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

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

237-185-4

EC Name:

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

Cas Number:

13680-35-8

Molecular formula:

C21H30N2

IUPAC Name:

4,4'-methylenebis(2,6-diethylaniline)

Test material form:

solid: crystalline

Details on test material:

- Physical state: see above
- Appearance: see above

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.

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

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).