Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-12-17 to final report date
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 870.3700
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
June 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
The study was performed following design described in final decision number CCH-D-2114453649-36-01/F.
An extended one-generation reproductive toxicity study (annex X, according to OECD 443) in rats, oral route with the registered substance was performed including: ten weeks premating exposure duration for the parental (P0) generation; dose level setting shall aim to induce systemic toxicity at the highest dose level; cohort 1A; cohort 1B without extension to mate the cohort 1B animals to produce the F2 generation

Test material

Constituent 1
Chemical structure
Reference substance name:
3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide
EC Number:
939-581-9
Cas Number:
1471314-81-4
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Amides, C12-C18 (even numbered), N-[3-(dimethylamino)propyl], N'-oxides
- Substance type: Clear liquid
- Lot/batch No.: BLBX017090
- Storage condition of test material: Room temperature in the dark
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Amides, C12-C18 (even numbered), N [3 (dimethylamino) propyl], N’-oxides
- Substance type: Almost clear colorless liquid
- Physical state: liquid
- Analytical purity: 35.2% in water
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: S019341917
- Expiration date of the lot/batch: 28 March 2021
- Action of test substance: no data
- purity/weighing factor: none
- total correction factor: 2.85, based on purity

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed container, 2 to 8°C, protected from light
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Formulations of 0.2 and 100 mg/mL AI were stable for 15 days when stored refrigerated (2 to 8°C) or for 14 days when stored at room temperature (15 to 25°C) and formulations of 0.1 mg/mL AI were stable for 1 day when stored at room temperature (15 to 25°C) in Covance Study 8410552.
Formulations of 0.1 and 100 mg/mL AI were confirmed to be homogenous in Covance Study 8410552.

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Residual samples were taken, retained for up to 15 days and stored refrigerated (2 to 8°C), and then discarded.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) rats
Details on species / strain selection:
The Crl:WI(Han) rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics. The rat is normally the preferred species because of the extent of background data and the comparability to general toxicity tests. The strain used is not known for low fecundity or high incidence of spontaneous developmental defects.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: One hundred male and one hundred female Crl:WI(Han) rats from Charles River Laboratories, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at animal arrival: 28 or 31 days old
- Weight at study initiation: (P) Males: 0,1362 - 0,2004 kg; Females: 0,1069 - 0,153 kg
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes (Home Office, 2014).
- The F0 generation animals were housed in groups of up to four by sex and dose group (pre-pairing phase for both sexes and post-pairing phase for males). During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed, or until the end of the pairing phase. Following mating, females were housed individually during gestation or post-pairing and with their litter during the lactation phase.
- During the maturation phase, the F1 generation animals were housed in groups of up to four by sex and dose group.
- Bedding was provided at least weekly to each cage by use of clean Aspen wood chips or European Softwood bedding during gestation and lactation phases (Datesand Ltd; Manchester, United Kingdom). Each batch of bedding was analyzed for specific constituents and contaminants. No contaminants were present in the bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
- Diet (e.g. ad libitum): ad libitum, VRF1 (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants.
No contaminants were present in the diet at levels that might have interfered with achieving the objective of the study. Results are retained on file at Covance.
A 50-g sample of each batch of diet used in the study was collected and stored at ambient temperature until study finalization.
- Water (e.g. ad libitum): ad libitum, Water from the main tap supply was provided via water bottles. The water is periodically analyzed for specific contaminants.
No contaminants were present in the water at levels that might have interfered with achieving the objective of the study. Results are retained on file at Covance.

- Acclimation period: Animals were acclimated for 13 or 11 days, and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 40% to 70%
- Air changes (per hr): The rooms were air conditioned to provide a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark.

IN-LIFE DATES: From: 2019-12-30 To: 2020-08-07

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: The test article was formulated as a suspension in purified water following dispensary SOPs and the formulation method (Method 8410555_O_01D), as maintained in the study data.
- Frequency of preparation: weekly
- Storage of formulation: Refrigerated (2 to 8°C) in a sealed container protected from light.
- Formulations prepared for use on the first dosing occasion of the F0 generation, the start of dosing for the F1 generation, and the last day of the F1 generation were analyzed to determine the achieved concentration. Triplicate samples were taken from the middle of the test article formulations and were analyzed. A single sample was taken from the middle of the control formulations and was analyzed.
- Formulations (excluding vehicle control article) were stirred continuously for at least 30 minutes before and throughout dosing.

- Test item was prepared at the appropriate concentration as solution in purified water.

VEHICLE
- Justification for use and choice of vehicle: not applicable (vehicle is water)
- Concentration in vehicle: 0, 5, 10, 15 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed, or until the end of the pairing phase.
- Proof of pregnancy: Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. The day on which mating was confirmed was designated as GD 0.
- After successful mating each pregnant female was caged (how): individual
- Any other deviations from standard protocol: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations prepared for use on the first dosing occasion of F0 generation, the start of dosing for the F1 generation and the last day of the F1 generation were analyzed to determine the achieved concentration. The mean % nominal concentration should be between 90 to 110% and with a relative standard deviation (RSD) ≤5.0%. Results were within these criteria. Test article was not detected in the Group 1 control samples.
The analytical procedure was validated within Covance study 8280017.
Duration of treatment / exposure:
Males: 119 consecutive days (10 weeks prior to pairing, 2 weeks during pairing, and 35 days post-pairing) , Females: up to 121 days (10 weeks prior to pairing, during pairing, throughout gestation, and up to LD 21).
Frequency of treatment:
Daily, at approximately the same time each day +/- 3.5 hours.
Details on study schedule:
xxx (experimental completion date is not included in draft report)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 males and 24 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The high-dose level of 150 mg/kg/day AI was anticipated to induce minimal parental toxicity (i.e., changes in red blood cell parameters and local irritancy of the stomach) without causing death or affecting parturition, pup growth, and mating of the first generation. The intermediate-dose level of 100 mg/kg/day AI was anticipated to be a no observed adverse effect level (NOAEL) for parental toxicity. The low-dose level of 50 mg/kg/day AI was anticipated to be a no observed effect level (NOEL) for parental and offspring toxicity.

