3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two key studies are performed with the test substance.

*The test substance was administered to rats for a period up to 121 consecutive days at dose levels up to 150 mg/kg bw/day (active ingredient, AI), in an extended one-generation reproductive toxicity study performed according to OECD guideline 443 (Barraclough N, 2021). The NOAEL for systemic toxicity is 150 mg/kg bw/day AI. The NOEL for reproductive toxicity and the NOEL for offspring growth and development are considered 150 mg/kg bw/day AI.

*The test substance was administered to rats for a period of up to 54 consecutive days at dose levels up to 100 mg/kg bw/day, in a screening test for reproduction/developmental toxicity performed according to OECD guideline 421 (Marr, 2010). The NOEL for systemic and reproductive toxicity was considered 100 mg/kg bw/day (based on 31.8% purity).

Link to relevant study records

Reference

Reference 1

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 2021-10-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

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

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.

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

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:

- F0 males were dosed for 119 consecutive days (10 weeks prior to pairing, 2 weeks during pairing, and 35 days post-pairing); females were dosed for up to 121 days (10 weeks prior to pairing, during pairing, throughout gestation or the post-pairing phase, and up to LD 21).
- All F1 offspring were dosed from PND 14 to 21; prior to this, pups received test article through the milk. Following selection into appropriate cohorts, the selected F1 animals were administered once daily

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:

- 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.
- 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.
Background information:
* In a 28-day, repeat-dose toxicity study in the Sprague-Dawley rat (Safepharm Laboratories Limited Study number 625/051), animals were administered 15, 150, or 1000 mg/kg bw/day AI of the test article by oral gavage. Clinical observations of toxicity and functional incidents, such as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption, and reduced body weight gain, were observed in animals administered 1000 or 150 mg/kg bw/day AI. Test article-related changes in the liver and spleen were detected in animals administered 1000 or 150 mg/kg bw/day AI. Histopathological changes were observed in the kidneys, urinary bladder, and stomach of animals administered the high dose. Hematological changes (hemolytic anemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time) and elevations in plasma aspartate aminotransferase, alanine aminotransferase, and bilirubin were observed in animals administered 1000 or 150 mg/kg bw/day AI. Therefore, the no observed effect level (NOEL) for this study was considered to be 15 mg/kg bw/day AI.
* In an OECD 421 reproduction/developmental toxicity screening study in the Han Wistar rat (Harlan Laboratories Ltd Study 2833-003), administration of 15, 40, or 100 mg/kg bw/day AI did not result in any test article-related effects on reproduction or offspring development up to PND 5.
* In a 90-day repeat-oral dose toxicity study in the Sprague-Dawley rat (Covance Study 8280017), animals were administered 50, 150, or 500 mg/kg bw/day AI of the test article by oral gavage. One male administered 500 mg/kg bw/day AI was found dead during Week 12 and this was considered test article related. Clinical observations at this dose included salivation, staining around the mouth, and noisy breathing and, in combination with high water consumption values, this was indicative of an irritant or unpalatable test article formulation. Decreased hemoglobin, red blood cell counts, mean cell hemoglobin concentration, hemoglobin distribution width, and red cell distribution width and increased reticulocyte counts and percentage, mean cell, volume and mean cell hemoglobin were noted. Microscopic spleen changes of hemosiderin deposition were noted, together with an increase in spleen weight, in animals administered 500 mg/kg bw/day AI. Microscopic examination revealed adverse findings in animals administered 500 mg/kg bw/day AI, including squamous cell hyperplasia of the forestomach and transitional cell hyperplasia of the urinary bladder, which was considered a result of localized irritancy properties of the test article. Findings in animals administered 150 mg/kg bw/day AI included low body weight gains, and as no effect on food consumption was noted, this was considered nonspecific toxicity. Forestomach squamous cell hyperplasia was noted in a single male administered 150 mg/kg bw/day AI and was considered adverse. Test article-related red blood cell changes were noted but considered nonadverse. The only test article-related change observed in males administered 50 mg/kg bw/day AI were statistically relevant minor changes in the red blood cell parameters, which were considered test article-related but not adverse in nature; therefore, the no observed effect level (NOEL) for this study was considered to be 50 mg/kg bw/day AI.
* In a recent pre- and postnatal development dose range-finding study (Covance Study 8410554) performed in the pregnant female Han Wistar rat at dose levels of 75, 100, or 125 mg/kg bw/day AI, no unscheduled deaths occurred as a result of the test article. During gestation, incidences of mouth rubbing were noted after dosing in animals administered 100 or 125 mg/kg bw/day AI, although no test article effects on body weights or food consumption were evident. At sacrifice on GD 21, uterine contents were not adversely affected, although gravid uterus weights and total weight change were marginally higher in animals administered 125 mg/kg bw/day compared with controls. No effect on fetal or placenta weights were noted. Female fetal ano-genital distance was reduced for female fetuses from the group administered 125 mg/kg bw/day AI, compared with control (-13.5%), although the etiology of this is unclear. One fetus from a 125 mg/kg bw/day AI litter exhibited malformations of omphalocele, short trunk, malrotated hindlimb, and micromelia of the hind and forelimb. The fetus was also recorded with variations of edema of the head and neck and small genital tubercle. This was isolated to one fetus, remaining fetuses did not exhibit any variations or malformations, and the etiology of this finding is unclear; however, as no effects were noted in the pups following parturition in this group, this was considered an incidental finding. In the postnatal subgroup, isolated instances of mouth rubbing and excessive salivation were noted for females administered 125 mg/kg bw/day AI during lactation. One female administered 75 mg/kg bw/day AI had a total post-natal litter loss on LD 0. The litter, consisting of three pups, was confirmed to have been born alive, but had no milk in the stomach at necropsy. In isolation, this finding was considered to have arisen incidentally. No test article-related effects on litter data or pup weights were observed, and no test article-related clinical observations were noted. Ano-genital distance recorded on PND 4 was marginally lower for female pups maternally administered 125 mg/kg bw/day AI, compared with controls (-6%). At necropsy, one pup maternally administered 125 mg/kg bw/day AI was recorded with pelvic dilatation of the kidneys, and one was recorded with a tail sore. No other macroscopic observations were noted for the pups. For the adults, thyroid weights were lower in all test article-treated groups, compared with controls, although a dose response was not evident (-3, -20, or -10% for animals administered 75, 100, or 125 mg/kg bw/day AI). Pup thyroid weights, taken on PND 22 were increased by +16, -30, or +22% for absolute weights and +25, +24, or +27% for body weight relative, following maternal exposure of 75, 100, or 125 mg/kg bw/day AI. Test article-related macroscopic findings noted in the adult females were confined to a single incidence of pelvic dilatation of the right kidney noted for one female administered 125 mg/kg bw/day AI, and a uterine mass on the right uterine horn for one female administered 100 mg/kg bw/day AI.

Positive control:

not applicable

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

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: 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.

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

Key result

Critical effects observed:

no

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.
 

Behaviour (functional findings):

not examined

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.

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-24 - 2009-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Physical state: pale amber liquid
- Analytical purity: 31.8% (99.9% w/w active ingredient), 35.46% (based on solid substance)
- Lot/batch No.: PA19100368
- Storage condition of test material: room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: 12 weeks old
- Weight at study initiation: males: 305-358 g; females: 189-219 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages.
Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum; pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: September 15, 2009 To: November 8, 2009

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test material formulations were determined. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.

Details on mating procedure:

- M/F ratio per cage: on Day 15, all animals were paired on a 1 male:1 female basis within each dose group
- Length of cohabitation: Maximum of 14 days
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
- After successful mating each pregnant female was caged (how): Females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken and analysed for concentration. The test material formulations were diluted with water to give a final, theoretical test material concentration of approximately 3 mg/ml. Standard solutions of test material were prepared in water at a nominal concentration of 3 mg/ml.

The concentration was determined by high performance liquid chromatography (HPLC) using an external standard technique.
HPLC : Agilent Technologies 1100 or 1200, incorporating autosampler and workstation
Column : Gemini 3µ C18 (100 x 4.6 mm id)
Column temp : 40 °C
Mobile phase : Eluent A:acetonitrile with 0.1% trifluoroacetic acid; Eluent B:water with 0.1% trifluoroacetic acid
Flow-rate : 1 ml/min
UV detector wavelength: 205 nm
Injection volume : 25 µl
Retention time : Profile of peaks from ~ 1.4 to 10.5 mins

The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery. The results indicate that the prepared formulations were within ± 2% of the nominal concentration.

Duration of treatment / exposure:

54 days

Frequency of treatment:

Daily

Details on study schedule:

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
iii) Following evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Dose / conc.:

0 mg/kg bw/day

Remarks:

actual ingested

Dose / conc.:

15 mg/kg bw/day

Remarks:

actual ingested, incorporating a correction factor for 31.8% purity, converted to 16.7 mg/kg bw/day considering correction for 35.46% purity

Dose / conc.:

40 mg/kg bw/day

Remarks:

actual ingested, incorporating a correction factor for 31.8% purity, converted to 44.5 mg/kg bw/day considering correction for 35.46% purity

Dose / conc.:

100 mg/kg bw/day

Remarks:

actual ingested, incorporating a correction factor for 31.8% purity, converted to 111.35 mg/kg bw/day considering correction for 35.46% purity

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1 - 4).

WATER CONSUMPTION AND COMPOUND INTAKE:
Water intake was observed daily by visual inspection of water bottles for any overt changes. Inter-group differences did not indicate any need for more formal gravimetric measurements.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded and offspring were individually identified within each litter by a tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Days 1 and 4 post partum

All live offspring were assessed for surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy were killed after Day 26 post coitum.

GROSS NECROPSY
All adult animals including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

HISTOPATHOLOGY
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where indicated:
Coagulating gland, Prostate, Epididymides, Seminal vesicles, Ovaries, Testes, Mammary tissue, Uterus/Cervix, Pituitary, Vagina.
The tissues from control and 100 mg/kg/day dose group animals, and any animals which failed to achieve a pregnancy, were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from control and 100 mg/kg/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

GROSS NECROPSY
Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change, Food consumption for females during gestation and lactation, Litter data, Sex ratio, Implantation losses and viability indices, Offspring bodyweight and bodyweight change, Offspring surface righting, Adult absolute and bodyweight relative organ weights.

Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnetts test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test. The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

In addition, histopathological findings were analysed (excluding non-mated females and females not producing a pregnancy/litter) using the following methods: Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Reproductive indices:

- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices: mating index (%) and pregnancy index (%)
- Gestation length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition index

Offspring viability indices:

- Implantation losses (%)
- Live birth and viability indices
- Sex ratio (% males)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant findings were observed throughout the study. Sporadic episodes of increased salivation were evident in animals of either sex treated with 100 mg/kg/day, one male from this treatment group also had red/brown staining around the mouth. One 40 mg/kg/day female had noisy respiration. Generalised fur loss was detected in one 15 mg/kg/day and one control female. Increased salivation is often recorded following the oral administration of an unpleasant tasting test material formulation and is considered not to be indicative of systemic toxicity. The remaining observations are considered incidental and not to represent an adverse effect of treatment. No such effects were detected in the 40 or 15 mg/kg/day males.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically significant effect on bodyweight development was detected for treated animals, in comparison to controls. A slight but statistically significant increase in bodyweight gain (P<0.05) was evident in the 40 and 100 mg/kg/day males during Weeks 5 and 6. This increase was also seen in the 15 mg/kg/day males during Week 6 only. There was no adverse effect on overall bodyweight gain between the treated and control animals. In the absence of an effect on dietary intake or supporting histopathological correlates this finding is considered to be of no toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effect on dietary intake was detected for treated animals, in comparison to controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Water consumption was monitored daily by visual inspection of the water bottles. There was no adverse effect on water consumption.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No adverse effect on mating performance was detected. All animals from the control and treated groups mated within four days of pairing with the exception of one 15 mg/kg/day female which mated after thirteen days of pairing.

Key result

Dose descriptor:

NOEL

Remarks:

for reproductive toxicity

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

NOEL is 111.35 mg/kg bw/day based on 35.46% purity

Key result

Dose descriptor:

NOEL

Remarks:

for systemic toxicity

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

NOEL is 111.35 mg/kg bw/day based on 35.46% purity

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs were detected

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect on total litter weights, offspring bodyweight development or surface righting reflex was detected in treated animals, in comparison to controls.

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At terminal kill, one female treated with 15 mg/kg/day and one treated with 100 mg/kg/day produced a litter with one pup which showed a dark left testis. One 40 mg/kg/day female produced a litter with one pup which showed a dark right testis. There were no abnormalities detected in the remaining animals. The incidental signs detected were considered to be of no toxicological importance.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

VIABILITY (OFFSPRING)
There were no obvious differences in the number of corpora lutea or implantation sites from treated females when compared to controls; and pre-implantation and postimplantation losses from treated animals were comparable to controls. Furthermore, no obvious effect on litter size, sex ratio and offspring viability were detected for treated animals, in comparison to controls.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

NOEL is 111.35 mg/kg bw/day based on 35.46% purity

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The oral administration of the test material to rats for a period of up to fifty-four consecutive days at dose levels of up to 100 mg/kg/day, did not result in any treatment-related effects. The No Observed Effect Level (NOEL) for systemic toxicity was therefore considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). The No Observed Effect Level (NOEL) for reproductive toxicity was considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). However, taking into account the purity based on solid substance (35.46%) is recommended and based on this purity and expert judgement by the registrant, and thus factor 2.82, the NOEL for systemic and reproductive toxicity can be considered to be 111.35 mg/kg.

Executive summary:

The study was performed to screen for potential adverse effects of the test material on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study complies with the recommendations of the OECD Guideline No. 421 (Reproduction/Developmental Toxicity Screening Test). The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han:HsdRccHan:WIST strain rats, for up to fifty-four consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 15, 40 and 100 mg/kg/day (incorporating a correction factor for 31.8% purity). A control group of ten males and ten females was dosed with vehicle alone (Distilled water). Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, and all surviving females and offspring on Day 5 post partum. Any females that failed to achieve pregnancy were killed after Day 26 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. The oral administration of the test material to rats for a period of up to fifty-four consecutive days at dose levels of up to 100 mg/kg/day, did not result in any treatment-related effects.

