Phenol, isopropylated, phosphate (3:1)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

None

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March 2020 to 23 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

OECD guideline for the Testing of Chemicals Number 443, Extended One-Generation Reproductive Toxicity Study (adopted 25 June 2018).

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Species Selection and Dose Administration 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.

Background Information
In a previous prenatal development study in the rat (MPI Research Study 399-243; MPI Research, 2014), pregnant female rats were administered the test article at a dose level of 0, 100, 200, or 400 mg/kg/day from GD 6 to 19 and were sacrificed on GD 20. All animals survived to their scheduled termination. Salivation was observed for all test article treated groups. No adverse test article effects were observed following administration of ≤200 mg/kg/day. Following administration of 400 mg/kg/day, maternal toxicity was restricted to early in the dosing period (GD 0 through 3) and included clinical observations (i.e., decreased activity, red material around the nose/mouth, hunched posture, thin appearance, discolored brown hair on the face, and discolored brown hair on the forelimbs), low gestation weight gain, and low food consumption. No adverse test article effect was observed on pregnancy indices; uterine implantation data; fetal sex ratios; fetal body weights; or fetal external, visceral, or skeletal evaluations. In the group administered 400 mg/kg/day, low incidences of red or white foci and swollen mucosa of the non glandular portion of the stomach were observed during F0 female macroscopic evaluations and were considered test article related. The no observed adverse effect level (NOAEL) for maternal toxicity was 200 mg/kg/day and the no observed effect level (NOEL) for developmental toxicity was 400 mg/kg/day.
In a previous 90-day repeat dose study (MPI Research Study 399-241; MPI Research, 2015), male and female rats were administered the test article at a dose level of 0, 25, 100, or 325 mg/kg/day. No adverse effects were noted in-life. Test article related clinical pathology changes included reversible increases in cholesterol for males administered 100 mg/kg/day and both sexes administered 325 mg/kg/day; reversible increases in fibrinogen and globulin for males administered 325 mg/kg/day (suggestive of an inflammatory response); and reversible increases in urea nitrogen for males administered ≥100 mg/kg/day. No test article related effects were observed for either sex administered 25 mg/kg/day. Macroscopic findings included tan discoloration and enlargement of the adrenal glands in all test article treated groups. Test article-related adrenal gland weight changes were noted in males administered ≥100 mg/kg/day and in females at all dose levels. Macroscopic and organ weight changes correlated with diffuse vacuolation of the zona fasciculata, observed microscopically, and was characterized by enlarged cells with foamy cytoplasm. In addition, the zona fasciculata cells noted near the zona reticularis had large, single, clear, vacuoles that expanded outwardly, depending on severity. The adrenal gland changes were considered adverse, based on the severity and the marked increases in adrenal gland weights in females. At recovery, the diffuse vacuolation was not present; however, cells near the zona reticularis had large, single, cytoplasmic vacuoles. Test article-related organ weight changes were noted for the liver of both sexes administered ≥100 mg/kg/day, thyroid gland for males administered 325 mg/kg/day, and ovary weights for females at all dose levels. Organ weights for the liver, thyroid gland, and ovary correlated microscopically with centrilobular or pablobular hypertrophy in the liver, follicular cell hypertrophy in the thyroid glands, and interstitial cell vacuolation in the ovaries. Additionally, lower terminal body weight in males administered 325 mg/kg/day resulted in higher organ:body weights, lower absolute organ weights, and/or lower organ:brain weights for the brain, epididymides, heart, and pituitary gland. A NOEL or NOAEL was not established for that study.
In a previous OECD 421 study (MPI Research Study 1038-004; MPI Research, 2005), male and female rats were administered the test article at a dose level of 0 or 400 mg/kg/day. Following administration of 400 mg/kg/day to rats, mean pup survival after parturition and up to PND 4 was lower than controls. Microscopically, test article-associated fatty changes were observed in the adrenals of males and females administered 400 mg/kg/day. Fatty changes were similar in the adrenals of males and females and were characterized by cells in the zona fasciculata and zona reticularis that were enlarged by the presence of increased small or large clear vacuoles. Vacuolation of interstitial cells of the ovaries and centrilobular hepatocellular hypertrophy of livers was also seen in females administered 400 mg/kg/day of the test article.

Dose Level Selection
The high-dose level of 100 mg/kg/day was expected to elicit some toxicity without causing undue stress or death.
The intermediate-dose level of 25 mg/kg/day was anticipated to be a no observed adverse effect level (NOAEL).
The low-dose level of 5 mg/kg/day was anticipated to be a no observed effect level (NOEL).
The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.

Specific details on test material used for the study:

No further details specified in the study report

Species:

rat

Strain:

Wistar

Details on species / strain selection:

104 male and 104 female Crl:WI(Han) rats were obtained from Charles River Laboratories, Margate, United Kingdom, in order to provide sufficient animals for F0 generation study selection.
Upon arrival, animals were 5 to 6 weeks of age. At the start of dosing, F0 animals were 6 to 7 weeks of age, and males weighed between 149.0 and 217.6 g; females weighed between 107.2 and 171.4 g.
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for 13 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Environmental Conditions, Diet, and Water
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).
F0 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, F1 animals were housed in groups of up to four by sex, dose group, and cohort. For Cohort 1B, 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, males were housed in groups of up to four by sex and dose group (post-pairing phase); females were housed individually during gestation or post-pairing, and with their litter during the lactation phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
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 Labcorp.

Water
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. Results are retained on file at Labcorp.

Diet
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. Results are retained on file at Labcorp.
Four batches of diet were used on this study. A 50 g sample of each batch of diet used on study was collected and stored at ambient temperature until study finalization.

Environment
Animals were housed in exclusive rooms. Rooms were air conditioned to provide a minimum of 15 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 19 to 25 deg C and 40 to 70%, respectively.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark.

Environmental Enrichment
Animals were provided with wooden Aspen chew blocks, rodent retreats and paper wool nesting materials (gestation and lactation phases only) as forms of environmental enrichment. The paper wool nesting material was retained at bedding changes between GD 18 and LD 4.

Animal Identification and Assignment to the Study
Upon arrival, F0 animals were assigned to groups based on a total randomization procedure.
Animals were individually identified by electronic implant. Pups were uniquely identified from PND 1 via a back mark with an indelible pen.
During FOB assessments, applicable F1 generation, Cohort 2A animals were identified by tail marks.
F1 Cohort 2B animals were uniquely identified by tail mark with an indelible pen.
For F0 animals, following the first full weight collection during the predose phase, group mean body weights and standard deviations were calculated and inspected to ensure no unacceptable differences occurred between groups.
Cages were placed in group order across the batteries of cages, except during FOB assessments.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Formulations were prepared daily for the first 2 days and then weekly thereafter.
The test article was formulated as a suspension in corn oil following dispensary SOPs and the formulation method (Method 8415993_O_01D), as maintained in the study data.
Formulations were stored refrigerated (2 to 8 deg C) in a sealed container, protected from light.

Details on mating procedure:

F0 Generation
During the pairing phase, one male was housed for up to 14 days with one female of the same group.
Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon the confirmation of mating, vaginal washing was discontinued, and the male was removed from the cage. The day of mating confirmation was considered GD 0.

F1 Generation - Post weaning
During the pairing phase, one Cohort 1B male was housed for up to 14 days with one female of the same cohort/dose group.
Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon the confirmation of mating, vaginal washing was discontinued, and the male was removed from the cage. The day of the confirmation of mating was considered GD 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Procedure
The samples were analyzed in accordance with the validated Labcorp Analytical Procedure (DFA/M032/20).
The analytical method involved extraction and dilution in Acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 µg/mL to 100 µg/mL. Standard and sample solutions were all spiked with a known amount of Tetradecane (internal standard), and the peak area response ratio of the test item:internal standard was used for quantitation.

Concentration of Dose Formulations
The formulations for the first, middle and last dosing occasion preparations were sampled. For all groups, 1 × 5 mL was sampled at random from the formulation by Labcorp Harrogate Dose Formulation personnel.
Two aliquots from all groups were analyzed in accordance with the analytical procedure. The remainder of the sample was retained for contingency. On the first and last dosing occasion duplicate additional aliquots were taken from the samples and analysed following out of specification results being achieved in the initial analysis. Samples were disposed of once satisfactory results were achieved.
Procedural recoveries were prepared as a quality control measure and were not used to correct for the analytical results.

Duration of treatment / exposure:

F0 males were dosed once daily for 126 consecutive days (at least 10 weeks prior to pairing, during pairing, and 41 days post-pairing); females were dosed for up to 128 days (10 weeks prior to pairing, during pairing, throughout gestation, and up to LD 21).
All F1 offspring were dosed once daily from PND 14 through 21; prior to this, pups were maternally administered test article through milk. Following selection into appropriate cohorts, selected F1 animals were administered test article once daily.

Frequency of treatment:

Daily

Details on study schedule:

F0 Generation Procedures
Test Article Administration
Formulations (excluding vehicle control group) were stored at room temperature and stirred continuously for at least 30 minutes before and throughout dosing.
The test article was administered orally by gavage.
F0 males were dosed once daily for 126 consecutive days (at least 10 weeks prior to pairing, during pairing, and 41 days post-pairing); females were dosed for up to 128 days (10 weeks prior to pairing, during pairing, throughout gestation, and up to LD 21).
Dosing was omitted on one occasion for the following females as they were in or near parturition at the time of daily dosing.
• Animals R0404, R0418, R0420, R0421, and R0425 (F0 generation, Group 1)
• Animals R0502, R0508, R0516, and R0524 (F0 generation, Group 2)
• Animals R0604, R0608, R0609, R0615, and R0621 (F0 generation, Group 3)
• Animals R0701, R0706, R0715, R0717, R0719, R0720, and R0725 (F0 generation, Group 4)
All F1 offspring were dosed once daily from PND 14 through 21; prior to this, pups were maternally administered test article through milk. Following selection into appropriate cohorts, selected F1 animals were administered test article once daily.
Formulations were administered at a constant dose volume of 4 mL/kg. Dose volumes were based on the most recently recorded body weight for each animal.

F1 Generation Procedures - Post Weaning
Test Article Administration
Formulations (excluding vehicle control group) were stirred continuously for at least 30 minutes before and throughout dosing.
The test article was administered orally by gavage.
Following selection into appropriate cohorts, selected F1 animals were administered once daily as follows:
• Cohort 1A: For up to 73 days
• Cohort 1B: For up to 104 consecutive days for males (up to 75 days prior pairing, during pairing and 15 days post pairing) or for up to 108 days for females (up to 75 days prior to pairing, during pairing, throughout gestation and up to LD 3
• Cohort 2A: For 54 days
• Cohort 2B: For 1 day
Dosing was omitted for the following Cohort 1B females as they were in or near parturition at the time of daily dosing.
• Animals R1222, R1227, and R1230 (F1 generation, Group 1)
• Animals R1326 and R1328 (F1 generation, Group 2)
• Animals R1424 and R1426 (F1 generation, Group 3)
• Animals R1522, R1523, R1526, R1528, and R1532 (F1 generation, Group 4).
Formulations were administered at a constant dose volume of 4 mL/kg. Dose volumes were based on the most recently recorded body weight for each animal.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0 Generation: 25/sex per dose
F1 Generation, Cohort 1A & 1B: 20/sex per dose
F1 Generation, Cohort 2A & 2B: 10/sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The high-dose level of 100 mg/kg/day was expected to elicit some toxicity without causing undue stress or death.
The intermediate-dose level of 25 mg/kg/day was anticipated to be a no observed adverse effect level (NOAEL).
The low-dose level of 5 mg/kg/day was anticipated to be a no observed effect level (NOEL).
The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.

Parental animals: Observations and examinations:

F0 Generation
Clinical Observations
Health Monitoring
All animals were observed in the home cage at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity.

Clinical Examinations
Each animal was given a detailed physical examination once daily from the start of dosing. An individual record was maintained of the clinical condition of each animal.

Weekly Detailed Clinical Observations
All animals were assessed by detailed clinical observations within the home cage, in the hand, and in an open field arena for approximately 30 seconds. 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. For one of the females with no confirmation of mating but which was pregnant (Animal R0404 [F0 generation, Group 1]), the data collected during the post-pairing phase were back entered to the gestation phase.
Each animal was observed and evaluated for the following:
Activity, Aggression to cage mate, Alertness, Approach response, Acoustic startle response, Bar test, Behaviour – other & stereotype, Body tone, Discharge, Excretion, Extensor thrust, Eye closure, Eyes, Gait, Grasping loss, Grip strength, Involuntary movement, Lacrimation, Pain response, Palpebral reflex, Pelage, Pinna response, Posture, Proprioception (right hind leg), Pupil status, Pupillary response, Reactivity to handling, Respiration, Restlessness, Righting reflex, Salivation. Skin colour, Tail, Visual response, Vocalisation, Waxy ridgity.


Postdose Observations
Animals were observed daily for the first 7 days of dosing at approximately 0.5 hours postdose.

Body Weights
Body weights were recorded once during acclimation (Predose Day 1[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.
Where an animal had completed parturition before the daily dose on LD 0, a body weight was recorded and was used to calculate the correct dose volume to be administered to avoid overdosing; then, dosing occurred.
Body weights for females with no confirmation of mating were recorded weekly during the post-pairing phase. For one of the females with no confirmation of mating but which was pregnant (Animal R0404 [Group 1] and R0510 [Group 2]), the data collected during the post pairing phase were back-entered to the gestation phase.

Food Consumption
Male food consumption was recorded twice weekly during the pre-pairing and post pairing phases.
Female 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 consumption was not recorded during pairing (both sexes) or post-pairing (females), between GD 21 and LD 0, due to parturition, and from LD 14, due to the start of pup weaning.
Food consumption was calculated as g/animal/day.
Food conversion efficiency (body weight gain per gram of food consumed) was also calculated.


F0 Generation Clinical Laboratory Procedures
Clinical Pathology
Sample Collection and Handling
Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.6 mL prior to the issue of Protocol Amendment 05 [2 x 0.5 mL pots were used], then 1 x 0.5 mL thereafter [trisodium citrate], nominal), clinical chemistry (1 x 0.5 mL prior to the issue of Protocol Amendment 05, then 1 x 0.6 mL thereafter [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 on Post Pairing Day 42 (Week 19 of the study) for at least 10 randomly selected males within each group and at necropsy on LD 22 for at least 10 randomly selected females within each group. Samples were collected after animals were fasted overnight.
Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring blood travelled 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 blood travelled all the way to the top and bottom of the tube each time to mix with the clot activator.
Blood samples for thyroid hormone analysis from unselected F1 generation pups sacrificed on PND 22 (2 x 0.6 mL [serum separator tubes], nominal) were withdrawn by cardiac puncture from two male pups/litter and two female pups/litter. Samples from one male and one female sample were analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis are 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.
Each blood sample for thyroid hormone analysis was gently inverted several times (approximately 10), ensuring blood travelled 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).
During sample processing, each blood sample obtained from adults 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 are retained in storage until report finalization, after which, they will be discarded.
Urine samples from the first six males/group from batch 1 and 2 (total of 12 males per group) and up to 13 females/group which littered first (across batch 1 and 2) were collected overnight the day prior to necropsy. Food was removed during collection.

Hematology Tests
hemoglobin, red blood cell count, packed cell volume (hematocrit), mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, hemoglobin distribution width, total and differential white cell count, platelet count*, platelet crit, mean platelet volume, platelet distribution width
*Includes platelet clump assessment. Clump counts below 100 are considered none detected; clump counts over 100 are considered platelet clumps present and are confirmed by review of Advia cytogram or blood film examination.

Blood smears were prepared from each hematology specimen and used when necessary to confirm results produced by the analyzer.

Coagulation Tests
prothrombin time, fibrinogen, activated partial thromboplastin time

Clinical Chemistry Tests
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, enzymatic creatinine, urea, glucose, blood urea nitrogen

Urinalysis Tests
volume, color, turbidity, specific gravity, pH*, protein*, glucose*, ketones*, urobilinogen*, bilirubin*, blood*, microscopy of sediment
*Determined semi quantitatively.

Thyroid Hormone Tests
thyroxine (Total T4), thyroid stimulating hormone (TSH)

Bone Marrow Smear Evaluation
Bone marrow smears were prepared at necropsy from the femur for F0 animals. Tissues were taken into bovine serum albumin, smear-prepared, air-dried, and fixed in methanol, but not examined.

F1 Generation Procedures - Post Weaning
Clinical Observations
Health Monitoring
All animals were observed in the home cage at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity.

Clinical Examinations
Each animal was given a detailed physical examination once daily from the start of dosing. An individual record was maintained of the clinical condition of each animal.

Weekly Detailed Clinical Observations
All animals of Cohorts 1A, 1B, and 2A were assessed by detailed clinical observations within the home cage, in the hand, and in an open field arena for approximately 30 seconds. Both sexes were assessed once weekly from Maturation Day 22, and Cohort 1B females were assessed on GD 0, 6, 14, and 20 and on LD 4.
Each animal was observed and evaluated for the following:
Activity, Aggression to cage mates, Alertness, Approach response, Acoustic startle response, Bar test, Behaviour – other & stereotype, Body tone, Discharge, Excretion, Extensor thrust, Eye closure, Eyes, Gait, Grasping loss, Grip strength, Involuntary movement, Lacrimation, Pain response, Palpebral reflex, Pelage, Pinna response, Posture, Proprioception (right hind leg), Pupil status, Pupillary response, Reactivity to handling, Respiration, Restlessness, Righting reflex, Salivation, Skin colour, Tail, Visual response, Vocalisation, Waxy rigidity.

Postdose Observations
Animals of Cohorts 1A, 1B, and 2A were observed daily for the first 7 days of dosing at approximately 0.5 hours postdose.

Body Weight
Body weights for Cohorts 1A, 1B (males), and 2A were recorded on PND 22, 24, 26, 28, 30, 32, 34, and 36 and then weekly thereafter.
Body weights for Cohorts 1B females were recorded on PND 22, 24, 26, 28, 30, 32, 34, and 36; weekly thereafter (maturation/pre-pairing phase); on GD 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and on LD 1 and 4.
Body weights for Cohort 2B were recorded on PND 21 and 22 (terminal body weight).
For Cohorts 1A, 1B, and 2A, body weights were recorded on the day balano preputial separation or vaginal opening occurred.

Food Consumption
Food consumption was recorded twice weekly from Maturation Day 22, until the day prior to necropsy for Cohorts 1A and 2A. See Protocol Deviations.
Food consumption was recorded twice weekly from Maturation Day 22 until pairing (both sexes); then twice weekly during post-pairing (males); every 2 days during gestation, and from LD 1 to 4 for Cohort 1B.
Food consumption was not recorded during pairing and between GD 21 and LD 0, due to parturition.
Food 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.

Oestrous cyclicity (parental animals):

F0 Generation
Daily vaginal lavage (washings) was undertaken from females for 14 days prior to pairing and until confirmation of mating, and on the day of necropsy.

F1 generation - Post weaning
Daily vaginal lavage (washings) were undertaken for Cohort 1A 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.
Daily vaginal lavage (washings) were undertaken for Cohort 1B females after vaginal opening until the first cornified smear was observed, then from Maturation Day 75 until confirmation of mating, and on the day of necropsy for females that littered.

Sperm parameters (parental animals):

Sperm Assessments
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 (vas deferens and epididymis cauda). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from all males for each group were read for morphological changes. The left epididymis was discarded following seminology investigations.

Cohort 1A
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 (vas deferens and epididymis cauda). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from all males for each group were read for morphological changes. The left epididymis was discarded following seminology investigations

Litter observations:

F0 Generation
Parturition
Animals were observed three times each day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female littered. Females that were confirmed as mated but did not litter were sent to necropsy on GD 26; females that did not have a confirmation of mating and did not litter were sent to necropsy on Post-Pairing Day 25.
Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, 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 F0 female 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).

F1 Offspring Procedures - Pre-Weaning
Litter Size and Sex Determination
Litter size and pup sex were recorded on PND 0, 1, 4, 7, 14, and 21. See Protocol Deviations.
Daily records of mortality and changes in litter sizes were maintained. Pups found dead or in a moribund condition were given a macroscopic necropsy for cause of death and possible birth defects.

Clinical Observations
A detailed clinical examination was recorded for each pup on PND 1, 4, 7, 14, and 21.

Postdose Observations
All pups were observed daily from PND 14 through 21 at approximately 0.5 hours postdose.

Body Weights
Pup body weights were recorded on PND 1, 4, 7, 13, 14, 16, 18, and 20.

