Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a study performed according to OECD 421 guideline, the substance was administered daily by oral administration (gavage) to male and female rats from before mating, through mating and gestation until Day 6 post-partum at dose levels of 100, 300 and 1000 mg/kg bw/day. The NOAEL for the substance was 1000 mg/kg/day (the limit dose) for reproductive performance and offspring growth and survival.


 

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 March 2010 to 4 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1994
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: approximately 62 to 69 days of age
- Weight at study initiation: Males: 310-365 g; Females: 206-257 g
- Fasting period before study: no
- Housing: See table 7.8.1/1
- Diet: standard rodent diet (SDS VRF1 Certified) ad libitum
- Water: potable water taken from the public supply ad libitum
- Acclimation period: 6 days before dosing started

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 28 April 2010 To: 23 June 2010
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% w/v aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation (frequency): weekly
- Storage conditions : stored refrigerated (approximately at 2-8°C)

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance is poorly soluble in water
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After unsuccessful pairing replacement of first male by another male with proven fertility: not needed in this study
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): see table 7.8.1/1
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability of the dosage forms: homogeneity of the test substance in 1% methylcellulose formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 100 mg/mL. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 6% of the initial time zero value and the coefficient of variation was less than 5%.

Test item concentrations: the test item concentrations in the administered dosage forms analyzed in the first and last weeks of treatment remained within an acceptable range of +10% to -15% when compared to the nominal values.
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- until sacrifice in week 6,

In the females:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- during pregnancy,
- during lactation until day 6 post-partum inclusive

Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: approximately 10-12 weeks
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels used in this study (0, 100, 300 and 1000 mg/kg/day) were selected on the basis of the results of a 7-day repeated- dose preliminary study in the CD rat. In that study, dose levels of 100, 300 and 1000 mg/kg/day were used. There were no test material-related mortalities and no signs observed, bodyweight performance and food intake were unaffected by administration of the test material and there was no clear effect on organ weights and no macroscopic abnormalities detected. The high dose of 1000 mg/kg/day is considered in most circumstances to be the limit dose for an OECD 421 study and this was selected as no toxicity had been detected in the preliminary study. The low dose of 100 mg/kg/day was chosen anticipated No Observed Adverse Effect Level and the intermediate dose of 300 mg/kg/day was set at a logarithmic interval between the low and high doses.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to dosing.
A detailed physical examination was performed on the first day of dosing and weekly thereafter for F0 males until termination, and weekly for F0 females prior to pairing and on Days 0, 7, 14 and 20 after mating and Days 1 and 7 of lactation to monitor general health.

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: daily in relation to dose administration during the first week of dosing, twice weekly during the second to fourth weeks of dosing and weekly from Week 5 until termination for males. After mating, these observations were conducted for females on Days 0, 7, 14 and 20 after mating and on Day 4 lactation.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males were weighed before dosing commenced (Day -1), on the first day of dosing (Week 0) and weekly throughout dosing until termination. F0 females were weighed before dosing commenced (Day -1), on the first day of dosing (Week 0) and weekly until mating was detected. The females were weighed subsequently on Days 0, 6, 13 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the start of dosing until the animals were paired for mating. From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase, for each cage.
For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-5, 6-12 and 13-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/animal/day) was calculated for each animal.

WATER CONSUMPTION AND COMPOUND INTAKE: No

PARTURITION OBSERVATIONS AND GESTATION LENGTH
From Day 20 after mating, F0 females were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored.
The duration of gestation was calculated as the time elapsing between the detection of mating and commencement of parturition.
Oestrous cyclicity (parental animals):
For 15 days before pairing (including the day of pairing), daily vaginal smears were taken from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
Not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Number and sex of pups, stillbirths, live births
- Clinical signs: Daily records were maintained for evidence of ill health or reaction to parental dosing; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and 7 of age.
- Bodyweight: Individual offspring bodyweights were recorded on Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS:
Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring killed or dying before Day 7 of age were examined. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were terminated after Day 7 of lactation of the females.
- Maternal animals: All surviving animals were killed on Day 7 of lactation, after confirmation that a second mating was not required.

GROSS NECROPSY
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.
For F0 females, the numbers of corpora lutea on each ovary and the numbers of implantation sites in each uterine horn were counted.

ORGAN WEIGHTS
Epididymides (Left and Right)
Ovaries (Left and Right)
Testes (Left and Right)

HISTOPATHOLOGY
For Groups 1 (Control) and 4 (1000 mg/kg/day) the epididymides, ovaries, and testes were examined for parental animals sacrificed on completion of the scheduled dosing period and for the parental animals killed during the study.
Tissues reported at macroscopic examination as being grossly abnormal were examined for all parental animals in line with current practice.
Special emphasis was placed on the stage of spermatogenesis and interstitial testicular cell structure.

Postmortem examinations (offspring):
GROSS NECROPSY
All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including pre-coital interval and mating performance and fertility, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. For adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population
Reproductive indices:
Percentage mating = Number animals mating / animals paired x 100
Conception rate (%) = Number animals achieving pregnancy / animals mated x 100
Fertility index (%) = Number animals achieving pregnancy / animals pairing x 100
Gestation index (%) = Number of live litters born / Number pregnant x 100
Offspring viability indices:
Post-implantation survival index (%) = Total number offspring born / Total number uterine implantation sites x 100
Live birth index (%) = Number live offspring on Day 1 after littering / Total number offspring born x 100
Viability index (%) = Number live offspring on Day 7 after littering /Number live offspring on Day 1 after littering x 100
Percentage males = Number males in litter / Total number offsprings in litter x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of adverse reaction to administration of the test substance during the pre-pairing,
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female, assigned to the group receiving 100 mg/kg/day was killed for welfare reasons on Day 4 of dosing due to trauma to the eye, which may have arisen from interaction with cage mates or the cage environment. The finding was considered incidental to administration of the test substance and the cause of the trauma is unclear.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Administration of the test substance had no clear effect on bodyweight during the pre-pairing, gestation and lactation phases.
Bodyweight gain amongst males receiving 300 mg/kg/day and females receiving 100 and 300 mg/kg/day was slightly high during the pre-pairing phase when compared with Controls. However, in the absence of a clear dose response and a consistent effect between the sexes the significance of this is unclear.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no clear effect of administration of the test substance on mean food consumption during the pre-pairing period or for females during the gestation or lactation phases.
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
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histopathological findings considered to be related to administration of the test substance.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Oestrous cycle activity, pre-coital interval, mating performance and fertility were considered to be unaffected by administration of the test substance. A number of animals across all three dose groups receiving the test material showed slightly extended cycles (classified as irregular cycles) or prolonged acyclic periods (presumed pseudopregnancy) from early in the dosing period but there was no indication that this was associated with dosing and the incidences were considered fortuitous.
There was no evidence of dystocia, and all females successfully gave birth to live young. There was no clear effect of administration of the test substance on gestation length, all being within the expected range of 22 to 23 days. The gestation index for all groups was 100%.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test material-related clinical signs recorded amongst the offspring.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Parental dosing with the test substance had no effect on mean litter size or offspring survival to Day 7 of age at any dose level investigated.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean offspring birth weights were unaffected by parental dosing with the test substance. At 1000 mg/kg/day, bodyweight gain to Day 7 was marginally lower than Controls for both males and females (92% and 88% of control respectively). Much of this difference could be attributed to the lower bodyweight gain of offspring in the larger litters at the high dose level and was considered to be unrelated to dosage withthe test substance.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Of the few offspring that died during the early post-natal period, most showed an absence of milk in the stomach as the predominant necropsy finding. This finding is common among offspring that die at an early age, and there were no findings observed which indicated a specific reason why these pups failed to thrive.
The type and distribution of findings at necropsy of the offspring that survived to scheduled termination on Day 7 of age did not suggest any adverse effect of dosing of the parental animals with the test substance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was no clear effect on sex ratio. The percentage of males in the group receiving 100 mg/kg/day was slightly high, however, sex ratio varied widely from 3M:12F to 12M:2F within the study and apparent intergroup differences in mean values are not uncommon in small studies. In the absence of a similar finding in the other dose groups no toxicological significance is attached to this finding.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for the substance was 1000 mg/kg/day (the limit dose) for reproductive performance and offspring growth and survival in the CD rat following oral gavage administration in a standard screening test.
Executive summary:

A reproduction/developmental toxicity screening test was conducted with the test substance according to the OECD test guideline No. 421 and in compliance with Good Laboratory Practice.


Crl:CD®(SD) rats (10/sex/dose level) were dosed once daily by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day (dose volume of 10 mL/kg bw in 1% methylcellulose) for 15 days prior to pairing through to Day 6 after birth of the F1 generation.


During the study, data was recorded on clinical condition, bodyweight, food consumption, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken on the adult animals. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed before macroscopic pathology investigations were undertaken at necropsy.


There were no test material-related mortalities and no signs considered to be related to administration of the test substance


There was no clear effect on bodyweight during the pre-pairing, gestation and lactation phases. Amongst males receiving 300 mg/kg bw/day and females receiving 100 and 300 mg/kg bw/day bodyweight gain was slightly high during the pre-pairing phase, and to termination for males, when compared with Controls. However, in the absence of a clear dose response and a consistent effect between the sexes the significance of this is unclear. Food consumption was unaffected during the pre-pairing period for both sexes and for females during the gestation and lactation phases. 


Oestrous cycle length, mating performance, fertility, parturition, reproductive capacity and gestation length were unaffected by administration of the test substance at all dose levels investigated. Bodyweight gain, survival and development of the offspring to Day 7 of age were also unaffected.


There was no test material related effect on the weights of the testes and epididymides or ovaries, and no test material related macroscopic or microscopic abnormalities were detected in the reproductive organs.


Based on the results of this reproduction/developmental toxicity screening study, it was concluded that for the test substance a dose level of 1000 mg/kg bw/day represented the No Observed Adverse Effect Level (NOAEL) for reproduction/developmental toxicity in the CD rat.


 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
The study in rat is considered to be reliable (klimisch score of 1).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A reproduction/developmental toxicity screening test was conducted with the test substance according to the OECD test guideline No. 421 and in compliance with Good Laboratory Practice.


Crl:CD®(SD) rats (10/sex/dose level) were dosed once daily by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day (dose volume of 10 mL/kg bw in 1% methylcellulose) for 15 days prior to pairing through to Day 6 after birth of the F1 generation.


There were no test material-related mortalities and no signs considered to be related to administration of the test substance.


There was no clear effect on bodyweight during the pre-pairing, gestation and lactation phases. Amongst males receiving 300 mg/kg bw/day and females receiving 100 and 300 mg/kg bw/day bodyweight gain was slightly high during the pre-pairing phase, and to termination for males, when compared with Controls. However, in the absence of a clear dose response and a consistent effect between the sexes the significance of this is unclear. Food consumption was unaffected during the pre-pairing period for both sexes and for females during the gestation and lactation phases. 


Oestrous cycle length, mating performance, fertility, parturition, reproductive capacity and gestation length were unaffected by administration of the test substance at all dose levels investigated. Bodyweight gain, survival and development of the offspring to Day 7 of age were also unaffected.


There was no test material related effect on the weights of the testes and epididymides or ovaries, and no test material related macroscopic or microscopic abnormalities were detected in the reproductive organs.


Based on the results of this reproduction/developmental toxicity screening study, it was concluded that for the test substance a dose level of 1000 mg/kg bw/day represented the No Observed Adverse Effect Level (NOAEL) for reproduction/developmental toxicity in the SD rat.

Effects on developmental toxicity

Description of key information

A developmental toxicity study in rat is available, no effect on development was observed up to 1000 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 Jan 2022 to 02 Mar 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)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 to 6 months.
- Weight at study initiation: between 3080 and 4335 g.
- Housing: The animals were individually housed in noryl cages. For psychological / environmental enrichment, animals were provided with dumbells, hay and as music.
- Diet: Pelleted complete diet (Diet Reference No. 3409, KLIBA) and compacted hay pellets (SSNIFF) were provided ad libitum. During the acclimation period, when reduced food consumption was noted (=50 g/animal/day), carrots were distributed. Carrots were also given to two animals showing little or no food consumption during the treatment period and already noted before treatment initiation.
- Water: municipal tap water provided in water bottles, ad libitum.
- Analyses confirmed the absence in feed and water of known contaminants that could have interfered with the outcome of the study.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS (TARGET)
- Temperature: 15 to 21°C.
- Humidity: 30 to 70%.
- Air changes: Approximately 5 to 15 filtered, non-recycled air changes per hour.
- Photoperiod (hrs dark / hrs light): 8-hrs/16-hrs

IN-LIFE DATES: From 24 Jan 2022 to 02 Mar 2022.
Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) Methyl Cellulose in drinking water treated by reverse osmosis.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations were prepared daily, on the day of each administration and used within 4 hours after the end of their preparation. Dosing formulation were prepared based on Test Facility Study No. 48783 VAS.