The 'No Observed Effect Level' (NOEL) for systemic toxicity was therefore considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). The 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). However, taking into account the purity based on solid substance (35.46%) is recommended and based on this purity and thus factor 2.82, the NOEL for systemic and reproductive toxicity can be considered to be 111.35 mg/kg. 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study: Extended One Generation Reproductive Toxicity Study performed according to OECD guideline 443; GLP-compliant study.

Once-daily oral gavage administration of 142.5, 285, or 427.5 mg/kg bw/day (equivalent to 50, 100, or 150 mg/kg bw/day AI) of the test item resulted in test article-related systemic effects at all dose levels investigated and in both generations (Barraclough, 2021). Four groups of 24 male and 24 female Crl:WI(Han) rats were allocated to the study as the parental (F0) generation, and were administered 0, 50, 100, or 150 mg/kg bw/day AI. Formulations were administered by oral gavage for up to 119 consecutive days for males (10 weeks prior to pairing, 2 weeks during pairing, and 35 days post-pairing) or up to 121 days for females (10 weeks prior to pairing, during pairing, throughout gestation, and up to Lactation day 21. Direct dosing to the F1 offspring was initiated from PND 14. Formulations were administered at a constant dose volume of 10 mL/kg based on most recently recorded body weight, and the control article (vehicle) was purified water. Doses were administered at approximately the same time each day. 

Assessment of toxicity for the F0 generation was based on mortality, clinical observations, body weights and body weight changes, food consumption, estrous cycles, pregnancy indices, parturition, offspring development, and clinical pathology (hematology, coagulation, clinical chemistry, and urinalysis) and anatomic pathology evaluations. Litters were observed from birth, and the number of pups, sexes, body weights, and any clinical observations were monitored. Ano-genital distance was measured on PND4, and areolae (nipple) counts were recorded for male offspring on PND13. F0 males were sacrificed during week 18, and females were sacrificed on LD22, along with pups not selected for the next phase of the study. Blood samples were taken for thyroid hormone assessments; selected organs were weighed, and macroscopic examinations were performed on the unselected offspring. 

Pups were selected from F1 litters to form two cohorts of the F1 generation and were administered the test article until the day prior to necropsy for each cohort (cohort 1A, up to PND92, or cohort 1B, up to PND112). Assessment of toxicity for the F1 generation was based on mortality, clinical observations, body weights, food consumption, sexual maturation and estrous cycle assessments, macroscopic examinations, and selected organ weights were recorded. In addition, ovarian follicle counts, seminology analysis, and clinical and anatomic pathology assessments were performed for cohort 1A. 

Parental (F0) generation:

No test article-related unscheduled deaths occurred. 

A test article-related clinical and postdose observation of mouth rubbing was noted for all males and females administered 100 or 150 mg/kg bw/day AI, and a dose response was evident, with fewer incidences observed following 50 mg/kg bw/day AI administration. A high incidence of excessive salivation was also noted for males and females administered 150 mg/kg bw/day AI and for some males administered 100 mg/kg bw/day AI. Three females administered 150 mg/kg bw/day AI were recorded with paddling behavior, and three males and three females administered 100 mg/kg bw/day AI. Such observations are common in gavage studies and are considered due to the unpalatable taste or irritant nature of the test article formulation rather than an indication of test article toxicity; therefore, they were considered nonadverse. 

No test article-related observations were noted during the weekly detailed examinations. No test article-related effects on mean body weight, body weight gain, or food consumption were noted following administration of all dose levels throughout the study. Nonadverse and dose-related test article-changes in the red cell parameters were evident for males and females administered at all dose levels, compared with controls. These included reductions in mean hemoglobin, red blood cell counts, red cell distribution width, and mean cell hemoglobin, compared with controls. Associated increases in reticulocyte counts and mean cell volume were evident at all dose levels, compared with controls; and a clear dose response was evident.

Nonadverse test article-related reductions in potassium levels were evident for males and females administered 150 mg/kg bw/day AI compared with controls, with the effect extending to females administered 100 mg/kg bw/day AI. No test article-related effects on thyroxine or thyroid stimulating hormone were observed for adults, and no test article-related effects were noted for urinalysis parameters. Microscopically, an increased severity of extramedullary hemopoiesis and pigmented macrophages was noted in the spleen for animals at all dose levels, which generally correlated with the macroscopic findings of enlarged spleen observed for four females and three males administered 150 mg/kg bw/day AI and increases in spleen weights observed throughout the dose groups. 

No test article-related effects on estrous cycle assessments were noted for number and duration of estrous cycles prior to pairing. 

Mating, fecundity, or fertility indices were unaffected by the test article, and no effect on gestation length was noted. Furthermore, no test article-related effects on the sperm production or quality were noted.

After completion of parturition, 20, 20, 19, or 21 females administered 0, 50, 100, or 150 mg/kg bw/day AI delivered a live litter, respectively. No test article-related effects were observed on gestation length, parturition, pup survival, or litter size.

F1 Generation Offspring (pre-weaning):

Test article-related clinical observations were confined to the presence of mouth rubbing observed immediately after direct dosing from PND14 for some animals administered 100 or 150 mg/kg bw/day AI, which was considered associated with the unpalatable nature of the test article formulations and was considered nonadverse. No test article-related effect on sex ratio, body weights, or ano-genital distance was noted, and no presence of nipple retention was noted for any male pup. Thyroxine and thyroid stimulating hormones were unaffected for pups on PND 22. 

F1 Generation Offspring (post-weaning):

No test article-related unscheduled deaths occurred. Nonadverse test article-related clinical observations associated with the unpalatable nature of the test article formulations were observed. Mouth rubbing was observed at all dose levels in a dose-dependent manner. Transient instances of paddling and excessive salivation were also recorded for three females and one male administered 150 mg/kg bw/day AI and two males administered 100 mg/kg bw/day AI. Isolated instances of excessive salivation were also evident for males and two females administered 150 mg/kg bw/day AI and one female administered 100 mg/kg bw/day AI. No test article-related body weight, body weight gains, or food consumption changes were evident. No test article-related effect on sexual maturation was noted, and the estrous cycle was unaffected by administration of the test article at any dose level. 

Hemoglobin, red blood cell counts, and mean cell hemoglobin concentration were significantly reduced for males and females administered at all dose levels, compared with controls, with a clear dose response. Associated increases in percentage reticulocytes (counts and percentage), mean cell volume and mean cell hemoglobin were also observed, although these changes were considered not to represent an adverse effect of the test article. Nonadverse decreased glucose, triglyceride, globulin, and albumin levels, with associated increases in albumin:globulin ratio, were confined to males administered 150 mg/kg bw/day AI, compared with controls. No test article-related thyronine or thyroid stimulating hormones changes were observed. Splenic immunophenotyping for CD3+, CD3+CD4+, and CD3+CD8 exhibited moderate increases for all female dose groups, compared with controls, and increases in CD3-CD161a+ for all test article treated males, compared with controls. However, due to variability, these differences were considered not to represent an adverse immunotoxic response at any dose level. No test article-related effects were observed on the sperm parameters or ovarian follicle counts. An increase in the severity of extramedullary hemopoiesis and of pigmented macrophages was noted microscopically in the spleen from F1 cohort 1A animals of both sexes treated at all dose levels, compared with controls, which generally correlated with the macroscopic findings of enlarged spleen and increases in spleen weights. 

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 bw/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 bw/day AI in this study.
No effects on the developing offspring were noted at any dose level; as such, the NOEL for offspring growth and development is 150 mg/kg bw/day AI.

Key study: Screening test for reproduction/developmental toxicity performed according to OECD guideline 421; GLP-compliant study.

The oral administration of the test material to rats for a period of up to fifty-four consecutive days at dose levels of up to 100 mg/kg bw/day, did not result in any treatment-related effects (Marr, 2010). The No Observed Effect Level (NOEL, and thus also NOAEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day (incorporating a correction factor for 31.8% purity; converted to 111.35 mg/kg bw/day based on 35.46% purity). The No Observed Effect Level (NOEL) for reproductive toxicity was considered 100 mg/kg bw/day (incorporating a correction factor for 31.8% purity; converted to 111.35 mg/kg bw/day based on 35.46% purity).

Effects on developmental toxicity

Description of key information

- Key study: A key prenatal development study in rabbits is performed according to OECD guideline 414 and according to GLP requirements (Barraclough, 2021; Klimisch 1). The NOAEL for embryo-fetal survival, development and growth is set at 125 mg/kg bw/day when the test substance is administered during organogenesis in the rabbit. 

- Key study: A key prenatal development study in rat is performed according to OECD guideline 414 and according to GLP requirements (Barraclough, 2021; Klimisch 1). The NOAEL for embryo-fetal survival, development and growth is set at 250 mg/kg bw/day when the test substance is administered during organogenesis in the rat. To facilitate dose level selection, a dose range finder in pregnant rats was performed.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019-10-17 to 2020-02-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline required

Principles of method if other than guideline:

The study was non-specific but was expected to meet the requirements of the relevant agencies.

GLP compliance:

no

Remarks:

The study is a non-regulatory study for which a claim of GLP compliance was not made. However, the laboratory procedures used were consistent with International Standards of GLP.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.: S019341917
- Substance type: Almost clear colorless liquid
- Physical state: liquid
- Molecular weight: 316 g/mol
- Analytical purity: 35.2% in water
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Expiration date of the lot/batch: 28 March 2021
- Purity/weighing factor: none

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 refrigerated (2 to 8° C), or for 14 days at room temperature (15 to 25°C) in Covance Study 8410552, Covance Laboratories, 2020c).

OTHER:
- Correction factor: 2.85, based on purity

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sixty time-mated female Crl:WI(Han) rats were obtained from Charles River Laboratories, Margate, United Kingdom.
- Age at study initiation: approx. 9 weeks old at the time of mating.
- Weight at study initiation: at least 140 g at time of mating. Animals were delivered to Covance by GD 3 and weighed between 171.6 and 252.2 g at the start of dosing.
- Fasting period before study: not indicated
- 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). Females were housed individually during gestation and with their litter during lactation. Bedding was provided on a weekly basis to each cage by use of European Softwood bedding or clean Aspen wood chips (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. Animals were provided with wooden Aspen chew blocks and nesting materials as forms of environmental enrichment.
- Diet (e.g. ad libitum): Animals had ad libitum access to 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 which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels which might have interfered with achieving the objective of the study.
- Acclimation period: Acclimation was limited by mated status, 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 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2019-10-21 To: 2020-02-11

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulation: formulations were prepared weekly. The test article was formulated as a suspension in purified water.
- Formulations were stored refrigerated (2 to 8 °C) in a sealed container, protected from light.
- Formulations of 0.2 and 100 mg/mL AI were stable for 15 days refrigerated (2 to 8 °C), or for 14 days at room temperature (15 to 25 °C) in Covance study 8410552.
- Formulations were stirred continuously from at least 30 minutes prior to and throughout dosing (excluding the control group). On GD8, group 5, subgroup 1 animals R0802 and R0805 (1000 mg/kg/day AI) were not doses due to significant body weight loss.
- A dose volume of 10 mL/kg was used. Individual dose volumes were based on the most recent individual body weight.

VEHICLE
- Amount of vehicle (if gavage): 10 mL

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: Mating for Crl:WI(Han) rats (overnight at Charles River Laboratories, Margate, United Kingdom) was confirmed by the presence of a vaginal plug in situ, or other evidence of mating, if necessary. The day on which mating was confirmed was designated as Gestation Day (GD) 0.

Duration of treatment / exposure:

- Prenatal development (Subgroup 1) animals were dosed once daily from GD 6 through 20.
- Postnatal development (Subgroup 2) animals were dosed once daily from GD 6 to Lactation Day (LD) 21.

Frequency of treatment:

Once daily

Duration of test:

114 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1; Control
Subgroup 1 and 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low)
Subgroup 1 and 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate)
Subgroup 1 and 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Group 4 (High)
Subgroup 1 and 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 5 (High II)
Subgroup 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 6 (Intermediate II)
Subgroup 1

No. of animals per sex per dose:

- Prenatal Development (Subgroup 1): 6 female animals/dose group (6 groups). Dosing was omitted if an animal was in or near parturition at the time of daily dosing. This included Animals R0501, R0505 and R0506 (Group 2); R0603 (Group 3); and R0703 (Group 4).
- Prenatal Development (Subgroup 2): 6 female animals/dose group (4 groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Species selection rationale: The 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. Historical control data are available at the test facility for this strain of animal.

- Administration route selection rationale: The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.

