Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April to 24 June, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2018
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
4 weeks acclimation instead of 2; chemical analysis and prothrombin analysis software were different; vaginal smear not taken from 1 control female sacrificed early; 3 females with total litter loss were killed after 6-14 days not immediately
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

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 because there are ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Lecco, Italy
- Females: nulliparous, virgin and non-pregnant at study commencement
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: males: 217 to 238 g; females: 220 to 234 g
- Fasting period before study: no
- Housing, pre-mating period: groups of 5 animals of one sex in clear polysulfone solid bottomed cages measuring 59.5 × 38.0 × 20.0 cm (Techniplast Gazzada S.a.r.l., Buguggiate (VA), Italy); nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing, during mating: one male and one female in clear polysulfone cages measuring 42.5 × 26.6 × 18.5 cm with a stainless steel mesh lid and floor (Techniplast – Gazzada S.a.r.l., Buguggiate (VA), Italy); each cage tray held absorbent material which was inspected and changed daily
- Housing, after mating: males: recaged as they were prior to mating; females: individual solid bottomed cages for the gestation period, birth and lactation periods (measuring 42.5 × 26.6 × 18.5 cm); nesting material was provided inside suitable bedding bags; suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week
- Diet: ad libitum; a commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milane2se (MI), Italy); certificates and quality analysis included in report
- Water: ad libitum , via water bottles, except in the case of urinalysis investigations
- Acclimation period: four weeks, from time of arrival until beginning of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 2 °C
- Humidity: 55 % ± 15 %
- Air changes: 15 to 20 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
10 ml/kg bw
Vehicle:
water
Remarks:
softened
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
- The required amount of test item was dissolved in the vehicle (softened water) in order to create concentrations of 10, 30 and 80 mg/ml. The formulations were prepared daily. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Volume of vehicle/test item: 10 ml/kg of body weight based on most recently recorded body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (RTC Study no. A3095). Samples of the formulations prepared during the current study (the first and the last week of treatment) were analysed to check the concentrations. Chemical analysis was carried out by the Analytical Chemistry Department at RTC. The software used for this activity was Analyst 1.6.2. Results of formulation analyses were within the acceptability limits (90-110%), with the exception of Group 2 during the last week. Analysis was successfully repeated and results were within the acceptability limits.
Duration of treatment / exposure:
- males: 34 days in total, from two weeks prior to pairing until day before necropsy, 14 days after the mating of all females
- females: between 50 and 60 days in total; from two weeks prior to pairing until day 13 post-partum
- 2 weeks prior to mating: dosing began in male and female animals
- mating period: usually 2 to 3 days
- gestation period: from day of successful mating (day 0 post coitum) until day of parturition (day 0 post partum), usually 22 days; females which did not give birth after 25 days post coitum were sacrificed shortly after; all animals gave birth after 22 to 23 days
- post partum period: from day of parturition (day 0 post partum) until sacrifice (day 14 post partum)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low-dose group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
mid-dose group
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
high-dose group
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected in consultation with the Sponsor, based on information from a preliminary, non-GLP compliant study (RTC no. E0244).
- Rationale for animal assignment: on the day of allocation all animals were weighed. Animals at the extremes of the weight distribution and animals showing irregular cycle were excluded to leave the required number of animals. The rats were allocated to groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 animals of one sex per cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were distributed to minimise possible environmental effects. No replacements occurred after the first dose was administered.
- Route of exposure: the test item was administered orally, by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.

Examinations

Observations and examinations performed and frequency:
MORTALITY AND CAGE SIDE OBSERVATIONS
- Time schedule: daily; all animals were checked early in each working day and again 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
- Severely debilitated animals were observed carefully; animals judged to be in extremis were killed
- A complete necropsy was performed as detailed below

DETAILED CLINICAL OBSERVATIONS
- Time schedule: once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed approximately at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.
- Once before commencement of treatment and once a week thereafter, 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). All observations were recorded for individual animals.
- Clinical observations checked in Table 1: Clinical observations according to the Functional Observation Battery Tests, in "Other information on materials and methods" were performed. All observed parameters, with the exception of the pre-dose observations, are reported in a group incidence table (Table 2: Clinical signs of females - group incidence, in "Other information on results"). Individual data were not included in the report. Data were reported until Week 5 of study for males and Week 7 of study for females. All other data were not tabulated in the report but archived together with all raw data.

BODY WEIGHT
- Time schedule for examinations: males: weighed weekly from allocation to termination; females: weighed weekly from allocation to positive identification of mating and on gestation days 0, 7, 14 and 20. Females which did not give birth were also weighed just prior to necropsy (data not presented in the report but archived with study raw data). Dams were also weighed on Days 1, 4, 7 and 13 post partum and just prior to 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 gestation 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 collection was performed for hormone determination fromall animals at termination under conditions of food deprivation.
- Blood samples for haematology, clinical chemistry and coagulation were collected by random selection from 5 males and 5 females (females with viable litters if possible) under condition of food deprivation. No samples were taken for animals sacrificed for humane reasons.
- Males: blood samples for haematological and biochemical investigations and hormone determination were collected under isofluorane anaesthesia from the retro-orbital sinus. Blood samples for coagulation test (food available) were collected at necropsy from the vena cava under isofluorane anaesthesia. The order of collection was equalised between groups. Blood samples taken from male nos. X0930026 and X0930078 were unsuitable for haematological investigations (clotting blood).
- Females: as a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the abdominal vena cava under isofluorane anaesthesia. The order of collection was equalised between groups.
The blood samples collected were divided into tubes as follows:
- EDTA anticoagulant for haematological investigations
- Heparin anticoagulant for biochemical tests
- Citrate anticoagulant for coagulation tests
- No anticoagulant for hormone assay

HAEMATOLOGY
- Analysed by Siemens Advia 120: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocites, eosinophils, basophils, monocytes, large unstained cells), and platelets

COAGULATION
- Using Instrumentation Laboratory ACL Elite Pro: prothrombin time

CLINICAL CHEMISTRY
- Using Siemens Advia 1200: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, bile acids, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G Ratio, sodium, potassium, calcium, chloride and inorganic phosphorus

URINALYSIS
- Urinalyses was performed only randomly selected males, using Menarini AUTION MAX 4280
- During the last week of treatment, individual overnight urine samples were also collected from the same animals selected for clinical pathology investigations (5 males/group), randomly selected. 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.
- The measurements performed on urine samples are: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, and blood.
- The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, and other abnormal components.