Route of administration rationale: The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type. The oral route is a potential route of accidental human exposure to be investigated in hazard and risk assessment.

Animal species selection rationale: The Crl:WI(Han) rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics. The rat is normally the preferred species because of the extent of background data and the comparability to general toxicity tests. The strain used is not known for low fecundity or high incidence of spontaneous developmental defects.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
VIABILITY CHECKS
- Time schedule: All animals were observed in the home cage twice daily at the beginning and end (nominal) of the working day for signs of ill health or overt toxicity.

CAGE SIDE AND DETAILED OBSERVATIONS
- General Appearance: Each animal was given a detailed physical examination once daily from the start of dosing. The examinations were performed after dosing (after returning the animal to the home cage) from the issue of Protocol Amendment 2 (Pre-Pairing Day 2 or 16). An individual record was maintained of the clinical condition of each animal.
- Postdose Observations: The first batch of animals (arrived on 17 December 2019) were observed daily for the first 7 days approximately 1 hours postdose. The second batch of animals (arrived on 02 January 2020) was observed daily for the first 7 days immediately after dosing, upon return to the home cage.
- Clinical observations: All animals were assessed by detailed clinical observations for approximately 30 seconds in an open field arena. Males were assessed once weekly from the start of dosing. Females were assessed once weekly during the pre-pairing and post pairing phases; on GD 0, 6, 14, and 20; and on LD 1, 7, 14, and 21.
- Maternal observations: Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered.

BODY WEIGHT
Frequency: Body weights were recorded once during acclimation (Day 1 of the predose phase [Day-3]) for all animals.
Male body weights were recorded weekly from the first day of dosing (Pre-Pairing Day 1).
Female body weights were recorded weekly from the first day of dosing (Pre-Pairing Day 1); on GD 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and on LD 1, 4, 7, 14, and 21. Body weights recorded during the pairing phase were not reported. Body weights for females with no confirmation of mating were recorded weekly during the post-pairing phase.

FOOD CONSUMPTION
Frequency
- Males: food consumption was recorded twice weekly during pre- and post-pairing.
- Females: food consumption was recorded twice weekly during pre-pairing; every 2 days during gestation; and from LD 1 to 4, 4 to 7, and 7 to 14.
- Food consumptions were not recorded during pairing, between GD 21 and LD 0, due to parturition, and from LD 14, due to the start of weaning of pups.

CLINICAL PATHOLOGY
- Sample collection: Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), clinical chemistry (1 x 0.6 mL [serum separator tubes], nominal) and thyroid hormone analysis (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta at necropsy for at least 10 randomly selected males and females within each group from Cohort 1A.
- Animals fasted: Samples were collected after animals were fasted overnight.

HEMATOLOGY and COAGULATION:
- Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time followed by at least 5 minutes on an automatic mixer. Blood samples for clinical chemistry were gently inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator.
- Hematology Tests: hemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, total and differential white cell count, platelet count
- Coagulation Tests: prothrombin time, activated partial thromboplastin time, fibrinogen
- Clinical Chemistry Tests: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transferase, total cholesterol, total bilirubin, total protein, albumin
globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, triglycerides, creatinine, urea, glucose

THYROID ANALYSES:
- During sample processing, each blood sample for thyroid hormone analysis was split into two equal aliquots. One aliquot was analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis were retained in storage until report finalization, after which, they will be discarded.
Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until placed in frozen storage. See Protocol Deviations.
- Each blood sample for thyroid hormone analysis was gently inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator, and was stored at room temperature (15 to 25°C) for at least 30 minutes prior to processing to allow samples to clot. Each sample was then centrifuged at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at < 10°C (nominal -20°C).
- Parameters: thyroxine (Total T4), thyroid stimulating hormone (TSH)

URINALYSIS
- Sample collection: Urine samples from 10 randomly selected males and females in each group from Cohort 1A were collected overnight the day prior to necropsy. Food was removed during collection.
- Parameters: volume, color, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, microscopy of sediment.


Oestrous cyclicity (parental animals):
Daily vaginal lavage (washings) were undertaken from females for 14 days prior to pairing, and until the confirmation of mating, and on the day of necropsy.
Sperm parameters (parental animals):
For each scheduled male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from the left epididymis (cauda epididymis and vas deferens). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from males in Groups 1 and 4 were read for morphological changes. The left epididymis was discarded following seminology investigations.
Litter observations:
VIABILITY CHECKS
- Preweaning: Litter size and pup sex were recorded on PND 0, 1, 4, 7, 14, and 21. Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition were given a macroscopic necropsy for cause of death and possible birth defects. All pups were observed daily from PND 14 to 21 immediately after dosing and upon return to the home cage.
- Postweaning: All animals were observed in the home cage twice daily at the beginning and end (nominal) of the working day for signs of ill health or overt toxicity.

CLINICAL OBSERVATIONS
- Preweaning: A detailed clinical examination was recorded for each pup on PND 1, 4, 7, 14, and 21.
- Postweaning: Each animal was given a detailed physical examination once daily from the start of dosing, performed immediately after dosing, upon return to the home cage. An individual record was maintained of the clinical condition of each animal. All animals were assessed by detailed clinical observations for approximately 30 seconds in an open-field arena. Animals were assessed once weekly from Maturation Day 22.

BODY WEIGHTS
- Preweaning: Pup body weights were recorded on PND 1, 4, 7, 13, 14, 16, 18, and 20.
- Postweaning: Body weights were recorded on Maturation Day 22, 24, 26, 28, 30, 32, 34, and 36, then weekly thereafter. In addition, body weights were recorded on the day balano preputial separation or vaginal opening occurred.