- Dose selection rationale:
*High II Dose: In the absence of any obvious test article-related effects noted in the lower dose levels to establish a suitable high-dose level for the subsequent OECD test guideline 414 and 443 rat studies, and considering the findings of the previous 28-day study, the limit dose of 1000 mg/kg/day active ingredient (AI) was selected as the high II dose level, to observe for maternal toxicity and effects on prenatal development.
*High Dose: The high-dose level of 125 mg/kg/day AI was expected to elicit some toxicity without causing death.
* Intermediate II Dose: Due to the deaths of two animals noted following administration of 1000 mg/kg/day AI on GD 8, and significant body weight losses, the dose level was considered excessive and the remaining animals were terminated on the same day. The dose level of 500 mg/kg/day AI was considered suitable for exploration in this study.
*Intermediate Dose: An intermediate-dose level of 100 mg/kg/day AI was expected to show minimal test article-related effects.
*Low Dose: A dose level of 75 mg/kg/day AI was selected as the low dose, as it was expected to be well tolerated.
*Background information:
**In a 28-day repeated dose toxicity study in the Sprague-Dawley rat (Safepharm Laboratories Limited Study number 625/051), animals were administered 15, 150, or 1000 mg/kg/day AI by oral gavage. Clinical observations indicative of toxicity; functional incidents, such as hunched posture; sensory reactivity; reduced food consumption; increased water consumption; and reduced body weight gain were observed for animals administered 1000 or 150 mg/kg/day AI. Treatment-related changes in liver and spleen were observed following administration of 1000 or 150 mg/kg/day AI. Microscopic changes were also observed in the kidneys, urinary bladder, and stomach of high-dose animals. Hematological changes (hemolytic anemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time) and elevations in plasma aspartate aminotransferase, alanine aminotransferase, and bilirubin were observed in animals administered 1000 or 150 mg/kg/day AI. Therefore, the no observed effect level (NOEL) for this study was considered 15 mg/kg/day AI.
**In a 90-day repeated oral dose toxicity study in Sprague-Dawley animals were administered 50, 150, or 500 mg/kg/day AI by oral gavage (Covance Study 8280017). At the high dose of 500 mg/kg/day AI, one male was found dead during Week 12; this death was considered treatment-related. Clinical observations at this dose included salivation, staining around the mouth and noisy breathing, in combination with high water consumption values; this indicated irritant test materials and the animals not liking the taste of the formulation. Microscopic examination revealed adverse findings in the (fore) stomach (squamous cell hyperplasia) and urinary bladder (transitional cell hyperplasia), which were considered a result of the irritating properties of the test article. Findings for animals administered 150 mg/kg/day AI included low body weight gains; as no effect on food consumption was observed, this was considered non-specific toxicity. Squamous cell hyperplasia of the forestomach was noted in one male administered 150 mg/kg/day AI and, as this finding matched those recorded for those administered 500 mg/kg/day AI, it was considered adverse. The only treatment-related changes noted in males administered 50 mg/kg/day AI were statistically relevant minor changes in the red blood cell parameters, which were considered treatment-related but not adverse in nature; therefore, the NOEL for this study was considered 50 mg/kg/day AI.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed in the cage twice daily, at the beginning and end of the working day for signs of ill health or overt toxicity. Animals were observed daily immediately after dosing upon return to the home cage, and approximately 1 hour (±30 minutes) postdose. Postdose observations for subgroup 2 animals were not recorded from GD 20 to LD 4, to limit disturbing the litter. Character and timing of reactions to treatment were recorded, together with all observations (including absence of observations but were not reported).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination outside of the cage, daily from the start of dosing until necropsy. An individual record was maintained of the clinical condition of each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded for Subgroup 1 animals on GD 3, 6, 7, 8, 9, 12, 15, 17, 19, 20, and 21. Body weights were recorded for Subgroup 2 animals on GD 3, 6, 7, 8, 9, 12, 15, 17, 19, 20 and 21 and on LD 1, 4, 7, 14, 17, and 22.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Daily food consumptions were recorded for all animals from GD 6 through 21, and from LD 0/1 through 14 for Subgroup 2 animals only. Consumption was calculated as g/animal/day.

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

Terminal procedure - maternal animals:
All animals that died prior to their scheduled sacrifice were examined macroscopically for abnormalities of the thoracic and abdominal viscera.
- Subgroup 1 and 2 females were sacrificed in replicate order. Food was not removed prior to scheduled necropsies.
- Subgroup 1 females were sacrificed on GD21 by cervical dislocation following isoflurane anesthesia; death was confirmed by exsanguination.
- Subgroup 2: Females were sacrificed on LD 22 by isoflurane anesthesia; once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.

POST-MORTEM EXAMINATIONS: Yes
- Subgroup 1: Animals were examined macroscopically. All lesions were recorded and retained in the relevant fixative.
* Organs examined: ovaries and uteri
* The uterus of apparently non-pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.
- Subgroup 2:
*A macroscopic examination was performed, and all lesions were recorded. The following tissues from each animal were preserved in 10% neutral-buffered formalin unless otherwise indicated.
* Organs examined: cervix, thyroid, gross lesions, uterus , ovaries, vagina, pituitary
* The uterus of all mated and littered females and any apparently non-pregnant female was immersed in 10% ammonium sulfide solution, and implantation sites were counted.
The thyroid was dissected free from fat and other contiguous tissue and weighed postfixation and retained in 10% buffered formalin.

OTHER:
- Parturition:
Subgroup 2 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, or until a potential GD 25, whichever was earlier. Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation was recorded, where possible, and marked the end of gestation; when not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made. Abnormal observations of nesting, parturition, or nursing were recorded. It was considered important that the dam was disturbed as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).
- Estrous Stage Determination: The stage of the estrous for Subgroup 2 animals was recorded on LD 22 prior to necropsy

Ovaries and uterine content:

The uterus and ovaries were examined for implantations and corpora lutea, respectively. The extent of development of the implantation sites was determined as near as possible.
Maternal Necropsy - Subgroup 1:
- The ovaries and uteri were removed and examined, and the following data were recorded:
* Pregnancy status
* Gravid uterus weight
* Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported)
* Number of corpora lutea
* Number and intrauterine position of implantations subdivided into:
* Live fetuses
* Early intrauterine deaths
* Late intrauterine deaths
* Dead fetuses
- Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or fetal tissue, in addition to placental tissue. Dead fetuses were classified as those which appeared to have died shortly before necropsy.

Blood sampling:

not applicable

Fetal examinations:

Fetal Examinations:
- Litter Size and Sex Determination: On Postnatal Days (PND) 1, 4, 7, 14, 17, and 22, litter size and pup sex were recorded.
- Clinical Observations: Each animal was given a detailed clinical examination on PND 1, 4, 7, 14, 17, and 22.
- Body Weights: On PND 1, 4, 7, 14, 17, and 22, pup body weights were recorded.
- Ano-genital Distance: The Ano-genital distance of all pups was recorded on PND 4.
- Nipple/ Areolae Count: The number of nipples/areolae for male pups was counted on PND 13.
- 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.

Fetal necropsy:
- Live fetuses were sacrificed by a subcutaneous injection of sodium pentobarbitone followed by confirmation of cessation of circulation.
- Individual fetal and placental weights were recorded, and fetuses were examined externally and externally sexed.
- Fetal abnormalities were classified as malformations (rare and/or potentially lethal defects) or variations (commonly occurring non-lethal abnormalities).

Pup Necropsy:
- Subgroup 2 pups were sacrificed on PND 22 by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. - A macroscopic examination was performed for all pups, including decedents, with particular attention to reproductive organs.
- Thyroids were taken from one pup/sex/litter and dissected free from fat and other contiguous tissue and weighed post fixation and retained in 10% buffered formalin.

Statistics:

See section Any other information on materials and methods incl. tables

Indices:

- Corrected body weight (Carcass Weight) = (Terminal body weight - uterine weight) X 100
- Corrected body weight change = (Carcass weight - GD 6 body weight) X 100
- Total weight change = (GD 21 body weight - GD 6 body weight) X 100
- % male fetuses = (Number of male fetuses/Number of fetuses of determined sex) X 100
- % pre-implantation loss = ((Number of corpora lutea - number of implantations)/Number of corpora lutea) X 100
- % post-implantation loss = ((Number of implantations - number of live embryos)/Number of implantations) X 100
- % male pups = (Number of male pups Day 1/Number of pups of determined sex) X 100
- Gestation index = (Number of females with live pups/Number of pregnant females) X 100
- % post implantation survival index = (Number of pups born/Number of implantation sites) X 100
- % live birth index = (Number of pups alive Day 1/Number of pups born) X 100
- % Day 22 viability index = (Number of pups alive Day 22/Number of pups born alive) X 100

Historical control data:

Historical control data are available at the test facility for this strain of animal.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Test article-related clinical observations of raised hair, hunched posture, irregular respiration, cold to touch body, and/or soft/small/brown feces were noted prior to sacrifice on GD 8 for two animals administered 1000 mg/kg/day AI (Animals R0802 and R0805). These observations in this group were considered adverse. Incidences of raised hair, hunched posture, head shaking, irregular and/or noisy respiration were observed for animals administered 500 mg/kg/day AI. These observations were mostly confined to individual animals, and were considered nonadverse. No test article-related clinical observations were noted for animals administered ≤125 mg/kg/day AI.
Prior to their unscheduled sacrifice, test article-related postdose observations for animals administered 1000 mg/kg/day AI were confined to a single incidence of mouth rubbing upon return to the home cage on GD 8 (Animal R0804). This finding is generally associated with the unpalatable nature or unpleasant taste of the test article formulation, and in isolation was considered not to have represented a toxic effect of the test article. A test article-related increase in the incidence of mouth rubbing (from GD 11 or 12) was observed for animals administered ≤100 mg/kg/day AI, with isolated incidences of paddling behavior or excessive salivation also noted for one or two animals administered 500 mg/kg/day AI. These observations are generally associated with the unpalatable nature or unpleasant taste of test article formulation rather than an indication of test article toxicity. No test article-related postdose observations were noted for animals administered 75 mg/kg/day AI.

Postnatal Development (Subgroup 2): No test article-related clinical observation were noted. The isolated clinical observations recorded were confined to one or two animals within each group, including controls, and were considered low incidence findings occasionally observed in studies of this type and unrelated to the test article. Test article-related postdose observations were confined to a few incidences of mouth rubbing upon return to home cage during gestation (100 or 125 mg/kg/day AI) and lactation (125 mg/kg/day AI), with two animals from the latter dose group (Animals R0701 and R0702) also observed with isolated incidences of excessive salivation. Postdose observations of mouth rubbing and salivation are common following gavage administration and were considered associated with the unpalatable nature of unpleasant taste of the test article formulation, rather than an indication of test article toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Prenatal Development (Subgroup 1): Test article-related mortality occurred following administration of 1000 mg/kg/day AI. One female administered 1000 mg/kg/day AI (Animal R0806) was found dead on GD 8. The remaining animals from this group were also sacrificed on that same day because this dose level was considered excessive due to marked body weight losses and reduced food consumption. No unscheduled deaths occurred for the remaining groups.

Postnatal Development (Subgroup 2): No unscheduled deaths occurred as a consequence of test article toxicity. One female administered 75 mg/kg/day AI (Animal R0501) was sacrificed on LD 0 due to total litter loss. Only three live pups were noted in the litter, and in the absence of similar observations at the higher dose levels, this finding was considered incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Adverse, test article-related marked mean body weight loss was observed, between GD 7 and 8 following administration of 1000 mg/kg/day AI (mean body weight loss - 16.9 g; P < 0.01), and, as such, this dose level was considered excessive for further dosing.
Initial test article-related body weight losses were observed between GD 6 and 8 for females administered 500 mg/kg/day AI, and lower mean body weight gain was noted between GD 8 and 9 for these animals, with mean overall body weight gain approximately -19%, compared with controls over the dosing duration (GD 6 to 21). Overall mean body weights were not overtly affected, compared with controls; as such, the initial body weight losses were considered nonadverse. No test article-related effect on body weights was noted following administration of ≤125 mg/kg/day AI.

Postnatal Development (Subgroup 2): A test article-related reduction in mean body weight gain was noted during lactation (LD 4 to 14) for animals administered 125 mg/kg/day AI, compared with controls. Lower mean body weight gains were observed for animals administered 125 mg/kg/day AI compared with controls, between LD 4 and 7 (-56%), and the effect was still evident between LD 7 and 14 (-30%, compared with controls). This resulted in a lower overall mean body weight gain for these animals during lactation (approximately -35%). No test article-related effect on body weights was observed during gestation following administration of 125 mg/kg/day AI, compared with controls. No effect on body weights was noted following 75 or 100 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Adverse, test article-related reductions in food consumption were observed following administration of 1000 mg/kg/day AI. Mean food consumption between GD 6 and 8 was statistically significantly lower than controls (up to -83%; P < 0.05 or P < 0.001) prior to the unscheduled sacrifice of this dose group. Test article-related reductions in food consumption were also observed following administration of 500 mg/kg/day AI, compared with controls, with an up to 40% reduction in mean food consumption between GD 6 and 9, and statistically significant reductions between GD 7 and 9 (P < 0.05 or P < 0.01). Some improvement in food consumption was noted thereafter; however, mean food consumption values remained lower than controls throughout the remainder of the in-life phase. Overall food consumption (GD 6 to 21) was approximately -16% lower than controls. In the absence of any associated adverse changes, these small reductions in food consumption were considered nonadverse at this dose level. No test article-related effect on food consumption was noted following administration of ≤125 mg/kg/day AI.

Postnatal Development (Subgroup 2): No test article-related effect on food consumption was observed at any dose level investigated. Any intergroup differences observed during gestation or lactation were considered to have arisen incidentally and not as a consequence of test article administration.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Postnatal Development (Subgroup 2): No test article-related effect on maternal thyroid weights was noted. Any minor intergroup differences were not dose related and considered incidental.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Test article-related macroscopic changes were noted in the intestinal tract for animals administered 1000 mg/kg/day AI. Following the unscheduled sacrifice of this group, abnormal contents in the jejunum, ileum, cecum, and colon were observed, with one female (Animals R0804) noted with slight to moderate distension of the jejunum, ileum, and colon. No test article-related macroscopic observations were noted in the remaining animals at scheduled sacrifice.

Postnatal Development (Subgroup 2): No test article-related macroscopic observations were noted for the maternal animal at scheduled sacrifice.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Postnatal Development (Subgroup 2):
Estrous Stage Determination: No test article-related effect on the stage of estrus was observed at scheduled sacrifice. Most females across all dose groups were in di-estrus.

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Prenatal Development (Subgroup 1): All uterine parameters, including the mean number of implantations, live fetuses, and post-implantation losses, for test article-treated animals were comparable with controls. Any minor differences were not dose-related and considered incidental.

Postnatal Development (Subgroup 2): The mean number of live pups born following test article administration at all dose levels was slightly lower than controls, and post-implantation loss was slightly higher than controls. However, no dose-relationship was evident; taking into consideration the small group sizes, this was considered incidental. One female administered 75 mg/kg/day AI (Animal R0501) was sent to necropsy on LD 0 due to the death of all three live born pups. In the absence of similar findings in groups administered 100 or 125 mg/kg/day, this finding was considered incidental.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

Prenatal Development (Subgroup 1): At the terminal necropsy, all remaining animals (five controls, six animals administered 75 mg/kg/day, five animals administered 100 mg/kg/day AI, six animals administered 125 mg/kg/day AI, and six animals administered 500 mg/kg/day AI) had live fetuses.