BLOOD COLLECTION AND THYROID HORMONE DETERMINATION (T3, T4 AND TSH)
- Samples were taken from all parental males from all groups
- Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots and stored at -80°C pending analysis
- Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K) Estrous cyclicity (parental animals)

VAGINAL SMEARS AND OESTROUS CYCLE
- Stock females: oestrous cycle was monitored by vaginal smears in all stock females for at least 2 weeks before allocation, in order to exclude from the study females with irregular cycles. These data are not tabulated in this report but will be archived with the raw data.
- Females allocated to groups: vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data were examined to determine a) anomalies of the oestrous cycle and b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating). Vaginal smears were also taken from all females, with the exception of female no. X0930037, before despatch to necropsy. No vaginal smears were taken from female no. X0930013 sacrificed for humane reasons.
Sacrifice and pathology:
SACRIFICE
- Parental animals and those that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia; one animal sacrificed for humane reasons was killed with carbon dioxide
- Parental males: males were killed after the mating of all females or after at least 28 days of treatment period
- Parental females: females with live pups were killed on Day 14 post partum; the females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after (within 3 days), with the exception of 3 females which were sacrificed between 6 to 14 days from the total litter loss occurrence
- The females which did not give birth were killed on post coitum Days 27-28

GROSS NECROPSY
- The clinical history of the males and females of the parental generation 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 (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination
- Parental females: all females were examined also for number of visible implantation sites (pregnant animals) and number of corpora lutea (pregnant animals); uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation

ORGAN WEIGHTS
- All parental animals completing the scheduled test period: organs indicated in Table 2: Tissue processing, in "Any other information on materials and methods", were dissected free of fat and weighed; the ratios of organ weight to body weight were calculated for each animal
- Samples of all the tissues listed in Table 2 were fixed and preserved in 10% neutral buffered formalin, except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol

HISTOPATHOLOGY
- After dehydration and embedding in paraffin wax, sections of the tissues listed in Table 2 were cut at 5 micrometer thickness and stained with haematoxylin and eosin; in addition, the testes and epididymides 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 also performed
- Clinical pathology investigations were performed on the tissues (Table 2) of the following animals:
- 5 males and 5 females randomly selected from the control group (killed at term);
- 5 high-dose group males (killed at term);
- 1 high-dose group female (one surviving female with live pups was killed at term on Day 14 post partum and subjected to clinical pathology investigations; the others as follows: one not pregnant killed on Day 28 of gestation, the other three females with total litter loss sacrificed between Day 3 and 14 post partum);
- one female (no. X0930013) killed for humane reasons during the treatment period; and
- all abnormalities in all groups

Postmortem examinations (offspring):

SACRIFICE
- Pups killed for humane reasons or those that had completed the scheduled test period (Day 4 or Day 14 post partum) were euthanised by intraperitoneal injection of Thiopenthal

GROSS NECROPSY
- Pups on Day 4 post partum: all pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities; all culled pups sacrificed at Day 4 post partum were subjected to an external examination; sex was determined by internal gonads inspection
- Pups on Day 14 post partum: all live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonads inspection; all pups with abnormalities were retained in a 10% neutral buffered formalin
- Nipple retention at Day 14 post partum: no nipples/areolae were found in male pups

ORGAN WEIGTHS
- Thyroid was weighed from one male and one female from each litter (if possible the same pup sel-ected for serum hormone determination) and preserved for possible histopathological examination; the thyroid weight was determined after fixation
Other examinations:
Litter observations:

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

CULLING AND PUP SELECTION FOR BLOOD COLLECTION (SERUM HORMONE) AT NECROPSY
- On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter; partial adjustment (for example, 5 males and 3 females) was acceptable
- At least one culled male and one culled female were selected for hormone determination
- If litters did not have extra pups to be culled (ie: litters with 8 pups or less), at least 1 female pup was sacrificed for hormone determination in order to retain more male pups for nipple retention on Day 14 post partum; however, retained female pups in each litter were not below 2; this means that for litters with 6 males and 2 females, no pups were selected for hormone determination

ANOGENITAL DISTANCE (AGD) OF PUPS
- The AGD of each pup was measured on Day 1 post partum; the AGD was normalised to the cube root of body weight collected on Day 1 post partum

NIPPLE COUNT
- On Day 13 post partum all live male pups were observed for the presence of nipple areolas: no nipple areolas were seen

