4,4'-methylenebis[2,6-diethylaniline]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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

Studies on rats and rabbits were conducted

Developmental toxicity on rats

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

Developmental toxicity on rabbits

This developmental toxicity study (OECD No. 414) was performed to assess the effects of the test item on the embryonic and foetal development (including the organogenesis period) of New Zealand White rabbits in their first pregnancy. The does (one control and three test item treated groups) were treated daily by oral (gavage) administration, from Gestation Day (GD) 6 up to and including Gestation Day (GD) 27, where the day of insemination was counted as Day 0 of pregnancy (GD 0). Control does were treated with the vehicle (1% (w/v) aqueous methyl cellulose solution) only. Caesarean sections, necropsy of does and examination of uterine contents were performed on GD 28.

The dose levels were set by the Sponsor / Study Monitor based on the available data and information from previous experimental work, including the results of a Dose Range Finding (DRF) toxicity study in pregnant New Zealand White rabbits performed at the Test Facility.

Based on the results from the DRF study, doses of 100, 30 and 10 mg/kg bw/day were selected for the main study (designated High, Mid and Low dose, respectively). The aim was to use the highest dose of 100 mg/kg bw/day to induce toxic effects, but ideally no death or suffering, and to obtain a NOAEL at the lowest dose level.

Test item formulations were analysed for concentration and homogeneity twice during the treatment period using a validated HPLC-UV (High Performance Liquid Chromatography method with UV detection) method. Simultaneously, vehicle control formulations were also analysed for the test item concentration.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

The number of confirmed pregnant, evaluated does was 19, 16, 16, 16 in the Control, Low, Mid and High dose groups, respectively.

All test item formulations were within the range of 94-105% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

A total of 10 animal were found dead or pre-terminally euthanized in the study generally as a result of gavage accident or early abortion; there were no mortalities ascribed to test item effects.

There was a transient initial body weight loss with low food consumption in the first few days of the treatment period at the High dose only, but no effect on the overall body weight gain of rabbits. The only clinical sign associated with treatment was reduced faeces in this High dose group. The weight and clinical sign effects confirm the selected High dose was close to the MTD.

There were no treatment related findings at necropsy.

There were no statistically significant differences in the intrauterine parameters in the test item treated animals when compared to the controls. There was no significant difference in number of foetuses, or in the sex distribution of foetuses between the control and treatment groups.

The number of foetuses with retarded body weight per. There was no statistical difference in the number of runts between the control and treated groups.

There were no test item related external, visceral or skeletal malformations in the study. In case of one High dose foetus a malformation of the skull was recorded (at both external and skeletal evaluation). Based on the single occurrence it was considered as isolated and incidental, ascribed to individual variability, not being related to the test item treatment. All other findings correspond or occur in the study control or historical control data or considered to be isolated and incidental, ascribed to individual variability.

At least 16 litters were evaluated in each treatment group, hence the study was considered to be valid.

In conclusion, LZ 596, when administered daily by oral gavage to pregnant New Zealand White rabbits from gestation days GD6 to GD27 at up to 100 mg/kg bw/day resulted in transient effects on body weight and food intake, but no clearly adverse test item related mortality, clinical signs, or overall body weight or food consumption effects. No on the embryotoxic or foetotoxic effects were observed. There were no test item related external, visceral or skeletal malformations.

The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAELmaternal toxicity:               100 mg/kg bw/day

Based on the lack of adverse findings in the High dose group for the overall study duration.

NOAELembryotoxicity:                  100 mg/kg bw/day

Based on the lack of intrauterine effect in the High dose group.

NOAELfoetotoxicity:                       100 mg/kg bw/day

Based on the lack of intrauterine effect in the High dose group.

NOAELteratogenecity:                    100 mg/kg bw/day

Based on lack of treatment related abnormalities in the High dose group.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - September 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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
- Purity: 99.8%
- Batch no.: 1852
- Date of production: 09 September 2020

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: 09 September 2023
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Species and strain: New Zealand White rabbit.
- Source: S&K-Lap Kft. (Address: 2173 Kartal, Császár út 135, Hungary).
- Hygienic level: Standard laboratory conditions.
- Number/sex of animals: 89 female animals, 22 inseminated female animals in the Control, Low and Mid dose groups; 23 inseminated female animals in the High dose group. The sufficient number of spare animals were ordered
- Number of groups: 4 groups (one control and 3 test item-treated groups)
- Age of animals: Young adult female rabbits, nulliparous and non-pregnant, approximately 4-5 months old at insemination
- Body weight: 3829 – 4936 g at start of the treatment, did not exceed ± 20% of the mean weight at onset of treatment
- Acclimatisation time: 5 days before insemination
- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbits in adjoining cages.
- Water: tap water
- Food: The animals received standard diet for rabbits (Batch number: 0008038307 / 0008211304 / 0008321719, Expiry date: 13 April 2022 / 22 June 2022 / 02 August 2022) produced by Cargill Takarmány Zrt. (Address: H-5300 Karcag, Madarasi road, Hungary), ad libitum. The Supplier provided analytical certificates for the batches used, which are included in the raw data and will be archived at the Test Facility. The food was routinely analysed, and it was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal Screening
The health status of the animals assigned to study was verified by the clinical Veterinarian. Females were nulliparous and non-pregnant at receipt.

