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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-09-05 to 2021-05-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Polyetheramine D 230
- Physical state: Colorless liquid
- Analytical purity: 100% as stated by sponsor
- Lot/batch No.: 000STD77L0
- Storage condition of test material: Room temperature
- Other: Test substance receipt-2010-09-16
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Reaction products of di-, tri- and tetra-propoxylated propane-1,2-diol with ammonia, Polyoxypropylenediamine, POPDA
- Physical state: Clear, colorless liquid
- Lot/batch No.: 9D410
- Expiration Date: 31 Jul 2021
- Purity: 99.6 %
- Correction factor: 1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept at controlled room temperature, blanketed under nitrogen
- Stability under test and storage conditions: not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: test substance is stable in R.O. deionized water when prepared and stored under the same conditions at concentrations bracketing those used in the present study (CRL Study 01318004).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: One hundred-eleven (53 male and 58 female) virgin Crl:CD(SD) Sprague-Dawley rats from Charles River Laboratories, Inc
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at animal arrival: 52 days
- Weight at study initiation:
- males: 202 - 238 g on day of transfer; 202 - 231 g at randomization
- females: 131 - 181 g on day of transfer; 201 - 241 g at randomization

- Housing:
Animals were socially housed in solid-bottomed cages by dose group (2 per cage/sex) until cohabitation. During the cohabitation period, one male rat and one female rat were pair housed in the male rat’s solid-bottomed cage. After cohabitation, female rats were individually housed until delivery or scheduled euthanasia. Male rats were returned to the same sex housing that was established prior to the mating period. Each dam and delivered litter were housed in a common nesting box. Control group animals were housed on a separate rack from the test substance-treated animals.
Nesting material (Bed-o'Cobs®) was provided throughout the course of the study. Nesting material was changed as often as necessary to keep the animals dry and clean. Analyses for possible contamination were conducted on each lot of nesting material.
There were no known contaminants in the nesting material that would interfere with the objectives of the study.
Rats were provided with Crink-l’Nest™ and a pelleted food. Analyses for possible contamination were conducted on each lot of enrichment devices. There were no known contaminants in the enrichment devices that would interfere with the objectives of the study.

- Diet (e.g. ad libitum): ad libitum, pelleted Certified Rodent Diet® #5002 (PMI® Nutrition International), available from individual feeders. The food was analyzed for environmental contaminants.
There were no known contaminants in the food that would interfere with the objectives of the
study.

- Water (e.g. ad libitum): ad libitum, All water was from a local source and passed through a reverse osmosis membrane before use. Chlorine was added to the processed water as a bacteriostat; processed water was expected to contain no more than 1.2 ppm chlorine at the time of analysis. Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility.
There were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: After receipt at the Testing Facility, the rats were acclimated for at least 4 days prior to initiation of estrous cycling (female rats) or initiation of dose administration (male rats).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Air changes (per hr): a minimum of 10 changes per hour of fresh air that had been passed through 99.97% HEPA filters.
- Photoperiod (hrs dark / hrs light): automatically controlled 12-hour light/12-hour dark cycle was maintained (± 30 minutes), except during designated procedures. On 3 occasions, the lights were turned on during the dark period (up to 19 minutes) to facilitate study activities.

IN-LIFE DATES: From: 2019-09-05 To: 2019-11-18

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
R.O. deionized
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Preparation of Control Substance:
The control substance was dispensed daily for administration to Group 1 animals. An adequate
amount of the control substance was dispensed once weekly into daily aliquots, which were
stored in a refrigerator set to maintain 4°C until use. The aliquots were removed from the
refrigerator and allowed to warm to room temperature and stirred for at least 30 minutes before
dosing. The dosing formulation was also stirred continuously during dosing.
- Preparation of Test Substance:
Test substance dosing formulations were prepared based on Sponsor instructions at appropriate
concentrations to meet dose level requirements. The dosing formulations at least once weekly,
blanketed under nitrogen, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and allowed to warm to room temperature and stirred for at least 30 minutes before dosing. The dosing formulations were also
stirred continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): R.O. deionized water
- Storage conditions: Kept at ambient temperature
- Concentration in vehicle: 0, 15, 30, 60, 120 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: Acclimatization period: 2 per cage/sex until cohabitation. Cohabitation period: one male rat and one female rat were pair housed. After cohabitation: female rats were individually housed until delivery or scheduled euthanasia. Male rats were returned to the same sex housing that was established prior to the mating period. Each dam and delivered litter were housed in a common
nesting box.
- Length of cohabitation: maximum of 7 days
- Proof of pregnancy: Females with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis:
- at first preparation: all groups (concentration), groups 2 and 5 (homogeneity)
- last or second to last preparation: all groups (concentration)