BLOOD COLLECTION AND THYROID HORMONE DETERMINATION (T3, T4 AND TSH)
- Blood collection from pups on days 4 and 14 post partum: on Day 4 post partum, as part of the necropsy procedure, blood samples of approximately 0.2 ml were taken from 2 pups (1 male and 1
female, where possible)
- On Day 14 post partum, as part of the necropsy procedure, blood samples of approximately 0.5 ml were taken from 2 pups (1 male and 1 female, where possible)
- Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture)
- Blood samples from different pups were pooled where necessarynecessary. The order of collection was equalised between groups
- Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots and stored at -80 °C, pending analysis
- Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K)
Statistics:
- Standard deviations were calculated as appropriate. For variables such as body weight, food consumption, clinical pathology parameters and organ weight 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.
- 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 nonparametric version of the Williams test. The criterion for statistical significance was p < 0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- Males (Table 2 of "Any other information on results"): no toxicological significance was attributed to the signs seen in the few treated males with signs and symptoms: scabbing on the dorsum was occasionally seen in one male in the control group and one male in the low dose group (duration of one and two days, respectively), and swollen/damaged hindlimb was seen in one mid-dose male (five days)
- Females (Tables 3 and 4 of "Any other information on results"): no toxicologically relevant changes were seen at the daily observations in treated females. One control female showed signs of suffering, piloerection and pallor on Day 22 of gestation during delivery, so was sacrificed for humane reasons. Piloerection, pallor and staining (vagina) were seen in one female treated at 300 mg/kg/day within the first 2-5 days of post partum period, after that a complete recovery was observed. Few female animals from the control group and the low and mid dose groups experienced hairloss on the forelimb after mating.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- Males: no male animals died before the end of the study period
- Females (Table 1 of "Any other information on results"): one control female was humanely sacrificed on Day 22 of gestation for difficulty in delivery: the dam had a total of 11 pups, of which 2 were found dead at birth and the remaining 9 were sacrificed with the dam. No milk in the stomach was recorded for pups and no macroscopic findings were seen at necropsy of the dam, whereas at histopathology inflammatory cell foci and extramedullary haemopoietis were observed in the liver.
- A statistically significant increase in terminal body weight was observed in high-dose females (approximately 9%) compared to controls.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Males (Tables 9 and 11 of "Any other information on results"): no differences were observed in treated males compared to controls during the study.
- Females (Tables 10 and 12 of "Any other information on results"): no differences were observed in treated females (all treated groups) compared to controls during the pre-mating and the gestation periods. No effects on body weight were observed during the post partum period in females in the low- and mid-dose groups compared to controls. A 24 % decrease in body weight (mean) was noted in females in the high-dose group compared to controls during the last week of the gestation period. This reduction was not attributed to maternal toxicity but considered due to foetal toxicity during the last period of pregnancy.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Males (Table 13 of "Any other information on results"): no effects on food consumption were observed in treated males during the pre-pairing phase
- Females (Table 14 of "Any other information on results"): no effects on food consumption were observed in treated females (all groups) during the pre-pairing and gestation phases. Slight decreases (up to 12 %) were seen in females of the low- and mid-dose groups, compared to controls, during the post partum phase.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Males (Table 17 in "Any other information on results"): compared with controls, high-dose males showed a slight decrease in erythrocytes, haemoglobin and haematocrit (approximately 17% below controls). The severity of changes was insufficient in magnitude to represent an adverse anaemia. In addition, monocytes and eosinophils were lower than controls in many treated males. This was mostly due to the high values of some control males, therefore these differences were not considered to be of toxicological relevance.
- Females (Table 18 in "Any other information on results"): no significant differences were observed between the control group and any treatment group.
- Coagulation (Table 19 in "Any other information on results"): no changes in coagulation parameters were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Males: males in the high-dose group showed an increase in urea (42%), sodium, chloride and albumin to globulin ratio; and a decrease in cholesterol (47%) and triglycerides (36%).
- Females: the remaining female of the high-dose group showed increase of protein (15%), albumin (19%), calcium (10%) and bile acids (4.3 fold) above mean control values. A slight increase in protein and albumin was also recorded in some females of the other treated groups, showing minimal severity (2% to 7%).
- Compared with controls, fluctuations of some metabolic parameters were recorded in treated animals, mainly those receiving the highest dose. The other statistically significant differences between control and treated animals (protein, albumin, chloride and sodium in males, alanine and aspartate aminotransferase in females) were not dose-related and/or of minimal severity, therefore they were considered to be of no toxicological relevance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
- Males: no toxicologically relevant changes were observed.
- Females: not measured.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Functional Observation Battery Tests (Tables 5 to 8 of "Any other information on results"): the evaluation of the functional observation battery tests did non indicate any relevant differences between treated groups and controls, in both sexes. Observation of treated animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item compared to controls. Rearing occurred in multiple male and female animals of each treatment and control group, between 4 and 30 times, for durations of betwen 1 and 5 days, however the incidence was not significant between any treatment group and the control group regarding number of times, number of animals or duration, and there was no dose-response relationship. Males and females were both found to defecate and urinate however no important difference in number of times or number of animals was observed between control and treament groups and there was no dose-response relationship.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- No relevant changes were observed in absolute and relative organ weight of treated animals compared to controls.
- The statistically significant changes observed in some organs were not considered toxicologically relevant. Specifically, significantly significant changes included:
- increased absolute and relative male adrenal weight (approximately 23% for relative weight),
- increased absolute and relative male and female liver weight (approximately 16% for male and 20% for female relative weight),
- increased absolute and relative prostate and seminal vesicle weight (approximately 29% for relative prostate weight and 38% for relative seminal vesicle weight),
- decreased relative female heart weight (approximately 10% of relative weight) of the high-dose group,
- decreased absolute and relative male thymus weight (approximately 26% for high dose, 23% for mid-dose and 19% for low dose group for relative weight).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Animals that completed the treatment period and killed at termination did not show relevant macroscopic changes that could be considered treatment-related.
- Two high-dose males showed enlarged adrenals; one high-dose male showed an enlarged seminal
vesicle and one high-dose female showed swollen and dark pituitary glands; however these findings were considered sporadic, spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- No treatment-related changes were noted in animals sacrificed at the end of the treatment period.
- The sporadic lesions such as epithelial vacuolation of the non glandular stomach of one high-dose male, congestion of mesenteric lymph nodes in three high-dose males or hepatic vacuolation observed in control and high-dose treated females, were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- Motor activity, grip strength and sensory reactivity to stimuli (Tables 15 and 16 in "Any other information on results"): no relevant differences were observed in motor activity in treated animals compared to controls (both sexes). Slight decreases in grip strength were observed in treated males (second trial only), which were statistically significant in the mid- and high-dose groups and had a negative dose-response relationship. A slightly statistically significant decrease in landing foot splay (second trial only) was seen in high-dose males. These changes were not seen in treated female groups of the low- and mid-dose groups only (female high-dose group was not considered as it contained only one animal).

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Fate of females - group incidence.

Group number 1 2 3 4
Initial group size 10 10 10 10
Not pregnant 1 1 1 1
Humane kill 1 0 0 0
Total litter loss 0 0 1 7
Total resorption 0 0 0 1
With live pups on day 14post partum 8 9 8 1

Table 2: Clinical signs of males - group incidence - day 1 pre-mating phase to day 21 after pairing.

Group number 1 2 3 4
No. animals observed 10 10 10 10
no. animals no. days no. animals no. days no. animals no. days no. animals no. days
No significant signs 10 34.9 10 34.8 10 34.0 10 35.0
Appearance             
- scabs 1 1.0 1 2.0 0 0.0 0 0.0
- swelling 0 0.0 0 0.0 1 5.0 0 0.0
- damaged hindlimb 0 0.0 0 0.0 1 5.0 0 0.0

Table 3: Clinical signs of females - group incidence - days 1 to 15 pre-mating.

Group number 1 2 3 4
No. animals observed 10 10 10 10
no. animals no. days no. animals no. days no. animals no. days no. animals no. days
No significant signs 10 15.0 10 14.8 10 15.0 10 15.0
Eye/Ear/Mouth             
- tooth/teeth cut 0 0.0 1 2.0 0 0.0 0 0.0

Table 4: Clinical signs of femalespost coitumandpost partumperiods - group incidence (day 0 gestation to day 14post partum).

Group number 1 2 3 4
No. animals observed 10 10 10 10
no. animals no. days no. animals no. days no. animals no. days no. animals no. days
Appearance             
- staining 0 0.0 0 0.0 1 10.0 0 0.0
- piloerection 1 10.0 0 0.0 1 10.0 0 0.0
- hair loss 2 20.0 1 10.0 1 10.0 0 0.0
Respiratory/Cardiovascular           
- pale 1 10.0 0 0.0 1 10.0 0 0.0

Table 5: Clinical observations during treatment - behavioural examination - removal of animals from the home cage - group incidence - males.