Animal Identification
After insemination, each animal included in the study was identified by a number unique within the study, tattooed on the ear, written on the ear using a permanent marker, and cross referenced to the Animal Master File of the Test Facility. The animal numbers consisted of 4 digits, the first digit being the group number, the second digit was 5 for the females (or 6, in case of a replacement), and the last 2 digits were the animal number within the group, as indicated in the Experimental design section. The cages were marked with individual identity cards, with information at least about the study code, sex, dose group, cage number, animal number, date of insemination and Caesarean section/necropsy date. Cages were arranged to minimise any possible effects due to cage placement. The litters were identified at necropsy with litter numbers. The flasks used for fixation and all sheets used for recording contained these numbers only up to the end of foetal examinations (facilitating blind examination of foetuses). The foetuses were identified individually during the Caesarean section by means of waterproof plastics with the litter number and according to their implantation sites. The heads from the half of each litter were removed and identified by ear punching and fixed in Sannomiya mixture for Wilson-sections.

ENVIRONMENTAL CONDITIONS:
- Illumination: Artificial light, from 6 a.m. to 6 p.m.
- Temperature: 16.7 - 26.7°C
- Relative humidity: 36 - 70%
- Ventilation: 15-20 air exchanges/hour
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 formulated in the selected vehicle (1% (w/v) aqueous methyl cellulose solution, abbreviated as 1% MC) as a visibly stable suspension at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. As agreed with the Study Monitor, no correction for purity of the test item was applied during formulation. Formulations were prepared fresh every day prior to administration to animals. Based on the results of the analytical method validation study, test item formulations in the concentration range of 2-200 mg/mL were proven stable for at least 3 days when stored at room temperature. Formulations were prepared in glass containers*. The appropriate amount of test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and/or magnetic stirring) with the required amount of vehicle to reach homogeneity by visual observation. Mortar and pestle were applied as necessary to aid homogeneity and dissolution. The formulations were stirred with a magnetic stirrer from the time of preparation until completion of each treatment.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

APPARATUS
- HPLC system with UV detection: Knauer Azura HPLC-UV system (inner ID: KN2)
P6.1L HPLC pump, Serial No.: FBE161400001
AS 3950 Autosampler, Serial No.: AZA153700010
UV 2.1L UV Detector, Serial No.: FOA161300001
CT 2.1 Column thermostat, Serial No.: FCA160100006
- Knauer Azura HPLC-UV system (inner ID: KN3)
P6.1L HPLC pump, Serial No.: FBE161400003
AS 3950 Autosampler, Serial No.: AZA153700003
UV 2.1L UV Detector, Serial No.: FOA161300003
CT 2.1 Column thermostat, Serial No.: FCA160100016
- Balances: Sartorius BP221S, Sartorius BCE224I-1S
- Magnetic stirrer: FS RT Basic Strirrer 170, Heidolph MR 3001
- Ultrasonic bath (US): Elma, Elmasonic S300
- Density meter: Anton Paar DMA500
- Water purification system: Millipore, Direct-Q 8 UV
- Refrigerator: Zanussi (2–8°C)

HPLC-CONDITIONS:
- Column: ACE 5 C18 150 × 4.6 mm, 5 μm
- Column temperature: 25°C
- Mobile Phase (eluent):
“A”: Acetonitrile (ACN)
“B”: 30 mM NaH2PO4 buffer (adjusted to pH = 3, with H3PO4)
- Elution mode: Isocratic, A / B = 70 / 30
- Flow: 1.0 mL/min
- Detector (UV): 250 nm
- Injection volume: 30 μL
- Retention time: ~5.7 minutes
- Total run time: 8 minutes

Details on mating procedure:

Insemination Procedure
Synchronisation of the oestrus cycle of the does was performed 48 hours prior to insemination by administration of PMSG (gonadotropin) hormone (40 IU/female, sc). The female rabbits were artificially inseminated. The insemination procedure was performed in batches by the breeder at the Test Facility, with sperm originated from New Zealand White male rabbits from the same source as the females. Each female was inseminated with diluted sperm containing at least 2 million spermatozoa. At the same time as the artificial insemination was performed, ovulation was stimulated with 1 mL buserelin-based compound (0.2 mL/animal, i.m.). The day of insemination was regarded as Gestation Day 0 (GD0) for each female.

Selection, Assignment, Replacement, and Disposition of Animals
The inseminated, assumed pregnant female rabbits were randomly allocated to the dose groups on each insemination day in such a way that the group averages of the body weight were as similar as possible. This process was controlled by the software Provantis v9.3, to verify the homogeneity/variability between/within the groups to ensure that animals of all test groups are as nearly as practicable of a uniform weight. Due to gavage accident, a High dose female (#4501) was replaced by a spare animal from the same shipment of animals which were kept under the same environmental conditions. The replacement animal received an updated number (# 4601). Any unused, spare animals were moved back to the stock colony soon after the fourth week of treatment.

Duration of treatment / exposure:

The test item was administered daily to pregnant animals from implantation (Gestation Day (GD) 6) to one day prior to the day of scheduled kill (Gestation Day (GD) 27), which should be as close as
possible to the normal day of delivery without risking loss of data resulting from early delivery. The day of artificial insemination was regarded as Gestation Day (GD) 0 for each animal. Caesarean section and necropsy with macroscopic examination was performed on Gestation Day (GD) 28.

For detailed dosing and treatment information, see tables "Any other information on materials and methods incl. tables".

Frequency of treatment:

daily

Duration of test:

see above

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

22-23 mated 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 Rabbits by Oral Administration.

