,5-dicyano-
2-(trifluoromethyl)imidazolate, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring, were investigated in this study.
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.
Oestrous cycle, fertility index, reproductive parameters and mating performance were similar between the treated groups and control animals.
Decrease in the live litter size at birth and increase in the pre-natal loss were seen in high dose group when compared with control values. In addition, increased incidence of pup loss at birth was recorded in high dose females resulting in a total litter loss within Day 3 of post partum phase for all dams of the high dose group. Litter data did not show any treatment related effects in low and mid-dose groups.
Clinical signs in pups and sex ratio did not reveal any treatment related effects. An increased incidence of found dead and/or missing pups was noted in high dose group.
No nipples were found in male pups at Day 13 of lactation.
No treatment-related effects were observed in anogenital distance measured in male and female pups of the low and mid-dose groups.
At Day 4 post partum, T3 and T4 were decreased in pups from all groups, with the exception of T4 in females dosed at 5 mg/kg/day with no dose-relation.
At Day 14 post partum, T4 was decreased in male and female pups of groups receiving 5 and 10 mg/kg/day showing dose-relation. In addition, T3 showed a decrease in male pups of group receiving 10 mg/kg/day.
Since no other related changes were recorded (TSH increase and/or increased thyroid weight), the changes noted in thyroid hormones were not considered to be adverse.
Necropsy findings in deceased and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.
A slight decrease in pups thyroid absolute weight was noted in mid-dose group.
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.
Regular layering in the germinal epithelium was noted in the seminiferous tubules of the observed high dose males.

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.
The NOAEL for reproductive toxicity for parental males and females was 25 mg/kg/day.
The NOAEL for pup developmental toxicity was 10 mg/kg/day.