1-[bis[3-(dimethylamino)propyl]amino]propan-2-ol

Endpoint summary

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

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-07 - 2012-08-16

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.

Reason / purpose for cross-reference:

reference to same study

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: 345-395 grams for the males and 211-258 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 accommodate 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: 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

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*, ovaries*, 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 - cervical*, spinal cord - midthoracic*, spinal cord - lumbar*, lacrimal glands; other: unique animal identifier (not for evaluation), gross findings*

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.

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; these 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.

Endocrine findings:

no effects observed

Description (incidence and severity):

No effects were observed in adrenal glands, pituitary and parathyroid/thyroid glands.

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;

Key result

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:

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (nominal)

System:

hepatobiliary

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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

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.

Reference 2

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

Quality of whole database:

GLP-compliant study, based on OECD guideline

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 in both rats (as first species) and rabbits (as second species). There are no effects observed that would trigger performance of an extended one-generation reproductive toxicity study. 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

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021/02/28 - 2021/12/28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Dose range finding study

GLP compliance:

no

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 to 7 Months
- Weight at study initiation: 2,5 - 3kg
- Fasting period before study: no
- Housing: The rabbits will be housed individually (except during cohabitation when the female rabbits will be cohabited with males in 1:1 ratio)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: From: To: 28/02/2021 - 05/04/2021

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until visual conformation of mating
- Proof of pregnancy: visual confirmation of mating referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 6 to GD 28 of presumed gestation

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

45 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

Remarks:

decreased to 120mg/kg bw/d from GD 9 onwards

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for clinical signs were performed twice a day, pre-dose and post-dose (30 minutes to 2 hour post dose, approximately) during treatment period and at least once on other days.
Each rabbit was observed twice daily during treatment for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the assessment if there wereno clinical signs of concern, then the observation was carried out once during weekend and public holidays.

DETAILED CLINICAL OBSERVATIONS: No

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

FOOD CONSUMPTION : Yes
- A known weight of food (food input) will be provided on Day 0. The food left over will be recorded and replenished to a known weight on Days 3, 6, 9, 12, 15, 18, 21, 24 and 27 and food output on Day 29 of presumed gestation will be recorded.

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, will be performed on all animals in the study including dead, moribund and those sacrificed pre-term or at term

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: No
- Skeletal examinations: No
- Head examinations: No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs or mortalities at the doses of 45 and 90 mg/kg/day. At the dose of 180 mg/kg/day (G4), there were 2/6 mortalities on GD 8. Hence the dose was reduced to 120 mg/kg/day
from GD 9 onwards. On GD 17, 2 rabbits were found dead.

Mortality:

mortality observed, treatment-related

Description (incidence):

At the dose of 180 mg/kg/day (G4), there were 2/6 mortalities on GD 8. Hence the dose was reduced to 120 mg/kg/day from GD 9 onwards. On GD 17, 2 rabbits were found dead.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean maternal body weights of rabbits at
tested dose of 45 mg/kg/day were statistically comparable to vehicle control group.
At 90 mg/kg/day, the mean body weights of rabbits were statistically comparable to vehicle control group. However, there was a tendency towards reduction in body weights and the body weight gain was significantly reduced during GD27-29 and the adjusted body weight gain was significantly reduced as compared to vehicle control group.
Due to only 2 remaining animals in the high dose group the analysis of body weight (change) was not done.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 45 mg/kg/day, the mean food consumption was statistically comparable to vehicle control group.
At 90 mg/kg/day, the food consumption was reduced (from -24 to 65%) during different periods of gestation with statistical significance at GD21-24, 24-27, 27-29, 6-29 and 0-29.
The reduction in body weight and food consumption at 90 mg/kg/day was considered treatment related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no gross pathological findings in any females of all dose levels, sacrificed at caesarean section except for an incidence of pale liver of all lobes in a dead rabbit at 180/ 120 mg/kg/day.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Mean or individual values of maternal parameters comprising of uterine weight, number of corpora lutea, implantations, early and late resorptions and pre and post implantation loss at 45 and 90 mg/kg/day were comparable to the vehicle control group. Gross evaluation of placenta revealed no findings. Due to only two surviving animals in the high dose group the statistical analysis was not possible.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 90 mg/kg/day, there was significant reduction in mean fetal weights of males (-14%), females (-18%) and male and female (-15%) as compared to vehicle control group. All other litter data parameters comprising of total number of fetuses, number of live fetuses were statistically comparable to vehicle control group at 45 and 90mg/kg bw/day.
Due to only one litter remaining at the high dose 180/120 mg/kg bw/d the data was not analysed statistically.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

No abnormality was noticed on external observations of fetuses at any of the dose levels.

