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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Waltz (2013) performed a combined repeated dose toxicity study with reproduction/developmental toxicity screening test in rats according to OECD guideline 422 (GLP-compliant). A NOAEL of < 25 mg/kg/day was derived based on maternal and paternal hepatotoxicity and a NOAEL of 100 mg/kg bw/day was derived for reproductive toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 June 2012 - 16 August 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented GLP study performed according to OECD Guideline 422. However, dose formulations were not analyzed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
No dose formulation analysis has been performed
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffcat ZR-50
- Substance type: Clear orange liquid
- Physical state: Liquid
- Lot/batch No.: PFW100119
- Storage condition of test material: Room temperature, 20.4 to 22°C
- Composition and purity is documented by the Sponsor and communicated to Calvert in the form of a Certificate of Analysis
- Stability: no information is provided

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: A minimum of 13 weeks old at initiation of cohabitation
- Weight at study initiation: >= 300 grams for the males and >=200 grams for the females at initiation of cohabitation
- Fasting period before study: No
- Housing: Upon arrival and until randomization, males and females were group-housed, sexes separate. Following randomization and until cohabitation, males and females were housed individually. During cohabitation, one female was placed with a male breeder from the same group. Following cohabitation, males and females were housed individually. No later than gestation day 17, mated female animals were placed in totes with bedding. The room in which the animals were kept were documented in the study records. No other species were kept in the same room.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Study animals were acclimated to their housing for a minimum of 7 days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 to 21.4 °C
- Humidity (%): 40.1 - 71.5%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights were turned on during the dark cycle to accommdate blood sampling or other study procedures.
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Dose preparation:
The test article and vehicle control preparations were prepared weekly or additionally as needed by diluting the test article in vehicle (w/v) to reach the proper concentrations.

- Dose formulation samples:
On the first day of dosing, at the beginning of cohabitation and at the last day of dosing, duplicate 1-mL samples were obtained from top, middle, and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test article in vehicle. These samples were stored at room temperature, approximately 20.4 to 22°C. Dose formulation samples were not analysed and discarded at the finalisation of the study.
Details on mating procedure:
Animals were mated by placing one male and one female from the same dose group overnight in a breeding cage until evidence of copulation was noted, after which the male animal was separated. Animals remained in cohabitation for a maxmium of three weeks.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Male animals were dosed for a total of 35 days (starting two weeks prior to the cohabitation period). Treatment continued for the males during the same-group cohabitation period and until the day before scheduled euthanasia on day 21 of cohabitation. Female animals were dosed once daily for 15 days prior to cohabitation, during cohabitation, throughout pregnancy and up to including day 3 of lactation.
Frequency of treatment:
daily

Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
actual ingested
No. of animals per sex per dose:
10 for all dose groups and 5 for the recovery groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based upon previously conducted toxicity studies.


Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
Throughout the treatment phase, a minimum of twice daily, prior to dose administration and a minimum of once following dosing. On non-dosing days, a minimum of once daily.

BODY WEIGHT: Yes
Males: Animals were weighed at the time of randomization/selection, on the first day of dosing and weekly thereafter. A fasted terminal body weight was recorded prior to scheduled euthanasia.
Females: Animals were weighed at the time of randomization/selection, on the first day of dosing, weekly thereafter, and on gestation days 0, 4, 7, 14 and 20, 23 and 26, and on day 0 and 4 of lactation. A fasted terminal body weight was recorded prior to scheduled euthanasia.

FOOD CONSUMPTION:
Males and females: Full feeder weights and/or feeder weigh backs were recorded once weekly prior to cohabitation, on gestation days 0-4, 4-7, 7-10, 10-14, 14-17, 17-20, 20-23 and 23-26 and on day 0 and 3 lactation. During cohabitation food consumption was not recorded.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

Clinical pathology evaluation (groups 1-6)
Sample collection: Blood samples for evaluation of serum chemistry, hematology and coagulation parameters were collected from five animals/sex in all groups prior to terminal sacrifice. Animals were anesthetized by CO2 inhalation prior to blood collection. Immediately following exsanguination by cardiocentesis for terminal blood collection, rats were returned to the CO2 chamber to ensure euthanasia. Animals were fasted overnight (approximately 12-24 hours) prior to blood collection for clinical pathology evaluation.

HAEMATOLOGY: Yes
Method of collection: cardiocentesis
Anticoagulant: K2-EDTA
Parameters analyzed: red blood cell count and morphology, white blood cell count*, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, hematocrit, hemoglobin, reticulocyte count
*: total and different white blood cell counts, including neutrophils, basophils, eosinophils, monocytes, lymphocytes and large unstained cells
Coagulation:
Method of collection: cardiocentesis
Anticoagulant: sodium citrate
Coagulation parameters: activated partial thromboplastin time, prothrombin time

CLINICAL CHEMISTRY: Yes
Method of collection: cardiocentesis
Anticoagulant: none
Parameters analyzed: Alanine aminotransferase, albumin, albumin/globulin ratio (calculated), alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatinine, globulin (calculated), glucose, phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea nitrogen

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
The functional observational battery was assessed for five rats/sex/group once during the study (toward the end of the dosing periods). Each rat was placed in a fixed environment considering of a Plexiglas enclosure, fitted with a lid. The enclosure was placed on absorbent paper which detects excretions. In this environment, rats were free to move about. The rats were observed for signs of pharmacological or toxicological activity following treatment and the results recorded. Observations for the following symptoms were made:
abnormal posture, ataxia, awareness reaction, body tremors, corneal reflex, decreased abdominal tone, decreased grip strength, decreased respiration, excretion, immobility, increased secretion, irritability, loss of righting, motor activity, nociceptive (pain) response, piloerection, pinnal reflex, pupil size, seizures/convulsions, startle response, sterotypy, vocalization

OTHER:
Organ weights:
For all male group 1-6 animals, the following organs were weighed before fixation, after dissection of excess fat and other excess tissues. Organ weights were not recorded for animals found dead.
Organs weighed: epididymides, testes,
For five male and female groups 1-6 animals, at scheduled sacrifice, the following organs (when present) were weighed before fixation, after dissection of excess fat and other excess tissues. Organ weights were not recorded for animals found dead. Paired organs were weighed together unless gross abnormalities were present, in which case they were weighed separately.
Organs weighed: adrenals, heart, kidneys, liver, thymus, spleen, brain
Organ to body weight ratios were calculated (using the final body weight obtained prior to necropsy), as well as organ to brain weight ratios.
Oestrous cyclicity (parental animals):
Vaginal examination: estrous cycle evaluation was performed daily for 2 weeks prior to cohabitation and daily during the treatment and cohabitation periods. Day 0 of gestation was determined by evidence of copulation which was determined by the examination of vaginal smears made daily to determine if sperm are present in a smear of vaginal contents or by the presence of a copulatory plug in situ. Examination of vaginal smears was performed at approximately the same time each day (early morning) throughout the cohabitation period.
Sperm parameters (parental animals):
Epididymides and testes weight
Litter observations:
All neonates were sexed and eximined as soon as possible after delivery for litter size, still births, liver births and any gross anomalies. In addition, all pups were observed daily and weighed within 24 hours of parturition and on day 4 post-partum.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Tissue collection and preservation (groups 1-6):
All tissues for all adults were examined. For all animals necropsied, the tissues listed below were preserved in 10% neutral buffered formalin (except for the epididymides and testes that were retained in modified Davidson's fixative for optimum fixation).
Tissues collected: Cardiovascular: aorta*, heart; digestive: salivary gland(s), tongue, esophagus, stomach*, small intestine* (duodenum, jejunum, ileum); urogenital: kidneys*, urinary bladder*, orvaries*, uterus*, cervix*, vagina*, testes*, epididymides*, prostate*, seminal vesicles*; endocrine: adrenals*, pituitary, thyroid/parathyroid*; large intestine* (cecum, colon, rectum), pancreas, liver*; respiratory: trachea*, larynx, lung with mainstem bronchus*; lymphoid/hematopoietic: sternum with bone marrow*, thymus*, spleen*, lymph nodes* (mandibular, mesenteric); skin/musculoskeletal: skin, mammary gland, skeletal muscle, femur with articular surface; nervous/special sense: eye with optic nerve, sciatic nerve*, brain*, spinal cord - cervica*l, spinal cord - midthoracic*, spinal cord - lumbar*, lacrimal glands; other: unique animal identifier (not for evaluation), gross findings*
HISTOPATHOLOGY: Yes
Histology (groups 1-6):
Tissues for evaluation were processed to paraffin blocks and prepared to slides. Slides were stained with hematoxylin and eosin. Occasionally, other stains were required by the study pathologist to aid in the diagnosis of lesions; theser were documented in the final report.

Slides were prepared for five animals/sex in group 1, 4, 5 and 6 for all tissues marked with * above.

If test article-related lesions were noted, additional slides were prepared on those tissues from groups 2 and 3 at additional cost to the Sponsor, following the Sponsor's consent.


