Registration Dossier

Administrative data

Description of key information

In a dose-range-finding study, the test item was administered by oral gavage to Wistar rats for 7 consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day in propylene glycol at a dose volume of 4 mL/kg bw (CitoxLab, 2015). There were no adverse effects observed which could clearly be related to test item administration. Therefore, based on the observation of this DRF study, the dose levels for the main study were 0, 100, 300 and 1000 mg/kg bw/day.

In the repeated dose oral toxicity study according to OECD 422 with Wistar rats exposed to 100, 300 and 1000 mg/kg bw/day, the test item caused some mortality in the adults at 1000 mg/kg/day. Minor changes in the liver in the Mid and High dose groups of the parental generation, as well as changes in kidneys in the High dose group were not considered to be adverse.

It is considered that the no observed adverse effect levels (NOAEL) in this study for the parental/adult is 300 mg/kg bw/day under the current test conditions.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015-07-01 to 2015-07-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
no guideline followed
Version / remarks:
As a preliminary study, this study does not follow a specific guideline and is designed to allow selection of appropriate dose levels for use in the upcoming studies.
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany), from SPF colony
- Strain: Crl:WI Wistar rats
- Sex. male and female
- Age: Young adult rats, at least 10 old at the start of the treatment
- Weight at study initiation: Males: 333-371 g, females: 221-260 g
- Housing:group-housed, 5 animals of the same sex and group/cage
- Diet (e.g. ad libitum): Ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water (e.g. ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-26.0 °C
- Humidity (%): 40 - 68 %
- Air changes (per hr): 15 - 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
The test item was formulated in the vehicle at concentrations of 25, 75 and 250 mg/mL to achieve dose levels of 100, 300 and 1000 mg/kg bw/day using a dose volume of 4 mL/kg.
The test item was formulated in the vehicle, as a visibly stable homogenous formulation (solution) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd. Formulations were prepared daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulation samples of 1 – 250 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 7 days in room temperature.
Analysis of test item formulations for concentration was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Five samples were taken from test item formulations once in duplicate, one set to analyze and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.

The measured concentrations of test item evaluated for each test item-dose group varied between 91% and 98% of the nominal contents. No test item was detected in the control samples. These results were within acceptable ranges (90% - 110%) and are acceptable for the study purposes.
Analysis of test item formulation samples of 1 – 250 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 7 days in room temperature.
Duration of treatment / exposure:
7 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were set by the Sponsor based on available data and information from previous experimental work, including the results of an acute oral toxicity study (Acute Toxic Class Method, OECD 401, in which the test item was proven to be non- toxic after a single oral administration at the dose level of 2000 mg/kg bw). Single Dose Phase was not performed and Repeat Dose Phase started (up to 3 dose levels). A control group was treated concurrently with the vehicle only (PG).

Five males and five females/group were treated daily for 7 days in order to obtain preliminary information on the potential toxicity of the test item following repeated administration at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group was treated concurrently with the vehicle only (PG). The first day of dosing of each animal was regarded as Day 0. The individual volume of the treatment was based on the most recent individual body weight of the animals.
Positive control:
not required
Observations and examinations performed and frequency:
Clinical observations and mortality
- Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
- Clinical observations were made twice daily, before and after treatment, at the beginning and towards the end of the working day as practical, as no clinical changes were noted after the dose administration.
- The animals were monitored for any clinical signs, including pertinent behavioural changes, signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma

Body weight measurement
- Body weight of each animal was recorded with precision of 1 g at randomization, then on Days 0, 2, 4, 6 and Day 7 (prior to necropsy, fasted).

Food consumption measurement
- Food was recorded with precision of 1 g on Days 0, 2, 4 and 6.

CLINICAL PATHOLOGY
- On Day 7, blood samples for clinical pathology evaluation (haematology and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia.
- After an overnight period of food deprivation of animals, 2 blood samples were collected, for haematology (in K3-EDTA tubes, 1.6 mg/mL blood) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Haematology and blood clotting times
PARAMETERS (UNITS) METHODS
Haematology
RBC Red Blood Cell (erythrocyte) count, (1012/L) M/μL Automatic laser cell count
WBC White Blood Cell (leukocyte) count, (109/L) K/μL Automatic laser cell count
Hgb Haemoglobin concentration, (g/dL) Determination of cyan-methemoglobin absorbance
Hct Haematocrit (relative volume of erythrocytes) (%) Computed by equipment
MCV Mean Corpuscular (erythrocyte) Volume (fL) Laser cell volume determination
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg) Computed by equipment
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL) Computed by equipment
RDW Red Cell (erythrocyte) volume (%) Distribution Width Laser detection
Plt Platelet (thrombocyte) count (109/L) K/μL Automatic laser cell count
MPV Mean Platelet Thrombocyte volume (fL) Cell volume determination by laser
RETIC % Reticulocyte count (%) Comparative value based on laser light detection
NE % Neutrophil (%) Cell differentiation based on myeloperoxidase activity
LY % Lymphocyte (%) Cell differentiation based on myeloperoxidase activity
MO % Monocyte (%) Cell differentiation based on myeloperoxidase activity
BA % Basophil (%) Cell differentiation based on myeloperoxidase activity
EO % Eosinophil (%) Cell differentiation based on myeloperoxidase activity
LUC % Large Unstained Cells (%) Cell differentiation based on myeloperoxidase activity