Executive summary:

The objective of this preliminary study was to select the doses for the
definitive prenatal developmental toxicity study in New Zealand white rabbits. This study evaluated the maternal and developmental toxicity of the test item when administered orally to pregnant New Zealand white rabbits during gestation days (GD) 6 to 28. In this dose range finding study, 24 mated female rabbits were assigned to four groups (G1: Vehicle control, G2: low dose, G3: mid dose and G4: High dose). GD 0 for each individual female rabbit in the study was considered as the day on which mating had occurred. The rabbits in the high dose group were treated at 180 mg/kg/day from GD6 to GD8. Due to test item related mortalities, the dose was reduced to 120 mg/kg/day from GD 9 onwards.

Based on the mortalities of 4/6 females in the 180/120 mg/kg/day this dose is above the highest tolerable dose. Based on the reduction in body weight and food consumption and reduction in mean fetal weights at 90 mg/kg/day, the following doses are proposed for the definitive study: 90, 45, 15 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-08-19 - 2022-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other 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: XXXXXXXXXXXX
- 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: 2021-08-19 To: 2021-09-24

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

XXXXXXXXXXXXXXXXXXX

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: XXXXXXXXXX
- 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:

Gestation day 6 until Gestation day 28

Frequency of treatment:

daily

Duration of test:

XXXXXXXXXX

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 GD 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.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2017-09-28 to 2017-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was performed to investigate the effects of the test item on embryonic and fetal development, following repeated administration by gavage at dose levels 25, 100 and 250 mg/kg bw/day to the Sprague-Dawley Crl:CD(R) (SD) IGS BR strain rat during gestation, including the period of organogenesis. The results provide information for the selection of dose levels for use in further investigation of pre-natal development in the rat.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: PFW160721
- Physical State/Appearance : clear colorless liquid
- Expiry Date : 2018-12-30
- Purity: ca. 98.32%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at ambient temperature/humidity in darkness; may be used/formulated in light
- Stability and homogeneity of the test material in the vehicle under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item formulations were previously determined by Envigo Research Limited, Shardlow, UK Analytical Services under Envigo Study Number: 41500461. Results showed the formulations to be stable at concentrations of 0.5 and 10 mg/mL for up to 21 days when stored at approximately 4°C, in the dark. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled Water.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD® (SD) IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: thirty-two time-mated female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: Not indicated. Animals were delivered in one batch containing females prior to Day 1 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
- Weight at study initiation: On arrival, the females weighed 186g to 266g.
- Fasting period before study: not indicated
- Housing: The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The animals were housed in a single air-conditioned room within the test facility.
- Diet: ad libitum, a pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used.
- Water: ad libitum, mains drinking water was supplied from polycarbonate bottles attached to the cage.
- The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15 air changes/hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2017-10-06 To: 2017-10-23

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared at the appropriate concentrations as a solution in distilled water
- Formulations at the low and intermediate dose levels were prepared once and stored at approximately 4 °C in the dark. High dose formulations were prepared daily and administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.

VEHICLE
- Concentration in vehicle: 0, 2.5, 10, 25 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item formulations were previously determined by Envigo Research Limited, Shardlow, UK Analytical Services under Envigo Study Number: 41500461. Results showed the formulations to be stable at concentrations of 0.5 and 10 mg/mL for up to 21 days when stored at approximately 4°C, in the dark. Formulations at the low and intermediate dose levels were therefore prepared once and stored at approximately 4 °C in the dark. High dose formulations were prepared daily and administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Details on mating procedure:

- The day that positive evidence of mating was observed was designated Day 0 of gestation.

Duration of treatment / exposure:

From Day 3 to Day 19 of gestation

Frequency of treatment:

daily

Duration of test:

18 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control treatment group

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:

8 female rats/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen in collaboration with the Sponsor and based on previous toxicity data.
- Route of administration rationale: The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- 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. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
- the rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral change 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. Additional observations were also performed four hours following dosing (not at weekends).

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was recorded for each individual animal for the periods Days 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST-MORTEM EXAMINATIONS: Yes
- All animals were euthanized 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

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

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: No
- The fetuses were killed by subcutaneous injection of a suitable barbiturate.

Statistics:

No statistics available.