Anogenital Distance
The anogenital 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. See Protocol Deviations.

Weaning
Pups were weaned on PND 21, and 240 pups/sex were randomly selected from available litters (any obvious runts [pups with a body weight more than two standard deviations below the mean pup weight of the respective litter] were not included in selection). Animals of Cohorts 1A and 1B were allocated, based on a random selection of 20 animals/sex/group from the available litters (one male and one female from each litter). Animals of Cohorts 2A and 2B were allocated, based on a random selection of 10 animals/sex/group from the available litters (one male and one female from each litter).
On PND 22, a blood sample for thyroid hormone analysis was withdrawn from all pups not selected to continue into the F1 generation. Following blood sampling, pups were sacrificed and necropsied.

F1 Generation - Post weaning
Parturition
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 littered. Females confirmed as mated but that did not litter were sent to necropsy on GD 26.
Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, 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).

F2 Offspring Procedures
Litter Size and Sex Determination
Litter size and pup sex were recorded on PND 0, 1, and 4.
Daily records of mortality and changes in litter sizes were maintained. Pups found dead or in a moribund condition were given a macroscopic necropsy for cause of death and possible birth defects.

Clinical Observations
A detailed clinical examination was recorded for each pup on PND 1 and 4.

Body Weights
Pup body weights were recorded on PND 1 and 4.

Anogenital Distance
The anogenital distance of all pups was recorded on PND 4.

Neurotoxicity Testing
Cohort 2A animals were assessed for acoustic startle on Maturation Day 24 (±1 day).
Each animal was evaluated for startle habituation using a Kinder Auditory Startle Chamber. Prior to habituation testing, each animal was acclimatized to the chamber for 4 minutes under white noise conditions. Each animal underwent one habituation session, which consisted of 50 standard trials of 115 dB bursts of noise, with mixed inter-trial intervals.
On a separate day, prior to pre-pulse inhibition (PPI) testing, each animal was acclimatized to the chamber for 4 minutes under white noise conditions. Animals underwent testing, which consisted of 41 randomized trials. Thirty were pre-pulse trials (10 trials each of 74, 78, or 86 dB pre-pulse, followed by 115 dB startle), and 11 were standard trials (115 dB startle, with no pre-pulse), with mixed inter-trial intervals and decibel levels. The first standard trial (no pre-pulse) for each animal was used for acclimatization and was not analyzed. Data were reported as percent pre pulse inhibition for the PPI session.
Data collected for habituation included the mean maximum amplitude (five blocks of 10 trials for each group). Pre-pulse inhibition was calculated as 100 - ((maximum startle amplitude on pre-pulse trials / maximum startle amplitude on 115 dB standard trials) x 100) for each animal.

Functional Observational Battery
Cohort 2A animals underwent FOB assessments by detailed clinical observations, quantitative assessments, and elicited responses.
The FOB assessments were undertaken in a manner so the observer did not know the dose group of animals during testing. Observations were performed at approximately the same time on each occasion.

Detailed Clinical Observations
Cohort 2A animals were assessed for detailed clinical observations on Maturation Day 69 (±6 days). Males were assessed on Maturation Days 66, and 68 through 71. Females were assessed on Maturation Days 67, and 69 through 72.
Animals were observed within the home cage, in the hand, and in the arena for approximately 2 minutes. Each animal was observed and evaluated for the following.
Activity, Aggression to cage mates, Alertness, Approach response, Acoustic startle response, Bar test, Behaviour – other & stereotype, Body tone, Discharge, Excretion, Extensor thrust, Eye closure, Eyes, Gait, Grasping loss, Grip strength, Involuntary movement, Lacrimation, Pain response, Palpebral reflex, Pelage, Pinna response, Posture, Proprioception (right hind leg), Pupil status, Pupillary response, Reactivity to handling, Respiration, Restlessness, Righting reflex, Salivation, Skin colour, Tail, Visual response, Vocalisation, Waxy rigidity.
Numerical observations: Faecal boli, Rears, Urine pools, Latency to first step.

Quantitative Assessments
Cohort 2A animals were assessed for quantitative assessments on Maturation Day 69 (±6 days). Males were assessed on Maturation Days 66, and 68 through 71. Females were assessed on Maturation Days 67, and 69 through 72.
Quantitative assessment parameters are as follows.
Hindlimb foot splay, Forelimb and hindlimb grip strength.

Motor Activity
Cohort 2A animals were assessed for motor activity on Maturation Day 69 (±6 days).
Motor activity was assessed in an automated photocell activity recorder (Kinder Motor Monitor system) for 60 minutes.
Motor activity was recorded with the room lights turned off and white noise switched on. The white noise generator was maintained (level recorded at the beginning and end of each session) at an appropriate level so that a sound meter registered between 60 and 80 dB (Decibels), during the course of the scheduled sessions.
Animals were acclimated in their holding cages to the white noise for at least 10 minutes prior to any procedure taking place in the room.
Motor activity data are reported over 10-minute intervals.
The following parameters were assessed:
Basic Movement: Count of all horizontal beam breaks
Fine Movement: Measure of small movements, such as grooming and head movements; the animal remained in a fixed point (i.e., a single beam break with no other beams affected).
Total Ambulations: Measure of large movements; when a new beam was blocked and the anchor beam was broken (i.e., animal has relocated its whole body [e.g., a step forward]).
Total ambulation = X ambulation + Y ambulation
Total Distance Travelled: Record of distance travelled around the cage.
Total distance travelled = High-Density Periphery Zone Distance + Center Zone Distance
Rearing Event: Beam break on top grid (rearing)

F1 Generation Cohort 1A Clinical Laboratory Procedures
Clinical Pathology
Sample Collection and Handling
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 on Maturation Day 92, 93, 94, or 95 for at least 10 randomly selected males within each group and at necropsy on Maturation Day 92, 93, or 94 for at least 10 randomly selected females within each group. Samples were collected after animals were fasted overnight.
Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring that the blood travelled 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 blood travelled all the way to the top and bottom of the tube each time to mix with the clot activator.
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 blood travelled 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).
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 are retained in storage until report finalization, after which, they will be discarded.
Urine samples from at least 10 randomly selected males and females in each group were collected overnight the day prior to necropsy. Food was removed during collection.

Hematology Tests
hemoglobin, red blood cell count, packed cell volume (hematocrit), mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, hemoglobin distribution width, total and differential white cell count, platelet count*, platelet crit, mean platelet volume, platelet distribution width
*Includes platelet clump assessment. Clump counts below 100 are considered none detected; clump counts over 100 are considered platelet clumps present and are confirmed by review of Advia cytogram or blood film examination.
Blood smears were prepared from each hematology specimen and used when necessary to confirm results produced by the analyzer.

Coagulation Tests
prothrombin time, activated partial thromboplastin time, fibrinogen

Clinical Chemistry Tests
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, enzymatic creatinine, urea, glucose, blood urea nitrogen

Urinalysis Tests
volume, color, turbidity, specific gravity, pH*, protein*, glucose*, ketones*, urobilinogen*, bilirubin*, blood*, microscopy of sediment
*Determined semi quantitatively.

Thyroid Hormone Tests
thyroxine (Total T4), thyroid stimulating hormone (TSH)

Bone Marrow Smear Evaluation
Bone marrow smears were prepared at necropsy from the femur for F1 Cohort 1A animals. Tissues were taken into bovine serum albumin, smear-prepared, air-dried, and fixed in methanol, but not examined.

Postmortem examinations (parental animals):

F0 Generation Terminal Procedures
With the exception of fasting, these procedures were also followed for unscheduled sacrifices.

Adult Necropsy
F0 males were sacrificed by isoflurane anesthesia on Post-Pairing Day 42 (Week 19) after an overnight period without food. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
F0 females were sacrificed by isoflurane anesthesia after the completion of weaning on LD 22 (those that littered) or GD 26 (those with confirmation of mating that did not litter) or on Post-Pairing Day 25 (those with no confirmation of mating that did not litter). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Females sacrificed on LD 22 were starved overnight prior to necropsy.
Females with the same day of mating/littering were sacrificed in a controlled, randomization order (i.e. one animal from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Females that did not litter were sacrificed in animal identification order.
The number of implantation 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.
Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded. Macroscopic examinations were performed under the general supervision of a pathologist. For tissue collection.
Organ weights, were recorded at each scheduled sacrifice, excluding decedents and paired females that mated but did not litter. Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
Tissue List
Adrenal1 [WE], animal identification, bone marrow smear (femur)2, brain [WE], cecum [E], colon [E], duodenum [E], epidydymis1,3 [WE], eye4 [E], femur with bone marrow and femorotibial joint [E], gross lesions [E], head, heart [WE], ileum [E], jejunum [E], kidney1 [WE], liver [WE], lungs with main stem bronchi and bronchioles [E], mammary gland [E], muscle, biceps femoris [E], nerve, optic [E], nerve, sciatic [E], ovary1 [WE], oviduct [W5E], pituitary [WE], prostate [WE], rectum [E], seminal vesicle with coagulating glands and fluid [WE], spinal cord, cervical [E], spinal cord, lumbar [E], spinal cord, thoracic [E], spleen [WE], stomach [E], testis1,3 [WE], thymus [WE], thyroid with parathyroid6 [WE], trachea [E], urinary bladder [E], uterus with cervix [WE], vagina [E].
E = Processed and examined microscopically; W = Weighed
Note: bone designated for microscopic examination was decalcified using Kristenson’s fluid.
1 Tissue weighed as a pair
2 Smear prepared, air dried, the fixed in methanol
3 Tissue taken into Modified Davidsons fixative and processed to at least the block stage
4 Tissue taken into Davidsons fixative
5 Weighed with uterus
6 Tissues weighed together

Reproductive and Endocrine Tissue List
Animal identification, epididymis1 [E], gross lesions [E], mammary gland [E], ovary [E], oviduct [E], pituitary [E], prostate [E], seminal vesicle [E], testis1 [E], uterus with cervix [E], vagina [E].
E = Processed and examined microscopically
1 Tissue taken into Modified Davidsons fixative and processed to at least the black state


Histology and Microscopic Examinations
All tissues denoted by E in the Table from Groups 1 and 4 and all decedents and the adrenal glands 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 denoted by E in the table 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.
Due to suspected test article-related effects in Group 4, thyroids, liver, spleen (both sexes), ovaries (females), and kidneys (males), for all Group 2 and 3 animals, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
Qualitative assessment of spermatogenesis and testicular interstitial cell structure was performed for all males, where appropriate.

F1 Generation Terminal Procedures
With the exception of fasting, these procedures were also followed for unscheduled sacrifices.

Cohort 1A
Animals were sacrificed by isoflurane anesthesia between Maturation Days 92 and 95 (Week 11) after an overnight period without food. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Upon sacrifice, macroscopic examinations were conducted and all lesions were recorded. Macroscopic examinations were performed under the general supervision of a pathologist. For tissue collection, see Text Table.
Organ weights, as indicated in Text Table, were recorded at each scheduled sacrifice. Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
Animal R1103 (Group 4, F1 Cohort 1A male) was found dead and replaced. Tissues as indicated in Text Table 3.8 were retained in 10% neutral buffered formalin, unless otherwise indicated. Tissues were not processed or examined microscopically by the Study Pathologist.
Tissue List
Adrenal1 [WE], animal identification, bone marrow smear (femur)2, brain [WE], cecum [E], colon [E], duodenum [E], epidydymis1,3 [WE], eye4 [E], femur with bone marrow and femorotibial joint [E], gross lesions [E], head, heart [WE], ileum [E], jejunum [E], kidney1 [WE], liver [WE], lungs with main stem bronchi and bronchioles [E], lymph node, mandibular8 [WE], lymph node, mesentric8 [WE], mammary gland [E], muscle, biceps femoris [E], nerve, optic [E], nerve, sciatic [E], ovary1 [WE], oviduct [W5E], pituitary [WE], prostate [WE], rectum [E], seminal vesicle with coagulating glands and fluid [WE], spinal cord, cervical [E], spinal cord, lumbar [E], spinal cord, thoracic [E], spleen7 [WE], stomach [E], testis1,3 [WE], thymus [WE], thyroid with parathyroid6 [WE], trachea [E], urinary bladder [E], uterus with cervix [WE], vagina [E].
E = Processed and examined microscopically; W = Weighed
Note: bone designated for microscopic examination was decalcified using Kristenson’s fluid.
1 Tissue weighed as a pair
2 Smear prepared, air dried, the fixed in methanol
3 Tissue taken into Modified Davidsons fixative and processed to at least the block stage
4 Tissue taken into Davidsons fixative
5 Weighed with uterus
6 Tissues weighed together
7 Tissue weighed for all animals. For at least 10 animals/sex/group, half of the spleen was provided to Labcorp Immunology and Immunotoxicity then used for splenic lymphocyte subpopulation analysis.
8 Tissue weighed and examined for 10/sex/group (males with the lowest identification number; females with the highest identification number.

Histology and Microscopic Examinations
All tissues denoted by E in Text Table 3.8 from Groups 1 and 4, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
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 were processed.
Additional sections of testes and epididymides were also stained with periodic acid Schiff (PAS) for spermatogenic staging for all males in Groups 1 and 4 and all decedents.
Qualitative assessment of spermatogenesis and testicular interstitial cell structure was performed for all males, where appropriate.
Qualitative assessment of the stages of follicular development to regression from small growing follicles to corpora lutea was performed in the ovaries for all females.
Due to suspected test article-related effects in Group 4, thyroids, liver (both sexes), ovaries (females), and spleens from remaining males and females, where not processed as part of immunotoxicity assessments, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
All processed tissues were examined microscopically by the Study Pathologist.

Immunotoxicity Assessments
Prenatally and postnatally 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 groups.

Cohort 1B
Males were sacrificed by isoflurane anesthesia on Post Pairing Day 16 (Week 15), but food was not removed. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). 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 4 (those that littered) or GD 26 (those with confirmation of mating that did not litter). Food was not removed prior to sacrifice. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
Females with the same day of mating/littering were sacrificed in a controlled randomization order (i.e. one animal from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Females that did not litter were sacrificed in animal identification order.
The number of implantation sites and corpora lutea 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.
Decedent animals were sacrificed by isoflurane anesthesia. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Tissues from decedent animals were retained as indicated in Text Table. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
Upon sacrifice, macroscopic examinations were conducted and all lesions were recorded. Macroscopic examinations were performed under the general supervision of a pathologist.
Organ weights were recorded at each scheduled sacrifice, excluding decedents. Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.

Tissue List for Cohort 1B
Animal identification, epidudymis1,2 [WE], gross lesions [E], mammary gland [E], ovary [WE], oviduct [W3E], pituitary [WE], prostate [WE], seminal vesicle [WE], testis1,2[WE], uterus with cerviz [WE], vagnia [E].
E=Processed and examined microscopically
W=Weighed
1 Tissue weighed as a pair
2 Tissue taken into Modified Davidsons fixative and processed to at least the block stage
3 Weighed with uterus

3.13.2.1 Histology Processing and Microscopic Examinations
All tissues denoted by E in Text from males in Groups 1 through 4 that did not sire and females in Groups 1 through 4 that were not pregnant were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
Processed tissues were not examined microscopically by the Study Pathologist.
The ovary from all scheduled females in Groups 1 through 4 were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin. The tissues were examined microscopically by the Study Pathologist.

Postmortem examinations (offspring):

Pup Necropsy
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, the major blood vessels were severed to exsanguinate the animal. Sacrifices were carried out in group order.
Full macroscopic examinations with particular attention to the reproductive genitals, were conducted for all decedents and pups sacrificed on PND 22.
Organ weights, were recorded from each pup sacrificed on PND 22, and were dissected free from fat and other contiguous tissue and weighed before fixation.
The following tissues from each pup sacrificed on PND 22 were preserved in 10% neutral buffered formalin. Tissues are retained until report finalization, then discarded.
Adrenal glands [W], brain [W], gross lesions, kidneys [W], mammary tissue, spleen [W], stomach, thymus [W].
W = Weighed

3.13.3 F2 Generation Pup Necropsy
Pups on PND 4 were sacrificed 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. Sacrifices were carried out in a controlled randomization order, (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed).
Macroscopic examinations of the abdominal cavity, adrenal, brain, gastrointestinal tract, gross lesions, head, kidney, leg, tail and thoracic cavity were conducted for all decedents and pups sacrificed on PND 4. Macroscopic examinations were performed under the general supervision of a pathologist.
The kidney weight was recorded for each pup sacrificed on PND 4, and was dissected free from fat and other contiguous tissue and weighed before fixation.
The adrenal glands, kidneys and animal identification from each pup sacrificed on PND 4 were preserved in 10% neutral buffered formalin. Tissues are retained until report finalization, then discarded.

3.13.4 Cohort 2A
Animals were sacrificed on Maturation Day 76, 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; food was not removed. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed).
Upon sacrifice, macroscopic examinations were conducted (which included all orifices, cranial cavity, external surfaces of the brain and spinal cord, cervical tissues and organs, thoracic, abdominal and pelvic cavities and viscera, external surface of the body and nasal cavity and paranasal sinuses), and all lesions were recorded. Macroscopic examinations were performed under the general supervision of a pathologist.
The brain from all animals was weighed and retained in 10% neutral buffered formalin, prior to fixed tissue dissection to remove tissues. Animals were subjected to whole body perfusion fixation with 50% Karnovsky’s fluid.

Tissue List for Cohort 2A
Olfactory bulb [H&E*; MSSa*], Forebrain (including hippocampus [H&E*; MSSa*], Basal ganglia (including caudate nucleus) [H&E*; MSSa*], Hypothalamus/thalamus [H&E*; MSSa*], Midbrain [H&E*; MSSa*], Brain stem (including pons and medulla oblongata) [H&E*; MSSa*], Cerebellum [H&E*; MSSa*], Cerebral cortex [H&E*; MSSa*], Entire spain cord (including cervical [to include enlargement at C4 level], thoracic and lumbar [to include enlargement at L4/5 level] regions) [H&E* – Left TS and LS from all three sections], Cervical dorsal root ganglia [H&E*; TB*† ], Lumbar dorsal root ganglia [H&E*; TB*†], Trigeminal ganglia [H&E*], Eye including retina [H&E*], Optic nerve [H&E*], Proximal and distal sciatic nerve [H&E* – Left TS and LS; TB – Right TS*† ], Proximal and distal tibial nerve [H&E* – Left TS and LS; TB – Right TS*† ], Sural nerve [H&E* – Left TS and LS; TB – Right TS*† ], Anterior tibialis muscle (left side) [H&E*], Gastrocnemius muscle (left side) [H&E*], Macroscopic lesions [H&E*]
H&E = Hematoxylin and eosin; TB = Toluene Blue; MSS = Myelin and Silver Stain; LS = Longitudinal section; TS = Transverse section.
a Luxol fast blue and Cresyl violet stains
The tissue listed were collected from all animals
The animal identification was also preserved

Histology and Microscopic Examinations
Those tissues indicated by * in Text were trimmed, processed, and embedded in paraffin wax (all animals); however, neuropathological evaluation by hematoxylin and eosin (H&E) was performed on Groups 1 and 4 only.
Those tissues indicated by † in Text were trimmed, post-fixed in osmium tetroxide, processed, and embedded in epoxy resin (all animals); however, neuropathological evaluation by toluidine blue was performed on Groups 1 and 4 only.

Cohort 2B
Animals were sacrificed on Maturation Day 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. Sacrifices were carried out in a controlled randomization order (i.e. one cage of animals from Groups, 1, 4, 2 then 3 and repeated in the same sequence until all animals sacrificed). Food was not removed.
Upon sacrifice, macroscopic examinations were conducted (which included all orifices, cranial cavity, external surfaces of the brain and spinal cord, cervical tissues and organs, thoracic, abdominal and pelvic cavities and viscera, and external surface of the body), and all lesions were recorded and retained in 10% neutral buffered formalin. Macroscopic examinations were performed under the general supervision of a pathologist.
The brain from all animals was weighed and then retained in 10% neutral buffered formalin, prior to fixed tissue dissection to remove tissues .