Postnatal Development (Subgroup 2): The mean number of live pups born following test article administration at all dose levels was slightly lower than controls, and post-implantation loss was slightly higher than controls. However, no dose-relationship was evident; taking into consideration the small group sizes, this was considered incidental. One female administered 75 mg/kg/day AI (Animal R0501) was sent to necropsy on LD 0 due to the death of all three live born pups. In the absence of similar findings in groups administered 100 or 125 mg/kg/day, this finding was considered incidental.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Pregnancy was unaffected by administration of up 500 mg/kg/day AI. All animals administered 1000 mg/kg/day AI and prematurely terminated on GD 8 were pregnant.
One animal administered 100 mg/kg/day AI (Animal R0202) littered prior to necropsy on GD 21. This is a low incidence finding occasionally observed in studies of this type, was considered related to the time of mating of animals at the animal supplier, and did not represent an effect of the test article.

Postnatal Development (Subgroup 2): No test article-related effect on parturition or litter data was observed up to 125 mg/kg/day AI. Four controls and all six females from each test article-treated group completed parturition with live offspring. No impact on the gestation length, gestation index, number of still births or live births, and live birth or Day 22 viability indices was observed for test article-treated females compared with controls

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Prenatal Development (Subgroup 1): Pregnancy was unaffected by administration of up 500 mg/kg/day AI. All animals administered 1000 mg/kg/day AI and prematurely terminated on GD 8 were pregnant.
Of the surviving dose groups, all animals were pregnant, except one control animal (Animal R0004) and one animal administered 100 mg/kg/day AI (Animal R0204); these females were supplied non- pregnant from the animal supplier, which was not test article related. One animal administered 100 mg/kg/day AI (Animal R0202) littered prior to necropsy on GD 21. This is a low incidence finding occasionally observed in studies of this type, was considered related to the time of mating of animals at the animal supplier, and did not represent an effect of the test article.

Postnatal Development (Subgroup 2): No test article-related effects on pregnancy were observed up to a dose level of 125 mg/kg/day AI. Of the six females in each group, five controls and all six females from each group administered 75, 100, or 125 mg/kg/day AI were pregnant. One control female (Animal R0402) was noted as non-pregnant, while one pregnant control female (Animal R0405) did not litter and was observed with no viable fetuses on GD 26. These findings were confined to the control group and considered a low incidence finding, with no relationship to test article administration.

Other effects:

not specified

Key result

Dose descriptor:

dose level: 500 mg/kg/day AI

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: suitable high dose in OECD414 study

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal Observations: A possible test article-related effect on fetal weights was observed following 500 mg/kg/day, compared with controls. Mean fetal weights, adjusted for litter size, following maternal exposure of 500 mg/kg/day AI was -8% lower than controls, although statistical significance was not attained. The etiology of this finding was unclear, and a relationship to test article administration could not be ruled out. Nevertheless, this was considered nonadverse. Mean corrected body weight, mean corrected body weight change, and mean total weight change for dams administered 500 mg/kg/day AI were -6, -48, and -13% lower than controls, respectively. In view of the marginally lower mean body weight gains observed in-life, this was considered a nonadverse test article-related effect.

Pup Observations: No test article-related effect on offspring body weight was observed throughout lactation.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Prenatal Development (Subgroup 1): All uterine parameters, including the mean number of implantations, live fetuses, and post-implantation losses, for test article-treated animals were comparable with controls. Any minor differences were not dose-related and considered incidental.

Postnatal Development (Subgroup 2): The mean number of live pups born following test article administration at all dose levels was slightly lower than controls, and post-implantation loss was slightly higher than controls. However, no dose-relationship was evident; taking into consideration the small group sizes, this was considered incidental. One female administered 75 mg/kg/day AI (Animal R0501) was sent to necropsy on LD 0 due to the death of all three live born pups. In the absence of similar findings in groups administered 100 or 125 mg/kg/day, this finding was considered incidental.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Fetal observations: Fetal sex was unaffected by test article administration.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal observations: Mean gravid uterine weight in the groups administered 500 mg/kg/day AI was slightly higher than controls; no dose-relationship was evident, and this was considered likely due to slightly lower litter size in the control group, and unrelated to the test article.

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal observations: No test article-related effect on fetal ano-genital distance was noted. Minor decreases in ano-genital distance, noted for the female fetuses maternally administered the test article up to a dose level of 500 mg/kg/day AI were not statistically significant and considered to have arisen incidentally.

Pup observations: No test article-related effect on pup ano-genital distance was observed. Statistical analysis of the data did not show any statistically significant intergroup differences.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Pup observations: No test article-related mortality or clinical observations were noted for any offspring. The mortality or clinical observations recorded were low incidence findings occasionally observed in rat offspring of this age and were considered not related to maternal administration of the test article.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal observations: No test article-related fetal malformations or variations were noted on GD 21. One fetus from the litter of a single animal administered 125 mg/kg/day AI (Animal R0302-L3 Male) had a number of external malformations and variations, including omphalocele, short trunk, malrotated right hind limb at the ankle joint, micromelia of the left forelimb and right hind limb, small entire genital tubercle, edema of the head/neck, and subcutaneous hematoma of the abdomen. These observations were confined to a single fetus from one litter and were not recorded for the litters maternally exposed to 500 mg/kg/day; as such, they were considered unrelated to the test article.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Fetal observations: No test article-related fetal malformations or variations were noted on GD 21.

Visceral malformations:

no effects observed

Description (incidence and severity):

Fetal observations: No test article-related fetal malformations or variations were noted on GD 21.

Other effects:

no effects observed

Description (incidence and severity):

Pup Nipple/ Areolae Count: Maternal administration of the test article had no effect on mean nipples/areolae count in male pups.

Pup macroscopy: No test article-related macroscopic observations were noted for offspring at scheduled sacrifice. Any macroscopic observations were low incidence findings occasionally observed in rat offspring of this age and were considered not related to maternal administration of the test article.

Pup Organ Weights: No test article-related effect on pup thyroid weights was noted. An increase in mean absolute and body weight-related thyroid/parathyroid weights was noted for male pups maternally exposed to the test article at all dose levels, compared with controls. No dose- relationship was observed, and the corresponding values in females were similar to controls. Taking into consideration the small groups size, this was considered to have arisen incidentally.

Key result

Dose descriptor:

dose level: 500 mg/kg/day AI

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: suitable high dose in OECD414

Conclusions:

Once daily oral (gavage) administration of Amides, C12-C18 (even numbered), N-[3- (dimethylamino) propyl], N’-oxides at a dose level of 75, 100, 125, 500, or 1000 mg/kg/day AI to pregnant female rat to assess prenatal development, resulted in mortality following administration of 1000 mg/kg/day AI. The nature and severity of these observations demonstrated that this dose level exceeded the maximum-tolerated dose, and this dose level was considered excessive. A dose level of 500 mg/kg/day AI was better tolerated, with nonadverse postdose observations, minor reduction in body weight gain and food consumption, reduced fetal weight, and reduced corrected body weight and corrected body weight change. These were considered nonadverse, and a dose of 500 mg/kg/day AI was considered a suitable high-dose level for further exploration in a subsequent prenatal development study in the rat (Covance Study 8410555, [Covance Laboratories Ltd, 2020a]).
For assessment of postnatal development, once daily oral (gavage) administration of Amides, C12-C18 (even numbered), N-[3-(dimethylamino) propyl], N’-oxides at dose levels of 75, 100 or 125 mg/kg/day AI to the female rat from Gestation Day 6 through to Lactation Day 21 was well tolerated with only minor test article-related effects noted. The effects were confined to postdose observations which were considered attributable to the nature of the test article formulations and not a direct toxic effect of the test article, and lower mean body weight gains observed following 125 mg/kg/day AI during lactation. These effects were considered nonadverse. Furthermore, no effect on the subsequent offspring was noted. In view of the overall findings noted in this study, and taking into consideration the findings from previous studies performed on this test article over a much longer duration (Covance Study 8280017; Covance Laboratories Ltd, 2014), a dose level of 150 mg/kg/day AI was selected as the high-dose level for the subsequent OECD 443 study (Covance Study 8410556). This dose 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, or mating of the first generation.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-03-11 to 2021-05-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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: Appearance: Almost clear colorless liquid
- Physical state: Liquid
- Analytical purity: Purity: 35.2% in water
- Purity test date: no data
- Lot/batch No.: S019341917
- Expiration date of the lot/batch: 28 March 2021
- action of test substance: no data
- correction factor: 2.85, based on purity
- purity/weighing factor: none

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 confirmed to be stable and homogenous for 15 days when stored refrigerated (2 to 8°C) or for 14 days when stored at room temperature (15 to 25°C) in Covance Study 8410552.


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

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hra:(NZW) SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Eighty time-mated female New Zealand White Hra:(NZW) SPF rabbits from Envigo Global Services Inc., Denver, Pennsylvania, United States of America.
- Age at start of study (day 0 of gestation): at least 20 weeks
- Weight at day 0 (after mating): at least 2.7 kg
- Fasting period before study: no data
- Housing: Animals were individually 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).
Suitable tray liners were provided and changed at least weekly.
- Diet (e.g. ad libitum): ad libitum, LabDiet 5322 rabbit diet (PMI Nutritional International). Each batch of diet was analyzed for specific constituents and contaminants.
Upon arrival and on some occasions when low food consumption was observed, animals were provided with moist hay or approximately 50 g of moistened diet. This was not included in any food consumption calculations. 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.
- Water (e.g. ad libitum): ad libitum, Water from the main tap supply was provided ad libitum 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: Acclimation was limited by mated status.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 13-23°C
- Humidity (%): not less than 45%
- Air changes (per hr): The room was 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, with the exception of when experimental procedures dictated.

IN-LIFE DATES: From: 2020-03-16 To: 2020-05-20

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.
- Test item accounting: Formulations prepared for use on GD 6 and 28 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 were stirred continuously from at least 30 minutes prior to and throughout dosing (excluding the control group). Formulations were administered at room temperature (15 to 25 °C).

- 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, 12.5, 17.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations prepared for use on Gestation Day 6 and 28 of the study 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.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: Each female was mated with one proven male at the supplier’s laboratory.
- M/F ratio per cage: 1:1, using identified stock New Zealand White bucks
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: natural mating observed referred to as day 0 of pregnancy

Duration of treatment / exposure:

days 6 to 28 after mating (22 days)

Frequency of treatment:

once daily, at a similar time on each occasion.

Duration of test:

30 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

175 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 time-mated female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Route of administration rationale: The oral route of administration was chosen because it is an acceptable and commonly used route of 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 New Zealand White Hra:(NZW) SPF rabbit was selected because it is a readily available non-rodent species acceptable to the regulatory authorities. Historical control data are available at the test facility for this strain of animal.

- Dose selection rationale:
*The high-dose level of 175 mg/kg/day active ingredient (AI) was based on the findings from the preliminary dose range-finding study in pregnant rabbits (Covance Study 8410557). This dose level was considered the highest dose level to exhibit some toxicity (i.e., changes in body weight, food consumption, and fetal weights) without causing death.
*The intermediate-dose level of 125 mg/kg/day AI was selected, minimal changes in food consumption were expected, and it was anticipated to represent a no observed adverse effect level (NOAEL).
*The low-dose level of 50 mg/kg/day AI has been selected as the low dose, as it was expected to be well tolerated and was anticipated to represent a no observed effect level (NOEL).
*Background information:
A preliminary study was performed in the non-pregnant rabbit (Covance Study 8410553). Dose levels of 50, 150, and 200 mg/kg/day AI were administered for 7 consecutive days; a correction factor of 2.85 was used. Small reductions in mean food consumption were observed following administration of 50 mg/kg/day AI, compared with controls (-11% between Days 1 and 4, and -8% between Days 5 and 8). A dose level of 150 mg/kg/day AI resulted in body weight losses up to 60 g for all animals and approximately 25% lower mean food consumption compared with controls. All animals administered 200 mg/kg/day AI exhibited body weight losses between Days 1 and 4 (-10 g, - 120 g and -150 g), with body weight losses still evident for two animals between Days 5 and 8 (-90 g and -80 g), with corresponding reductions in mean food consumption, compared with controls (Days 1 and 4, -49%; Days 5 and 8; -74%).
Based on the finding of the non-pregnant rabbit study, a preliminary dose range-finding study was performed in the pregnant rabbit (Covance Study 8410557). In this study, dose levels of 25, 75, 125, and 175 mg/kg/day AI were investigated. All dose levels were well tolerated. Small body weight losses were observed in a few animals up to GD 12 following administration of 175 mg/kg/day AI, and body weight gains remained lower until the end of the study, with mean body weight change from GD 7 to 29 as more than half of that observed for the controls. Lower mean food consumption of more than 15% was observed on the first day of dosing following administration of 175 mg/kg/day AI, with the reduction becoming more prominent as the study progressed. By GD 19, animals from this group were eating approximately 40% less than controls, and by GD 25, mean values were approximately 60% lower than controls. Mean percentage change from GD 7 to 29 was 37% lower than controls. At the scheduled sacrifice, corrected weight change and total weight change following administration of 175 mg/kg/day AI was lower than controls. Lower mean food consumption was also observed in the group administered 125 mg/kg/day AI, with 30% lower food consumption observed between GD 15 and 17. Overall mean food consumption was 13% lower than control of the study duration following administration of 125 mg/kg/day AI. Marginally lower overall mean food consumption was also observed following administration of 75 mg/kg/day AI, compared with controls (-11%), with the highest reduction noted between GD 17 and 19 (-24%). No test article-related effect on uterine parameters was noted at any dose level examined. Mean fetal weights following administration of 175 mg/kg/day AI were -9.4% for female fetuses or -10% for male fetuses, compared with controls. Mean placenta weight in this group was 5% lower than controls. No test article-related fetal malformations or variations were noted.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination outside the home cage daily from GD 4 until necropsy. An individual record of the clinical condition of each animal was maintained.
- Animals were observed daily at 1 hour (± 30 minutes) postdose.

BODY WEIGHT: Yes
- Body weights for each animal were recorded on GD 4, 6, 7, 8, 9, 12, 15, 17, 19, 22, 25, 28, and 29.
Unscheduled body weights were recorded on GD 24, 26, and 27 for welfare reasons.