ANOGENITAL DISTANCE
- Preweaning: Prior to the culling procedure on PND 4, the ano-genital distance of all pups was measured.
Procedure:

NIPPLE PRESENCE - ALL MALE ANIMALS
- Preweaning: The number of nipples/areolae for all male pups was recorded on PND 13.

FOOD CONSUMPTION
- Frequency: Food consumption was recorded twice weekly from Maturation Day 22 until the day prior to necropsy for Cohort 1A and until the day of necropsy for Cohort 1B.
- Procedure: Consumption was calculated as g/animal/day.

SEXUAL MATURATION
- Animals were observed for vaginal opening (females) from Maturation Day 28 and cleavage of the balano-preputial gland (males) from Maturation Day 37.
- Observations lasted until these milestones were attained. Body weights were recorded on the day balano-preputial separation or vaginal opening occurred.

ESTROUS CYCLE EVALUATIONS in F1 generation
Daily vaginal lavage (washings) were undertaken for Cohort 1A and 1B females after vaginal opening until the first cornified smear was observed, then from Maturation Day 75 to 89, inclusive, and on the day of necropsy.

CLINICAL PATHOLOGY
- Sample collection: Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), clinical chemistry (1 x 0.6 mL [serum separator tubes], nominal) and thyroid hormone analysis (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta at necropsy for at least 10 randomly selected males and females within each group from Cohort 1A.
- Samples were collected after animals were fasted overnight.

HEMATOLOGY and COAGULATION:
- Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time followed by at least 5 minutes on an automatic mixer. Blood samples for clinical chemistry were gently inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator.
- During sample processing, each blood sample for thyroid hormone analysis was split into two equal aliquots. One aliquot was analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis were retained in storage until report finalization, after which, they will be discarded.
- Hematology Tests: hemoglobin, red blood cell count, packed cell volume, mean cell volume
mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, total and differential white cell count, platelet count.
- Coagulation Tests: prothrombin time, activated partial thromboplastin time, fibrinogen
- Clinical Chemistry Tests: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transferase, total cholesterol, total bilirubin, total protein, albumin
globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, triglycerides, creatinine, urea, glucose.

THYROID ANALYSES:
- Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until placed in frozen storage.
- Each blood sample for thyroid hormone analysis was gently inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator, and was stored at room temperature (15 to 25C) for at least 30 minutes prior to processing to allow samples to clot. Each sample was then centrifuged at 2300g for 10 minutes at approximately 4C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at < 10°C (nominal -20°C).

URINALYSIS
- Urine samples from 10 randomly selected males and females in each group from Cohort 1A were collected overnight the day prior to necropsy. Food was removed during collection.
- Parameters: volume, color, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, microscopy of sediment.
Postmortem examinations (parental animals):
SACRIFICE:
- Sacrifice / method of euthanasia: Males were sacrificed by isoflurane anesthesia on Post-Pairing Day 36 (Week 18) after an overnight period without food. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
Females were sacrificed by isoflurane anesthesia on LD 22 (those that littered) or 26 days post coitum (those with confirmation of mating that did not litter) or Post Pairing Day 36 (those with no confirmation of mating that did not litter). Females sacrificed on LD 22 had food removed overnight prior to necropsy.

- Unscheduled death: Rats that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions.

- Scheduled euthanasia:
Females with the same day of mating/littering were sacrificed in a controlled randomization order, when possible. Females that did not litter were sacrificed in animal identification order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.

OVARIAN AND UTERINE EXAMINATIONS
- The number of implantations sites for all paired females were recorded. The uterus of any apparently non pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

NECROPSY
- Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded. Organ weights were recorded at each scheduled sacrifice, excluding decedents.

ORGAN WEIGHTS
- Organ weights were recorded at each scheduled sacrifice, excluding decedents. Organs were dissected free from fat and other contiguous tissue and were weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
- weighed organs: adrenal, brain, epididymis (right), heart, ovary, oviduct, pituitary, prostate, seminal vesicle with coagulating, spleen, testis, kidney, liver, thymus, thyroid with parathyroid and uterus with cervix

TISSUE COLLECTION AND PRESERVATION
- Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated: adrenal nerve, nerve, sciatic, bone marrow smear (femur), ovary, brain, oviduct, cecum, pituitary, colon, prostate, duodenum, rectum, epididymis (right), seminal vesicle with coagulating glands and fluid, eye, spinal cord, cervical, femur with bone marrow and femerotibial joint, spinal cord, lumbar, gross lesions, spinal cord, thoracic, head, spleen, heart, stomach, ileum, testis, jejunum, thymus, kidney, thyroid with parathyroid, liver, trachea, lungs with main stem, bronchi and bronchioles, urinary bladder, mammary gland, uterus with cervix, muscle, biceps femoris and vagina
- The uterus of any apparently non pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

HISTOLOGY
- All tissues (adrenal nerve, nerve, sciatic, bone marrow smear (femur), ovary, brain, oviduct, cecum, pituitary, colon, prostate, duodenum, rectum, epididymis (right), seminal vesicle with coagulating glands and fluid, eye, spinal cord, cervical, femur with bone marrow and femerotibial joint, spinal cord, lumbar, gross lesions, spinal cord, thoracic, head, spleen, heart, stomach, ileum, testis, jejunum, thymus, kidney, thyroid with parathyroid, liver, trachea, lungs with main stem, bronchi and bronchioles, urinary bladder, mammary gland, uterus with cervix, muscle, biceps femoris and vagina) from Groups 1 and 4 and all decedents, and the spleen from Groups 2 and 3, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
- All tissues ( animal identification, pituitary, epididymis (right), prostate, gross lesions, seminal vesicle, mammary gland, testis, ovary, uterus with cervix, oviduct and vagina) from males in Groups 2 and 3 that did not sire and females in Groups 2 and 3 that were not mated and/or not pregnant were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
- Additional sections of testes and epididymides were also stained with periodic acid Schiff (PAS) for spermatogenic staging for all males from Groups 1 and 4 and all decedents.