Tissue List for Cohort 2A
Olfactory bulb [H&E*; MSSa*], Forebrain (including hippocampus [H&E*; MSSa*], Basal ganglia (including caudate nucleus) [H&E*; MSSa*], Hypothalamus/thalamus [H&E*; MSSa*], Midbrain (including tectum, tegmentum and cerebral peduncles) [H&E*; MSSa*], Brain stem (including pons and medulla oblongata) [H&E*; MSSa*], Cerebellum [H&E*; MSSa*], Cerebral cortex [H&E*; MSSa*], Macroscopic lesions [H&E*]
H&E = Hematoxylin and eosin; MSS = Myelin and Silver Stain
a Luxol fast blue and Cresyl violet stains
The tissue listed were collected from all animals
The animal identification was also preserved

Histology and Microscopic Examinations
Those tissues indicated by * in Text were trimmed, processed, and embedded in paraffin wax (all animals); however, neuropathological evaluation by H&E was performed on Groups 1 and 4 only.

Statistics:

See below under "any other information"

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Nonadverse, test article related clinical observations were confined to instances of salivation noted following administration of 100 mg/kg/day, with one instance also noted for one male administered 25 mg/kg/day. This was considered attributable to test article palatability and was not a toxic effect of the test article.
Instances of increased salivation were noted for 11 of 25 females administered 100 mg/kg/day. Isolated instances of increased salivation were noted for two males administered 100 mg/kg/day (Animal R0306 on Pre-Pairing Day 71 and Pairing Days 1 and 2; Animal R0321 on Pre-Pairing Days 57 and 60 to 65) and one male administered 25 mg/kg/day on Pre-Pairing Day 57 (Animal R0225). Such observations are occasionally observed following gavage administration of a slightly irritant or unpalatable test article formulation; as such, this finding was considered not to have represented toxicity.
Remaining clinical observations were generally associated with pelage changes or skin lesions which were evident throughout groups, with no dose relationship and are commonly observed in studies of this type. These were not related to the test article.
No observations were recorded postdose or during the weekly detailed clinical examinations.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Animal Fate
No test article-related unscheduled deaths occurred.
A total of seven unscheduled deaths occurred in the F0 generation. The cause of demise for these decedents was not attributed to the test article, and the findings noted in these animals were generally similar to those observed for animals that survived to the scheduled sacrifice.
One control female (Animal R0409) was sacrificed on Pre-Pairing Day 17 following clinical observations of labored and audible respiration. Macroscopic examinations revealed abnormal contents in the thoracic cavity and an esophageal rupture, which were consistent with gavage dosing trauma.
One female administered 25 mg/kg/day (Animal R0602) was sacrificed on Pre Pairing Day 21 following clinical observations of hunched posture, labored respiration, and decreased activity. Macroscopic findings included abnormal contents in the oral cavity, which were considered procedure related and not a consequence of the test article.
One male administered 25 mg/kg/day (Animal R0202) was sacrificed on Post-Pairing Day 24 following whole body convulsions that lasted less than 1 minute. The whole body convulsions were also observed on Post-Pairing Day 11. The animal was, therefore, sacrificed for humane reasons in accordance with test facility welfare standards. In isolation at this dose level, this was not test article related. One female administered 5 mg/kg/day (Animal R0522) was sacrificed on LD 19 following a whole body convulsion that lasted more than 1 minute. The animal was, therefore, sacrificed for humane reasons in accordance with test facility welfare standards. In the absence of a dose response, the isolated instances in these dose groups, was considered unrelated to the test article.
One female administered 5 mg/kg/day (Animal R0516) and one female administered 25 mg/kg/day (Animal R0609) were sacrificed on LD 0 following clinical observations of dystocia (difficulty in parturition). Macroscopic findings for Animal R0516 included abnormal contents in the stomach, with appearance of placental/pup tissue. The two incidences in two groups were isolated, and dystocia is occasionally observed in studies of this type and the finding was not observed in the high dose group; as such, in the absence of a dose response, these findings were considered incidental in nature, with no relationship to the test article.
One female administered 100 mg/kg/day (Animal R0715) was sacrificed on LD 1 following total postnatal litter loss. This is occasionally observed in studies of this type, and in isolation, was unrelated to the test article.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test article-related effects on mean body weight or body weight gain were noted.
The occasional statistically significant intergroup differences in mean body weight gains did not show any trends and were inconsistent across phases and sexes; as such, they were considered to have arisen incidentally, with no relationship to the test article.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test article-related effect on food consumption was observed.
The occasional statistically significant intergroup differences did not show any trends and were inconsistent across phases and sexes; as such, they were considered to have arisen incidentally, with no relationship to the test article.

Food efficiency:

no effects observed

Description (incidence and severity):

No overt differences in food conversion efficiency were evident for test article-treated animals, compared with controls.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related hematology changes were noted at any dose level.
White blood cells, specifically in the neutrophil, lymphocyte fractions, were marginally higher for all test article treated male groups, compared with controls; however, in the absence of a convincing dose response or the absence of similar changes noted for females, these intergroup differences were considered to have arisen incidentally and were not associated with the test article.
Females administered 100 mg/kg/day exhibited increased red cell distribution width and decreased packed cell volume (P< 0.05), compared with controls. Remaining red cell parameters were unaffected, and a similar change was not evident for males. In the absence of a clear hematopoietic effect, these intergroup differences were considered to have arisen incidentally and were not test article related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related clinical chemistry changes were noted at any dose level.
Total protein was significantly increased for males administered 100 mg/kg/day, compared with controls (P< 0.05). In the absence of any supporting data or similar changes noted for females, this was considered to have arisen incidentally, with no association with the test article.
Females administered 100 mg/kg/day were noted with decreased glucose levels on LD 22, compared with controls (P < 0.05). A similar change was not evident for F0 males. In the absence of any supporting data to suggest an effect of the test article, this was considered to have arisen incidentally.

Thyroid Hormone Analysis
No test article-related changes in T4 or TSH were noted at any dose level, compared with controls.
The statistically significant increase in TSH was observed for males administered 25 mg/kg/day, compared with controls, but was not supported by a concurrent and expected reduction in T4 levels, and similar changes were not evident for females. Furthermore, no thyroid weight changes were noted at this dose level, and, as such, these were considered to have arisen incidentally.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test article-related changes were noted in the urine parameters investigated.
Statistical analysis of the quantitative data did not show any statistically significant intergroup differences.

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 adrenal glands, ovary, thyroid glands, liver, spleen, and kidneys.
An increased incidence and severity of diffuse zona glomerulosa vacuolation was recorded for the adrenal glands of F0 animals from all test article-treated groups, compared with controls. A similar increased incidence and severity of diffuse zona fasciculata vacuolation was recorded for both sexes administered 25 or 100 mg/kg/day. Zona glomerulosa vacuolation was characterized by cells within this zone having an increased number and/or size of large, pale, cytoplasmic vacuoles, which increased the depth of this zone at a moderate grade. Zona fasciculata vacuolation was characterized at lower grades by cells within this zone with increased numbers of pale, cytoplasmic vacuoles and at higher grades by expansion of the zone by cells with large, pale, cytoplasmic vacuoles and/or large, clear vacuoles distorting the border with the zona reticularis. These findings generally correlated with the organ weight changes and macroscopic finding of large adrenal glands.
Slight to moderate ovarian interstitial cell vacuolation was recorded for all F0 females administered 25 or 100 mg/kg/day and was characterized by interstitial cells with moderate to large, pale, cytoplasmic vacuoles. This finding generally correlated with the changes in organ weights and organ weight ratios for females administered 100 mg/kg/day.
Follicular cell hypertrophy was recorded for the thyroid glands of males administered 5 mg/kg/day and both sexes administered 25 or 100 mg/kg/day (more notably in F0 males). Follicular cell hypertrophy was characterized by thyroid follicular cells with increased amounts of pale cytoplasm, without any associated microscopic changes in colloid production. This finding generally correlated with the changes in organ weights and organ weight ratios and the macroscopic finding of large thyroids.
In addition, cystic follicular cell hyperplasia was recorded for the thyroid gland from one male (Animal R0118) and one female (Animal R0723), administered 5 and 100 mg/kg/day, respectively, and a benign follicular cell adenoma was recorded for one F0 male administered 100 mg/kg/day (Animal R0309).
Centrilobular hepatocellular hypertrophy/eosinophilia was recorded for some F0 animals administered 100 mg/kg/day. This finding was characterized by centrilobular hepatocytes with increased amounts of eosinophilic cytoplasm, occasionally causing an increase in cellular size. This finding generally correlated with the changes in organ weights and organ weight ratios and the macroscopic finding of large liver.
An increase in the incidence and severity of pigmented macrophages was recorded for the spleen of males administered 25 or 100 mg/kg/day, compared with controls, and was characterized by macrophages with increased amounts of yellow-brown, granular pigment within the red pulp. Pigmented macrophages were noted in spleens from F0 males. This finding may be observed as a background change in rats. It was only observed in one sex in the F0 generation; as such, it was considered of unlikely toxicological significance, and, therefore, considered nonadverse.
An increased severity of renal hyaline droplet accumulation was recorded for F0 males administered 100 mg/kg/day, compared with controls. Hyaline droplet accumulations were characterized by renal cortical tubular cells with small to moderate amounts of granular, eosinophilic, cytoplasmic material, without any associated changes in cellular structure, tubular architecture, or inflammation. This finding generally correlated with the macroscopic finding of pale kidney.
No other test article-related microscopic findings were recorded for F0 animals.
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, age, and at this stage of reproduction.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles were unaffected by the test article at any dose level prior to pairing or prior to scheduled sacrifice on Lactation Day 22.
Statistical analysis of the data did not show any statistically significant intergroup differences.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects were noted on sperm analysis or sperm morphology parameters at any dose level.
Straightness of sperm was significantly lower for males administered 100 mg/kg/day, compared with control males (P < 0.001), with mean values outside the HCD range (65 to 73%). Straight line velocity was also reduced by 11%, compared with controls, although mean values did not attain statistical significance and were within the HCD range (85.4 to 118.2 um/sec). These reductions were due to two zero (0) values for Animals R0302 and R0310) and the mated female partners achieved a pregnancy, therefore, and as such, did not represent an effect of the test article.
Caudal epididymis weights used for sperm analysis were statistically significantly lower than those of controls (P < 0.01), although mean values were within the HCD range (0.224 to 0.270 g). Individual data across groups showed some values that were higher and lower than the HCD range; five control values were lower than the HCD and six control values were higher than the HCD range, which may have resulted in the statistically significant intergroup differences. In the absence of any supporting testes or epididymis weight changes at the terminal sacrifice, this change in caudal weights was considered to have arisen incidentally and unrelated to the test article.
Total abnormal sperm counts were increased in a dose-related manner for all male groups, compared with controls, although a large standard deviation was evident and resulted in a lack of statistical significance in the group administered 100 mg/kg/day. Statistically significant increases in percentage abnormal sperm were evident following administration of 5 or 25 mg/kg/day, compared with controls (P < 0.01 or P < 0.001, respectively). The percentage change in the group administered 100 mg/kg/day was also increased in a dose related manner, although the large standard deviation resulted in a lack of statistical significance at this dose level. The number of normal sperm was unaffected at any dose level; as such, these intergroup differences were considered to have arisen incidentally.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A test article-related reduction in fertility was evident following administration of 100 mg/kg/day, compared with controls. Fertility index was reduced, compared with controls, and was slightly lower than the historical control data (HCD) range (85 to 100%), although values did not attain statistical significance, compared with controls.
Following mating, 23, 23, 23, and 21 females administered control article (vehicle) or 5, 25, or 100 mg/kg/day, respectively, were pregnant.
No test article-related effects on mating performance were noted. The majority of animals mated within 4 days of pairing.
The lower fertility index observed following administration of 100 mg/kg/day, resulted in a lower fecundity index, compared with controls, although the value was within the HCD range (85 to 100%); as such, this was considered not to have represented an effect of the test article. Of the four non-pregnant females from the group administered 100 mg/kg/day, Animals R0718, R0721, and R0722 mated on the day of pairing. Animal R0718 was in estrus from the previous night, therefore it was considered likely that a decaying estrus was the cause of the lack of pregnancy following mating and unrelated to the test article. Animal R0716 mated on the second day of pairing. One, two, and one non-pregnant females, respectively, were in the groups administered control article (vehicle) or 5 or 25 mg/kg/day.

Parturition and Litter Data
Following parturition, 23, 22, 22, and 21 females administered control article (vehicle) or 5, 25, or 100 mg/kg/day, respectively, produced a live litter.
Adverse test article-related increased numbers of stillborn pups were noted following administration of 100 mg/kg/day compared with controls. However, there was no effect of the test article on implantation losses in utero or on pup survival after PND 1 until weaning; values were similar to controls. A total of 14 stillborn pups were observed in litters from the group administered 100 mg/kg/day, compared with controls. Although values did not attain statistical significance, the number of stillborn pups was outside the HCD range (0 to 6 pups). These stillborn pups were from five litters of the group administered 100 mg/kg/day; this number was also outside the HCD range (0 to 4 litters).
Two stillborn pups from one control article (vehicle) litter, seven stillborn pups from two 5 mg/kg/day litters and five stillborn pups from two 25 mg/kg/day litters were observed. Although a dose response was evident following administration of 25 mg/kg/day, these numbers were within the historical control data ranges, and, as such, were considered to have arisen incidentally.
No effect on the duration of gestation was noted. Two females (Animal R0516 administered 5 mg/kg/day and Animal R0609 administered 25 mg/kg/day), were sacrificed early due to clinical observations of dystocia, and the number of pups born was small, however at necropsy, no fetuses were found in utero. In the absence of a dose response and no increase in the duration of gestation, these findings were considered related to difficulties during parturition for these females and unrelated to test article administration.
The increased incidence of stillborn pups, resulted in a lower live birth index for litters of the group administered 100 mg/kg/day, compared with controls, although mean values were within the HCD range (92.9 to 100%).

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 Generation Offspring Observations (Pre-weaning)
Pup Clinical Observations
No test article-related clinical observations were noted for pups from all groups.
The incidence of missing pups was slightly higher in litters of the group administered 100 mg/kg/day, compared with controls (four versus one), although this was within the HCD range (0 to 7 pups) and, as such, was considered incidental.
No postdose observations were noted following direct dosing from PND 14.

F1 Generation
No postdose observations were noted.
No test article-related clinical observations or weekly detailed observations were noted.
Excessive salivation was observed during the maturation phase for two females administered 100 mg/kg/day (Cohort 1A Animal R1502 on Maturation Day 77, Animal R1503 on Maturation Day 59). These were isolated incidences and, taking into the consideration the duration of the dosing phase, were considered to have arisen incidentally.
Detailed weekly clinical observations showed isolated incidences of high stepping gait, generally for females, with incidences also noted for controls. This was evident on occasion during the maturation, pairing, and gestation phases. Isolated incidences of elevated tail were noted for a few animals in each group, including controls. In the absence of a convincing dose relationship, these findings were considered transient in nature as they were not evident on each weekly testing occasion; as such, no relationship to the test article was noted.

F2 Pup
No test article-related clinical observations were noted for pups at any dose level.
The minor observations noted were considered low incidence findings, and, as such, were considered to have arisen incidentally.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F1 Generation Offspring Observations (Pre-weaning)
No test article-related effect on postnatal pup survival was observed.

F1 Generation
No test article-related unscheduled deaths occurred.
One Cohort 1A male administered 100 mg/kg/day (Animal R1103) was found dead on PND 22. A cause of demise was not determined for this animal as it was not examined microscopically; however, the macroscopic finding was consistent with findings in rats of this strain and age and, in isolation, this death was considered to have arisen incidentally, with no relationship to the test article.
One Cohort 1B male administered 5 mg/kg/day (Animal R0928) was sacrificed on PND 71 following a convulsion that lasted more than 1 minute. The cause of demise was not confirmed as it was not examined microscopically. Another Cohort 1B male administered 5 mg/kg/day (Animal R0931) was sacrificed on Pairing Day 14. This was considered attributable to a gavage dosing error, as confirmed by esophageal rupture of the lung and fluid present in the thoracic cavity upon macroscopic examination.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 Generation Offspring Observations (Pre-weaning)
No test article-related effects on pup body weights were noted at any dose level, compared with controls.
Statistical analysis of the data did not show any statistically significant intergroup differences.

F1 Generation
No test article-related effects on body weights or body weight gains were observed.
Mean body weights on PND 22 were essentially similar across groups and remained similar throughout the dosing period.
Mean body weights were essentially similar to control for females during gestation; however, mean body weight gain was higher for all test article-treated groups over the lactation phase (LD 1 to 4), and a dose relationship was evident. However, this was most likely attributable to variation within the control group, as evidenced by a larger standard deviation value, compared with remaining groups.

F2 Pup
No test article-related effect on pup body weights was evident on PND 1 and 4.
Statistical analysis of the data did not reveal any significant intergroup differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F1 Generation
No test article-related effects on food consumption were observed at any dose level.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.

Food efficiency:

no effects observed

Description (incidence and severity):

No overt differences in food conversion efficiency were evident for test article-treated animals, compared with controls.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 Generation
No test article-related hematology changes were noted.
Statistically significant increases in white cell counts were evident for females at all dose levels and were supported by statistically significant increases in lymphocyte counts for females administered 25 or 100 mg/kg/day, compared with controls, and a similar trend was evident for males. A similar pattern was evident following the immunophenotyping from the spleen. However, a dose response was not evident, and in the absence of any microscopic correlates, these increases were considered not to have represented an immune response and were, therefore, nonadverse in nature.
Females administered 100 mg/kg/day were noted with an increase in fibrinogen, compared with controls (P < 0.05). This was an isolated finding and, in the absence of similar changes for males, was considered to have arisen incidentally.
Females administered 25 or 100 mg/kg/day were noted with a dose-related increase in percentage red cell distribution width (RDWG %), compared with controls (P < 0.05). In the absence of any supporting changes in red cell mass, and because similar changes not observed for the males, these intergroup differences were considered to have arisen incidentally and were unrelated to the test article.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

F1 Generation Offspring Observations (Pre-weaning)
No test article-related changes in T4 or TSH were evident following maternal or direct administration of the test article at any dose level, compared with controls.
Statistical analysis of the data did not show any statistically significant intergroup differences. Marginal increases in TSH were not accompanied by a reduction in T4 levels, and were, therefore, considered to have arisen incidentally.

F1 Generation
Test article-related increases in globulin levels were noted for animals administered 100 mg/kg/day and males administered 25 mg/kg/day and increases in cholesterol levels were noted for females administered 100 mg/kg/day, compared with controls.
Total protein levels were elevated for females administered 100 mg/kg/day (P < 0.05), characterized by an increase in globulin level (P < 0.01). A:G ratio was consequently reduced (P < 0.01), compared with controls.
Globulin levels were increased for males administered 25 or 100 mg/kg/day (P < 0.01 or P < 0.05, respectively), and A:G ratio was reduced, although statistical significance was only attained following administration of 25 mg/kg/day, with no associated increase in total protein. These were considered to be nonadverse, test article-related changes.
Cholesterol levels were increased for females administered 100 mg/kg/day, compared with controls (P < 0.01). A similar change was evident in a previous 90-day toxicity study (MPI Research Study 399-241; MPI Research, 2015), and, as such, was considered test article related.
Reduced glucose levels were evident for males administered 100 mg/kg/day, compared with controls (P< 0.01). This was not evident for F1 females; as such, it was considered to have arisen incidentally.

F1 Generation: Thyroid Hormone Analysis
No test article-related TSH and T4 changes were noted at any dose level.
The statistically significant increase in TSH for males administered 25 mg/kg/day did not show an expected reduction in T4 levels. Furthermore, a similar change was not evident for females or at the high dose level and was not supported by any thyroid weight changes; as such, this was considered to have arisen incidentally, with no relationship to the test article.

Urinalysis findings:

no effects observed

Description (incidence and severity):

F1 Generation
No test article-related changes were evident in urine parameters measured.
Statistical analysis of the quantitative data did not show any statistically significant intergroup differences.

Sexual maturation:

no effects observed

Description (incidence and severity):

No test article-related effects on age of sexual maturity or body weights at attainment, were evident at any dose level.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

F1 Generation Offspring Observations (Pre-weaning)
No test article related effects on anogenital distance were noted for males or females from all test article-exposed litters, compared with controls.
Statistical analysis of the data did not show any statistically significant intergroup differences.

F2 Pup
No test article-related effects were evident on anogenital distance.
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):

F1 Generation Offspring Observations (Pre-weaning)
Nipple retention was not observed for any male at any dose level.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 Generation Offspring Observations (Pre-weaning)
No test article-related organ weight changes were recorded for PND 22 offspring from F0 animals, compared with controls.
Organ weight and organ weight ratio changes for these animals were attributed to biological variation.

F2 Pup
No changes in kidney weight or kidney:body weight ratio were noted for F2 offspring from test article-treated F1 animals.
Changes in organ weights and organ weight ratios for these animals were attributed to biological variation.

