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Administrative data

Description of key information

Under the conditions of the present study, LZ 596 caused slight reduction in body weight development and food consumption, changes in serum biochemical parameters (elevated gamma glutamyltransferase activity and cholesterol concentration – male and female – liver weight alterations along with cytoplasmic vacuolization in the liver lobes (male and female) following a consecutive 90 days oral administration at 50 mg/kg bw/day to Hsd.Brl.Han:Wistar rats. At 25 mg/kg bw/day, slightly elevated cholesterol concentration (female) and changes in liver weights and cytoplasmic vacuolization in the liver lobes (male and female) were detected. At 10 mg/kg bw/day, slightly elevated cholesterol concentration (female) and changes in liver weights (male and female) were observed. Based on the observations made in this toxicity study the dose levels for the No Observed Adverse Effect Levels (NOAEL) was determined as follows:

- NOAEL for systemic toxicity of male rats: 10 mg/kg bw/day

- NOAEL for systemic toxicity of female rats: 10 mg/kg bw/day

The effects observed at dose levels of 25 and 10 mg/kg/day on body weights, food consumption, biochemistry and organ weights were treatment-related, however the changes were not severe and do not justify a classification for STOT Rep. Exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January - May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Specific details on test material used for the study:
TEST MATERIAL
- Substance identification/name in the report: LZ 596
- Molecular formula: C21H30N2
- Batch no.: 1229
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is commonly used species for toxicological studies in accordance with international recommendations. The Wistar strain is a well-known laboratory model with sufficient historical data.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS:
- Species / Strain: Rat, Hsd.Brl.Han: of Wistar origin
- Source: Toxi-Coop Zrt. Cserkesz u. 90. Budapest, H- 1103-Hungary
- Hygienic level: SPF at arrival and kept in good conventional environment during the study. The breeder certified the healthy status of animals
- Age at the treatment: Young adult rats, males: 55 – 60 days old / females: 55 – 60 days old
- Body weights at commencement of the treatment: male animals: between range of 226 and 274 g / female animals: between range of 143 and 175 g
- Acclimatization time: 20 days
- Water: tap water
- Food: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany

HOUSING:
- Housing: 2 or 3 animals per cage
- Cage type: Type III polypropylene/polycarbonate.
- Bedding: Certified laboratory wood bedding (Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany). The cages and bedding were changed twice a week.

ENVIRONMENTAL CONDITIONS:
- Illumination: Artificial light, from 6 a.m. to 6 p.m.
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70 %
- Ventilation: 10-15 air exchanges/hour
Environmental conditions were maintained by an air-condition system. Temperature and relative humidity were verified and recorded daily during the study.
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally. The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.
Vehicle:
methylcellulose
Details on oral exposure:
The test item and vehicle were administered to the appropriate animals by daily oral gavage from Day 0 to Days 89/90 (males/females, respectively). The dose volume for each animal was based on the most recent body weight measurement. Recovery animals were administered in the same way from Day 0 up to and including Day 89.
The test item was administered orally (by gavage) to Wistar rats (15 male and 16 female animals in the control and n = 15 animals/sex in the high dose group,
n = 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control = methylcellulose), 10, 25 and 50 mg/kg bw/day doses corresponding to concentrations of 0, 1, 2.5 and 5 mg/mL, applied in a dose volume of 10 mL/kg bw for 90/91 days. 5 male and 4 female animals in the control and 5 animals/ sex in the high dose groups were observed without administration for additional four weeks (recovery observations).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified beforehand. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item proved to be stable at 1 and 200 mg/mL concentrations at room temperature for two days and in refrigerator for five days. Concentrations of the test item in the dosing formulations varied from 97 % to 108 % of nominal concentrations at each analytical occasion, thereby confirming proper dosing.

APPARATUS:
- HPLC system: Hitachi LaChrom Elite
L-2000 Organizer, No.: 22E82-042
L-2130 HPLC pump, No.: 22E50-029
L-2200 Autosampler, No.: 22E81-005
L-2400 UV Detector, No.: 22E80-018
L-2350 Column Oven, No.: 2274-034
- Balances: BL 120S Sartorius, No.: 15307011
Mettler Toledo, PG 203 S, No.: 1122082152
Mettler Toledo, AB54-S, No.: 1122092721
- pH meter: HACH, HQ40d, No.: 090400030482
- Vortex mixer: Assistent, Reamix 2789, No.: 730170
- Ultrasonic bath: Branson, 3210E-MT, No.: A700343D
- Water purification system: MILLIPORE, DIRECT Q5, No.: F0DA13956K