Group number 1 2 3 4
No. animals observed 10 10 10 10
no. animals no. days no. animals no. days no. animals no. days no. animals no. days
Removal              
- easy 10 5.0 10 5.0 10 5.0 10 5.0
Handling Reactivity             
- normal 10 5.0 10 5.0 10 5.0 10 5.0
Lachrymation           
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Palpebral Closure             
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Salivation               
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Piloerection             
- absent 10 5.0 10 5.0 10 5.0 10 5.0

Table 6: Clinical observations during treatment - behavioural examination - removal of animals from the home cage - group incidence - females.

Group number 1 2 3 4
No. animals observed 10 10 10 10
no. animals  no. days  no. animals  no. days  no. animals  no. days  no. animals  no. days 
Removal               
- easy 10 6.7 10 6.9 10 7.0 10 6.6
Handling Reactivity             
- normal 10 6.7 10 6.9 10 7.0 10 6.6
Lachrymation             
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Palpebral Closure             
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Salivation               
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Piloerection             
- absent 10 6.7 10 6.9 10 7.0 10 6.6

Table 7: Clinical observations during treatment - behavioural examination - open arena - group incidence - males - 1 to 5 weeks.

Group number 1 2 3 4
No. animals observed 10 10 10 10
no. animals  no. days  no. animals  no. days  no. animals  no. days  no. animals  no. days 
Rearing
- rearing 4-7 0 0.0 0 0.0 1 2.0 0 0.0
- rearing 11-14 5 2.6 4 5.0 7 4.3 6 4.5
- rearing 15-20 9 4.0 6 5.0 5 3.6 5 4.6
- rearing 21-30 1 1.0 0 0.0 0 0.0 0 0.0
Spasms
- absent  10 5.0 10 5.0 10 5.0 10 5.0
Myclonia
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Gait
- normal 10 5.0 10 5.0 10 5.0 10 5.0
Mobility Impairment
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Arousal
- normal 10 5.0 10 5.0 10 5.0 10 5.0
Vocalisation
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Stereotypies        
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Unusual Respiration        
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Bizarre Behaviour        
- absent 10 5.0 10 5.0 10 5.0 10 5.0
Urination         
- absent 10 4.9 10 4.8 10 4.9 10 4.7
- urination 1-3 1 1.0 2 1.0 1 1.0 2 1.5
Defecation             
- absent 10 5.0 10 4.8 10 5.0 10 4.9
defecation 1-3 0 0.0 1 2.0 0 0.0 1 1.0
Tremors               
- absent 10 5.0 10 5.0 10 5.0 10 5.0

Table 8: Clinical observations during treatment - behavioural examination - open arena - group incidence - females - 1 to 7 weeks.

Group number 1 2 3 4
No. animals observed 10 10 10 10
no. animals  no. days  no. animals  no. days  no. animals  no. days  no. animals  no. days 
Rearing
- rearing 4-7 0 0.0 0 0.0 0 0.0 1 1.0
- rearing 8-10 7 2.7 2 2.0 6 2.0 4 3.0
- rearing 11-14 10 4.2 10 5.8 10 5.1 10 5.0
- rearing 15-20 4 1.5 4 1.8 4 1.8 3 1.0
Spasms
- absent  10 6.7 10 6.9 10 7.0 10 6.6
Myclonia
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Gait
- normal 10 6.7 10 6.9 10 7.0 10 6.6
Mobility Impairment
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Arousal
- normal 10 6.7 10 6.9 10 7.0 10 6.6
Vocalisation
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Stereotypies
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Unusual Respiration
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Bizarre Behaviour
- absent 10 6.7 10 6.9 10 7.0 10 6.6
Urination
- absent 10 5.6 10 6.2 10 5.2 10 5.4
- urination 1-3 8 1.3 6 1.0 8 2.0 6 1.7
urination 4-6 1 1.0 1 1.0 1 2.0 1 2.0
Defecation
- absent 10 6.6 10 6.8 10 6.8 10 6.0
- defecation 1-3 1 1.0 1 1.0 1 2.0 0 0.0
Tremors
- absent 10 6.7 10 6.9 10 7.0 10 6.6

Table 9: Body weight of males - group mean data.

Group Body weight (g)
day 1 pre-test   day 1 pre-mating   day 8 pre-mating day 15 pre-mating day 8 after pairing day 15 after pairing
1 no. 10 10 10 10 10 10
mean 391.39 408.96 435.99 463.51 473.55 493.54
s.d. 17.60 29.99 31.42 34.53 37.21 40.60
2 no. 10 10 10 10 10 10
mean 393.03 407.50 438.51 462.46 465.16 489.03
s.d. 20.72 35.75 36.71 36.21 43.97 49.02
3 no. 10 10 10 10 10 10
mean 391.68 414.43 442.64 461.31 474.20 489.64
s.d. 18.08 24.24 28.06 28.72 31.78 34.35
4 no. 10 10 10 10 10 10
mean 391.45 416.93 445.27 467.57 474.86 496.98
s.d. 19.71 23.02 23.87 23.27 21.72 22.37

Table 10: Body weight of females - group mean data.

Group Body weight (g)
day 1 pre-test   day 1 pre-mating   day 8 pre-mating day 15 pre-mating day 0 gestation day 7 gestation day 14 gestation day 20 gestation day 1postpartum day 4postpartum day 7postpartum day 13postpartum
1 no. 10 10 10 10 9 9 9 9 8 8 8 8
mean 281.33 289.12 285.46 299.99 305.19 350.76 384.91 471.52 366.18 378.83 382.13 399.00
s.d. 9.17 11.50 11.21 16.80 24.03 20.42 21.39 30.83 28.00 24.12 25.27 23.64
2 no. 10 10 10 10 9 9 9 9 9 9 9 9
mean 282.15 285.59 280.43 292.80 296.67 344.17 377.76 460.35 361.94 373.41 377.87 397.59
s.d. 8.66 15.51 20.44 24.32 18.19 14.89 23.89 27.63 23.78 19.26 18.98 22.92
3 no. 10 10 10 10 9 9 9 9 9 8 8 8
mean 282.59 292.71 295.87 306.05 308.42 351.94 389.59 478.24 368.63 383.56 393.65 416.52
s.d. 8.82 21.37 28.70 30.32 27.58 26.44 29.23 39.17 29.84 37.42 33.40 29.28
4 no. 10 10 10 10 9 9 9 9 8 - - -
mean 281.71 287.30 296.61 309.00 315.62 354.77 390.42 456.43 378.86 - - -
s.d. 7.34 11.16 12.00 12.03 15.85 13.49 12.50 25.34 16.78 - - -

Table 11: Body weight gain per day of males - group mean data.