F2 Pup
Test article-related changes in organ weights and organ weight ratios were recorded for the adrenal glands and liver of Cohort 1A animals. Test article-related ovary weight changes were recorded for Cohort 1A and 1B females.
Increased adrenal weights and/or adrenal weight ratios and liver weights and/or liver weight ratios were recorded for both sexes administered 100 mg/kg/day, compared with concurrent controls. Increased ovarian weights and ovarian weight ratios were recorded for F1 Cohort 1A females administered 100 mg/kg/day, compared with concurrent controls. The observed changes generally correlated with findings noted macroscopically and/or microscopically.
Test article-related increases in ovarian weights and ovarian weight ratios were recorded for F1 Cohort 1B females administered 25 or 100 mg/kg/day, compared with concurrent controls, which generally correlated with microscopic findings .
All other organ weight and organ weight ratio changes noted for Cohort 1A and 1B animals were attributed to normal 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.
No test article-related brain weight changes were recorded for Cohort 2A and 2B animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 Generation Offspring Observations (Pre-weaning)
No test article-related macroscopic abnormalities were noted for any tissues on PND 22.
An increase in the number of stillborn pups was noted for F1 offspring (more notable in female pups) from F0 females administered 100 mg/kg/day, compared with controls. However, no consistent, associated macroscopic findings were noted to suggest an underlying cause.
Most tissues in pups that died or were sacrificed between PND 0 and 22 were macroscopically unremarkable, and/or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age and at this stage of development.

F2 Pup
No test article-related macroscopic findings were recorded for F2 offspring from test article-treated F1 animals.
Most tissues in pups that died or were sacrificed between PND 0 and 4 were macroscopically unremarkable and/or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age and at this stage of development.

Test article-related macroscopic findings were noted in the adrenal glands, liver, and ovaries of Cohort 1A animals.
Large adrenal glands and large ovaries were noted in F1 Cohort 1A females administered 100 mg/kg/day, which generally correlated with increased organ weights/weight ratios and microscopic findings.
Large liver was noted for F1 Cohort 1A males administered 100 mg/kg/day, which also generally correlated with increased organ weights/weight ratios and microscopic findings.
No other test article-related macroscopic findings were recorded.
Other tissues from Cohort 1A animals were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
No test article-related macroscopic findings were recorded for Cohort 1B animals. Tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain, age, and at this stage of reproduction.
No test article-related macroscopic findings were recorded for Cohort 2A or 2B animals. Most 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):

Test article-related microscopic findings were recorded for the adrenal glands, thyroid glands, and liver of Cohort 1A animals. Test article-related microscopic changes were also recorded for the ovaries of F1 Cohort 1A and 1B females.
An increased incidence and severity of diffuse zona glomerulosa vacuolation of the adrenal gland was recorded for F1 Cohort 1A animals administered 25 or 100 mg/kg/day, compared with controls. A similar increased incidence and severity of diffuse zona fasciculata vacuolation was recorded for F1 Cohort 1A animals from all test article-treated groups. Zona glomerulosa vacuolation was characterized by cells within this zone having an increased number and/or size of large, pale, cytoplasmic vacuoles, which increased the depth of this zone at a moderate grade. Zona fasciculata vacuolation was characterized at lower grades by cells within this zone with increased numbers of pale, cytoplasmic vacuoles and at higher grades by expansion of the zone by cells with large, pale, cytoplasmic vacuoles and/or large, clear vacuoles distorting the border with the zona reticularis. These findings generally correlated with the organ weight changes and macroscopic findings.
An increased incidence and severity of follicular cell hypertrophy was recorded for the thyroid glands of males administered 25 mg/kg/day and both sexes administered 100 mg/kg/day, more notably in F1 Cohort 1A males. Follicular cell hypertrophy was characterized by thyroid follicular cells, with increased amounts of pale cytoplasm, without any associated microscopic changes in colloid production.
Centrilobular hepatocellular hypertrophy/eosinophilia was recorded for both sexes administered 100 mg/kg/day. Animals administered 5 or 25 mg/kg/day were generally comparable with controls. This finding was characterized by centrilobular hepatocytes with increased amounts of eosinophilic cytoplasm, occasionally causing an increase in cellular size.
Slight to marked ovarian interstitial cell vacuolation was recorded for all F1 Cohort 1A females administered 25 or 100 mg/kg/day. Interstitial cell vacuolation was characterized by ovarian interstitial cells with moderate to large, pale, cytoplasmic vacuoles and generally correlated with the organ weight changes and macroscopic finding of large ovaries.
No other test article-related microscopic findings were recorded for F1 Cohort 1A animals. 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, age, and at this stage of development.
Minimal to marked ovarian interstitial cell vacuolation was noted for all terminal sacrifice F1 Cohort 1B females administered 25 or 100 mg/kg/day, which generally correlated with the observed organ weight and organ weight ratio changes recorded for this cohort.
No other tissues were examined from terminal sacrifice F1 Cohort 1B females.
In addition, moderate interstitial cell vacuolation was noted for the ovaries of one F1 Cohort 1B female administered 25 mg/kg/day, and slight to moderate interstitial cell vacuolation was noted in the ovaries from three F1 Cohort 1B females administered 100 mg/kg/day, which were examined due to failure to conceive. This finding generally correlated with the organ weight and weight ratio changes for these groups.
No other test article-related microscopic findings were recorded for the limited tissue list examined in F1 Cohort 1B females that did not conceive, as microscopic findings in the examined reproductive tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain, age, and at this stage of reproduction.
No test article-related microscopic findings were recorded for Cohort 2A and 2B animals. Microscopic findings 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, treatment-related

Description (incidence and severity):

F1 Generation
Estrous Cycles
No test article-related effects on the number or duration of the estrous cycles were evident.
The statistically significant increase in mean cycle length for females administered 25 mg/kg/day (P < 0.01) did not show an expected reduction in the number of cycles, and in the absence of a dose response, was considered to have arisen incidentally.

Fertility and Reproductive Performance
No test article-related effects on mating were evident at any dose level.
All animals mated, with the majority of females within each dose group mating within the first 6 days of pairing.
Fertility and fecundity indices were slightly lower following administration of 100 mg/kg/day, compared with controls. Seventeen of 20 females administered 100 mg/kg/day were pregnant, compared with 19 of 20 from each of the remaining groups. This resulted in lower fecundity and fertility indices at this dose level, which were just within the HCD range for both indices (85 to 100%).
Three non-pregnant females were noted in the group administered 100 mg/kg/day (Animals R1530, R1533, and R1535), compared with one each from the remaining groups. Animals R1530 and R1535 mated on the day of pairing and were in estrus from the previous night, therefore it was considered likely that a decaying estrus was the cause of the lack of pregnancy following mating for these animals, and unrelated to the test article. One female administered 25 mg/kg/day (Animal R1440) mated on Pairing Day 9. The remaining non-pregnant animals did not mate on the first day of pairing and were not in estrous on the day of mating; as such, the slightly higher non pregnancy rate in the group administered 100 mg/kg/day was considered to have arisen incidentally and was not related to the test article as non-pregnancies are occasionally observed in studies of this type.
One control female (Animal R1239) was pregnant but had a total in utero litter loss, which is occasionally observed in studies of this type.

Parturition and Litter Data
Parturition, gestation length, and gestation index were unaffected.
Live birth index and prenatal and postnatal pup survival were unaffected. No stillborn pups were evident following administration of 5 or 100 mg/kg/day. Four stillborn pups were evident from two control litters and two stillborn pups observed from two litters from the group administered 25 mg/kg/day. Pup mortality (missing or dead) was limited to four, one, two, or four pups from groups administered control article (vehicle) or 5, 25, or 100 mg/kg/day, respectively; therefore, the PND 4 Viability index was similar between control and the group administered 100 mg/kg/day. One control female (Animal R1239) was sent to necropsy on GD 24. This animal had an in utero total litter loss. No total postnatal litter losses were evident at any dose level.
Litter size and sex ratio were unaffected by the test article.
No test article-related effect on the number of implantation sites was evident. Post implantation loss was higher following administration of 100 mg/kg/day, compared with controls (mean number 1.71 and 14.16% for the group administered 100 mg/kg/day, compared with 0.94 or 8.57% for controls), although these values were within the historical control data range (0.23 to 2.67 implantations losses; 1.7 to 19.83 post implantation loss) and, as such, were considered to have arisen incidentally.
A total of 18, 19, 19, or 17 females administered control article (vehicle) or 5, 25, or 100 mg/kg/day, respectively, survived to their scheduled sacrifice.

F1 Generation
Seminology Evaluations
No test article-related changes in sperm analysis or sperm morphology parameters were observed at any dose level.
A statistically significant reduction in sperm motility was evident for males administered 100 mg/kg/day, compared with control (P < 0.05), although mean values were within the historical control data range (66 to 93%), which was attributable to seven lower than expected values at this dose level, compared with three low and two high values from the control group. In the absence of any supporting changes to suggest an effect on sperm, this intergroup difference was considered to have arisen incidentally and was not associated with the test article.

Ovarian Follicle Count Evaluations
Test article-related reductions in ovarian follicle counts, primarily primordial follicles, were evident following administration of 25 or 100 mg/kg/day, compared with controls.
Small reductions in primordial ovarian follicles were evident for F1 females administered 25 or 100 mg/kg/day, compared with controls, although a dose relationship was not evident. This resulted in statistically significant reductions in total follicle counts (P < 0.05) following administration of 25 or 100 mg/kg/day, compared with controls.
Small reductions in primordial ovarian follicles were evident following administration of 5 mg/kg/day, compared with controls, although statistical significance was not attained for the overall ovarian counts, and as such, this was considered to be incidental and unrelated to the test article.

Brain Histomorphometry Evaluations
No test article-related changes in brain histomorphometry were noted on PND 22 (Cohort 2B) or PND 76 (Cohort 2A) for animals administered 100 mg/kg/day, compared with controls.
The statistically significant changes in some histomorphometry measurements between controls and animals administered 100 mg/kg/day generally affected different brain regions and were inconsistent between sexes and between F1 Cohort 2A and Cohort 2B animals. Outliers within the data were noted and were probably due to slightly oblique or damaged sections. Therefore, in the absence of behavioral or clinical neurological observations and no corresponding macroscopic or microscopic findings, these changes were considered to have arisen incidentally, with no relationship to the test article.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

F2 Pup
Functional Observational Battery
No test article-related effects were observed following the FOB assessments.
Statistical analysis of the quantitative data did not reveal any statistically significant intergroup differences.

Motor Activity
No test article-related effects on locomotor activity parameters were evident.
The statistically significant intergroup differences detected for males, females, and combined sex throughout groups, compared with controls, did not show any dose relationship, and, in the absence of any supporting data to suggest a neurotoxic effect of the test article, were considered to have arisen incidentally.

Acoustic Startle
No test article-related deficits in pre-pulse inhibition or startle habituation were noted, compared to controls.
The statistically significant increase in startle habituation in 1 of 5 blocks (Block 3) for males administered 5 mg/kg/day, compared with controls, was minimal (P< 0.05) and were not part of a dose response; as such, the increase was considered to have arisen incidentally

Developmental immunotoxicity:

effects observed, treatment-related

Description (incidence and severity):

F2 Pup
An immunotoxic response was not evident at any dose level.
Test article-related increases in T lymphocytes, Helper T lymphocytes, Cytotoxic T lymphocytes, and B lymphocyte populations were evident at all dose levels. These increases, however, did not show a dose response and, at the magnitude observed, were considered not immunotoxic and, therefore, nonadverse in nature.
In F1 Cohort 1A animals administered 5, 25, or 100 mg/kg/day, total T lymphocytes (CD3+) and Helper T lymphocytes (CD3+CD4+) had slight to marked increases in absolute numbers on Maturation Days 92, 3, 4, or 5, compared to controls. The latter effects were not consistently reflected within the relative frequencies or were dose dependent in nature. A similar level of a test article-related increase was also observed in the Cytotoxic T lymphocyte population (CD3+CD8+), compared to controls, and the highest increase in absolute numbers was noted in F1 Cohort 1A animals administered 100 mg/kg/day, although a dose-dependent effect was observed for this population in males only.
The spleen natural killer (NK) lymphocytes (CD3-CD161a+) of Reofos 35-treated F1 Cohort 1A animals were generally comparable to those of controls. Moderate increases in absolute NK numbers (and slight increases in relative frequencies) were observed in F1 Cohort 1A females administered 5 mg/kg/day and were associated with high inter-animal variability and considered not biologically significant.
Splenic B lymphocyte (CD45RA+) absolute numbers (and, for the most part, relative frequencies) were slightly to moderately increased in F1 Cohort 1A males administered >5 mg/kg/day, compared to controls. Similarly, B lymphocyte absolute numbers and relative frequencies typically increased to a slight extent in F1 Cohort 1A females administered >5 mg/kg/day, compared to controls, with the exception of B lymphocyte numbers in F1 Cohort 1A females administered 25 mg/kg/day, which were comparable to those of controls. Aforementioned differences in B lymphocyte populations of Reofos 35-treated groups and controls were independent of dose.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

developmental immunotoxicity

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

F0 Generation

Summary incidence of the stage of estrus on Lactation Day 22

	Diestrus	Estrus	Proestrus
Control	19	4	0
5 mg/kg/day	16	3	2
25 mg/kg/day	20	0	1
100 mg/kg/day	18	1	1

 

Summary of Organ Weight % Difference Compared with Concurrent Controls – Terminal Sacrifice

		Males			Females
		2M	3M	4M	2F	3F	4F
Tissue	Level (mg/kg/day)	5	25	100	5	25	100
		% difference
Adrenal	Group mean absolute weights	0.147	8.516	16.630***	-1.741	13.564*	32.485***
	Body weight ratio	-1.475	8.055	17.507***	1.740	19.127**	33.431***
Thyroid/parathyroid	Group mean absolute weights	8.830	7.161	22.382***	-1.177	-8.101	-3.836
	Body weight ratio	6.418	6.293	23.222***	1.331	-4.480	-3.608
Liver	Group mean absolute weights	3.863	7.797**	19.194***	-2.322	-2.129	1.316
	Body weight ratio	1.461	7.353**	19.622***	0.374	2.232	2.042
Kidney	Group mean absolute weights	2.162	2.914	6.837	-2.076	-2.905	2.522
	Body weight ratio	-0.202	2.236	6.919*	1.045	1.581	3.717
Ovary	Group mean absolute weights	-	-	-	-7.985	1.689	34.323***
	Body weight ratio	-	-	-	-5.332	5.884	34.881***
F = Female; M = Male * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001 Note: Organ weights were not taken from decedents or excluded animals, based on reproductive status

 

Incidence of Selected Findings: Adrenal Gland – Terminal Sacrifice

		Males				Females
		1M	2M	3M	4M	1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100	0	5	25	100
Adrenal gland	No. examined	25	25	24	25	23	21	21	20
Vacuolation, zona glomerulose, increased, diffuse	Grade -	20	11	2	2	19	9	0	0
	1	5	13	10	5	4	9	5	0
	2	0	1	12	15	0	3	12	12
	3	0	0	0	3	0	0	4	8
Vacuolation, zona fasciculata, increased diffuse	Grade-	16	11	1	0	23	21	4	0
	1	9	13	9	4	0	0	11	6
	2	0	1	12	7	0	0	6	10
	3	0	0	2	12	0	0	0	4
	4	0	0	0	2	0	0	0	0
- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; 4 = Marked; F = Female; M = Male

 

Incidence of Selected Findings: Ovary – Terminal Sacrifice

		Females
		1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100
Ovary	No. examined	23	21	21	20
Vacuolation, interstitial cell	Grade -	23	21	1	0
	1	0	0	5	0
	2	0	0	12	6
	3	0	0	3	14
- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; 4 = Marked; F = Female

 

Incidence of Selected Findings: Thyroid Gland – Terminal Sacrifice

		Males				Females
		1M	2M	3M	4M	1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100	0	5	25	100
Thyroid Gland	No. examined	25	25	24	25	23	21	21	20
Hypertrophy, follicular cell	Grade -	25	19	6	5	23	21	16	15
	1	0	5	16	6	0	0	5	4
	2	0	1	2	14	0	0	0	1
- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male

 

Incidence of Selected Findings: Liver – Terminal Sacrifice

		Males				Females
		1M	2M	3M	4M	1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100	0	5	25	100
Liver	No. examined	25	25	24	25	23	21	21	20
Hypertrophy/eosinophilia, hepatocyte, centrilobular	Grade -	25	25	24	19	22	21	20	12
	1	0	0	0	3	0	0	1	4
	2	0	0	0	3	1	0	0	4
- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male

 

Incidence of Selected Findings: Spleen – Terminal Sacrifice

		Males				Females
		1M	2M	3M	4M	1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100	0	5	25	100
Spleen	No. examined	25	25	24	25	23	21	21	20
Pigmented macrophage, increased	Grade -	5	6	1	0	1	5	2	1
	1	14	13	7	7	13	8	9	8
	2	6	5	15	13	9	7	8	9
	3	0	1	1	5	0	1	2	2
- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; F = Female; M = Male

 

Incidence of Selected Findings: Kidney – Terminal Sacrifice

		Males				Females
		1M	2M	3M	4M	1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100	0	5	25	100
Kidney	No. examined	25	25	24	25	23	0	0	20
Accumulation, hyaline droplet	Grade -	0	1	0	0	23			1
	1	20	24	23	4	0			0
	2	5	0	1	21	0			0
- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male

 

Summary of Organ Weight % Difference Compared with Concurrent Controls – Terminal Sacrifice (Cohort 1A)

		Males			Females
		2M	3M	4M	2F	3F	4F
Tissue	Level (mg/kg/day)	5	25	100	5	25	100
		% difference
Adrenal	Group mean unadjusted weights	2	1	8	-8	5	24***
	Body weight ratio	2	3	12*	-6	7	28***
	Brain weight ratio	3	3	8	-7	7	25***
Liver	Group mean unadjusted weights	2	6	15***	-3	0	10
	Body weight ratio	2	8	20***	0	3	13***
	Brain weight ratio	3	7	15***	-1	3	11*
Ovary	Group mean unadjusted weights	-	-	-	14	7	28***
	Body weight ratio	-	-	-	18	10	32***
	Brian weight ratio	-	-	-	15	10	29***
F = Female; M = Male * = P ≤ 0.05; *** = P ≤ 0.001 Note: Organ weights were not taken from decedents.

 

Summary of Organ Weight % Difference Compared with Concurrent Controls – Terminal Sacrifice (Cohort 1B)

		Females
		2F	3F	4F
Tissue	Level (mg/kg/day)	5	25	100
		% difference
Ovary	Group mean unadjusted weights	9	15*	19**
	Body weight ratio	11	19**	20**
F = Female * = P ≤ 0.05; ** = P ≤ 0.01 Note: Organ weights were not taken from decedents or excluded animals.

