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

Toxicity to reproduction

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-07 to 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 & purity: documented by the Sponsor, communicated to Calvert as 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 initation 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 oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose preparation: The test article formulations 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.

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.
- Oral route was chosen as it is the route affording the maximum exposure, based on results of previously conducted acute studies

Positive control:

Not applicable

Observations and examinations performed and frequency:

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

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

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

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

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

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

URINALYSIS: No

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

Sacrifice and pathology:

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.

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 was 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).

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.

Adult clinical observations:
Females: All female group 1 animals appeared normal during the study. Female group 2 findings were limited to animal #0363 with hair loss around the muzzle with associated moderate, occasional swelling between cohabitation phase day 1 and gestation phase day 13. Three group 3 animals exhibited instances of food crumbling on some days between dosing day 2 and gestation day 5. All but one group 4 animals exhibited clinical signs including but not limited to, pale skin, food crumbling, loose feces, noisy respiration, nasal discharge and/or piloerection. Some of these clinical signs were seen as early as dosing phase day 2 in some animals and lasted up to lactation phase day 3.
Males: All male groups 1-4 animals appeared normal during the dosing and cohabitation phase of the study. Only the incidental missing of the upper incisors was observed in one group 2 and two group 3 animals.

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 histopathological effect was observed in adrenals, 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

Description (incidence and severity):

Females: Similar to the groups 3-4 findings above, unmated female group 6 absolute liver weights and liver-to-body and liver-to-brain weights were statistically significantly increased. Female group 6 liver weights were 116% above control group 5 values. In addition, group 6 kidney weights were statistically significantly increased.
Males: Male group 6 absolute liver weights, liver-to-body and liver-to-brain weights were statistically significantly increased. Group 6 liver weights were 59% above control group values. In addition, group 6 body weights were statistically significantly decreased, and group 6 testes-, spleen- and kidney-to-body weight ratios were statistically significantly increased.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Females: At the end of the 16 day recovery phase all female group 6 animals had enlarged and pale livers at necropsy. Microscopically, these findings correlated to slight to moderate vacuolation of the hepatocytes, predominantly centrilobular.
Males: All male group 6 animals had enlarged and pale livers at necropsy. Microscopically, these findings also correlated to slight to moderate vacuolation of the hepatocytes, predominantly centrilobular. One male group 5 animal had an incidentally enlarged prostate with no microscopic correlation.

Neuropathological findings:

not examined

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 the 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 specified

Details on results:

Male and female rats were dosed orally once daily at 0, 25, 100 and 250 mg/kg to test for potential toxic effects/disturbances resulting from the administration of the test substance. 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. 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 effects 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 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 groups 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.
Additional five animals/sex/groups, dosed orally once daily at 0 and 250 mg/kg, were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity. 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 instances of food crumbling, piloerection, staining on the head (cranial) 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 to Groups 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.

Key result

Dose descriptor:

NOAEL

Effect level:

< 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Maternal and paternal hepatotoxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw (total dose)

System:

hepatobiliary

Organ:

liver

Treatment related:

not specified

Conclusions:

Based on the results of this 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 hepatotoxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

266-587-2

EC Name:

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

Cas Number:

67151-63-7

Molecular formula:

C13H31N3O

IUPAC Name:

1-Bis(3-(dimethylamino)propylamino)-2-propanol

Test material form:

liquid

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

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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;

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (nominal)

System:

hepatobiliary

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

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

Applicant's summary and conclusion

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.