Clinical chemistry
The following parameters were evaluated in all surviving animals:
PARAMETERS (UNITS) METHODS
Glucose Blood sugar concentration (mmol/L) Colorimetric test (540 nm)
T-BIL Total Bilirubin concentration (μmol/L) End-point colorimetric (dual-wavelength) test (400 & 460nm)
Urea Urea concentration (mmol/L) Colorimetric test (670 nm)
Chol. Cholesterol concentration (mmol/L) Colorimetric test (540 nm)
Creat. Creatinine concentration (μmol/L) Two-point rate test (670 nm)
Tot. Prot. Total Protein concentration (g/L) Colorimetric test (540 nm)
Alb. Albumin concentration (g/L) Colorimetric test (630 nm)
AST/GOT Aspartate Aminotransferase activity (U/L) Multiple-point rate test (340 nm)
ALT/GPT Alanine Aminotransferase activity (U/L) Multiple-point rate test (340 nm)
ALKP Alkaline. Phosphatase – activity (U/L) Multiple-point rate test (400 nm)
GGT Gamma Glutamyltransferase -activity (U/L) γ-Glutamyl-p-nitroanilid e+ Glycylglycine. Increase in p-nitroaniline-monitored at 400nm

Sacrifice and pathology:
PATHOLOGY EVALUATION
Terminal procedures and macroscopic evaluation
- Gross necropsy was performed on each animal irrespective of the date of death, including the animal found dead in extremis.
- Terminally on Day 7, surviving animals were euthanised under pentobarbital anaesthesia by exsanguination.
- After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically.
- Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Organ weight measurements
With precision of 0.01g
Brain
Epididymides
Heart
Kidneys
Liver
Prostate
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus
Uterus including cervix

With precision of 0.001g
Adrenals
Ovaries
Thyroids with parathyroids

Histopathology
On completion of the macroscopic examination the following tissues and organs were retained from animals.

Gross findings
Adrenals
Animal identification 1
Aorta10
Brain 2
Epididymis
Extraorbital lachrymal gland
Eye with the optic nerve 7
Femur with marrow
Harderian gland
Heart 3
Kidney
Large intestine 4
Liver12
Lungs with bronchi 5
Lymph node 6
Oesophagus
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular, sublingual and parotid glands)
Sciatic nerve
Seminal vesicle with coagulating gland
Skeletal muscle (quadriceps)
Skin, subcutis with mammary gland (inguinal)
Small intestine 8
Spinal cord11
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland 7
Tongue
Trachea
Urinary bladder
Uterus 9
Vagina

1. Fixation and preservation only.
2. Section according to the STP recommendations.
3. Section including both ventricles and atria, septum with papillary muscle.
4. Caecum, colon and rectum.
5. Lungs of euthanized animals were infused with formalin; 3 lobes, left, right cranial, right caudal.
6. Mandibular and mesenteric.
7. Where applicable, parathyroids and optic nerves will be examined histologically only if present in routine sections.
8. Duodenum, ileum and jejunum with Peyer’s patches.
9. Horns, body and cervix.
10. Aorta thoracic and abdominal
11. Transverse sections, 3 levels -cervical, thoracic and lumbar.
12. Liver, 3 lobes, left lateral, right medial, caudate

The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution. Organs showing macroscopic lesions of animals were preserved at the discretion of the attending pathologist and/or the study director to elucidate abnormal findings.
No histopathology evaluation was performed.


Other examinations:
no other examinations
Statistics:
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During the study, one high dose female was found dead on Day 7, with symptoms of hunched back position and slight decreased activity after the first treatment, on Day 0 but was symptom free for the duration of the study. For this individual animal, the only observation of clinical signs was on day 0, with an unexpected death on day 7. However, all other animals in the same group were symptom free during the total treatment period and had no effects in clinical pathology, necropsy and no clearly adverse effects for organ weights. In absence of toxicologically relevant symptoms/observations in the monitored parameters of this animal (body weight, food intake, clinical signs after day 0, necropsy) the relationship to treatment can not be excluded or confirmed.
No test item-related adverse effects or systemic clinical signs were noted following treatment at the dose levels of 30 and 100 mg/kg bw/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
one high dose female was found dead on Day 7, with symptoms of hunched back position and slight decreased activity after the first treatment, on Day 0 but was symptom free for the duration of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically significant or adverse effects observed on the animal body weights or body weight gains during the study.
There was a transient body weight reduction in high dose animals at the beginning of the treatment period, which caused a statistically significant difference in the body weight in females on Day 2 (p<0.05) and in case of males in the body weight gain between Days 0 and 2 (p<0.05), however it was recovered by the end of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item related differences in the mean daily food consumption in any test item treated group when compared to the controls.
Haematological findings:
no effects observed
Description (incidence and severity):
When compared to the controls, there were no differences that could be considered toxicologically significant in the treated animals.
The following statistically significant variations were noted:
In males, decreases in Platelet (thrombocyte) count was seen in low dose (p<0.05). In females, minimal decreases in Haemoglobin concentration and in Haematocrit (p<0.05) in mid dose and increases in White Blood Cell (leukocyte) count in the low dose (p<0.01) were noted.
These findings, in the absence of consistent patterns or other correlates were regarded as incidental or individual findings, generally within the historical control range, and were probably unrelated to treatment and/or with no toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
When compared to the controls, there were no differences that could be considered toxicologically significant in the treated animals.
Compared to control, the Total Protein concentration was decreased in low dose females with attaining statistical significance (p<0.01). In the absence of consistent patterns or other correlates were regarded as incidental or individual findings, generally within the historical control range, and was probably unrelated to treatment and/or with no toxicological significance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Compared to control, test item related absolute and relative liver weight changes were detected in high dose males and females and in mid dose females on Day 7, with attaining statistical significance.
The relative kidney weights of high dose males also were statistically higher than the control. This change was ascribed to biological variability or to individual, incidental changes and considered unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
During scheduled necropsy, no test item-related macroscopic changes were observed in the surviving animals dosed at 100, 300 and 1000 mg/kg bw. Minor changes, like enlarged spleen, or small testes or epididymis were considered as incidental.
At the necropsy on the found dead animal, the stomach, jejunum, duodenum, ileum was dilated, the lungs were dark red. These findings were not considered test item related.
Details on results:
There were statistically and/or toxicologically significant differences to control in the absolute or relative organ weights recorded in the males and females. Compared to the controls, the liver weights of mid dose females and of high dose males and females were higher up to by approximately 29% (absolute and relative to body weight and brain values).
Conclusions:
In conclusion, the test item administered by oral gavage to Wistar rats for 7 consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day in Propylene Glycol at a dose volume of 4 mL/kg bw, was not clearly associated with overt adverse effects that could be related to test item administration.
Based on the observation of this DRF study, the dose levels selected by the Sponsor in consultation with the Study Director for the main study were 0, 100, 300 and 1000 mg/kg bw/day.