FOOD CONSUMPTION: Yes
- The amount of food was determined once daily from GD 4 to necropsy.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: on GD 29 after mating
- Females were killed by an intravenous injection (overdose) of sodium pentobarbitone.
- Females were sacrificed in replicate order.
- Macroscopic pathology: Following confirmation of death of the females that were scarified on GD 29, the major blood vessels were severed. All macroscopic lesions were dissected, freed from fat and other contiguous tissue, recorded, and retained in the relevant fixative. Animals that were sacrificed moribund or found dead prior to the scheduled sacrifice were examined macroscopically for abnormalities of the thoracic and abdominal viscera.

OTHER:
MORTALITY:
- Moribund animals were sacrificed by an intravenous injection (overdose) of sodium pentobarbitone. Following confirmation of death, the major blood vessels were severed.

Ovaries and uterine content:

- The uterus and ovaries were examined for implantations and corpora lutea, respectively. The extent of development of the implantation sites was determined as near as possible. An evaluation of the uterus was made to indicate dead or live implantations.

- The ovaries and uteri were removed and examined, and the following data were recorded.
• Pregnancy status
• Gravid uterus weight
• Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported)
• Number of corpora lutea
• Number and intrauterine position of implantations subdivided into:
• Live fetuses
• Early intrauterine deaths
• Late intrauterine deaths
• Dead fetuses

- Early intrauterine deaths were classified as those that exhibited decidual or placental tissue only. Late intrauterine deaths exhibited embryonic or fetal tissue, in addition to placental tissue. Dead fetuses were classified as those that appeared to have died shortly before necropsy.
- The uterus of apparently non pregnant females was immersed in a 10% ammonium sulphide solution to reveal any evidence of implantation.

Fetal examinations:

- External examinations: Yes: [all fetuses per litter ]
- Internal examinations: Yes: [all fetuses per litter ]
- Soft tissue examinations: Yes [all viable fetuses per litter ]
- Skeletal examinations: Yes [all viable fetuses per litter ], examined in 50% glycerol and retained in glycerol/propylene glycol.
- Head examinations: Yes: [appr. one half of eviscerated fetuses per litter], fixed in Bouin's fluid. Heads were then examined by the Wilson sectioning method. Fetal head sections were retained in the relevant fixative.
- Fetal abnormalities were classified as malformations (rare and/or potentially lethal) or variations (commonly occurring non lethal abnormalities).
- Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose), followed by confirmation of cessation of circulation.
- Dead fetuses were examined externally and sexed, then discarded.

Statistics:

See any other information on materials and methods incl. tables.

Indices:

- % Pre-Implantation Loss = ((Number of corpora lutea - number of implantations) / Number of corpora lutea) X 100
- % Post-Implantation Loss = ((Number of implantations - number of live embryos) / Number of implantations) X 100
- Corrected Body Weight (Carcass Weight) = Terminal body weight - uterine weight
- Corrected Weight Change = Carcass weight - GD 7 body weight
- Total Weight Change = GD 29 body weight - GD 7 body weight
- % Male Fetuses = (Number of male fetuses / Number of fetuses of determined sex) X 100

Historical control data:

Historical control data will be routinely supplied for selected observations where this information is considered to assist interpretation of study data, and will normally include both fetal and litter incidences.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test article-related clinical observations were noted following administration of 125 or 175 mg/kg/day AI.
Clinical observation of increased incidence of yellow pelage around the feet, associated with increased urination, was observed for five females administered 175 mg/kg/day AI. Four females administered 175 mg/kg/day AI were also recorded with thin appearance, and decreased body tone was evident for one female (Animal B03015) which littered early, following administration of 175 mg/kg/day AI. The effects following 175 mg/kg/day AI were considered adverse, due to the mortality observed at this dose.
The increased incidence of yellow pelage around the feet was observed for five females administered 125 mg/kg/day AI was considered nonadverse.
No test article-related clinical observations were observed following 50 mg/kg/day AI and no postdose observations were noted at any dose level administered.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two test article-related unscheduled deaths occurred following administration of 175 mg/kg/day AI.
One animal administered 175 mg/kg/day AI (Animal B0319) was sacrificed on GD 26 due to body weight loss of 13% between GD 9 and GD 26, clinical observations of thin appearance, red staining on the cage tray liner, and mucoid feces. At necropsy, the animal was recorded with one early resorption and eight dead fetuses. The animal had a slightly pale and mottled liver and thin appearance. Another animal administered 175 mg/kg/day AI (Animal B0313) aborted on GD 28 and was subsequently terminated. Body weight loss was evident from GD 12 onwards. By GD 27, the animal had lost 530 g (-15% compared with the GD 12 body weight). The animal had a slightly pale and mottled liver and thin appearance at necropsy.
No further unscheduled deaths occurred as a result of test article toxicity. One animal administered 125 mg/kg/day AI (Animal B0209) died following a whole body convulsion immediately after dosing on GD 22. At necropsy, the animal was recorded with a rupture to the right lobe of the lung and red fluid in the oral and thoracic cavities. These findings were indicative of inappropriate dosing, and this was not test article related. The animal was confirmed as pregnant.
One control female (Animal B0014) was sacrificed on GD 26 following clinical observations of thin appearance and progressive body weight loss (17% between GD 12 and GD 26). The animal was confirmed as pregnant with nine live fetuses. This death was considered incidental in nature as this was a control animal.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test article-related lower mean body weights were evident following administration of 175 mg/kg/day AI, compared to controls.
Overall mean body weight gain was -37% compared with controls, from GD 6 to 29, however this value included decedent Animals B0313 and B0319 and was considered to reflect the intragroup variability in response to test article administration at 175 mg/kg/day. The effects following administration of 175 mg/kg/day AI were considered adverse, due to these mortalities. For surviving animals administered 175 mg/kg/day AI, mean body weights were 7% lower than controls on GD 29.
Animal B0315 administered 175 mg/kg/day AI showed body weight losses from GD 17 onwards. The body weight losses were considered to have resulted in the early parturition on the day of scheduled sacrifice. This was considered to be test article related.
No test article-related body weight changes were noted following administration of 50 or 125 mg/kg/day AI.
Total weight change following administration of 175 mg/kg/day AI was 25% lower than controls, and corrected weight change was 142% lower than controls. Gravid uterus weight following administration of 175 mg/kg/day AI was 10% lower than controls, although values did not attain statistical significance, and mean values were within the historical control data ranges (420.3 to 559.9 g).
Placenta weights, adjusted for litter size, were statistically significantly lower following maternal exposure of 175 mg/kg/day AI compared to controls (P < 0.05), and mean unadjusted placenta weights were also lower following administration of 175 mg/kg/day AI, compared with controls (-8%).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test article-related lower mean food consumption was observed following administration of 125 or 175 mg/kg/day AI.
Marginally lower mean food consumption was evident for females administered 175 mg/kg/day AI between GD 12 and 15 (-14%), with mean values more than 20% lower than control from GD 15 onwards, with statistically significant reductions observed between GD 22 and 25 (P < 0.05) and GD 25 and 28 (P < 0.01). Overall mean food consumption from GD 6 to 29 was -16% lower than control and attained statistical significance (P < 0.05).
Marginally lower mean food consumption was observed between GD 9 and 12 ( 13%) and GD 12 to 15 ( 18%) for females administered 125 mg/kg/day AI, with changes up to -26% of control between GD 22 and 25. Overall mean food consumption from GD 6 to 29 was -16% lower than controls and attained statistical significance (P < 0.05).
The effects following administration of 175 mg/kg/day AI were considered adverse, due to mortality observed at this dose. The effects noted following administration of 125 mg/kg/day AI were considered nonadverse.
No test article-related effects were evident following administration of 50 mg/kg/day AI.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test article-related macroscopic findings were noted for four females administered 175 mg/kg/day AI.
At scheduled sacrifice, one female administered 175 mg/kg/day AI was recorded as thin (Animal B0320); this finding was also recorded for two decedent females administered 175 mg/kg/day AI (Animals B0313 and B0319). Two females administered 175 mg/kg/day AI were recorded with mottled livers (Animals B0315 and B0317). This was also observed for two decedent females from this group (see Section 4.2.1) and was considered test article related.
No test article-related macroscopic findings were noted for females administered 50 or 125 mg/kg/day AI.
Animal B0315 administered 175 mg/kg/day AI littered on the day of scheduled necropsy; this was considered to be test article-related, as the animal showed body weight losses from GD 17 onwards.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Test article-related effects were evident following the cesarean section examination, for females administered 125 or 175 mg/kg/day AI.
Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

gross pathology

mortality

number of abortions

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Nonadverse test article-related fetal variations were observed for fetuses from females administered 125 or 175 mg/kg/day AI. Female fetal weights, adjusted for litter size were 8% lower than controls (P < 0.05) following maternal exposure of 175 mg/kg/day AI. Male fetal weights, adjusted for litter size were 5% lower than controls, although values did not attain statistical significance following maternal exposure of 175 mg/kg/day AI. Mean fetal weight values for eight litters were outside of the historical control data ranges for combined sex fetal weights (36.4 to 43 g). The magnitude of the change in fetal weight was small (≤10%), and as such, was considered nonadverse. Test article-related reductions for female fetal weights, adjusted for litter size were evident following maternal exposure of 125 mg/kg/day AI; mean values were 7% lower than controls (P < 0.05) and within the historical control data ranges (35.4 to 42.8 g). No such changes were evident for male fetal weights. Mean combined fetal weight values for eight litters were lower than the historical control data range for the combined sexes (36.4 to 43 g). Mean unadjusted placenta weights were also lower following administration of 125 mg/kg/day AI, compared with controls (-6%); although placenta weights adjusted for litter size were not affected. These changes were considered to be attributable to the effects of the test article on the maternal animal, and did not represent an adverse effect on fetal development.
No test article-related effects were noted following administration of 50 mg/kg/day AI.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Following maternal exposure to the test article, no effect on pregnancy parameters, characterized by the mean number of implantation sites, percentage implantation loss or number of live fetuses was noted at any dose level. It was however noted that one female administered 175 mg/kg/day AI (Animal B0313) aborted and another female from this dose group (Animal B0315) littered early with one dead fetus recorded in utero. These findings were considered adverse test article-related effects.

Changes in sex ratio:

not specified

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Total weight change following administration of 175 mg/kg/day AI was 25% lower than controls, and corrected weight change was 142% lower than controls. Gravid uterus weight following administration of 175 mg/kg/day AI was 10% lower than controls, although values did not attain statistical significance, and mean values were within the historical control data ranges (420.3 to 559.9 g).
Female fetal weights, adjusted for litter size were 8% lower than controls (P < 0.05) following maternal exposure of 175 mg/kg/day AI. Male fetal weights, adjusted for litter size were 5% lower than controls, although values did not attain statistical significance following maternal exposure of 175 mg/kg/day AI. Mean fetal weight values for eight litters were outside of the historical control data ranges for combined sex fetal weights (36.4 to 43 g). The magnitude of the change in fetal weight was small (≤10%), and as such, was considered nonadverse.
Placenta weights, adjusted for litter size, were statistically significantly lower following maternal exposure of 175 mg/kg/day AI compared to controls (P < 0.05), and mean unadjusted placenta weights were also lower following administration of 175 mg/kg/day AI, compared with controls (-8%).
Test article-related reductions for female fetal weights, adjusted for litter size were evident following maternal exposure of 125 mg/kg/day AI; mean values were 7% lower than controls (P < 0.05) and within the historical control data ranges (35.4 to 42.8 g). No such changes were evident for male fetal weights. Mean combined fetal weight values for eight litters were lower than the historical control data range for the combined sexes (36.4 to 43 g). Mean unadjusted placenta weights were also lower following administration of 125 mg/kg/day AI, compared with controls (-6%); although placenta weights adjusted for litter size were not affected. These changes were considered to be attributable to the effects of the test article on the maternal animal, and did not represent an adverse effect on fetal development.
No test article-related effects were noted following administration of 50 mg/kg/day AI.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related malformations were recorded. The malformations observed were from disparate organ systems and indicated no dose dependent trend, with similar malformations also present in litters from the control group or previously observed within the historical control data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Nonadverse test article-related fetal variations were observed for fetuses from females administered 125 or 175 mg/kg/day AI.
The majority of the variations observed following maternal exposure of 125 or 175 mg/kg/day AI were associated with the extent of ossification of the fetal skeleton. None of the incidences are found in 100% of fetuses providing clear evidence that ossification of both the appendicular and axial skeleton was progressing and the higher incidences observed were considered delayed development associated with reduced female fetal weight and not a developmental change. A higher incidence of unossified metacarpals was noted in the litters of females administered 125 or 175 mg/kg/day AI achieving statistical significance. Peripheral bone ossification is later in development and this finding was considered to correlate with the lower fetal weights observed for females in these dose groups and associated with a delay in development. A higher percentage of litters of females administered 125 or 175 mg/kg/day AI included fetuses with unilateral ossification of the sternebra, compared with controls, however mean values at the high and low dose fell within the historical control range and in the absence of statistical significance at the high dose and no dose response, this finding was considered incidental.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Small thyroid was noted for two fetuses in a single litter maternally administered 175 mg/kg/day AI (Animal B0303 male fetus L1 and female fetus L3). This finding was not present in the concurrent control group or the historical control data. The change was marginal and only affected two fetuses; therefore, this was classified as a variation and considered nonadverse.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: limb

external: tail

skeletal: skull

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: vertebra

skeletal: hindlimb

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: central nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: eye

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

125 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Formulation analysis report:

The mean % nominal concentration should be between 90 to 110% and with a relative standard deviation (RSD) ≤ 5.0%. Results were within these criteria (see Text Table 3.1 and Text Table 3.2).

The test article was not detected in the Group 1 control samples.

Text Table 3.1:    Gestation Day 6

Group	Concentration mg/mL	Results as % Nominal Concentration			Mean (%)	RSD (%)
2	5.0	100	100	99	100	0.48
3	12.5	104	102	102	103	0.74
4	17.5	101	101	101	101	0.32
Mean and RSD are calculated from the data base values, not the rounded values presented above.

 

Text Table 3.2:    Gestation Day 28

Group	Concentration mg/mL	Results as % Nominal Concentration			Mean (%)	RSD (%)
2	5.0	104	104	105	104	0.23
3	12.5	104	103	106	105	1.27
4	17.5	107	106	108	107	0.78
Mean and RSD are calculated from the data base values, not the rounded values presented above.