 

Incidence of Selected Findings: Adrenal Gland – Terminal Sacrifice (Cohort 1A)

		Males				Females
		1M	2M	3M	4M	1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100	0	5	25	100
Adrenal gland	No. examined	20	12	13	20	20	11	16	20
Vacuolation, zona glomerulosa, increased, diffuse	Grade -	12	1	0	1	14	5	1	0
	1	8	9	8	7	6	6	7	2
	2	0	1	5	12	0	0	7	18
	3	0	1	0	0	0	0	1	0
Vacuolation, zona fasciculata, increased diffuse	Grade-	20	6	3	0	18	6	1	0
	1	0	5	5	7	2	5	5	1
	2	0	1	5	12	0	0	10	12
	3	0	0	0	1	0	0	0	7
- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; F = Female; M = Male

 

Incidence of Selected Findings: Thyroid Gland – Terminal Sacrifice (Cohort 1A)

		Males				Females
		1M	2M	3M	4M	1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100	0	5	25	100
Thyroid Gland	No. examined	20	20	20	20	20	20	20	20
Hypertrophy, follicular cell	Grade -	18	18	10	6	20	19	19	13
	1	2	2	8	7	0	1	1	6
	2	0	0	2	7	0	0	0	1
- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male

 

Incidence of Selected Findings: Liver – Terminal Sacrifice (Cohort 1A)

		Males				Females
		1M	2M	3M	4M	1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100	0	5	25	100
Liver	No. examined	20	20	20	20	20	20	20	20
Hypertrophy/eosinophilia, hepatocyte, centrilobular	Grade -	17	16	14	2	20	20	17	5
	1	3	4	6	15	0	0	3	8
	2	0	0	0	3	0	0	0	7
- = Finding not present; 1 = Minimal; 2 = Slight; F = Female; M = Male

 

Incidence of Selected Findings: Ovary – Terminal Sacrifice (Cohort 1A)

		Females
		1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100
Ovary	No. examined	20	20	20	20
Vacuolation, interstitial cell	Grade -	20	19	0	0
	1	0	1	3	0
	2	0	0	14	1
	3	0	0	3	12
	4	0	0	0	7
- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; 4 = Marked; F = Female

 

Incidence of Selected Findings: Ovary – Terminal Sacrifice (Cohort 1B)

		Females
		1F	2F	3F	4F
Tissue and finding	Level (mg/kg/day)	0	5	25	100
Ovary	No. examined	18	19	19	17
Vacuolation, interstitial cell	Grade -	18	19	0	0
	1	0	0	7	0
	2	0	0	12	3
	3	0	0	0	8
	4	0	0	0	6
- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; 4 = Marked; F = Female

 

 

Conclusions:

Following, once daily oral gavage administration of 5, 25, or 100 mg/kg/day Reofos 35 to Han Wistar rats for up to two consecutive generations, test article related effects were noted at all dose levels investigated.
Fertility was reduced following administration of 100 mg/kg/day to the F0 generation, which was accompanied by an increased incidence of stillborn pups and litters with stillborn pups at this dose level. Fertility was also slightly reduced for the F1 generation following administration of 100 mg/kg/day. Furthermore, microscopic examination of the ovaries revealed interstitial cell vacuolation for F0 and F1 generations following administration of 25 or 100 mg/kg/day, and fewer ovarian follicles (specifically, primordial follicles) were evident following administration of 25 or 100 mg/kg/day. In the absence of an effect on fertility following administration of 5 or 25 mg/kg/day, the no observed adverse effect level (NOAEL) for reproductive toxicity is 25 mg/kg/day. Postnatal pup development was unaffected for F1 and F2 generations at any dose level; as such, the no observed effect level (NOEL) for offspring growth and development is 100 mg/kg/day.
No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.
Systemic effects were confined to microscopic changes in the adrenal glands, which were considered to have represented toxicity at all dose levels. Based on this finding, neither a no observed effect level (NOEL) nor a no observed adverse effect level (NOAEL) was established for systemic toxicity. Reversibility of these findings was not investigated

Executive summary:

The objective of the study was to determine the effects of the test article, Reofos 35, on the gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, lactation, weaning and the growth and development of the offspring when administered orally, by gavage, to two successive generations. Developmental neurotoxicity was also assessed in the F1 offspring.

 

Methods

For the parental (F0) generation, male and female Crl:WI(Han) rats were assigned to four groups and administered 0 (control article [vehicle]), 5, 25, or 100 mg/kg/day Reofos 35 via oral gavage, for up to 126 consecutive days for males (at least 10 weeks prior to pairing, during pairing and 41 days post-pairing) or up to 128 days for females (10 weeks prior to pairing, during pairing, throughout gestation, and up to Lactation Day [LD] 21), until the day before necropsy. Direct dosing to the F1 offspring was initiated from Postnatal Day (PND) 14. The control article (vehicle) was corn oil, and a constant dose volume of 4 ml/kg was used.

Assessment of toxicity for the F0 generation was based on mortality, clinical observations, body weights, food consumption, estrous cycles, pregnancy indices, parturition, offspring development, and clinical and anatomic pathology. Litters were observed from birth, and the number of pups, sexes, body weights, and any clinical observations were monitored. Anogenital distance was measured on PND 4, and areolae (nipple) counts were recorded for male offspring on PND 13. F0 males were sacrificed during Week 19 (Study Day 127), and F0 females and F1 pups not selected for the next study phase were sacrificed on LD 22 (PND 22). Blood samples were taken for thyroid hormone assessments; selected organs were weighed, and macroscopic examinations were performed on the unselected offspring.

For the F1 generation, male and female Crl:WI(Han) rats were assigned to four groups in four cohorts and dosed as indicated in the following table: Animals were dosed via oral gavage at a volume of 4 mL/kg, once daily from PND 14 until the day before necropsy. The control article (vehicle) was corn oil.

 

Cohort 1A – Systemic and Reproductive Toxicity

Group	Dose Level (mg/kg/day)	Number of Animals/Group F1 generation Cohort 1A
		Males	Females
1 (Control)	0	20	20
2 (Low)	5	20	20
3 (Intermediate)	25	20	20
4 (High)	100	20	20

 

Cohort 1B – Reproductive Toxicity

Group	Dose Level (mg/kg/day)	Number of Animals/Group F1 generation Cohort 1B
		Males	Females
1 (Control)	0	20	20
2 (Low)	5	20	20
3 (Intermediate)	25	20	20
4 (High)	100	20	20

 

Cohort 2A – Developmental Neurotoxicity

Group	Dose Level (mg/kg/day)	Number of Animals/Group F1 generation Cohort 2A
		Males	Females
1 (Control)	0	10	10
2 (Low)	5	10	10
3 (Intermediate)	25	10	10
4 (High)	100	10	10

 

Cohort 2B – Developmental Brain Neuropathology Assessments

Group	Dose Level (mg/kg/day)	Number of Animals/Group F1 generation Cohort 2B
		Males	Females
1 (Control)	0	10	10
2 (Low)	5	10	10
3 (Intermediate)	25	10	10
4 (High)	100	10	10

 

Mortality, clinical observations, and body weights were recorded for F1 animals. Food consumption and sexual maturation were assessed for Cohorts 1A, 1B, and 2A. In addition, estrous cycles, ovarian follicle counts, seminology analysis, ovarian follicle counts, and clinical and anatomic pathology assessments were performed for Cohort 1A. Estrous cycles, pregnancy indices, parturition, offspring development, and anatomic pathology were assessed for Cohort 1B. Acoustic startle, motor activity, learning behavior, and brain histomorphometry were assessed for Cohort 2A. Brain histomorphometry was performed for Cohort 2B.

 

Results

Parental (F0) Generation

No test article-related unscheduled deaths occurred.

Test article-related clinical observations were confined to instances of salivation noted following administration of 100 mg/kg/day, with one instance also noted for one male administered 25 mg/kg/day.

No test article-related effects on body weights, body weight gain, or food consumption were noted.

Estrous cycles were unaffected, and no effect on mating performance was observed at any dose level.

A test article-related reduction in fertility was evident following administration of 100 mg/kg/day, compared with controls, and an increased incidence of stillborn pups was also evident at this dose level.

Hematology, clinical chemistry, and urine parameters were unaffected by the test article, and no test article-related changes in thyroxine (T4) or thyroid stimulating hormone (TSH) were evident for F0 males or females.

Sperm assessments did not reveal any test article-related effects for F0 males.

Increased adrenal weights and weight ratios were recorded for F0 animals administered 100 mg/kg/day and F0 females administered 25 mg/kg/day, compared with concurrent controls. Increased liver weights and weight ratios were recorded for F0 males administered 25 or 100 mg/kg/day. Increased thyroid weight and weight ratios and kidney:body weight ratios were recorded for F0 males administered 100 mg/kg/day, and increased ovarian weight and weight ratios were recorded for F0 females administered 100 mg/kg/day, compared with concurrent controls.

Macroscopically, an increased incidence of enlarged adrenals were recorded for both sexes from all test article-treated groups, more notably in females, and an increased incidence of pale adrenal was recorded for both sexes administered 25 or 100 mg/kg/day. An increased incidence of large thyroid gland and pale kidney was recorded for F0 males administered 100 mg/kg/day, compared to controls, and an increased incidence of large liver was recorded for males administered 25 or 100 mg/kg/day, compared to controls.

Microscopically, an increased incidence and severity of diffuse zona glomerulosa vacuolation was noted for the adrenal glands of F0 animals from all test article‑treated groups, compared with controls, and an increased incidence and severity of diffuse zona fasciculata vacuolation was recorded for F0 animals administered 25 or 100 mg/kg/day. Slight to moderate ovarian interstitial cell vacuolation was recorded for all F0 females administered 25 or 100 mg/kg/day.

Also microscopically, an increased incidence and severity of thyroid follicular cell hypertrophy was recorded for the thyroid glands of males administered 5 mg/kg/day and both sexes administered 25 or 100 mg/kg/day. Centrilobular hepatocellular hypertrophy/eosinophilia in the liver were recorded for F0 animals administered 100 mg/kg/day. An increased incidence and severity of pigmented macrophages was recorded for the spleen of F0 males administered 25 or 100 mg/kg/day, and an increased severity of renal hyaline droplet accumulation was recorded for F0 males administered 100 mg/kg/day.

 

F1 Offspring (Pre-Weaning)

Postnatal pup survival was not affected, and no clinical observations were associated with the test article. Furthermore, pup body weights and anogenital distance were unaffected. Nipple retention was not evident for male pups from any group.

No test article-related changes in T4 or TSH were evident, and no test article-related macroscopic changes or organ weight changes were evident for the PND 22 pups.

 

F1 Generation (All Cohorts)

No test article-related unscheduled deaths occurred.

No postdose observations were noted. No test article-related clinical observations or weekly detailed observations were noted.

Body weights, body weight gains, and food consumption were not affected by the test article.

No test article-related effects on age of sexual maturity or body weights at attainment were evident at any dose level. The number or duration of estrous cycles was also not affected.

 

F1 Generation Systemic and Reproductive Toxicity

No test article-related changes to urine parameters were evident for Cohort 1A animals.

Nonadverse test article-related increases in cholesterol, total protein, and globulin, and reductions in albumin:globulin (A:G) ratio were evident for Cohort 1A animals following administration of 100 mg/kg/day, compared with controls.

No test article-related TSH and T4 changes were noted for Cohort 1A animals.

Mating was unaffected by the test article for all Cohort 1B animals.

Fertility and fecundity indices were marginally lower for Cohort 1B animals administered 100 mg/kg/day, compared with controls.

Parturition, gestation length, and gestation index were unaffected, and no test article‑related effects on litter size or sex ratio were evident for Cohort 1B animals.

No test article-related clinical observations or effects on body weight or anogenital distance were noted for F2 pups, and no test article-related macroscopic findings or changes in kidney weight or kidney:body weight ratio were evident.

No test article-related changes to sperm analysis or sperm morphology parameters were observed for Cohort 1A males at any dose level.

Test article-related statistically significant reductions in ovarian follicle counts, primarily in primordial follicles, were evident for Cohort 1A females following administration of 25 or 100 mg/kg/day, compared with controls.

Increased adrenal weights and/or adrenal weight ratios and increased liver weights and/or liver weight ratios were recorded for F1 Cohort 1A animals administered 100 mg/kg/day, compared with concurrent controls.

Increased ovarian weights and ovarian weight ratios were recorded for F1 Cohort 1A and 1B females administered 100 mg/kg/day, compared with concurrent controls. Increases in ovarian weights and ovarian:body weight ratios were also recorded for F1 Cohort 1B females administered 25 mg/kg/day, compared with concurrent controls.

Macroscopically, enlarged adrenal glands and large ovaries were noted in F1 Cohort 1A females administered 100 mg/kg/day, and large liver was noted in F1 Cohort 1A males administered 100 mg/kg/day.

Microscopically in the adrenal glands, an increased incidence and severity of diffuse zona glomerulosa vacuolation was noted for F1 Cohort 1A animals administered 25 or 100 mg/kg/day, compared with controls, and an increased incidence and severity of diffuse zona fasciculata vacuolation was observed for F1 Cohort 1A animals from all test article-treated groups.

Slight to marked ovarian interstitial cell vacuolation was recorded for all F1 Cohort 1A females, and minimal to marked interstitial cell vacuolation was noted for the ovaries of F1 Cohort 1B females administered 25 or 100 mg/kg/day.

Thyroid follicular cell hypertrophy was recorded for F1 Cohort 1A males administered 25 mg/kg/day and both sexes administered 100 mg/kg/day, and centrilobular hepatocellular hypertrophy/eosinophilia was noted in the liver of F1 Cohort 1A animals administered 100 mg/kg/day.

No adverse test article-related differences in lymphocyte populations were evident in spleens, and no evidence of an immune response were evident following microscopic examinations from selected tissues of Cohort 1A animals.

 

F1 Generation Neurotoxicity and Neuropathology

No test article-related effects on functional observation battery (FOB) or motor activity parameters were evident for Cohort 2A animals, and no test article-related deficits in pre-pulse inhibition or startle habituation were noted for test article treated animals, compared with controls.

No test article-related brain weight changes or macroscopic or microscopic findings were recorded for F1 Cohort 2A or 2B animals, and no test article-related changes in brain histomorphometry were noted on PND 22 (Cohort 2B) or PND 76 (Cohort 2A) for F1 animals administered 100 mg/kg/day, compared with controls.

 

Conclusion

Following, once daily oral gavage administration of 5, 25, or 100 mg/kg/day Reofos 35 to Han Wistar rats for up to two consecutive generations, test article‑related effects were noted at all dose levels investigated.

Fertility was reduced following administration of 100 mg/kg/day to the F0 generation, which was accompanied by an increased incidence of stillborn pups and litters with stillborn pups at this dose level. Fertility was also slightly reduced for the F1 generation following administration of 100 mg/kg/day. Furthermore, microscopic examination of the ovaries revealed interstitial cell vacuolation for F0 and F1 generations following administration of 25 or 100 mg/kg/day, and fewer ovarian follicles (specifically, primordial follicles) were evident following administration of 25 or 100 mg/kg/day. In the absence of an effect on fertility following administration of 5 or 25 mg/kg/day, the no observed adverse effect level (NOAEL) for reproductive toxicity is 25 mg/kg/day. Postnatal pup development was unaffected for F1 and F2 generations at any dose level; as such, the no observed effect level (NOEL) for offspring growth and development is 100 mg/kg/day.

No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.

Systemic effects were confined to microscopic changes in the adrenal glands, which were considered to have represented toxicity at all dose levels. Based on this finding, neither a no observed effect level (NOEL) nor a no observed adverse effect level (NOAEL) was established for systemic toxicity. Reversibility of these findings was not investigated.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 October 2005 - 2 May 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted by a recognised facility in compliance with GLP to a recent regulatory test guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

This study was conducted in accordance with the protocol, except for the following deviations.

On one occasion, the humidity in the animal room was 74%, which was outside the protocol specified range of 30 to 70%.

On Day 7, the postdose observations for two mpIPTPP males (animal numbers 281 and 282) were conducted one minute prior to the protocol-specified window of 60-90 minutes postdose.

On Day 10, the postdose observations for seven control males (animal numbers 209,211,212,213,214,215, and 217), two Reofos 35 males (animal numbers 218 and 219), one Reofos 65 male (animal number 236), seven Reofos 65 washed males (animal numbers 248, 249, 250, 251, 252, 253, and 255), and three mpIPTPP males (animal numbers 271, 273, and 274) were conducted one to four minutes prior to the protocol-specified window of 60-90 minutes postdose.

On Day 10, the postdose observation for one Reofos 65 washed female (animal number 337) was conducted 7 minutes past the protocol-specified window of 60-90 minutes postdose.

On Day 16, test article administration for one control male (animal number 201) was conducted 1 minute prior the protocol-specified window of 1 hour from the Day 1 dose.

On Day 20, the postdose observations for six control males (animal numbers 201, 202,205,207,208, and 211), and one control female (animal number 288) were conducted 1 minute past the protocol-specified window of 60-90 minutes postdose.

On Day 22, the postdose observations for one Reofos 35 male (animal number 227) and one Reofos 65 female (animal number 314) were conducted 9 and 2 minutes, respectively, beyond the protocol-specified window of 60-90 minutes postdose.

On Day 23, the postdose observations for six Reofos 120 females (animal numbers 341,342,344,345,346, and 348) were conducted 1 to 2 minutes prior to the protocol-specified window of 60-90 minutes postdose.

On Day 35, the postdose observation for one recovery control male (animal number 214) was conducted 24 minutes past the protocol-specified window of 60-90 minutes postdose.

On Day 42, test article administration for all animals was conducted from 1 hour and 23 minutes to 1 hour and 42 minutes past the protocol-specified window of 1 hour from the Day 1 dose.

A terminal body weight for one mpIPTPP male (animal number 271) was not collected at necropsy.

The cranial cavity was not examined at necropsy for all main study females.

The uterus for one control recovery female (animal number 297) was stained in ammonium sulfide solution for detection of implantation sites, and saved in 10% neutral buffered formalin.

In the opinion of the Study Director, these minor deviations did not affect the quality or integrity of the study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina.
- Age at study initiation: 12 weeks
- Weight at study initiation: No data. Changes in body weight are listed in the results tables, which can be found under 'Attached backgound documents.'
- Fasting period before study:
- Housing: In stainless steel, wire-mesh type cages, except during pairing, near partrition, and during lactation. During pairing, animals were cohabited, one male and one female within the same treatment group. On approximately Day 20 of gestation, females were individually housed in plastic cages containing wood chip bedding.
- Diet (e.g. ad libitum): meal Lab Diet Certified Rodent Diet #5002, PMI Nutrition International, Inc.); ad libitum.
- Water (e.g. ad libitum): tap water; ad libitum.
- Acclimation period: 3 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 65-76°F
- Humidity (%): 30 to 74%
- Air changes (per hr): No data.
- Photoperiod (hrs dark / hrs light): approximately 12 hours light per day.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The basal diet will be meal Lab Diet Certified Rodent Diet #5002, PMI Nutrition International, Inc.
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food: No data.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil - no justification given.
- Concentration in vehicle: Control animals administered 400 mg/kg/day and a dose volume of 5 mL/kg of the vehicle.
- Amount of vehicle (if gavage): No data - The test aricles were diluted with the vehicle in order to achieve the desired dose volume.
- Lot/batch no. (if required): TH1937, TH1938, TM0158 and TO0383.
- Purity: No data: The Study Director is not aware of any potential contaminants likely to be present in the certified diet that would interfere with the results of this study. Therefore, no analyses other than those routinely performed by the feed supplier will be conducted.
-Preparation: To prepare the vehicle, the required amount of corn oil was dispensed into amber glass containers prior to handling the test articles. The vehicle was dispensed weekly and stored refrigerated.

Details on mating procedure:

- M/F ratio per cage: 1 Male to 1 Female
- Length of cohabitation: After evidence of mating was observed, the female was returned to an individual cage for the remainder of the study. The maximum pairing period was 14 days.
- Proof of pregnancy: Evidence of a copulatory plug in the vagina and/or sperm in the vaginal lavage referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing, any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia.
- After successful mating each pregnant female was caged: The female was returned to an individual cage for the remainder of the study.
- Any other deviations from standard protocol: On one occasion, the humidity in the animal room was 74%, which was outside the
protocol specified range of 30 to 70%. In addition, several postdose observations and test article administrations were administered late (between 1 minute and 1 hour and 42 minutes late). A terminal body weight for one mpIPTPP male (animal number 271) was not
collected at necropsy. The cranial cavity was not examined at necropsy for all main study females. The uterus for one control recovery female (animal number 297) was stained inammonium sulfide solution for detection of implantation sites, and saved in 10% neutral buffered formalin. All of these minor deviations were considered by the Study Director not to affect the quality or integrity of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity: Duplicate samples (5 mL/sample) from the vehicle and each test article formulation prepared for Week 1 were collected from the top, middle, and bottom of the mixing container using a syringe, while stirrng, and placed in amber glass bottles. The samples were delivered to MPI Analytical at room temperature for homogeneity analysis of the test article formulations.
- Stability: Samples (5 mL/sample) from each test aricle formulation from Week 1 were collected from the container with a syrnge, while stirrng, and placed in amber glass bottles. The samples were stored refrgerated for up to 14 days, and delivercd to MPI Analytical on wet ice after 7
and 14 days for analysis of the stability of the test articles in the vehicle.
- Concentration: Samples (5 mL/sample) from each test article formulation were collected from the preparations for Weeks 2 through 4 and 8 of study. Samples collected for homogeneity analysis were also used to verify the Week 1 concentrations. The samples were collected from the container, while stirring, with a syrnge, placed into amber glass bottles, and stored at room temperature or refrgerated until analyzed for test article concentrations.
- Analyses: All analytical work was conducted by MPI Analytical, a division ofMPI Research, using a Sponsor-supplied analytical method validated under MPI Research Study Number 1038-005.
- Reserve Sample and Test Article Disposition: A reserve sample from each batch of test article used in this study was taken and archived.The remaining test article will be returned to a Sponsor-designated location after completion of the study.

