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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Effects on reproductive organs have been evaluated based on the subchronic repeated dose toxicity study in rats:

The screening study for reproductive / developmental toxicity 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). Regular or periodic observation of mortality, clinical parameters, body weights and food consumption were performed. Subsequently gross pathology was done on every experimental animal including organ weights and histopathological examinations. 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 and female rats: 10 mg/kg bw/day. No adverse effects on reproductive organs of males and females were reported in the study. NOAEL for the fertility based of absence of the adverse effects on the reproductive organs was determined to be 10 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Justification for type of information:
A 90-day subchronic toxicity test and a pre-natal developmental toxicity study were performed instead.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Groups of twenty four sperm-positive female Hsd. Brl. Han: WIST Rats were treated with the test item by oral administration at three dose levels of 5, 15 and 50 mg/kg bw/day and one control group from day 5 up to and including day 19 post coitum daily. The control animals were given the vehicle (1% methylcellulose) alone. The treatment volume was 5 mL/kg/bw. Formulation analytics (checking of homogeneity and achieved concentrations of the test item in the dosage forms) was performed two times during the treatment period using a validated HPLC/UV method. During the study animals were checked for mortality and clinical signs. Body weight and food consumption of the dams were also recorded. The day of detection of sperm in the vaginal smear of females was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. Organs of the dams were examined macroscopically. Organs and tissues with undiagnosed macroscopic findings were fixed in 4 % buffered formalin solution at necropsy for possible histological examination. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The placentas were weighed and examined externally. The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method. After double staining, the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 89 dams with live fetuses at termination on gestation day 20, (23, 24, 21 and 21 in the control, 5, 15 and 50 mg/kg bw/day groups respectively. Results The formulations were proved to be homogeneous. Measured concentrations of LZ 596 were in the range of 93-107% of the nominal concentrations at all the three concentration levels for both investigated series.

There was no mortality and were no clinical signs observed during the in-life phase. Enlarged or slightly enlarged liver was found at necropsy in the majority of the dams in the 50 mg/kg bw/day group which was attributed to the treatment of the dams. No treatment related findings were recorded in the 5 and 15 mg/kg bw/day dose groups.

Reduction in the food consumption as well as body weight gain and corrected body weight gain of the dams was observed in the 50 mg/kg bw/day dose group which was considered to be due to the treatment with the test item.

No treatment related changes were indicated in the 5 and 15 mg/kg bw/day groups. There were no significant differences in the mean values of corpora lutea, implantations, early- and late embryonic death, dead fetuses, viable fetuses and and the sex distribution of fetuses in the experimental groups. Intrauterine parameters There was no significant differences in the body weight of the fetuses and placental weight. Fetal- and placental weight The number of litters with malformed fetuses was two in each group during the fetal examinations. Fetal examination No test item effect was indicated. External examination There were no malformations recorded in the 15 and 50 mg/kg bw/day groups. One fetus was found with meningocele and short tail in the control group and one fetus with a malrotated forelimb in the 5 mg/kg bw/day dose group which was not confirmed at skeletal examination. Incidence of body weight retarded fetuses and other variations was not influenced by the treatment. Malformation of the brain was found in one fetus in the 5 and one in the 15 mg/kg bw/day dose group. Considering that no malformation of the brain was recorded for the fetuses in the 50 mg/kg bw/day dose group and that brain malformations occur sporadically with low incidence unrelated to the treatment according to the historical control data and background data another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993), this was judged not to be a consequence of the treatment of the dams with LZ 596. Visceral examination Situs inversus totalis in one fetus in the 15 mg/kg bw/day group was considered to be without any test item relationship. Statistically significant increase was indicated in the incidence of fetuses with variations and abnormalities due to the increase of fetuses with bilateral hydroureter in the 50 mg/kg bw/day group which is a common finding in prenatal developmental toxicity studies according to the background data of this laboratory (Appendix XXIV/A, B) and another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993).

The percentage of bilateral hydroureter was not higher than 7% in this study and the incidence of associated variation dilated renal pelvis was with a very low incidence and did not increase in the test item treated groups which suggests that this variation was without a toxicological relevance. Misaligned tracheal cartilage rings as another type of variation were found without any dose response. No test item effect was indicated. Skeletal examination None of the variations or malformations increased significantly in the test item treated groups. Conclusion Based upon these data, treatment of pregnant Hsd. Brl. Han: WIST Rats from gestational day 5 to 19 by oral administration of LZ 596, caused maternal toxicity such as reduced food consumption and body weight gain as well as enlargement of the livers at the dose level of 50 mg/kg bw/day. The test item did not result in external and skeletal abnormalities or visceral malformations. The slightly higher incidence of visceral variation hydroureter at 50 mg/kg bw/day was judged to be without a toxicological relevance. The doses of 5 and 15 mg/kg bw/day of LZ 596 did not cause any maternal or fetal effects.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

