Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-(2-aminomethylethyl)-.omega.-(2-aminomethylethoxy)-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

- In a screening reproductive toxicity study (according to OECD guideline 421; Calvert Laboratories Inc, 2011; key; Klimisch 1), rats were exposed to 3, 10, 30 mg/kg (nominal in water) via topical application. No systemic, reprotoxic or developmental effects were observed: a NOAEL of 30 mg/kg bw/day was derived.

 

- In a screening reproductive toxicity study (according to OECD guideline 421; Charles River Laboratories Inc., 2021; key; Klimisch 1), rats were exposed to 0, 15, 30, 60, 120 mg/kg/day (in R.O. deionized water) via oral gavage. 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. The reproductive NOAEL was 300 mg/kg/day and the NOAEL for viability and growth in the offspring was 600 mg/kg/day.

 

- In a key, K1 extended one-generation reproductive toxicity study (performed according to OECD guideline 443 and compliant to GLP requirements), rats were exposed to 50, 150 and 450 mg/kg bw/day via oral gavage (Barnett, 2020). Based on draft results, the NOAEL for general, systemic toxicity for the P and F1 generation is considered to be 150 mg/kg bw/day. The reproductive and developmental NOAEL was set at 450 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

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.

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

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: 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.

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

Key result

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

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

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.

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-10-11 to 2010-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)-
- Physical state: clear colorless liquid
- Lot/batch No.: OF502
- Storage condition of test material: at room temp (10-24.6°C)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan
- Age at study initiation: (P) x a minimum of 13 weeks at initiation of cohabitation
- Weight at study initiation: (P) Males: >/=300 g; Females: >/=200 g at initiation of cohabitation
- Fasting period before study:
- Housing: Upon arrival and until randomization, males and females were group-housed, sexes separate. Following randomization and until cohabitation, males and females were individually housed. During cohabitation, one female was placed with a male breeder from the same group. Following cohabitation, males and females were housed individually.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): All animals had access to Harlan Teklad Rodent Diet (certified) or equivalent ad libitum.
- Water (e.g. ad libitum): Water was available ad libitum via an automatic watering device.
- Acclimation period: Study animals were acclimated to their housing for a minimum of 7 days prior to the first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights were turned on during the dark cycle to accommodate blood sampling or other study procedures.

IN-LIFE DATES: From: To:

Route of administration:

dermal

Vehicle:

other: deionized water

Details on exposure:

TEST SITE
- Area of exposure: 4cm x 4cm
- % coverage:
- Type of wrap if used:
- Time intervals for shavings or clipplings: The pelage covering the interscapular and dorsal thoracic regions of the body of the study animals were clipped at least 48 hours prior to the first topical dose and additionally as needed.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): In case of excessive test substance buildup the application site was cleansed after consultation with the sponsor.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 ml/kg (concentrations of 20, 50 and 80 mg/ml) of the test substance was distributed with a syringe as evenly as possible over the exposure area of the intact skin of each animal.
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 ml/kg

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; During the dosing period the rats were fitted with BiteNot collars 8 hours a day to prevent the animal from licking the test substance.

Details on mating procedure:

- M/F ratio per cage: 1 male to 1 female from the same group
- Length of cohabitation: Males and females remained together until evidence of copulation was noted or for a maximum of three weeks.
- Proof of pregnancy: Day 0 of gestation was determined by evidence of copulation which was determined by the examination of vaginal smears made daily to determine if sperm were present in a smear of vaginal contents or by the presence of a copulatory plug in situ. Examinations of vaginal smears were performed at approximately the same time each day (early morning) through the cohabitation period.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: After three weeks of cohabitation, the Study director was able to elect to move certain females with other males from the same dose group, in an attempt to expedite mating.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): Following cohabitation, the females were housed individually.
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first day of dosing, at the beginning of cohabitation and at the last day of dosing, duplicate 1-ml samples were obtained from top, middle and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test substance in vehicle. In addition, samples were tested for stability. These samples were stored at room temperature, approximately 10 to 30 degrees C.

Duration of treatment / exposure:

The males were treated once daily for a minimum of four weeks (starting two weeks prior to cohabitation). Treatment continued during the same-group cohabitation period and until the day before sacrifice (Day 28/euthanasia Day 29). Females were treated once daily for a minimum of 15 days prior to cohabitation, during cohabitation and from presumed Gestation Days 0 through Day 19 of gestation.

Frequency of treatment:

once daily

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/group (80 animals)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based upon previously conducted toxicity studies. A dermal dose range finding study was performed by Calvert labs (report 0325RH11.001, dated March 21, 2011). Test article was dosed by topical application to 80 SD rats (40 males, 40 females) approximately 11 weeks old at initiation. Four dose levels used: control, 100 mg/kg, 250 mg/kg, 400 mg/kg. Due to necrosis and thickening of the skin at the test article application site, dosing for group 3 and 4 was discontinued on day 2 and animals were euthanised. On day 4, similar effects were observed in the dosing group 2. Due to these severe skin effects observed in the 100-400 mg/kg/d dose groups, 24 additional animals were added to the study in order to evaluate dermal effects at lower dose levels: 8 animals (4 male/4 female) were divided over 3 dose groups: 10 mg/kg, 25 mg/kg and 75 mg/kg. The dose was applied once daily for 7 days. Dose administration was discontinued for the 75 mg/kg dose group on day 3 (necrosis). Surviving animals (dose group 10 and 25 mg/kg) were sacrificed on day 7.
Gross necropsy was performed (examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents; no tissues were retained).
Clinical signs in animals dosed at 25 mg/kg were limited to very slight to well defined erythema and fissuring at the dosing site of some male and female animals. The NOAEL was considered to be 10 mg/kg.

- Rationale for animal assignment: Using a random number generator, study animals were assigned a random number, sorted based upon the random number assignments, and then assigned to groups. Using SYSTAT version 9.0.1, an Analysis of Variance followed by Dunnett's test was performed to confirm that there were no significant differences in mean body weights between the study groups. If the group assignments showed statistical significane, animals were allowed to be reassigned within the order to improve the similarity of group mean body weights.

Positive control:

N/A

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was observed twice daily (a.m. and p.m.), once prior to scheduled sacrifice. Clinical observations were made throughout the treatment phase, a minimum of twice daily, prior to dose administration and a minimum of once following dosing. On non-dosing days, observations were made a minimum of once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed once weekly prior to initiation of cohabitation, during cohabitation and at terminal sacrifice. A final body weight was also obtained for all males sacrificed moribund. Females were weighed once weekly prior to initiation of cohabitation, on Gestation Days 0, 4, 7, 14 and 20, and on Day 0 and 4 of lactation. A final body weight was also obtained for all females showing signs of premature delivery or sacrificed moribund.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Full feeder weights and/or feeder weight backs were recorded once weekly, except during mating.