Duration of treatment / exposure:

The main study males were treated for at least 42 consecutive days, while the main study females were treated for up to 54 days, depending on
reproductive performance.

Frequency of treatment:

The test and control articles will be administered once per day at approximately the same time (± one hour from the Day 1 start of dosing) each day.

Details on study schedule:

After 14 days of treatment, the main study males were randomly cohabited, one male to one female, with the corresponding females in the treatment and control group. Each female was housed in the cage of a male during mating. Positive evidence of copulation was established by daily inspection for a copulatory plug in the vagina and/or sperm in the vaginal lavage.
The day on which positive evidence of copulation was observed was considered Day 0 of gestation. After evidence of mating was observed, the female was returned to an individual cage for the remainder of the study. The maximum pairing period was 14 days, at the end of which any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia.

After two weeks post-administration of vehicle or test article, recovery males and females were paired (1: 1) for two weeks or until evidence of mating was observed, as described above.

Parturition and F 1 Litter Observations

Beginning on GD 19, main study and recovery females were examined twice daily for signs of partrition. The mated females were allowed to give birth (F1). The duration of gestation was calculated, and any difficulties occurring at parturition were recorded. The day on which all pups were delivered was designated as LD O. The litters were examined as soon as possible after delivery and parameters including litter size, number of dead and live pups, and gross abnormalities of the pups were recorded. Any intact pups found dead at birth or during lactation were preserved in Bouin's solution for possible future visceral evaluation. Pups were weighed, sexed, and examined for abnormalities on LD 0 and 4. Any abnormal behavior observed in the pups was recorded daily. On LD 4, the pups were euthanized via intraperitoneal injection of sodium pentobarbital solution and preserved intact in Bouin's solution for possible future visceral evaluation.

Remarks:

Doses / Concentrations:
400 mg/kg/day at a dose volume of 5 ml/kg
Basis:
actual ingested

No. of animals per sex per dose:

12 male and female.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose level for each test article was selected by the Sponsor on the basis of available data from previous studies.
- Rationale for animal assignment (if not random): Prior to selection for study, each animal was given a detailed clinical examination, body weights were measured and recorded, and sex was verified. Animals considered suitable for study were randomized, by sex, using body weights, into treatment groups using a simple randomisation procedure. All animals placed on study had body weights that fell within ± 20% of the mean body weight for each sex. Extra animals obtained for this study, but not placed on study, were euthanized via carbon dioxide inhalation and discarded. Eighty two male (weighing 350 to 409 g at randomization) and 82 female rats (weighing 221 to 261 g at randomization) were assigned to the control or treated groups as described in the table in the method section.

Positive control:

No data.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily
- Cage side observations: Morbidity, mortality, signs of injury, and availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The onservations were conducted once, at approximately 60-90 minutes postdose.
- Clinical Observations included: changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and reactivity to handling, as well as the presence of clonic or tonic movements, stereotypics (e.g., excessive grooming, repetitive circling), diffcult or prolonged partrition or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights for the males and females were recorded the day after arrval, prior to randomization, at initiation of test article administration, weekly during the premating period, weekly for males, unmated females, and recovery animals (both sexes) through to termination of the main study animals. Recovery animals continued to be weighed weekly during the two-week premating period, mating and postmating periods (males and unmated females). All mated main study and recovery females were weighed on GD 0, 7, 14, and 20, and on LD 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
- Time schedule for examinations: Individual food consumption was measured and recorded weekly for all main and recovery
animals during the study except during the respective mating period. Food consumption was not measured during the mating period, because the animals were cohabited. Following the cohabitation period, food consumption was recorded weekly for males and unmated females until euthanasia. Food consumption was recorded for all mated females on GD 0, 7, 14, and 20 and LD 0 and 4.

Oestrous cyclicity (parental animals):

Not examined. Historical Control Fertility and Reproduction Data for theCRL:CD(SD) IGS BR Rats is present in the report appendices under 'Attached Background Material.'

Sperm parameters (parental animals):

Not examined.

Litter observations:

STANDARDISATION OF LITTERS: No data

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: The litters were examined as soon as possible after delivery and parameters including litter size, number of dead and live born pups, and gross abnormalities of the pups were recorded.
- Time schedule for observations: Pups were weighed, sexed, and examined for abnormalities on LD 0 and 4. Any abnormal behavior observed in the pups was recorded daily. On LD 4, the pups were euthanized via intraperitoneal injection of sodium pentobarbital solution and preserved intact in Bouin's solution for possible future visceral evaluation.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE:
- Male animals: All surviving animals at test termination.
- Maternal animals: All surviving animals at test termination.

GROSS NECROPSY:
All main study and recovery animals were euthanized via carbon dioxide inhalation and examined carefully for external abnormalities, including palpable masses. The skin was reflected from a ventral midline incision and any subcutaneous abnormalities were identified
and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities, and the organs were removed and examined. Special emphasis was placed on organs ofthe reproductive system. Implantation sites (scars) along the uterus were counted and recorded. All tissues were fixed in neutral buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues Adrenal, Epididymis, Gonads, ovary, testis, Gross lesions, Liver (3 sections collected; 2 examined) and Uterus (both homs)/Cervix were prepared for microscopic examination and weighed, respectively. Body weights and protocol-specified organ weights were recorded at scheduled necropsy and appropriate organ weight ratios were calculated (relative to body weights). Organs weighed were: Adrenal, Epididymis, Gonads: ovary, testis and Liver. Paired organs were weighed together. Organs were not weighed for animals dying spontaneously.

Postmortem examinations (offspring):

A full body visceral evaluation was conducted on a total of 22 selected pups found dead or euthanized on LD 4. Five pups were selected from the control group, one pup found dead at birth (litter number 284), one pup found dead during the 4-day lactation period (litter number 289), and three randomly selected pups from those that survived and were euthanized on LD 4. Nine pups were selected from the Reofos 65 washed group, one pup dead at birth (litter number 333), two pups randomly selected from the eight pups found dead at birth (litter number 335), three randomly selected pups (litter numbers 335, 337, and 339) from the 22 pups that died over the 4-day lactation period, and three randomly selected pups from those that survived and were euthanized on LD 4. Eight pups were selected from the mpIPTPP group, one dead pup at birth (one each from litter numbers 353,355, and 360), two pups that died during LD 0-4 (one each from litter numbers 362 and 364), and three randomly selected pups from those that survived to LD 4 and were euthanized. The pups were evaluated for visceral irregularities using a free-hand razor blade sectioning technique similar to that described by Wilson for the evaluation of term rat fetuses.

Statistics:

Tests used are listed below. The methods are detailed in the method section.
- T-Test Comparisons
- Arcsin-Square-Root Transformation
- Fisher's Exact Test
- Covariate Analysis

MPI Research Computer Systems:
Data Collection System: Provantis ™
HPLC and GC Analysis: Millenium32
Statistical Analysis: SAS
Reporting: SAS and Microsoft Office Professional

Reproductive indices:

- Mating Index - Males:
((No. males with evidence of mating) / ( No. males paired)) x 100

-Mating Index - Females:
((No. females with evidence of mating) / (No. females paired)) x 100

- Gestation Index:
((No. females delivering at least 1 live pup) / (No. females pregnant)) x 100

Offspring viability indices:

- Live Birth Index:
((No. live pups at birth) / (No. pups born)) x 100

- Dead Day 0 Index:
((No. dead pups on Day 0) / (No. pups born)) x 100

- Viabilty Index - Day 4:
((No. pups surviving 4 days (preculling)) / (No. live pups at birth)) x 100

- Lactation Index - Day 21:
((No. pups surviving 21 days (weaning)) / (No. live pups at 4 days (postculling))) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

MALES: During the daily clinical examinations of male rats, the only effect seen from treatment with the various isopropylated triphenyl phosphate mixtures tested was an increased incidence of salivation.
FEMALES: Similar to the males, salivation was also seen with increased frequency in female rats treated with the various isopropylated triphenyl phosphate mixtures tested. This was a prominent finding during the premating/mating and gestation phases of study and became less obvious during lactation (LD 0-4).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Reofos 35: Male body weights in the Reofos 35 group were significantly lower than controls at Weeks 3 through 8. In the Reofos 35 females, mean body weights and body weight gain during the premating, gestation, and lactation periods were comparable to controls and unaffected by treatment.
- Reofos 65: Mean body weights for the Reofos 65 males were slightly lower than controls throughout the study and only over the last two weeks (Weeks 7-8), when body weights were about 7% lower than controls, were differences statistically significant. In the Reofos 65 females, mean body weights and body weight gain during the premating, gestation, and lactation periods were considered comparable to controls and unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Even though the term fatty change was used for adrenal gland and vacuolation was used for the interstitial cells of the ovary, the material that accumulated to cause the change was probably the same in each organ and may be cholesteroI. Whereas both males and females had increased liver weight, only females had a distinct centrilobular hypertrophy in which cells had a small increase in acidophilic cytoplasm.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

An adverse effect on fertility and reproductive performance was seen in the Reofos 65 group.
Fertility and fecundity indices were 50% in this group and statistically lower than controls (50% vs. 100%). These effects were reversible, however, and were not apparent in the recovery phase of the study. Although not statistically lower than controls, a slight decrease of 25% for both fertility and fecundity indices were seen in the Reofos 35 group. This difference was considered suggestive of an effect on fertility and reproductive performance. No effects on fertility or reproductive performance were seen in the remaining treated groups.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): MALES: During the daily clinical examinations of male rats, the only effect seen from treatment with the various isopropylated triphenyl phosphate mixtures tested was an increased incidence of salivation. This was seen at least once during the study in most of the males from each treatment group, but not among the control males. Swollen lips were seen in some males in several of the treatment groups. A single exception (Reofos 120 male number 263 Days 9 and 10) of this was seen only during one day of study (i.e., Day 9). It was not seen in the control males or males from the Reofos 35 group and its occurrence in the remaining treatment groups ranged from 2/12 males in the Reofos 65 group to 9/12 animals in the mpIPTPP group. Because the finding was transient and not seen either prior to this day or for the remainder of the study, it was not considered toxicologically meaningful or related to treatment. Clinical examinations of males in the Reofos 65 group retained into the recovery phase of study were unremarkable. Salivation prevalent during the treatment period in these males (5/5 males) was seen in just one animal on a single occasion during the recovery phase when animals were not being treated.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): FEMALES: Similar to the males, salivation was also seen with increased frequency in female rats treated with the various isopropylated triphenyl phosphate mixtures tested. This was a prominent finding during the premating/mating and gestation phases of study and became less obvious during lactation (LD 0-4). Other findings seen among the treated females throughout the study occurred at low incidence or with similar frequency as controls or were transient (i.e. swollen lips) and not considered related to treatment. These findings were considered consistent with those seen commonly in this laboratory with this strain and age of animal. Clinical examinations of females in the Reofos 65 group retained into the recovery phase of study were unremarkable. Salivation prevalent during the treatment period in these animals (5/5 females) was not seen during the recovery phase, when animals were not being treated.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- Reofos 35: Male body weights in the Reofos 35 group were significantly lower than controls at Weeks 3 through 8. Body weights over this period ranged from about 6% lower than control at Week 4 to about 11 % lower at Week 8. These animals also gained less weight than control during the study and for many of these weekly intervals these differences were statistically significant. These changes in body weight and weight gain for these males did not, however, correlate with any change in food consumption. In the Reofos 35 females, mean body weights and body weight gain during the premating, gestation, and lactation periods were comparable to controls and unaffected by treatment.
- Reofos 65: Mean body weights for the Reofos 65 males were slightly lower than controls throughout the study and only over the last two weeks (Weeks 7-8), when body weights were about 7% lower than controls, were differences statistically significant. Weekly body weight gain for these males was generally comparable to controls throughout the study. The only statistically significant change from controls was a lower weight gain for Week 6-7. In the Reofos 65 females, mean body weights and body weight gain during the premating, gestation, and lactation periods were considered comparable to controls and unaffected by treatment. Mean weight gain in this group over the second week of the premating period was statistically higher than controls, but considered spurious and unrelated to treatment.
- Mean body weights and body weight gain for the Reofos 65 washed, Reofos 120 and mpIPTPP males and females were considered comparable to controls and unaffected by treatment. No effect of treatment with Reofos 65, Reofos 65 washed, Reofos 120 or mpIPTPP was evident from food consumption data.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): An adverse effect on fertility and reproductive performance was seen in the Reofos 65 group.
Fertility and fecundity indices were 50% in this group and statistically lower than controls (50% vs. 100%). These effects were reversible, however, and were not apparent in the recovery phase of the study. Although not statistically lower than controls, a slight decrease of 25% for both fertility and fecundity indices were seen in the Reofos 35 group. This difference was considered suggestive of an effect on fertility and reproductive performance.No effects on fertility or reproductive performance were seen in the remaining treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS): In males, adrenal and liver weights absolute and relative to body weight were increased in the Reofos 35 (exclusive of absolute liver weight), Reofos 65, Reofos 65 washed, and Reofos 120 groups. In females, the liver weights, absolute and relative to body weight, were increased for these same groups and in the mpIPTPP group. Additionally, in females, the adrenal weights, absolute and relative to body weight, were increased in the Reofos 35, Reofos 65, Reofos 65 washed, and Reofos 120 groups. The increased adrenal weights in males and females and liver weights in females were associated with microscopic changes in these organs. In the recovery phase, adrenal to body weight ratio for the Reofos 65 females was decreased.

GROSS PATHOLOGY AND HISTOPATHOLOGY (PARENTAL ANIMALS): Test article-associated fatty change was observed in the adrenal glands of males and females in Groups 2, 3,4, and 5; vacuolation of interstitial cells of the ovaries in Groups 2,3,4,5; and centrilobular hepatocellular hypertrophy oflivers of females in Groups 2,3,4, and 5. Fatty change was similar in the adrenal glands of males and females and was characterized by cells in the zona fasciculata and zona reticularis that were enlarged by the presence of increased small or large clear vacuoles. In a similar fashion, the cytoplasm of interstitial cells in the medulla of the ovaries was expanded by numerous small clear vacuoles. Even though the term fatty change was used for adrenal gland and vacuolation was used for the interstitial cells of the ovary, the material that accumulated to cause the change was probably the same in each organ and may be cholesteroI. Whereas both males and females had increased liver weight, only females had a distinct centrilobular hypertrophy in which cells had a small increase in acidophilic cytoplasm. Males may have had diffuse liver cell enlargement rather than a zonally recognizable type of enlargement. Other microscopic observations were of low incidence, were not present in a dose-response pattern, and are incidental or usual in rats.

Dose descriptor:

NOAEC

Effect level:

< 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

haematology

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of treatment with the various isopropylated triphenyl phosphate mixtures tested was evident from pup clinical findings at birth or LD 4. All 12 pups in the litter of Reofos 65 washed female number 333 were described as cold (i.e., skin cold to touch) at birth, but this was not noted in the 10 pups that survived to LD 4 in this litter.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- Reofos 35: Five of the nine litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls.
- Reofos 65: Four of the six litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls..
- Reofos 65 washed: Six of the 10 litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls.
- Reofos 120:. Six of 11 litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls. Two litters experienced particularly high pup mortality over LD 0-4.
-mpIPTPP: No effect of treatment was evident from this treatment on pup survival to LD 4.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Reofos 65: In this group, mean pup body weights, distinguished by sex and as a combined sex, were comparable to controls at LD O. On LD 4 pup weights were about 7-8% lower than controls and these differences were statistically significant for females and the combined sexes.
- Reofos 65 washed: Mean pup body weights, distinguished by sex and as a combined sex, in the Reofos 65 washed group were comparable to controls at LD 0 but notably lower than controls (14-18%) at LD 4. Only for female pups, however, was this difference (18%) at LD 4 statistically significant.
- Reofos 35, mpIPTPP, Reofos 120: Mean pup body weights, distinguished by sex and as a combined sex, in these groups at LD 0 and 4 did not differ statistically from controls and were considered comparable between the two groups.

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING):
- Reofos 35: The mean pup survival index over LD 0-4 in the Reofos 35 group (92.43%) was statistically lower than controls (99.36%). Five of the nine litters in this group experienced somc pup mortality over this period in comparison to two of 12 litters in controls.
- Reofos 65: The mean pup survival index over LD 0-4 in the Reofos 65 group (86.63%) was statistically lower than controls (99.36%). Four of the six litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls. In the Reofos 65 recovery phase, the pup survival index for LD 0-4 was 100% and 100% in controls. If the lower pup survival to LD 4 in the main study was treatment-related it was not
apparent in the recovery animals.
- Reofos 65 washed: The mean pup survival index over LD 0-4 in the Reofos 65 washed group (77.95%) was statistically lower than controls (99.36%). Six of the 10 litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls. All 17 pups in the litter of female number 336 from the Reofos 65 washed group died over this 4-day period.
- Reofos 120: The mean pup survival index over LD 0-4 in the Reofos 120 group (78.63%) was lower than controls (99.36%) but this difference was not statistically significant. Six of 11 litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls. Two litters experienced particularly high pup mortality over LD 0-4. Female number 342 had 11 live pups at Day 0 and only one survived to LD 4, and female number 343 had 13 pups at birth and none survived to LD 4.
-mpIPTPP: The mean pup survival index over LD 0-4 in the mpIPTPP group was 96.81 % and comparable to controls (99.36%). No effect of treatment was evident from this treatment on pup survival to LD 4.

CLINICAL SIGNS (OFFSPRING): No effect of treatment with the various isopropylated triphenyl phosphate mixtures tested was evident from pup clinical findings at birth or LD 4. All 12 pups in the litter of Reofos 65 washed female number 333 were described as cold (i.e., skin cold to touch) at birth, but this was not noted in the 10 pups that survived to LD 4 in this litter. Other findings seen among the live pups in the treated groups at birth or LD 4 occurred at low incidence and were considered unrelated to treatment.

BODY WEIGHT (OFFSPRING):
- Reofos 65: In this group, mean pup body weights, distinguished by sex and as a combined sex, were comparable to controls at LD O. On LD 4 pup weights were about 7-8% lower than controls and these differences were statistically significant for females and thc combined sexes. In the Reofos 65 recovery phase, mean pup weights in the treated group were higher than the recovery controls at LD 0 and 4 and some of these differences were statistically significant. If the lower pup weights on LD 4 in the main study were treatment-related it was not
apparent in the recovery animals.
- Reofos 65 washed: Mean pup body weights, distinguishcd by sex and as a combined sex, in the Reofos 65 washed group were comparable to controls at LD 0 but notably lower than controls (14-18%) at LD 4. Only for female pups, however, was this difference (18%) at LD 4 statistically significant.
- Reofos 35, mpIPTPP, Reofos 120: Mean pup body weights, distinguished by sex and as a combined sex, in these groups at LD 0 and 4 did not differ statistically from controls and were considercd comparable between the two groups.

GROSS PATHOLOGY AND HISTOPATHOLOGY (OFFSPRING):
No effect of treatment with the various isopropylated triphenyl phosphate mixtures tested was evident from external macroscopic examinations of pups found dead during lactation or at scheduled LD 4 euthanasia. The few findings seen among pups from the treated groups occurred at low incidence and were considered spurious and unrelated to treatment. No effect of treatment with Reofos 65 washed or mpIPTPP was evident from the pup visceral evaluations. Folded retina was the only finding seen during these special examinations and was observed with similar frequency among the control and treated pups. It was seen with greatest frequency among pups found dead on LD 0 or over the LD 0-4 period, but was also seen in a few pups euthanized on LD 4.

Dose descriptor:

other: Not determined

Generation:

F1

Remarks on result:

not measured/tested

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Reproductive effects observed:

not specified

Results:

The majority of the results tables can be found in the 'Attached Background Material Section' in the document 'Reofos 35 Oral Repro-Developmental Tox Rat Results Tables.pdf.'

Homogeneity of formualtions used in study:

Homogeneity
Test Article	Nominal Concentration (mg/mL)	Concentration Found (mg/mL)		Percent Recovery (a)		RSD (%)
		Mean	Range	Mean	Range
Reofos 35	80.0	79.6	78.6-80.4	99.5	98.3-100.5	0.7
Reofos 65	80.0	81.7	80.7-82.8	102.0	100.9-103.6	0.9
Reofos 65 washed	80.0	80.7	78.6-82.0	100.8	98.2-102.5	1.4
Reofos 120	80.0	80.9	79.2-81.8	101.1	99.0-102.2	1.2
mpIPTPP	80.0	79.7	78.8-80.2	99.6	98.5-100.2	0.7

a: Mean Percent Recovery was calculated from the nominal concentration.