HPLC-CONDITIONS:
- Column: Luna C18(2) 150*4.6 mm, 3μ, No: 628031-10
- Mobile Phase: Acetonitrile : Water : Buffer = 7 : 2 : 1 (v/v/v)
- Flow: 1 mL/min
- Injection volume: 10 μL
- Temperature: 25 °C
- Detector: UV, 250 nm
Duration of treatment / exposure:
90 days of treatment, followed by a 28 days recovery period
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- 0 mg/kg/day: 15 males (incl. 5 males for recovery group) / 15 females (incl. 5 females for recovery group)
- 10 mg/kg/day: 10 males / 10 females
- 25 mg/kg/day: 10 males / 10 females
- 50 mg/kg/day: 15 males (incl. 5 males for recovery group) / 15 females (incl. 5 females for recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
The dose setting at 10, 25 and 50 mg/kg/day was based on data obtained from a previously performed 14-Day Repeated Dose Oral Toxicity Study of the test item in rats. In this study, the test item induced significant liver weight increase at 25, 50 and 100 mg/kg bw/day associated with increased activity of gamma glutamytransferase at 100 mg/kg bw/day (male and female) and elevated serum level of cholesterol at 25, 50 and 100 mg/kg bw/day (female) associated with
cytoplasmic vacuolation in the hepatocytes at 50 and 100 mg/kg bw/day. Doses are selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
Positive control:
not tested
Observations and examinations performed and frequency:
MORTALITY:
Inspection for signs of morbidity and mortality was made twice daily at the beginning and at the end of the working day during the treatment and recovery periods. Dead animals were processed in the same way as the animals of the terminal necropsy except histological processing and evaluation in one of them due to the autolysis of organs.

CLINICAL OBSERVATIONS:
General clinical observations were made cage-side once a day, after treatment at approximately the same time. General clinical observations were also made cage-side once a day for recovery animals during the course of the post-treatment period,
On the day prior to the first treatment with the test item, and approximately once weekly thereafter, a detailed observation was conducted while handling the animal on days that the animals were weighed and food consumption measurements were taken. Potential signs noted included but were not limited to: changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, and unusual respiratory pattern). Likewise, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotype activities (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., selfmutilation, walking backwards) were recorded. Detailed clinical observations were conducted in the same way during the recovery period. All observations and/or mortality checks were recorded In the last exposure week, functional observations were conducted for all animals. Sensory reactivity to stimuli of different types, assessment of grip strength and motor activity assessment were examined. General physical condition and behavior of animals were tested.
A modified Irwin test was performed. (Irwin, S. Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Functional observation was not performed at the end of the recovery period because treatment related changes were not detected at the termination of the treatment.

BODY WEIGHT:
Individual body weights were recorded two times during acclimation. Animals involved in the study were weighed on Day 0 (prior to study start) and twice weekly during weeks 1-4 and once weekly thereafter (weeks 5 - 13 and during the recovery period) with a precision of 1 g. The animals were also weighed immediately prior to sacrifice in order to calculate organ to body weight ratios. Decedents were also weighed. Individual body weight changes were calculated according to the days of measurements and for the study overall.

FOOD CONSUMPTION:
Food consumption was determined with the measurement of non-consumed diet with a precision of 1 g once weekly to coincide with body weight measurement.
Sacrifice and pathology:
CLINICAL PATHOLOGY:
Clinical pathology examinations including hematology, blood coagulation and clinical chemistry were conducted in all animals at the end of the treatment period (i.e. one day after the last dosing) and at the end of the recovery period. Clinical pathology examinations were conducted on Days 90 and 91 (male and female, respectively), as well as on Day 118 (recovery groups). Animals were food deprived for approximately 16 hours prior to blood collection.

NECROPSY:
Gross pathology was performed on every experimental animal just after the death, at termination of the treatment (i.e. one day after the last dosing), or at termination of the recovery period. Surviving animals were euthanized by exsanguination after verification of narcosis with Isofluran CP®. The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the
tissues and organs was observed macroscopically. All observations were recorded with details of the location, color, shape and size.
Unscheduled sacrifice: Any rats that died were examined for the cause of death on the day the observation was made.
Rats were evaluated for gross lesions. Organs and tissues were excised, weighed (except for animals found dead), and preserved as described for those animals sacrificed by design.