VEHICLE
- Justification for use and choice of vehicle: the test item showed to be soluble and stable in the selected vehicle, and non-toxic to animals.
- Concentration in vehicle: 83, 167 and 333 mg/mL for the low-, mid- and high-dose groups, respectively.
- Amount of vehicle (if gavage): 3 mL/kg bw/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On GD6 and on GD 28 samples for concentration analysis were collected (from middle part) from all groups, while samples for homogeneity (from bottom, middle and top parts) analysis were collected from low- and high-dose groups only.
Analyses described were performed by HPLC/UV using a validated analytical procedure.
For concentration, mean sample concentration results shall be within or equal to 15% of theoretical concentration to meet acceptance criteria. For homogeneity, the relative standard deviation (RSD) of concentrations shall be of =10% for each group to comply with the acceptance criteria.
Details on mating procedure:
- Impregnation procedure: purchased time-mated.
- Other: Day 0 post-coitum is the day of successful mating. Untreated females were between GD0 and GD1 on arrival at the Test Facility.
Duration of treatment / exposure:
From GD6 to GD28.
Frequency of treatment:
Daily.
Duration of test:
24 days (From GD6, first day of administration, to GD29, day of scheduled necropsy).
Dose / conc.:
250 mg/kg bw/day
Remarks:
Low-dose group
Dose / conc.:
500 mg/kg bw/day
Remarks:
Mid-dose group
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High-dose group
No. of animals per sex per dose:
22 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of exposure was selected because this is one of the routes of administration which is requested by the Regulatory Authorities for this type of test item.
The dose levels were selected, based on the results of the following previous study: Dose-Range finding Study No. 48785 RSL where New Zealand White female rabbits (8 mated females per group) received 600, 800 or 1000 mg/kg bw/day, from GD6 to GD28, inclusive. No deaths, clinical signs or relevant effects on body weight or food consumption attributed to the test item were noted. No test item-related macroscopic post-mortem findings were observed in the dams. There were no effects on mean numbers of pre- and post-implantation losses or on the number of live fetuses per dam in any groups. The mean fetal body weight was unaffected. At external examinations, there were no variations or malformations considered to be related to the test item treatment. Based on the results of this study, 1000 mg/kg bw/day was selected as the high-dose level.
The low-dose and mid dose were selected using a ratio representing approximately a 2-fold interval (i.e. 250 and 500 mg/kg bw/day).

- Justification of test system and number of animals: At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study was designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Mortality: at least twice daily beginning upon arrival through termination (on days of receipt and necropsy at least once daily).
- Clinical Observations: at least once daily; beginning upon arrival through termination, before dosing when applicable. In addition to the daily cageside observation, animals were observed between 1 to 3 hours post-dose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A full clinical examination was performed once during acclimation, on each weighing day during dosing phase and on the day of euthanasia. Net body weight (carcass weight) was also determined subtracting the gravid uterine weight to the body weight recorded on GD29. Finally, the net body weight change was determined subtracting the gravid uterine weight and the body weight on GD6 to the body weight recorded on GD29.

BODY WEIGHT: Yes
- Time schedule for examinations: Each female was weighed on GD2, GD5, GD6, GD9, GD12, GD15, GD19, GD21, GD24, GD27 and GD29.

FOOD CONSUMPTION: Yes
- Food consumption for each animal was recorded daily between GD2 and GD 29.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD29 by an intravenous injection of sodium pentobarbital.
- Organs examined: all animals (including animals euthanised prematurely) were subjected to a macroscopic examination of the abdominal and thoracic cavities to determine pregnancy status, number of corpora lutea and numbers and types of uterine implantations. All macroscopic abnormalities observed were recorded and preserved in 10% formalin (or in another appropriate fixative).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Pregnancy status: Yes
- Gravid uterus weight: Yes, for each pregnant female (with at least one live fetus) at hysterectomy. The uterus of apparently non-gravid females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Each fetus was examined for external defects and all live fetuses were euthanized by intraperitoneal injection of sodium pentobarbital.
Dead fetuses were examined externally and preserved in Harrison's fixative but not examined further.
The following examinations were performed in all live fetuses on GD29:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter.
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, all per litter (The head of one half of the fetuses was fixed in Harrison's fluid for subsequent examination by serial sectioning. In the other half of the fetuses, the brain of each fetus was sampled and fixed in Harrison's fluid)
Statistics:
Body weight, food consumption and reproductive data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher's exact probability test (proportions).
Indices:
CONSTRUCTED VARIABLES:
- Total number of resorptions: Sum of uterine scars + early resorptions + late resorptions
- % of dead fetuses per litter: (Total No. of dead fetuses in litter / No. of implantation sites) x 100
- % of live fetuses per litter: (Total No. of live fetuses in litter / No. of implantation sites) x 100
- Pre-Implantation Loss (%): [(No. of corpora lutea – No. of implantations) / No. of corpora lutea] x 100
- Post-Implantation Loss (%): [(No. of implantations – No. of live fetuses) / No. of implantations] x 100
Historical control data:
HCD was collected from 2019-2020 from a total of 5 studies and reported in the results section. Please also refer to relevant tables under section "Any other information on results incl. tables".
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All clinical signs recorded in surviving females (all pregnant, except for two high dose females) were considered to be unrelated to the test item treatment as they were recorded both in control and test item-treated animals (abnormal feces and/or absent/decreased/soft) and/or are routinely observed in pregnant females (red discharge from the vagina or in the bedding and skin lesions/scabs).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female was euthanized on GD29 due to premature delivery. Two dead fetuses, one live fetus and three placentas with red discharge were found in the bedding. Abnormal gait was noted from GD26, associated with a body weight loss between GD24 and 29 (-8%) and reduced food consumption from GD26 (on average 42 g/day). Four scars were observed in the uterine horns.
A mid-dose female was euthanized on GD28 due to premature delivery. One fetus and two placentas were found in the bedding. A body weight loss was recorded between GD24 and 27 (-5%). Eleven scars were observed in the uterine horns.
A high-dose female was prematurely euthanized on GD24 due to abortion. Four dead fetuses and two placentas were found in the bedding. A body weight loss was recorded between GD21 and 24 (-8%), associated with little food consumption on GD23 (5 g/day vs 147 g in controls). Five scars were observed in the uterine horns.
No macroscopic post-mortem findings were recorded in any of these females.
These isolated cases of abortion/premature delivery, noted irrespective of the dose level, were considered to be incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects were noted on mean body weight or mean body weight gain at any dose level.
The slightly lower mean body weight gain recorded at 250 and 500 mg/kg bw/day between GD19 and 21 (+6 and +5 g, respectively, vs +16 g in controls) were considered to be incidental as this was mainly due to the contribution of two isolated females (one at each dose level) and recorded with no dose-level relationship.
No effects on mean carcass weights or mean net body weight changes were observed at any dose level.
Please also refer to Tables 2 and 4 under section "Any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Please also refer to Table 3 under section "Any other information on results incl. tables".
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects on mean gravid uterus weights were observed at any dose level.
Please also refer to Table 4 under section "Any other information on results incl. tables".
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
None of the macroscopic findings observed were considered to be related to the test item administration as they were noted with no dose-relationship and are findings routinely observed in female rabbits of this strain and age.
Please also refer to Table 5 under section "Any other information on results incl. tables".
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Three females of the control, mid-dose and high-dose groups were euthanized due to prematurely deliveries on GD29, GD28 and GD24, respectively. For more details please refer also to section "mortality".
These isolated cases of abortion/premature delivery, noted irrespective of the dose level, were considered to be incidental.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The higher mean number of post-implantation loss in the low-dose group (11.3 vs 9.1 in controls), was considered to be unrelated to the test item treatment as the differences were not dose-related and mainly due to the contribution of two low-dose females with a high number of early resorptions.
Please also refer to Table 6 under section "Any other information on results incl. tables".
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No total litter loss was observed in any of the experimental groups.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The higher mean number of resorptions recorded at 250 mg/kg/day (1.1 vs 0.5 in controls), particularly early, leading to a higher mean post-implantation loss percentage (11.3% vs 9.1% in controls), was considered to be unrelated to the test item treatment as the differences were not dose-related and mainly due to the contribution of two females.
Please also refer to Table 6 under section "Any other information on results incl. tables".
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Incidences of dead fetuses were 3.5%, 0.0%, 0.0% and 0.0% for the control, low-, mid- and high-dose groups, respectively, achieving statistical significance in all treated groups. Considering that dead fetuses were observed only in the control group, the finding is considered of no toxicological relevance and not attributable to the test-item administration.
Please also refer to Table 6 under section "Any other information on results incl. tables".
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At hysterectomy on GD29, 19/22, 21/22, 20/22 and 19/22 females were pregnant with live fetuses in the groups treated at 0, 250, 500 and 1000 mg/kg bw/day, respectively.
Please also refer to Table 1 under section "Any other information on results incl. tables".
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: Absence of adverse effects up to and including the highest dose level tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal weight was unaffected by the test item treatment at any dose level.
Please also refer to Table 7 under section "Any other information on results incl. tables".
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was unaffected by the test item treatment at any dose level.
Please also refer to Table 7 under section "Any other information on results incl. tables".
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related external malformations were observed at any dose level.
Iniencephaly was the only malformation observed in one control fetus.
Please also refer to Table 8 under section "Any other information on results incl. tables".