Dose formulation samples have been analyzed by high performance liquid chromatography
method using charged aerosol detection (HPLC-CAD) for the determination of Diaminopolypropylene glycol (MW > 230 g/mol), using a validated analytical procedure (1318.DIW1.02).
Duplicate (1 mL) sets of top, middle, and bottom test substance samples (duplicate middle only for Groups 1, 3, and 4) for the sampling time points were collected; triplicate sets of top, middle, and bottom test substance samples (triplicate middle only for Groups 1, 3, and 4) were collected in the same manner and stored in a refrigerator set to maintain 4°C, blanketed under nitrogen, at the Testing Facility as backup samples and were discarded after Study Director authorization. On days where only concentration analysis was required, the formulations were only sampled from the middle. At the last preparation sampling, 5 sets of top, middle, and bottom samples were inadvertently collected for the Group 2 dose level. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤ 5% for each group.

The dose formulations were within specification. Homogeneity testing showed that the
formulation technique used produced homogeneous preparations.
Duration of treatment / exposure:
Males: 62-63 days, beginning 28 days before cohabitation with females, during cohabitation and continuing through the day before euthanasia.
Females: 49-56 days, beginning 15 days before cohabitation with males and continuing through postpartum day 12 (rats that delivered a litter) or gestation day 24 (rats that did not deliver a litter). Any dam in the process of parturition was not given the test substance or the control substance formulations until the following work day.
F1 generation pups were not directly given the test substance or control substance formulations but were possible exposed to the test or vehicle control substance formulations during maternal gestation (in utero exposure) or via maternal milk during the lactation period.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of Route and Dose Levels:
The oral (gavage) route was selected for use because this is the intended route of human exposure. The doses were selected based on previous dietary toxicity studies conducted in rats and rabbits. In these studies, doses up to 150 mg/kg/day were generally well tolerated in both species. Therefore, the high dose of 600 mg/kg/day was selected as it was anticipated to show some signs of toxicity. The low dose of 75 mg/kg/day was selected as it was anticipated to result in no signs of overt toxicity. The intermediate doses of 150 and 300 mg/kg/day were the approximate geometric means of the low and the high doses and were anticipated to provide dose response relationships.

Justification for the choice of species:
The test system was selected because: 1) it is a standard species accepted for use in fertility and early embryonic development studies; 2) this species and strain has been demonstrated to be sensitive to reproductive toxicants; and 3) historical data and experience exist at the Testing Facility.

Deviations from standard protocol:
- On 08 Oct 2019, P generation female 340 (Group 4) was found in the nesting box with male 238 and female 339 (Group 4). As female 340 was observed with a vaginal plug, it cannot be determined who this female mated with during cohabitation. This deviation did not impact the outcome of the study because this rat will be excluded from summarization and statistical analyses.
- On DG 2 (14 Oct 2019), P generation female 326 (Group 3) was found to still be housed with P generation male 226 (Group 3) after previously being confirmed mated on 12 Oct 2019. The pair were separated and placed into single housing at this time. This deviation did not impact the outcome of the study because this female had previously mated and had a confirmed date of mating.
Positive control:
Not applicable.

Examinations

Parental animals: Observations and examinations:
VIABILITY CHECKS: Yes
- Time schedule: Viability was observed at least twice daily.

CAGE SIDE AND DETAILED OBSERVATIONS: Yes
- General Appearance: once (males) or 4 times (females) during the acclimation period and daily during the dose and postdose periods.
- Postdose Observations: between 1 to 2 hours after dose administration.
- Maternal Observations: Daily during the postpartum period.
- Natural delivery observations: Beginning on GD 17, the dam was observed at least twice daily.

BODY WEIGHT: Yes
- Males: body weights were recorded on the day of transfer from the Testing Facility’s general population and daily during the dose and postdose periods.
- Female: body weights were recorded on the day of transfer from the Testing Facility’s general population, 3 times during the acclimation period and daily during the dose and postdose periods.

FOOD CONSUMPTION: Yes
- Males: food consumption values were recorded at least twice weekly during the dose period up until the initiation of cohabitation (food left value).
- Females: At least twice weekly during the dose period up until the initiation of cohabitation, on DGs 0, 4, 7, 11, 14, 17, 20 and 25 (as applicable) and on DL 0, 4, 7, 10 and 13.