 

 

 

Conclusions:

Once daily oral (gavage) administration of 142.5, 356.25 or 498.75 mg/kg/day Amides, C12-C18 (even numbered), N-[3-(dimethylamino) propyl], N’-oxides (equivalent to 50, 125, or 175 mg/kg/day AI) to pregnant female rabbits, resulted in test article-related effects, including mortality and a single abortion following administration of 175 mg/kg/day AI. As such, 175 mg/kg/day AI was considered
adverse for maternal toxicity. However, the maternal toxicity did not result in any developmental effects on the fetus, and as such. the no observed adverse effect level (NOAEL) for developmental toxicity of the fetus was established as 175 mg/kg/day AI.
Test article-related effects following administration of 125 mg/kg/day AI were confined to nonadverse small reductions in food consumptions, lower mean female fetal and placental weights and fetal variations. These effects were considered to be attributable to the maternal effects and not a direct effect on fetal development, as these changes did not adversely affect the growing fetus. The NOAEL for maternal toxicity was established as 125 mg/kg/day AI.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-03-11 to 2021-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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 were confirmed to be homogenous in Covance Study 8410552.


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

Species:

rat

Strain:

other: Crl:WI(Han) rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Eighty time-mated female Crl:WI(Han) rats Charles River Laboratories, Margate, UK.
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: at least 0.140 kg
- Fasting period before study: no
- Housing: Animals were individually 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). Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding (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.

Number of animals per cage : during acclimatization no data, during pairing: no data, during gestation: one female.

- Diet: ad libitum, SDS Rat and Mouse Breeder Diet VRF1 (Special Diets Services Ltd, Witham, United Kingdom). No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
- Water: ad libitum, Water from the main tap supply via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
- Acclimation period: Acclimation was limited by mated status.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: from 2020-03-16 to: 2020-04-03

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.
- Test item accounting: Samples for analysis were taken from formulations prepared for use on GD 6 and 20 (a total two occasions).
- Formulations were stirred continuously from at least 30 minutes prior to and throughout dosing (excluding the control group).

- 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, 25, 50, 70 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean percent nominal concentration should have been between 90 and 110%, with a relative standard deviation (RSD) ≤ 5.0%. Results were within these criteria.
The test article was not detected in the control samples. The analytical procedure was validated within Covance study 8280017.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: overnight (at the supplier’s laboratory).
- M/F ratio per cage: no data
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: presence of a vaginal plug in situ.
- Any other deviations from standard protocol: Not applicable

Duration of treatment / exposure:

days 6 to 20 after mating

Frequency of treatment:

once daily

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- 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) Wistar Han rat was selected because it is known to be accepted by various regulatory authorities. Historical control data are available at the test facility for this strain of animal.

- Dose selection rationale:
* High-dose Level: A high-dose level of 700 mg/kg/day AI was selected based on the findings from the preliminary dose range-finding study in pregnant rats (Covance Study 8410554; Covance Laboratories Ltd, 2020) where 1000 mg/kg/day AI was considered excessive and 500 mg/kg/day AI was considered non-adverse. A dose level of 700 mg/kg/day AI was expected to elicit some toxicity (i.e., body weight loss, reduced food consumption and lower fetal weights), without causing death.
* Intermediate-dose Level: A dose level of 500 mg/kg/day AI was selected as a suitable mid-dose level at which minimal changes in adult and fetal body weight were expected to be seen. This was anticipated to represent a no observed adverse effect level.
* Low-dose level: A low-dose level of 250 mg/kg/day AI was expected to be well tolerated, and anticipated to represent a no observed effect level.
* Background information:
**In a 28-day repeated dose toxicity study in the Sprague-Dawley rat, animals were administered 15, 150 or 1000 mg/kg/day AI by oral gavage (Safepharm Laboratories Limited Study number 625/051). Clinical signs of toxicity, functional incidents such as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption and reduced body weight gain were observed following administration of 1000 and 150 mg/kg/day AI. Treatment-related changes in liver and spleen were observed following administration of 1000 or 150 mg/kg/day AI. Histopathological changes were also observed in kidneys, urinary bladder, and stomach of animals at the high dose. Hematological changes (hemolytic anemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time) and elevations in plasma aspartate aminotransferase, alanine aminotransferase and bilirubin were observed following administration of 1000 and 150 mg/kg/day. Therefore, the no observed effect level (NOEL) for this study was considered to be 15 mg/kg/day AI.
**In a 90-day repeated oral dose toxicity study in the Sprague-Dawley rat (Covance Study 8280017 [Covance Laboratories Ltd, 2014]), animals were administered 50, 150 or 500 mg/kg/day AI by oral gavage. Following 500 mg/kg/day AI administration, one male was found dead during Week 12 and this death was considered to be test article related. Clinical observations at this dose included salivation, staining around the mouth and noisy breathing and in combination with high water consumption values, this indicates irritant test materials with the animals not liking the taste of the formulation. Microscopic examination revealed adverse findings in the (fore) stomach (squamous cell hyperplasia) and urinary bladder (transitional cell hyperplasia) which was considered a result of the irritating properties of the test article. Findings for animals following administration of 150 mg/kg/day AI included low body weight gains and as no effect on food consumption was observed, this was considered non-specific toxicity. Forestomach squamous cell hyperplasia was noted in a single male at 150 mg/kg/day AI and as this finding was consistent with those recorded at 500 mg/kg/day AI and considered adverse. The only test article-related change evident for males administered 50 mg/kg/day AI were minor changes in the red blood cell parameters which were considered test article related, but not adverse in nature; therefore, the no observed effect level (NOEL) for this study was considered to be 50 mg/kg/day.
**In a dose range-finding study in the pregnant rat (Covance Study 8410554 [Covance Laboratories Ltd, 2020]), dose levels of 75, 100, 125, 500 and 1000 mg/kg/day AI were investigated. Dosing commenced at GD 6. On GD 8, one female in the 1000 mg/kg/day group was found dead and the remaining animal from this dose group were terminated as this dose level was considered excessive (substantial body weight losses and reduced food consumption). Following administration of 500 mg/kg/day AI, initial small body weight loses were noted for 4 of 6 females; however, mean body weights on GD21 were only 3% lower than controls. Lower mean food consumption was also noted during the first few days of dosing; however, overall mean food consumption was only -16% compared with controls. Mean corrected body weight (carcass weight) was marginally lower in the 500 mg/kg/day AI group, mean corrected weight change and total weight change were also lower in the 500 mg/kg/day AI group compared with controls. No test article-related macroscopic findings were noted at this dose and no effect on uterine contents was noted. No effect on litter size or sex ratio were evident. Mean fetal weights, adjusted for litter size were lower for 500 mg/kg/day litters compared with controls (-8%). No such effect was observed at the lower dose levels. No test article-related fetal malformation or variations. No test article-related effects were noted in the other dose levels investigated. As such, based on the findings of this study, a dose level 500 mg/kg/day AI was considered as a no observed adverse effect level and a dose level of 1000 mg/kg/day AI was considered excessive. Based on these findings, a high-dose level of 700 mg/kg/day was anticipated to show some toxicity without causing maternal death.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for a clinical examination, twice a day for postdose observation (upon return to home cage and at 1 hour (±30 minutes) postdose).
- All animals were observed in the cage twice daily, at the beginning and end of the working day for signs of ill health or overt toxicity.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination outside of the cage daily from GD 6 to necropsy. An individual record of the clinical condition of each animal was maintained. Animals were observed daily, upon return to home cage and at 1 hour (±30 minutes) postdose. Character and timing of reactions to treatment were recorded, together with all observations (including absence of observations).


BODY WEIGHT: Yes
- Body weights for each animal were recorded on GD 4, 6, 7, 8, 9, 12, 15, 17, 19, 20, and 21. Unscheduled body weights were recorded on GD 10 and 11 for welfare reasons.

FOOD CONSUMPTION: Yes
- The amount of food consumed was determined from GD 6 to 7, 7 to 8, 8 to 9, 9 to 12, 12 to 15, 15 to 17, 17 to 19, 19 to 20, and 20 to 21. Food consumption was calculated as g/animal/day.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: on day 20 or 21 after mating.
- Females were sacrificed by controlled randomization (i.e., one animal from Groups 1, 2, and then 3 and repeated in the same sequence until all animals were sacrificed).
- Females were sacrificed by exsanguination following isoflurane anesthesia. All lesions were recorded and retained in the relevant fixative.

- Macroscopic pathology:

- All animals that died prior to their scheduled sacrifice and all Group 4 animals were examined macroscopically for abnormalities of the thoracic and abdominal viscera.
- The thyroid from all scheduled sacrifice Group 1, 2, and 3 animals was dissected free from fat and other contiguous tissue and weighed post-fixation
- Thyroid tissue and gross lesions from all Group 1 through 4 animals, including decedents, were embedded in paraffin wax BP, sectioned at a nominal 5 μm, stained with hematoxylin and eosin, and examined microscopically by the Study Pathologist.


OTHER:
MORTALITY:
Group 4 animals were sacrificed early as the dose level was deemed too excessive to continue dosing following the deaths of Animals R0303 and R0308; these animals were found dead on GD 9 and 8, respectively. Therefore, Animals R0301, R0302, R0304, and B0305 were sacrificed early on GD 10; Animals R0306, R0307, R0309, and R0310 were sacrificed early on GD 9; Animals R0311 through R0315 were sacrificed early on GD 8; and Animals R0316 through R0320 were sacrificed early on GD 7.

Ovaries and uterine content:

- The uterus and ovaries were examined for implantations and corpora lutea, respectively. The extent of development of the implantation sites was determined as near as possible. An evaluation of the uterus was made to indicate dead or live implantations.

- The ovaries and uteri were removed and examined, and the following data were recorded for Group 1, 2, and 3 animals.
• Pregnancy status
• Gravid uterus weight
• Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported)
• Number of corpora lutea
• Number and intrauterine position of implantations subdivided into:
• Live fetuses
• Early intrauterine deaths
• Late intrauterine deaths
• Dead fetuses

- Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or fetal tissue, in addition to placental tissue. Dead fetuses were classified as those which appeared to have died shortly before necropsy. The uterus of any apparently non pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

Blood sampling:

- Blood samples (1 x 0.5 mL [EDTA]) were withdrawn from the abdominal aorta of all Group 1, 2, and 3 animals at necropsy on GD 20 or 21.
- Blood sample collection occurred by controlled randomization (i.e. one animal from Groups 1, 4, 2 then 3 and were repeated in the same sequence until all animals are sampled).
- Hematology Tests:
hemoglobin concentration: red blood cell count
packed cell volume: reticulocyte count
mean cell volume: mean cell hemoglobin
mean cell hemoglobin concentration: red cell distribution width
total and differential white cell count : platelet count
- Blood samples were fully inverted several times (approximately 10) to ensure 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 prior to processing.

- Blood samples for thyroid hormone analysis (2 x 0.8 mL [serum separator tubes; one primary and one spare]) were withdrawn from the abdominal aorta of all Group 1, 2, and 3 animals at necropsy on GD 20 or 21.
- Parameters: Total Triiodothyronine (T3), Total Thyroxine (T4), Thyroid Stimulating Hormone (TSH)
- Blood samples for thyroid hormone analysis were gently inverted several times (approximately 10) to ensure that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator. Samples were allowed to clot for at least 30 minutes at ambient temperature prior to centrifugation. Samples were then centrifuged and divided into uniquely labeled amber polypropylene tubes (one primary sample and one spare sample) within 2 hours of blood sampling and were protected from light until frozen at <-10°C (-20°C nominal). Only the primary sample was analyzed in the first instance by Covance; the second sample was stored as a spare for future analysis if required.

Fetal examinations:

- External examinations: Yes: [all viable fetuses per litter ]
- Internal examinations: Yes: [all viable fetuses per litter ]
- Soft tissue examinations: Yes [one half per litter ]
- Skeletal examinations: Yes [one half per litter ], fixed in potassium hydroxide and stained with Alizarin Red S, retained in glycerol/propylene glycol, and examined for skeletal abnormalities.
- Head examinations: Yes [one half per litter ]
- Remaining fetuses will be eviscerated and fixed in Bouin’s fluid, the viscera were examined, and the heads were serially sectioned for examination of the internal head structures. The fetuses fixed for visceral examination were retained in the relevant fixative.
- Fetal abnormalities were classified as malformations (rare and/or potentially lethal defects) or variations (commonly occurring non-lethal abnormalities).
- Individual fetal and placental weights were recorded, and fetuses were examined externally and sexed (externally and internally).
- The AGD of all fetuses was recorded.

Statistics:

See any other information on materials and methods incl. tables.

Indices:

% Pre-Implantation Loss = ((Number of corpora lutea - number of implantations) / Number of corpora lutea) X 100
% Post-Implantation Loss = ((Number of implantations - number of live embryos) / Number of implantations) X 100
Corrected Body Weight (Carcass Weight) = Terminal body weight - uterine weight
Corrected Weight Change = Carcass weight - GD 6 body weight
Total Weight Change = GD 21 body weight - GD 6 body weight
% Male Fetuses = (Number of male fetuses / Number of fetuses of determined sex) X 100

Historical control data:

Historical control data will be routinely supplied for selected observations where this information is considered to assist interpretation of study data, and will normally include both fetal and litter incidences.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test article-related clinical observations were noted at all dose levels.