Maternal examinations:

MORTALITY:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Additional time points were included as necessary (documented in the raw data). Any animal which showed clinical signs considered severe were isolated and/or sacrificed to prevent suffering, cannibalism and/or autolysis, and was processed in the same way as the animals subjected to terminal necropsy.
BODY WEIGHTS:
Body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Body weight gain was calculated for each interval, including GD 0-6, GD 6-27 and GD 0-27.
FOOD CONSUMPTION:
Food was measured with precision of ±1 g on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Food consumption was calculated for each interval, including GD0-3, GD3-6, GD 0-6, GD6-9, GD9-12, GD12-15, GD15-18, GD18-21, GD21-24, GD24-27, GD 6-27 and GD 0-27.
CLINICAL OBSERVATIONS:
Detailed clinical observations were made on all animals at the start (GD 0), at the onset of treatment (GD 6), then at least weekly and on the day of necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated included, but not be limited to, 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) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Cage Side Observations
General (routine) clinical observations were made twice daily: in the morning (am) and in the afternoon (pm). Between GD6 - GD27, the observations were made in the morning before the treatment (am) and in the afternoon after the treatment (pm). Only one clinical observation was made in the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, no routine, but one detailed clinical observation was made on necropsy days. When signs of severe toxicity were noted, animals were observed more frequently.

Organ Weights
The uterus including the cervix was weighed (with a precision of ±1 g) and examined for early and late embryonic or foetal deaths and for the number of live foetuses. Organ weights were not measured in animals found dead during the study.

Tissue Collection and Preservation
The viscera of the does were examined macroscopically for any structural abnormalities or pathological changes for possible future evaluation. All the gross findings or the relevant tissues / organs were retained in 10% buffered formalin for possible future evaluation. Thereafter the carcass of the does were discarded without further examination.

Ovaries and uterine content:

The ovaries and uterus were removed, and the pregnancy status ascertained. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and held in front of a light source to clearly identify the implantation sites. Uteri that appear non-gravid were further examined to confirm the non-pregnant status (i.e. by Salewski staining or a suitable alternative method). The corrected body weight was calculated (body weight on GD28 minus weight of the gravid uterus). The ovaries and uterus were examined for early and late embryonic or foetal deaths and for the number of live foetuses.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percent of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

After ensuring humane death, each fully developed foetus was weighed individually (with a precision of ±0.1 g), and the litter mean was calculated. The crown-rump length of foetuses was measured (accuracy of ±1 mm) and the litter mean was calculated. All foetuses were externally and viscerally examined, and sex was determined (internal sex organs). All external abnormalities found during the foetal examinations were recorded; additional observations were recorded as noted during caesarean section and necropsy. Additionally, photographic records were made about foetal malformations.
The heads from approximately half of the animals from each litter were removed and processed for evaluation of soft tissue alterations (including eyes, brain, nasal passages and tongue), using fixation in Sannomiya mixture for Wilson-sections. After fixation the head was examined by Wilson's free-hand razor blade method. Then all foetuses were prepared for skeletal examination. The skeletons were examined after double staining with acetic Alcian blue + alkalic Alizarin red (cartilage and bone staining). All abnormalities (external, visceral and skeletal malformations and variations) found during the foetal examinations were recorded; photographic records were made additionally where the Study Director considered it appropriate.

Statistics:

At the Test Facility, the statistical evaluation of data (labelled as † in the lists in Section 14.1) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS 9.2 (built in Provantis System) and was documented in the raw data.
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then 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 Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of (co)variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences, identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was re

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Most of the observations were considered to be incidental; the lack or reduced amount of faeces was considered as treatment related and was expected based on the dose selection at 100 mg/kg/day being close to the MTD. However, in the absence of other adverse findings in the High dose group in this main study, it was not considered to be a clear sign of significant maternal toxicity

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of 10 animals were found dead or pre-terminally euthanized during the study, majority of the deaths/terminations were between GD23 and GD28 (late pregnancy).
One Mid dose (#3511) and one High dose (#4522) animals were found dead but based on the isolated occurrence these facts were considered as not showing a test item related effect on mortality. Another High dose animals (#4501) had a gavage accident and was pre-terminally euthanized, this fact was not related to the test item treatment.
In seven cases, animals aborted (1, 2, 2, 2 cases in the Control, Low, Mid and High dose groups, respectively) and were therefore pre-terminally euthanized according to the Study Plan. For those animals, the number of coloured (red) discharge on the vulva or on the floor of the cages was recorded. No test item effect was concluded.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A transient test item-related effect was observed in the body weight and body weight gain during the first few days of the treatment period at the High dose only.
At the start of the treatment (GD 6-9) there was a bodyweight loss in High dose animals, but animals showed a similar body weight gain profile to the Control animals after that and there were no statistically significant differences in the overall body weight or body weight gain of the test item treated and Control animals at the end of the treatment period. The transient effect was not sufficient to be clearly adverse but demonstrated that a higher High dose level would not be feasible. The body weight gain of the High dose group by the end of the study was approximately 8% lower than Control, but no statistical significance was reached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A transient test item related effect on the food consumption was observed in the first few days of the treatment period at the High dose only.
There was a statistically significantly decreased food consumption (by 16.0%, p>0.05) observed at the start of the treatment (GD 6-9) in the High dose group when compared to Control. But this difference was later reduced (to 8.6%), and there was no statistical significance when the food consumption value calculated for the entire treatment period was compared between High dose and Control group.

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, non-treatment-related

Description (incidence and severity):

Macroscopic findings in the evaluated pregnant animals were generally observed with low incidence without suggestion of any test item-related effects.
All changes were considered to be incidental or a common background. In case of non-pregnant animals, no abnormalities were noted. In case of early deaths/euthanasia, no treatment related changes were observed.

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:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 89 females (22 in the Control, Low and Mid dose group, and 23 in the High dose group, respectively) were inseminated in the study. The number of confirmed pregnant, evaluated does was 19, 16, 16 and 16 in the Control, the Low, Mid and High dose group, respectively. There were a total of 12 non-pregnant females in the study, but no dose related trend was noted, so this fact was considered as animal variability. The total number of evaluated females was 19, 16, 16 and 16 in the Control, Low, Mid and High dose groups, which is sufficient for the requirements of the OECD No. 414 guideline as a valid study.
There were some animals with lower number (≤ 5) of implantation sites (2, 1, 3, 4 in the Control, Low, Mid and High dose groups), it is considered there was no relationship with treatment. These litters were included in the data evaluations.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

One external malformation (Craniochisis) was observed in the High dose group (1/141 foetuses; #4504/4), which corresponded to multiple malformed skull recorded at skeletal evaluation. Although the current Historical Control database does not contain this specific observation, it looks an isolated occurrence that was considered incidental, ascribed to biological variability. No external malformation was recorded in any other groups.No external variation was observed during external valuation.