HAEMATOLOGY
Thyroid Analyses:
- Blood samples (0.5 mL to 1.5 mL) were collected from each rat at scheduled euthanasia via the
inferior vena cava and placed into serum separator tubes.
- The blood samples were allowed to clot at room temperature for at least 20 minutes (not exceeding 1 hour) and then centrifuged for 15 minutes at 3500 rpm in a centrifuge set to
maintain 21°C. The resulting serum was separated and divided into two aliquots with 250μL of serum for TSH analysis and the remaining for T4 analysis, and frozen immediately on dry ice until stored in a freezer set to maintain -20°C (TSH samples) or -80°C. T4 serum samples were shipped (frozen on dry ice) to Charles River Laboratories, Ashland, LLC for analysis.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by
vaginal lavage for 14 consecutive days before initiation of dose administration, for
14 consecutive days beginning with the day after the first dose administration, and then until
spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was
observed in situ during the cohabitation period. On DL 13, an examination of vaginal cytology
was performed prior to scheduled euthanasia to determine the stage of estrous cycle.
Sperm parameters (parental animals):
No data.
Litter observations:
VIABILITY CHECKS
- Natural delivery observations: Beginning on GD 17, the number and vital status of the pups were recorded
- Litters were observed for dead pups at least twice daily and the pups in each litter were counted
once daily.

CLINICAL OBSERVATIONS
- Clinical observations were recorded daily.

BODY WEIGHTS
- Body weights were recorded on Days 0 (birth) 4, 7, 10 and 13 postpartum.

ANOGENITAL DISTANCE
- On Day 0 postpartum, the measurement was taken using a calibrated stereomicroscope, micrometer and ruler. For male pups, the anogenital distance was measured from the cranial edge of the anus to the base of the anogenital aperture. For females, the anogenital distance was
measured from the cranial edge of the anus to the base of the urinary aperture.

NIPPLE PRESENCE - ALL MALE ANIMALS
- On Day 12 postpartum, nipple presence and number were evaluated for all F1 generation male
pups.

HEMATOLOGY
Thyroid Analyses:
- Blood samples were collected via cardiac puncture following euthanasia on Day 4 postpartum (0.01 mL to 0.3 mL) from the culled pups (pooled by litter) and on Day 13 postpartum (0.01 mL
to 1.0 mL) from up to 2 pups/sex/litter, when possible, and placed into serum separator tubes.
- The blood samples were allowed to clot at room temperature for at least 10 minutes and then
centrifuged for 10 minutes at 3000 g in a centrifuge set to maintain 4°C within 30 minutes of collection. For Day 4 postpartum, the resulting serum was transferred into uniquely labeled polypropylene tubes and immediately frozen on dry ice until transferred into a freezer set to maintain -80°C. For Day 13 postpartum, the resulting serum was transferred into 2 uniquely labeled polypropylene tubes and frozen immediately on dry ice until stored in a freezer set to maintain -20°C (TSH samples) or -80°C .
- T4 serum samples were shipped (frozen on dry ice) to Charles River Laboratories, Inc., Ashland,
OH for analysis.


Postmortem examinations (parental animals):
SACRIFICE:
- Sacrifice / method of euthanasia: P generation rats were euthanized by carbon dioxide asphyxiation.
- Unscheduled death: The rats that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions.
- Scheduled euthanasia:
Female rats:
- Females that did not deliver a litter were euthanized on DG 25 and examined. Blood
samples were collected.
- On Day 13 postpartum, surviving P generation female rats were euthanized and examined. Blood samples were collected.
Male rats:
- On DS (= day of study) 63 or 64, surviving P generation male rats were euthanized and examined. Blood samples were collected.

OVARIAN AND UTERINE EXAMINATIONS
The reproductive tract was dissected from the abdominal cavity. The number and distribution of
implantation sites and corpora lutea were recorded. A full uterine examination was conducted on
P generation female 310 in the 0 mg/kg/day dose group (Group 1). The uteri of the apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites.

NECROPSY
The rats were examined for gross lesions, and subjected to a gross necropsy of the thoracic,
abdominal, and pelvic viscera. Images were generated for illustration of or consultation on gross observations. Generation of such images was documented. Images and associated documentation were retained and were archived.