Postmortem examinations (offspring):
All pups were euthanized by an intrathoracic injection of a barbiturate overdose on day 4 of lactation. All surviving neonates were euthanized by an intrathoracic injection of a barbiturate overdose on lactation day 4.
Statistics:
Statistical evaluation was performed on in-life, clinical pathology, and organ weight numerical data. For in-life and clinical pathology parameters, the software determined statistical significance by the following decision tree. First, the homogeneity of the data was determined by Barlett's test. If the data were homogeneous, a one-way analysis of variance was performed to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test were used to determine the degree of significance from the control means (p<0.05, p<0.01 and p<0.001). If the data is non-homogeneous, the Kruskal-Wallis non-parametric analysis ws performed to assess statistical signficance. If statistically significant differences between the means were found (p<0.05, p<0.01 and p<0.001), the Mann-Whitney U-Test was used to determine the degree of significance from the control means (p<0.05, p<<0.01 and p<0.001). For necropsy organ weight data, the evaluation of the equality of means were made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test was used to determine the degree of significance from the control means (p<0.05 and p<0.01).
Reproductive indices:
Pre-cotial interval (in days): (sum of days until successful copulation)/(number of presumed pregnant animals)
Copulation index (%): (number of presumed pregnant animals/number of paired animals) x 100
Fertility index (%): (number of pregnant animals/number of presumed pregnant animals) x 100
Preimplantation loss (%): [(no. of corpora lutea - number of implantations)/(number of corpora lutea)] x 100
F0 gestation index (%): (no. of females with live pups/no. of pregnant animals) x 100
Offspring viability indices:
F0 live birth index (%): (no. of pups born alive/no. of pups born) x 100
F0 viability index (%): (no. of pups alive on day 4/no. of pups alive at birth) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Females: Clinical observations in Group 6 animals were limited to two animals with instances of food crumbling between days 2-12 of the dosing phase. In addition, one group 6 animal had piloerection between days 12-13 and staining on the head (cranial) between days 12-15.

Males: Male Group 6 animal #0346 had noisy respiration between days 3-4. On day 28, it also exhibited piloerection, had red discharge around the muzzle and nares, and had stained forepaws. All other male Groups 5-6 animals appeared normal during the dosing and recovery phase.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All male and female animals survived until their scheduled sacrifice on day 57 (17 days after their respective last day of dosing).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females: Statistically significantly lower body weights were observed between days 42-56 in unmated Group 6 females compared to concurrent control Group 5 animals. In addition, body weight gains were statistically significantly higher for Group 6 on Dosing Day 42.
Males: Statistically significantly lower body weights were observed between days 35-56 in Group 6 males compared to Group 5. In addition, body weight gains were statistically significantly lower for Group 6 on dosing day 21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females: Female Group 6 food consumption was statistically significantly increased during the recovery phase on days 49 and 56.
Males: Food consumption was statistically significantly reduced among Group 6 animals on days 7, 35 and 42, and statistically significantly increased at the end of the recovery phase on day 56.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Females: Similar to mated Group 4 animals, at the end of the recovery period on day 57 unmated female Group 6 leukocyte counts (WBC) were statistically significantly increased and red blood cell (RBC) hemoglobin (HGB) and hematocrit values (HCT) were statistically significantly decreased. In addition, Group 6 absolute basophil (#BASO) and large unstained cell levels (#LUC) were statistically significantly increased. All red blood cells in both dose groups were normocytic and normochromic.
Males: Male Group 6 platelet values (PLT) were statistically significantly increased. All red blood cells in both dose groups were normocytic and normochromic.
Coagulation:
Females: Group 6 had statistically significantly decreased prothrombin times (PT) when compared to Group 5 animals. However, the slightly reduced PT were not considered adverse.
Males: No statistically significant effects on prothrombin times (PT) and activated partial thromboplastin times (APTT) were detected at the end of the recovery phase on day 57.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Females: Group 6 aspartate aminotransferase values (AST) were 33% increased. In addition, Group 6 inorganic phosphorus (PHOS), cholesterol (CHOL) and chloride levels (CL) were statistically significantly increased, and creatinine levels (CREAT) and albumin globulin ratios (A/G) were statistically significantly decreased.
Males: Significant changes in liver enzyme chemistry were also seen in Group 6 animals at the end of the recovery phase on day 57. Group 6 aspartate aminotransferase values (AST) were 107% and alanine aminotransferase values (ALT) were 156% increased, respectively. In addition, Group 6 total protein (TP), globulin (GLOB) and albumin level (ALB) were all statistically significantly decreased.

Since vacuolation and fibrosis was still present in the liver of all male and female Group 6 animals, the associated changes in clinical chemistry parameters were still present at the end of the recovery period on day 57.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Females: There were no treatment-related findings for any of the qualitative functional observational battery tests peformed on dosing day 37.
Males: There were no treatment-related findings for any of the qualitative functional observational battery tests performed on dosing day 37.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article toxic effects were still present in the parathyroid glands, trachea, lungs, liver and spleen of males and females euthanized 15 days after the last day of dosing with Jthe test substance at 250 mg/kg.
Females and males: Parathyroid gland: minimal to slight vacuolation of the parathyroid chief cells was still present in 4 of 5 males and 5 of 5 females from the highest dose group (group 6).
Trachea: minimal vacuolation of the tracheal epithelial cells still occurred in 1 of 5 males and 2 of 5 females from the highest dose group (group 6).
Lungs: minimal to slight epithelial vacuolation was still present within the bronchi and bronchioles in all males and females previously treated with the test substance at 250 mg/kg (group 6). In addition, minimal to slight vacuolation of the media layer was still present in the pulmonary blood vessels in 2 of 5 males and 5 of 5 females from the previously treated highest dose group (group 6).
Liver: vacuolation of the hepatocytes and parenchymal fibrosis were still present in the liver of all male and female rats previously treated with the test substance at 250 mg/kg (group 6). The hepatocyte vacuolation was still predominantly centrilobular but with reduced severity grade when compared to livers from mated animals. The severity grade ranged from slight to moderate. Fibrosis was still present within the centrilobular regions and was increased in severity when compared to livers from mated animals. In addition, to increased severity in group 6 animals, fibrosis often bridged among centrilobular areas, a feature not seen in group 4 animals.
Spleen: foam cells were still present in the splenic red pulp of 1 of 5 males and 5 of 5 females previously treated with the highest dose of the test substance (group 6). However, the severity was decreased. Minimal to slight decrease of the cellularity of the marginal zone was still present in 3 of 5 male and 1 of 5 female rats previously treated with the test substance at 250 mg/kg (group 6).
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
Pregnancy status:
Two group 1 (#0353, #0356), two group 2 (#0363, #0370) and two group 4 dams (#0383, #0389) were determined to be non-gravid. Group 2 dam #0362 was gravid as determined at necropsy. However, no delivery as well as no pups were observed for this dam.

The mean pre-coital interval (in days) as well as the duration of the gestion period (days) was statistically significantly longer for group 2 and 3 dams, respectively. However, no dose dependent trend was observed. The fertility index (%) and the number of gravid dams was comparable among groups.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Group 4 testes-to-body-weight ratios were statistically significantly increased, and group 4 epididymides weight ratios were statistically significantly decreased;
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
< 25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Maternal and paternal hepatotoxicity
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Remarks on result:
not measured/tested
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Most viable groups 1 and 2 neonates appeared normal, with milk present in the stomach and signs of nesting. Clinical observations including, but not limited to coolness to touch, discoloration of the muzzle and/or paleness were slightly more frequent among group 3 neonates. In addition, some neonates exhibited clincial signs including, but not limited to coolness to touch, discolored/missing tail tips, discolored hind paws, and/or discolored muzzle.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Group 4 dams had a statistically significantly lower number of neonates delivered compared to group 1 control dams (113, 91, 108 and 52, for groups 1-4 respectively). Only approximately 56% of all the group 4 neonates delivered on day 0 were viable, compared to 88%, 96% and 84% for groups 1-3. However, the % survival of those neonates that were viable on day 0 was comparable among groups (even though the absolute number in group 4 was much lower). Most viable groups 1 and 2 neonates appeared normal, with milk present in the stomach and signs of nesting. A large number of group 4 neonates were found missing (presumably cannabalized) or only partial bodies were found.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group 2 male mean neonate body weights were statistically significantly decreased on day 0 of lactation and group 3 male and female mean neonate body weights were statistically significantly increased on day 4 of lactation compared to group 1 neonates. The biological significance of this finding is unknown. However, the number of viable neonates (on lactation day 0 and 4) that could be included for analysis was significantly lower for group 4 compared to groups 1-3.
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):
No treatment-related malformations were observed for neonates examined on day 4 of lactation. Besides the frequent observation of no milk in the stomach, no morphological malformations were observed for the early deaths that were not cannibalized.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Remarks on result:
not measured/tested
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day
Conclusions:
Based on the results of the study, the no observed adverse effect level (NOAEL) for male and female rats exposed to the test substance is considered to be less than 25 mg/kg/day, based on maternal and paternal hepatoxicity. The NOAEL for reproductive toxicity is considered to be 100 mg/kg/day.
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
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 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test is performed in rats, in which male and female rats are exposed to 0 (vehicle), 25, 100, 250 mg/kg bw/d via gavage (K1, GLP, OECD 422; Calvert, 2013).

The NOAEL for oral repeated dose toxicity in Sprague-Dawley rats is considered to be < 25 mg/kg bw/day (actual dose received) for both sexes.