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:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were recorded in all groups. Majority of the observed malformations (multiple malformed skull, fused sternum / sternebra, branched rib, vertebrae / hemivertebrae, split vertebrae) were comparable with study control data and/or have isolated occurrence, thus these findings were considered incidental, ascribed to individual variability and not related to treatment.
In case of fused rib malformation, 2 cases were recorded in 2 Low dose litters. This observation was not included in the current study control group but there was a case for this finding in the historical control database. Based on the low number and the lack of dose response and as no occurrence was noted in the two higher concentration dose groups, it was considered as biological variability and not related to the treatment.
All the skeletal variations (misaligned sternum, unossified sternum / sternebra (2), misshapen sternum / sternebra, interrupted ribs, dumbbell ossification of a vertebrae) corresponded to the current historical control or study control data or were considered to be incidental findings without dose response.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformation (a cyst in oviduct) was recorded in the Control (1/193 foetus), Mid dose (1/156 foetus) and High dose (2/141 foetuses in 1 litter) groups. All the observed findings were comparable with study control data and have isolated occurrence, thus these findings were considered incidental, ascribed to individual variability and not related to treatment.
Visceral variations were detected in all groups. All the observed findings (thymic cord, discoloured liver and supernumerary renal vein) were comparable with historical control or current study control data, or have isolated occurrence that was considered incidental, ascribed to individual variability and not related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the study conditions, the following no-observed-adverse-effect (NOAEL) levels were derived: (a) NOAEL maternal toxicity:100 mg/kg bw/day. Based on the lack of adverse findings in the High dose group for the overall study duration (b) NOAEL embryotoxicity: 100 mg/kg bw/day. Based on the lack of intrauterine effect in the High dose group (c) NOAEL foetotoxicity:100 mg/kg bw/day. Based on the lack of intrauterine effect in the High dose group (d) NOAEL teratogenicity: 100 mg/kg bw/day. Based on lack of treatment related abnormalities in the High dose group.

Executive summary:

This developmental toxicity study (OECD No. 414) was performed to assess the effects of the test item on the embryonic and foetal development (including the organogenesis period) of New Zealand White rabbits in their first pregnancy. The does (one control and three test item treated groups) were treated daily by oral (gavage) administration, from Gestation Day (GD) 6 up to and including Gestation Day (GD) 27, where the day of insemination was counted as Day 0 of pregnancy (GD 0). Control does were treated with the vehicle (1% (w/v) aqueous methyl cellulose solution) only. Caesarean sections, necropsy of does and examination of uterine contents were performed on GD 28. The dose levels were set by the Sponsor / Study Monitor based on the available data and information from previous experimental work, including the results of a Dose Range Finding (DRF) toxicity study in pregnant New Zealand White rabbits performed at the Test Facility. Based on the results from the DRF study, doses of 100, 30 and 10 mg/kg bw/day were selected for the main study (designated High, Mid and Low dose, respectively). The aim was to use the highest dose of 100 mg/kg bw/day to induce toxic effects, but ideally no death or suffering, and to obtain a NOAEL at the lowest dose level. Test item formulations were analysed for concentration and homogeneity twice during the treatment period using a validated HPLC-UV (High Performance Liquid Chromatography method with UV detection) method. Simultaneously, vehicle control formulations were also analysed for the test item concentration. Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically. The number of confirmed pregnant, evaluated does was 19, 16, 16, 16 in the Control, Low, Mid and High dose groups, respectively. All test item formulations were within the range of 94-105% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes. A total of 10 animal were found dead or pre-terminally euthanized in the study generally as a result of gavage accident or early abortion; there were no mortalities ascribed to test item effects. There was a transient initial body weight loss with low food consumption in the first few days of the treatment period at the High dose only, but no effect on the overall body weight gain of rabbits. The only clinical sign associated with treatment was reduced faeces in this High dose group. The weight and clinical sign effects confirm the selected High dose was close to the MTD. There were no treatment related findings at necropsy. There were no statistically significant differences in the intrauterine parameters in the test item treated animals when compared to the controls. There was no significant difference in number of foetuses, or in the sex distribution of foetuses between the control and treatment groups. The number of foetuses with retarded body weight per. There was no statistical difference in the number of runts between the control and treated groups. There were no test item related external, visceral or skeletal malformations in the study. In case of one High dose foetus a malformation of the skull was recorded (at both external and skeletal evaluation). Based on the single occurrence it was considered as isolated and incidental, ascribed to individual variability, not being related to the test item treatment. All other findings correspond or occur in the study control or historical control data or considered to be isolated and incidental, ascribed to individual variability. At least 16 litters were evaluated in each treatment group, hence the study was considered to be valid. In conclusion, LZ 596, when administered daily by oral gavage to pregnant New Zealand White rabbits from gestation days GD6 to GD27 at up to 100 mg/kg bw/day resulted in transient effects on body weight and food intake, but no clearly adverse test item related mortality, clinical signs, or overall body weight or food consumption effects. No on the embryotoxic or foetotoxic effects were observed. There were no test item related external, visceral or skeletal malformations. The following no-observed-adverse-effect (NOAEL) levels were derived: (a) NOAEL maternal toxicity:100 mg/kg bw/day. Based on the lack of adverse findings in the High dose group for the overall study duration (b) NOAEL embryotoxicity: 100 mg/kg bw/day. Based on the lack of intrauterine effect in the High dose group (c) NOAEL foetotoxicity:100 mg/kg bw/day. Based on the lack of intrauterine effect in the High dose group (d) NOAEL teratogenicity: 100 mg/kg bw/day. Based on lack of treatment related abnormalities in the High dose group (Hargitai, 2023).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June - November 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