ORGAN WEIGHTS:
The following organs were weighed and recorded for all animals. Paired organs were weighed together.
- With precision of 0.01g: Liver, kidneys, testes, epididymides, uterus and fallopian tubes, thymus, spleen, brain and heart.
- With precision of 0.001g: Adrenals, ovaries, thyroid/parathyroid

HISTOPATHOLOGY:
Histological examination was performed on the preserved organs and tissues of the animals from both the control and high dose groups (Groups 1 and 4, respectively, including recovery animals), as well as from one animal that died during the course of the study. Autolysis in the second animal that was found dead was too advanced for histological examination. In addition, histopathological examination of the liver was performed in all animals in groups of 10 and 25 mg/kg bw/day due to histological findings at 50 mg/kg bw/day, for a better estimation of the No Observed Adverse Effect Level (NOAEL).
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
- Body weight
- Food consumption
- Hematology and blood coagulation
- Clinical chemistry
- Organ weight
The heterogeneity 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 was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test. Frequency of toxic response and pathological findings by sex and dose was calculated.
Clinical signs:
no effects observed
Description (incidence and severity):
Toxic signs related to the test item were not detected at any dose level at the daily and detailed weekly clinical observations and in the course of the functional observation battery. The behavior and physical condition of the animals was normal at each dose level (50, 25 and 10 mg/kg bw/day) during the entire treatment period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item related mortality. Two control female rats died during the study. One of fatalities was caused by application error.
There were no preceding clinical signs or body weight changes in this animal and histological examination revealed findings (alveolar edema in the lung, and acute hemorrhage in the heart and thymus) in connection with probable suffocation as the cause of death. Prior to death of the other animal, body weight decrease, piloerection, hunched back, decreased activity, narrow eye aperture, brownish fur around the nose and pale limbs were detected suspiciously indicative of individual disease. Histological examination was not feasible due to the autolysis of organs and tissues.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight and the mean body weight gain lowered in male and female animals at 50 mg/kg bw/day and in female animals at 25 mg/kg bw/day, increasingly so towards the end of the treatment period. The summarized body weight gain was above the control in male and female animals during the post-treatment period however the differences in the mean body weight were only partially reversible at 50 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was affected by the treatment with the test item in male and female animals at 50 mg/kg bw/day and in female animals at 25 mg/kg bw/day in accordance with the body weight changes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Test item-related pathologic changes were not detected in the investigated hematology or blood coagulation parameters
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry investigations revealed slight changes referring to test item effect on hepatic function in male and female animals at 50 mg/kg bw/day. Higher activity of gamma glutamyltransferase (male and female at 50 mg/kg bw/day) and higher serum level of cholesterol (male and female at 50 mg/kg bw/day, female animals at 10 and 25 mg/kg bw/day) were observed. Slight changes in elevated mean alanine-amino transferase activity (male) and total bilirubin concentration (male and female) might also be indicative a test item influence on hepatic function at 50 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The liver weights (absolute and relative to body and brain weights) were significantly elevated in male and female animals at 10, 25 and 50 mg/kg bw/day with respect to controls. The changes in the liver weights ceased at 50 mg/kg bw/day up to the end of post-treatment period
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Nutmeg-like pattern of the liver was observed in male animals of mid and high dose groups (1/10 and 5/10, respectively) at 25 and 50 mg/kg bw/day at the termination of the treatment period but not at the end of recovery period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histological examination revealed cytoplasmic vacuolization in the liver lobes in male and female animals at 25 and 50 mg/kg bw/day. This liver alteration ceased during the course of post-treatment period as cytoplasmic vacuolation was not detected in the liver in the recovery animals (male or female) of 50 mg/kg bw/day group.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Key result
Critical effects observed:
no
Conclusions:
Based on the observations made in this toxicity study the dose levels for the No Observed Adverse Effect Levels (NOAEL) was determined as follows:
- NOAEL for systemic toxicity of male rats: 10 mg/kg bw/day
- NOAEL for systemic toxicity of female rats: 10 mg/kg bw/day
Executive summary:

This toxicity study was performed in accordance with test methods EC B.26 and OECD 408 over a 90 -day period on male and female Wistar rats. Recovery animals in the control and in the high dose groups were used for observation of reversibility, persistence, or delayed occurrence of toxic effects during the 28 days post treatment. The test item was administered orally (by gavage) to Wistar rats once a day at 0 (vehicle control), 10, 25 and 50 mg/kg bw/day doses corresponding to concentrations of 0, 1, 2.5 and 5 mg/mL, applied in a dose volume of 10 mL/kg bw for 90/91 days. Additional animals in the control and in the high dose groups were observed without administration for additional four weeks (recovery observations). The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified beforehand. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item proved to be stable at 1 and 200 mg/mL concentrations at room temperature for two days and in refrigerator for five days.