Male and female animals dosed at 100 and 250 mg/kg showed significant effects of toxicity. One female group 4 dam dosed at 250 mg/kg was found dead on lactation day 1 and all its neonates were found dead. All other female animals survived until their scheduled sacrifice. Besides adverse clinical signs and effect on body weight and food consumption, groups 3 and 4 animals exhibited adverse microscopic and macroscopic liver findings, effects on liver weights, BUN levels, and liver enzymes including AST and ALT. Microscopic liver findings included enlargement and pallor secondary to hepatocyte vacuolation and centrilobular fibrosis, which severity increased proportionally with dose levels. In addition at 100 and 250 mg/kg the test substance induced cytoplasmic vacuolation also occurred in the chief cells of the parathyroid gland (high dose groups only), tracheal, bronchial and bronchiolar epithelium, and within the media of the pulmonary vasculature. Additional findings included foam cells in the splenic red pulp (high dose groups only), decreased cellularity of the splenic marginal zone (high dose group only), and erosion on the glandular gastric mucosa. At 25 mg/kg microscopic findings were limited to the gastric mucosa of one animal and the liver of another animal.

The administration of the test substance also effected gestation, viability and related parameters. The mean number of neonates delivered per dam was significantly lower for group 4 females and the total number of neonates/dam alive at birth was significantly lower. Further, the total number of surviving group 4 neonates on day 4 of lactation was significantly lower.

After the last day of dosing on day 40 the satellite animals stayed on study for an additional 16 days without dosing to observe the reversibility, persistence or delayed occurrence of systemic toxic effects.

All male and female animals survived until their scheduled sacrifice on day 57 (17 days after their respective last day of dosing). Clinical observations in group 6 animals included instance of food crumbling, piloerection, staining on the head (crainal) and red discharge around the muzzle and nares with associated stained forepaws. Body weights and food consumption was variable between male and female satellite animals. Similar group 3 and 4 animals, unmated group 6 satellite animals dosed at 250 mg/kg exhibited adverse microscopic and macroscopic liver findings, effects on liver weights and liver enzymes.

All microscopic findings described for mated groups 3 and 4 animals above were also present after the 16 day recovery period in the unmated group 6 animals. The severity was similar or reduced grade except for hepatic fibrosis, which was more preeminent in livers from recovery groups.

In conclusion, the toxic effects seen at 250 mg/kg in mated group 4 animals were not reversible after a 16 day recovery period without dosing in the unmated group 6 satellite animals.

Extended one-generation reproductive toxicity study: No signs of toxicity to fertility or to reproductive organs or tissues were noted up to and including 100 mg/kg bw/day in a combined repeated dose toxicity with reproduction/developmental screening test (OECD 422), nor in the 90 -days repeated dose toxicity study (OECD 408). Effects in the liver and kidney are observed at 25 and 100 mg/kg bw/d. A prenatal developmental toxicity study (OECD guideline 414) has been performed. For these reasons, no EOGRTS is performed.


Effects on developmental toxicity

Description of key information

Allt (2018) performed a pre-natal development toxicity study via oral gavage in female Sprague-Dawley rats according to OECD Guideline 414 (K1, GLP-compliant). A NOAEL of 100 mg/kg bw/day was determined for the pregnant female (reductions in body weight gain and food consumption throughout the treatment period in females treated with 250 mg/kg bw/day. No treatment related changes were detected in the offspring parameters measured or on embryofetal development. The NOAEL for developmental toxicity was therefore considered to be 250 mg/kg bw/day.


 


Latha (2022) performed a pre-natal development toxicity study via oral gavage in female New Zeland White rabbits according to OECD Guideline 414 (K1, GLP-compliant). In this study, prenatal developmental toxicity of the test item was evaluated following daily administration at 0, 15, 45, 90 mg/kg/day during gestation days 6 to 28.


The test item at 15 and 45 mg/kg/day was without effect on maternal body weights, weight gain, food consumption, and the maternal and litter parameters were comparable to vehicle control group. Gross evaluation of the placenta revealed no findings. Treatment with the test item at 90 mg/kg/day resulted in treatment-related reduction in maternal body weight gain and food consumption indicating maternal toxicity. There were no gross pathological changes at any dose level. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity up to the highest dose of 90 mg/kg/day. Based on the above findings, under the test conditions used in this study, the NOAEL for maternal toxicity was considered at 45 mg/kg/day  and the NOAEL for developmental toxicity and teratogenicity was considered at 90 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:
19/08/2021 - 24/06/2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
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
- Source: KCC BIO LABS
- Age at study initiation: 6-10 months
- Weight at study initiation:
- Fasting period before study: no
- Housing: individually except during mating where 1 male and 1 female will be housed together
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 19/08/2021 - 24/09/2021
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Proof of pregnancy: visual confirmation of mating referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
GD6- GD28
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
45 mg/kg bw/day (nominal)
Dose / conc.:
90 mg/kg bw/day (nominal)
No. of animals per sex per dose:
23 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A preliminary dose-range-finding (DRF) in pregnant rabbits was carried out using 6 rabbits per group with the test item. The rabbits were treated at doses of 45, 90 and 180/120 mg/kg/day along with the concurrent vehicle control group at a dose volume of 4 mL/kg body weight from GD 6 to 28 and observed for clinical signs and mortality. Due to treatment related mortalities at 180 mg/kg/day, the dose was reduced to 120 mg/kg/day from GD 9 onwards.

The results were as follows:

Due to treatment related deaths (4/6 rabbits), dose of 180/120 mg/kg/day was considered to have exceeded the maximum tolerable dose.
At the dose of 90 mg/kg/day there was reduction in body weight, food consumption and reduction in fetal weights. No abnormality was noticed on external observations of fetuses.

Based on the results of DRF, the following doses are selected for the definitive study :

Vehicle control - 0 mg/kg/day
Low dose - 15 mg/kg/day
Mid dose - 45 mg/kg/day
High dose - 90 mg/kg/day

- Rationale for animal assignment (if not random):
Pregnant rabbits on day 0 (day of coitus) obtained each day were randomly distributed to different groups by body weight stratification method using ProvantisTM software (Instem).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for clinical signs was performed twice a day, pre-dose and post-dose during treatment period and at least once on other days.

BODY WEIGHT: Yes
- Time schedule for examinations: The rabbits were weighed on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Gross necropsy, which involves an external observation and thoracic and abdominal viscera including uterine contents, was performed on all animals in the study including dead, moribund and those sacrificed pre-term or at term (caesarean section).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Gross evaluation of placenta
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
Data will be captured using the ProvantisTM laboratory information management system (LIMS), parameters such as body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss , number of implantations, sex ratio, number of corpora lutea, early and late resorptions will be evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. If data found to be homogeneous and of normal distribution, will be analysed by analysis of variance (ANOVA). If data found to be nonhomogeneous or of nonnormal data will be subjected for transformation and ANOVA will be done on transformed data. When ANOVA will be significant, pairwise comparisons of treated groups to the control group will be made using a parametric test, Dunnett, to identify statistical differences.

Fetal weight for male and female will be analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss will be analyzed using Kruskal Wallis test for group comparison. Mann-Whitney / Wilcoxon pairwise comparison of the treated groups with the control group is performed, when the group differences are significant.

The incidence of dams with resorptions will be tested for using Chi-square test followed by Fisher’s exact test for group association.

The incidence of fetuses and litter (incidence and percent) observations for external,visceral and skeletal will be tested for the Cochran-Armitage trend test and the pairwise comparison will be tested by Fisher’s exact test for group association.

All hypothesis testing will be carried out at the 5% (2-sided) significance level unless otherwise specified.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean maternal body weights, body weight gain and adjusted body weights during the different days of gestation in test item treated groups were statistically comparable to vehicle control group. However, there was a non significant reduction in body weight gains during different days of gestation (GD6-9, GD9-12 and GD27-29) at 90 mg/kg/day as compared to vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
As compared to the vehicle control group, there was no change in food consumption of rabbits dosed at 15 and 45 mg/kg/day except for significant increase in food consumption at 15 mg/kg/day during GD 3 to 6, which was considered an incidental finding.

At 90 mg/kg/day, there was a significant reduction in food consumption by 11 to 29 % during GD 6-9, 9-12, 12-15, 18-21, 21-24 and 27-29 as compared to vehicle control group. The reduction in food consumption at 90 mg/kg/day was considered treatment related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The gravid uterine weight was statistically comparable between the vehicle control and rabbits treated at 15, 45 and 90 mg/kg/day.
Gross pathological findings:
no effects observed
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female abortion at 15 mg/kg/day on GD27, which was considered as an incidental finding.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The maternal parameters comprising pre and post implantation loss were statistically comparable between the vehicle control and rabbits treated at 15, 45 and 90 mg/kg/day.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The maternal parameters comprising early and late resorptions were statistically comparable between the vehicle control and rabbits treated at 15, 45 and 90 mg/kg/day.
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 2, 2, 3, 3 non pregnant females in the control, 15, 45 and 90mg/kg bw/d groups respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test-item-related malformation was observed during external observation of the fetuses at any of the doses. Anomaly of umbilical hernia (1/144 fetuses) at 15 mg/kg/day and (3/136 fetuses) at 45 mg/kg/day and small fetus (1/144 fetuses) at 15 mg/kg/day were observed. Malformations of cranium excencephaly in vehicle (1/153 fetuses) control group and right forelimb bent inwards (1/119 fetuses) at 90 mg/kg/day were observed. These findings were randomly distributed across the treatment groups and were considered incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test-item-related skeletal malformations observed in fetuses of does treated up to 90 mg/kg/day. Majority of variations and anomalies observed in various skeletal components across treated groups were comparable to the vehicle control group.