HISTOPATHOLOGY
All processed tissues were examined microscopically by the Study Pathologist. Qualitative assessments of stages of spermatogenesis and interstitial testicular cell structure were also performed for males from Groups 1 and 4.
Postmortem examinations (offspring):
SACRIFICE:
- Method of Euthanasia:
-Pups culled on PND 4, unselected pups on PND 22, and pups in a moribund condition were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, major blood vessels were severed to exsanguinate the animal. Sacrifices were carried out in group order, where possible.
- Cohort A: Animals were sacrificed by isoflurane anesthesia on Maturation Day 90 to 93 after an overnight period without food. Sacrifices were carried out in a controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
- Cohort B: Animals were sacrificed by isoflurane anesthesia on Maturation Day 108 to 113 after an overnight period without food. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.

- Unscheduled death: With the exception of fasting, these procedures were also followed for unscheduled sacrifices and deaths.

MALE REPRODUCTIVE ASSESSMENT
For each scheduled male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from the left epididymis (cauda epididymis and vas deferens). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from males in Groups 1 and 4 were read for morphological changes. The left epididymis was discarded following seminology investigations.

NECROPSY
Pups: Full macroscopic examinations, with particular attention to the reproductive genitals, were conducted for all decedents and pups sacrificed on PND 4 or PND 22.
Cohort A and B: Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

ORGAN WEIGHTS
Organ weights were recorded at each scheduled sacrifice, excluding decedents. Organs were dissected free from fat and other contiguous tissue and were weighed before fixation. Left and right organs were weighed together.
- Cohort A: adrenal, brain, epididymis (right), heart, kidney, liver, lymph node (mesenteric and mandibular), ovary, oviduct, pituitary, prostate, seminal vesicle with coagulating, spleen, testis
thymus, thyroid with parathyroid and uterus with cervix.
- Cohort B: epididymis, ovary, oviduct, pituitary, prostate, seminal vesicle, testis and uterus with cervix

TISSUE COLLECTION AND PRESERVATION
Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
- Cohort A: adrenal, muscle, biceps femoris, nerve (optic and sciatic), bone marrow smear (femur),
brain, ovary, cecum, oviduct, colon, pituitary, duodenum, prostate
epididymis (right), rectum, eye, seminal vesicle with coagulating glands and fluid, femur with bone marrow and femerotibial joint, spinal cord, cervical, gross lesions, spinal cord, lumbar, head spinal cord, thoracic, heart, spleen, ileum, stomach, jejunum, testis, kidney, thymus, liver, thyroid with parathyroid, lungs with main stem bronchi and bronchioles, trachea, lymph node, mesenterice, urinary bladder, lymph node, mandibulare, uterus with cervix, mammary gland and vagina
- Cohort B: pituitary, epididymis, prostate, gross lesions, seminal vesicle, mammary gland, testis, ovary, uterus with cervix, oviduct and vagina