Executive summary:

The objective of the study was to determine the Maximum Tolerated Dose (MTD) and to obtain preliminary information on the toxicity of the test item when administered to Wistar rats at 100, 300 and 1000 mg/kg bw/day after daily oral (gavage) administration for up to 7 days by oral gavage, to select dose levels for a main Combined Repeated Dose Toxicity Study with Reproduction/ Developmental Toxicity Screening Test.

 

The dose levels were set by the Sponsor based on available data and information from previous experimental work, including the results of an acute oral toxicity study (Acute Toxic Class Method, OECD 401, in which the test item was proven to be non-toxic after a single oral administration at the dose level of 2000 mg/kg bw). Single Dose Phase was not performed and Repeat Dose Phase was run (3 dose levels). A control group was treated concurrently with the vehicle only (Propylene Glycol, abbreviation: PG).

The test item was formulated in the vehicle at concentrations of 25, 75 and 250 mg/mL to achieve dose levels of 100, 300 and 1000 mg/kg bw/day using a dose volume of 4 mL/kg.

Clinical and mortality observations were performed twice daily. Body weight and food consumption were measured on Days 0, 2, 4 and 6, with a fasted body weight recorded prior to scheduled necropsy, on Day 7. Following repeated dose administration daily for 7 days, blood samples were collected for clinical pathology at necropsy. Gross macroscopic examination was performed at necropsy, one day after the last treatment. Selected organs were weighed. Tissues were preserved in fixative but not processed for further histopathological evaluation.

Results:

The measured concentrations of test item evaluated for each test item-dose group varied between 91 and 98%. No test item was detected in the control samples. These results were within acceptable ranges and considered suitable for the study purposes.

During the study, one high dose female was found dead on Day 7. In absence of toxicologically relevant symptoms/observations in the monitored parameters of this animal (body weight, food intake, clinical signs after day 0, necropsy) the relationship to treatment can not be excluded or confirmed.

No other unscheduled mortality occurred during the study. Test item caused no systemic adverse effects following administration by oral gavage daily for up to 7 days, at dose levels of 100 and 300 mg/kg bw/day in Wistar rats.

There were no toxicologically significant or adverse effects observed on the animal body weights or body weight gain. The mean body weights were similar or slightly higher than the controls in both males and females.

There were no adverse effects on the animal food consumption.

Clinical pathology parameters evaluated at the completion of the 7-day treatment period did not show any clear toxicologically significant, adverse effects or test item-related changes.

No test item related internal or external macroscopic observations were noted.

There were statistically and/or toxicologically significant differences to control in the absolute or relative organ weights recorded in the males and females. Compared to the controls, the liver weights of mid dose females and of high dose males and females were higher up to by approximately 29% (absolute and relative to body weight and brain values).

In conclusion, the test item administered by oral gavage to Wistar rats for 7 consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day in Propylene Glycol at a dose volume of 4 mL/kg bw, was not clearly associated with overt adverse effects that could be related to test item administration. Based on the observation of this DRF study, the dose levels selected by the Sponsor in consultation with the Study Director for the main study were 0, 100, 300 and 1000 mg/kg bw/day.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-08-26 to 2015-10-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany), from SPF colony
- Strain: Crl:WI Wistar rats
- Sex. male and female
- Age: Young adult rats, 10-11 weeks old at the start of the treatment and 12-13 weeks old at the start of mating
- Weight at study initiation: Males: 360-445 g, females: 220-265 g
- Housing:group-housed, with up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period
- Diet (e.g. ad libitum): Ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 - 25.4 °C
- Humidity (%): 30 - 68 %
- Air changes (per hr): 15 - 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the previous experimental work propylene glycol (abbreviated as PG in the raw data) was selected for vehicle of the study in agreement with the Sponsor. The same vehicle was used in the Dose Range Finding (DRF) study
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL
- Dose volume: 4 mL/kg bw
- Lot/batch no. (if required): Propylene glycol (1,2-Propanediol, PG), Manufacturer: Sigma-Aldrich Co., Lot number: SZBD1280V / SZBE1770V