As a non-GLP preliminary study, this study has been performed in accordance with the study plan, and Standard Operating Procedures.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

As a preliminary study, this study did not follow strictly a specific guideline, but is based on the principles of OECD Guideline no. 414

GLP compliance:

no

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
- Purity: 99.8%
- Batch no.: 1852
- Date of production: 09 September 2020

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: 09 September 2023
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Species / Strain: New Zealand White rabbit
- Source: S&K-Lap Kft., 2173 Kartal, Császár út 135, Hungary
- Age of animals at arrival: females: Young adult female rabbits, nulliparous and non-pregnant, approximately 4 - 5 months old at mating
- Body weights at arrival: females: 4063-4895 g at start of the treatment; was not exceed ± 20% of the mean weight at insemination
- Number of animals: two times 24 female animals: 6 inseminated female animals/group, 8 groups (one control and 3 test item-treated groups twice)
- Acclimatization time: environmental acclimation period for the study was 5 days for both sets of animals.
- Housing: rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages are of an open wire structure and cages were placed together to allow some social interaction
- Cage type: Type II polypropylene/polycarbonate with stainless steel covers equipped by self-feeding baskets
- Water. tap water
- Food: standard diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út, Hungary, ad libitum (Batch numbers: 0007413528 / 0007514001 / 0007591782 / 0007757248, Expiry dates: 11 August 2021 / 22 September 2021 / 23 October 2021 / December 2021).

ENVIRONMENTAL CONDITIONS:
- Illumination: Artificial light, from 6 a.m. to 6 p.m.
- Temperature: 16.7-22.6°C
- Relative humidity: 42-70%
- Ventilation: 15-20 air exchanges/hour

Route of administration:

oral: gavage

Vehicle:

other: 1% Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was freshly formulated in the selected vehicle (1% MC) prior to administration, as a visibly stable suspension at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of Charles River Laboratories Hungary Kft. The formulation was stirred by magnetic stirrer until completion of treatment. As agreed with the Study Monitor, no correction for purity of the test item was applied during formulation. Formulations were prepared freshly every day. The calculated amount test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual

VEHICLE:
Justification for use and choice of vehicle (if other than water): The test item is not soluble in water therefore 1% Methylcellulose was used as the vehicle. The test item was suitable for oral administration when suspended in 1% Methylcellulose.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

APPARATUS
- HPLC system with UV detection: Knauer Azura HPLC-UV system (inner ID: KN2)
P6.1L HPLC pump, Serial No.: FBE161400001
AS 3950 Autosampler, Serial No.: AZA153700010
UV 2.1L UV Detector, Serial No.: FOA161300001
CT 2.1 Column thermostat, Serial No.: FCA160100006
- Knauer Azura HPLC-UV system (inner ID: KN3)
P6.1L HPLC pump, Serial No.: FBE161400003
AS 3950 Autosampler, Serial No.: AZA153700003
UV 2.1L UV Detector, Serial No.: FOA161300003
CT 2.1 Column thermostat, Serial No.: FCA160100016
- Balances: Sartorius BP221S, Sartorius BCE224I-1S
- Magnetic stirrer: FS RT Basic Strirrer 170, Heidolph MR 3001
- Ultrasonic bath (US): Elma, Elmasonic S300
- Density meter: Anton Paar DMA500
- Water purification system: Millipore, Direct-Q 8 UV
- Refrigerator: Zanussi (2–8°C)

HPLC-CONDITIONS:
- Column: ACE 5 C18 150 × 4.6 mm, 5 μm
- Column temperature: 25°C
- Mobile Phase (eluent):
“A”: Acetonitrile (ACN)
“B”: 30 mM NaH2PO4 buffer (adjusted to pH = 3, with H3PO4)
- Elution mode: Isocratic, A / B = 70 / 30
- Flow: 1.0 mL/min
- Detector (UV): 250 nm
- Injection volume: 30 μL
- Retention time: ~5.7 minutes
- Total run time: 8 minutes

Details on mating procedure:

INSEMINATION PROCEDURE:
Synchronisation of the oestrus cycle of the does was performed 48 hours prior to insemination by administration of PMSG (gonadotropin) hormone (40 IU/female, sc). The female rabbits were artificially inseminated. The insemination procedure was performed in batches by the breeder at Charles River Laboratories Hungary Kft., with sperm originated from New Zealand White male rabbits from the same source as the females. Each female was inseminated with diluted sperm containing at least 2 million spermatozoa. At the same time as the artificial insemination was performed, ovulation was stimulated with 1 ml buserelin-based compound (0.2 mL/animal, i.m.). The day of insemination was regarded as Gestation Day 0 (GD0). The inseminated, assumed pregnant female rabbits were allocated to the dose groups on each insemination day in such a way that the group averages of the body weight were as similar as possible; Provantis software was used in order to verify homogeneity/variation among/within groups.