HISTOLOGY 
- Cohort A:
- All tissues (adrenal, muscle, biceps femoris, nerve (optic and sciatic), bone marrow smear (femur), brain, ovary, cecum, oviduct, colon, pituitary, duodenum, prostate
epididymis (right), rectum, eye, seminal vesicle with coagulating glands and fluid, femur with bone marrow and femerotibial joint, spinal cord, cervical, gross lesions, spinal cord, lumbar, head spinal cord, thoracic, heart, spleen, ileum, stomach, jejunum, testis, kidney, thymus, liver, thyroid with parathyroid, lungs with main stem bronchi and bronchioles, trachea, lymph node, mesenterice, urinary bladder, lymph node, mandibulare, uterus with cervix, mammary gland and vagina) from Groups 1 and 4 and all decedents, and the spleen from Groups 2 and 3, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin. See Protocol Deviations.
- For at least 10 animals/sex from Groups 2 and 3, the mandibular and mesenteric lymph nodes, adrenals, thymus, spleen, and bone marrow (femur) were processed for immunotoxicity assessments. All macroscopic lesions and the spleen were processed for remaining animals.
- Additional sections of testes and epididymides were also stained with periodic acid Schiff (PAS) for spermatogenic staging for all males from Groups 1 and 4 and all decedents.
- Pre- and post-natally induced immunotoxic effects were evaluated by assessment of the mandibular and mesenteric lymph nodes, adrenals, thymus, spleen, and bone marrow (femur) from at least 10 animals/sex/group from all Cohort 1A dose groups. Approximately half the spleen was used for flow cytometry analysis. The excised sample was weighed and the weight recorded. Absolute values were calculated from the recorded weights. The samples were placed in an appropriate cell culture media, then prepared into a single cell suspension as soon as possible by means of mechanical disruption of the spleen section.
- Cohort B: All tissues (pituitary, epididymis, prostate, gross lesions, seminal vesicle, mammary gland, testis, ovary, uterus with cervix, oviduct and vagina) from all Cohort 1B animals, including decedents, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
All processed tissues were examined microscopically by the Study Pathologist. Qualitative assessments of stages of spermatogenesis and interstitial testicular cell structure were also performed for males from Groups 1 and 4.
Qualitative assessment of the stages of follicular development to regression from small growing follicles to corpora lutea was performed in the ovaries.
Statistics:
See: Any other information on materials and methods incl. tables
Reproductive indices:
- Female Mating index = Number of mated females / Number of females cohabited (excluding females sacrificed during Cohabitation) X 100
- Male mating index = Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female X 100
- Female fecundity index=Number of pregnant females / Number of females mated (excluding females with an undetermined pregnancy status) X 100
- Male fecundity index=Number of males impregnating at least 1 female / Number of males mating with at least 1 female X 100
- Female fertility index =Number of pregnant females / Number of females cohabited (excluding females sacrificed during Cohabitation or with an undetermined pregnancy status) X 100
- Male fertility index=Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female X 100
- % post-implantation loss=(Number of implantations - number of live embryos) / Number of implantations X 100
- Gestation index=Number of females with live born pups / Number of pregnant females X 100
Offspring viability indices:
- Livebirth index = Number of live born pups / Number of pups born X 100
- Day 4 viability index=Number of pups alive on PND 4 precull / Number of pups born alive X 100
- Weaning index =Number of pups alive at weaning / Number of pups alive on PND 4 post cull X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Nonadverse, test article-related clinical postdose observations of mouth rubbing were noted for all males and females administered 150 mg/kg/day AI.
A high incidence of excessive salivation was also noted for 16 of 24 males and 14 of 24 females from this dose group. Isolated instances of paddling behavior were also recorded for three females prior to pairing (Animal R0701 on Day 44 and Animals R0716 and R0724 on Day 52) and were also evident for two females during lactation (Animal R0708 on LD 3 and 8, and Animal R0707 on LD 4).
Test article-related, clinical postdose observations of mouth rubbing were also observed for all males and females administered 100 mg/kg/day AI, with isolated instances of excessive salivation reported for seven males. An isolated incidence of paddling behavior was also noted in three males from this dose group (Animals R0214 and R0218 on Day 62 and Animal R0219 on Day 70). An isolated incidence of paddling behavior was recorded for three females during gestation (Animals R0601 and R0612 on GD 1 and Animal R0601 on GD 4).
Isolated instances of postdose mouth rubbing were also observed 14 of 24 males and 3 of 24 females administered 50 mg/kg/day AI.
Such observations are common in gavage studies and are considered due to the unpalatable taste of the test article formulation, rather than an indication of the toxicity of the test article; therefore, they were considered nonadverse.
High incidences of abnormalities of the pelage (discoloration, hair loss, and thin hair) were observed for 6 of 24 males administered 150 mg/kg/day AI during the pre-pairing phase, although this was not so obvious as the study progressed; as such, these findings were considered to have arisen incidentally and were not test article-related.
No test article-related observations were noted during the weekly detailed examinations.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test article-related mortality occurred.
One female administered 150 mg/kg/day AI (Animal R0719) was sacrificed on Lactation Day 0 following a complete litter loss, which consisted of one stillborn pup and one pup death (born alive). In isolation, this was considered to have arisen incidentally with no relationship to the test article.
One control male (Animal R0010) was found dead on Day 17. The animal was recorded with a sore/lesion below the right ear and dark focus on the thymus. One control female (Animal R0410) was found dead and cannibalized on Day 20 and reported with a cannibalized uterus following microscopic examinations. One control female (Animal R0415) was found dead on Day 29. At macroscopic examinations, the animal was reported with a missing tail tip. The cause of demise was not confirmed for these animals, however, as only the control article (vehicle) was administered to the controls, no association with the test article occurred.
One control male (Animal R0011) was sacrificed on Post-Pairing Day 27 after the animal was observed with loss of limb movement, swelling, and blue coloration of the left hindlimb. Macroscopic examinations revealed a hindlimb fracture below the stifle joint and mottled lobes of the liver. The cause of demise was a physical injury.
One female administered 150 mg/kg/day AI (Animal R0717) was found dead on Day 49. Macroscopic examinations revealed an esophageal rupture and abnormal contents in the thoracic cavity. These findings were consistent with a dosing trauma and not test article related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects on mean body weight or body weight gain were noted for males throughout the study or for females during the pre-pairing, gestation, or lactation phases.
Transient instances of statistically significant differences in mean body weight gains were observed throughout the dose groups, with no relationship to the test article; as such, they were considered to have arisen incidentally.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test article-related effects on mean food consumption were observed for males throughout the study duration or for females during the pre-pairing, gestation, or lactation phases.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.
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):
Nonadverse, dose-related test article-related changes in the red cell parameters were evident at all dose levels investigated at the end of the dosing phase for parental animals.
Dose-related reductions in mean cell hemoglobin concentration (MCHC), hemoglobin and red cell distribution and red blood cell count (RBC) were observed for males administered at all dose levels, compared with controls. Statistical significance was attained for all these parameters for males administered 100 or 150 mg/kg/day AI, and extended to 50 mg/kg/day AI males for RBC and MCHC (P < 0.001). These changes were accompanied by statistically significantly increased reticulocyte counts and percentage reticulocytes (P < 0.05 to P < 0.001) compared with controls. Statistically significant increases in mean cell volume (MCV) was also evident at all dose levels (P< 0.001) and mean cell hemoglobin levels (MCH) were also increased for males administered 100 or 150 mg/kg/day AI (P < 0.05 or P < 0.001, respectively), compared with controls.
Similar changes in red cell mass were evident for the females. RBC, RDW, and MCHC were reduced for females administered at all dose levels, with statistical significance attained for females administered 100 or 150 mg/kg/day AI, (P < 0.05 to P < 0.001) compared with controls. MCH, MCV reticulocytes (counts and percentages) were also increased for females at all dose levels with a clear dose relationship with increasing dose (from not statistically significant to P< 0.001) compared with controls.
Monocyte counts were reduced (P < 0.01), and percentage monocytes were decreased (P < 0.05) for males administered 150 mg/kg/day AI, compared with controls. Large unstained cell (LUC) was reduced for males administered 50 or 150 mg/kg/day AI, although a dose relationship was not evident. The absence of similar changes observed for the females and no associated supporting data to suggest that these intergroup differences were attributable to test article administration, these minor changes in the white cell parameters were considered to have arisen incidentally and were of no toxicological importance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Nonadverse test article-related reductions in potassium levels were evident following administration of 100 or 150 mg/kg/day AI, compared with controls.
Potassium levels were significantly reduced for males (P < 0.01) and females (P < 0.001) administered 150 mg/kg/day AI, compared with controls, with the reduction extending into females administered 100 mg/kg/day AI, compared with controls (P < 0.05). No such effect was evident for males administered 100 mg/kg/day or animals of either sex administered 50 mg/kg/day AI.
Alkaline phosphatase activity was reduced for females administered 50 or 100 mg/kg/day AI, compared with controls. The significance attained was minimal (P < 0.05), and in the absence of a similar change observed at the highest dose levels and the lack of a similar response observed for males, these reductions were considered to have arisen incidentally with no relationship to the test article.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects on thyroxine or thyroid stimulating hormone were observed.
A small but statistically significant increase in T4 was evident for males administered 50 mg/kg/day AI, compared with controls (P < 0.05). In the absence of a dose relationship, and in isolation, this increase was considered to have arisen incidentally.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test article-related effects were noted for the urine parameters assessed for males or females.
Statistical analysis of the urine volume or specific gravity data did not reveal any significant intergroup differences.
Behaviour (functional findings):
not examined
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):
Upon microscopic examination, test article-related findings were recorded for the spleen.
A dose-related increase in the severity of extramedullary hemopoiesis and pigmented macrophages were noted for F0 animals administered 50, 100, or 150 mg/kg/day AI, compared with controls, more notably in males. Extramedullary hemopoiesis was characterized by increased numbers of hemopoietic cells from all three cell lines within the splenic red pulp. Pigmented macrophages were characterized by increased numbers of macrophages with golden-brown cytoplasmic pigment within the splenic red pulp.
No other test article-related microscopic findings were recorded.
Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings rats of this strain, age, and stage of reproduction.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects on estrous cycle assessments were noted for the mean number and duration of estrous cycles prior to pairing.
Statistically significant increases in mean length of the cycles was noted for groups administered 100 or 150 mg/kg/day AI, compared with controls. However, a dose relationship was not evident, and no impact on mating or fertility occurred; as such, these statistically significant intergroup differences were considered to have arisen incidentally and not as a consequence of test article administration.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects on the seminology data were observed.
Sperm concentration was significantly lower for males administered 100 or 150 mg/kg/day AI, compared with controls; however, a dose relationship was not observed (P < 0.01 or P < 0.05, respectively). All other parameters were unaffected; in the absence of an effect on male fertility in this study, these intergroup differences were considered to have arisen incidentally.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating, fecundity, or fertility indices were unaffected by administration of the test article.
Most paired animals mated within 5 days of pairing.
Fecundity and fertility indices were increased in the group administered 150 mg/kg/day AI, compared with controls, which was considered to have arisen incidentally.
Of the 21, 21, 22, or 22 paired females administered 0, 50, 100, or 150 mg/kg/day AI, respectively, 20, 20, 19, or 22 females were pregnant.
No test article-related effects on the litter parameters was evident.
Of the 20, 20, 19 or 22 pregnant females administered 0, 50, 100 or 150 mg/kg/day AI, one animal administered 150 mg/kg/day AI (Animal R0702) had no viable fetuses. Another animal from this group (Animal R0719) was sacrificed on Lactation Day 0 (Section 4.2.1) due to total litter loss (one pup born alive and one stillborn). Both events were considered to have arisen incidentally with no relationship to the test article. All remaining pregnant females had surviving litters on Lactation Day 21.
No test article-related effect on gestation length was noted.
Litter size and survival index was unaffected. Sex ratio was unaffected by test article administration at any dose level.
An increased incidence of stillborn pups was noted following administration of 150 mg/kg/day AI, compared with controls. Animal R0719 had one pup born alive and one stillborn pup. Animal R0719 also had a gestation length of 25 days, with a high post-implantation loss of 12 implants, compared with one stillborn and one pup born alive, which resulted in the total litter loss. The remaining litters with stillborn pups did not exhibit large post-implantation losses or larger than expected litters. A marginally higher post-implantation loss and reduced gestation index was noted for females administered 150 mg/kg/day AI, compared with controls. This was attributable to the in utero litter loss (Animal R0702), which, in isolation, was considered to have arisen incidentally. Seven stillborn pups were evident from five litters administered 150 mg/kg/day AI, compared with four stillborn pups from three litters for controls. This was slightly higher that the historical control day range (HCD; 0 to 6 stillborn pups). However, the percentage of stillbirths in this dose group (0.33%) was within the HCD (0 to 3%), the mean number of pups born in each litter was also within the HCD range (4.7 to 15 pups), and no effect on mean post implantation loss occurred; as such, the marginally higher number of stillborn pups was considered to have arisen incidentally, as it was not supported by any other changes in offspring survival.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pup Clinical Observations: Test article-related clinical observations were confined to the presence of mouth rubbing, observed immediately after direct dosing from PND 14 for some animals administered 100 or 150 mg/kg/day Al, and a dose response was evident. This was not observed for pups administered 50 mg/kg/day AI or the control article (vehicle) alone.
The remaining clinical observations noted for the pups during the lactation phase were observed throughout the dose groups, including controls, with no dose relationship; as such, they were considered to have arisen incidentally.