There was no test item related mortality. Two control female rats died during the study. One of fatalities was caused by application error. There were no preceding clinical signs or body weight changes in this animal and histological examination revealed findings (alveolar edema in the lung, and acute hemorrhage in the heart and thymus) in connection with probable suffocation as the cause of death. Prior to death of the other animal, body weight decrease, piloerection, hunched back, decreased activity, narrow eye aperture, brownish fur around the nose and pale limbs were detected suspiciously indicative of individual disease. Histological examination was not feasible due to the autolysis of organs and tissues. Toxic signs related to the test item were not detected at any dose level at the daily and detailed weekly clinical observations and in the course of the functional observation battery. The behavior and physical condition of the animals was normal at each dose level (50, 25 and 10 mg/kg bw/day) during the entire treatment period.

The mean body weight and the mean body weight gain lowered in male and female animals at 50 mg/kg bw/day and in female animals at 25 mg/kg bw/day, increasingly so towards the end of the treatment period. The summarized body weight gain was above the control in male and female animals during the post-treatment period however the differences in the mean body weight were only partially reversible at 50 mg/kg bw/day.

The food consumption was affected by the treatment with the test item in male and female animals at 50 mg/kg bw/day and in female animals at 25 mg/kg bw/day in accordance with the body weight changes.

Test item-related pathologic changes were not detected in the investigated hematology or blood coagulation parameters.

Clinical chemistry investigations revealed slight changes referring to test item effect on hepatic function in male and female animals at 50 mg/kg bw/day. Higher activity of gamma glutamyltransferase (male and female at 50 mg/kg bw/day) and higher serum level of cholesterol (male and female at 50 mg/kg bw/day, female animals at 10 and 25 mg/kg bw/day) were observed. Slight changes in elevated mean alanine-amino transferase activity (male) and total bilirubin concentration (male and female) might also be indicative a test item influence on hepatic function at 50 mg/kg bw/day.

Nutmeg-like pattern of the liver was observed in male animals of mid and high dose groups (1/10 and 5/10, respectively) at 25 and 50 mg/kg bw/day at the termination of the treatment period but not at the end of recovery period.

The liver weights (absolute and relative to body and brain weights) were significantly elevated in male and female animals at 10, 25 and 50 mg/kg bw/day with respect to controls. The changes in the liver weights ceased at 50 mg/kg bw/day up to the end of post-treatment period.

Histological examination revealed cytoplasmic vacuolization in the liver lobes in male and female animals at 25 and 50 mg/kg bw/day. This liver alteration ceased during the course of post-treatment period as cytoplasmic vacuolation was not detected in the liver in the recovery animals (male or female) of 50 mg/kg bw/day group.

Under the conditions of the present study, LZ 596 caused slight reduction in body weight development and food consumption, changes in serum biochemical parameters (elevated gamma glutamyltransferase activity and cholesterol concentration – male and female – liver weight alterations along with cytoplasmic vacuolization in the liver lobes (male and female) following a consecutive 90 days oral administration at 50 mg/kg bw/day to Hsd.Brl.Han:Wistar rats. At 25 mg/kg bw/day, slightly elevated cholesterol concentration (female) and changes in liver weights and cytoplasmic vacuolization in the liver lobes (male and female) were detected. At 10 mg/kg bw/day, slightly elevated cholesterol concentration (female) and changes in liver weights (male and female) were observed. Based on the observations made in this toxicity study the dose levels for the No Observed Adverse Effect Levels (NOAEL) was determined as follows:

- NOAEL for systemic toxicity of male rats: 10 mg/kg bw/day

- NOAEL for systemic toxicity of female rats: 10 mg/kg bw/day

The effects observed at dose levels of 25 and 10 mg/kg/day on body weghts, food consumption, biochemistry and organ weights were treatment-related, however the changes were not severe and do not justify a classification for STOT Rep. Exposure.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1, GLP-study

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

Justification for classification or non-classification

According to the Globally Harmonised System of Classification and Labelling of Chemicals, the test item does not require any classification/labelling.