There was significant increase in extra 8 th lumbar centra and arch at 45 and
90 mg/kg/day and delayed ossification of 6th sternum at
45 mg/kg/day.However, these findings were not dose related and were randomly distributed across the treatment groups and hence were considered non-adverse.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test-item-related visceral malformations observed in fetuses of dams treated up to 90 mg/kg/day. Anomalies such as gall bladder hypoplastic in test-item-treated groups and hyperplastic gall bladder and bi-lobed gall bladder in vehicle control group were observed. Incidence of liver median lobe extra lobation at 90 mg/kg/day and slight kidney renal pelvis dilation at 15 mg/kg/day was observed. These findings were not considered adverse as these observations commonly occur in animals of this test model and the incidence of occurrence was consistent with vehicle control group.
Key result
Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Developmental effects observed:
no

Table 1: Summary maternal data



































































































































































































Sex: Female


 



G1


0


mg/kg/day



G2


15


mg/kg/day



G3


45


mg/kg/day



G4


90


mg/kg/day



Group size



 



 



23



23



23



23



Number dams aborted



 



 



0



1



0



0



Number sacrificed at C/S



 



 



23



22



23



23



Pregnant at C/S



 



N



21



20



20



20



Gravid Uterus


Weight (g)



[a]



Mean


SD



425.8


115.7



444.2


111.5



392.1


87.7



359.6


96.2



Number of


Corpora Lutea



[a]



Mean


SD


Sum



10.33


2.01


217.00



10.20


2.33


204.00



10.00


2.99


200.00



9.20


2.17


184.00



No. of


Implantation



[a1]



Mean


SD


Sum



7.67


2.33


161.00



7.50


2.44


150.00



7.20


1.94


144.00



6.60


2.48


132.00



Dams with Early resorption



 



N



1



3



3



3



Number of


Early Resorptions



[a1]



Mean


SD


Sum



0.0


0.2


1.0



0.2


0.4


3.0



0.2


0.5


4.0



0.2


0.4


3.0



% Early Resorption


/Animal



[a1]



Mean


SD



0.48


2.18



2.50


6.54



2.72


7.90



2.39


6.01



Dams with Late resorption



 



N



6



2



4



10



Number of


Late Resorptions



[a1]



Mean


SD


Sum



0.3


0.6


7.0



0.2


0.5


3.0



0.2


0.4


4.0



0.5


0.5


10.0



% Late Resorptions


/Animal



[a1]



Mean


SD



5.09


8.68



2.08


6.55



2.39


5.06



12.01


15.87



Dams with Resorptions



[f]



N



7



5



6



12



Total Number of Resorption (early + late)



[f]



Mean


SD


Sum



0.4


0.6


8.0


.



0.3


0.6


6.0


.



0.4


0.7


8.0


 



0.7


0.6


13.0


 



Pre-implantation


Loss/Animal



[a1]



Mean


SD



2.67


1.96


56.00



2.70


1.38


54.00



2.80


2.26


56.00



2.60


1.96


52.00



% Pre-implantation


Loss



[a]



Mean


SD



25.72


17.94



27.47


13.57



24.99


18.21



28.60


21.50



Post-implantation


Loss/Animal



[a1]



Mean


SD



0.38


0.59


8.00



0.30


0.57


6.00



0.40


0.68


8.00



0.65


0.59


13.00



% Post-implantation


Loss (%)



[a1]



Mean


SD



5.57


8.66



4.58


8.65



5.11


9.21



14.39


16.42


         

C/S: Cesarean section


 


[a] - Anova & Dunnett


[a1] - Anova & Dunnett (Rank)


[f] - Chi-Squared & Fisher's Exact


 


Table 2: Summary litter data


 

























































































































Sex: Female


 



G1


0


mg/kg/day



G2


15


mg/kg/day



G3


45


mg/kg/day



G4


90


mg/kg/day



Total Number of fetuses



 



Sum


 



153



144



136



119



Total Number of litters


 



 



Sum



21



20



20



20



Number of dead fetuses



 



Sum


 



0



0



0



0



Total number of live fetuses



 



Sum



153



144



136



119



Mean litter size


 


 



[a]



Mean


SD


N



7.3


2.4


21



7.2


2.6


20



6.8


1.8


20



6.0


2.8


20



No. of Live Male fetuses



 



Sum


 



71



80



65



60



% Male Fetus



 



Mean


SD


 



45.2


17.6



56.1


22.1



47.7


18.1



50.9


24.8



Mean Fetal Weight - Male


(g)



[c]



Mean


SD


 



40.836


5.758



41.944


3.431



38.849


5.728



38.526


7.595



No. of Live Female fetuses



 



Sum


 



82



64



71



59



% Female Fetus



 



Mean


SD


 



54.8


17.6



43.9


22.1



52.3


18.1



49.1


24.8



Mean Fetal Weight - Female


(g)



[c1]



Mean


SD



39.794


5.501



42.204


5.808



38.737


4.739



38.770


4.847



Mean Fetal Weight- Male+Female (both)


(g)



[c2]



Mean


SD



40.423


4.636



42.172


3.627



38.854


5.122



38.213


6.617



 


[a] - Anova & Dunnett (Log)


[c] - Ancova/Anova & Dunnett: {Covariate(s): Number of Live Male Fetuses}


[c1] - Ancova/Anova & Dunnett: Covariate(s): Number of Live Female Fetuses}


[c2] - Ancova/Anova & Dunnett: Covariate(s): Number of Live Fetuses}

Conclusions:
In this study, prenatal developmental toxicity of the test item was evaluated in New Zealand White rabbits following daily administration by oral gavage at 0, 15, 45, 90 mg/kg/day during gestation days 6 to 28.

The test item at 15 and 45 mg/kg/day was without effect on maternal body weights, weight gain, food consumption, and the maternal and litter parameters were comparable to vehicle control group. Gross evaluation of the placenta revealed no findings.

Treatment with the test item at 90 mg/kg/day resulted in treatment-related reduction in maternal body weight gain and food consumption indicating maternal toxicity.

There were no gross pathological changes at any dose level. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity up to the highest dose of 90 mg/kg/day.

Based on the above findings, under the test conditions used in this study, the following NOAEL values were derived:

• NOAEL for maternal toxicity was considered at 45 mg/kg/day as reduction in body weight gain and food consumption was observed at 90 mg/kg/day.

• NOAEL for developmental toxicity and teratogenicity was considered at
90 mg/kg/day since the other maternal and litter parameters were unaffected by treatment and there was no evidence of test item related fetal findings up to the dose of 90 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of the test item in New Zealand white rabbits. This study evaluated the developmental and maternal toxicity of the test item administered to pregnant rabbits by oral route during gestation days (GD) 6 to GD 28.  This study provided a rational basis for risk assessment in humans. The results of this study were used to establish the No Observed Adverse Effect Level (NOAEL) / No Observed Effect Level (NOEL) for maternal and developmental toxicity of the test item in rabbits.


The dose levels for this study were selected based on Dose Range Finiding study (DRF) study under study number N5369. Dose levels of 0, 45, 90 and 180/120 mg/kg/day were evaluated in pregnant rabbits (6/group) treated with the test item or vehicle from GD 6 to GD 28 at the dose volume of 2 mL/kg body weight. Due to test item related mortalities at 180 mg/kg/day, the dose was reduced to 120 mg/kg/day from GD 9 onwards.There was  reduction in body weight and food consumption and reduction in mean fetal weights at 90 mg/kg/day . Based on the results of of dose range finding study in pregnant rabbits, high dose of 90 mg/kg/day was selected for the definitive study. Mid dose of 45 mg/kg/day and low dose of
15 mg/kg/day was selected to provide a graded response to the test item.


In the definitive study, 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred. Test item dssolved in vehicle (Milli-Q water) was administered at 0, 15, 45 and 90 mg/kg/day at the dose volume of 10 mL/kg body weight. The control group received the vehicle only.


The following parameters and end points were evaluated in this study: Clinical signs, body weight, body weight gain, food consumption, caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses. The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed, and examined for external malformations. All the live fetuses were examined for visceral and skeletal variations and malformations.


Dose formulations were analysed twice i.e once during initiation and once at termination of treatment, the results of analysis indicated  within ± 10.0% of the nominal concentration and the relative standard deviation (%RSD) was less than 10%.


Main findings from the study are summarized below:



  • Mortality, clinical signs, and gross necropsy changes: There were no unscheduled deaths, clinical signs, or any gross pathological findings that could be attributed to the test item.

  • Body weight: No toxiclogically significant changes in maternal body weight or maternal body weight gain was observed at the doses of 15 and 45 mg/kg/day. At 90 mg/kg/day, there was a non-significant reduction in maternal body weight gain as compared to vehicle control group.

  • Food intake: No toxiclogically significant changes in maternal food intake were observed at 15 and 45 mg/kg/day. At 90 mg.kg/day there was a significant reduction of food consumption by 17 to 29% associated with reduction in maternal body weight gain indicating maternal toxicity.

  • Maternal developmental parameters: No significant changes in any maternal developmental toxicity endpoint were observed at 15, 45 and
    90 mg/kg/day. No gross pathological changes in the placenta were observed in any of the animals.


Litter Parameters: The total number of fetuses was 153, 144, 136 and 119 at 0, 15, 45, and 90 mg/kg/day, respectively. No dead fetuses were observed at any dose level. No significant changes in litter size, sex ratio, or fetal weight were observed up to the dose of 90 mg/kg/day.