No test item-related external variations were observed at any dose level.
The few findings recorded were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals with the same incidence.
Please also refer to Table 11 under section "Any other information on results incl. tables".
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related skeletal malformations were observed at any dose level.
The higher incidence of fused sternebrae at 1000 mg/kg bw/day (three fetuses from two litters vs one fetus in controls) was not attributed to the test item treatment as the difference was noted with no statistical significance and the incidence remained near the limits of our HCD range.
The other findings recorded were considered to be incidental as they were noted both in control and test item-treated animals and/or were noted in isolated fetuses and/or remained within the range of our HCD.
Please also refer to Table 9 under section "Any other information on results incl. tables".

No test item-related effects on cartilages were observed at any dose level.
The higher incidence of non-ossified caudal vertebrae and metacarpal bone (only the respective cartilages were present) at 500 mg/kg bw/day, when compared to controls, was not attributed to the test item treatment as the incidence was not dose-related and/or was near the limits of our HCD range.
The other findings recorded were considered to be incidental as they were noted both in control and test item-treated animals and/or were noted in isolated fetuses and/or remained within the range of our HCD.
Please also refer to Table 13 under section "Any other information on results incl. tables".

No test item-related skeletal variations were observed at any dose level.
The higher incidence of unossification/incomplete ossification of the caudal vertebra centrum and 1st metacarpal bone at 500 mg/kg bw/day, when compared to controls, was not attributed to the test item treatment as the incidence was not dose-related and/or was within or near the limits of our HCD range.
The other findings recorded were considered to be incidental as they were noted both in control and test item-treated animals and/or were noted in isolated fetuses and/or remained within the range of our HCD.
Please also refer to Table 14 under section "Any other information on results incl. tables".
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related visceral malformations were observed at any dose level.
At 1000 mg/kg bw/day, one litter had one fetus with malpositioned right kidney associated with a short right ureter (and dilated renal pelvis, see variations). As these malformations were isolated and noted in a single fetus, a relationship to the test item treatment cannot be ascertained.
The other findings were considered to be incidental and not related to the test item treatment as they were noted in isolated fetuses with no dose-relationship.
Please also refer to Table 10 under section "Any other information on results incl. tables".

No test item-related visceral variations were observed at any dose level.
The higher incidence of hemorrhagic ovary at 1000 mg/kg bw/day (six fetuses from four litters vs one fetus in controls) was not attributed to the test item treatment as this variation was isolated and the incidence remained within the range of our HCD.
The other findings were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals and/or with no dose-relationship and/or are common findings in fetuses of this strain.
Please also refer to Table 12 under section "Any other information on results incl. tables".
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Absence of adverse effects up to and including the highest dose level tested.
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Chemical Analyses of the Dose Formulations


The dose formulations prepared on GD6 and GD28, were all found to be within the acceptance criteria (measured concentrations at ± 15% of the theoretical concentrations). No test item was detected in the control dose formulation.


 


Table 1. Pregnancy Status
















































Dose level (mg/kg bw/day)



0



250



500



1000



Number of females



22



22



22



22



Pregnant females



20



21



21



20



Females with live fetuses at termination



19



21



20



19



Female prematurely euthanized (pregnant)



1



0



1



1



Non-pregnant females



2



1



1



2



 


Table 2. Mean body weights/mean body weight changes (g)































































































































































Dose level (mg/kg bw/day)



0



250



500



1000



Body weight (g)



GD 6



3639



3686



3685



3642



GD 9



3694



3740



3749



3698



GD 12



3762



3800



3793



3741



GD 15



3842



3913



3873



3838



GD 19



3883



3940



3905



3874



GD 21



3899



3946



3910



3915



GD 24



3956



3985



3956



3938



GD 27



4007



4045



4015



3982



GD 29



4057



4081



4060



4018



percentage difference vs. controls (%)



 



+1



0



-1



Body weight change (g)



GD 6-9



+55



+55



+64



+56



GD 9-12



+68



+59



+44



+43



GD 12-15



+80



+113



+80



+98



GD 15-19



+41



+27



+32



+36



GD 19-21



+16



+6



+5



+42



GD 21-24



+57



+39



+46



+23



GD 24-27



+50



+60



+59



+52



GD 27-29



+50



+36



+45



+36



GD 6-29



+417



+395



+386



+407



percentage difference vs. controls (%)



 



-5



-7



-2



No statistically significant differences versus controls.


 


Table 3. Mean food consumption (g/animal/day)














































































































































































Dose level (mg/kg bw/day)



0



250



500



1000



GD 6-7



171



183



173



180



GD 7-8



181



185



187



186



GD 8-9



175



179



176



180



GD 9-10



182



191



180



163



GD 10-11



179



186



173



154



GD 11-12



175



178



168



156



GD 12-13



166



169



154



144



GD 13-14



155



166



141



138



GD 14-15



145



167



148



143



GD 15-16



149



185



160



161



GD 16-17



159



174



171



170



GD 17-18



159



166



172



188



GD 18-19



171



178



169



185



GD 19-20



161



157



150



170



GD 20-21



165



157



152



178



GD 21-22



164



155



154



166



GD 22-23



155



150



150



170



GD 23-24



147



137



137



137



GD 24-25



151



127



140



134



GD 25-26



147



125



135



136



GD 26-27



132



136



139



136



GD 27-28



138



126



135



144



GD 28-29



145



135



152



146



No statistically significant differences versus controls.


 


Table 4. Gravid Uterus and Carcass Weights (g)









































Dose level (mg/kg bw/day)



0



250



500



1000



Gravid uterus weight



576.3



541.6



548.8



542.8



percentage difference vs. controls (%)



 



-6



-5



-6



Carcass weight



3517.7



3539.3



3510.8



3475.3



Net weight change from GD6.