Analytical verification of doses or concentrations:
yes
Remarks:
The samples were diluted into the calibrated range with dichloromethane then analysed by GC.
Details on analytical verification of doses or concentrations:
- Stability of the test item in the vehicle (PG) was assessed in the conditions employed on the study during the validation study (according to CiToxLAB study code 15/163-316AN, [4]).
- According to the results, the test item is stable in vehicle in concentration range of 1 mg/mL- 250 mg/mL for 7 days when stored at room temperature.
- Analysis of test item formulations for concentration was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Five samples were randomly taken and analysed from test item formulations and all concentrations.
- Sample analysis was performed on 3 occasions and all prepared formulations were analysed for concentration.
- One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test item.
Duration of treatment / exposure:
- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
- Females were dosed for 14 days pre-mating, for up to 14 days of mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum.
Frequency of treatment:
daily on a 7 days/week basis
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
Twelve male and twelve female Wistar rats/group (with the exception of the High dose group, where 13 animals per sex was treated)
Control animals:
yes, concurrent vehicle
Details on study design:
- Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day PPD4.
Positive control:
not required
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS:
- Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day. General clinical observations were made once a day.
- Animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.
- More detailed examinations were made once prior to treatment (to allow for within-subject comparisons), then weekly in the morning.
Animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT:
- All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, twice a week thereafter and at termination.
- Parent females were weighed on gestation days (GD) 0, 7, 10, 14, 17 and 20 and on postpartal days (PPD) 0 (within 24 hours after parturition) and PPD4 (before termination).

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g or 0.1 g at least weekly (on the days of body weight measurements).

NEUROTOXICITY:
- Assessment of any potential test item related neurotoxicity was performed during the last week of treatment (performed on Day 23 for males and PPD4 for females).

CLINICAL PATHOLOGY
Evaluation of clinical pathology blood and urine samples was conducted for 5 male and 5 female animals in each group and an additional male in the High dose group (the same animals selected for neurotoxicity assessment).

Haematology parameters examined (UNITS):
RBC Red Blood Cell (erythrocyte) count, (1012/L) M/µL
WBC White Blood Cell (leukocyte) count, (109/L) K/µL
Hgb Haemoglobin concentration, (g/dL)
Hct Haematocrit (relative volume of erythrocytes) (%)
MCV Mean Corpuscular (erythrocyte) Volume (fL)
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg)
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL)
RDW Red Cell (erythrocyte) volume (%)
Plt Platelet (thrombocyte) count, (109/L) K/µL
MPV Mean Platelet Thrombocyte volume (fL)
RETIC % Reticulocyte count (%)
NE % Neutrophil (%)
LY % Lymphocyte (%)
MO % Monocyte (%)
BA % Basophil (%)
EO % Eosinophil (%)
LUC % Large Unstained Cells (%)

Coagulation (blood clotting) parameters examined (UNITS):
APTT Activated Partial Thromboplastin Time (sec)
PT Prothrombin Time (sec)

Clinical chemistry (UNITS):
Glucose Blood sugar concentration (mmol/L)
T-BIL Total Bilirubin concentration (μmol/L)
Urea Urea concentration (mmol/L)
Chol. Cholesterol concentration (mmol/L)
Creat. Creatinine concentration (μmol/L)
Phos. Phosphorus concentration (mmol/L)
Na+ Sodium concentration (mmol/L)
K+ Potassium concentration (mmol/L)
Ca++Calcium concentration (mmol/L)
Cl- Chloride concentration (mmol/L)
Tot. Prot. Total Protein concentration (g/L)
Alb. Albumin concentration (g/L)
A/G Alb/glob ration
AST/GOT Aspartate Aminotransferase activity (U/L)
ALT/GPT Alanine Aminotransferase activity (U/L)
ALKP Alkaline. Phosphatase – activity (U/L)
Bile acids (µmol/L)

Urinalysis
Urine samples were collected for 16 hours during an overnight period of food deprivation during the last week of the study (Day 27-28 for males and PPD4-5 for female animals, respectively) from each animal by placing the animals in metabolic cages. The evaluation of the urine samples were performed by observation (e.g. appearance, colour) and test strips.
PARAMETERS (UNITS):
LEU / Leukocyte
NIT / Nitrite
pH
PRO / Protein
GLU / Glucose
UBG / Urobilinogen
BIL / Bilirubin
KET / Ketones
BLD / ERY Blood/Erythrocytes
SG / Specific Gravity
SED / Sediment
VOL / Volume
Colour/Appearance
Clarity/Appearance
Sacrifice and pathology:
SACRIFICE
Terminally, all surviving an one preterminally euthanized adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.

GROSS NECROPSY
Gross necropsy was performed on all animals, irrespective of the date of death.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically.
Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the females as applicable.


HISTOPATHOLOGY / ORGAN WEIGHTS
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Paired organs were weighed together except testes and epididymides which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

Tissue preservation and microscopic evaluation
The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, the testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution.
In addition, on completion of the macroscopic examination the following tissues and organs were retained from all animals:
Gross findings
Adrenal gland
Animal identification
Aorta
Brain
Epididymis
Eye with the optic nerve
Oesophagus
Femur with marrow
Heart
Kidney
Large intestine
Extraorbital lachrymal gland
Harderian gland
Liver
Lungs with bronchi
Lymph node
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular, sublingual and parotid glands)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin, subcutis with mammary gland (inguinal)
Skeletal muscle (quadriceps)
Small intestine
Spinal cord
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland7
Tongue
Trachea
Urinary bladder
Uterus
Vagina
The retained tissues and organs were embedded in paraffin wax; sections were cut at
4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscopy.

For the adult animals, detailed histological examination was performed first as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• additionally, due to the Study Director’s request on #4004 High dose male,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the control and high dose group and of all males that failed to sire and all females that failed to deliver healthy pups.

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Based on the histopathology findings observed in the liver of the examined High dose group animals, slides of the liver samples of all Mid dose and Low dose animals, furthermore the rest of High dose group animals were processed and examined additionally.