Duration of treatment / exposure:

The test item was administered daily to pregnant animals from implantation (Gestation Day (GD) 6) to one day prior to the day of scheduled kill (Gestation Day (GD) 27), which should be as close as possible to the normal day of delivery without risking loss of data resulting from early delivery. The day of artificial insemination was regarded as Gestation Day 0 (GD0). Caesarean section and necropsy with macroscopic examination were performed on Gestation Day (GD) 28.
The first group of six females was treated at 1000 mg/kg bw and 3 females of the Control group were treated with the vehicle followed by 16 days-long observation period. The dose levels of another two groups were set based on the symptoms observed. The dose levels were 500 and 250 mg/kg bw/day. The control group was treated with the vehicle only (1% Methylcellulose). Because there were serious test item related adverse effects in each dose groups, lower dose levels 0f 25, 50 and 100 mg/kg bw/day were initiated. The first group of six female was treated at 25 mg/kg bw and females of the Control group were treated with the vehicle followed by 19 days-long observation period. The dose levels of another two groups were setThe control group was treated with the vehicle only (1% MC). based on the symptoms observed. The dose levels were 50 and 100 mg/kg bw/day.

For detailed dosing and treatment information, see tables "Any other information on materials and methods incl. tables".

Frequency of treatment:

daily

Duration of test:

see above

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

additional dose group due to serious effects in first dosing with 250, 500 and 1000 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

additional dose group due to serious effects in first dosing with 250, 500 and 1000 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

additional dose group due to serious effects in first dosing with 250, 500 and 1000 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 sperm-positive females/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected with agreement of the Sponsor.

Maternal examinations:

MORTALITY:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Additional time points were included (documented in the raw data and reported). Animal which showed clinical signs considered severe was sacrificed to prevent suffering and was processed in the same way as the animals subjected to terminal necropsy.
BODY WEIGHT:
Body weight of each animal was recorded with precision of 1 g at least on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 or at death.
FOOD CONSUMPTION:
Food consumption was measured with precision of 1 g at least on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Food consumption was calculated for each interval, including GD0-3, GD3-6, GD6-9, GD9-12, GD12-15, GD15-18, GD18-21, GD21-24 and GD24-27.
CLINICAL OBSERVATIONS:
Detailed clinical observations were made on all animals at the day of insemination (GD 0) then weekly and at necropsy. Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated included, but not be limited to, 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) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Ovaries and uterine content:

The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. The corrected body weight was calculated (body weight on GD28 minus weight of the gravid uterus). Organ weights was not measured in animals found dead during the study.

Fetal examinations:

All animals (including those that died during the test or were removed from the study for animal welfare reasons) were subjected to a necropsy and a macroscopic examination. Before expected delivery, on GD 28, Caesarean section was performed on each, survived,
treated doe. Euthanasia of the does was performed by intramuscular injections of pentobarbital sodium. Females showing signs of abortion or premature delivery prior to scheduled necropsy were euthanized and subjected to a thorough macroscopic examination.
The number of corpora lutea in each ovary and of implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted; the number and percentage of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and held in front of a light source to clearly identify the implantation sites. Uteri that appear non-gravid were further examined to confirm the non-pregnant status. The live foetuses were identified, externally examined and weighed individually (accuracy, 0.1 g), and the litter mean was calculated. The crown-rump length of foetuses was measured (accuracy, 1 mm) and the litter mean was calculated. All external abnormalities found during the foetal examinations were recorded. Thereafter the carcass of the does and foetuses were discarded without further examination.

Statistics:

At the Test Facility, the statistical evaluation of data was performed with the program package SAS 9.3 (built in Provantis System) to be documented in the raw data. In case of the SAS 9.3 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
The limit for growth retarded foetuses was calculated from the average body weight of the vehicle control foetuses. A foetus was considered as growth retarded if the deviation from the mean control values was greater than minus two-fold standard deviation of all control foetuses. Non-pregnant females and females showing signs of abnormal pathology and/or misdosing were excluded from statistical analysis; in-life individual data is reported as applicable.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the preterminal or found dead animals the following symptoms were observed:
Alopecia (abdominal area) was observed for one 500 mg/kg bw/day dose female from Day 17
to Day 27. The longevity of the symptom was 11 days. Decreased activity was observed for one 100 mg/kg bw/day dose female from Day 18 to Day 20, for three 250 mg/kg bw/day dose females from Day 11 to Day 23, for three 500 mg/kg bw/day dose females from Day 21 to Day 24 and for five 1000 mg/kg bw/day dose females from Day 17 to Day 27. The longevity of the symptom was 11 days. Few faeces were observed for one 100 mg/kg bw/day dose female on Day 16, for one 250 mg/kg bw/day dose female from Day 13 to Day 19, for six 500 mg/kg bw/day dose females
from Day 14 to Day 27 and for five 1000 mg/kg bw/day dose females from Day 14 to Day 20.
The longevity of the symptom was 11 days. No faeces were observed for one 100 mg/kg
bw/day dose females from Day 13 to Day 20, for four 1000 mg/kg bw/day dose females from
Day 18 to Day 21. The longevity of the symptom was 3 days. Laboured respiration was observed for two 250 mg/kg bw/day dose females on Day 11 and on Day 23.Prostration was observed for one 250 mg/kg bw/day dose female on Day 23, for two 500 mg/kg bw/day dose females on Day 21 and Day 22 and for one 1000 mg/kg bw/day dose female on Day 20. Decreased respiratory rate was observed for two 500 mg/kg bw/day dose females on Day 21 and Day 22, respectively and for two 1000 mg/kg bw/day dose females on Day 15 and Day 20, respectively.
Wasted was observed for one 250 mg/kg bw/day dose female from Day 12 to Day 19 and for
one 1000 mg/kg bw/day dose female on Day 20. The longevity of the symptom was 8 days.
Lethargy was observed for one 1000 mg/kg bw/day dose female on Day 20. Mucoid colour (nose / snout) was observed for one 1000 mg/kg bw/day dose female on Day 20. Cold to touch (ears and paws) was observed for one 1000 mg/kg bw/day dose female from Day 18 to Day 19.
Cachexia was observed for one 1000 mg/kg bw/day dose female on Day 15. Coloured discharge (nose / snout) was observed for 1000 mg/kg bw/day dose female on Day 15. Red liquid (oral cavity) was observed for 1000 mg/kg bw/day dose female on Day 12.