Oestrous cyclicity (parental animals):

Estrous cycle evaluation was performed daily for 2 weeks prior to treatment initiation and daily during the treatment and cohabitation periods.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed by CO2 asphyxiation 2 weeks post-mating.
- Maternal animals: All surviving animals were sacrificed by CO2 asphyxiation on Day 4 of lactation.

GROSS NECROPSY
For all males and females sacrificed moribund or found dead prior to scheduled sacrifice, a complete gross necropsy was performed. The necropsy included the examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The ear tag, all gross lesions, ovaries, uterus, cervix, vagina and prostate were retained in 10% neutral buffered formalin for possible histopathological evaluation. Additionally, for male rats, the testes and epididymides were retained in modified Davidson's fixative for possible histopathological evaluation. Where applicable, the total number of implantation sites and the total number of corpora lutea for each ovary were recorded. Where applicable, the number of viable and non-viable fetuses were also recorded. All fetuses were discarded.
For the terminally sacrificed males (2 weeks post-mating), necropsy included the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
For the terminally sacrificed females (Day 4 of lactation), necropsy included the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents. The total number of corpora lutea were determined for each ovary, and the total number of implantation sites, viable and non-viable fetuses, and early or late resorptions were also determined. For presumed non-gravid females, the uterus was stained with 10% ammonium sulfide to assess the presence of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The epididymides and testes were weighed before fixation, after dissection of excess fat and other excess tissues, for all males surviving until the scheduled sacrificed day. Organ to body weight ratios were calculated (using the final body weight obtained prior to necropsy), as well as organ to brain weight ratios.
All tissues for all animals were examined. For all animals necropsied, the following tissues were preserved in 10% neutral buffered formalin (except for the epididymides and testes that were retained in modified Davidson's fixative for optimum fixation): ovaries, uterus, cervix, vagina, testes, epididymides, prostate, seminal vesicles, and gross findings. Special emphasis on stages of spermatogenesis and histopathology of the interstitial testicular structure were give.
Tissues for evaluation were processed to paraffin blocks and prepared to slides. Slides were stained with hematoylin and eosin. Occasionally, other stains may have been required to aid in the diagnosis of lesions; these were documented when used.
Slides were prepared for all tissues for all rats in the vehicle and high dose groups and for all animals that died early. If test substance related lesions were noted, additional slides were prepared on those tissues from the low and mid dose groups.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were euthanized by an intraperitoneal injection of a barbiturate overdose on day 4 of lactation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

Statistical analysis was performed on In-Life, Clinical Pathology, and Necropsy data when 3 or more animals were present in 2 or more dose groups. Statistical analyses were not performed if N<3 animals per group. For In-Life and Clinical Pathology parameters, the homogeneity of the data was determines by Bartlett¿s Test. If the data was homogeneous, a one-way analysis of variance was performed to assess statistical significance. If statistically significant differences between the means were found, Dunnett¿s test was used to determine the degree of significance from the control means (p<0.05 and p<0.01). If the data was non-homogeneous, the Kruskal-Wallis non-parametric analysis was performed to assess statistical significance. If statistically significant differences between the means were found (p<0.05, p<0.01), the Mann-Whitney U-Test was used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups were present for evaluation, the Mann-Whitney U-test was used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means were made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, Dunnett¿s test was used to determine the degree of significance from the control means (p<0.05 and p<0.01).

Reproductive indices:

1. Pre-coital interval (in days): sum of days until successful copulation/# of presumed pregnant animals
2. Copulation index (%): # of presumed pregnant animals/# of paired animals x 100
3. Fertility index (%): # of pregnant animals/# of presumed pregnant animals x 100
4. Preimplantation loss (%): # of corpora lutea-# of implantations/# of corpora lutea x 100

Reproductive performance:

no effects observed

Description (incidence and severity):

More details on the reproductive indices are in data tables attached.

No effects were observed, except some local irritating effects at the site of administration at 30 mg/kg bw.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: male and female reproductive performance

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: parental clinical signs

Key result

Critical effects observed:

no

No effects were observed.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no treatment-related effects on pregnancy status; gestation, viability and related parameters; fetal weights and fetal morphological observations. There were no test article related changes in fetal sex ratios.

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

No systemic effects were observed at the highest dose administrated. Therefore a NOAEL of 30 mg/kg bw/d is derived for male and female reproductive performance. The NOAEL for parental clinical signs was considered to be 10 mg/kg bw/d. There were no treatment-related effects on pregnancy status; gestation, viability and related parameters; fetal weights and fetal morphological observations.

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-12-03 to final report date

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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 10 weeks (70 days), to cover the full spermatogenesis and folliculogenesis before mating (assessment of effects on fertility). There is no other substance specific information supporting shorter premating exposure time.

- Basis for dose level selection: Dose level selections were made based on the results of the OECD 421 study (Charles River Study No. 20209537). Mortality, adverse clinical signs, reduced body weight gain and food consumption were observed in the female rats at 600 mg/kg/day. Therefore, the dose of 450 mg/kg/day was selected to show some signs of toxicity. The highest dose level aims to induce systemic toxicity, but not death or severe suffering of the animals, to allow comparison of reproductive and systemic toxicity. The intermediate and low dose were selected in anticipation of showing dose response relationships.

- Inclusion/exclusion of extension of Cohort 1B: extension of cohort 1B is excluded as this is the standard information requirement laid down in column 1 of 8.7.3, Annex X of the REACH regulation. Criteria to extend the cohort 1B are not met.

- Route of administration and dose levels: The oral (gavage) route was selected for use because this is the intended route of human exposure

- Test system and number of animals: 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.
The number of animals chosen for this study is the smallest number considered necessary to provide the minimum number of pregnancies recommended by the applicable guidelines.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: Reaction products of di-, tri- and tetra-propoxylated propane-1,2-diol with ammonia
- Source and lot/batch number of test material: 9D410
- Expiration date of the lot/batch: 2021-07-31
- Appearance: clear, colorless liquid
- 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.