RSD: Relative Standard Deviation

 

Stability of formulations used in the study:

Stability
Test Article	Nominal Concentration (mg/mL)	Mean Found Concentration (mg/mL) (Day)			Mean % Recovery from Day 0
		0a	7	14	7 days(b)	14 Days(b)
Reofos 35	80.0	79.6	79.8	80.6	100.2	101.3
Reofos 65	80.0	81.7	80.6	81.1	98.8	99.3
Reofos 65 washed	80.0	80.7	80.3	81.2	99.6	100.7
Reofos 120	80.0	80.9	81.6	81.9	100.8	101.2
mpIPTPP	80.0	79.7	81.3	82.0	102.0	102.9

a: Values expressed are the mean homogeneity concentrations.

b: Day 7 and 14 mean % recovery values were calculated from the respective Day 0 value

 

Analysis of doses used in the study:

Periodic Analysis of Dosing Formulations Used on Study
Test Article	Nominal Concentration (mg/mL)	Found Concentration (a) [mg/mL] (% of nominal)
		Week 1 (b)	Week 2	Week 3	Week 4	Week 8
Vehicle	0	0 (NA)	0 (NA)	0 (NA)	0 (NA)	0 (NA)
Reofos 35	80.0	79.6 (99.5)	79.6 (99.5)	79.9 (99.8)	79.5 (99.4)	78.1 (97.6)
Reofos 65	80.0	81.7     (102.1)	81.0 (101.3)	79.5 (99.3)	81.5 (101.8)	79.2 (99.0)
Reofos 65 washed	80.0	80.7    (100.8)	81.2 (101.5)	81.8 (102.3)	80.8 (101.1)	78.6 (98.3)
Reofos 120	80.0	80.9     (101.1)	79.3 (99.1)	80.1 (100.1)	79.2 (99.0)	80.5 (100.6)
mpIPTPP	80.0	79.7       (99.6)	80.3 (100.4)	80.8 (101.0)	79.5 (99.4)	74.6 (93.2)

a: Mean of duplicate analyses

b: Mean of the six homogeneity samples

NA: Not Applicable

 

Conclusions:

Each test article (Reofos 35, Reofos 65, Reofos 65 washed, Reofos 120, and mpIPTPP) evaluated was administered orally by gastric intubation at a dose level of 400 mg/kg/day and dose volume of 5 mL/kg in a study following OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test). Salivation was seen commonly among all the treated groups. Body weight and weight gain effects (lower) were seen in the Reofos 35 and Reofos 65 males. Poor reproductive performance was seen only in the Reofos 65 group but the response was reversible, and not apparent in a mating of recovery animals. Reproductive performance, parturition data, and litter size data for the remaining test groups were considered unaffected by treatment. Test article-associated organ weight effects were seen for the adrenals and liver and microscopically, test article-associated fatty changes were observed in thc adrenals of males and females in the Reofos 35, Reofos 65, Reofos 65 washed, and Reofos 120 groups.

Executive summary:

Each test article (Reofos 35, Reofos 65, Reofos 65 washed, Reofos 120, and mpIPTPP) evaluated was administered orally by gastric intubation at a dose level of 400 mg/kg/day and dose volume of 5 mL/kg in a study following OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test). Salivation was seen commonly among all the treated groups. Body weight and weight gain effects (lower) were seen in the Reofos 35 and Reofos 65 males. Poor reproductive performance was seen only in the Reofos 65 group but the response was reversible, and not apparent in a mating of recovery animals. Reproductive performance, parturition data, and litter size data for the remaining test groups were considered unaffected by treatment. Test article-associated organ weight effects were seen for the adrenals and liver and microscopically, test article-associated fatty changes were observed in thc adrenals of males and females in the Reofos 35, Reofos 65, Reofos 65 washed, and Reofos 120 groups.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

See discussion below

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are two 28-day screening studies available for the substance that assess reproductive toxicity. Adverse effects on fertility were noted in that effects on fertility and reproductive performance were documented during the study. These effects were reversible, however, and were not apparent in the recovery phase of the study. Test article-related reductions in male fertility and copulation indices were noted. Decreased epididymal weights were considered test article-related.   

A further OECD 443 extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B) was also conducted. Fertility was reduced following administration of 100 mg/kg/day to the F0 generation, which was accompanied by an increased incidence of stillborn pups and litters with stillborn pups at this dose level. Fertility was also slightly reduced for the F1 generation following administration of 100 mg/kg/day. Furthermore, microscopic examination of the ovaries revealed interstitial cell vacuolation for F0 and F1 generations following administration of 25 or 100 mg/kg/day, and fewer ovarian follicles (specifically, primordial follicles) were evident following administration of 25 or 100 mg/kg/day. In the absence of an effect on fertility following administration of 5 or 25 mg/kg/day, the no observed adverse effect level (NOAEL) for reproductive toxicity is 25 mg/kg/day. Postnatal pup development was unaffected for F1 and F2 generations at any dose level; as such, the no observed effect level (NOEL) for offspring growth and development is 100 mg/kg/day.
No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.
Systemic effects were confined to microscopic changes in the adrenal glands, which were considered to have represented toxicity at all dose levels. Based on this finding, neither a no observed effect level (NOEL) nor a no observed adverse effect level (NOAEL) was established for systemic toxicity. Reversibility of these findings was not investigated.

The effects triggered classification under the CLP Regulation (EC No 1272/2008) as follows:

Repr. Cat 2, H361f: Suspected of damaging fertility or the unborn child < Fertility - effects on ovaries; decreased live litter sizes

Effects on developmental toxicity

Description of key information

Developmental toxicity only is discussed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 25, 2014 to April 28, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & US EPA test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

A total of 112 time-mated female CD® [Crl:CD®(SD)] rats (approximately 8 to 10 weeks of age) were received from Charles River Laboratories, Portage, Michigan on February 25 and 26, 2014. The animals were randomly assigned to study and dosing began on GD 0. Prior to the initiation of dose administration, the Study Director reviewed the GD 0 body weight and detailed observation data for all animals and gave final approval for assignment to study.

Randomization, Assignment to Study, and Maintenance
Using a standard, by weight, measured value randomization procedure, 100 female animals (weighing 179 to 254 g, at randomization) were assigned to the control and treatment groups identified in the following table.
Group Assignments
Group Number Dose Level (mg/kg/day) Number of Time-mated Females
1 0 25
2 100 25
3 200 25
4 400 25

Animals assigned to study had body weights within ±20% of the mean body weight. Extra animals obtained, but not placed on study, were euthanized via carbon dioxide inhalation. Euthanasia was confirmed via exsanguination of the abdominal vena cava, and the carcasses were discarded.
Each animal was assigned an animal number to be used in the Provantis™ data collection system and was implanted with a microchip bearing a unique identification number. The individual animal number, implant number, and study number comprised a unique identification for each animal. Each cage was identified by the animal number, study number, group number, and sex.
The animals were individually housed in solid bottom cages with nonaromatic bedding in an environmentally controlled room. Animal enrichment was provided according to SOP. Fluorescent lighting was provided for approximately 12 hours per day. Temperature and humidity were continuously monitored, recorded, and maintained to the maximum extent possible within the ranges of 68 to 79°F and 30 to 70%, respectively. The actual temperature and humidity findings are not reported but are maintained in the study file.
Block Lab Diet® (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum. The lot number from each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer. Tap water was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP. The results of food and water analyses are retained in the Archives. The Study Director is not aware of any potential contaminants likely to be present in the diet or water that would have interfered with the results of the study. Therefore, no analyses other than those stated above were conducted.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The vehicle and test article were administered once a day from GD 0 to 19 at approximately the same time each day (±2 hours from the GD 0 dose) via oral gavage. The dose levels for the treated groups were 100, 200, and 400 mg/kg/day at a dose volume of 5 mL/kg/day. The control group received the vehicle in the same manner as the treated groups. The dosing formulations were stirred throughout administration. Individual doses were based on the most recent body weights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see following table) were collected using a positive displacement pipette, while stirring, and placed into amber glass bottles. Following acceptance of the analytical results (signing of the final report) by the Study Director, or at the discretion of the Study Director, backup samples will be discarded. Tabulated data is listed below under "any other information"

Samples were stored refrigerated at 2 to 8°C until shipped on gel packs to the MPI Research, Inc., site at State College, Pennsylvania, for analysis. All analytical work was conducted by MPI Research, Inc., using an analytical method developed by MPI Research, Inc.

Objective: To determine the homogeneity and concentration of Reofos 35 in dosing formulations.
Analytical Method Number and Title: 399-246-A-02 (V0008648-3): Determination of Reofos 35 in Corn Oil. Dose Formulations by GC-FID
Reference Standard: Reofos 35; Lot Number: 2012123125; MPI Research Inventory ID: SP0014320; Storage: Room temperature; Correction Factor: None
Data Collection and Analysis Software: Empower™ 2: Build 2154 (Waters Technologies Corporation®). ExyLIMS Version 3.0 (MPI Research, Mattawan, Michigan)
GC Conditions: Gas chromatography system equipped with a Hewlett Packard HP-1 column, 30 m x 0.25 μm, 0.25 μm thickness with a flame ionization detector.
Analysis Description: Prior to analysis, samples were diluted with 2-propanol (diluent) to within the range of the calibration curve. Vehicle samples were diluted with diluent using a dilution factor of 8. An aliquot of each sample was injected into the GC-FID system for analysis.
Regression Type: Linear unweighted
Study Sample Receipt(s): Number of Samples: 48; Date(s) of receipt by Analytical Department:
February 26, 2014 to March 12, 2014
Study Sample Storage Conditions: Refrigerated (2 to 8°C)
Storage Stability: All samples were analyzed within the established storage stability determined under MPI Research Study Number 399-243.
Analysis Period: March 6, 2014 to March 27, 2014

Run Acceptance Criteria
System Suitability Test Standards:
1. Injection repeatability (peak area and retention time) ≤5% RSD (relative standard deviation)
2. Resolution between the analyte peak and any adjacent peaks must be ≥1.5
3. Tailing Factor (T) ≤2
4. Theoretical Plates (N) ≥2000
Calibration Standards:
1. Accuracy within ±10% of the nominal concentration
2. Coefficient of determination (R2) ≥0.995
Performance Check Standards (Same preparation as the System Suitability Standard):
1. Periodically injected so that no more than 10 samples are bracketed by performance check standards. Samples are considered valid if bracketed by passing performance check standard injections.
2. Accuracy within ±10% of the nominal concentration
Blank Injections: ≤20% of the limit of quantitation (LOQ)

Assessments
Homogeneity:
1. Average concentration within ±10% of the nominal concentration
2. Precision ≤5% RSD
Concentration:
1. Average concentration within ±10% of the nominal concentration
2. Precision ≤5% RSD
3. Vehicle (control) samples < LOQ

Results and Discussion
Conclusion: A total of 36 samples were analyzed for Reofos 35 in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results. In dataset 030614, there was a programming error in the analytical sequence, which resulted in some samples (Group 1 Week 1 and Group 1 Week 2) not being injected and other samples (Group 2 Week 1) being injected multiple times. All data generated in this dataset is reported; therefore, there are additional replicates for the homogeneity and concentration analyses for the Group 2 Week 1 samples. All samples met the acceptance criteria, so there is no suspected impact.
Deviations: This study phase was conducted in accordance with the protocol with the exception of the following deviations:
Standard Operating Procedure (SOP) Deviations:
SOP deviations were acknowledged by the Study Director and documented in the raw data.
In the opinion of the Study Director, none of the SOP deviations were considered to have affected the quality or integrity of the study.

Details on mating procedure:

Time-mated females used in the study - no details specified in the study report.

Duration of treatment / exposure:

19 days

Frequency of treatment:

Daily

Duration of test:

20 days

Remarks:

Doses / Concentrations:
0, 100, 200, and 400 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Fresh vehicle, corn oil, was dispensed for use on study weekly and was stored refrigerated at 2 to 8°C when not in use.
The test article, Reofos 35, was used as received from the Sponsor, and no adjustment was made for purity. Formulations of the test article were prepared weekly at nominal concentrations of 20, 40, and 80 mg/mL, and were stored refrigerated at 2 to 8°C when not in use.

Analysis of Dosing Formulations
Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see table in Any other information) were collected using a positive displacement pipette, while stirring, and placed into amber glass bottles. Following acceptance of the analytical results (signing of the final report) by the Study Director, or at the discretion of the Study Director, backup samples will be discarded.
Stability has been established for at least 10 days under refrigerated (2 to 8°C) conditions under MPI Research, Inc., Study Number 399-246.

Justification for Route of Administration
The oral route is one of the potential routes of human exposure to this test article.

Justification of Dose Levels
The dose levels were selected by the Sponsor on the basis of available data from a recently conducted pilot prenatal developmental toxicity study (MPI Research, Inc., Study Number 399-245) and available data from a previously conducted reproduction/developmental screening study that included Reofos 35 (MPI Research, Inc., Study Number 1038-004). In the pilot study (MPI Research, Inc., Study Number 399-245), mortality (60%), lower body weights and body weight gain, lower food consumption, and reduced fetal body weights were observed in time-mated females treated daily from GD 0 through GD 20 with Reofos 35 at 500 mg/kg/day.
In the screening study (MPI Research, Inc., Study Number 1038-004), animals were treated daily for 8 weeks with Reofos 35 at 400 mg/kg/day. Clinical findings (salivation), lower body weights and body weight gain, reproductive effects, organ weight changes, and microscopic observations were observed.
Based on the available data, dose levels up to 400 mg/kg/day were evaluated in time-mated females from GD 0 to 20. The low- and mid-dose are at approximate log intervals and were chosen to provide a graded response.

Maternal examinations:

In-life Examinations
Cage-side Observations: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily. On occasion, veterinary consultations were conducted during the course of the study. All treatments and observations were recorded. The medical treatments and observations are not reported but are maintained in the study file.
Detailed Clinical Observations: Daily from GD 0 through 20 (60 to 90 minutes post dose on dosing days), each animal was removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior.
Body Weights and Body Weight Changes: Body weights for all animals were measured and recorded on GD 0, 3, 6, 9, 12, 15, 18, and 20. Individual body weight change was calculated for the following GD intervals: 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20, and 0-20. Adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to 20) were also calculated.
Food Consumption: Food consumption was measured and recorded on the corresponding body weight days and calculated for the same.

Postmortem Study Evaluations
Maternal Necropsy: A complete necropsy was performed on all dams under procedures approved by a veterinary pathologist. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy. The presence of lesions or other abnormal conditions in the dam were noted and described in the study records.

Ovaries and uterine content:

Ovarian and Uterine Examination
On GD 20, each female was euthanized by carbon dioxide inhalation, followed by exsanguination of the abdominal vena cava and immediately subjected to a cesarean section. Dams had uterine examinations conducted without knowledge of the treatment group. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous masses. The abdominal cavity was then opened, and the uterus was exposed. The uterus was excised, and the gravid uterine weight was recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable fetuses, early and late resorptions for each uterine horn, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded.
Each implant was categorized according to the following criteria.
Viable fetuses responded to touch. Nonviable fetuses did not respond to touch and had no signs of autolysis. Late resorptions were characterized by recognizable fetal form, but undergoing autolysis. Early resorptions were characterized as implantation sites that had no recognizable fetal characteristics.
The fetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each fetus was gently removed, and each fetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were examined grossly.
Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. If no foci were detected, the female was considered to be non-pregnant.

Fetal examinations:

Fetal Examinations
Each fetus was individually weighed, sexed, tagged, and examined for external malformations and variations. Fetuses were then euthanized by intraperitoneal injection of euthanasia solution. Fetal external, visceral, and skeletal examinations were conducted without knowledge of the treatment group.
Approximately one-half of the fetuses in each litter were placed in Bouin’s solution and the remaining fetuses were fixed in alcohol. All fetuses fixed in Bouin’s solution were examined for soft tissue defects using the Wilson razor-blade sectioning technique. The fetuses fixed in alcohol were macerated in potassium hydroxide, stained with Alizarin Red S, and cleared with glycerin by a method similar to that described by Dawson for subsequent skeletal examination. Fetal findings were classified as malformations or developmental variations under procedures approved by a developmental toxicologist. On occasion, additional information to clarify or identify a visceral or skeletal observation was documented. These comments are not reported, but are maintained in the study data. Mechanical artifacts (i.e., tail removed and discarded) occurred during examination and processing of the fetuses.
These artifacts are not reported, but are maintained in the study data.

Statistics:

See Any other information for Statistics data.

Indices:

Fetal and litter incidences are reported, but only the litter incidences were statistically analyzed.

Historical control data:

Historical Control Developmental Toxicity Data
Sprague Dawley Rat
07/2006 to 07/2011
Number of Studies: 50
Number of Litters Evaluated: 1230
Number of Fetuses Evaluated – External: 14180
Number of Fetuses Evaluated – Visceral: 7072
Number of Fetuses Evaluated – Skeletal: 7106

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. It was observed at least once in 4, 8 and 15 animals in the 100, 200, and 400 mg/kg/day dose groups, respectively. Its occurrence on study was likely a pharmacologic response to the test article and was not considered adverse. Other clinical findings observed at dose levels ≤200 mg/kg/day occurred at low incidence or with similar frequency as controls and considered unrelated to the test article. In the 400 mg/kg/day dose group, additional clinical findings observed within the first several days of dosing (GD 1 to 3) and considered test article related included decreased activity (one animal), red material around the nose/mouth (nine animals), hunched posture (one animal), thin appearance (two animals), hair on the face discolored brown (two animals), and hair on the forelimbs discolored brown (two animals). These clinical findings were transient and were not observed among the 400 mg/kg/day animals for the remainder of the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 400 mg/kg/day dose group, a mean body weight gain of 0.1 g over GD 0 to 3 differed statistically from the mean weight gain of 14.0 g in the control group. This low weight gain in the 400 mg/kg/day dose group was considered test article related and adverse correlating with a period of low food consumption.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 200 mg/kg/day dose group, mean food consumption throughout gestation was higher relative to mean control values and these differences were statistically significant over GD 3 to 6 (+23%), GD 6 to 9 (+18%), GD 9 to 12 (+17%), GD 12 to 15 (+10%), GD 18 to 20 (+10%), and GD 0 to 20 (+13%). The increase in food consumption in the 200 mg/kg/day dose group may have been test article related but the effect was not considered adverse and only over GD 0 to 20 did it correlate with statistically higher body weight gain relative to controls. In the 400 mg/kg/day dose group, mean food consumption over GD 0 to 3 at 9.1 g/day was statistically lower than the mean control value of 13.6 g/day. This was considered test article related and adverse correlating with a period of statistically lower body weight gain.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: red or white foci and swollen mucosa of the nonglandular portion of the stomach

Details on maternal toxic effects:
In-life Examinations
Mortality
All animals survived to scheduled termination on GD 20.

Detailed Clinical Observations
Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. It was observed at least once in 4, 8 and 15 animals in the 100, 200, and 400 mg/kg/day dose groups, respectively. Its occurrence on study was likely a pharmacologic response to the test article and was not considered adverse. Other clinical findings observed at dose levels ≤200 mg/kg/day occurred at low incidence or with similar frequency as controls and considered unrelated to the test article. In the 400 mg/kg/day dose group, additional clinical findings observed within the first several days of dosing (GD 1 to 3) and considered test article related included decreased activity (one animal), red material around the nose/mouth (nine animals), hunched posture (one animal), thin appearance (two animals), hair on the face discolored brown (two animals), and hair on the forelimbs discolored brown (two animals). These clinical findings were transient and were not observed among the 400 mg/kg/day animals for the remainder of the study.

Body Weights and Body Weight Changes
No test article effect at the 100 and 200 mg/kg/day dose levels was observed on gestation body weights or body weight gain. A statistically significant increase in mean weight gain over GD 0 to 20 in the 200 mg/kg/day dose group relative to the control mean value (170.7 g vs.156.7 g in controls) was considered unrelated to the test article but did correlate with statistically higher food consumption relative to the controls over this period. In the 400 mg/kg/day dose group, a mean body weight gain of 0.1 g over GD 0 to 3 differed statistically from the mean weight gain of 14.0 g in the control group. This low weight gain in the 400 mg/kg/day dose group was considered test article related and adverse correlating with a period of low food consumption. However, the effect was transient and over the ensuing intervals of GD 3 to 6 and GD 6 to 9, mean body weight gains in the 400 mg/kg/day dose group of 22.7 g and 22.8 g were statistically higher than the mean body weight gains of 15.2 g (49% higher) and 19.5 g (17% higher) respectively, in the control group. For the remainder of gestation and over the entire GD 0 to 20 period, mean weight gains in the 400 mg/kg/day dose group were comparable to mean control values.