Group Body weight gain (g/day)
day 1 pre-mating   day 8 pre-mating day 15 pre-mating day 8 after pairing day 15 after pairing
1 no. 10 10 10 10 10
mean 2.510 3.862 3.931 1.434 2.857
s.d. 2.826 1.423 1.168 1.330 0.913
2 no. 10 10 10 10 10
mean 2.067 4.429 3.423 0.385 3.409
s.d. 3.007 1.236 1.220 2.460 1.378
3 no. 10 10 10 10 10
mean 3.251 4.029 2.668* 1.840 2.207
s.d. 1.272 0.818 0.698 1.942 1.862
4 no. 10 10 10 10 10
mean 3.640 4.048 3.185 1.042 3.008
s.d. 0.880 0.895 0.935 1.189 1.316

* = mean value of group is significantly different from control at p < 0.05

Table 12: Body weight gain per day of females - group mean data.

Group Body weight gain (g/day)     
day 1 pre-mating   day 8 pre-mating day 15 pre-mating day 7 gestation day 14 gestation day 20 gestation day 4postpartum day 7postpartum day 13postpartum
1 no. 10 10 10 9 9 9 8 8 8
mean 1.112 -0.523 2.076 6.509 4.879 14.436 4.216 1.101 2.811
s.d. 0.948 1.303 1.503 1.514 1.243 2.492 3.448 2.388 1.571
2 no. 10 10 10 9 9 9 9 9 9
mean 0.491 -0.737 1.768 6.786 4.799 13.764 3.821 1.486 3.288
s.d. 1.453 1.113 1.553 1.198 1.308 2.244 2.762 1.828 2.433
3 no. 10 10 10 9 9 9 8 8 8
mean 1.447 0.451 1.454 6.217 5.379 14.774 3.811 3.363 3.811
s.d. 2.275 1.345 0.703 1.772 1.430 2.857 6.126 4.144 1.398
4 no. 10 10 10 9 9 9 - - -
mean 0.799 1.330** 1.769 5.593 5.093 11.002* - - -
s.d. 1.081 0.884 1.540 1.090 1.167 2.394 - - -

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Table 13: Food consumption of males - group mean data, based on food consumed over the previous period starting from Day 1 of dosing.

Group Food consumption (g/animal/day)
day 8 pre-mating day 15 pre-mating
1 no. 2 2
mean 29.27 30.68
2 no. 2 2
mean 28.41 29.49
3 no. 2 2
mean 27.90 29.04
4 no. 2 2
mean 28.40 28.41

Table 14: Food consumption of females - group mean data.

Group Food consumption (g/animal/day)
day 8 pre-mating* day 15 pre-mating* day 7 gestation** day 14 gestation** day 20 gestation** day 7postpartum*** day 13post partum***
1 no. 2 2 9 9 9 8 8
mean 20.42 22.00 28.42 30.18 32.30 51.62 69.75
s.d. - - 2.15 2.67 1.73 3.86 3.73
2 no. 2 2 9 9 9 9 9
mean 19.08 21.74 27.65 27.89 30.32 45.98 65.03
s.d. - - 1.70 2.54 3.57 6.77 9.26
3 no. 2 2 9 9 9 8 8
mean 20.44 22.37 27.77 29.85 31.62 45.39 66.49
s.d. - - 1.91 2.14 1.76 8.32 8.74
4 no. 2 2 9 9 9 - -
mean 20.31 22.38 27.43 28.94 32.88 - -
s.d. - - 2.07 2.19 1.54 - -

* based on food consumed over the previous period starting from Day 1 of dosing.

** based on food consumed over the previous period starting from Day 1 of gesation.

*** based on food consumption over the previous period starting from Day 1post partum.

Table 15: Motor activity - group mean data.

Males;

mating phase 

Control Group 2 Group 3 Group 4
mean s.d. number mean s.d. number mean s.d. number mean s.d. number
counter display: 852.2 172.1 5 1022.2 84.2 5 942.0 92.1 5 976.0 155.1 5

Females;

post partumphase 

Control Group 2 Group 3 -
mean s.d. number mean s.d. number mean s.d. number
counter display: 945.2 184.7 5 809.2 277.2 5 1009.2 67.1 5

Table 16: Sensory reactivity tostimuli- group mean data.

Males;

mating phase 

Control Group 2 Group 3 Group 4
mean s.d. number mean s.d. number mean s.d. number mean s.d. number
Grip strength 1 (g) 14.16 1.44 5 15.35 5.06 5 14.54 2.61 5 15.23 5.35 5
Grip strength 2 (g) 20.16 2.17 5 16.30 3.85 5 14.56* 2.35 5 13.64** 2.26 5
Mean grip strength (g) 17.160 1.099 5 15.830 4.249 5 14.550 2.244 5 14.480 3.430 5
Landing foot splay 1 (cm) 5.10 1.29 5 4.78 1.73 5 4.92 2.98 5 4.62 2.43 5
Landing foot splay 2 (cm) 5.82 1.32 5 5.84 1.38 5 4.36 2.35 5 3.26* 0.55 5
Mean landing foot splay (cm) 5.460 0.948 5 5.310 1.493 5 4.640 2.646 5 3.940 1.286 5
Females; 

post partumphase 

Control Group 2 Group 3 -
mean s.d. number mean s.d. number mean s.d. number
Grip strength 1 (g) 12.52 2.49 5 14.26 2.71 5 14.32 3.01 5
Grip strength 2 (g) 13.26 1.70 5 13.34 2.22 5 13.02 1.94 5
Mean grip strength (g) 12.890 2.020 5 13.800 2.369 5 13.670 2.017 5
Landing foot splay 1 (cm) 4.36 1.11 5 5.50 1.77 5 5.38 1.72 5
Landing foot splay 2 (cm) 4.88 0.59 5 5.10 1.47 5 5.50 1.62 5
Mean landing foot splay (cm) 4.620 0.720 5 5.300 1.575 5 5.440 1.604 5

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Table 17: Haematology - males - group mean data.