  • Fetal examination: The results from external, visceral, and skeletal examinations did not reveal any significant effects that could be attributed to the test item.


 


Based on the above findings, under the test conditions used in this study, the following NOAEL values were derived:



  • NOAEL for maternal toxicity was considered at 45 mg/kg/day as reduction in body weight gain and food consumption was observed at 90 mg/kg/day.

  • NOAEL for developmental toxicity and teratogenicity was considered at
    90 mg/kg/day since the other maternal and litter parameters were unaffected by treatment and there was no evidence of test item related fetal findings up to the dose of 90 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 November 2017 - 15 March 2018
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:
Adopted 22 January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guideline for Toxicology studies, 12 NouSan No 8147
Version / remarks:
24 November 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW160721
- Expiration date of the lot/batch: 30 December 2018
- Purity : ca. 98.32%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at ambient temperature/humidity in darkness; may be used/formulated in light
- Solubility and stability of the test substance in the solvent/vehicle: For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Distilled Water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services. The formulations were stable for at least 21 days.

OTHER SPECIFICS: No correction for purity was made.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD) IGS BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: no data
- Weight at study initiation: At the start of treatment the females weighed 204 to 299g.
- Fasting period before study: no data
- Housing: The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): ad libitum; A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used.
- Water (e.g. ad libitum): ad libitum, Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 01 December 2017 (first day of treatment) To: 20 December 2017 (final day of necropsy)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations are stable for at least 21 days and were therefore prepared twice and stored at approximately 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 0, 2.5, 10 and 25 mg/mL
- Treatment volume 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for test item in distilled water formulations at nominal concentrations of 2.5 mg/mL and 25 mg/mL when stored refrigerated for 21 days.
The mean concentrations of test item in test formulations analyzed for the study were within ± 10% of nominal concentrations, confirming accurate formulation.
The results indicate that the prepared formulations were within 3% of the nominal concentration.
Details on mating procedure:
Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
between Days 3 and 19 of gestation
Frequency of treatment:
daily
Duration of test:
From Day 3 to Day 20 of gestation.
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
low treatment group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Intermediate treatment group
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
high treatment group
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen in collaboration with the Sponsor and were based on previous toxicity data (Envigo Research Limited, Study Number XX96MF). The preliminary oral (gavage) pre-natal development toxicity study in the rat was performed as dose range finder for the OECD 414 study. Following dose levels were recommended for use in the main prenatal developmental toxicity study: 0 (Control), 25, 100 and 250 mg/kg bw/day. Based on the results of the study, dose levels of 0 (Control), 25, 100 and 250 mg/kg bw/day were recommended for use in the planned prenatal developmental toxicity study.
The highest dose level should produce an effect on body weight gain and the low and intermediate dose levels follow the recommendations of the OECD 414 guideline for a 2 to 4-fold interval between dose levels.

- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION: Yes
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION: Yes
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST-MORTEM EXAMINATIONS: Yes
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

Ovaries and uterine content:
The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):
Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus
Fetal examinations:
The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Percentage pre-implantation loss was calculated as:
[(number of corpora lutea-number of implantations)/number of corpora lutea] x 100

Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses)/number of implantations] x 100

Sex ratio:
Sex ratio was calculated as:
% male fetuses (sex ratio) = (number of male fetuses/total number of fetuses) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant adverse clinical signs evident in treated females.
One female treated with 250 mg/kg bw/day showed increased salivation post dosing on Day 5 only. A further female from this treatment group had pilo-erection on Day 8 only. In isolation, these findings were considered to be incidental and of no toxicological importance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females treated with 250 mg/kg bw/day showed a statistically significant reduction (p<0.01-0.001) in body weight gain between Days 4 and 11 of gestation. Body weight for these females was also statistically significantly reduced (p<0.05-0.01) from Day 8 onwards. Cumulative body weight gain was statistically significantly reduced (p<0.05-0.001) in these females throughout the treatment period and body weight and body weight gain when adjusted for gravid uterus weight was also statistically significantly reduced (p<0.05 and p<0.001 respectively) when compared to controls.
A statistically significant reduction (p<0.05) in body weight gain was evident in females treated with 100 mg/kg bw/day between Days 8 and 11 of gestation. A statistically significant reduction (p<0.05) in cumulative body weight gain was also evident in these females between Days 3 and 11 and Days 3 and 17. Body weight gain for these females during the remaining periods was comparable to controls.
Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by treatment at 25 mg/kg bw/day. See section 'Additional information on results' for more details. Historical control data for the parameters is described, when available.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females treated with 250 mg/kg bw/day showed a statistically significant reduction (p<0.01-0.001) in food consumption throughout the treatment period.
No differences as compared to the control group were detected for food consumption in females treated with 100 or 25 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No differences as compared to the control group were detected for water consumption.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related macroscopic abnormalities were detected in females treated with 250, 100 or 25 mg/kg bw/day.
One female treated with 100 mg/kg bw/day had a scab on the right shoulder and one control female had an enlarged right kidney. These findings were considered to be incidental findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
At 100 mg/kg bw/day, treatment was associated with slightly lower body weight gains between Days 8 and 11 of gestation and lower cumulative body weight gain between Days 3 and 11 and Days 3 and 17 of gestation. Body weight gain for these females during the remaining periods was comparable to controls. No macroscopic necropsy findings were apparent for adult females at this dosage. Given the transient nature of the effects on body weight gain, this dosage is considered to represent a No Observed Adverse Effect Level (NOAEL) for the pregnant female.

Number of abortions:
no effects observed
Description (incidence and severity):
There was no treatment-related effect observed in the pre- and post-implantation loss in all dose groups, when comparing to the control group:- preimplantation loss: 13.3%, 13.6%, 10.5%, 13.2% at 0, 25, 100 and 250 mg/kg bw/day-post-implantation loss: 0.5%, 0.9%, 1.4%, 1.2% at 0, 25, 100 and 250 mg/kg bw/day.All data was within the historical control range available for this species and strain. See section 'Additional information on results' for more details
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no treatment-related effect observed in the pre- and post-implantation loss in all dose groups, when comparing to the control group:
- preimplantation loss: 13.3%, 13.6%, 10.5%, 13.2% at 0, 25, 100 and 250 mg/kg bw/day
- post-implantation loss: 0.5%, 0.9%, 1.4%, 1.2% at 0, 25, 100 and 250 mg/kg bw/day.
All data was within the historical control range available for this species and strain. See section 'Additional information on results' for more details.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no total litter losses by resorption observed. See Section 'Additional information on results' for more details.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on early or late resorptions observed. See Section 'Additional information on results' for more details.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the number of dead fetuses observed. See Section 'Additional information on results' for more details.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There was no treatment-related, toxicologically relevant effect on the number of pregnant dams per dose group. The number of pregnant females per dose group were 24/24, 23/24, 23/24 and 22/24 at 0, 25, 100 and 250 mg/kg bw/day, resp. No historical control data is available for this parameter. See section 'Additional information on results' for more details
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There was no treatment-related, toxicologically relevant effect on the number of pregnant dams per dose group. The number of pregnant females per dose group were 24/24, 23/24, 23/24 and 22/24 at 0, 25, 100 and 250 mg/kg bw/day, resp. No historical control data is available for this parameter. See section 'Additional information on results' for more details
Other effects:
not specified
Details on maternal toxic effects:
There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio or pre- or post-implantation losses at 25, 100 or 250 mg/kg bw/day.
Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 25, 100 or 250 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There is no treatment-related effect on the mean fetal body weight, mean male fetal body weight or mean female fetal body weight. See section 'Additional information on results' for further details. Available historical control data is described.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There is no treatment-related effect on the number of live offspring. The percentage of live offspring was calculated as the number of live implants - the number of implants x 100. This means for the dose groups: 99.3%, 99.3%, 98.6% and 98.5% for the control, 25, 100 and 250 mg/kg bw/day. See section 'Additional information on results' for further details. The available historical control data is described.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no treatment-related effect observed on sex ratio. See Section 'Additional information on results' for more details. Historical control data is described, when available.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no treatment-related effects observed on litter weight or litter size. See Section 'Additional information on results' for more details. Historical control data is described, when available.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was no treatment-related effects observed on postnatal survival. See Section 'Additional information on results' for more details. Historical control data is described, when available.
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related effect on external malformations was observed in all dose groups. See section 'additional information on results' for further details. Available historical control data is described.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase (p<0.05-0.01) in the number of fetuses/litters showing incomplete ossification of the parietal, interparietal, hyoid, sacral arch, sternebra and femur were evident at 250 mg/kg bw/day. The effect on the sternebra also extended to fetuses/litters at 100 and 25 mg/kg bw/day. A statistically significant increase (p<0.05) in the number of fetuses/litters showing incomplete ossification of the zygomatic process of squamosal were evident at 25 and 250 mg/kg bw/day. A statistically significant increase (p<0.05) in the number of fetuses/litters showing incomplete ossification of the hyoid was evident at 100 mg/kg bw/day. With the exception of the sacral arch and sternebra, true dose related responses were not evident in the remaining parameters and group mean values were generally within historical control ranges (excluding interparietal, sacral arch and femur). In the absence of any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses and in the absence of an effect on mean fetal weight, the observation of isolated affected skeletal structures can be considered unlikely to represent true developmental abnormalities.
See Section 'Additional information on results' for further details. Available historical control data is described.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related effect on visceral malformations was observed in all dose groups. See section 'additional information on results' for further details. Available historical control data is described.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related changes up to 250 mg/kg bw/day
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: Summary of Female Performance: number of pregnant and non-pregnant dams per dose group