- NOAEL maternal toxicity: 15 mg/kg bw/day

- NOAEL developmental toxicity: 50 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2015 - February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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 test animals and environmental conditions:
TEST ANIMALS:
- Species / Strain: Hsd. Brl. Han: WIST Rats
- Source: TOXI-COOP ZRT. 1103 Budapest, Cserkesz u. 90.
- Hygienic level: SPF at arrival and kept in good conventional environment during the study
- Number of animals: 133 females and 80 males
- Number of animals involved in the study: animals needed to achieve at least 24 spermium positive females/group
- Age of animals at arrival: females 7-9 weeks / males 8-9 weeks
- Body weight of animals at arrival: females 120-160 g / males 260-310 g
- Age at start of mating: females 12-14 weeks / males 14-17 weeks
- Body weight of females at mating: 182-249 g
- Acclimatisation time: 33 days for females / 40 days for males
- Housing:
1. pre-mating period: 2-3 females /cage & 2-3 males/cage
2. during mating hours: 1 male with 1- 3 females
3. during pregnancy: 2-3 sperm positive females/cage
- Cage type: Type II polypropylene/polycarbonate with stainless steel covers equipped by self-feeding baskets
- Food: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: tap water

ENVIRONMENTAL CONDITIONS:
- Illumination: Artificial light, from 6 a.m. to 6 p.m.
- Temperature: 21-23 °C
- Relative humidity: 30 - 36 %
- Ventilation: 10-15 air exchanges/hour by central air-conditioning system.
Environmental conditions are maintained by an air-condition system. Temperature and relative humidity were verified and recorded daily during the study.
Route of administration:
oral: gavage
Vehicle:
other: 1% Methylcellulose in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered at appropriate concentrations, prepared with the vehicle. Preparation of the test item formulations was made with a frequency of 1, 2 or 3 days, using a magnetic stirrer. LZ 596 in formulations was stored at 2-8 °C according to the results of the Validation of the Analytical Method for the Determination of LZ 596.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling for analytical control of dosing was made on the first and last week of treatment. The formulations were proved to be homogeneous. Measured concentrations of LZ 596 were in the range of 93-107% of the nominal concentrations at all the three concentration levels for both investigated series.

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: Sartorius, BL 120S, No.: 15307011
Mettler Toledo, PG 203 S, No.: 1122082152
Mettler Toledo, AB54-S, No.: 1122092721
- pH meter: HACH, HQ40d, No.: 090400030482
- Water purification system: MILLIPORE, DIRECT Q5, No.: F0DA13956K
- Ultrasonic bath: Branson, 3210E-MT, No.: A700343D

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
Details on mating procedure:
The females were paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females / group achieves twenty four. Vaginal smears were prepared from each female, stained with 1 % aqueous methylene blue solution and examined for presence of sperm and for estrus cycle. The day of mating was regarded as day 0 of pregnancy (vaginal plug and/or sperm in the vaginal smear). Sperm positive females were separated and caged in groups of 1 to 3 animals, however individual caging was avoided if possible.
Duration of treatment / exposure:
The test item was administered orally (by gavage) from gestational day 5 to 19, daily. The route of application is selected in compliance with international guidelines.
Frequency of treatment:
daily
Duration of test:
see above
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 sperm-positive females/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of the Dose Range Finding Prenatal Developmental Toxicity Study of LZ 596 in Rats by Oral Administration.
Maternal examinations:
CLINICAL OBSERVATIONS:
General clinical observations of the sperm positive females were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. When signs of toxicity were observed, animals were checked more frequently. Individual observation included a check of behavior and general condition. Duration and severity of the clinical signs were recorded.

MORTALITY:
Observations for signs of morbidity and for mortality were made twice daily, at the beginning and before the end of the working period. Moribund animals or animals obviously in pain or showing signs of severe and enduring distress were euthanized. Dead and moribund animals were processed in the same way as the animals at terminal necropsy. In these females, implantations and corpora lutea were counted but fetuses and placentas were not measured and examined.