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

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The test system was selected because it is a standard species accepted for use in fertility and early embryonic development studies. This species and strain has been demonstrated to be sensitive to reproductive toxicants. Historical data and experience exist at the Testing Facility.
The number of animals chosen for this study is the smallest numbered considered necessary to provide the minimum number of pregnancies recommended by the applicable guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:CD(SD) Sprague-Dawley rats from Charles River Laboratories, Inc
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at animal arrival: approx 52 days
- Weight at study initiation: (P) Males: 179 - 245 g; Females: 139 - 191 g
- Housing:
- P generation: Animals were socially housed (where possible) in solid-bottomed cages by dose group (no more than 3 per cage/sex) until cohabitation, unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. 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 pair, where possible, 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.
- F1 Generation: After weaning, the F1 generation rats were socially housed in solid-bottomed cages by dose group throughout the study period (no more than 3 per cage per sex), unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. Control group animals where housed on a separate rack from the test substance-treated animals.
- Nesting material was provided. Nesting material was changed as often as necessary to keep the aimals dry and clean. Analyses for possible contamination are conducted on each lot of nesting material. There were no known contaminants in the nesting material considered to interfere with the objectives of the study.
- For psychological enrichment, animals were provided with items and/or a chewing object. Analysis for possible contamination are conducted on each lot of enrichment devices. There were no known contaminants in the enrichment devices considered to interfere with the objectives of the study.
- Diet (e.g. ad libitum): ad libitum, pelleted Certified Rodent Diet, available from individual feeders. The food is analyzed for environmental contaminants. Rats selected for clinical pathology blood collection were fasted on the day before scheduled euthanasia (no longer than 24 hours; water was available ad libitum). There were no known contaminants in the food considered to interfere with the objectives of the study.
- Water (e.g. ad libitum): ad libitum, from an automatic watering access system and/or individual bottles attached to the nesting boxes. 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. There were no known contaminants in the water considered to interfere with the objectives of the study.
- Acclimation period: at least 5 days prior to initiation of dose administration (male rats) or estrous cycle evaluation (female rats).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C (actual range 18°C -25°C)
- Humidity (%): 30% to 70% (actual range as low as 20%)
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: 2019-12-03 To: 2020-08-30

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

R.O. deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preparation of control substance: The control substance was dispensed daily for administration to Groups 1 and 5 animals. An adequate amount of the control substance was prepared at least once weekly and dispensed 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: The dosing formulations were prepared 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 stirred for at least 30 minutes before dosing. The dosing formulation was also stirred continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): R.O. deionized water
- Storage conditions: Ambient
- Concentration in vehicle: 0,10, 30, 90 mg/ml for control, G1, G2, G3, G4 groups respectively.
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: one male per one female. Within each dose group, consecutive order was used to assign rats to cohabitation (i.e., pairing)
- Length of cohabitation: maximum of 14 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 and assigned to individual housing.
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility: Females that did not mate with a male within the first 7 days of cohabitation were assigned an alternate male that did mate with a female (same dose group) and remained in cohabitation for a maximum of 7 additional days.
- Further matings after two unsuccessful attempts: Females not mated after completion of the 14-day cohabitation period were considered to be at DG 0 on the last day of cohabitation and assigned to individual housing.
- After successful mating each pregnant female was caged (how): individual
- Any other deviations from standard protocol: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The following dose formulation samples were collected for analysis:
- P generation First preparation: concentration (all groups), homogeneity (groups 2 and 4)
- P generation Last preparation: concentration (all groups)
- F1 generation First preparation: concentration (all groups), homogeneity (groups 6 and 8)
- F1 generation Last preparation: concentration (all groups)
The homogeneity results obtained from the top, middle and bottom for the groups 2 and 4 or groups 6 and 8 preparations were averaged and utilized as the concentration results.
Analyses were performed by GC using a validated analytical procedure.
Duplicate (1 mL each) top, middle, and bottom sets of samples (duplicate middle only for Groups 1, 3, 5 and 7) for each sampling time point were sent to the analytical laboratory. On days when only concentration analysis was required, the formulations were only sampled from the middle. Triplicate top, middle, and bottom sets of samples were also collected in the same manner and were retained and stored in a refrigerator set to maintain 4°C, blanketed under nitrogen at the Testing Facility as backup samples. 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%. Homogeneity results were considered acceptable at a relative standard deviation (RSD) of concentrations of < = 5% for each group. After acceptance of the analytical results, remaining backup samples will be discarded.

Duration of treatment / exposure:

Males: 113 -116 days
Females: 104 -108 days

Frequency of treatment:

daily

Details on study schedule:

Age at mating of the mated animals in the study: approx 17 weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

P generation - G1 (Control vehicle)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

P generation - G2 (low dose)

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

P generation - G3 (mid dose)

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

P generation - G4 (high dose)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

F1 generation - G5 (Control vehicle)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

F1 generation - G6 (low dose)

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

F1 generation - G7 (mid dose)

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

F1 generation - G8 (high dose)

No. of animals per sex per dose:

25 males and 25 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Justification of route: The oral (gavage) route was selected for use because this is the intended route of human exposure
- Justification of dose levels: Dose level selections were made based on the results of the OECD 421 study (Charles River Study No. 20209537). Mortality, adverse clinical signs, reduced body weight gain and food consumption were observed in the female rats at 600 mg/kg/day. Therefore, the dose of 450 mg/kg/day was selected to show some signs of toxicity. The intermediate and low dose were selected in anticipation of showing dose response relationships.

Positive control:

not applicable

Parental animals: Observations and examinations:

VIABILITY CHECKS: Yes
- Time schedule: At least twice daily during the study

CAGE SIDE AND DETAILED OBSERVATIONS: Yes
- General Appearance: At least weekly 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.

BODY WEIGHT: Yes
Frequency: At least weekly during acclimation and daily during the dose and postdose periods.

FOOD CONSUMPTION: Yes
Frequency
- 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.

CLINICAL PATHOLOGY
- Sample collection: On the day of scheduled euthanasia, blood samples were collected from 10 rat/sex/dose group Blood samples were collected from the inferior vena cava while under isoflurane/oxygen anesthesia. Rats were euthanized via exsanguination, following blood sample collection.
- timing of sample collection: DS 114-115 for males groups 1-4, DL22 for females groups 1-4
- Animals fasted: Animals were fasted on the day before scheduled euthanasia (no longer than 24 hours; water was available ad libitum).

- Hematology: Blood samples (0.5 mL each) were analyzed for the following parameters: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Red Blood Cell Distribution Width, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells, Other cells (as appropriate).
Blood smears were prepared from each hematology sample. Two slides per animal were prepared at the Testing Facility and stained by CR-ASH. Slide review was only performed on samples that met flagging criteria to confirm accurate hematology data.
- Coagulation: Blood samples (0.9 mL each) were processed for plasma, and plasma was analyzed for the following parameters: Activated partial thromboplastin time, Fibrinogen, Prothrombin time.
- Clinical Chemistry: Blood samples (0.5 mL each) were processed for serum, and the serum was analyzed for the following parameters: Alanine aminotransferase, Aspartate aminotransferase
Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin (When total bilirubin was >0.5 mg/dL, direct bilirubin was also measured and indirect bilirubin was calculated), Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride, Sample Quality (appearance includes a degree of hemolysis, lipemia, and icterus.