Other examinations:
no other examinations
Statistics:
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item related adverse effects were considered to appear in the High dose group (1000 mg/kg bw/day):
• Red discharge was detected for male #4006 on Day 9, noisy respiration and slightly decreased activity was also recorded for this animal which was later found dead on Day 12.
• Red discharge was also detected for two males (#4010 and #4011) on Day 3 and another male (#4004) on Days 18-20, other symptoms like slightly or moderately decreased activity, slight or moderate noisy respiration, piloerection and/or hunched back was also observed during the treatment period in those cases, however these animals survived until the scheduled necropsy.
´• For one female (#4504), piloerection and slightly decreased activity was observed on Day 2, then noisy respiration / laboured respiration / decreased respiratory rate and prostration were recorded, this animal was found dead on Day 3.
• Female (#4509) showed prostration and laboured respiration on Day 5, and from Day 36 tonic convulsions, fasciculation, discharge coloured, increased salivation, extremely decreased activity were observed, the animals was cold to touch and died on Day 39.

Female (#4502) was euthanized on Day 14 due to its general conditions (noisy respiration / laboured respiration, slightly / moderately decreased activity, distended abdomen, hunched back position, animals was also loosing weight).

Observations considered to be incidental and not directly test item related symptoms includes:
• In female #4509 a 4-cm wound, scar, fur thin were observed from Day 14.
• Minor signs in other animals in the High dose group: increased salivation was observed for three males on Day 2, a small scar was recorded for one male on Days 1-2 and one female on Days 18-26, noisy respiration was detected for five male animals on some days in the period of Days 3-27.

The treatment related clinical signs were restricted to the High dose group. The signs were generally non-specific indicators of toxicity, with several cases of laboured breathing which may be related to reflux of test item.

Scar (1-3 cm) was recorded for three female animals in the Control group in the period of Days 14-25. Fur thin (for three males in the period of Days 5-20) and slight noisy respiration (for one female animal between Days 19-21) were recorded in the Low dose group (100 mg/kg bw/day). Slight or medium noisy respiration was observed for two males and one female in the Mid dose group (300 mg/kg bw/day) on some days in the period of Days 2-24, fur thin was also detected for one female in the period of Days 6-20. These findings were considered to be unrelated to the test item.

Mortality:
mortality observed, treatment-related
Description (incidence):
One male in the High dose group (#4006) was found dead on Day 12, two females in the High dose group (#4504 and #4509) were found dead on Day 3 and Day 39, respectively. Furthermore one High dose female (#4502) was preterminally euthanized on Day 14. There was no mortality in the Control, Low and Mid dose groups during the study.
Notes:
1. With clinical signs of piloerection, hunched back, reduced activity, more than 20% weight loss over 3 days, laboured respiration, pallor on the visible mucosal membranes and an increased, soft palpable abdominal mass, High dose female #4502 was euthanized after consultation with the Clinical Veterinarian.
2. According to the macroscopic findings at necropsy, the most probable cause of death for female #4504 was treatment method and not test item related.
3. Female #4504 was replaced by female #4604, and male #4101 was added to the study as a pair for female #4604.

Test item related mortality (approximately 8% for males and 23% for males) and clinical adverse effects were observed in the High dose group animals (1000 mg/kg bw/day).

MORTALITY
One High Dose female (4502) was preterminally euthanized on Day 14 due to slightly laboured respiration, moderate decreased activity and hunched presence clinically observed. Additionally, two High Dose females (#4504 and #4509) and one High Dose male (#4006) were found dead.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was measured in the body weight data in males or females during the experiment when compared to the Control group. However, body weight averages in both sexes were the lowest in the High dose group (1000 mg/kg bw/day) among the four examined groups.

Significantly (p < 0.05) lower body weight gain value in the period of Days 0-7 was observed in the High dose males (1000 mg/kg bw/day) when compared to the Control, although this effect lost the significance when observed on the whole treatment period.

Increased body weight gain was measured during GD7-GD14 in the Low (100 mg/kg bw/day) dose female group when compared to the Control (p < 0.01), this effect was considered to be incidental.

Decreased body weight gain was measured during the first two weeks and the whole gestation period (GD0-7, GD7-14 and GD0-20) in the High (1000 mg/kg bw/day) female group when compared to the Control (p < 0.05). Body weight gain decrease between GD20 and PP0 was smaller in the High (1000 mg/kg bw/day) female group when compared to the Control (p < 0.05), indicating a lower litter weight.

In summary, there were no statistically significances in body weight in any of the treated animals. High dose males showed what appeared to be an adverse effect on body weight gain, at the beginning of the study, which seems to have almost recovered by the study termination. Clear adverse effect on body weight gain was observed during the pregnancy of High dose female animals.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slightly, but statistically significant lower mean food consumption (p < 0.05) was noted in the Mid and High dose (300 and 1000 mg/kg bw/day, respectively) males at the beginning of the treatment (D0-D7). During the later periods, food consumption was stabilized and was comparable to other groups.

No test item related adverse effect was present in the mean food consumption of the females; although in the second week significant values were recorded for the Mid and High dose groups (300 and 1000 mg/kg bw/day, respectively). As the actual food consumption slightly increased (Mid dose) or showed no difference (High dose), these differences were considered to be only statistical, and not biological differences, due to the individual variances.
It is considered that there were no biologically significant treatment related effects on food intake in either sex.

Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to the control means, the following main differences were observed during haematology examination.
• Statistically significant decrease (p<0.05) in the mean cell haemoglobin value in the males of the High dose group was observed, but the observed mean values in all groups were within the historical control range.
• Statistically significant decrease (p<0.05) in the relative monocyte ratio in the males of the High dose group was observed. In the female animals from the same dose group there was no effect. All values were within the normal historic range, indicating that the values are individual variations and not related to any test item effects.
• A dose-dependent, statistically significant (p < 0.05) decrease of relative large unclassified cell (LUC) ratio in all treated male groups (Low, Mid and High dose) was observed. There was no effect in female treated groups. Furthermore, the concurrent study control was high compared to the historical control database, the values in all groups were considered to be normal; the observed statistical differences were not considered to be an effect of test item.

In conclusion these findings, in the absence of consistent patterns or other correlates were regarded as incidental or individual findings, within the historical control range, and were considered to be unrelated to treatment and with no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to the control means, the following main differences were observed at the evaluation of the clinical chemistry data (detailed data are shown in Table 13):
• A statistically significant increase (p<0.01) in the AST concentration was seen in High dose males. However, no similar observations were made in the female animal groups. Moreover, all values for both sexes were within the Historical Control range. The statistical difference is not considered to be an effect of test item.
• An apparently statistically significant increase (p<0.01) in the bile acid concentration in the Mid and High dose males. The Control group mean was below the range of the Historical Control, the other groups all had a relatively high standard deviation with no trend related to treatment (a single high dose male had a high value affecting the mean). The range of individual values in the female control group was similar to the high dose males. Taking account of the individual variability, it is considered that the statistical differences in the Mid and High dose males was not related to test item.
• Total protein concentration was significantly higher than control in the Low and High groups (p<0.01) and at p<0.05 for the Mid dose group. There was no clear dose response in males, female values were clearly unaffected and all groups were in the normal historical control range.
• Albumin concentration followed the same pattern as total protein, which is to be expected when all animals are normal and not affected by treatment.
• Calcium was significantly lower (p<0.05) in the High dose female animals when compared to the control, but values are well within the Historical Control range. No similar changes could be seen in the male animals.
• Phosphorus concentrations showed statistical significance (p < 0.05) in the High dose animals, but even this High dose value is lower than the female Control group mean. No similar change was seen in the female animals.
• Sodium concentrations showed a statistical significance (p < 0.05) in the High dose animals, but all values were within the normal biological and Historical Control ranges. No similar changes were seen in the female animals.

These findings, in the absence of consistent patterns or other correlates were regarded as incidental or individual findings, generally within the historical control range, and were probably unrelated to treatment and/or with no toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant effects were noted in the evaluated urine parameters (volume, pH and specific gravity) of the test item treated male and female animals at any dose groups.
Urine sediment analysis in all groups showed similar results to the control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition or in the reactions to different type of stimuli (Irwin test) in the control or treated groups in males and females (performed on Days 23 and PPD4, respectively).

The landing foot splay values were comparable with the control mean in all treated groups in males and females.

There were no statistical differences in the grip-strength values measured in the forelimbs or hind limbs in any dose groups.
The main objective of the automatic locomotor activity evaluation is to show that the normal behaviour of relatively high exploring activity is seen initially, with a reduced activity with time, to an approximate plateau at about 20-30 minutes. All groups in the study had a normal pattern of activity over the one hour observation period, showing a lack of effect of the test item.

Immunological findings:
no effects observed
Description (incidence and severity):
There was no effect on α2µ-globulin levels.
The positive areas detected with antibodies against α2μ-globulin were deemed to be representative within the kidney tissue and demonstrated a wide inter- and intra-individual variation. The mean α2μ-globulin levels (assessed as relative areas of anti- α2μ-globulin positivity) in the kidney tissue of high dose males (450 mg/kg bw/d) as compared with the respective controls when considering left kidney alone, right kidney alone and both kidneys combined. The differences between control and test item-treated animals (groups 3 and 4) was statistically significantly lower (P=0.0062).
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically increased liver weight was present in both sexes, in the High dose females (in absolute weight or when adjusted for body or brain weights) and Mid and High dose males (in absolute weight or when adjusted for body or brain weights). Livers adjusted for body weights were also significantly higher in the Low dose male group but the individual data were close to the historical control mean, so it is considered that the Low dose group was not affected by treatment. When considering the observations at histopathology, these hepatic weight differences are considered to be non-adverse, treatment related effects.

Statistically significant increase in the kidney weight was observed in the Mid and High dose males (absolute and adjusted for body weight and brain weight). However the dose response was not clear as the Mid dose had higher values than the High group for absolute and brain adjusted means. A statistical difference in kidney weight in the Low dose group (only when adjusted for body weight) was within the historic normal range and not considered an effect of treatment. In females, the High dose group kidney weight, only when adjusted for body weight, was statistically higher than control (p<0.05). Although the dose-response was not clear, the male kidney weigh changes correspond with histopathological changes in the Mid and High males. Statistical differences in females and Low dose males are not clearly related to treatment.

Compared to controls, statistically significant changes in the adrenal glands were observed the absolute weight or when adjusted for body or brain weights in the Mid dose group (decrease) and High dose group (increase) females. However, in the male animals only the High dose group showed a statistically significant decrease in the absolute weight of the organ. All values were in the normal historic range, there was no histopathology suggesting any adrenal effect, and the numeric values showed no consistence with dose level or sex; hence these statistical differences are not considered to reflect an adverse effect in the adrenal glands at any dose level.

A dose dependent decrease of spleen weight (absolute and adjusted to brain weight) reached significance in the High dose group females. However, in the males, spleen weight adjusted to body weight showed significant increase in the High dose group. Without any related histopathological observations, and with all data in the historical control range, these findings were not considered to be a clear effect of the test item.