Group  1 2 3 4
red blood cell count (106µl) mean 9.174 8.935 8.820 7.743**
s.d. 0.421 0.307 0.241 0.225
no. 5 4 5 4
haemaglobin (g/dl) mean 16.16 15.90 15.44 13.48**
s.d. 0.72 0.76 0.51 0.74
no. 5 4 5 4
haematocrit (%) mean 50.84 50.15 47.82 41.53**
s.d. 2.74 2.82 1.88 1.77
no. 5 4 5 4
mean red blood cell volume (fl) mean 55.44 56.15 54.24 53.60
s.d. 1.02 2.86 0.84 1.76
no. 5 4 5 4
mean corpuscular haemaglobin (pg) mean 17.62 17.78 17.48 17.40
s.d. 0.41 0.63 0.18 0.68
no. 5 4 5 4
mean corpuscular haemaglobin (g/dl) mean 31.80 31.70 32.22 32.50
s.d. 0.48 0.55 0.38 0.50
no. 5 4 5 4
reticulocytes (%) mean 1.934 1.773 1.912 2.575
s.d. 0.729 0.381 0.335 0.214
no. 5 4 5 4
reticulocytes (109/l) mean 175.68 157.63 168.80 199.43
s.d. 61.02 28.68 31.18 17.19
no. 5 4 5 4
white blood cell count (103/µl) mean 8.410 5.778 7.538 6.473
s.d. 2.132 2.704 1.932 0.681
no. 5 4 5 4
neutrophils (103/µl) mean 1.044 0.775 0.748 0.788
s.d. 0.203 0.083 0.256 0.168
no. 5 4 5 4
lymphocytes (103/µl) mean 6.946 4.748 6.526 5.533
s.d. 1.866 2.690 1.785 0.704
no. 5 4 5 4
monocytes (103/µl) mean 0.204 0.110 0.120 0.073*
s.d. 0.105 0.022 0.042 0.010
no. 5 4 5 4
eosinophils (103/µl) mean 0.114 0.085 0.078 0.040**
s.d. 0.037 0.033 0.031 0.022
no. 5 4 5 4
basophils (103/µl) mean 0.066 0.040 0.044 0.025
s.d. 0.044 0.029 0.018 0.006
no. 5 4 5 4
large unstained cells (103/µl) mean 0.036 0.015 0.024 0.018
s.d. 0.027 0.010 0.013 0.010
no. 5 4 5 4
neutrophils (%) mean 12.76 18.10 10.32 12.25
s.d. 3.05 14.14 3.82 3.18
no. 5 4 5 4
lymphocytes (%) mean 82.28 76.38 86.16 85.38
s.d. 3.49 17.63 4.27 3.25
no. 5 4 5 4
monocytes (%) mean 2.44 2.33 1.64 1.15*
s.d. 0.76 1.14 0.39 0.13
no. 5 4 5 4
eosinophils (%) mean 1.40 2.35 1.06 0.60*
s.d. 0.45 2.64 0.36 0.27
no. 5 4 5 4
basophils (%) mean 0.76 0.60 0.54 0.35
s.d. 0.38 0.23 0.11 0.06
no. 5 4 5 4
large unstained cells (%) mean 0.42 0.28 0.32 0.25
s.d. 0.22 0.13 0.08 0.13
no. 5 4 5 4
platelets (103/µl) mean 968.8 956.3 923.0 837.8
s.d. 187.1 96.0 58.9 102.0
no. 5 4 5 4

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Table 18: Haematology - females - group mean data.

Group: 1 2 3
red blood cell count (106/µl) mean 7.590 7.574 7.114
s.d. 0.339 0.283 0.467
no. 5 5 5
haemaglobin (g/dl) mean 14.80 14.58 14.14
s.d. 0.63 0.43 0.29
no. 5 5 5
haematocrit (%) mean 44.66 44.54 43.00
s.d. 1.31 2.35 1.58
no. 5 5 5
mean red blood cell volume (fl) mean 58.90 58.74 60.62
s.d. 1.82 1.32 3.67
no. 5 5 5
mean corpuscular Hb (pg) mean 19.52 19.28 19.94
s.d. 0.29 0.29 1.14
no. 5 5 5
mean corpuscular Hb (g/dl) mean 33.16 32.82 32.88
s.d. 0.67 1.14 0.56
no. 5 5 5
reticulocytes (%) mean 2.150 2.466 3.008
s.d. 0.519 0.499 0.772
no. 5 5 5
reticulocytes (109/l) mean 162.08 187.76 216.64
s.d. 33.70 48.55 67.56
no. 5 5 5
white blood cell count (103/µl) mean 3.398 2.946 3.406
s.d. 1.405 1.566 0.659
no. 5 5 5
neutrophils (103/µl) mean 0.656 0.554 0.464
s.d. 0.219 0.172 0.134
no. 5 5 5
lymphocytes (103/µl) mean 2.550 2.246 2.810
s.d. 1.132 1.411 0.657
no. 5 5 5
monocytes (103/µl) mean 0.116 0.082 0.074
s.d. 0.054 0.069 0.041
no. 5 5 5
eosinophils (103/µl) mean 0.030 0.040 0.022
s.d. 0.019 0.032 0.013
no. 5 5 5
basophils (103/µl) mean 0.022 0.012 0.012
s.d. 0.019 0.008 0.008
no. 5 5 5
large unstained cells (103/µl) mean 0.020 0.012 0.022
s.d. 0.014 0.008 0.011
no. 5 5 5
neutrophils (%) mean 20.88 21.24 13.94
s.d. 6.65 6.54 4.27
no. 5 5 5
lymphocytes (%) mean 73.70 74.06 82.14
s.d. 5.34 6.81 5.06
no. 5 5 5
monocytes (%) mean 3.40 2.62 2.26
s.d. 1.36 1.48 1.10
no. 5 5 5
eosinophils (%) mean 0.82 1.26 0.64
s.d. 0.38 0.34 0.41
no. 5 5 5
basophils (%) mean 0.62 0.32 0.42
s.d. 0.31 0.19 0.16
no. 5 5 5
large unstained cells (%) mean 0.54 0.46 0.64
s.d. 0.33 0.30 0.26
no. 5 5 5
platelets (103/µl) mean 987.0 1178.4 1158.6
s.d. 123.6 134.5 142.0
no. 5 5 5

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Table 19: Coagulation - group mean data.

Males  Group 1 Group 2 Group 3 Group 4
mean s.d. number mean s.d. number mean s.d. number mean s.d. number
Prothrombin time (sec) 23.02 1.05 5 22.86 1.19 5 24.2 1.11 5 24.6 1.07 5
Females  Group 1 Group 2 Group 3 -
mean s.d. number mean s.d. number mean s.d. number
Prothrombin time (sec) 22.52 0.65 5 22.46 1.27 5 22.7 0.69 5

Table 20: Clinical chemistry - males - group mean data.