URINALYSIS:
- Sample collection: Urine samples were collected overnight for evaluation on the night prior to scheduled euthanasia.
- Samples were maintained at ambient conditions until analysis at the Testing Facility for the following parameters: Color, Appearance/Clarity, Specific gravity, Total Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood.

Thyroid Analyses:
- Blood samples (1.5 mL each) were collected from the same male and female rats selected for clinical pathology samples. Blood samples were collected from the inferior vena cava while under isoflurane/oxygen anesthesia for analysis of thyroxine (T4), and TSH. Rats were euthanized via exsanguination, following blood sample collection.
- Blood samples were placed into serum separator tubes, allowed to clot for at least 20 minutes (not exceeding 1 hour) and centrifuged at room temperature at 3500 rpm for 15 minutes. 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. Samples designated for TSH analysis were stored in a freezer set to maintain -20ºC. Samples designated for T4 analysis were stored in a freezer set to maintain -80°C until shipment for analysis.
- Analyses: Total serum thyroxine (T4) was measured in rat srrum using a validated analytical method. The thyroid stimulating hormone (TSH) was measured in rat serum using a validated analytical method.

OTHER:
- Natural delivery observations: Beginning on DG 17, the dam will be observed at least twice daily.

Oestrous cyclicity (parental animals):

- Frequency: Samples were collected for 14 consecutive days before initiation of the cohabitation period and then until spermatozoa was observed in a smear of the vaginal contents and/or a copulatory plug is observed in situ during the cohabitation period.
Samples were collected prior to scheduled euthanasia on DG 25, DL 21 or DL 22.
- Procedure: Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.

Sperm parameters (parental animals):

To assess the potential toxicity of the test substance on male reproductive system, the endpoints listed were evaluated for each male that survived to scheduled euthanasia
- Sperm motility: evaluated using computer-assisted sperm analysis (CASA). Motility was evaluated following dispersion, into an appropriate medium, of sperm from the left vas deferens.
- Sperm concentration: A homogenate was prepared from the left cauda epididymis for evaluation to determine sperm concentration (sperm per gram of tissue weight). Sperm concentration was evaluated using computer-assisted sperm analysis (CASA).

Litter observations:

VIABILITY CHECKS
- Preweaning: litters were observed for dead pups at least twice daily and the pups in each litter were counted once daily during the preweaning period starting on Day 0 postpartum
- Postweaning: at least twice daily during the study

CLINICAL OBSERVATIONS
- Preweaning: At least once daily starting on Day 0 postpartum
- Postweaning: general appearance once daily, postdose observations between 1 to 2 hours after dose administration

BODY WEIGHTS
- Preweaning: Days 0 (birth) 4, 7, 10, 13, 17 and 21 postpartum
- Postweaning: Daily

ANOGENITAL DISTANCE
- Preweaning: on day 0 postpartum (birth)
Procedure: using a calibrated stereomicroscope, micrometer and ruler. The anogenital distance was measured from the cranial edge of the anus, which comes to a point, to the base of the genital tubercle.

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

FOOD CONSUMPTION
- Frequency: At least once weekly.
- Procedure: Food consumption will be quantitatively measured per cage, where appropriate

SEXUAL MATURATION
- Once daily beginning on Day 27 postpartum (females) or once daily beginning on Day 38 postpartum (males) until the criterion was achieved.
- A body weight was recorded on the day that sexual maturation was achieved

ESTROUS CYCLE EVALUATIONS in F1 generation
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Samples were collected on the day after the vagina is observed to be patent continuing until 1 estrous stage has been identified. Samples were also be collected for at least 14 consecutive days beginning on Day 74 postpartum through the day of scheduled necropsy.

CLINICAL PATHOLOGY
- Sample collection: On PNDs 88 to 91, blood samples were collected from 10 rats/sex/dose group. Blood samples were collected from the inferior vena cava while under isoflurane/oxygen anesthesia. Rats were euthanized via exsanguination, following blood sample collection.
- Rats were fasted on the day before scheduled euthanasia (no longer than 24 hours; water was available ad libitum).
- Hematology: Blood samples were analyzed for the following parameters: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Red Blood Cell Distribution Width, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells, Other cells (as appropriate). Blood smears were prepared from each hematology sample. Two slides per animal were prepared at the Testing Facility and stained by CR-ASH. Slide review was only performed on samples that met flagging criteria to confirm accurate hematology data.
- Coagulation: Blood samples (1.8 mL each) were processed for plasma, and plasma was analyzed for the following parameters: Activated partial thromboplastin time, Fibrinogen, Prothrombin time.
- Clinical Chemistry: Blood samples (0.5 mL each) were processed for serum, and the serum was analyzed for the following parameters: Alanine aminotransferase, Aspartate aminotransferase
Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin (When total bilirubin was >0.5 mg/dL, direct bilirubin was also measured and indirect bilirubin was calculated), Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride, Sample Quality (appearance includes a degree of hemolysis, lipemia, and icterus.

URINALYSIS
Urine samples were collected overnight for evaluation from rats on the night prior to scheduled euthanasia. Samples were maintained at ambient conditions until analysis at the Testing Facility for the following parameters: Color, Appearance/Clarity, Specific gravity, Total Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood.

Thyroid Analyses:
- On PND 4 postpartum, blood samples were collected via cardiac puncture following euthanasia from all of the culled pups and pooled by litter.
- On PND 21, blood samples were collected via vena cava following euthanasia from 9 to 11 pups/sex/group not selected for continued observation and dosing.
- On PND 88-91 day, blood samples were collected from the same male and female rats selected for clinical pathology samples. Blood samples were collected from the inferior vena cava while under isoflurane/oxygen anesthesia. Rats were euthanized via exsanguination, following blood sample collection.
- Target Volume: As much as possible, when possible
- Anticoagulant: None (Serum separator tubes)
- Blood was collected and immediately placed into serum separator tubes, allowed to clot at room temperature for at least 10 minutes and then centrifuged for approximately 10 minutes in a refrigerated centrifuge set to maintain 4°C at 3000 g within 30 minutes of collection. Sera samples were split into two approximately equal aliquots. Samples designated for TSH analysis were stored in a freezer set to maintain -20ºC. Samples designated for T4 analysis were stored in a freezer set to maintain -80°C until shipment for analysis.
- T4 and TSH were measured in rat serum using a validated analytical method