Food Consumption
No adverse effect of test article at the 100 and 200 mg/kg/day dose levels was observed on gestation food consumption. Mean food consumption throughout gestation in the 100 mg/kg/day dose group was comparable to mean control values. In the 200 mg/kg/day dose group, mean food consumption throughout gestation was higher relative to mean control values and these differences were statistically significant over GD 3 to 6 (+23%), GD 6 to 9 (+18%), GD 9 to 12 (+17%), GD 12 to 15 (+10%), GD 18 to 20 (+10%), and GD 0 to 20 (+13%). The increase in food consumption in the 200 mg/kg/day dose group may have been test article related but the effect was not considered adverse and only over GD 0 to 20 did it correlate with statistically higher body weight gain relative to controls. In the 400 mg/kg/day dose group, mean food consumption over GD 0 to 3 at 9.1 g/day was statistically lower than the mean control value of 13.6 g/day. This was considered test article related and adverse correlating with a period of statistically lower body weight gain. The effect on food consumption at 400 mg/kg/day was transient and for the remainder of gestation, mean food consumption in this group was 4% to 17% higher than the mean control values with statistical significance identified over GD 3 to 6 (+15%), GD 9 to 12 (+17%), and GD 12 to 15 (+13%).

Postmortem Study Evaluations
Uterine and Ovarian Examinations
The pregnancy indices were 96%, 96%, 96%, and 92% in the control, 100, 200, and 400 mg/kg/day dose groups providing 24, 24, 24, and 23 GD 20 litters with fetuses for evaluation, respectively. No test article effect was observed from GD 20 uterine implantation data. In the 100 mg/kg/day dose group, the mean pre-implantation loss index was 14.63% and statistically higher than the 5.01% in controls but in the absence of a similar increase in this index at the higher dose levels, this was considered incidental and unrelated to the test article. All other mean uterine implantation parameters (corpora lutea, implantation sites, viable fetuses, post-implantation loss, litter size, and resorption sites [early, late, and total]) in the treated groups were comparable to mean control values. Mean gravid uterine weights in the 100 and 400 mg/kg/day dose groups were comparable to mean control values. In the 200 mg/kg/day dose group, the mean gravid uterine weight at 80.26 g was statistically higher than the mean control value of 71.24 g. This was considered related to the slightly greater number of fetuses in utero, and unrelated to the test article. The mean adjusted GD 20 body weights (GD 20 body weight minus the gravid uterine weight) and adjusted body weight change GD 0 to 20 in the treated groups were comparable to mean control values.

Maternal Macroscopic Observations
No test article effect at 100 and 200 mg/kg/day was observed from the maternal macroscopic examinations. Macroscopic findings observed among these animals occurred at low incidence (single animals affected), and were considered incidental and unrelated to the test article. In the 400 mg/kg/day dose group, red or white foci and swollen mucosa of the nonglandular portion of the stomach were observed in 3/25 animals. Their occurrence in the 400 mg/kg/day group was considered test article related as similar findings were not observed among control animals or animals at the lower dose levels. No other macroscopic findings were observed among the 400 mg/kg/day animals.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal Evaluations
Fetal Sex Ratio
No test article effect was observed from fetal sex ratios (% male fetuses/animal). Mean fetal sex ratios in the treated groups ranged from 45.4% to 53.4% and were comparable to the mean of 54.0% in the control group.
Fetal Body Weights
No test article effect was observed on fetal body weights. In the 100 mg/kg/day dose group, fetal body weights, distinguished by sex and for the combined sexes, were comparable to mean control values. In the 200 and 400 mg/kg/day dose groups, mean fetal body weights were higher than mean control values and although the differences were slight (5% to 7%) in most instances they were statistically significant and outside the ranges of recent historical control data for the laboratory. The increase in mean fetal body weights observed in the 200 and 400 mg/kg/day dose groups although statistically significant relative to control values, were still relatively slight 5 to 7% and not considered adverse or indicative of a test article-related response.
External Examinations
No test article effect was observed from the fetal external examinations. No malformations or developmental variations were observed among the control or treated fetuses from the external examinations.
Visceral Examinations
No test article effect was observed from the fetal visceral examinations. No visceral malformations were observed among the control or treated fetuses. The only visceral developmental variation observed was dilated ureter. This variation has a high historical presence in this laboratory (Appendix O, maximum litter incidence of 20.8%) and its occurrence in a single fetus in the 400 mg/kg/day dose group (litter incidence 4.3%) was considered incidental and unrelated to the test article.
Skeletal Examination
No test article effect was observed from the fetal skeletal examinations. The few skeletal malformations observed among fetuses in the treated groups (one fetus in the 200 mg/kg/day dose group and two fetuses from a single litter in the 400 mg/kg/day dose group) were dissimilar in appearance and considered unrelated to the test article. No skeletal malformations were observed in fetuses from the 100 mg/kg/day dose group; one control fetus had a skeletal malformation. Skeletal developmental variations observed in the treated groups occurred at low incidence or with similar frequency as in controls and no effect of the test article on skeletal development was indicated from these data.

Dose descriptor:

other: Not determined

Remarks on result:

not measured/tested

Abnormalities:

not specified

Developmental effects observed:

not specified

 Homogeneity

Analysis of the low- and high-dose formulations (20.0 and 80.0 mg/mL, respectively) used for dosing the first week of study confirmed they were homogeneous as prepared and therefore met the laboratory’s acceptance criteria of 100±10% of nominal, and percent relative standard deviation (RSD) ≤5. These analytical results are summarized in the table below.

Homogeneity
Dose Level (mg/kg/day)	Nominal Concentration (mg/mL)	Average Calculated Concentration (mg/mL)	Average %Recovery	%Relative Standard Deviation^c
100 400	20.0 80.0	18.7372^a 81.6908^b	93.7^a 102.1^b	1.9 1.5
^aRepresents an average of seven samples from the mixing container (two top, two middle and three bottom. Samples were collected while the formulation was stirring. ^bRepresents an average of six samples from the mixing containers (two top, two middle and two bottom). Samples were collected while the formulation was stirring. ^cAverage %recovery was calculated from the nominal concentration.

Concentration

Mean concentrations of formulations used for dosing each of the three weeks of study ranged between 93.2 and 102.1% of nominal confirming that animals were receiving the appropriate dose levels when the dose was administered at 5 mL/kg. No test article was found in the control samples. These results are summarized in the table below.

Concentration
Dose Level (mg/kg/day)	Nominal Concentration (mg/mL)	Average Calculated Concentration^a(mg/mL)	Average %Recovery^a,b
0 100 200 400	0.0 20.0 40.0 80.0	BLQ 18.6350 – 19.1496 37.4113 – 38.8650 81.0502 – 81.6550	NA 93.2 – 95.7 93.5 – 97.2 101.3 – 102.1
^aResults are the range of values determined during Weeks 1, 2 and 3. ^bAverage %recovery was calculated from the nominal concentration. BLQ – Below the Limit of Quantification (<0.4 mg/mL) NA – Not Applicable

 

Summary of Gestation Detailed Clinical Observations+

Days 0 to 20

Observation	0 mg/kg/day	100 mg/kg/day	200 mg/kg/day	400 mg/kg/day
Number of Animals Observed	25	25	25	25
Behavior/Activity                Activity decreased                Salivation	0/0 0/0	0/0 6/4	0/0 36/8	2/1 85/15
External Appearance                Discharge, Red, Anogenital region                Ear/portion of ear missing, Ear/left                Material around mouth, Red                Material around nose, Red                Posture hunched                Thin	0/0 6/1 0/0 0/0 0/0 0/0	0/0 0/0 0/0 0/0 0/0 0/0	1/1 5/1 0/0 0/0 0/0 0/0	0/0 0/0 1/1 11/9 2/1 3/2
Pelage/Skin                Hair, discolored, Brown, Face                Hair, discolored, Brown, Forelimb/left                Hair, discolored, Brown, Forelimb/right                Hair, discolored, Black, Thoracic region                Hair, sparse, Abdominal region                Hair, sparse, Cervical region                Hair, sparse, Forefoot/left                Hair, sparse, Forefoot/right                Hair, sparse, Forelimb/left                Hair, sparse, Forelimb/right                Hair, sparse, Hind foot/left                Hair, sparse, Hind foot/right                Hair, sparse, Hind limb/left                Hair, sparse, Hind limb/right                Hair, sparse, Inguinal region/left                Hair, sparse, Lumbar region                Hair wet, Anogenital region                Scabbed area, Cervical region                Scabbed area, Forelimb/right	0/0 0/0 0/0 0/0 14/1 0/0 47/7 42/6 1/1 5/1 9/2 9/2 7/1 7/1 0/0 1/1 0/0 0/0 0/0	0/0 0/0 0/0 0/0 0/0 0/0 10/3 19/4 12/1 12/1 4/1 4/1 0/0 0/0 6/1 9/1 0/0 2/1 0/0	0/0 0/0 0/0 0/0 0/0 30/3 20/2 23/4 54/7 42/5 0/0 0/0 6/1 10/1 0/0 5/1 0/0 0/0 8/1	4/2 3/2 3/2 1/1 21/3 0/0 1/1 5/1 35/3 14/1 0/0 0/0 27/3 14/1 0/0 9/1 2/1 0/0 6/2

+ Number of times observed/Total number of animals affected.

 

Summary of Gestation Body Weight Values

Endpoint	Study Interval (Day)	0 mg/kg/day			100 mg/kg/day			200 mg/kg/day			400 mg/kg/day
		Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Body Weight Values g
	0	214.1	18.94	24	214.8	18.47	24	215.3	20.20	24	215.0	20.55	23
	3	228.2	17.73	24	230.3	17.49	24	231.4	20.78	24	215.2	20.71	23
	6	243.3	18.60	24	245.3	18.44	24	248.9	22.21	24	237.9	21.35	23
	9	262.9	19.04	24	265.9	18.52	24	270.5	23.94	24	260.7	21.19	23
	12	283.3	20.42	24	285.6	18.85	24	292.8	27.78	24	284.6	22.81	23
	15	301.1	20.34	24	304.3	18.85	24	313.6	29.01	24	303.9	25.00	23
	18	336.2	26.04	24	338.9	21.33	24	350.7	33.29	24	336.3	27.33	23
	20	370.8	30.31	24	372.3	23.66	24	386.0	36.84	24	368.8	28.85	23

N – Number of measures used to calculate mean

SD – Standard Deviation

 

Summary of Gestation Body Weight Change Values

Endpoint	Study Interval (Day)	0 mg/kg/day			100 mg/kg/day			200 mg/kg/day			400 mg/kg/day
		Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Body Weight Values g
	0 – 3	14.0	4.47	24	15.5	5.49	24	16.1	8.51	24	0.1^b	9.69	23
	3 – 6	15.2	4.09	24	15.0	4.91	24	17.5	4.27	24	22.7^b	5.86	23
	6 – 9	19.5	4.67	24	20.6	3.86	24	21.6	5.24	24	22.8^a	4.01	23
	9 – 12	20.4	4.78	24	19.7	4.70	24	22.4	5.45	24	23.8	4.77	23
	12 – 15	17.8	5.30	24	18.7	3.29	24	20.8	4.29	24	19.3	4.47	23
	15 – 18	35.1	7.86	24	34.7	6.49	24	37.1	5.99	24	32.4	4.75	23
	18 - 20	34.6	6.70	24	33.3	5.62	24	35.3	5.29	24	32.5	4.50	23
	0 - 20	156.7	19.12	24	157.5	17.40	24	170.7^a	21.25	24	153.7	18.35	23

N – Number of measures used to calculate mean        ^aSignificantly different from control; (p<0.05)

SD – Standard Deviation                                                   ^bSignificantly different from control; (p<0.01)

 

Summary of Gestation Food Consumption Values

Endpoint	Study Interval (Day)	0 mg/kg/day			100 mg/kg/day			200 mg/kg/day			400 mg/kg/day
		Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Food Consumption g/animal/day
	0 – 3	13.6	1.89	24	13.8	2.13	24	14.2	3.48	24	9.1^b	3.23	23
	3 – 6	17.1	2.31	24	19.2	1.73	24	21.1^b	2.52	24	19.6^a	4.92	23
	6 – 9	18.5	2.27	24	19.4	1.43	24	21.9^b	3.30	24	20.5	4.27	23
	9 – 12	19.1	2.15	24	20.1	1.64	24	22.4^b	2.87	24	22.3^b	2.66	23
	12 – 15	22.3	2.04	24	23.3	1.56	24	24.6^b	3.03	24	25.3^b	3.22	23
	15 – 18	24.5	3.17	24	25.1	2.21	24	26.6	3.32	24	25.6	3.17	23
	18 - 20	23.1	3.35	24	24.8	2.12	24	25.4^a	3.56	24	24.2	2.85	23
	0 - 20	19.6	1.97	24	20.6	1.34	24	22.2^b	2.66	24	20.8	2.70	23

N – Number of measures used to calculate mean        ^aSignificantly different from control; (p<0.05)

SD – Standard Deviation                                                   ^bSignificantly different from control; (p<0.01)

 

Summary of Maternal Developmental Observations at Uterine Examination

Endpoint		0 mg/kg/day	100 mg/kg/day	200 mg/kg/day	400 mg/kg/day
No. Females on Study		25	25	25	25
No. Not Pregnant		1	1	1	2
No. Pregnant		24	24	24	23
Pregnancy Index Percent		96.0	96.0	96.0	92.0
No. Females with Viable Fetuses Day 20 Gestation		24	24	24	23
Corpora Lutea                No. per Animal	Mean SD N	12.9 2.08 24	13.5 2.08 24	13.5 1.53 24	13.8 2.84 23
Implantation Sites                No. per Animal	Mean SD N	12.3 2.09 24	11.3 1.57 24	12.7 1.95 24	12.3 1.42 23
Preimplantation Loss                % per Animal	Mean SD N	5.01 7.506 24	14.36^b 13.246 24	5.67 7.263 24	9.51 11.254 23
Viable Fetuses                No. per Animal	Mean SD N	11.5 2.04 24	10.8 1.84 24	12.0 1.43 24	11.4 1.50 23
Fetal Sex Ratio                % Males per Animal	Mean SD N	54.0 16.36 24	49.8 15.02 24	53.4 16.60 24	45.4 18.37 23
Postimplantation Loss                % per Animal	Mean SD N	6.27 7.070 24	4.73 7.509 24	4.94 7.831 24	6.79 5.383 23
Nonviable Fetuses                No. per Animal	Mean SD N	0.0 0.00 24	0.0 0.00 24	0.0 0.00 24	0.0 0.00 23
Litter Size                No. per Animal	Mean SD N	11.5 2.04 24	10.8 1.84 24	12.0 1.43 24	11.4 1.50 23
Resorptions: Early + Later                No. per Animal	Mean SD N	0.8 0.88 24	0.5 0.78 24	0.6 1.01 24	0.8 0.65 23
Resorptions: Early                No. per Animal	Mean SD N	0.8 0.85 24	0.5 0.78 24	0.6 0.97 24	0.8 0.65 23
Resorptions: Late                No. per Animal	Mean SD N	0.0 0.00 24	0.0 0.00 24	0.0 0.00 24	0.0 0.00 23

No. – Number                                                                                      ^bSignificantly different from control; (p<0.01)

N – Number of measured used to calculate mean

SD – Standard Deviation

Conclusions:

This study was conducted to evaluate the potential adverse effects of the test article, Reofos 35, on systemic toxicity, female reproductive performance, and development of the fetus following repeated exposure to pregnant rats. Dose levels evaluated were 0 (corn oil vehicle), 100, 200, or 400 mg/kg/day and animals were treated orally with a single dose daily from GD 0 to 19. All animals survived to scheduled termination. The No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was 200 mg/kg/day and the No-Observed-Effect Level (NOEL) for developmental toxicity was 400 mg/kg/day, the highest dose level evaluated. Reofos 35 was not teratogenic in the rat at the dose levels tested.

Executive summary:

The study was conducted for Chemtura Corporation to evaluate the potential adverse effects of the test article, Reofos 35, following repeated exposure to pregnant animals from Gestation Day (GD) 0 to 19, including systemic toxicity, female reproductive performance, and evaluation of the fetuses.

The study was conducted in accordance with Standard Operating Procedures (SOPs) and the protocol as approved by the Sponsor. All SOP deviations were acknowledged by the Study Director and documented in the raw data. In the opinion of the Study Director, none of the SOP deviations affected the quality or integrity of the study. This study was based on Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011, the United States EPA, Office of Prevention, Pesticides, and Toxic Substances, Guideline 870.3700, Prenatal Developmental Toxicity, August 1998, and the OECD 414 Guideline for the Testing of Chemicals, January 2001.

 

Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination on GD 20. All fetuses were given the appropriate external, visceral, and/or skeletal examination.

Homogeneity of the dosing formulations was confirmed at the low- and high-dose levels in Week 1 and concentrations of test formulations used for dosing study ranged from 93.2 to 102.1% of nominal. No test article was found in control samples.

All control and treated animals survived to scheduled termination. Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. Its occurrence was attributed to a pharmacologic response to the test article and was not considered adverse. At dose levels ≤200 mg/kg/day, no adverse effects of the test article were observed from maternal clinical findings, gestation body weights, body weight change, food consumption, or macroscopic examinations. At 400 mg/kg/day, maternal toxicity was restricted to early in the treatment period (GD 0 to 3) and included clinical findings (i.e., decreased activity, red material around the nose/mouth, hunched posture, thin appearance, hair on the face discolored brown, and hair on the forelimbs discolored brown), low gestation weight gain, and low food consumption. No adverse effect of treatment with Reofos 35 at the dose levels evaluated was observed on pregnancy indices, uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, or skeletal evaluations. In the 400 mg/kg/day dose group, a low incidence of red or white foci and swollen mucosa of the nonglandular portion of the stomach was observed in the maternal macroscopic evaluations and considered test article-related.

Thus, in this rat oral prenatal developmental toxicity study with Reofos 35, the No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was 200 mg/kg/day and the No-Observed–Effect Level (NOEL) for developmental toxicity was 400 mg/kg/day, the highest dose level evaluated. Reofos 35 was not teratogenic in the rat at the dose levels tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

See discussion below

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A NOAEL of 200 mg/kg/day is documented for this endpoint on the basis of the maternal toxicity observed.

 

Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination on GD 20. All fetuses were given the appropriate external, visceral, and/or skeletal examination.

 

All control and treated animals survived to scheduled termination. Salivation was observed in each of the test substance-treated groups and considered a test article-related effect. Its occurrence was attributed to a pharmacologic response to the test article and was not considered adverse. At dose levels ≤200 mg/kg/day, no adverse effects of the test article were observed from maternal clinical findings, gestation body weights, body weight change, food consumption, or macroscopic examinations. At 400 mg/kg/day, maternal toxicity was restricted to early in the treatment period (GD 0 to 3) and included clinical findings (i.e., decreased activity, red material around the nose/mouth, hunched posture, thin appearance, hair on the face discolored brown, and hair on the forelimbs discolored brown), low gestation weight gain, and low food consumption. No adverse effect of treatment with the test substance at the dose levels evaluated was observed on pregnancy indices, uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, or skeletal evaluations. In the 400 mg/kg/day dose group, a low incidence of red or white foci and swollen mucosa of the nonglandular portion of the stomach was observed in the maternal macroscopic evaluations and considered test article-related.

 

Thus, in this rat oral prenatal developmental toxicity study with the test substance, the No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was 200 mg/kg/day and the No-Observed–Effect Level (NOEL) for developmental toxicity was 400 mg/kg/day, the highest dose level evaluated. The test substance was not teratogenic in the rat at the dose levels tested.

Justification for selection of Effect on developmental toxicity: via oral route:

See discussion below; based on maternal toxicity observed.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP and in compliance with agreed protocols. Some of the reports do not detail a specific method; however it documents dose levels and responses in detail, so is deemed appropriate for use in the support of a formal registration.  Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds in the absence of definitive data for this endpoint.

Justification for classification or non classification

Without further clarification, the reproduction toxicity triggered classification under the CLP Regulation (EC No 1272/2008) as follows:

Repr. Cat 2, H361f: Suspected of damaging fertility or the unborn child < Fertility - effects on ovaries; decreased live litter sizes