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 was calculated for each litter value using the following formula:
% 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):

No treatment related adverse clinical signs were evident in females treated with 25, 100 or 250 mg/kg bw/day.
One female treated with 250 mg/kg bw/day showed an isolated incidence of increased salivation on Day 7 only.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Females treated with 250 mg/kg bw/day showed reduced body weight gains between Days 3 and 11, with five females showing actual body weight losses at some point during this period. Recovery was evident thereafter, however, as a consequence of the initial reduced gains; overall body weight gain was lower than controls. Gravid uterus weight for these females was comparable to controls; however, body weight gain for these females when adjusted for gravid uterus weight was reduced when compared to controls. It should be noted that for one female from this treatment group, body weight loss may have been attributed to overgrown teeth, which were assessed and trimmed on Day 9 and may have been affecting the animal's ability to eat.

Females treated with 100 mg/kg bw/day showed a reduction in body weight gain between Days 3 and 4. Improvement was evident thereafter; however, slightly lower overall body weight gains were evident in these females. The lower overall body weight gain may have been a consequence of slightly lower gravid uterus weights evident in these females; however, body weight gain for these females when adjusted for gravid uterus weight was also reduced when compared to controls.

No such effects were evident in females treated with 25 mg/kg bw/day.

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 lower food intake when compared to controls between Days 3 and 14. Food consumption for these females was comparable to controls between Days 14 and 17, however reduced intake was again observed between Days 17 and 20.

No such effects were evident 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):

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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:

no effects observed

Description (incidence and severity):

No abnormalities were detected during the macroscopic examination of the pregnant females at termination on Day 20 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no obvious adverse effects of maternal treatment on litter data as assessed by pre and post-implantation losses at 25, 100 or 250 mg/kg bw/day.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no obvious adverse effects of maternal treatment on litter data as assessed by the early and late embryonic/fetal deaths, live fetuses at 25, 100 or 250 mg/kg bw/day.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Key result

Dose descriptor:

dose level:

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: proposed high dose in prenatal development toxicity study

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Intergroup differences for mean fetal weights did not indicate any obvious effects of maternal treatment at 25, 100 or 250 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no obvious adverse effects of maternal treatment on litter data as assessed by the live fetuses at 25, 100 or 250 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no obvious adverse effects of maternal treatment on litter data as assessed by the sex ratio as assessed by percentage male at 25, 100 or 250 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Intergroup differences for mean litter weights did not indicate any obvious effects of maternal treatment at 25, 100 or 250 mg/kg bw/day.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 25, 100 or 250 mg/kg bw/day.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Intergroup differences for placental weights did not indicate any obvious effects of maternal treatment at 25, 100 or 250 mg/kg bw/day.

Key result

Dose descriptor:

dose level:

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Treatment of females at 250 mg/kg bw/day was associated with effects on body weight gain between Days 3 and 11, however, recovery was evident thereafter. Gravid uterus weight for these females was comparable to controls; however, body weight gain for these females when
adjusted for gravid uterus weight was reduced when compared to controls. Females treated with 100 mg/kg bw/day also showed effects on bodyweight gain (including when adjusted for gravid uterus weight), although these were restricted to Days 3 and 4 of gestation and recovery at this lower dose level was much quicker than at 250 mg/kg bw/day. During the majority of the treatment period, food consumption for females treated with 250 mg/kg bw/day was lower when compared to control. Body weight gains and food consumption in the remaining females was unaffected by treatment.
The oral administration of the test substance to pregnant rats by oral gavage during gestation, at dose levels of 25, 100 and 250 mg/kg bw/day, resulted in initial reductions in body weight gain and food consumption at 250 mg/kg bw/day. A dose level of 250 mg/kg bw/day showed a treatment related maternal effect, but not a fetal development effect. These effects, however, were not considered significantly adverse to exclude the use of this dose level from further studies of this duration. Therefore, dose levels of 0 (Control), 25, 100 and 250 mg/kg bw/day are recommended for use in the planned prenatal developmental toxicity study.

Conclusions:

The oral administration of the test substance to pregnant rats by oral gavage during gestation, at dose levels of 25, 100 and 250 mg/kg bw/day, resulted in initial reductions in body weight gain and food consumption at 250 mg/kg bw/day. A dose level of 250 mg/kg bw/day showed a treatment related maternal effect, but not a fetal development effect. These effects, however, were not considered significantly adverse to exclude the use of this dose level from further studies of this duration. Therefore, dose levels of 0 (Control), 25, 100 and 250 mg/kg bw/day are recommended for use in the planned prenatal developmental toxicity study.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-07 - 2018-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other 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.
See data tables for detailed information.