F1 Clinical Observations: Nonadverse test article-related clinical observations were noted after dosing at all dose levels.
Transient and test article-related clinical observations of mouth rubbing were observed for all animals administered 100 or 150 mg/kg/day AI (Cohorts 1A and 1B). Isolated instances of mouth rubbing were also recorded for 14 of 40 males (two from Cohort 1A and 12 from Cohort 1B) and 12 of 40 females (five from Cohort 1A and seven from Cohort 1B) administered 50 mg/kg/day AI. A dose response was evident.
Isolated instances of paddling behavior were recorded for three females administered 150 mg/kg/day AI (Cohort 1A: Animal R1513 on PND 81; Cohort 1B: Animal R1533 on PND 102 and Animal R1535 on PND 104). Isolated instances of paddling behavior were also recorded on PND 101 and 102 for one Cohort 1B male administered 150 mg/kg/day AI (Animal R1135), and this was also evident for two Cohort 1B males administered 100 mg/kg/day AI (Animal R1028 on PND 29 and Animal R1133 on PND 101 and 102).
Excessive salivation was recorded on PND 67 and 80 for one Cohort 1A male (Animal R1106) administered 150 mg/kg/day AI. This was also reported on four occasions between PND 97 and 102 for one Cohort 1B male (Animal R1131) administered 150 mg/kg/day AI. Animal R1132, administered 150 mg/kg/day AI, was recorded with this on PND 102, and Animal R1137, administered 150 mg/kg/day AI, was reported with this finding on PND 96 and 97. One female (Animal R1435) administered 100 mg/kg/day AI also exhibited excessive salivation on PND 102. Animal R1534, administered 150 mg/kg/day AI, exhibited this finding on PND 104, and Animal R1539, administered 150 mg/kg/day AI, exhibited excessive salivation between PND 97 and 102.
An instance of jaw chomping was recorded on PND 31 for one Cohort 1B male (Animal R1124) administered 150 mg/kg/day AI. In isolation, this was considered to have arisen incidentally and not test article related.
The remaining clinical observations were observed throughout the dose groups, including the control; as such, they were considered to represent low incidence findings in animals of this age and arisen incidentally.
No observations were noted during the weekly detailed clinical examinations at any dose level.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test article-related unscheduled deaths have occurred. The following deaths were considered to have arisen incidentally.
One Cohort 1B male administered 100 mg/kg/day AI (Animal R1038) was sacrificed on PND 51. The animal was recorded with hunched posture, decreased activity, piloerection, thin and pale, cold to the touch, and with excessive red colored lacrimation from both eyes. Pale adrenals and thymus were recorded. The spleen was recorded as thin. The liver was recorded as mottled and pale. The jejunum included abnormal contents and was distended. The ileum was impacted. The pancreas had an abnormal texture with adhesion to the stomach. The abdominal cavity was fluid filled. A cause of demise could not be determined for this animal based on the macroscopic observations recorded. In the absence of similar mortality in the intermediate or in the high dose group, this death was considered to have occurred incidentally.
One Cohort 1A female administered 150 mg/kg/day AI (Animal R1506) was sacrificed on PND 75 following the presence of protruding eyes. In the absence of similar observations in test article treated animals, this death was considered to have occurred incidentally.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Pup Body Weights: No test article-related effect on body weights was noted.
Mean body weights were essentially similar across all dose groups at the end of the pre weaning phase, and statistical analysis of the data did not reveal any statistically significant intergroup differences.