Category

Number of Females at Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Initial Group Size

24

24

24

24

Non-Pregnant

0

1

1

2

Pregnant

24

23

23

22

° No historical data available

Table 2: Group Mean Litter Data Values: pre- and postimplantation loss, number and percent

Dose Level (mg/kg bw/day)

 

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Implantation Loss
%

       Number of live implants

Early

Late

Total

Pre

Post

 Male  Female  Total

0 (Control)

mean

16.3

14.0

0.0

0.0

0.1

13.3

0.5

 6.0 7.9  13.9 

sd#

2.1

1.6

0.2

0.2

0.3

8.2

1.8

 1.8 2.0  1.5 

number of animals

24

24

24

24

24

24

24

 24 24  24 

25

mean

16.7

14.4

0.1

0.0

0.1

13.6

0.9

 6.7 7.6  14.3 

sd#

1.8

1.8

0.3

0.0

0.3

7.9

2.5

 1.7 1.9  1.9 

number of animals

23

23

23

23

23

23

23

 23 23  23

100

mean

15.9

14.2

0.2

0.0

0.2

10.5

1.4

 7.3 6.7  14.0 

sd#

1.5

1.5

0.5

0.2

0.6

7.6

3.9

 1.9 1.7 1.5

number of animals

23

23

23

23

23

23

23

 23 23  23 

250

mean

15.9

13.7

0.1

0.1

0.2

13.2

1.2

 6 7.5  13.5 

sd#

2.1

2.0

0.3

0.3

0.5

8.9

3.2

 2.6 2.2  1.9 

number of animals

22

22

22

22

22

22

22

 22 22  22 
Historical data  range$ 11 - 20  9 - 18 0 - 2  0 - 4 0 - 4  0 - 31.8  0 - 6.9   3 - 10 3 - 10  10 - 17 
 mean 15   14 11.4  1.3   6 13 
 sd# 2  0 10.2  2.8   2
 number of animals 194  200  210 210  210  203  198   201 196  203 

# sd: range = minimum to maximum

$ range = mean + / - 2 standard deviations

pre-implantation loss = number of corpora lutea - number of implants; pre-implantation loss % = pre-implantation loss / number of corpora lutea x 100; post-implantation loss = number of implants - number of live implants (total); postimplantation loss % = post implantation loss / number of implants x 100

Table 3: Group Mean Body Weight Values:

Dose Level (mg/kg bw/day)

 

Body Weight (g) on Day of Gestation

3

4

5

8

11

14

17

20

0 (Control)

mean

252.8

257.2

263.0

276.1

292.8

311.9

339.1

382.5

sd

23.1

24.4

25.8

26.7

28.8

29.2

31.8

33.6

number of animals

24

24

24

24

24

24

24

24

25

mean

251.8

258.4

263.7

276.7

293.3

312.8

338.0

383.3

sd

16.4

19.4

19.0

18.7

20.4

21.0

21.2

24.4

number of animals

23

23

23

23

23

23

23

23

100

mean

248.6

253.4

257.7

269.1

283.1

302.4

326.8

370.7

sd

13.2

15.3

15.3

15.5

17.0

16.7

17.9

21.0

number of animals

23

23

23

23

23

23

23

23

250

mean

252.0

253.6

254.3

260.1*

268.7**

290.5**

316.1**

358.9*

sd

18.6

18.2

17.5

18.6

18.9

19.1

20.6

26.5

number of animals

22

22

22

22

22

22

22

22

Historical data  range  204 - 291 / °  214 - 306 225 - 321  243 - 341  258 - 361  283 - 396  322 - 447 
 mean 247  / ° 260  273  292  310  340  385 
 sd  22 / ° 23  24  25  26  28  31 
 number of animals  288 / ° 290  290  289  290  289  290 

° For Day 4 no historical data is available

° *p<0.05; ** p<0.01

Table 4: Group Mean Body Weight Change Values

Dose Level (mg/kg bw/day)

 

Body Weight Change (g) during Days of Gestation

3 to 4

4 to 5

5 to 8

8 to 11

11 to 14

14 to 17

17 to 20

0 (Control)

mean

4.4

5.8

13.1

16.8

19.1

27.2

43.4

sd

4.1

3.7

3.4

4.1

3.5

4.9

5.9

number of animals

24

24

24

24

24

24

24

25

mean

6.6

5.3

13.1

16.5

19.6

25.2

45.3

sd

3.7

3.0

4.4

3.4

3.8

3.5

6.8

number of animals

23

23

23

23

23

23

23

100

mean

4.9

4.3

11.3

14.0*

19.3

24.4

43.9

sd

3.9

2.9

5.0

5.6

4.7

4.8

5.8

number of animals

23

23

23

23

23

23

23

250

mean

1.6

0.6***

5.8***

8.6**

21.9

25.6

42.7

sd

3.9

4.9

5.2

16.4

17.3

6.5

10.6

number of animals

22

22

22

22

22

22

22

° * p<0.05; ** p<0.01; *** p<0.001

Table 4a: Group Mean Body Weight Change Values - Historical Data

   

 Days 3 -5

 Days 5 - 6

Days 6 - 7 

 Days 7 - 8

 Days 8 - 11

 Days 11 - 14

 Days 14 - 18

 Days 17 - 20

Historical data: body weight change (g)

 range

 3 - 22

-5 - 10 

-2 - 13 

-2 - 11 

 10 - 29

10 - 27 

20 - 40 

32 - 53 

 mean

 12

19 

18 

30 

44 

 sd

 5

 number of animals

 286

128 

205 

298 

293 

288 

288 

289 

Table 5: Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values

Dose Level (mg/kg bw/day)

 

Body Weight (g) on Days of Gestation

Body Weight Change (g) during Days of Gestation

Gravid Uterus Weight
(g)

Adjusted
Body Weight (g)
 Day 20

Adjusted
Body Weight Change (g)
3-20

3

20

3-20

0 (Control)

mean

252.8

382.5

129.7

82.880

299.6

46.8

sd

23.1

33.6

12.9

8.641

29.4

10.1

number of animals

24

24

24

24

24

24

25

mean

251.8

383.3

131.4

85.789

297.5

45.6

sd

16.4

24.4

10.7

9.994

22.1

10.7

number of animals

23

23

23

23

23

23

100

mean

248.6

370.7

122.1

82.770

287.9

39.4

sd

13.2

21.0

14.0

7.550

17.4

10.7

number of animals

23

23

23

23

23

23

250

mean

252.0

358.9

106.9

78.508

280.4*

28.4***

sd

18.6

26.5

12.1

8.685

24.4

12.4

number of animals

22

22

22

22

22

22

°* p<0.05; ** p<0.01; *** p<0.001

° Available Historical Data:

- Gravid Uterus Weight (g):

~ Range: 63.56 - 106.59 ~ Mean: 85.08 +/- 10.76 ~ Number of animals: 286

- Adjusted Body Weight Day 20 (g):

~ Range: 247 - 353 ~ Mean 300 +/- 26 ~ Number of animals: 286

- Adjusted Body Weight Change Days 5 - 20 (g):

~ Range: 20 - 64 ~ Mean 45 +/- 11 ~ Number of animals: 126

Table 6: Mean Number and Percent of Live Offspring

    Dose Level (mg/kg bw/day)             Number of Live Implants  % Male Fetuses  total live implants / number of implants x 100
 Male Female  Total     
    0 (Control)  mean  6.0 7.9  13.9  43.1  99.3 
 sd 1.8  2.0  1.5  12.7 
    25  mean  6.7 7.6  14.3  46.8  99.3 
 sd  1.7 1.9  1.9  11.2   /
    100  mean 7.3  6.7  14.0  51.9  98.6 
 sd 1.9  1.7  1.5  11.7 
    250  mean 6.0  7.5  13.5  43.9  98.5 
 sd 2.6  2.2  1.9  17.1 
    Historical data  mean  6 13  48.5   /
 sd  2 31.1 

Table 7: Mean Foetal / Pup Body Weight by Sex and Sexes Combined

 Dose Level (mg/kg bw/day   Mean Male Fetal Weight (g)   Mean Female Fetal Weight (g)  Mean Fetal Weight (g)  Mean Placental Weight (g)  Litter Weight (g)  Total Placental Weight (g)
0 (Control)  mean 3.959  3.747  3.840  0.541  53.382  7.537 
 sd 0.201  0.152  0.168  0.046  5.739  1.081 
number of animals  24  24  24  24  24  24 
25 mean  3.923  3.733  3.825  0.548  54.584  7.803 
sd  0.314  0.270  0.287  0.053  7.193  1.117 
 number of animals 23  23  23  23  23  23 
100  mean 3.922  3.686  3.805  0.566  52.922  7.883 
 sd 0.282  0.313  0.290  0.071  5.116  1.234 
number of animals  23  23  23  23  23  23 
250  mean 3.778  3.618  3.691  0.560  49.832  7.475 
 sd 0.345  0.320  0.319  0.106  6.747  1.038 
 number of animals 22  22  22  22  22  22 
Historical data  range 3.64 - 4.59  3.36 - 4.45  3.46 - 4.54  0.4 - 0.73   35.43 - 71.14 5.24 - 9.71 
 mean 4.11   3.9 0.57  53.28  7.47 
 sd  0.24  0.27 0.27  0.08  8.93  1.12 
 number of animals 200  203   203 203  202   202