-128.4



-146.4



-163.2



-135.4



No statistically significant differences versus controls.


 


Table 5. Macroscopic Post-Mortem Examination























































Dose level (mg/kg bw/day)



0



250



500



1000



Thymus


Reddish color



 



1



 



 



Spleen


Irregular surface



1



 



1



1



Heart


Pericardium, translucent content



1



 



 



 



Liver


Granular consistency



 



2



 



 



Stomach


Depressed area



2



2



 



2



Female gonads


Serous cyst(s)


(connective tissue, periovarian region)



 



2



 



 



 


Table 6. Hysterectomy data





























































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD
min-max



Number of females with live fetuses at termination



19



21



20



19



100



Mean number of corpora lutea per animal



11.2



11.2



12.2



11.5



11.7-13.0



Mean number of implantation sites per animal



9.8



9.6



10.1



9.8



10.1-10.6



Mean percentage of pre-implantation loss (%)



12.1



14.1



16.9



13.9



9.4-21.7



Mean number of live fetuses per animal



8.8



8.5



9.6



9.1



8.9-9.9



Dead fetuses (%)



3.5



0.0**



0.0**



0.0**



0.00-0.39



Mean number of resorptions per female



0.5



1.1*



0.5



0.7



/



Mean number of early resorptions



0.2



0.6*



0.3



0.4



0.55-1.09



Mean number of late resorptions



0.4



0.5



0.3



0.3



Mean percentage of post-implantation loss (%)



9.1



11.3



4.8



7.8



7.3-13.2



Statistically significant vs. controls: *: p<0.05. **: p<0.01.


HCD: Historical Control Data (2019-2020), n= 5 studies; /: no data.


 


Table 7. Mean fetal body weight (g) and sex ratio






























Dose level (mg/kg bw/day)



0



250



500



1000



HCD
min-max



Mean fetal body weight (g)



42.5



42.5



39.2



40.9



37.8-42.3



Mean percentage of male fetuses (%)



52.5



55.5



52.0



52.9



42.3-55.3



No statistically significant differences versus controls.


HCD: Historical Control Data (2019-2020), n= 5 studies.


 


Table 8. Mean fetal (F) and litter (L) incidences of external malformations (%)























































Dose level (mg/kg bw/day)



0



250



500



1000



Number of litters



19



21



20



19



Number of fetuses



176



178



193



173



Trunk



 



 



 



 



. Iniencephaly, F (L)



0.6 (5.3)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



Litters affected, n (%)



1 (0.6)



0 (0.0)



0 (0.0)



0 (0.0)



Fetus affected, n (%)



1 (5.3)



0 (0.0)



0 (0.0)



0 (0.0)



n: number. No statistically significant differences vs. controls.


 


Table 9. Mean fetal (F) and Litter (L) incidences of skeletal malformations






























































Dose level (mg/kg bw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Sternebrae



 



 



 



 



 



. fused sternebrae , F (L)



0.0 (0.0)



0.6 (4.8)



0.0 (0.0)



1.7 (15.8)



0.5-1.4
(4.5-14.3)



Litters affected, n (%)



4 (21.1)



3 (14.3)



5 (25.0)



3 (15.8)



 



Fetus affected, n (%)



4 (2.4)



3 (1.7)



5 (2.6)



3 (1.7)



 



n: number.


No statistically significant differences vs. controls.


HCD: Historical Control Data (2019-2020), n= 5 studies


 


Table 10. Mean fetal (F) and litter (L) incidences of soft tissue malformations (%)






































































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Kidneys



 



 



 



 



 



. marked dilated renal pelvis, F (L)



0.0 (0.0)



0.6 (4.8)



0.0 (0.0)



0.0 (0.0)



0.0-2.9
(0.0-22.2)



. malpositioned kidney, F (L)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.6 (5.3)



/



Vessels



 



 



 



 



 



. dilated aortic arch, F (L)



0.0 (0.0)



0.0 (0.0)



0.5 (5.0)



0.0 (0.0)



0.0-0.6
(0.0-5.6)



Ureter



 



 



 



 



 



. short ureter, F (L)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.6 (5.3)



/



Litters affected, n (%)



0 (0.0)



1 (4.8)



1 (5.0)



1 (5.3)



 



Fetus affected, n (%)



0 (0.0)



1 (0.6)



1 (0.5)



1 (0.6)



 



n: number. No statistically significant differences vs. controls.


HCD: Historical Control Data (2019-2020), n= 5 studies; /: no data.


 


Table 11. Mean fetal (F) and litter (L) incidences of external variations (%)






























































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD


min-max



Number of litters



19



21



20



19



100



Number of fetuses



176



178



193



173



949



General



 



 



 



 



 



. abnormal color of amniotic fluid, F (L)



1.1 (10.5)



0.0 (0.0)



0.0 (0.0)



1.7 (10.5)



0.0-1.6
(0.0-5.0)



Paw and digit



 



 



 



 



 



. paw hyperflexion, F (L)



0.6 (5.3)



0.0 (0.0)



0.5 (5.0)



0.6 (5.3)



/



Trunk



 



 



 



 



 



. abdomen, blackish color, F (L)



0.6 (5.3)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.0-0.5
(0.0-5.0)



Litters affected, n (%)



4 (21.1)



0 (0.0)



1 (5.0)



2 (10.5)



 



Fetus affected, n (%)



4 (2.3)



0 (0.0)



1 (0.5)



4 (2.3)



 



n: number. No statistically significant differences vs. controls.


HCD: Historical Control Data (2019-2020), n= 5 studies; /: no data.


 


Table 12. Mean Fetal (F) and Litter (L) incidences of soft tissues variations (%)














































































































































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Spleen



 



 



 



 



 



. misshapen, F (L)



0.6 (5.3)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



/



Gall bladder



 



 



 



 



 



. small, F (L)



0.0 (0.0)



0.0 (0.0)



0.5 (5.0)



0.0 (0.0)



0.0-3.9
(0.0-18.2)



Kidneys



 



 



 



 



 



. dilated renal pelvis, F (L)



4.2 (31.6)



2.8 (19.0)



1.0 (10.0)



0.6 (5.3)



0.0-2.9
(0.0-22.2)



. enlarged kidney, F (L)



1.2 (10.5)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



/



Gonads



 



 



 



 



 



. hemorrhagic ovary, F (L)



0.6 (5.3)



0.0 (0.0)



0.5 (5.0)



3.5 (21.1)



0.0-3.5
(0.0-16.7)



Vessels



 



 



 



 



 



. absent brachiocephalic trunk, F (L)



36.3 (89.5)



50.0* (100.0)



34.7 (80.0)



41.6 (94.7)



29.3-36.0
(68.2-94.4)



. short innominate artery, F (L)



1.8 (15.8)



0.6 (4.8)



1.6 (15.0)



1.7 (10.5)



0.0-3.8
(0.0-23.8)



Trunk



 



 



 



 



 



. thin abdominal wall, F (L)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.6 (5.3)



0.0-0.5
(0.0-5.0)



Thymus



 



 



 



 



 



. enlarged, F (L)



0.6 (5.3)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



/



Litters affected, n (%)



18 (94.7)



21 (100)



17 (85.0)



18 (94.7)



 



Fetus affected, n (%)



70 (41.7)



94* (52.8)



71 (36.8)



77 (44.5)



 



n: number. Statistically significant vs. controls: *: p<0.05.