Pup Organ Weights: Nonadverse test article-related increases in spleen weights were evident following 50 or 150 mg/kg/day AI, compared with controls.
Spleen:body weight ratios were higher for F1 female pups from dams administered 50 mg/kg/day and F1 pups of both sexes from dams administered 150 mg/kg/day AI, compared to F1 control pups (P < 0.05).This was not observed for pups administered 100 mg/kg/day AI, compared with controls. Test article-related effects on the spleen were prevalent in this study; as such, the increase in spleen weight was considered test article related, although these increases were considered not to represent an adverse effect.


F1 Body Weights: No test article-related body weight or body weight changes have been noted.
Mean body weights for test article-treated groups were essentially similar to controls.
A small statistically significant reduction in mean body weight gain was evident for Cohort 1A males administered 150 mg/kg/day AI, compared with controls, between PND 22 to 24 (P<0.05). This intergroup difference was not observed again for the Cohort 1A males and was not evident for the Cohort 1B males administered at the same dose level; as such, this was considered to have arisen incidentally with no relationship to test article administration.

F1 Organ Weights: Test article-related changes in splenic weights and weight ratios were recorded for F1 Cohort 1A animals from all test article-treated groups.
A generally dose-related increase in group mean unadjusted splenic weights, spleen:body weight, and spleen:brain weight ratios was recorded for F1 Cohort 1A animals from all test article treated groups, compared with controls, which generally correlated with the macroscopic and microscopic findings. A minor, statistically significant increase in liver weights and weight ratios was recorded for F1 Cohort 1A females administered 150 mg/kg/day AI, but this occurred without a corresponding microscopic finding.
No test article-related changes in reproductive organ weights or weight ratios were recorded for Cohort 1B animals. Minor organ weight and organ weight ratio changes were attributed to biological variation.
All other organ weight and organ weight ratio changes were attributed to biological variation and were considered not test article related as they were small in magnitude, not dose-dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test article-related effects on mean food consumption was evident for test article treated groups, compared with controls.
Statistical analysis of the data did not reveal any significant intergroup differences.
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):
Nonadverse test article-related changes in red cell parameters were evident for males and females throughout the dose groups.
Hemoglobin, mean cell hemoglobin concentration, and red cell counts were reduced for males and females administered 150 mg/kg/day AI, compared with controls and mean cell hemoglobin, mean cell volume, and reticulocyte counts and % reticulocytes were increased (P < 0.001), compared with controls. Red cell distribution was reduced for males administered 150 mg/kg/day AI (P < 0.001), and packed cell volume was also reduced for females administered 150 mg/kg/day AI (P< 0.05), compared with controls.
Hemoglobin and red cell count were reduced for males and females administered 100 mg/kg/day AI (P < 0.001). Mean cell hemoglobin concentration was reduced for males (P < 0.001) and packed cell volume and mean cell hemoglobin concentration was also reduced for females (P < 0.01), compared with controls. Mean cell hemoglobin, mean cell volume, reticulocyte counts, and % reticulocytes were increased (P < 0.001), compared with controls.
Hemoglobin and mean cell hemoglobin concentration, red cell count, and red cell distribution was reduced for males administered 50 mg/kg/day AI (P < 0.05 to P < 0.001), compared with controls. Red blood cell counts and mean cell hemoglobin concentration were reduced for females administered 50 mg/kg/day AI (P < 0.01). Mean cell hemoglobin, mean cell volume, reticulocyte counts, and % reticulocytes were increased for males and females administered 50 mg/kg/day AI (P < 0.001), compared with controls.
Remaining statistically significant intergroup differences were observed in one sex or did not exhibit a dose relationship; as such, they were considered to have arisen incidentally.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Nonadverse test article-related clinical chemistry changes of decreased glucose, triglyceride, and globulin levels (P < 0.05 to P< 0.001) with associated increases in A:G ratio (P < 0.001) were observed for males administered 150 mg/kg/day AI, compared with controls. No associated organ changes were noted to suggest that these changes resulted in adversity; therefore, these changes were of little toxicological significance.
No such changes were evident for females administered 150 mg/kg/day AI or animals of both sexes administered 50 or 100 mg/kg/day AI.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test article-related effects were noted for the urine parameters assessed for males or females at any dose level investigated.
Statistical analysis of the urine volume or specific gravity data did not reveal any significant intergroup differences.
Sexual maturation:
no effects observed
Description (incidence and severity):
No test article-related effect on sexual maturation was noted.
Statistical analysis of the data did not reveal any significant intergroup differences.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test article-related effect on ano-genital distance was noted following administration of the test article at any dose level, compared with controls.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No presence of nipple retention was noted for any male pup following administration at any dose level.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article-related changes in splenic weights and weight ratios were recorded for F1 Cohort 1A animals from all test article-treated groups.
A generally dose-related increase in group mean unadjusted splenic weights, spleen:body weight, and spleen:brain weight ratios was recorded for F1 Cohort 1A animals from all test article treated groups, compared with controls, which generally correlated with the macroscopic and microscopic findings. A minor, statistically significant increase in liver weights and weight ratios was recorded for F1 Cohort 1A females administered 150 mg/kg/day AI, but this occurred without a corresponding microscopic finding.
No test article-related changes in reproductive organ weights or weight ratios were recorded for Cohort 1B animals. Minor organ weight and organ weight ratio changes were attributed to biological variation.
All other organ weight and organ weight ratio changes were attributed to biological variation and were considered not test article related as they were small in magnitude, not dose-dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Pup Macroscopic Observations: Nonadverse, test article-related enlarged spleens were recorded for one male pup each from a dam administered 50 or 150 mg/kg/day.
This was not observed for male pups administered 100 mg/kg/day AI or female pups at any dose level. Test article-related effects on the spleen were observed; as such, the incidences of enlarged spleens were considered to be test article related, although these increases were considered not to represent an adverse effect.
Other tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rat pups of this strain and age.