° range = mean + / - 2 standard deviations

Table 8: Summary Incidence of Fetal External Findings

External Findings

Dose level (mg/kg bw/day)

0 (Control)

25

100

250

Number of fetuses (litters) examined

334 (24)

329 (23)

321 (23)

298 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Small fetus

5

4

1.5

6

5

1.8

7

5

2.0

13

4

5.6

Small placenta

3

2

0.9

4

3

1.2

1

1

0.3

1

1

0.3

Pale fetus

1

1

0.3

0

0

0.0

3

3

0.9

1

1

0.3

Large placenta

0

0

0.0

1

1

0.3

5

2

1.5

12

2

5.4

Pale placenta

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.3

Total

8

5

2.4

8

6

2.3

14

8

4.1

15

5

6.4

 %†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

 Table 9: Summary Incidence of Fetal Visceral Findings

Visceral Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

172 (24)

169 (23)

166 (23)

156 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

External

 

 

 

 

 

 

 

 

 

 

 

 

Hemorrhage

1

1

0.6

1

1

0.5

0

0

0.0

0

0

0.0

Situs inversus

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Head

 

 

 

 

 

 

 

 

 

 

 

 

Tongue - short

0

0

0.0

0

0

0.0

1

1

0.7

0

0

0.0

Rugae - non-uniform patterning

5

5

2.8

11

8

6.6

7

7

4.3

13

9

7.9

Brain - olfactory ventricle - enlarged

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Brain - cerebral aqueduct - enlarged

0

0

0.0

0

0

0.0

2

1

1.1

0

0

0.0

Abdomen

 

 

 

 

 

 

 

 

 

 

 

 

Liver - additional lobe between right and left median

2

2

1.2

1

1

0.7

0

0

0.0

0

0

0.0

Liver - papillary process - reduced in size

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Umbilical artery - left-sided

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

Testis - partially undescended

1

1

0.5

1

1

0.6

0

0

0.0

1

1

0.6

Ureter - kinked

28

13

16.3

17

11

10.2

10

7

5.7

12

8

7.3

Ureter - dilated

23

11

13.4

12

8

7.4

7

4

4.0*

3

3

1.9**

Renal pelvic cavitation - increased

9

5

5.1

10

7

5.9

4

4

2.3

11

6

6.4

Renal papilla - absent

2

2

1.2

1

1

0.6

1

1

0.5

4

2

2.5

Renal medulla - reduced in size

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Renal medulla - absent

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Thorax

 

 

 

 

 

 

 

 

 

 

 

 

Thyroid - Isthmus thickened

0

0

0.0

0

0

0.0

2

1

1.1

0

0

0.0

Thyroid - parathyroid - enlarged

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Thymus - lobe partially undescended

9

5

4.9

10

6

6.0

4

4

2.4

13

9

8.0

Lungs - right lobe - single lobe present

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

* p<0.05; ** p<0.01

Table 9a: Summary Incidence of Fetal Visceral Findings Continued

Visceral Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

172 (24)

169 (23)

166 (23)

156 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Thorax (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Atrium - enlarged

1

1

0.6

0

0

0.0

1

1

0.6

0

0

0.0

Single atrioventricular valve

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Atrial septal defect

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Ventricle - enlarged

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Ventricular septal defect

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Total

45

17

25.7

37

18

22.2

25

16

14.8

39

15

23.7

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

Table 10: Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

162 (24)

160 (23)

155 (23)

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull

 

 

 

 

 

 

 

 

 

 

 

 

Fontanelle (anterior) - large

2

1

1.0

0

0

0.0

4

4

2.5

1

1

0.9

Nasal - incomplete ossification

15

8

8.9

23

13

14.3

14

8

8.7

32

14

21.1

Nasal/Frontal - sutural bone

1

1

0.6

1

1

0.5

0

0

0.0

0

0

0.0

Frontal - incomplete ossification

3

3

1.7

4

4

2.5

4

4

2.4

12

9

7.8

Frontal - unossified area

3

3

1.8

1

1

0.7

5

4

3.1

14

7

9.9

Frontal - fissure

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

Parietal - incomplete ossification

9

6

5.3

14

10

8.6

12

7

7.4

27

12

17.9*

Parietal/Interparietal - sutural bone

0

0

0.0

0

0

0.0

0

0

0.0

2

2

1.3

Interparietal - incomplete ossification

32

13

19.9

27

12

16.9

26

13

16.6

54

17

37.2*

Occipital (Supra-occipital) - incomplete ossification

26

12

16.1

20

12

12.6

22

13

14.2

46

16

30.7

Occipital (Supra-occipital) - unossified area(s)

11

9

7.1

10

8

6.3

9

6

5.7

6

6

4.0

Squamosal - incomplete ossification

18

9

11.3

22

11

13.6

18

9

11.5

33

13

22.1

Squamosal - unossified area(s)

0

0

0.0

1

1

0.5

2

2

1.2

4

3

2.5

Jugal - incomplete ossification

5

4

2.9

11

8

7.3

6

6

4.2

17

9

11.7

Zygomatic process of maxilla - incomplete ossification

9

7

5.5

19

11

12.4

18

10

11.7

25

11

17.4

Zygomatic process of maxilla -elongated

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Zygomatic process of squamosal - incomplete ossification

0

0

0.0

5

4

3.0*

1

1

0.6

9

5

6.2*

Zygomatic process of squamosal - fused to jugal

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Premaxilla - incomplete ossification

4

3

2.3

1

1

0.6

4

4

2.3

9

6

6.2

Hyoid - incomplete ossification

10

9

5.8

16

11

9.7

24

15

15.7*

17

8

11.1*

Hyoid - not ossified

21

12

12.5

17

12

9.7

26

13

16.6

30

14

19.2

Tympanic annulus - incomplete ossification

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

* p<0.05

Table 10a: Summary Incidence of Fetal Skeletal Findings - Continued

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

162 (24)

160 (23)

155 (23)

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Presphenoid - incomplete ossification

1

1

0.7

5

2

2.6

2

2

1.2

3

2

1.9

Presphenoid - not ossified

0

0

0.0

2

2

1.1

0

0

0.0

3

3

1.9

Vertebral column

 

 

 

 

 

 

 

 

 

 

 

 

Odontoid - ossification present

3

3

1.8

0

0

0.0

0

0

0.0

0

0

0.0

Ventral arch of vertebra 1 - ossification present

30

16

18.6

47

22

29.8

40

19

26.8

49

16

34.8

Cervical (neural) arch - incomplete ossification

13

7

7.5

10

7

6.7

9

8

5.7

8

6

5.4

Cervical (neural) arch - fused

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Thoracic centrum - incomplete ossification

5

4

2.9

5

4

3.4

2

2

1.2

5

4

3.5

Thoracic centrum - not ossified

1

1

0.6

0

0

0.0

2

2

1.2

0

0

0.0

Thoracic centrum - bipartite ossification

2

2

1.1

3

3

2.1

1

1

0.6

2

2

1.7

Thoracic centrum - dumb-bell-shaped

18

12

11.4

15

11

9.2

12

9

8.1

20

12

14.0

Thoracic centrum - asymmetrically ossified

4

2

2.3

2

2

1.6

4

4

2.7

2

2

1.2

Lumbar centrum - incomplete ossification

1

1

0.6

1

1

0.5

0

0

0.0

0

0

0.0

Lumbar (neural) arch - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Sacral centrum - incomplete ossification

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Sacral (neural) arch - incomplete ossification

25

12

15.0

28

12

17.7

33

16

21.2

51

17

34.9*

Sacral (neural) arch - not ossified

0

0

0.0

0

0

0.0

1

1

0.5

1

1

0.8

Caudal vertebrae - less than 4 ossified

39

18

23.6

39

15

23.9

43

15

27.4

59

16

39.6

Number of pre-sacral vertebrae = 25/27

2

2

1.3

0

0

0.0

1

1

0.6

0

0

0.0

Ribs

 

 

 

 

 

 

 

 

 

 

 

 

Ossification centre - associated with 7th cervical vertebra

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

14th rib - extra - associated with 1st lumbar vertebra

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Ossification centre - associated with 1st lumbar vertebra

5

5

3.2

9

3

5.8

6

5

4.0

2

2

1.3

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

* p<0.05

Table 10b: Summary Incidence of Fetal Skeletal Findings - Continued

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

162 (24)

160 (23)

155 (23)

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Ribs (continued)

 

 

 

 

 

 

 

 

 

 

 

 

One or more ribs - wavy

1

1

0.7

1

1

0.6

0

0

0.0

1

1

0.6

One or more ribs - thickened

1

1

0.7

0

0

0.0

1

1

0.6

0

0

0.0

Rib - short

1

1

0.7

1

1

0.5

0

0

0.0

0

0

0.0

Rib - rudimentary

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Rib - fused

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Rib - no articulation point

1

1

0.6

1

1

0.5

0

0

0.0

0

0

0.0

Costal cartilage - misaligned

4

4

2.4

2

2

1.0

2

2

1.2

1

1

0.6

Costal cartilage - not fused to sternebra

28

12

17.9

16

8

10.3

14

9

9.2

13

10

9.6

Sternebrae

 

 

 