HCD: Historical Control Data (2019-2020), n= 5 studies; /: no data.


 


Table 13. Mean fetal (F) and Litter (L) incidences of cartilages findings














































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Caudal vertebrae



 



 



 



 



 



. cartilage of caudal vertebra(e): present, F (L)



1.8 (15.8)



5.6 (23.8)



7.8* (40.0)



3.5 (26.3)



0.0-3.4
(0.0-27.3)



Metacarpal bone



 



 



 



 



 



. cartilage of metacarpal bone(s): present, F (L)



10.7 (52.6)



11.8 (42.9)



21.8** (55.0)



10.4 (42.1)



0.0-21.2
(0.0-52.4)



Litters affected, n (%)



19 (100)



21 (100)



20 (100)



19 (100)



 



Fetus affected, n (%)



147 (87.5)



159 (89.3)



168 (87.0)



150 (86.7)



 



n: number.


Statistically significant vs. controls: *: p<0.05, **: p<0.01.


HCD: Historical Control Data (2019-2020), n= 5 studies.


 


Table 14. Mean Fetal (F) and Litter (L) incidences of skeletal variations (%)






























































































Dose level (mg/kgbw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Caudal vertebrae



 



 



 



 



 



. incomplete ossification of centrum, F (L)



1.2 (10.5)    



5.1 (23.8)                               



7.8** (35.0)                               



3.5 (26.3)             



1.0-4.7
(4.8-30.0)



. unossified centrum, F (L)



0.6 (5.3)



0.6 (4.8)



5.2* (30.0)



1.2 (10.5)



0.0-3.4
(0.0-27.3)



Metacarpal bone



 



 



 



 



 



. unossified 1st metacarpal, F (L)



4.2 (21.1)



1.7 (14.3)



9.8* (30.0)



1.7 (15.8)



2.9-8.4
(18.2-38.1)



. incomplete ossification of 1st metacarpal, F (L)



6.5 (42.1)



10.1 (42.9)



14.5* (55.0)



8.7 (36.8)



8.3-20.7
(27.3-42.9)



Litters affected, n (%)



19 (100)



21 (100)



20 (100)



19 (100)



 



Fetus affected, n (%)



162 (96.4)



168 (94.4)



189 (97.9)



161 (93.1)



 



n: number.


Statistically significant vs. controls: *: p<0.05, **: p<0.01.


HCD: Historical Control Data (2019-2020), n= 5 studies.

Conclusions:
Based on the results obtained in this study, the No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg bw/day.
Executive summary:

The objective of this study was to provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism of 99422018 when administered by the oral (gavage) route from implantation to the day prior to the scheduled hysterectomy [from Gestation Day (GD) 6 to GD28].


Three groups of 22 time-mated female New-Zealand White rabbits received the test item, once daily by the oral route (gavage) at dose levels of 250, 500 or 1000 mg/kg/day from GD6 to GD28 inclusive. A control group of 22 time-mated females received the vehicle only (1% methylcellulose in drinking water) under the same experimental conditions. A constant dosage volume of 3 mL/kg/day was used.
The actual test item concentrations were determined in the dose formulations on two occasions, using a validated HPLC/UV analytical method.
The animals were checked daily for mortality and clinical signs. Body weight was recorded at designated intervals. Food consumption was recorded daily.
On GD29, the females were euthanized and a macroscopic post mortem examination was performed. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. Placentas were observed. The fetuses were weighed and sexed by internal examination of the gonads. They were subjected to detailed external, soft tissue and skeletal (bones + cartilages) examinations.


Actual concentrations of the test item in the dose formulations analyzed during the study were within the accepted range of ± 15% of the theoretical concentrations.
At hysterectomy on GD29, 19/22, 21/22, 20/22 and 19/22 females were pregnant with live fetuses in the groups treated at 0, 250, 500 and 1000 mg/kg/day, respectively.
No test item-related deaths or clinical signs were recorded.
Body weight, body weight change and food consumption were unaffected by the test item treatment.
At necropsy of the dams, no test item-related macroscopic findings were observed.
Gravid uterus weight, carcass weight and net body weight change were not impacted by the test item treatment. No test item-related effects on the numbers of corpora lutea, implantations or resorptions were noted at any dose level.
The fetal body weight and sex ratio were unaffected at all dose levels.
At external, soft tissue or skeletal examinations of the fetuses, no variations or malformations attributable to the test item treatment were noted.


The test item, 99422018, was administered daily to time-mated female NZW rabbits by the oral route (gavage) at the dose level of 250, 500 or 1000 mg/kg/day from GD6 to GD28, inclusive.
Based on the results obtained in this study, the No Observed Effect Level (NOEL) for maternal parameters and embryo-fetal development was considered to be 1000 mg/kg/day.


 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
A total of 110 Crl:CD Sprague Dawley (SD) virgin female rats, 9 weeks old (200-225 g) were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy. The male rats used were from the same supplier, and were at least 11 weeks old (at least 350 g). After arrival the weight range was determined (from 209.1 - to 227.7 g) and the females were uniquely identified by tattoo on the hind feet. A health check was then performed by a veterinarian. An acclimatisation period of approximately 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
Route of administration:
oral: gavage
Vehicle:
other:
Remarks:
1% methyl cellulose
Details on exposure:
The required amount of test item was suspended in the vehicle. The preparations were made daily (concentrations of 10, 30 and 100mg/mL) or up to approximately weekly interval based on the stability data obtained in ERBC study no. A3717. Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (ERBC Study no. A3717). Samples of the formulations prepared during the current study (the first day,
then the first week and the last week of treatment, where possible) were analysed to check the homogeneity and concentration.
Chemical analysis was carried out by the Analytical Chemistry Department. The software used for this activity was Empower® 2 Build No.
2154. The results of the analyses were within the acceptability criteria for content and homogeneity (85 to 115% of the theoretical concentration, with a CV < 10 %).
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in themorning from the day after pairing until a positive identification of mating is made. The day of mating, as judged by the presence of spermin the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
Duration of treatment / exposure:
All animals were dosed once a day from Day 5 through Day 19 post coitum.
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
group 1
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
group 4
No. of animals per sex per dose:
Each group comprised 24 mated female rats
Control animals:
yes, concurrent vehicle
Details on study design:
The effects of 99422018 were investigated in Sprague Dawley SD rats during pregnancy and embryo-foetal development. Female rats were mated with sexually mature males of the same strain and then assigned to complete 4 groups of 24 females each. Where possible, females inseminated by the samemale were evenly distributed across the groups. The schema of treatment and the number of animals per group is described below:
Groups Treatment Number of
(mg/kg/day) Female
1 0 24
2 100 24
3 300 24
4 1000 24

All femaleswere administered by oral gavage during the gestation period fromDay 5 through Day 19 post coitum at 100, 300 and 1000 mg/kg/day and the dose volume was set at 10 mL/kg body weight. Control females received the vehicle (1%Methyl cellulose) at the same dose volume during the same treatment period.
Maternal examinations:
Clinical sign:
All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice.