Postmortem examinations (parental animals):

SACRIFICE:
- Sacrifice / method of euthanasia: P generation rats selected for clinical pathology evaluation and/or hormone analysis were euthanized via exsanguination following blood collection under isoflurane/oxygen anesthesia. All other P generation rats were euthanized by carbon dioxide asphyxiation.
- Unscheduled death: Rats that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions.
- Scheduled euthanasia:
Female rats
- Females that did not deliver a litter were euthanized on DG 25 and were examined in Necropsy, Ovarian and Uterine Examinations. Organs were collected, weighted and preserved as described below.
- Dams with no surviving pups were euthanized after the last pup was found dead, missing (presumed cannibalized), or euthanized. The rats were examined in Necropsy, Ovarian and Uterine Examinations. Organs were collected, weighted and preserved as described below.
- On Day 21 postpartum, P generation female rats not selected for blood sample collection were euthanized and examined in Necropsy and Ovarian, Uterine Examinations. Organs were collected, weighted and preserved as described below.
- On Day 22 postpartum, P generation female rats selected for blood sample collection had blood samples collected as described above, and were euthanized. The rats were examined in Necropsy and Ovarian, Uterine Examinations. Organs were collected, weighted and preserved as described below.
Male rats:
- On DS 114 to 117, P generation male rats had blood samples collected as described above and/or were euthanized. The rats were examined in Necropsy, organ weights, and histology. Organs were collected, weighted and preserved as described below.

OVARIAN AND UTERINE EXAMINATIONS
The reproductive tract was dissected from the abdominal cavity. The number and distribution of implantation sites and corpora lutea was recorded. Uteri of apparently nonpregnant animals were examined while being pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries from all female rats were retained in 10% neutral buffered formalin.

NECROPSY
The rats were examined for gross lesions and were subjected to a gross (or complete necropsy for some male and female rats) necropsy of the thoracic, abdominal, and pelvic viscera. Images were generated for illustration of for consultation on gross observations.

ORGAN WEIGHTS
The organs listed were weighed at necropsy for all scheduled euthanasia rats. Organ weights were not recorded for rats found dead or euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
The following organs were weighed: Brain, Cervix, Epididymis, Epididymis - Cauda Left, Gland adrenals, Gland coagulating, Gland parathyroid, Gland pituitary, Gland prostate, Gland seminal vesicles, Gland thyroid, Heart, Kidneys, Liver, Ovaries, Oviducts, Pancreas, Spleen, Testes, Thymus, Uterus

TISSUE COLLECTION AND PRESERVATION
Representative samples of the following tissues were collected from all rats and preserved in 10% neutral buffered formalin: Bone marrow, Brain, Cervix, Epididymis, Epididymis - Cauda Left, Esophagus, Eye, Gland adrenals, Gland coagulating, Gland mammary, Gland parathyroid, Gland pituitary, Gland prostate, Gland seminal vesicles, Gland thyroid, Gross lesions/masses, Heart, Kidneys, Large intestine cecum, Large intestine colon, Large intestine, rectum, Liver, Lungs, Muscle skeletal, Nerve peripheral, Nerve optic, Ovaries, Oviducts, Pancreas, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum, Stomach, Spleen, Testes, Thymus, Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

HISTOLOGY
Tissues listed above were collected from rats that died or were euthanized before scheduled termination, all P generation rats in Groups 1 and 4, and target tissues for all rats, and rats with gross lesions 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.
Histological examination was performed on any rats that died or were euthanized before scheduled termination, all P generation rats in Groups 1 and 4, and all gross lesions from all rats. If lesions attributed to the test substance as determined by the Study Director and/or Veterinary Pathologist were observed in the rats exposed to the high test substance concentration, the same organs were examined histologically in all the rats exposed to the lower test substance concentrations until a no-effect level was established.

Postmortem examinations (offspring):

SACRIFICE:
- Method of Euthanasia:
- F1 generation rats/pups > PND 15 were euthanized by carbon dioxide asphyxiation.
- F1 generation pups ≤ PND 15 were euthanized by an intraperitoneal injection of sodium pentobarbital (390 mg/mL).
- F1 generation rats selected for clinical pathology evaluation were euthanized via exsanguination, following blood sample collection.
- Unscheduled death:
- Days 0 to 21 Postpartum: Pups that were found dead during delivery or at the completion of littering (Day 0 postpartum) were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sink were identified as stillborn; pups with lungs that float were identified as liveborn and to have died shortly after birth. Pups with gross lesions were preserved in Bouin's solution for possible future evaluation. Pups that die (Days 1 to 21 postpartum) or were euthanized (Days 0 to 21 postpartum) before scheduled termination were examined for gross lesions and the cause of death or condition as soon as possible after the observation is made. Pups with gross lesions euthanized or found dead on Days 0 to 3 postpartum were preserved in Bouin's solution for possible future evaluation. Gross lesions from pups found on Days 4 to 21 postpartum were preserved in 10% neutral buffered formalin.
- Postweaning (> Day 21 Postpartum): 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 is made. The rats were examined for gross lesions.
- Scheduled Euthanasia:
- Pups not selected for continued observation were euthanised on Day 21 postpartum. 9 to 11 pups/sex/group had blood samples collected. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed and carcasses were retained in 10% neutral buffered formalin for 10 pups/sex/group (one pup per litter, when possible) and any pups with gross lesions; all other tissues/carcasses will be discarded.
- Female rats: On Day 88-91 postpartum, F1 generation female rats had blood samples collected and/or were euthanized. The rats were examined as described in the sections Necropsy and Organ Weights. Tissues were collected, weighed, retained and/or evaluated; all other tissues were discarded.
- Male rats: On Day 90 postpartum ± 1 day, F1 generation male rats had blood samples collected and/or were euthanized. The rats were examined as described in the sections Necropsy, and Organ Weights. Tissues were collected, weighed, retained and/or evaluated; all other tissues were discarded.
Assessment of the male reproductive system was performed. A similar proportion of males from each group, as appropriate, were euthanized on any one day.

MALE REPRODUCTIVE ASSESSMENT
To assess the potential toxicity of the test substance on the male reproductive system, the endpoints listed below were evaluated for each male that survived to scheduled euthanasia.
- Sperm motility: Sperm motility was evaluated using computer-assisted sperm analysis (CASA). Motility was evaluated following dispersion, into an appropriate medium, of sperm from the left vas deferens.
- Sperm concentration: A homogenate was prepared from the left cauda epididymis for evaluation to determine sperm concentration (sperm per gram of tissue weight). Sperm concentration was evaluated using computer-assisted sperm analysis (CASA).