 

 

 

 

 

 

 

 

 

Sternebra - incomplete ossification

0

0

0.0

5

4

3.0*

5

5

3.1*

8

7

5.4**

Sternebra - not ossified

2

2

1.2

0

0

0.0

2

2

1.2

0

0

0.0

Sternebra - bipartite ossification

1

1

0.6

0

0

0.0

1

1

0.9

0

0

0.0

Sternebra - misaligned

2

2

1.2

3

3

1.7

6

6

4.0

5

4

3.6

Sternebra - misshapen

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Xiphoid cartilage - partially split

4

4

2.5

6

6

3.8

11

7

8.0

6

4

4.4

Pectoral Girdle

 

 

 

 

 

 

 

 

 

 

 

 

Scapula - bent

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Scapula - misshapen

3

3

1.8

0

0

0.0

1

1

0.7

4

4

2.7

Pelvic Girdle

 

 

 

 

 

 

 

 

 

 

 

 

Ischium - incomplete ossification

1

1

0.6

2

2

1.3

1

1

0.6

6

5

4.0

Pubis - not ossified

1

1

0.6

2

2

1.1

1

1

0.6

3

3

2.2

Pubis - incomplete ossification

8

6

4.6

5

5

3.1

12

7

7.6

15

11

9.9

Table 10c: Summary Incidence of Fetal Skeletal Findings - Continued

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

25

100

250

Number of Fetuses (litters) Examined

162 (24)

160 (23)

155 (23)

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Forelimbs

 

 

 

 

 

 

 

 

 

 

 

 

Metacarpal - not ossified

56

19

34.3

46

16

28.4

63

19

39.5

83

18

56.4

Metacarpal - incomplete ossification

2

2

1.2

2

2

1.2

3

3

1.9

9

6

5.8

Forepaw phalanges - 1 or more - ossified

8

4

4.8

14

7

8.5

10

6

6.3

8

6

5.6

Humerus - incomplete ossification

1

1

0.6

3

3

1.8

2

2

1.2

7

5

4.8

Humerus - hole

3

3

1.8

0

0

0.0

1

1

0.6

0

0

0.0

Humerus - bent

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

Hindlimbs

 

 

 

 

 

 

 

 

 

 

 

 

Metatarsal - 1st - ossified

1

1

0.8

1

1

0.5

0

0

0.0

0

0

0.0

Metatarsal - not ossified

0

0

0.0

0

0

0.0

2

2

1.2

0

0

0.0

Metatarsal - incomplete ossification

1

1

0.6

0

0

0.0

2

2

1.2

2

2

1.6

Femur - incomplete ossification

11

7

6.9

6

4

3.8

17

8

10.1

30

14

20.6**

Total

136

24

84.3

132

23

82.3

134

23

86.8

135

22

95.2

%†: Group mean percent

NF: Number of Fetuses

NL: Number of litters

* p<0.05; ** p<0.01

Table 11 Caesarea necropsy findings

   number of females  V  ED  LD  DF  NP
 control  24  22  1  1    
 25 mg/kg bw/d  24  20  3      1
 100 mg/kg bw/d  24  20  3  1    1
 250 mg/kg bw/d  24  19  2  1  1  2

V: viable fetuses; ED: early death (no visible distinction between placental/decidual tissue and embryonic tissue); LD: late death: seperate embryonic/fetal and placental tissue visible; DF: dead fetus: a fetus that died shortly before necropsy; NP: non pregnant

Conclusions:
The oral (gavage) administration of the test substance to pregnant rats during gestation at dose levels of 25, 100 and 250 mg/kg bw/day resulted in reductions in body weight gain and food consumption throughout the treatment period in females treated with 250 mg/kg bw/day. Although a slight reduction in body weight gain and cumulative body weight gain was noted for the 100 mg/kg bw/day dose group between Days 8 and 11 and Days 3 and 11 and 3 and 17 respectively, the effects were minimal and body weight gains for the remainder of the periods were comparable to controls. Consequently, 100 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.
No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
90 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
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

A pre-natal development toxicity study via oral gavage is performed in female Sprague-Dawley rats (OECD Guideline 414). The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 25, 100, and 250 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Distilled Water) to serve as a control. Clinical signs, body weight change, food and water consumptions were monitored during the study. All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.


There were no unscheduled deaths. There were no significant adverse clinical signs evident in treated females. Females treated with 250 mg/kg bw/day showed a reduction in body weight gain between Days 4 and 11 and body weight for these females was reduced from Day 8 onwards. Cumulative body weight gain was reduced in these females throughout the treatment period. Body weight and body weight gain, when adjusted for gravid uterus weight, was also reduced when compared to controls. A reduction in body weight gain was evident in females treated with 100 mg/kg bw/day between Days 8 and 11 of gestation. Improvement was evident thereafter, however, a reduction in cumulative body weight gain was evident in these females between Days 3 and 11 and Days 3 and 17. No such effects were evident in females treated with 25 mg/kg bw/day. Females treated with 250 mg/kg bw/day showed a reduction in food consumption throughout the treatment period. No differences as compared to the control group were detected for food consumption in females treated with 100 or 25 mg/kg bw/day. No differences as compared to the control group were detected for water consumption. No treatment related macroscopic abnormalities were detected in females treated with 250, 100 or 25 mg/kg bw/day.


The number of implantations, subsequent embryofetal survival, live litter size and sex ratio on Day 20 of gestation were considered to be unaffected by maternal treatment at 25, 100 or 250 mg/kg bw/day. Mean fetal, placental and litter weights were also considered to have been unaffected by maternal treatment at 25, 100 or 250 mg/kg bw/day. External examination of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 25, 100 or 250 mg/kg bw/day. Findings at detailed skeletal and visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 25, 100 or 250 mg/kg bw/day.


 


The oral (gavage) administration of the test substance to pregnant rats during gestation at dose levels of 25, 100 and 250 mg/kg bw/day resulted in reductions in body weight gain and food consumption throughout the treatment period in females treated with 250 mg/kg bw/day. Although a slight reduction in body weight gain and cumulative body weight gain was noted for the 100 mg/kg bw/day dose group between Days 8 and 11 and Days 3 and 11 and 3 and 17 respectively, the effects were minimal and body weight gains for the remainder of the periods were comparable to controls. Consequently, 100 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.


No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.


 


In the prenatal developmental toxicity in New Zealand white rabbits Latha (2022) evaluated the developmental and maternal toxicity of the test item administered to pregnant rabbits by oral route during gestation days (GD) 6 to GD 28.  This study provided a rational basis for risk assessment in humans. The results of this study were used to establish the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity of the test item in rabbits.


The dose levels for this study were selected based on Dose Range Finiding study (DRF) study under study number N5369. Dose levels of 0, 45, 90 and 180/120 mg/kg/day were evaluated in pregnant rabbits (6/group) treated with the test item or vehicle from GD 6 to GD 28. Due to test item related mortalities at 180 mg/kg/day, the dose was reduced to 120 mg/kg/day from GD 9 onwards.There was  reduction in body weight and food consumption and reduction in mean fetal weights at 90 mg/kg/day . Based on the results of of dose range finding study in pregnant rabbits, high dose of 90 mg/kg/day was selected for the definitive study. Mid dose of 45 mg/kg/day and low dose of
15 mg/kg/day was selected to provide a graded response to the test item.


In the definitive study, 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred. Test item dssolved in vehicle (Milli-Q water) was administered at 0, 15, 45 and 90 mg/kg/day at the dose volume of 10 mL/kg body weight. The control group received the vehicle only.


The following parameters and end points were evaluated in this study: Clinical signs, body weight, body weight gain, food consumption, caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses. The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed, and examined for external malformations. All the live fetuses were examined for visceral and skeletal variations and malformations.


Dose formulations were analysed twice i.e once during initiation and once at termination of treatment, the results of analysis indicated  within ± 10.0% of the nominal concentration and the relative standard deviation (%RSD) was less than 10%.


Main findings from the study are summarized below:


There were no unscheduled deaths, clinical signs, or any gross pathological findings that could be attributed to the test item. No toxiclogically significant changes in maternal body weight or maternal body weight gain was observed at the doses of 15 and 45 mg/kg/day. At 90 mg/kg/day, there was a non-significant reduction in maternal body weight gain as compared to vehicle control group. No toxiclogically significant changes in maternal food intake were observed at 15 and 45 mg/kg/day. At 90 mg.kg/day there was a significant reduction of food consumption by 17 to 29% associated with reduction in maternal body weight gain indicating maternal toxicity. No significant changes in any maternal developmental toxicity endpoint were observed at 15, 45 and 90 mg/kg/day. No gross pathological changes in the placenta were observed in any of the animals. No dead fetuses were observed at any dose level. No significant changes in litter size, sex ratio, or fetal weight were observed up to the dose of 90 mg/kg/day. The results from external, visceral, and skeletal examinations did not reveal any significant effects that could be attributed to the test item. Based on the above findings, under the test conditions used in this study, the NOAEL for maternal toxicity was considered at 45 mg/kg/day as reduction in body weight gain and food consumption was observed at 90 mg/kg/day. The NOAEL for developmental toxicity and teratogenicity was considered at 90 mg/kg/day since the other maternal and litter parameters were unaffected by treatment and there was no evidence of test item related fetal findings up to the dose of 90 mg/kg/day.


 

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, the substance should not be classified as toxic to reproduction.

Additional information