ORGAN WEIGHTS
The organs mentioned below were weighed at necropsy for all scheduled euthanized
P generation rats. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) were calculated.
The following organs were weighed: Cervix, Epididymis, Gland prostate, Gland seminal vesicles (with coagulating gland), Gland thyroid, Liver, Ovaries, Oviducts, Testes, Uterus.

TISSUE COLLECTION AND PRESERVATION
Representative samples of the tissues mentioned below were collected from all rats and
preserved in 10% neutral buffered formalin, unless otherwise indicated. A table of random units was used to select one control group rat per sex (male 206 and female 310) from which all tissues examined at necropsy were retained, in order to provide control tissues for any possible future evaluations of gross lesions.
Tissues collected from all rats and preserved in 10% neutral buffered formalin: Cervix, Epididymis, Gland, mammary, Gland prostate, Gland seminal vesicles (with coagulating gland), Gland thyroid, Gross lesions/masses, Liver, Ovaries, Oviducts, Testes, Uterus.

HISTOLOGY
Tissues identified (Epididymis, Gland prostate, Gland seminal vesicles (with coagulating gland), Gland thyroid, Gross lesions/masses, Ovaries, Testes) from all rats that died or were euthanized before scheduled termination, all rats in Groups 1 and 5 and all gross lesions from any rat were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Histopathological evaluation was performed by a board-certified veterinary pathologist on all
rats that died or were euthanized before scheduled termination, all rats in Groups 1 and 5, and all
gross lesions from all rats.
Postmortem examinations (offspring):
SACRIFICE
- Method of Euthanasia: F1 generation pups were euthanized by an intraperitoneal injection of sodium pentobarbital (390 mg/mL).
- Unscheduled death: Pups that were found dead before examination of the litter for pup viability (Day 0 postpartum) were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were identified as stillborn; pups with lungs that float were identified as liveborn and to have died shortly after birth. Pups that died (Days 1 to 8 postpartum) or were euthanized (Day 0 postpartum) before scheduled termination were examined for gross lesions and the cause of death or condition as soon as possible after the observation was made. The presence or absence of milk in the stomach was determined.
- Scheduled Euthanasia: Blood samples were collected. Gross lesions from all pups on
Day 13 postpartum and the thyroid/parathyroid on Day 13 postpartum were retained for
1 pup/sex/litter and preserved in 10% neutral buffered formalin for possible future evaluation. Additional thyroid/parathyroid were inadvertently retained for pups in litters 309 in the 0 mg/kg/day dose group (Group 1), 314 and 319 in the 75 mg/kg/day dose group (Group 2), and 330 in the 150 mg/kg/day dose group (Group 3).

NECROPSY
Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Carcasses were discarded without further evaluation. For pups that had a complete necropsy performed, a cross section of the head at frontal parietal suture was not completed.
Statistics:
See Any other information on materials and methods incl. tables section.
Reproductive indices:
• Gestation Length: the gestation length is calculated from GD 0 to the day the first pup is observed.
• Female Pregnancy Index: Number of Pregnant Females / Number of Females Paired
• Gestation Index: Percentage of pregnancies that result in birth of live litters
Number of Animals with Live Offspring x 100 Number of Animals Pregnant
Offspring viability indices:
• Live Birth Index: Percentage of pups born alive.
Number of Live Newborn Pups x 100 Number of Newborn Pups
• Viability Index: Percentage of pups born that survive 14 days postpartum

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: An increased incidence of abnormal breathing sounds was observed at 150, 300 and 600 mg/kg/day and excess salivation at 300 and 600 mg/kg/day in comparison with the control
group values. There was also an increased incidence of labored breathing and staining on the fur
at 600 mg/kg/day in comparison with the control group values.
All other clinical signs that were observed were considered unrelated to the test substance
because: 1) the observations were not dose-dependent; and/or 2) the observations occurred in
only one rat in a dose group and the clinical signs did not persist. These clinical signs included
localized tremors, suspected dehydration, hunched posture, wet fur, thin fur cover, a lesion on
the skin, a scab on the skin, hypersensitive, hyperreactive, abnormal consistency of the feces,
sneezing, broken teeth, low carriage, convulsions, domed head and decreased activity.