In the male animals, statistically significant increases were found in the heart (Mid and High dose group) and epididymides (High dose group) weight when adjusted to the body weight and compared to the Control group. Heart weight was also significantly higher when adjusted to the brain weight in the Mid dose group. Since there was no dose dependency, all values were considered to be in the normal range and no similar findings were found in female animals, these findings were not considered to be a clear effect of the test item.

Absolute weight of thyroid (with parathryroid) glands showed statistically significant increase in the Low and High dose female animals. This statistical significance in the High dose group was present in relative body weight values too. As no similar changes were observed in the Male animals, no dose response was present, and all means were within the Historical Control range, these finding were not considered to be a clear effect of the test item.

Statistically significantly decreased values were detected both in the absolute and adjusted to the brain, uterus weight values in the High dose group, but not when adjusted for body weight and all means were within the Historical Control range. The difference was not considered to be an adverse effect of test item.

Other organs had organ weights not significantly different to control.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic Findings

No test item-related macroscopic findings were observed up to the dose level of 1000 mg/kg bw/day.

Changes in the adrenals, ovary, kidneys, prostate, stomach and thymus were present in low incidence in control and/or dosed animals. Therefore were considered to be without relationship to administered test item.

FOUND DEAD, Macroscopic Findings
No test item-related macroscopic findings were seen in unscheduled deaths.
Non-collapsed lungs were noted at necropsy in the male 4006. Perforation of the stomach, small thymus, fibrinous material adhered to the liver, gas contents in the ileum and cecum, 2 ml of the liquid within the abdominal cavity were seen in the female 4504. Dark/red discoloration of the non-collapsed lungs, depressed red area and few red foci at the glandular stomach were observed in the female 4509.

Microscopic Findings
Test item-related findings were recorded in the liver and/or kidneys in two found dead females 4504 and 4509.

Microvesicular centrilobular/periportal vacuolation altered the liver with mild intensity (non-correlated with necropsy) in the female 4504 and moderate degree in the female 4509. Mild bilateral vacuolation of the cortical tubules appeared in the kidneys of the female 4509.

Other changes were considered to be without relationship to the administered test item. Minimal focal erosion of the stomach glandular mucosa and moderate agonal congestion of the lungs in female 4509 correlated with gross findings. In the female 4504, the perforation of the stomach recorded at necropsy was not confirmed by histopathological examination. Moderate decrease of size/cellularity of the thymic cortex manifested at necropsy as small thymus.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic Findings
Test item-related microscopic findings were noted in the liver (300 and 1000 mg/kg bw/day) and kidney (1000 mg/kg bw/day).
The liver changes were characterized as microvesicular vacuolation of centrilobular/periportal hepatocytes. The hepatocytes were filled by numerous small vacuoles (microvesicular form). The nuclei of hepatocytes were not displaced to the periphery. In the males, minimal severity was noted in 2/12 Mid Dose males and minimal to mild intensity was seen in 6/12 High Dose males.

Minimal to moderate vacuolation was present in 7/12 Mid Dose and 9/10 High Dose females. Minimal degree was observed at the Mid Dose level versus minimal to moderate change in the High Dose females.

No test item-related changes were seen in the liver at the dose level of 100 mg/kg bw/day.

Test item-related changes were observed in the cortical tubules of the kidneys. In the males only, minimal to mild bilateral diffuse accumulation of the eosinophilic droplets was recorded in 4/6 males. These rounded droplets with varying size appeared within the cytoplasm of the tubule cells or within the lumen of the tubules. In the females, minimal to mild bilateral vacuolation of the cortical tubules was observed in 4/5 animals. Microvesicular forms of intracytoplasmic clear vacuoles with variable size were microscopically detected.

All other changes in examined tissues and organs were incidental or regarded as common background observations in this rat strain.

Non pregnant females
Due to failed in delivery of healthy pups, detailed histopathological examination was performed on retained reproductive organs in following animals: 2504, 2505 and 4501. No test item-related changes were recorded by light microscopy in these females.

Histopathology Conclusion
In conclusion, the treatment produced test item-related microscopic changes in the liver (300 and 1000 mg/kg bw/day) and kidney (1000 mg/kg bw/day). These changes were considered to be non-adverse.
Minimal to moderate microvesicular vacuolation of centrilobular/periportal hepatocytes was observed in H&E/phloxine stained liver. In the males, minimal severity was noted in Mid Dose males and minimal to mild intensity was seen in High Dose males. In the females, minimal degree was observed at the Mid Dose while minimal to moderate vacuolation was recorded in the High Dose females. Higher incidence was observed in the female dosing groups comparing with treated males.

There were no meaningful differences in incidence or severity between treated males and females. In the kidneys stained with H&E/phloxine, test item-related microscopic findings were observed in the cortical tubules of High dose animals in both sexes. In the males only, minimal to mild bilateral diffuse accumulation of the eosinophilic droplets was observed. The rounded droplets with varying size appeared within the cytoplasm of the tubule cells or within the lumen of the cortical tubules. In the females, minimal to mild bilateral vacuolation of the cortical tubules was detected. Under the conditions of this study, test item did not cause an increased α2µ-globulin levels in male rats when immunohistochemically examined. In contrast, the values obtained by image analysis using previously a monoclonal antibody were generally lower as compared with control animals (statistically significant). The presence of α2µ-globulin in the kidneys of male Wistar rats of this strain is deemed to reflect the normal physiological situation. The differences in the measured values reflect the normal variation and hence, are incidental.

There was one High Dose female preterminally euthanized on Day 14 due to slightly laboured respiration, moderate decreased activity and rapid weight loss. Test item-related minimal microvesicular hepatocellular vacuolation of the liver (with centrilobular/periportal zonation) was microscopically noted in this animal. Two females and one male from the High Dose groups were found dead. Test item-related mild to moderate microvesicular centrilobular/periportal vacuolation of the liver was present in two unscheduled deaths. Test item-related mild bilateral vacuolation of the cortical tubules altered the kidneys of one found dead female.