F1 Macroscopic Observations: Upon macroscopic examination, test article-related findings were noted in the spleen.
Large spleen was recorded for occasional F1 Cohort 1A males from all test article treated groups, which generally correlated with microscopic findings. Large spleen was also recorded for one F1 Cohort 1B male (Animal R1135) administered 150 mg/kg/day AI.
Other tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Upon microscopic examination, test article-related findings were recorded for the spleen.
An increased incidence and/or severity of extramedullary hemopoiesis and pigmented macrophages was noted for F1 Cohort 1A animals administered 50, 100, or 150 mg/kg/day AI. No other test article-related microscopic findings were recorded. No effects attributable to the test article were noted in the examined lymphoid tissues.
Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.

Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Pup Thyroid Hormone Analysis: No test article-related changes in thyroxine or thyroid stimulating hormones were evident for pups on PND 22.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.

F1 Thyroid Hormone Analysis: No test article-related thyroxine or thyroid stimulating hormones changes were observed.
Males administered all dose levels attained statistically significant reductions in T4 levels, compared with controls (P < 0.05), although no convincing dose response was noted. Values were within the historical control data ranges (46-100 nmol/L), no correlating increase in TSH levels was observed, and a similar change was not evident for females; as such, this was considered to have arisen incidentally and unrelated to the test article.

F1 Estrous Cycle: No test article-related effects on the duration or mean number of estrous cycles was noted.The mean number and length of the estrous cycles was comparable across groups and statistical analysis of the data did not reveal any significant intergroup differences.

F1 Seminology Evaluations: No test article-related effects were observed for the sperm parameters investigated. The statistically significant intergroup differences observed did not exhibit a dose relationship or associated changes with other parameters; as such, they were considered to have arisen incidentally.

F1 Ovarian Follicle Counts: No test article-related changes were noted for ovarian follicles from test article-treated F1 females, compared with controls.
Statistical analysis did not reveal any statistically significant intergroup differences.
 

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
effects observed, treatment-related
Description (incidence and severity):
Nonadverse moderate increases in the absolute concentration of CD3+, CD3+CD4+, and CD3+CD8+ were noted in F1 generation Cohort 1A females administered 50 or 100 mg/kg/day AI; these increases were paralleled by a marginal increase in the relative frequencies of these immune cell populations.
No variation in the absolute concentration or relative frequency of CD45RA+ cells was observed for test article-treated F1 generation Cohort 1A animals.
Dose-dependent increases in the absolute concentration of CD3-CD161a+ cells, matched by negligible variations in frequency, were observed for test article-treated F1 generation Cohort 1A males.
Due to the variability in the absolute concentration and relative frequency observed in control and test article-treated F1 generation Cohort 1A animals, the contribution of the test article to the variations reported was deemed minor, and the test article was unlikely to contribute to immune toxicities; as such, it was considered nonadverse.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)

Applicant's summary and conclusion

Conclusions:
Once-daily oral gavage administration of 142.5, 285, or 427.5 mg/kg/day (equivalent to 50, 100, or 150 mg/kg/day AI) test substance resulted in test article-related systemic effects at all dose levels investigated and in both generations. Test article-related effects consisted of clinical observations noted after dosing, which were attributable to test article palatability and did not represent systemic toxicity. Changes in red cell parameters were also observed, together with an increased incidence and/or severity of extramedullary hemopoiesis and pigmented macrophages in the spleen, which corresponded with incidences of large spleens and increased spleen weights. These findings, however, did not have an impact on the overall health of the animals; as such, the no observed adverse effect level (NOAEL) for systemic toxicity is 150 mg/kg/day AI.
No adverse effects on mating performance, fertility, fecundity, gestation, parturition, or lactation were noted; as such, the no observed effect level (NOEL) for reproductive toxicity is 150 mg/kg/day AI in this study.
No effects on the developing offspring was noted at any dose level; as such, the NOEL for offspring growth and development is 150 mg/kg/day AI.