- Test article-related clinical observations noted for animals administered 700 mg/kg/day AI: Two of the 20 females administered 700 mg/kg/day AI were found dead: Animal R0308 was found dead on GD 8 and Animal R0303 was found dead on GD 9. Animal R0308 had red colored pelage around the muzzle, brown discharge around the nose, and mucoid feces at the time of death on GD 8. No clinical observations were recorded for Animal R0303 prior to the time of death. Test article-related clinical observations for animals administered 500 mg/kg/day AI included instances of excessive salivation (5 of 20), mucoid feces (4 of 20), audible respiration (6 of 20), thin appearance (4 of 20), piloerection (8 of 20), and hunched posture (4 of 20). There was also an increased incidence of teeth pallor (4 of 20) compared with controls. Test article-related clinical observations noted for animals administered 250 mg/kg/day AI were confined to instances of audible respiration for Animal R0112 on GD 14 and Animal R0117 on GD 20 and 21. In isolation, these were considered non adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two of the 20 females administered 700 mg/kg/day AI were found dead: Animal R0308 was found dead on GD 8 and Animal R0303 was found dead on GD 9. Due to these deaths and the extent of the body weight losses and reductions in food consumption observed for this group, remaining animals were sacrificed early (on GD 7 through 10 due to the staggered dosing regimen). One of the 20 females administered 500 mg/kg/day AI (Animal R0201) was sacrificed on GD 10 following the severity of the clinical observations. No further unscheduled deaths occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test article-related body weight losses and / or reduced body weight gains were evident from the start of dosing for all test article treated groups, compared with controls.
Substantial body weight loss was observed following 700 mg/kg/day AI prior to early sacrifice. Body weight losses between 3.9 to 46.5 g were observed, with progressive loss as the dosing continued up to GD 10.
Minor initial body weight losses were observed following administration of 500 mg/kg/day AI, with statistically significant lower mean body weights observed on GD 9 (P < 0.05) and GD 12 (P < 0.001), compared with controls. Mean body weight gains were significantly lower than controls from GD 6 to 8 (P < 0.001), compared with controls; overall mean body weight gain over the dosing period was 34% lower than controls (P < 0.001).
Nonadverse marginally lower mean body weights were observed following administration of 250 mg/kg/day AI, with statistical significance achieved on GD 12 (P < 0.05), compared with controls. Mean body weight gains were also reduced from the start of dosing until GD 15. Overall mean body weight gain over the dosing period was 13% lower than controls (P < 0.05).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test article-related reductions in food consumption were evident from the start of dosing for all test article treated groups, compared with controls, with a clear dose relationship.
Mean food consumption for females administrated 700 mg/kg/day AI was reduced by 47%, compared with control values after the first dose, with further reductions observed on GD 8 and 9, at which point administration of this dose level was terminated.
Statistically significant lower mean food consumption was observed from GD 7 until the end of doing for females administered 500 mg/kg/day AI. Mean overall food consumption over the dosing period was 26% lower than controls (P < 0.001).
Lower mean food consumption was also observed during the dosing period for females administered 250 mg/kg/day AI, with overall mean food consumption 12% lower than control (P < 0.001).

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):

Test article-related changes in red cell parameters were observed on GD 21 following administration of 250 or 500 mg/kg/day AI, and a clear dose relationship was evident.
Statistically significant reductions in hemoglobin levels were observed following 250 or 500 mg/kg/day AI (P < 0.001; -20.5% or -18.5% respectively), compared with controls). Red blood cell counts for 250 or 500 mg/kg/day AI were also -28.3% or 29.5% compared with controls (P < 0.001).
Mean cell hemoglobin concentration was reduced by 8% compared with controls following administration of 250 or 500 mg/kg/day AI (P<0.001), and packed cell volume was also reduced following administration of 250 or 500 mg/kg/day AI (P < 0.001; -13.5% or -11% respectively), compared with controls.
Statistically significant increases in reticulocytes (counts and percentage) were also observed following administration of 250 or 500 mg/kg/day (P < 0.001) with approximately 4-fold increases in absolute reticulocyte counts and approximately 3 fold increases in percentage reticulocyte counts, compared with controls.
Mean cell volume was significantly increased following administration of 250 or 500 mg/kg/day AI (P < 0.001; -21% or -26% respectively), and mean cell hemoglobin was also higher (P < 0.001; +11% or +15% Respectively), compared with controls. Increases in red cell distribution width were also observed following administration of 250 or 500 mg/kg/day AI, compared with controls (P < 0.001; +31% or +45% respectively). Finally, mean platelet counts were significantly higher for females administered 250 or 500 mg/kg/day AI, compared with control (P < 0.05 [+9%] and P < 0.01 [+21%], respectively).

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Test article-related changes in thyroid hormones were observed on GD 21 following administration of 250 or 500 mg/kg/day AI.
Females administered 500 mg/kg/day AI had a statistically significant reduction in total thyroxine (T4; -24%) levels and total triiodothyronine (T3; -15%), compared with controls (P < 0.05), and an increase in thyroid stimulating hormone (TSH; +44%), although statistical significance was not achieved for this parameter.
Small reductions in T4 and T3 were also observed following administration of 250 mg/kg/day AI, and a dose response was observed, compared with controls (-17% or -19% respectively), although statistical significance was not achieved.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test article-related thyroid weight differences were recorded.
Organ weight and organ weight ratio differences were attributed to normal biological variation and were considered not related to test article as they were small in magnitude, not dose-dependent, due to normal inter-animal variability, and/or lacked a microscopic correlate.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test article-related macroscopic findings were recorded.
Most tissues were macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test article-related thyroid weight differences were recorded.
Organ weight and organ weight ratio differences were attributed to normal biological variation and were considered not related to test article as they were small in magnitude, not dose-dependent, due to normal inter-animal variability, and/or lacked a microscopic correlate.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Test article-related changes in the uterine parameters were noted following administration of 250 or 500 mg/kg/day AI.
A higher percentage of pre-implantation loss was observed following administration of 500 mg/kg/day AI, compared with controls. Marginally higher mean post-implantation loss and percent post-implantation loss (early resorptions) was also noted, compared with controls. Three females administered 500 mg/kg/day AI had no viable fetuses: Animals R0202 and R0204 had one early resorption each, which resulted in the total in utero litter losses, and Animal R0209 had 11 early resorptions noted. Furthermore, one female administered 500 mg/kg/day AI (Animal R0216) had six early resorptions, and one female administered 500 mg/kg/day AI (Animal R0218) had five early resorptions. Seven other females administered 500 mg/kg/day AI were recorded with one early resorption.
Increases in post-implantation losses were also observed following administration of 250 mg/kg/day AI, with one female (Animal R0112) that had four early resorptions and two females (Animals R0116 and R0118) had one late resorption each. In total, 9 of 19 females administered 250 mg/kg/day AI had one or more resorptions (47%), compared with 8 of 18 control females (44%). Mean values for early and late resorption were, however, within the historical control data ranges (early: 0.0 to 2.0; late: 0.0 to 0.2). As such, this was considered unrelated to the test article.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Test article-related changes in the uterine parameters were noted following administration of 250 or 500 mg/kg/day AI.
A higher percentage of pre-implantation loss was observed following administration of 500 mg/kg/day AI, compared with controls. Marginally higher mean post-implantation loss and percent post-implantation loss (early resorptions) was also noted, compared with controls. Three females administered 500 mg/kg/day AI had no viable fetuses: Animals R0202 and R0204 had one early resorption each, which resulted in the total in utero litter losses, and Animal R0209 had 11 early resorptions noted. Furthermore, one female administered 500 mg/kg/day AI (Animal R0216) had six early resorptions, and one female administered 500 mg/kg/day AI (Animal R0218) had five early resorptions. Seven other females administered 500 mg/kg/day AI were recorded with one early resorption.
Increases in post-implantation losses were also observed following administration of 250 mg/kg/day AI, with one female (Animal R0112) that had four early resorptions and two females (Animals R0116 and R0118) had one late resorption each. In total, 9 of 19 females administered 250 mg/kg/day AI had one or more resorptions (47%), compared with 8 of 18 control females (44%). Mean values for early and late resorption were, however, within the historical control data ranges (early: 0.0 to 2.0; late: 0.0 to 0.2). As such, this was considered unrelated to the test article.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Test article-related changes in the uterine parameters were noted following administration of 250 or 500 mg/kg/day AI.
A higher percentage of pre-implantation loss was observed following administration of 500 mg/kg/day AI, compared with controls. Marginally higher mean post-implantation loss and percent post-implantation loss (early resorptions) was also noted, compared with controls. Three females administered 500 mg/kg/day AI had no viable fetuses: Animals R0202 and R0204 had one early resorption each, which resulted in the total in utero litter losses, and Animal R0209 had 11 early resorptions noted. Furthermore, one female administered 500 mg/kg/day AI (Animal R0216) had six early resorptions, and one female administered 500 mg/kg/day AI (Animal R0218) had five early resorptions. Seven other females administered 500 mg/kg/day AI were recorded with one early resorption.
Increases in post-implantation losses were also observed following administration of 250 mg/kg/day AI, with one female (Animal R0112) that had four early resorptions and two females (Animals R0116 and R0118) had one late resorption each. In total, 9 of 19 females administered 250 mg/kg/day AI had one or more resorptions (47%), compared with 8 of 18 control females (44%). Mean values for early and late resorption were, however, within the historical control data ranges (early: 0.0 to 2.0; late: 0.0 to 0.2). As such, this was considered unrelated to the test article.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Test article-related changes in the uterine parameters were noted following administration of 250 or 500 mg/kg/day AI.
A higher percentage of pre-implantation loss was observed following administration of 500 mg/kg/day AI, compared with controls. Marginally higher mean post-implantation loss and percent post-implantation loss (early resorptions) was also noted, compared with controls. Three females administered 500 mg/kg/day AI had no viable fetuses: Animals R0202 and R0204 had one early resorption each, which resulted in the total in utero litter losses, and Animal R0209 had 11 early resorptions noted. Furthermore, one female administered 500 mg/kg/day AI (Animal R0216) had six early resorptions, and one female administered 500 mg/kg/day AI (Animal R0218) had five early resorptions. Seven other females administered 500 mg/kg/day AI were recorded with one early resorption.
Increases in post-implantation losses were also observed following administration of 250 mg/kg/day AI, with one female (Animal R0112) that had four early resorptions and two females (Animals R0116 and R0118) had one late resorption each. In total, 9 of 19 females administered 250 mg/kg/day AI had one or more resorptions (47%), compared with 8 of 18 control females (44%). Mean values for early and late resorption were, however, within the historical control data ranges (early: 0.0 to 2.0; late: 0.0 to 0.2). As such, this was considered unrelated to the test article.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Test article-related changes in the uterine parameters were noted following administration of 250 or 500 mg/kg/day AI.
A higher percentage of pre-implantation loss was observed following administration of 500 mg/kg/day AI, compared with controls. Marginally higher mean post-implantation loss and percent post-implantation loss (early resorptions) was also noted, compared with controls. Three females administered 500 mg/kg/day AI had no viable fetuses: Animals R0202 and R0204 had one early resorption each, which resulted in the total in utero litter losses, and Animal R0209 had 11 early resorptions noted. Furthermore, one female administered 500 mg/kg/day AI (Animal R0216) had six early resorptions, and one female administered 500 mg/kg/day AI (Animal R0218) had five early resorptions. Seven other females administered 500 mg/kg/day AI were recorded with one early resorption.
Increases in post-implantation losses were also observed following administration of 250 mg/kg/day AI, with one female (Animal R0112) that had four early resorptions and two females (Animals R0116 and R0118) had one late resorption each. In total, 9 of 19 females administered 250 mg/kg/day AI had one or more resorptions (47%), compared with 8 of 18 control females (44%). Mean values for early and late resorption were, however, within the historical control data ranges (early: 0.0 to 2.0; late: 0.0 to 0.2). As such, this was considered unrelated to the test article.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

dead fetuses

early or late resorptions

endocrine findings

food consumption and compound intake

haematology

mortality

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean combined male and female fetal weights were significantly lower following maternal exposure to 500 mg/kg/day AI, compared with controls (P < 0.01).

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

At the scheduled sacrifice, 19, 19, or 16 females administered 0 (control), 250 or 500 mg/kg/day AI, respectively, were pregnant with live fetuses.
Three females administered 500 mg/kg/day AI had no viable fetuses: Animals R0202 and R0204 had one early resorption each, which resulted in the total in utero litter losses, and Animal R0209 had 11 early resorptions noted.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected.

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects on AGD were noted following maternal exposure to 250 or 500 mg/kg/day AI.
Ano-genital distance for male fetuses maternally exposed to 500 mg/kg/day AI were 9% shorter than control, and female fetuses maternally exposed to 500 mg/kg/day exhibited a 12% longer AGD, compared with concurrent controls. Similar changes in AGD were observed following maternal exposure to 250 mg/kg/day AI, with male AGD reduced by 7% and female AGD increased by 7%, compared with controls. However, statistical analysis of the data did not indicate any significant intergroup differences, and the majority of values were within the historical control data ranges for this parameter (males: 1.74 to 3.54 mm; females: 0.62 to 3.53 mm); as such, these minor intergroup differences were considered to have arisen incidentally.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related fetal malformations were noted.
Fetal examination revealed a single malformation of umbilical hernia for one fetus maternally exposed to 500 mg/kg/day AI (Animal R0203; Fetus R2). In isolation, this was considered incidental in nature, with no relationship to the test article.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Test article-related fetal variations were evident for fetuses from females administered 500 mg/kg/day AI, compared with controls.
Noteworthy fetal variations included an increased incidence of unossified or incomplete ossification of the cervical centrum and metatarsals and phalanx (hindlimb) for fetuses maternally exposed to 500 mg/kg/day AI, compared with controls and at the higher end of historical control data ranges. Test article-related reduced ossification was observed for the cervical vertebra centra and hind paws of fetuses maternally exposed to 500 mg/kg/day AI.
The ossification of the parietal skull bones of fetuses maternally exposed to 250 mg/kg/day AI were statistically significant compared with controls, however, overall fetal ossification was within expected parameters. Therefore, this finding at this dose level, in the absence of a dose relationship, was considered to have arisen incidentally.

Visceral malformations:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

reduction in number of live offspring

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Formulation analysis

The mean % nominal concentration should be between 90 to 110% and with a relative standard deviation (RSD) ≤  5.0%. Results were within these criteria (see Text Table 3.1 and Text Table 3.2).

The test article was not detected in the Group 1 control samples.

Text Table 3.1:    Gestation Day 6

Group	Concentration mg/mL	Results as % Nominal Concentration			Mean (%)	RSD (%)
2	25	100	99	99	99	0.52
3	50	95	95	97	96	0.81
4	70	94	95	95	95	0.56
Mean and RSD are calculated from the data base values, not the rounded values presented above.

 

Text Table 3.2:    Gestation Day 20

Group	Concentration mg/mL	Results as % Nominal Concentration			Mean (%)	RSD (%)
2	25	103	103	103	103	0.16
3	50	102	104	103	103	1.12
Mean and RSD are calculated from the data base values, not the rounded values presented above.