Group: 1 2 3 4
alkaline phosphate (U/l) mean 261.64 287.04 263.20 298.46
s.d. 49.56 35.75 29.45 44.92
no. 5 5 5 5
alanine amino-transferase (U/l) mean 34.40 31.46 32.94 34.78
s.d. 7.15 5.26 7.79 12.04
no. 5 5 5 5
aspartate amino-transferase (U/l) mean 87.88 83.36 91.82 96.36
s.d. 5.04 7.29 16.97 21.69
no. 5 5 5 5
gamma-glutamyl transferase (U/l) mean 0.3 - - 0.2
s.d. - - - -
no. 1 0 0 1
total bilirubin (mg/dl) mean 0.050 0.040 0.064 0.074
s.d. 0.012 0.023 0.022 0.035
no. 5 5 5 5
total cholesterol (mg/dl) mean 51.74 52.70 42.36 27.40**
s.d. 9.80 9.91 9.57 7.45
no. 5 5 5 5
triglycerides (mg/dl) mean 61.08 46.76 63.08 39.26
s.d. 26.76 14.07 24.20 11.18
no. 5 5 5 5
glucose (mg/dl) mean 91.12 94.56 98.68 102.10
s.d. 13.51 13.75 11.48 11.51
no. 5 5 5 5
bile acids (µmol/l) mean 7.34 8.30 6.36 11.60
s.d. 3.39 2.66 2.32 2.39
no. 5 5 5 5
total protein (g/dl) mean 6.58 6.72 6.82* 6.50
s.d. 0.18 0.26 0.04 0.31
no. 5 5 5 5
albumin (g/dl) mean 3.96 4.08 4.16* 4.08
s.d. 0.11 0.13 0.09 0.13
no. 5 5 5 5
globulin (g/dl) mean 2.62 2.64 2.66 2.42
s.d. 0.15 0.15 0.09 0.18
no. 5 5 5 5
albumin /globulin ratio mean 1.50 1.56 1.56 1.68**
s.d. 0.07 0.05 0.09 0.08
no. 5 5 5 5
urea (mg/dl) mean 23.90 24.54 26.74 34.02**
s.d. 3.03 3.66 3.35 3.85
no. 5 5 5 5
creatine (mg/dl) mean 0.306 0.306 0.274 0.294
s.d. 0.036 0.031 0.025 0.040
no. 5 5 5 5
chloride (mmol/l) mean 103.94 103.46 104.28 106.02**
s.d. 0.59 0.77 1.29 0.79
no. 5 5 5 5
inorganic phosphorus (mg/dl) mean 6.658 6.428 6.592 6.970
s.d. 0.226 0.447 0.352 0.238
no. 5 5 5 5
calcium (mmol/l) mean 2.728 2.770 2.808 2.796
s.d. 0.054 0.089 0.038 0.073
no. 5 5 5 5
sodium (mmol/l) mean 146.96 147.30 147.72 149.38**
s.d. 0.53 1.08 0.86 0.31
no. 5 5 5 5
potassium (mmol/l) mean 3.866 3.806 3.986 3.786
s.d. 0.228 0.248 0.192 0.238
no. 5 5 5 5

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Table 21: Clinical chemistry - females - group mean data.

Group: 1 2 3
alkaline phosphate (U/l) mean 186.98 160.04 244.26
s.d. 91.17 34.27 56.36
no. 5 5 5
alanine amino-transferase (U/l) mean 66.62 60.98 49.34*
s.d. 6.48 14.77 8.68
no. 5 5 5
aspartate amino-transferase (U/l) mean 92.48 86.06 73.48*
s.d. 13.53 19.66 3.55
no. 5 5 5
gamma-glutamyl transferase (U/l) mean 0.60 0.95 0.47
s.d. 0.26 0.35 0.25
no. 5 5 5
total bilirubin (mg/dl) mean 0.033 0.017 0.018
s.d. 0.019 0.015 0.016
no. 5 5 5
total cholesterol (mg/dl) mean 69.60 85.3 80.38
s.d. 11.35 17.42 13.86
no. 5 5 5
triglycerides (mg/dl) mean 74.72 59.74 55.02
s.d. 34.43 40.01 15.45
no. 5 5 5
glucose (mg/dl) mean 107.50 104.04 110.28
s.d. 31.68 24.04 35.23
no. 5 5 5
bile acids (µmol/l) mean 30.26 41.68 58.26
s.d. 11.81 24.33 23.35
no. 5 5 5
total protein (g/dl) mean 5.98 6.38* 6.44*
s.d. 0.27 0.18 0.23
no. 5 5 5
albumin (g/dl) mean 3.70 3.84 3.96**
s.d. 0.07 0.13 0.09
no. 5 5 5
globulin (g/dl) mean 2.28 2.54 2.48
s.d. 0.23 0.09 0.25
no. 5 5 5
albumin /globulin ratio mean 1.66 1.52 1.60
s.d. 0.15 0.11 0.17
no. 5 5 5
urea (mg/dl) mean 44.58 44.78 52.28
s.d. 12.31 4.96 8.77
no. 5 5 5
creatine (mg/dl) mean 0.422 0.412 0.414
s.d. 0.036 0.027 0.036
no. 5 5 5
chloride (mmol/l) mean 96.90 97.02 97.34
s.d. 2.21 1.97 3.68
no. 5 5 5
inorganic phosphorus (mg/dl) mean 6.140 5.429 5.782
s.d. 1.291 0.810 0.756
no. 5 5 5
calcium (mmol/l) mean 2.454 2.474 2.582*
s.d. 0.069 0.113 0.029
no. 5 5 5
sodium (mmol/l) mean 140.68 142.20 142.56
s.d. 1.58 1.76 1.51
no. 5 5 5
potassium (mmol/l) mean 3.632 3.226 3.620
s.d. 0.612 0.278 0.390
no. 5 5 5

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Table 22: Urinalysis - males - group mean data.

Group: 1 2 3 4
urine volume overnight (ml) mean 9.6 9.3 8.4 8.7
s.d. 2.84 1.89 2.19 3.65
n. 5 5 5 5

Table 23: Macroscopic observations - group incidence.

Males Females Unscheduled death; females
Group 1 2 3 4 1 2 3 4 1
No. in group 10 10 10 10 9 10 10 10 1
Adrenals
abnormal colour 0 0 0 0 1 0 0 0 0
abnormal size 0 0 0 2 1 0 1 1 0
Cervical Nodes
abnormal colour 0 1 0 1 0 0 0 1 0
Kidneys
abnormal area(s) 1 0 0 0 0 0 0 0 0
pelvic dilatation 3 1 0 1 0 0 1 0 0
Mesenteric Nodes
abnormal colour 0 1 0 3 1 0 0 0 0
Pituitary 
abnormal colour 0 0 0 0 0 0 0 1 0
abnormal shape 0 0 0 0 0 0 0 1 0
Seminal vesicles
abnormal size 0 0 0 1
Stomach
abnormal area(s) 0 0 0 0 5 5 1 0 0
Thymus
abnormal area(s) 0 1 0 0 0 0 0 0 0
abnormal size 0 0 0 0 3 0 1 0 0
Uterus
abnormal size   0 0 0 4 0
abnormal contents   0 0 0 3 0
mass(es)   0 0 1 0 0
cyst(s)   1 0 0 0 0
not pregnant   1 1 1 1 0
total resorption   0 0 0 0 0
Forelimbs
hairloss 0 0 0 0 1 1 0 0 0
Hindlimbs
hairloss 0 0 0 0 0 1 0 0 0
Lymph Nodes
abnormal colour 0 0 0 1 0 0 0 0 0
Skin
abnormal area(s) 0 1 0 0 0 0 0 0 0
Whole Animal
no abnormalities detected 6 7 10 4 2 3 5 4 1

Table 24: Microscopic observations - group incidence.