BODY WEIGHT:
The body weight of the male animals was not measured. The body weight of the female rats was measured at least once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). Corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION:
The food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).
Ovaries and uterine content:
EXAMINATION FOR SIGN OF IMPLANTATION:
On gestation days 13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding which indicates the implantation of conceptuses.
Fetal examinations:
All sperm positive females were sacrificed by decapitation under deep Isofluran anaesthesia on day 20 of gestation. The abdomen was opened, the uterus with cervix and left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed.
Organs and tissues with undiagnosed macroscopic findings were fixed in 4 % buffered formalin solution at necropsy for possible histological examination. Control organs were fixed in 4 % buffered formalin solution and preserved for comparison. The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted. Animals in which unambiguous implantation sites, but not fetuses, were found were considered as pregnant. Uteri that appear non-gravid were further examined to confirm the non-pregnant status. Fetuses were removed from the opened uterus and were sunk in a Petri-dish filled up with water. Spontaneous movement of fetuses was observed as a viability assessment. Euthanasia of the fetuses was performed by hypothermia. The fetuses were washed with tap water and randomly laid on a filter paper with written ordinal numbering. Bleeding from the umbilical cord after it is cut was observed as an indication of viability before euthanasia.
Each live fetus and its placenta was weighed individually (fetuses accuracy 0.01 g, placentas accuracy 0.001 g), and subjected to external examination. The gender of the fetuses was determined according to the anogenital distance. The fetuses were individually identified and about the half of each litter was subjected to visceral examination and the other half for skeletal examination. The body of those subjected to visceral examination was fixed in Sanomiya mixture. After fixation the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. The abdominal region of those subjected to skeletal examination was opened, the viscera and skin of fetuses were removed and the cadaver was fixed in alcian-blue-acetic acid-ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.
Statistics:
Statistical analysis was performed with SPSS PC+ software.
Clinical signs:
no effects observed
Description (incidence and severity):
There was no mortality and were no clinical signs observed during the in-life phase.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no mortality and were no clinical signs observed during the in-life phase.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduction in the food consumption as well as body weight gain and corrected body weight gain of the dams was observed in the 50 mg/kg bw/day dose group which was considered to be due to the treatment with the test item. No treatment related changes were indicated in the 5 and 15 mg/kg bw/day groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduction in the food consumption as well as body weight gain and corrected body weight gain of the dams was observed in the 50 mg/kg bw/day dose group which was considered to be due to the treatment with the test item. No treatment related changes were indicated in the 5 and 15 mg/kg bw/day groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no significant differences in the body weight of the fetuses and placental weight
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged or slightly enlarged liver was found at necropsy in the majority of the dams in the 50 mg/kg bw/day group which was attributed to the treatment of the dams. No treatment related findings were recorded in the 5 and 15 mg/kg bw/day dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
not measured/tested
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
dermal irritation
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
immunology
organ weights and organ / body weight ratios
gross pathology
neuropathology
histopathology: non-neoplastic
histopathology: neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
other:
Remarks on result:
not measured/tested
Key result
Abnormalities:
no effects observed
Localisation:
placenta
Description (incidence and severity):
No abnormalities were observed on the placentas of any animals in any examined
groups.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no significant differences in the body weight of the fetuses and placental weight
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item effect was indicated. There were no malformations recorded in the 15 and 50 mg/kg bw/day groups. One fetus was found with meningocele and short tail in the control group and one fetus with a malrotated forelimb in the 5 mg/kg bw/day dose group which was not confirmed at skeletal examination. Incidence of body weight retarded fetuses and other variations was not influenced by the treatment
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test item effect was indicated. None of the variations or malformations increased significantly in the test item treated groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformation of the brain was found in one fetus in the 5 and one in the 15 mg/kg bw/day dose group. Considering that no malformation of the brain was recorded for the fetuses in the 50 mg/kg bw/day dose group and that brain malformations occur sporadically with low incidence unrelated to the treatment according to the historical control data and background data another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993), this was judged not to be a consequence of the treatment of the dams with the test item.
Situs inversus totalis in one fetus in the 15 mg/kg bw/day group was considered to be without any test item relationship.
Statistically significant increase was indicated in the incidence of fetuses with variations and abnormalities due to the increase of fetuses with bilateral hydroureter in the 50 mg/kg bw/day group which is a common finding in prenatal developmental toxicity studies according to the background data of this laboratory and another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993). The percentage of bilateral hydroureter was not higher than 7% in this study and the incidence of associated variation dilated renal pelvis was with a very low incidence and did not increase in the test item treated groups which suggests that this variation was without a toxicological relevance. Misaligned tracheal cartilage rings as another type of variation were found without any dose response
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The number of litters with malformed fetuses was two in each group during the fetal examinations.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
other:
Remarks on result:
not measured/tested
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
No fetal effects were seen at external, visceral and/or skeletal examination of foetuses
in the study which could be related to the test item administration.
Key result
Developmental effects observed:
no
Conclusions:
Based upon these data, treatment of pregnant Hsd. Brl. Han: WIST Rats from gestational day 5 to 19 by oral administration of LZ 596, caused maternal toxicity such as reduced food consumption and body weight gain as well as enlargement of the livers at the dose level of 50 mg/kg bw/day. The test item did not result in external and skeletal abnormalities or visceral malformations. The slightly higher incidence of visceral variation hydroureter at 50 mg/kg bw/day was judged to be without a toxicological relevance. The doses of 5 and 15 mg/kg bw/day of LZ 596 did not cause any maternal or fetal effects. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
- NOAEL maternal toxicity: 15 mg/kg bw/day
- NOAEL developmental toxicity: 50 mg/kg bw/day
Executive summary:

Groups of twenty four sperm-positive female Hsd. Brl. Han: WIST Rats were treated with the test item by oral administration at three dose levels of 5, 15 and 50 mg/kg bw/day and one control group from day 5 up to and including day 19 post coitum daily. The control animals were given the vehicle (1% methylcellulose) alone. The treatment volume was 5 mL/kg/bw. Formulation analytics (checking of homogeneity and achieved concentrations of the test item in the dosage forms) was performed two times during the treatment period using a validated HPLC/UV method. During the study animals were checked for mortality and clinical signs. Body weight and food consumption of the dams were also recorded. The day of detection of sperm in the vaginal smear of females was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. Organs of the dams were examined macroscopically. Organs and tissues with undiagnosed macroscopic findings were fixed in 4 % buffered formalin solution at necropsy for possible histological examination. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The placentas were weighed and examined externally. The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method. After double staining, the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 89 dams with live fetuses at termination on gestation day 20, (23, 24, 21 and 21 in the control, 5, 15 and 50 mg/kg bw/day groups respectively. Results The formulations were proved to be homogeneous. Measured concentrations of LZ 596 were in the range of 93-107% of the nominal concentrations at all the three concentration levels for both investigated series.

There was no mortality and were no clinical signs observed during the in-life phase. Enlarged or slightly enlarged liver was found at necropsy in the majority of the dams in the 50 mg/kg bw/day group which was attributed to the treatment of the dams. No treatment related findings were recorded in the 5 and 15 mg/kg bw/day dose groups.

Reduction in the food consumption as well as body weight gain and corrected body weight gain of the dams was observed in the 50 mg/kg bw/day dose group which was considered to be due to the treatment with the test item.

No treatment related changes were indicated in the 5 and 15 mg/kg bw/day groups. There were no significant differences in the mean values of corpora lutea, implantations, early- and late embryonic death, dead fetuses, viable fetuses and the sex distribution of fetuses in the experimental groups. Intrauterine parameters: There was no significant differences in the body weight of the fetuses and placental weight. Fetal- and placental weight The number of litters with malformed fetuses was two in each group during the fetal examinations. Fetal examination No test item effect was indicated. External examination There were no malformations recorded in the 15 and 50 mg/kg bw/day groups. One fetus was found with meningocele and short tail in the control group and one fetus with a malrotated forelimb in the 5 mg/kg bw/day dose group which was not confirmed at skeletal examination. Incidence of body weight retarded fetuses and other variations was not influenced by the treatment. Malformation of the brain was found in one fetus in the 5 and one in the 15 mg/kg bw/day dose group. Considering that no malformation of the brain was recorded for the fetuses in the 50 mg/kg bw/day dose group and that brain malformations occur sporadically with low incidence unrelated to the treatment according to the historical control data and background data another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993), this was judged not to be a consequence of the treatment of the dams with LZ 596. Visceral examination Situs inversus totalis in one fetus in the 15 mg/kg bw/day group was considered to be without any test item relationship. Statistically significant increase was indicated in the incidence of fetuses with variations and abnormalities due to the increase of fetuses with bilateral hydroureter in the 50 mg/kg bw/day group which is a common finding in prenatal developmental toxicity studies according to the background data of this laboratory (Appendix XXIV/A, B) and another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993).

The percentage of bilateral hydroureter was not higher than 7% in this study and the incidence of associated variation dilated renal pelvis was with a very low incidence and did not increase in the test item treated groups which suggests that this variation was without a toxicological relevance. Misaligned tracheal cartilage rings as another type of variation were found without any dose response. No test item effect was indicated. Skeletal examination None of the variations or malformations increased significantly in the test item treated groups. Conclusion Based upon these data, treatment of pregnant Hsd. Brl. Han: WIST Rats from gestational day 5 to 19 by oral administration of LZ 596, caused maternal toxicity such as reduced food consumption and body weight gain as well as enlargement of the livers at the dose level of 50 mg/kg bw/day. The test item did not result in external and skeletal abnormalities or visceral malformations. The slightly higher incidence of visceral variation hydroureter at 50 mg/kg bw/day was judged to be without a toxicological relevance. The doses of 5 and 15 mg/kg bw/day of LZ 596 did not cause any maternal or fetal effects.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

- NOAEL maternal toxicity: 15 mg/kg bw/day

- NOAEL developmental toxicity: 50 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1; guideline study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

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.