Conclusions:

Once daily oral (gavage) administration of 250, 500, or 700 mg/kg/day AI of the test substance to pregnant female rats, resulted in adverse effects, resulting in early sacrifice of females administered 700 mg/kg/day AI.
Maternal toxicity was also evident following 500 mg/kg/day AI, however, no adverse effects were noted for the developing fetus. The transient delays in fetal maturation were associated with maternal toxicity and did not represent fetal toxicity. The no observed adverse effect level (NOAEL) for prenatal development of the fetus was established as 500 mg/kg/day AI.
Test article-related effects following administration of 250 mg/kg/day AI to the maternal animal consisted of isolated instances of audible respiration, mouth rubbing, and piloerection. Marginally lower mean body weights and associated reductions in food consumption were also evident, compared with controls. Hematology changes were confined to marginal changes in red cell parameters and thyroid hormone changes were also noted. Effects were confined to the maternal animal and no effects were evident for the developing fetus.
The NOAEL for maternal toxicity was established as 250 mg/kg/day AI.
The no observed effect level (NOEL) for prenatal development of the fetus was established as 250 mg/kg/day AI.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

 

Prenatal development toxicity study in rat - supporting dose range finder:

A Dose-Range-Finding study in rats (K1; Barraclough, 2020) was performed. Six groups of time-mated female Crl:WI(Han) rats were allocated to Subgroup 1 (prenatal development), or four groups of six-mated female Crl:WI(Han) rats were allocated to Subgroup 2 (postnatal development). Animals of Subgroup 1 were administered the test article at the dose levels 0 (control), 75 (low), 100 (intermediate), 125 (high), 1000 (high II), and 500 (intermediate II) mg AI/kg/day, once daily by oral gavage from Gestation Days (GD) 6 through 20, inclusive. Animals of Subgroup 2 were administered the test article at the dose levels 0 (control), 75 (low), 100 (intermediate), 125 (high) mg AI/kg/day, once daily from GD6 through Lactation Day (LD) 21. The control article (vehicle) was purified water. Assessment of toxicity was based on mortality, clinical and postdose observations, body weights, and food consumption for all animals. For assessment of prenatal development, necropsies were performed for all Subgroup 1 animals on GD21, and any macroscopic abnormalities were recorded. The progress and outcome of pregnancy were assessed. Fetuses were evaluated for external malformations and variations, and fetal ano-genital distances were recorded. For assessment of postnatal development (Subgroup 2), assessment of toxicity was also based on pregnancy indices, parturition, and offspring development. Necropsies were performed for all females on LD22, and for all pups, including pups found dead. Any macroscopic abnormalities were recorded.

Prenatal development (subgroup 1)

Test article-related mortality occurred following administration of 1000 mg/kg/day. One of six females was found dead on GD 8, and the remaining animals from this group were also sacrificed on that same day because this dose level was considered excessive due to marked body weight losses and reduced food consumption. No unscheduled deaths occurred for the remaining groups. Test article-related instances of raised hair, hunched posture, head shaking, irregular and/or noisy respiration were observed for animals administered 500 mg/kg/day active ingredient (AI). These observations were transient and considered nonadverse. Nonadverse, test article-related postdose  observations were confined to instances of mouth rubbing for all animals administered 100, 125 to 500 mg/kg/day AI. Isolated incidences of paddling behavior or excessive salivation were also observed following administration of 500 mg/kg/day AI, which were attributable to the nature of the test article formulations and not considered adverse. Initial test article-related body weight losses were observed for up to four females administered 500 mg/kg/day AI between GD 6 to 8, and lower mean body weight gain was noted between GD 8 and 9 for these animals, with lower overall mean body weight gains observed, compared with controls. This was associated with lower mean food consumption. At the terminal sacrifice, mean corrected body weight, mean corrected body weight change, and mean total weight change were observed for females administered 500 mg/kg/day AI, compared with controls. Given the small magnitude of the effects, these findings were considered nonadverse at the dose level of 500 mg/kg/day AI. Pregnancy was unaffected by administration of up to 500 mg/kg/day AI, and no test article-related macroscopic abnormalities were recorded for maternal animals. Lower mean fetal weights were observed following maternal exposure of 500 mg/kg/day AI, compared with controls. This was possible related to test article administration and was considered nonadverse. No test article-related fetal malformations or variations were noted, and no test article-related effect on fetal ano-genital distance was observed.

 

Postnatal development (subgroup 2)

No test article-related mortality or clinical observations were noted up to 125 mg/kg/day AI. The nonadverse, test article-related postdose observation of mouth rubbing was evident following administration of ≥100 mg/kg/day AI and during lactation (125 mg/kg/day AI), with isolated incidences of excessive salivation. Lower mean body weight gains were observed during lactation for animals administered 125 mg/kg/day AI, compared with controls. No test article- related effect on food consumption was observed. No test article-related effects on pregnancy or parturition were observed. No test article-related effects were noted for offspring following maternal exposure to the test article at any dose level. No test article-related macroscopic observations or changes in thyroid weights or ano-genital distance were noted for offspring at the terminal sacrifice. No test article-related macroscopic observations, thyroid weight changes, or changes in the stage of estrous were noted for maternal animals at the scheduled sacrifice. In conclusion, once daily oral (gavage) administration of Amides, C12-C18 (even numbered), N-[3-(dimethylamino) propyl], N’- oxides at a dose level of 75, 100, 125, 500, or 1000 mg/kg/day AI to pregnant female rat to assess prenatal development, resulted in mortality following administration of 1000 mg/kg/day AI. The nature and severity of these observations demonstrated that this dose level exceeded the maximum-tolerated dose, and this dose level was considered excessive. A dose level of 500 mg/kg/day AI was better tolerated, with nonadverse postdose observations, minor reduction in body weight gain and food consumption, reduced fetal weight, and reduced corrected body weight and corrected body weight change. These were considered nonadverse at this level, and a dose of 500 mg/kg/day AI was considered a suitable high-dose level for further exploration in a subsequent prenatal development study in the rat (Covance Study 8410555 [Covance Laboratories Ltd, 2020a]). 
For assessment of postnatal development, once daily oral (gavage) administration of Amides, C12-C18 (even numbered), N-[3-(dimethylamino) propyl], N’-oxides at dose levels of 75, 100 or 125 mg/kg/day AI to the female rat from Gestation Day 6 through to Lactation Day 21 was well tolerated with only minor test article-related effects noted. The effects were confined to postdose observations which were considered attributable to the nature of the test article formulations and not a direct toxic effect of the test article, and lower mean body weight gains observed following 125 mg/kg/day AI during lactation. These effects were considered nonadverse. Furthermore, no effect on the subsequent offspring was noted. In view of the overall findings noted in this study, and taking into consideration the findings from previous studies performed on this test article over a much longer duration (Covance Study 8280017 [Covance Laboratories Ltd, 2014]), a dose level of 150 mg/kg/day AI was selected as the highdose level for the subsequent OECD 443 study (Covance Study 8410556 [Covance Laboratories Ltd, 2020b). This dose 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, or mating of the first generation.

 

Prenatal developmental toxicity study in rat - OECD414

Once daily oral (gavage) administration of 712.5, 1425, or 1995 mg/kg bw/day (equivalent to 250, 500, or 700 mg/kg bw/day AI) of the test substance to the pregnant female rat resulted in test article‑related effects at all dose levels.

Two animals were found dead following administration of 700 mg/kg bw/day AI. Clinical observations included piloerection, thin appearance, hunched posture, discoloration of the pelage, audible respiration, partially closed eyes, and decreased activity. For surviving females, significant body weight losses and substantial reductions in food consumption were also observed. Due to the nature and severity of these observations, this dose level was considered excessive, and all surviving females administered this dose were sacrificed early on Gestation Day 7 through to 10.

Test article-related effects were also observed following administration of 500 mg/kg bw/day AI, with one unscheduled death noted on GD 10. This death occurred following clinical observations of mucoid feces, partially closed eyes, piloerection, hunched posture, and thin appearance. Similar clinical observation was noted for other females from this group. Initial body weight losses resulted in lower mean body weight gain and accompanying reductions in food consumption. At scheduled sacrifice, reduced corrected body weight (carcass weight), corrected body weight change, and total weight change were also lower than controls, which corresponded with the lower mean body weights observed in-life. Such effects were not observed in the dose range finding study at this dose level (Covance Study 8410554; Covance Laboratories Ltd, 2020), hence the rational for dose level selection for the current study. 

Hematology assessments from blood samples taken on the day of scheduled sacrifice revealed changes in the red cell parameters, consistent with an anemic response, which was expected as similar findings were observed in a previous 90‑day toxicity study (Covance Study 8280017; Covance Laboratories Ltd, 2014). Furthermore, thyroid hormone assessments indicated changes in the thyroid hormones which were consistent with hypothyroidism. However, no test article-related macroscopic abnormalities, changes in thyroid weights, or any microscopic thyroid changes were noted.

Cesarean section examinations revealed an increase in pre-implantation loss following administration of 500 mg/kg bw/day AI, which was considered attributable to the maternal toxicity observed, and not a consequence of developmental toxicity. However, the most significant effect was the increased incidence of post‑implantation losses, high incidence of females with no viable fetuses, and a higher incidence of females with high numbers of early resorptions. This resulted in smaller litter sizes and fewer fetuses from the group administered 500 mg/kg bw/day AI, compared with controls. In addition, fetal examinations revealed increased incidences of unossified or incomplete ossification of the cervical centrum, metatarsals, and phalanx of the hindlimbs, which corresponded with the lower mean fetal weights for this group. Reduced ossification parameters were indicative of a transient delay in fetal maturation and were considered nonadverse.

Similar changes were also noted following administration of 250 mg/kg bw/day AI, albeit to a lesser extent than observed following administration of 500 mg/kg bw/day AI. Clinical observations were confined to two instances of audible respiration, mouth rubbing, and an isolated instance of piloerection following dosing. Marginally lower mean body weights and reductions in food consumption were evident, and carcass weight, corrected weight change, and total weight change at scheduled sacrifice were also marginally lower than controls. Similar hematological and thyroid hormone changes were also noticed, but were much less pronounced. However, no effect on fetal weights or change in the incidence of fetal variations was noted, and, as such, 250 mg/kg bw/day AI is considered to represent the no observed adverse effect level (NOAEL) for maternal animals and prenatal development of the subsequent offspring.

Prenatal development toxicity study in rabbit - OECD414:

Four groups of 20 time-mated female New Zealand White Hra:(NZW) SPF rabbits were administered 0 (control article [vehicle]), 50, 125, or 175 mg/kg bw/day of the Active Ingredient (AI; equivalent to 142.5, 356.25, or 498.75 mg/kg bw/day of the test article) by oral gavage, once daily, from Gestation Day (GD) 6 through 28 at a volume of 10 mL/kg. The control article (vehicle) was purified water. 

Test article-related unscheduled deaths occurred following administration of 175 mg/kg bw/day AI for 2 of the 20 animals. One animal administered 175 mg/kg bw/day AI was sacrificed on GD 26 due to clinical observations of thin appearance, red staining on the cage tray liner, and mucoid feces and body weight loss. Another animal administered 175 mg/kg bw/day AI aborted on GD 28 and was subsequently terminated. Clinical observations of thin appearance was observed for four animals (including two decedents) administered 175 mg/kg bw/day AI. Yellow discoloration of the pelage around the feet was observed for five females administered 125 mg/kg bw/day AI and five females administered 175 mg/kg bw/day AI. Test article-related lower mean body weights (GD 29: -7%), body weight gains (GD 6 to 29; -37%) and food consumption (GD 6 to 29; -16%) were evident following administration of 175 mg/kg bw/day AI, compared with controls. These reductions correlated with the lower mean total weight change and corrected weight change evident at scheduled sacrifice, compared with controls. Nonadverse test article-related lower mean food consumption was also observed for females administered 125 mg/kg bw/day AI, compared with controls over the dosing duration (-16%). Test article-related macroscopic findings of thin appearance were recorded for three animals and mottled livers were recorded for four females (two of which were decedents) administered 175 mg/kg bw/day AI. Of the 20 females in each group, 19, 20, 18, or 18 females administered 0, 50, 125, or 175 mg/kg bw/day AI were pregnant with live fetuses at the scheduled sacrifice. One animal administered 175 mg/kg bw/day AI littered prior to scheduled sacrifice, which was considered associated with the test article-related body weight losses observed from GD 17 onwards. Lower mean gravid uterus weight following administration of 175 mg/kg bw/day AI, was noted compared with controls. Marginally lower adjusted mean fetal weights (-7%) and mean unadjusted placenta weights (-8%) were evident following maternal exposure of 175 mg/kg bw/day AI. Nonadverse test article-related lower adjusted mean female fetal weights (-7%) and mean unadjusted placenta weights (-6%) were evident following maternal exposure of 125 mg/kg bw/day AI, compared with controls. Test article-related fetal variations of higher incidence of unossified metacarpals was observed for fetuses from animals administered 125 or 175 mg/kg bw/day AI, compared with controls. Peripheral bone ossification is observed later in development and this finding was considered to correlate with the lower fetal weights observed for females in these dose groups. No test article‑related fetal malformations were noted.

In conclusion, once daily oral (gavage) administration of 142.5, 356.25 or 498.75 mg/kg bw/day of the test substance (equivalent to 50, 125, or 175 mg/kg bw/day AI) to pregnant female rabbits, resulted in test article-related effects, including mortality and a single abortion following administration of 175 mg/kg bw/day AI. As such, 175 mg/kg bw/day AI was considered adverse. Test article-related effects following administration of 125 mg/kg bw/day AI were confined to nonadverse small reductions in food consumptions, lower mean female fetal and placental weights and fetal variations. These effects were considered attributable to the maternal effects and not a direct effect on fetal development, as these changes did not adversely affect the growing fetus. The no observed adverse effect level (NOAEL) for maternal toxicity was established as 125 mg/kg bw/day AI. The NOAEL for developmental toxicity was established as 125 mg/kg bw/day AI.

Toxicity to reproduction: other studies

Description of key information

No other studies available

Justification for classification or non-classification

Based on the available data, the substance is not classified as a reproductive toxicant according to the criteria laid down in the CLP Regulation (EC) 1272/2008.