Females: An increased incidence of abnormal breathing sounds was observed at 300 and 600 mg/kg/day in
comparison with the control group value. At 600 mg/kg/day, there was an increased incidence of
excess salivation, labored breathing, thin body, erected fur, cold to touch and fur staining in
comparison with the control group values.
All other clinical signs that were observed were considered unrelated to the test substance
because: 1) the observations were not dose-dependent; and/or 2) the observations occurred in
only one rat in a dose group and the clinical signs did not persist. These clinical signs included
hunched posture, a scab on the skin, hypersensitive and red nostril discharge.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Males: Male 236 at 300 mg/kg/day was euthanized on DS 54 due to adverse clinical signs. This rat had
abnormal breathing sounds on DSs 6 to 32 and 37 to 51; excess salivation on DSs 9, 11 and 29;
low carriage on DSs 18 to 20; domed head on DSs 18 to 54; hypersensitive on DSs 27 and 28;
hunched posture on DSs 31 to 54; labored breathing on DSs 33 to 39; soft feces on DSs 45 and
46; suspected dehydration on DSs 49 and 50; red staining on the fur of the eyelid on DS 52; a
lesion on the skin on DSs 53 and 54; and decreased activity on DS 54. There were no test
substance related effects on body weight changes observed in this animal, and all tissues
appeared normal at necropsy. This death was considered to be test substance related due to the
number and severity of the clinical signs that were observed.

Females: One female at 300 mg/kg/day was found dead on DS 15. At 600 mg/kg/day, there were two
females that were found dead and another two females that were euthanized due to adverse
clinical signs. The two females that were euthanized at 600 mg/kg/day had issues with
respiration (abnormal breathing sounds and/or labored breathing) prior to being euthanized. One
of these females also had a rapid body weight loss prior to being euthanized. There were no
additional differences in body weight or body weight gain observed in these females in
comparison with the other females in this group. One of the females that was found dead had
numerous dark red foci on the thymus, and all of the other females appeared normal at necropsy.
The deaths observed at 300 and 600 mg/kg/day were considered to be test substance related.
Clinical and necropsy observations and body weight values for each of these rats either found
dead or humanely euthanized are described below. All other female rats survived until
scheduled euthanasia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: At 600 mg/kg/day, there were decreases or statistically significant decreases (p≤0.05 or p≤0.01)
in mean body weights beginning on DS 33 and continuing at all intervals thereafter in
comparison with the control group values. Statistically significant (p≤0.05) decreases in body
weight gains or body weight losses were observed at 600 mg/kg/day on DSs 9 to10, 14 to 15 and
34 to 35 in comparison with the control group values.
At the end of the dosing period (on DS 63), the mean body weights in the male rats were 100%,
98%, 100% and 88% of the vehicle control group value in the 75, 150, 300 and 600 mg/kg/day
dose groups, respectively. There was a statistically significant increase (p≤0.05) in body weight
gain observed at 75 mg/kg/day on DSs 1 to 2 in comparison with the control group value. On
DSs 33 to 34, there were statistically significant increases (p≤0.05 or p≤0.01) in body weight
gain observed at 150, 300 and 600 mg/kg/day in comparison with the control group value. There
were statistically significant (p≤0.05 or p≤0.01) decreases in body weight gain or body weight
loss observed on DSs 34 to 35 at 75 and 150 mg/kg/day in comparison with the control group
value. There were also statistically significant increases (p≤0.05) in body weight gain observed
on DSs 52 to 53 at 75, 300 and 600 mg/kg/day in comparison with the control group value.
These changes in body weight gain were not considered to be test substance related because they
were single occurrences and did not persist.

Females: At 600 mg/kg/day, there were decreases or statistically significant decreases (p≤0.05 or p≤0.01)
in mean body weights beginning on DS 9 and continuing through the pre-mating, gestation and
lactation periods in comparison with the control group values. There was a statistically
significant (p≤0.01) body weight loss observed at 600 mg/kg/day on DGs 10 to 11 in comparison
with the control group value.
Maternal body weights and body weight gains were unaffected by the test substance during the
pre-mating, gestation and lactation periods by doses up to and including 300 mg/kg/day. At
75 mg/kg/day, there was a statistically significant decrease (p≤0.05) in body weight gain
observed on DGs 4 to 5 in comparison with the control group value.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males: Food consumption was unaffected by doses of the test substances in the males at all intervals
prior to cohabitation at doses up to and including 600 mg/kg/day.