In the surviving animals, the following symptoms were observed:
No clinical symptoms were recorded in the Control group. Few faeces were observed for one 500 mg/kg bw/day dose female from Day 14 to end of study, two 100 mg/kg bw/day dose females on Day 16 and Day 23 and one 50 mg/kg bw/day dose female on Day 21. No faeces were observed for one 100 mg/kg bw/day dose female from Day 13 to Day 15. The longevity of the symptom was up to 15 days; this is a common finding in rabbits associated with treatment with test items.
Alopecia was observed for one 500 mg/kg bw/day dose female from Day 17 to end of treatment, one 25 mg/kg bw/day dose female on Day 28 and for one 50 mg/kg bw/day dose female from Day 21 to end of treatment. The longevity of the symptom was 11 days; this was not attributed to treatment.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

oral-gavage treatment

Mortality:

mortality observed, treatment-related

Description (incidence):

One 100 mg/kg bw/day dose female (#8505), one 250 mg/kg bw/day dose female (#2503) and one 500 mg/kg bw/day dose female (#3506) found dead on GD14, GD12 and GD 21, respectively.
Three 250 mg/kg bw/day dose animals (#2501, #2502, #2504), three 500 mg/kg bw/day dose animals (#3501, #3504, #3505) and one 1000 mg/kg bw/day dose animal (#4504) were preterminally euthanized following abortion. One 100 mg/kg bw/day dose animal (#8502) and four 1000 mg/kg bw/day dose animals (#4502, #4503, #4505, #4506) were preterminally euthanized following serious body weight decrease on GD15-GD21.
The large majority, or possibly all the early deaths/terminations were considered to be treatment related toxic effects of treatment. At 1000 and 500 mg/kg bw/day the early deaths or terminations were all considered to be treatment related and showed a dose response. At 250 mg/kg bw/day the animal found dead on day 12 (the first in the study) was considered not to be treatment related although the 2 abortions (GD 19 and GD 24) were in line with higher dose effects as was the preterminal euthanasia (GD 23). The single rabbit found dead on GD14 at 100 mg/kg bw/day occurred earlier than in animals at higher dose levels, which is not totally consistent with an expected dose response relationship, hence the significance of this death is equivocal. The single preterminal euthanasia animal at 100 mg/kg bw/day (GD21) did have clinical signs that were of similar timing and nature as seen at higher dose levels before termination, so was considered as treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals in the Control group gain weight during the study. The animals in the 1000, 500 and 250 mg/kg w/day groups gained weight before treatment from Day 0 to Day 6, but from Day 9, they lost weight (by-0.8%, by -2.7% and by -4.5%, respectively) compared to the Control. This weight loss was increased to Day 21 (by -3.4%, by -16.8% and by -19.1%, respectively). At Day 21 all the does died in the 1000 mg/kg bw/day dose group, and in the 250 mg/kg w/day and 500 mg/kg w/day dose group there were two at Day 21 and at Day 24 only one animal remained in the group.
Statistically significant body weight loss was observed (p<0.01) for the 500 mg/kg bw/day and
1000 mg/kg bw/day dose group between Day 6-9 period (by -172.5%, by -321.8% and by -389.3%, respectively). The reduced weight gain was even more intense reaching Day 18-21 period, where the decrease was -282.8%, -1041.3% and -1180.5%, respectively. Statistically significantly increased body weight (p<0.01) and body weight gain (p<0.05) was observed in the 25 mg/kg bw/day group during the whole study. Slowly increased body weight was observed in the 25, 50 and 100 mg/kg bw/day dose groups. From Day 9 the body weight gain in the 50 and 100 mg/kg bw/day groups was decreased compared to the control and the 25 mg/kg bw/day dose groups.
It is considered that there were no adverse effects on body weight at 25 and 50 mg/kg bw/day.
At 100 mg/kg bw/day there was a transient adverse effect on body weight from start of treatment to ~Day 15. At 250 to 1000 mg/kg bw/day there was a clearly adverse effect on body weight with a dose response.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced daily food consumption was observed during the study in the 100 mg/kg bw/day (transiently from Day6 to ~Day 15) and at 250 mg/kg bw/day the food intake was consistently low during the treatment period (~40% below control Days 24-28). The 500 and 1000 mg/kg
bw/day dose groups had statistically significant lower food consumed, at less than 50% of control from ~Day 9 at 1000 and ~Day 12 at 500 mg/kg bw/day.
Statistically significantly decreased food consumption (p<0.05) was observed between Day 6-9 period in the 500 mg/kg bw/day (by -42.0%) and 1000 mg/kg bw/day (by -47.7%) dose groups. Decreased food consumption was observed in the 250 mg/kg bw/day dose group as well without statistical significance (by -22.5%). This tendency was more severe in case of the 500 mg/kg bw/day and 1000 mg/kg bw/day dose groups where the food consumption was decreased by -78.8% and by -98.2% at Day 18-21 period of the study. For the 250 mg/kg bw/day dose group, the difference was smaller at Day 18-21 period (decreased by -18.5%). Examining the individual animal data for daily food intake, controls generally ate 100-250g each day, with occasional low values of >50g. In all groups, even at 1000 mg/kg bw/day, nonpregnant animals generally had intakes similar to controls. Pregnant does at 1000 and 500 mg/kg bw/day all had lower food intake, generally after ~ GD12. At 250 mg/kg bw/day, where 4 pregnant animals survived beyond GD14, 3/4 of them had significant periods of food intake below the control range (a single pregnant animal appeared normal). At 100 mg/kg bw/day, the single animal that was found dead early and the animal euthanised early both had unusually low food intake from ~ GD9, the other 3/5 had normal food intake throughout the treatment period.
This effect was considered to be a clearly test item related adverse effect at 250 to 1000 mg/kg bw/day, with some effect in a minority of does at 100 mg/kg bw/day.