In addition, there were two Low Dose and one High Dose females with failed delivery of healthy pups, therefore detailed histopathological examination was performed on retained reproductive organs. No test item-related changes were recorded by light microscopy in these females.
Details on results:
In summary, daily administration of test item by oral gavage to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the conditions of this study did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, clinical chemistry, haematology, coagulation or on urinalysis parameters.

Test item-related liver weight and microscopic changes (microvesicular vacuolation of centrilobular/periportal hepatocytes) were present in the liver of Mid and High dose animals in both sexes. The relatively low severity of these changes and in the absence of hepatic degenerative hepatic changes, these observations were considered to be non-adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: clinical effect/mortility
Organ:
other: mortility/clinical effects (see above)
Treatment related:
yes
Dose response relationship:
yes

Results of the formulation analysis

Nominal concentrations

Measured concentrations on 26 August 2015 (Day 0)

mg/mL

Percentage of the nominal

Control

not detectable

-

25 mg/mL

23.7 ± 2.99

95

75 mg/mL

68.5 ± 1.68

91

250 mg/mL

247 ± 13.1

99

Nominal concentrations

Measured concentrations on 23 September 2015 (Day 28)

mg/mL

Percentage of the nominal

Control

not detectable

-

25 mg/mL

23.1 ± 0.83

92

75 mg/mL

67.8 ± 1.49

90

250 mg/mL

239 ± 21.9

95

Nominal concentrations

Measured concentrations on 07 October 2015 (Day 42)

mg/mL

Percentage of the nominal

Control

not detectable

-

25 mg/mL

25.7 ± 0.79

103

75 mg/mL

69.2 ± 1.49

92

250 mg/mL

254 ± 14.7

102


 

Conclusions:
In conclusion, under the conditions of this study, at 1000 mg/kg/day test item caused some mortality in the adults. Minor changes in the liver in the Mid and High dose groups of the parental generation, as well as changes in kidneys in the High dose group were not considered to be adverse.

It is considered that the no observed adverse effect levels (NOAEL) in this study for the parental/adult were:
 
NOAELmaternal toxicity:                             300 mg/kg bw/day
NOAELpaternal toxicity:                              300 mg/kg bw/day
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the toxicity of the test item following repeated daily administration by oral gavage to Wistar rats. The study included a reproductive/developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum.

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day PPD4.

Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals.

For the adult animals, histopathological examination was performed on the selected list of retained organs in the Control and High dose groups, and in the liver and kidney samples in the Control and all treated groups. Furthermore, additional renal immunohistochemistry analyses were performed to detect alpha-microglobulin.

Results

In summary, daily administration of test item by oral gavage to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the conditions of this study did not result in adverse changes in neurological assessment, body weight, food consumption, clinical chemistry, haematology, coagulation or on urinalysis parameters.

Additionally, no test item-related effects were indicated on male and female reproductive performance nor on the development of the F1 offspring.

Adults/Parental animals

Test item related mortality (approximately 8% for males and 23% for females) and clinical adverse effects (red discharge, noisy respiration, slightly or moderately decreased activity, slight or moderate noisy respiration (or in some cases laboured respiration or decreased respiratory rate), prostration, tonic convulsion, increased salivation, decreased body temperature, piloerection and/or hunched back) were observed in the High dose group animals (1000 mg/kg bw/day).

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in any groups.

Test item-related liver weight and microscopic changes (microvesicular vacuolation of centrilobular/periportal hepatocytes) were present in the liver of Mid dose (300 mg/kg bw/day) and High dose (1000 mg/kg bw/day) animals in both sexes. The relatively low severity of these changes and in the absence of hepatic degenerative hepatic changes, these observations were considered to be non-adverse.

In the kidney cortical tubules of High dose (1000 mg/kg bw/day) animals, minimal to mild bilateral vacuolation in females and minimal to mild bilateral diffuse accumulation of the eosinophilic droplets in males, were observed. There was no effect onα2µ-globulin levels. Taking into account the lack of degenerative changes, and the type and severity of the changes, the observations are not considered to be adverse.

There were no test item-related microscopic changes in reproductive organs. All other changes in examined tissues and organs in any groups were incidental or regarded as common background observations in the applied rat strain.

Conclusion

 

In conclusion, under the conditions of this study, at 1000 mg/kg/day test item caused some mortality in the adults. Minor changes in the liver in the Mid and High dose groups of the parental generation, as well as changes in kidneys in the High dose group were not considered to be adverse.

It is considered that the no observed adverse effect levels (NOAEL) in this study for the parental/adult and F1/pups generations were:

 

NOAELmaternal toxicity:                             300 mg/kg bw/day

NOAELpaternal toxicity:                              300 mg/kg bw/day

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Species:
rat
Quality of whole database:
The study is a guideline study with Klimisch score 1 (reliable without restrictions).
System:
other: Mortality

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In light of the physicochemical properties of the substance, a lquid with a very low vapour pressure and with a low log Kow value, the registrant considers that testing by oral route is the most appropriate.

Furthermore, significant dermal or inhalation exposure is not expected and no toxicity is observed in the acute dermal toxicity study (Hüls AG, 1996) as was seen in the acute oral toxicity test (Hüls AG, 1996). Therefore, no repeated-dose toxicity tests have been performed for the dermal or inhalation route of exposure.

Justification for classification or non-classification

According to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008 and based on the results of the repeated dose toxicity studies the test substance is not classified.