Females: At 600 mg/kg/day, there were decreases in food consumption observed on DGs 4 to 7, 7 to 11
and 11 to 15 in comparison with the control group values.
Maternal food consumption values were unaffected by the test substance during the gestation and
lactation periods by doses up to and including 300 mg/kg/day. There were statistically
significant increases (p≤0.05) in food consumption observed on LDs 10 to 13 at 75 and
300 mg/kg/day in comparison with the control group value. These increases were not considered
to be test substance related because they were not dose dependent.
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, non-treatment-related
Description (incidence and severity):
There were no test substance-related microscopic findings noted in the males or the
LD 13 female rats. The microscopic findings observed were considered incidental, of the nature
commonly observed in this strain and age of rats, and/or were of similar incidence and severity
in control and treated animals and, therefore, were considered unrelated to administration of the test substance.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day, there were decreases in the number of cycles and an increase in the mean of
cycle lengths (days) observed during the estrous evaluation performed 14 days prior to the
cohabitation period in comparison with the control group value. There were also statistically
significant reductions (p≤0.05) in the number and percentage of the female fertility and
pregnancy indices in comparison with the control group values. All additional mating and fertility parameters were unaffected by doses of the test substance as
high as 300 mg/kg/day.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day, there were statistically significant reductions (p≤0.05) in the number and
percentage of the male fertility index and the number and percentage of the male pregnancy
index in comparison with the control group values.
All mating and fertility parameters were unaffected by doses of the test substance up to and
including 300 mg/kg/day.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical observations in the F1 generation pups were attributable to maternal doses of the test
substance as high as 600 mg/kg/day.
All clinical signs that were observed were considered unrelated to the test substance because:
1) the observations were not dose dependent; and/or 2) the observations occurred in only one pup
in a particular dose group. These clinical signs included thin appearance; pallor skin; suspected
dehydration; coldness to the touch; scab on the skin; constricted hindpaw; and purple skin.
Dermal irritation (if dermal study):
not examined
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No changes in body weights in the F1 generation pups were attributable to maternal doses of the
test substance as high as 600 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances for the male and female pups were comparable across all dose groups
and there was no statistically significant difference.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no male pups observed with the
presence of nipples on study.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Terminal body weights were not affected at any dose level. All organ weights (absolute and
relative) were unaffected by doses of the test substance up to and including 600 mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance related observations in the pups at the time of necropsy. During the
unscheduled necropsies, absent stomach content was observed in 2, 1 and 1 pups in the 75,
150 and 300 mg/kg/day maternal dose groups, respectively. There was 1 pup in the
300 mg/kg/day maternal dose group that had abnormal consistency in the right renal papilla at
the time of scheduled necropsy. These differences were not considered to be test substance
related because the changes were not dose dependent.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Endocrine findings: There were no test substance-related differences in T4 thyroid hormone levels in PND 4 pups, or PND 13 male or female pups. There were statistically
significant decreases (p≤0.05) in the mean T4 hormone levels in the PND 4 pups in the
75 mg/kg/day maternal dose group and the PND 13 female pups in the 150 mg/kg/day maternal
dose group as compared to the control group values. These differences were not considered to
be test substance related because they were not dose dependent.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Growth & development

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Dose Formulation Analyses
The dosing suspensions of the test substance prepared in R.O. deionized water were analyzed following the first and last preparations and were found to be within ± 10% of the target concentration and homogeneous under the conditions of this study.
The first preparation of the dosing suspensions of 15, 30, 60 and 120 mg/mL were analyzed and found to be 102%, 104%, 101% and 104% of the target concentrations, respectively. The homogeneity values obtained for the first preparation were 2.7% and 2.7% RSD for the 15 and 120 mg/mL formulations, respectively. The last preparation of the dosing suspensions of 15, 30,
60 and 120 mg/mL were analyzed and found to be 98.0%, 105%, 96.8% and 93.8% of the target concentrations, respectively.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general
toxicity was 150 mg/kg/day for the male and female rats. There was an increased incidence of
abnormal breathing sounds in the male rats at 150 mg/kg/day; however, this was not considered
to be adverse as there were no additional effects observed at this dose. Mortality and adverse
clinical signs were observed in the male and female rats at 300 and 600 mg/kg/day. Reductions
in body weight and body weight gain were observed in males and females at 600 mg/kg/day.
The reproductive NOAEL was 300 mg/kg/day as there were no test substance-related changes in
estrous cycling in the female and mating and fertility in the males or females. There were
effects on estrous cycling, mating and pregnancy observed at 600 mg/kg/day. The NOAEL for
viability and growth in the offspring was 600 mg/kg/day based on the growth and development
of the three litters at this dose.