NECROPSY
The rats were examined for gross lesions, and were subjected to a gross necropsy (or complete necropsy for some male and female rats) of the thoracic, abdominal, and pelvic viscera.
Images were generated for illustration of or consultation on gross observations.

ORGAN WEIGHTS
- The following organs were weighed at necropsy for all scheduled euthanasia rats: Brain, Cervix, Epididymis, Epididymis - Cauda Left, Gland - adrenals, Gland - coagulating, Gland - parathyroid, Gland - pituitary, Gland - prostate, Gland - seminal vesicles, Gland - thyroid, Heart, Kidneys, Liver, Ovaries, Oviducts, Spleen, Testes, Thymus, Uterus
- Organ weights were not recorded for rats found dead or euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

TISSUE COLLECTION AND PRESERVATION
- Representative samples of the tissues listed were collected from all rats and preserved in 10% neutral buffered formalin, unless otherwise indicated.
- in addition, F1 male rat 4079 (group 8, unscheduled euthanasia) head was retained when not required and F1 male rat 4004 (group 5, PND89) subcutis was retained in 10% NBF and examined when not required.
- list of tissues: Bone marrow, Brain, Cervix, Epididymis, Epididymis - Cauda Left, Esophagus, Eye, Gland - adrenals, Gland - coagulating, Gland - mammary, Gland - parathyroid, Gland - pituitary, Gland - prostate, Gland - seminal vesicles, Gland - thyroid, Gross lesions, Heart, Kidneys, Large intestine cecum, Large intestine - colon, Large intestine - rectum, Liver, Lungs, Muscle - skeletal, Nerve - peripheral, Nerve - optic, Ovaries, Oviducts, Pancreas, Small intestine - duodenum, Small intestine - ileum, Small intestine - jejunum, Stomach, Spleen, Testes, Thymus, Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

HISTOLOGY 
Tissues listed above were collected from rats that died or were euthanized before scheduled termination, all F1 generation rats in Groups 5 and 8, target tissues for all rats, and rats with gross lesions were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Entire left and right ovaries for the F1 generation rats assigned to Groups 5, 6, 7 and 8 were embedded in separate paraffin blocks. Consistency in orientation of each ovary was imperative, such that each ovary was placed into its own designated block. Each ovary was sectioned beginning at 200 microns within the ovary. The first section was collected and designated as section 1 for follicle counts. After advancing the microtome 100 microns, the next section was collected as section 2. This process continued until a total of five sections from each ovary were collected for follicle counts, plus one section collected (between follicle count sections 3 and 4) on a separate slide for routine histopathological evaluation by the study pathologist. The five sections were placed on one slide for each ovary. Section 1 was placed closest to the slide label, continuing in a linear pattern, until five sections were on the same slide (section 5 being most distal to the slide label).
Each section was quantitatively evaluated for primordial follicles to include small growing follicles. Presence or absence of corpora lutea was recorded for each rat.  Group means and standard deviations were calculated for each rat (left, right, and both ovaries combined). Where the sample size was appropriate (N> 2), group mean values were compared in order to detect any significant differences in the number of primordial follicles in left ovaries, right ovaries, and both ovaries combined. The rats assigned to Group 8 counts were ± 15% (number of follicles and/or presence of corpora lutea) of the control animals and were not statistically significant, therefore, no further evaluation was considered necessary.

HISTOPATHOLOGY
Histopathological evaluation was performed by a board-certified veterinary pathologist.
Histological examination was performed on any rats that died or were euthanized before scheduled termination, all F1 generation rats in Groups 5 and 8, and all gross lesions from all rats. If lesions attributed to the test substance as determined by the Study Director and/or Veterinary Pathologist were observed in the rats exposed to the high test substance concentration, the same organs were examined histologically in all the rats exposed to the lower test substance concentrations until a no-effect level was established.

Statistics:

Any data collected during the predose period was not tabulated, summarized or statistically analyzed. All statistical analyses were performed within the respective study phase, unless otherwise noted.
Clinical and necropsy observations data were summarized but no inferential statistical analysis was performed.
Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion or by litter.
- descriptive statistical analyses: means, standard deviations, percentages, numbers and/or incidences were reported as appropriate by dataset
- interferential statistical methods: all statistical tests were conducted at the 5% significance level/ All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest were: group 2 vs 1, group 3 vs 1, group 4 vs 1, group 6 vs 5, group 7 vs 5, group 8 vs 5
More details are described in the field 'Any other information on materials and methods'

Reproductive indices:

Gestation Length: The gestation length is calculated from DG 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/Number of Animals Pregnant )x 100

Offspring viability indices:

Number of offspring per litter: Live and dead pups
Number of implantation sites.
General condition of dam and litter during the postpartum period.
Live Birth Index: Percentage of pups born alive.
(Number of Live Newborn Pups/ Number of Newborn Pups ) x 100
Viability Index: Percentage of pups born that survive 21 days postpartum

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Males: An increased incidence of excess salivation and abnormal breathing sounds were observed at 450 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 or two rats in a dose group and the clinical signs did not persist. These clinical signs included suspected dehydration, hunched posture, fur loss, fur staining, thin fur cover, scab on the skin, splayed limbs, thin body, hypersensitive, hypersensitive, abnormal consistency of the feces, red discharge, broken teeth. decreased activity and lack of or impaired righting reflex.
- Females: During the precohabitation, gestation and/or lactation periods, there was an increased incidence of suspected dehydration, labored breathing, abnormal breathing sounds, shallow breathing, hunched posture, thin fur cover erect fur, hyperreactivity and decreased activity at 450 mg/kg/day in comparison with the control group values.
All other clinical signs that were observed were considered unrelated to the the test substance because: 1) the observations were not dose-dependent; and/or 2) the observations occurred in only one or two rats in a dose group and the clinical signs did not persist. These clinical signs included salivation, bent tail, fur loss, fur staining, scab on the skin, thin body condition, low carriage, ungroomed fur, swollen muzzle and abnormal consistency of the feces.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

