Registration Dossier

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:
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 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.

Link to relevant study records
Reference
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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted 22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
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 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
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
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.

 


 

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.