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 data tables for detailed information. 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.
See data tables for detailed information.

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 data tables for detailed information.

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 data tables for detailed information.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no total litter losses by resorption observed.
See data tables for detailed information.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on early or late resorptions observed.
See data tables for detailed information.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the number of dead fetuses observed.
See data tables for detailed information.

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 data tables for detailed information.

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 data tables for detailed information.

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

Key result

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 data tables for detailed information.

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 data tables for detailed information.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no treatment-related effect observed on sex ratio. See data tables for detailed information.

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 data tables for detailed information.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There was no treatment-related effects observed on postnatal survival. See data tables for detailed information.

External malformations:

no effects observed

Description (incidence and severity):

No treatment-related effect on external malformations was observed in all dose groups. See data tables for detailed information.

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 data tables for detailed information.

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment-related effect on visceral malformations was observed in all dose groups. See data tables for detailed information.

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

Quality of whole database:

GLP-compliant study, based on OECD guideline

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

Preliminary Oral study in rat (supporting)

The purpose of the study was to select dose levels for future investigative studies of prenatal development in rats. The study investigated the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis. 

The test item was administered by gavage to three groups each of eight time-mated Sprague-Dawley Crl:CD(R) (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 eight 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 euthanized 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 macroscopic appearance of fetuses were recorded. 

There were no unscheduled deaths. No clinical signs of toxicity were detected. A treatment related decrease in body weight gain and cumulative body weight without and with adjustment for gravid uterus weight was detected in females treated with 250 mg/kg bw/day but was considered to be secondary to reduction in food consumption for this group. Females treated with 250 mg/kg bw/day showed reduced body weight gains between Days 3 and 11; recovery was evident thereafter. As a consequence of the initial reduced gains, overall body weight gain was lower than controls. Gravid uterus weight for these females was comparable to controls; however, body weight gain for these females when adjusted for gravid uterus weight was reduced when compared to controls. Females treated with 100 mg/kg bw/day showed a reduction in body weight gain between Days 3 and 4. Improvement was evident thereafter; however, slightly lower overall body weight gains were evident in these females. No such effects were evident in females treated with 25 mg/kg bw/day. Females treated with 250 mg/kg bw/day showed lower food intake when compared to controls between days 3 to 14 and days 17 to 20. No such effects were evident in females with 100 or 25 mg/kg bw/day. No treatment related effects were observed for water consumption in females treated with 25, 100 or 250 mg/kg bw/day. No treatment related macroscopic abnormalities were detected in females treated with 25, 100 or 250 mg/kg bw/day. There was no treatment related effect 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 and pre and 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 treatment related effects at 25, 100 or 250 mg/kg bw/day. Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated a treatment related effect on fetal development at 25, 100 or 250 mg/kg bw/day. The oral administration of the test substance to pregnant rats by oral gavage during gestation, at dose levels of 25, 100 and 250 mg/kg bw/day, resulted in initial reductions in body weight gain and food consumption at 250 mg/kg bw/day. A dose level of 250 mg/kg bw/day showed a treatment related maternal effect, but not a fetal development effect. These effects, however, were not considered significantly adverse to exclude the use of this dose level from further studies of this duration. Therefore, dose levels of 0 (control), 25, 100 and 250 mg/kg bw/day are recommended for use in the prenatal developmental toxicity study.

Prenatal development toxicity study in rats (OECD414)

A prenatal 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.

 

Preliminary oral study in rabbits (supporting information)

The objective of this preliminary study was to select the doses for the
definitive prenatal developmental toxicity study in New Zealand white rabbits. This study evaluated the maternal and developmental toxicity of the test item when administered orally to pregnant New Zealand white rabbits during gestation days (GD) 6 to 28. In this dose range finding study, 24 mated female rabbits were assigned to four groups (G1: Vehicle control, G2: low dose, G3: mid dose and G4: High dose). GD 0 for each individual female rabbit in the study was considered as the day on which mating had occurred. The rabbits in the high dose group were treated at 180 mg/kg/day from GD6 to GD8. Due to test item related mortalities, the dose was reduced to 120 mg/kg/day from GD 9 onwards.

Based on the mortalities of 4/6 females in the 180/120 mg/kg/day this dose is above the highest tolerable dose. Based on the reduction in body weight and food consumption and reduction in mean fetal weights at 90 mg/kg/day, the following doses are proposed for the definitive study: 90, 45, 15 mg/kg/day.

Prenatal development toxicity study in rabbits (OECD 414)

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.