Body weight:
All animals were weighed on Days 0, 3, 5, 9, 12, 15, 18 and 20 post coitum.

Food consumption:
Food consumption was measured on Days 3, 5, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

Blood collection for thyroid hormone determination (T3, T4 and TSH):
On Day 20 post coitum, blood samples for thyroid hormones determination (approximately 0.5 mL), were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. This procedure was performed within a short time frame (e.g. two hours, as possible) in the morning of the day of necropsy. Samples were transferred into tubes containing no anticoagulant and
centrifuged at room temperature. The serum obtained was divided in two aliquots and stored at -80°C, pending analysis.

Immunoanalysis - Thyroid hormonedetermination (T3, T4 and TSH) (Delegated phase):
Immunoanalysis was carried out by the Test Site, BioVetim (Study Phase number: BIOVX1310), according to the following validated immunoanalytical methods:
– RV-T3/R/S-BKM/RIA-002 for T3
– RV-T4/R/S-BKM/RIA-002 for T4
– RV-TSH/R/S-IZO/RIA-002 for TSH

Samples from all females were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).

Number of samples: 96 × 2 aliquots

The aliquots were maintained at -80°C until transfer to the Test Site indicated below, where the immunoanalysis was carried out: BIOVETIM

Thyroid weight, fixation, preservation and examination:
From all animals completing the scheduled test period, the thyroid was dissected free of fat, fixed and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. The ratio of thyroid weight to body weight was calculated for each animal.
After dehydration and embedding in paraffin wax, sections of the tissue were cut at 5 micrometre thickness and stained with haematoxylin and eosin. Sections were examined for evaluation of pathological changes.
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
– Gravid uterine weight;
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements
and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.
Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible.
– late resorptions: placental and foetal remnants visible.
Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
All live foetuses were examined externally. In addition, the anogenital distance (AGD) was recorded in all male and females foetuses. Approximately one-half of the foetuses (i.e. routinely, every second live foetus) in each litter were preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal (single staining) examination. Skeletal and fixed-visceral examinations were performed in all groups.
Structural deviations were classified as follows:
Malformations
Major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies
Minor abnormalities that are detected relatively frequently.
Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
This type of classification was applied only for skeletal examination
Fixed visceral examination (Delegated phase):
Approximately 3 weeks after the last necropsy, the foetuses were transferred from Bouin’s solution into pots containing alcohol (70%). Fixed visceral examination of foetuses was performed at an external GLP-certified laboratory
The fixed visceral examination (Study Phase number: 12982 B) was performed according to the corresponding SOPs of the Test Site. The foetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys.
After examination, the tissue was preserved in a solution of glycerine/ethanol (one foetus per container). Descriptions of any malformation and variation was recorded electronically on Microsoft Excel for Microsoft 365 MSO (16.0.13001.20254), QC checked and QA audited
Statistics:
For continuous variables the significance of the differences amongst group means was
assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test and intergroup differences between the control and treated groups assessed by a
non-parametric version of theWilliams test.
Historical control data:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were recorded. A single case of hairloss and damaged
ear were seen in the control and at 100 mg/kg/day respectively, and considered incidental
occurrences.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Description (incidence and severity):
No effects on T3,T4 and TSH determination
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects were seen on thyroid weight.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted at histopathological examination of thyroid gland
from females at all dose levels.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The normalised anogenital distance (AGD) was found statistically significantly lower at
1000 mg/kg/day in male foetuses (2.55 vs 2.62 of control) and statistically significantly
higher at 300 mg/kg/day in female foetuses (1.68 vs 1.55 of control). The difference was
of low magnitude being -2.7 % in males and +8.3% in females. In the absence of a dose
relation, both in male and female foetuses, the statistical differences were not considered
of toxicological significance but rather due to biological variations.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
At termination, the number of live foetuses and the percentage of males respect to females
were comparable between groups.
The mean body weights of both males and females, as reported in the table below, were
statistically significantly higher at 100 and 1000 mg/kg/day compared to the control group.
These differences did not follow a dose dependent pattern and were low of magnitude. In
addition, the weight of all individual foetuses was within historical control data. therefore,
these changes were not considered of toxicological importance.
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
Anogenital distance of all rodent fetuses:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Several major defects in the skull bones ossification were recorded in two foetuses (two litters) at 1000 mg/kg/day, in one foetus at 300 mg/kg/day and in one control foetus. At the high dose, the anomalies included the unossified presphenoid bone (foetus no. 5; Dam no. 187) and the malposition of atlas, axis and exocipitals (foetus no. 9; Dam no. 183).
At mid-dose, the absence of pterygoid bone was recorded in the foetus no. 15 (Dam no. 105). The same foetus showed additional major abnormalities such as cleft palate and the maxillary bone fused. The unossified presphenoid bone was also observed in one control foetus (foetus no. 15; Dam no. 17). Moreover, another one high dose foetus (foetus no. 1; Dam no. 161) showed the non-ossification of pubis as malformation. Although these major abnormalities were not observed in the CRO’s internal control data, they occurred in single foetuses sometimes showing multiple malformations. Therefore, the incidence of affected litters was very low and did not follow a dose dependent pattern. Thus, these major abnormalities were considered unrelated to treatment.
Minor abnormalities or variations occurred in all groups and included, for example, incomplete ossifications of several bones of the skull, no ossification of the hyoid, incomplete ossification of thoracic/sacral vertebrae, asymmetrical ossification of sternebrae, incomplete ossifications of the pubis, metacarpals. The incidence of the affected litters in treated groups was similar or even lower than observed in the control group or without dose relation.
Therefore, the findings were considered unrelated to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no visceral malformation associated with treatment with the test item.
Malformation such as situs inversus was observed in one foetus (foetus no. 2; Dam no. 121)
at 300 mg/kg/day. This major finding occurred in one single foetus and it was considered
incidental and not related to the test item.
The overall incidences of foetuses or litters with any remaining finding were isolated incidences
and did not indicated any test item effect. Thus, they were considered not to be test
item-related.
Two single variations in form of thymus long at 100 mg/kg/day and in formof abdomen
internal haemorrhage at 100 mg/kg/day, showed high incidence when compared to the
concurrent control. These findings were unrelated to each other and there was no dose dependency.
Therefore, these findings were considered to be incidental and not test item related.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Treatment related:
no
Conclusions:
In conclusion, treatment with the test item did not cause maternal toxicity and
did not reveal teratogenic potential up to and including the dose level of 1000 mg/kg/day.
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level)
for maternal and developmental toxicity could be set at 1000 mg/kg/day.
Executive summary:

The effects of test item were investigated in Sprague Dawley SD rats during pregnancy and embryo-foetal development. Female rats were mated with sexually mature males of the same strain and then assigned to complete 4 groups of 24 females each. Where possible, females inseminated by the same male were evenly distributed across the groups.
All females were administered by oral gavage during the gestation period from Day 5 through Day 19 post coitum at 100, 300 and 1000 mg/kg/day and the dose volume was set at 10 mL/kg body weight. Control females received the vehicle (1%Methyl cellulose) at the same dose volume during the same treatment period.
All females were checked twice a day for mortality and at least once daily for clinical signs. Scheduled measurements of body weight and food consumption were performed during the in vivo phase. Before despatch to necropsy (Day 20 post coitum), blood samples were collected for hormones determination of T3, T4 and TSH. At term, females were caesarean sectioned on Day 20 post coitum and subjected to detailed post mortem examination.
The thyroid was weighed and preserved in fixative for histopathology. Gravid uterus was weighed and the uterine content examined for the number of implantations, intrauterine deaths and live foetuses. The ovaries were also examined and the corpora lutea counted.
Live foetuses were weighed, sexed and observed for external abnormalities. The foetuses were then processed for skeletal and fixed visceral examinations.

No unforeseen mortality occurred during the study. All females were sacrificed at termination and were found pregnant with the exception of a total of 4 females: one each in the control and high dose group (1000 mg/kg/day) and 2 in the mid-dose group (300 mg/kg/day).
No treatment-related clinical signs were recorded.
No changes in body weight/body weight gain were seen between groups throughout the study.
Food consumption was comparable between groups.
The serum levels of T3, T4 and TSH did not differ between treated and control groups.
Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.
Caesarian section data, litter data and sex ratios were unaffected by treatment.
The normalised anogenital distance was found lower in males at 1000mg/kg/day and higher in females at 300 mg/kg/day when compared to the respective control animals. These differences reached a statistical significance. However, the difference was of low magnitude being -2.7 % in males and +8.3% in females. In the absence of a dose relation, both in male and female foetuses, the statistical differences were not considered of toxicological significance but rather due to biological variations.
No treatment-related effect was seen on thyroid weight.
At post mortem, no treatment-related changes were noted.
No treatment-related changes were noted in thyroid gland examination of females receiving test item at low, medium and high dose.
No treatment-related findings were recorded at external examination of foetuses, at skeletal examination and at visceral examination.
In conclusion, treatment with the test item did not cause maternal toxicity and
did not reveal teratogenic potential up to and including the dose level of 1000 mg/kg/day.
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity could be set at 1000 mg/kg/day.


 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study in rat is considered to be reliable (klimisch score of 1).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity study in rat (ERBC, 2021)


The effects of test item were investigated in Sprague Dawley SD rats during pregnancy and embryo-foetal development. Female rats were mated with sexually mature males of the same strain and then assigned to complete 4 groups of 24 females each. Where possible, females inseminated by the same male were evenly distributed across the groups.


All females were administered by oral gavage during the gestation period fromDay 5 through Day 19 post coitum at 100, 300 and 1000 mg/kg/day and the dose volume was set at 10 mL/kg body weight. Control females received the vehicle (1%Methyl cellulose) at the same dose volume during the same treatment period.


No unforeseen mortality occurred during the study. All females were sacrificed at termination and were found pregnant with the exception of a total of 4 females: one each in the control and high dose group (1000 mg/kg/day) and 2 in the mid-dose group (300 mg/kg/day).


No treatment-related clinical signs were recorded. No changes in body weight/body weight gain were seen between groups throughout the study. Food consumption was comparable between groups.Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.


The serum levels of T3, T4 and TSH did not differ between treated and control groups.


Caesarian section data, litter data and sex ratios were unaffected by treatment.


The normalised anogenital distance was found lower in males at 1000mg/kg/day and higher in females at 300 mg/kg/day when compared to the respective control animals. These differences reached a statistical significance. However, the difference was of low magnitude being -2.7 % in males and +8.3% in females. In the absence of a dose relation, both in male and female foetuses, the statistical differences were not considered of toxicological significance but rather due to biological variations.


No treatment-related effect was seen on thyroid weight.


At post mortem, no treatment-related changes were noted.


No treatment-related changes were noted in thyroid gland examination of females receiving tset item at low, medium and high dose.


No treatment-related findings were recorded at external examination of foetuses, at skeletal examination and at visceral examination.


In conclusion, treatment with the test item did not cause maternal toxicity and
did not reveal teratogenic potential up to and including the dose level of 1000 mg/kg/day.
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity could be set at 1000 mg/kg/day.


 


Developmental toxicity study in rat (CRL, 2022)


The objective of this study was to provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism of 99422018 when administered by the oral (gavage) route from implantation to the day prior to the scheduled hysterectomy [from Gestation Day (GD) 6 to GD28].


Three groups of 22 time-mated female New-Zealand White rabbits received the test item, once daily by the oral route (gavage) at dose levels of 250, 500 or 1000 mg/kg/day from GD6 to GD28 inclusive. A control group of 22 time-mated females received the vehicle only (1% methylcellulose in drinking water) under the same experimental conditions. A constant dosage volume of 3 mL/kg/day was used.
The actual test item concentrations were determined in the dose formulations on two occasions, using a validated HPLC/UV analytical method.
The animals were checked daily for mortality and clinical signs. Body weight was recorded at designated intervals. Food consumption was recorded daily.
On GD29, the females were euthanized and a macroscopic post mortem examination was performed. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. Placentas were observed. The fetuses were weighed and sexed by internal examination of the gonads. They were subjected to detailed external, soft tissue and skeletal (bones + cartilages) examinations.


Actual concentrations of the test item in the dose formulations analyzed during the study were within the accepted range of ± 15% of the theoretical concentrations.
At hysterectomy on GD29, 19/22, 21/22, 20/22 and 19/22 females were pregnant with live fetuses in the groups treated at 0, 250, 500 and 1000 mg/kg/day, respectively.
No test item-related deaths or clinical signs were recorded.
Body weight, body weight change and food consumption were unaffected by the test item treatment.
At necropsy of the dams, no test item-related macroscopic findings were observed.
Gravid uterus weight, carcass weight and net body weight change were not impacted by the test item treatment. No test item-related effects on the numbers of corpora lutea, implantations or resorptions were noted at any dose level.
The fetal body weight and sex ratio were unaffected at all dose levels.
At external, soft tissue or skeletal examinations of the fetuses, no variations or malformations attributable to the test item treatment were noted.


The test item, 99422018, was administered daily to time-mated female NZW rabbits by the oral route (gavage) at the dose level of 250, 500 or 1000 mg/kg/day from GD6 to GD28, inclusive.
Based on the results obtained in this study, the No Observed Effect Level (NOEL) for maternal parameters and embryo-fetal development was considered to be 1000 mg/kg/day.

Justification for classification or non-classification

No effects on reproductive performance and develomental toxicity were observed at the limit dose of 1000 mg/kg bw/day in the available studies performed in rats and/or rabbits.


On the basis of these results and according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, no classification is warranted with respect to reproductive toxicity.


 

Additional information