- Males: There were no test substance-related mortalities observed in the P generation male rats. One male in the control group was euthanized due to adverse clinical signs on DS 82, a male at 150 mg/kg/day was found dead on DS 3 and a male at 450 mg/kg/day was euthanized due to adverse clinical signs on DS 36. Based on there being single occurrences in each dose group and there being no correlation in the death or moribundity, these deaths were not considered to be test substance related.
- Females: The only deaths that were considered to be test substance related occurred in the 450 mg/kg/day dose group. One female at 50 mg/kg/day and another at 150 mg/kg/day were euthanized due to no surviving pups. There were no test substance related clinical signs or effects on body weights observed in these females. At 450 mg/kg/day, two females were found dead and three females were euthanized due to adverse clinical signs. Common clinical signs in these females included hunched posture, decreased activity and respiratory issues (labored breathing and abnormal breathing signs). The females that were euthanized due to adverse clinical signs also experienced body weight loss prior to euthanasia. All other P generation female rats survived until scheduled euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Males: Body weights and body weight gains were unaffected by the test substance during the dose period by doses up to and including 150 mg/kg/day. At 50 mg/kg/day, there was a statistically significant decrease (p≤0.01) in body weight gain observed on DSs 91 to 92 in comparison with the control group value. This difference was not considered to be test substance related because it was not dose dependent. There were statistically significant increases (p≤0.01) in body weight gain on DSs 94 to 95 and statistically significant (p≤0.05 or p≤0.01) body weight losses on DSs 95 to 96 observed in the 50, 150 and 450 mg/kg/day dose groups, respectively. These differences were not considered to be test substance related because they were single occurrences that did not persist. At 150 mg/kg/day, there was a statistically significant increase (p≤0.01) in body weight gain observed on DSs 30 to 31, a statistically significant (p≤0.01) body weight loss on DSs 43 to 44 and a statistically significant increase (p≤0.01) in body weight gain observed on DSs 44 to 45 in comparison with the control group values. These differences were not considered to be test substance related because they were not dose dependent.
At 450 mg/kg/day, there were reductions or statistically significant reductions (p≤0.05) in mean body weights observed beginning on DS 106 and continuing for the remainder of study in comparison with the control group values. At 450 mg/kg/day, there was a statistically significant reduction (p≤0.05) in body weight gain on DSs 7 to 8 and a statistically significant (p≤0.05) body weight loss on DSs 108 to 109 in comparison with the control group values.

- Females: 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 150 mg/kg/day. At 50 mg/kg/day, there was a statistically significant decrease (p≤0.05) in body weight gain observed on DSs 48 to 49 in comparison with the control group value. There was also a statistically significant increase (p≤0.05) in body weight gain observed on DSs 66 to 67 and a statistically significant body weight loss (p≤0.05) at 50 mg/kg/day on DSs 68 to 69 in comparison with the control group values. At 450 mg/kg/day, there were statistically significant increases (p≤0.05 or p≤0.01) in body weight gain observed on DSs 18 to 19 and DLs 15 to 16 in comparison with the control group values. These differences were not considered to be test substance related because they were single occurrences that did not persist.
At 450 mg/kg/day, there were statistically significant reductions (p≤0.05) in mean body weights observed beginning on DS 20 and continuing for the remainder of the pre-mating period in comparison with the control group values. There were reductions or statistically significant reductions (p≤0.05) in mean body weights at 450 mg/kg/day on DGs 5, 19 and 21 in comparison with the control group values. There was a statistically significant (p≤0.01) body weight loss at 450 mg/kg/day on DSs 9 to 10 in comparison with the control group value.
More details are available in data tables attached.

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 substance in the males at all intervals prior to cohabitation at doses up to and including 450 mg/kg/day. There were statistically significant increases (p≤0.05 or p≤0.01) in the mean food consumption values observed on DSs 22 to 25 at 50, 150 and 450 mg/kg/day in comparison with the control group value. There was a statistically significant decrease (p≤0.01) in the mean food consumption value on DSs 60 to 64 at 150 mg/kg/day in comparison with the control group value. There were statistically significant increases (p≤0.05 or p≤0.01) in the mean food consumption values on DSs 64 to 67 at 50 and 150 mg/kg/day and on DSs 67 to 71 at 150 mg/kg/day in comparison with the control group values.
- Females: Food consumption values were unaffected by the test substance during the precohabitation, gestation and lactation periods by doses up to and including 150 mg/kg/day. At 150 mg/kg/day, there was a statistically significant decrease (p≤0.05) in the mean food consumption value observed on DGs 17 to 20 in comparison with the control group value. This decrease was not considered to be test substance related because it was not dose dependent.
At 450 mg/kg/day, there were decreases or statistically significant decreases (p≤0.05 or p≤0.01) in the mean food consumption values observed on DSs 8 to 11, 11 to 15 and at all intervals between 18 to 22 and 46 to 50 in comparison with the control group values.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Male and Female: No test substance-related differences in the hematology parameters occurred at any dose level. All differences were consistent with biological variation and were not considered to be test substance related.

Coagulation: No test substance-related differences in coagulation parameters occurred at any dose level in the F1 generation male or female rats. All differences in these parameters, regardless of statistical significance, were consistent with biological variation and were considered unrelated to the test substance

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Male and Female: No test item-related differences in the clinical chemistry occurred at any dose level. All differences were consistent with biological variation and were not considered to be test substance related.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Males: No test substance-related changes in urinalysis values were observed in the P generation male rats. At 450 mg/kg/day, there was a statistically significant decrease (p≤0.01) in the pH of the urine in comparison with the control group value. This difference was not considered to be test substance related as it was consistent with biological variation for this parameter.

- Females: No test substance-related changes in urinalysis values were observed in the P generation female rats at any dose. At 150 mg/kg/day, there was a statistically significant decrease (p≤0.05) in the volume of urine collected in comparison with the control group value. This difference was not considered to be test substance related because it was not dose dependent.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Males: Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. In two male rats at 450 mg/kg/day, segments of the nasopharyngeal epithelium were replaced by squamous metaplasia. Findings within the nasal cavity were considered test substance-related due to their presence in only treated animals and not controls. The described nasal cavity findings were considered to likely be secondary to irritation caused by nasal reflux of the administered test substance. Other 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.
- Females: Inflammation was seen within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. In three rats at 450 mg/kg/day, segments of the nasopharyngeal epithelium were replaced by squamous metaplasia. Findings within the nasal cavity were considered test substance-related due to their presence in only treated animals and not controls. The described nasal cavity findings were considered to likely be secondary to irritation caused by nasal reflux of the administered test substance. Other 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 examined

Other effects:

not specified

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related changes in the number of estrous stages per 14 day interval evaluated and the mean cycle lengths. At 150 mg/kg/day, there was a statistical significant increase (p≤0.05) in the mean cycle length (days) in comparison with the control group value. This increase was not considered to be test substance related because it was not dose dependent.
All additional fertility parameters were unaffected by doses of the test substance as high as 450 mg/kg/day.
More details are available in data tables attached.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no statistically significant or biologically important differences in the mean percentage of motile sperm and the mean cauda epididymal sperm density parameters observed at any dose level.