Males Females Unscheduled death; females
Group 1 2 3 4 1 2 3 4 1
Adrenals 5 0 0 7 5 0 1 5 0
vacuolisation 2 0 0 2 0 0 0 0 0
Brain 5 0 0 5 5 0 0 5 0
haemorrhage 1 0 0 0 0 0 0 0 0
Heart 5 0 0 5 5 0 0 5 0
inflammatory cell foci  1 0 0 0 0 0 0 1 0
Kidneys  6 1 0 5 5 0 1 5 0
pelvic dilatation 3 1 0 0 0 0 0 0 0
mineralisation  0 0 0 1 0 0 0 0 0
nephropathy  1 0 0 0 0 0 0 0 0
Liver 5 0 0 5 5 0 0 5 1
inflammatory cell foci 5 0 0 4 5 0 0 4 1
vacuolation 0 0 0 0 2 0 0 3 0
extramedullary haemopoiesis 0 0 0 0 0 0 0 3 1
clear cell change 0 0 0 0 0 0 0 1 0
Lungs 5 0 0 5 5 0 0 5 0
inflammatory cell foci 2 0 0 0 0 0 0 0 0
mineralisation 2 0 0 1 3 0 0 0 0
alveolar haemorrhage 0 0 0 0 1 0 0 0 0
Penis 5 0 0 5
inflammatory cell foci 0 0 0 1
Pituitary 5 0 0 5 5 0 0 4 0
congestion  0 0 0 0 0 0 0 1 0
Prostate  5 0 0 5
inflammatory cell foci 2 0 0 1
Skin  5 1 0 5 5 1 0 5 0
scab formation 0 1 0 0 0 0 0 0 0
ulceration 0 1 0 0 0 0 0 0 0
chronic inflammation  0 1 0 0 0 0 0 0 0
Stomach  5 0 0 5 7 5 0 5 0
epithelial vacuolation 0 0 0 1 0 0 0 0 0
mucosal erosion  0 0 0 0 5 3 0 0 0
glandular dilation  0 0 0 0 1 0 0 1 0
Thymus  5 1 0 5 5 0 1 5 0
atrophy  0 1 0 0 4 0 1 3 0
congestion   0 1 0 0 0 0 0 0 0
Thyroid  5 0 0 5 5 0 0 5 0
thyro-glossal duct remnant  0 0 0 1 2 0 0 0 0
Uterus   6 0 1 6 0
luminal dilatation   0 0 0 2 0
decidual formation   1 0 1 0 0
Mesenteric Nodes  5 0 0 7 4 0 0 5 0
congestion   0 0 0 3 1 0 0 0 0
Cervical Nodes  5 1 0 6 5 0 0 5 0
congestion   0 0 0 1 0 0 0 0 0
Lymph Nodes  0 0 0 1 0 0 0 0 0
congestion   0 0 0 1 0 0 0 0 0

Applicant's summary and conclusion

Conclusions:
The NOAEL for short-term repeated dose oral toxicity was found to be at least 800 mg/kg bw/day.
Executive summary:

The short-term repeated dose toxicity of the test item was evaluated in a repeated dose test, combined with a reproductive and developmental screening test, according to the OECD Guideline 422 (2016). Groups comprising 10 male and 10 female Sprague-Dawley rats each were administered 10 ml/kg bw test item in softened water daily by oral gavage, at concentrations of 0 (negative control), 100, 300 and 800 mg/kg bw/day. Males were treated for a total of 36 days including a two-week pre-pairing phase, a mating phase and a post coitum phase, after which they were sacrificed. Females were treated for between 50 and 60 days, including a pre-pairing phase (two weeks), mating phase (approximately 2 days), gestation (approximately 22 days) and post partum phase (14 days) after which all surviving dams were sacrificed along with their litter. The following investigations were performed in all groups: body weight (all), clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli; all), food consumption (two males and two females per group; performed weekly), haematology (5 animals/sex/group), clinical chemistry (5 animals/sex/group), urinalysis (males only; 5 animals/sex/group), macro- and microscopic observations (5 males and 5 females from the high-dose and control groups only), organ weights and histopathological examination (5 animals/sex from control and high-dose groups only).

No mortality occurred among male animals, whereas one female from the control group was sacrificed for humane reasons for difficulties in delivery. Subsequent histopathologic examination showed inflammatory cell foci and extramedullary haematopoiesis in the liver, and no macroscopic findings. No toxicologically relevant clinical signs were observed among treated animals compared to controls including observations of neurotoxicity assessment, motor activity and sensory reactivity to stimuli; one mid-dose female experienced piloerection, pallor and staining (vagina) on days 2 to 5 post partum, and a slight yet dose-related decrease in grip strength and landing foot splay was observed in treated males in the second trial only. No significant changes in body weight were observed, except a decrease of 24 % body weight during the last week of gestation among high-dose females, however the low body weight in high-dose females in the last week of gestation was thought to be due to foetal toxicity rather than maternal toxicity. Food consumption and urinalysis were unchanged between control and treatment groups. The small differences observed in macroscopic and microscopic observations were considered not treatment related and common in this species and age. The following statistically significant organ weight findings were recorded but were not considered toxicologically relevant: increased absolute and relative male adrenals weight (approximately 23%), increased absolute and relative male and female liver weight (ca. 16% for males and 20% for females), increased absolute and relative prostate and seminal vesicle weight (approximately 29% for prostate weight and 38% for seminal vesicle weight), decreased relative female heart weight (ca. 10%) of the high-dose group and decreased absolute and/or relative male thymus weight (ca. 26% for high dose, 23% for mid-dose and 19% for low-dose group.

In conclusion, the difference in body weight in high-dose females was thought to be a result of foetal toxicity and not dam toxicity; and other differences observed between control and treated groups were not found to be of toxicological importance. Based on these findings, the NOAEL for short-term repeated dose oral toxicity was considered to be at least 800 mg/kg/day in both male and female rats.