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

FOUND DEAD ANIMALS
There was one doe in the 100 mg/kg bw/day group (#8505), one doe in the 250 mg/kg bw/day dose group (#2503) and one doe in the 500 mg/kg bw/day dose group (#3506) found dead on GD14, GD12 and GD21, respectively. The deaths are considered to be treatment related except in case of #2503 doe where the death was probably not treatment related. Gavage accident could happen however at necropsy the cause of the death was undetermined.
PRETERMINALLY EUTHANIZED ANIMALS
Three 250 mg/kg bw/day dose females (#2501, #2502, #2504), three 500 mg/kg bw/day dose females (#3501, #3504, #3505) and one 1000 mg/kg bw/day dose female (#4504) were preterminally euthanized following abortion. Although spontaneous abortions do occasionally occur in rabbits, these all appear to be treatment related. One 100 mg/kg bw/day dose female #8502) and four 1000 mg/kg bw/day dose females (#4502, #4503, #4505, #4506) were preterminally euthanized following serious body weight decrease. The serious body weight loss in
case of the 100 mg/kg bw/day dose female was equivocal, it can not be considered as clearly test item related effect.
SURVIVING ANIMALS
Twelve Control, five 25 mg/kg bw/day dose females, six 50 mg/kg bw/day dose females, three 100 mg/kg bw/day dose females, one 250 mg/kg bw/day dose female, one 500 mg/kg bw/day dose female dose female were pregnant and survived the treatment. The effects at 1000, 500 and 250 mg/kg/day were clearly well above the MTD; at 100 mg/kg/day it was equivocal whether it was test item related or not.

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:

effects observed, treatment-related

Description (incidence and severity):

Forty-eight females (6 for each group) were artificially inseminated in the study. One animal
in each of the six test item-treated groups was non-pregnant except in the control and 50 mg/kg
bw/day groups where all 6 were pregnant. The number of surviving confirmed pregnant does
was 12/12, 5/5, 6/6, 3/5, 1/5, 1/5 and 0/5 in the Control, 25 mg/kg bw/day, 50 mg/kg bw/day,
100 mg/kg bw/day, 250 mg/kg bw/day, 500 mg/kg bw/day and 1000 mg/kg bw/day dose
groups, respectively.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

One 100 mg/kg bw/day dose female, three 250 mg/kg bw/day dose females, three 500 mg/kg bw/day dose females and four 1000 mg/kg bw/day dose females were preterminally euthanized during the study. The condition of the foetuses was corresponded to the normal foetuses at that age. The number of viable foetuses was decreased in the 500 and 250 mg/kg bw/day dose groups compared to the Control. The mean foetal bodyweight (male and female) in the surviving animals was similar to the Control group. However, when analysed the mean foetal weight per litter (the more relevant measure) there was lower weight observed in the 500 mg/kg bw/day dose group; the 250 mg/kg bw/day dose groups were comparable to the Control. There was no effect on the mean foetal body weight per litter in the 100, 50 and 25 mg/kg bw/day. The slightly reduced foetal body weight in the 500 mg/kg bw/day dose group was considered to be a secondary effect due to the observed maternal toxicity (lower food consumption and/or lower body weight parameters).

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:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Key result

Abnormalities:

no effects observed

Localisation:

other: see below

Key result

Developmental effects observed:

no

Conclusions:

Based on the above-mentioned results dose level of 50-100 mg/kg bw/day could be the High dose in case of the upcoming OECD 414 study.

Executive summary:

The test item, when administered daily by oral gavage to pregnant New Zealand rabbits from gestation days GD6 to GD27 up to 1000 mg/kg bw/day, was associated with the following findings: Five animals out of six in the 1000 mg/kg bw/day dose group were preterminally euthanized due to serious body weight decrease or abortion. Most terminations were from GD19 to GD21 (mid-late pregnancy). One animal was non-pregnant and showed no adverse findings. Statistically significantly decreased body weight, body weight gain and food consumption were observed. Based on the data this dose level is lethal for  pregnant rabbits. One out of 6 animal in the 500 mg/kg bw/day dose group was found dead and three animals were preterminally euthanized due to serious body weight decrease and / or abortion. One animal was non-pregnant and showed almost no adverse findings. Statistically significantly decreased body weight, body weight gain and food consumption were observed. With only one surviving litter test item related effects on the foetuses were not possible. This dose level is clearly above the MTD. One out of 6 animal in the 250 mg/kg bw/day dose group was found dead and three animals were preterminally euthanized due to serious body weight decrease and / or abortion. One animal was non-pregnant and showed reduced activity but no other clinical findings. With only one surviving litter it is not possible to conclude if there were effects on numbers of foetuses or if development was affected. This dose level is clearly above the MTD. One out of six female in the 100 mg/kg bw/day dose group found dead and one female was preterminally euthanized due to serious body weight decrease. One animal was nonpregnant. Decreased body weight gain and food consumption were observed in the 100 mg/kg bw/day dose group without statistical significance, although the dead and euthanised does both had unusually low food intake (all others were normal). There was no test item related effect observed on the foetal parameters in this group. There was no preterminal death in the 50 and 25 mg/kg bw/day dose groups. One animal was non-pregnant in the 25 mg/kg bw/day dose group. There was no test item related effect observed on the foetal parameters in the 25 and 50 mg/kg bw/day dose groups. Based on the above-mentioned results dose level of 50-100 mg/kg bw/day could be the High dose in case of the upcoming OECD 414 study.

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.