Reproductive performance:

no effects observed

Description (incidence and severity):

- All male mating and fertility parameters were comparable among the dose groups and did not significantly differ from the control group values.
- All female mating and fertility parameters were unaffected by doses of the test substance as high as 450 mg/kg/day.
- Natural Delivery and Litter Observations: A total of 23/25 (92%), 23/25 (92%), 22/25 (88%) and 17/20 (85%) treated female rats were pregnant and delivered a litter in the 0 (Control), 50, 150 and 450 mg/kg/day dose groups, respectively.
Natural delivery and litter observations were not affected by doses of test substance as high as 450 mg/kg/day.
No parameters evaluated at natural delivery or during preweaning period was affected by the test substance at doses as high as 450 mg/kg/day. These parameters included duration of gestation, gestation index, females completing delivery, dams with liveborn pups, dams with no liveborn pups, dams with stillborn pups, number of stillborn pups per litter, number of stillborn pups, number of liveborn pups, live birth index (%), live male pups per litter (%), implantation sites and post-implantation loss per litter. More details are available in data tables attached.

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

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- From birth through weaning:
No clinical observations in the F1 generation pups were attributable to maternal doses of the test substance as high as 450 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 2) the observations occurred in only one pup in a particular dose group. These clinical signs included thin appearance; pale skin; purple discoloration; a scab, lesion or laceration on the skin; swollen forelimb or hindlimb; ungroomed fur; thin fur cover; no milk band present; cold to touch; missing digit on the hindpaw; and mass present on the forelimb.

- F1 generation:
There was an increased incidence in abnormal breathing sounds observed at 150 and 450 mg/kg/day in the F1 generation female rats and at 450 mg/kg/day in the F1 generation male rats in comparison with the control group values. There was also an increased incidence of suspected dehydration, excess salivation and labored breathing in the F1 generation male rats 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 or two rats in a dose group and the clinical signs did not persist. These clinical signs included shallow breathing, hunched posture, thin fur, scab on the skin, abnormal fecal consistency, red or orange discharge, broken teeth and liquid discharge.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The only deaths that occurred in the F1 generation rats occurred at 450 mg/kg/day. At 450 mg/kg/day one male and one female were found dead during the dose period and three males were euthanized due to adverse clinical signs. Common clinical signs in the rats included respiratory issues (labored breathing and abnormal breathing signs) and suspected dehydration. The three males that were euthanized due to adverse clinical signs also experienced body weight loss prior to euthanasia. Male 4094 was given a dosing holiday on PND 61 due to potential blockage observed during dosing. This male was euthanized on PND 62 due to adverse clinical signs and had necropsy observations indicative of an intubation error. All other F1 generation male and female rats survived until scheduled euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- From birth through weaning:
No changes in body weights in the F1 generation pups were attributable to maternal doses of the test substance as high as 450 mg/kg/day. There was a statistically significant increase (p≤0.05) in mean male pup body weights on PND 10 in comparison with the control group value. This increase was not considered to be test substance related because it was not dose dependent.

- F1 generation:
There were no test substance-related differences in body weight and body weight gain observed in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.
There was a statistically significant reduction (p≤0.05) in body weight gain observed in the F1 generation male rats at 50 mg/kg/day on PNDs 80 to 81 in comparison with the control group value. In the F1 generation female rats at 50 mg/kg/day, there were statistically significant increases (p≤0.01) in body weight gain observed on PNDs 50 to 51, 70 to 71 and 74 to 75; statistically significant reductions (p≤0.05 or p≤0.01) in body weight gain observed on PNDs 60 to 61 and 71 to 72; and statistically significant (p≤0.01) body weight losses on PNDs 73 to 74 and 76 to 77 in comparison with the control group values. There were statistically significant reductions (p≤0.05 or p≤0.01) in body weight gains observed in the F1 generation male rats at 150 mg/kg/day on PNDs 51 to 52 and 67 to 68 in comparison with the control group values. In the F1 generation female rats at 150 mg/kg/day, there was a statistically significant increase (p≤0.05) in body weight gain observed on PNDs 37 to 38 and a statistically significant reduction (p≤0.01) in body weight gain observed on PNDs 67 to 68 in comparison with the control group values. These differences were not considered to be test substance-related because they were not dose dependent.
There was a statistically significant reduction (p≤0.05) in body weight gain observed in the F1 generation male rats at 450 mg/kg/day on PNDs 33 to 34 in comparison with the control group value. In the F1 generation male rats at 450 mg/kg/day, there was a statistically significant increase (p≤0.01) in body weight gain on PNDs 49 to 50 and a statistically significant reduction (p≤0.05) in body weight gain observed on PNDs 50 to 51 in comparison with the control group values. These differences were not considered to be test substance-related because they were single occurrences and did not persist.
More details are available in data tables attached.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance-related differences in food consumption observed in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test substance-related differences in the hematology and coagulation parameters occurred at any dose level in the F1 generation male or female rats. All differences in these parameters, regardless of statistical significance, were consistent with biological variation and were considered unrelated to the test substance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test substance-related differences in the clinical chemistry parameters occurred at any dose level in the F1 generation male or female rats. All differences in these parameters, regardless of statistical significance, were consistent with biological variation and were considered unrelated to the test substance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test substance-related differences in urinalysis observed in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on male or female sexual maturation at doses as high as 450 mg/kg/day. The average day of preputial separation and vaginal patency on which these parameters were achieved did not differ significantly among the dose groups.

- estrous cycle evaluation: The day the first estrus occurred after vaginal patency, the number of estrous cycles per 14 day interval evaluated and the mean cycle lengths were all comparable among the groups.

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 on the day of delivery and there were no statistically significant differences.

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:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related organ weight changes were noted in the F1 generation male or female rats. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related gross findings were noted in the F1 generation male or female rats. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. Segments of the nasopharyngeal epithelium were replaced by squamous metaplasia in one female at 450 mg/kg/day. Findings within the nasal cavity were considered test substance related due to their presence in only treated animals and not controls. The described nasal cavity findings were considered to likely be secondary to irritation caused by nasal reflux of the administered test substance.
Other 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.

Other effects:

no effects observed

Description (incidence and severity):

- Sperm evaluation: There were no statistically significant or biologically important differences in the mean percentage of motile sperm and the mean cauda epididymal sperm density parameters observed at any dose level in the F1 generation male rats.
- Thyroid hormone levels: There were no test substance-related differences in T4 or TSH thyroid hormone levels in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.
- Ovarion follicle evaluation: Significant increases were noted in the number of primordial follicles in left (50 and 450 mg/kg/day only), right (>=50 mg/kg/day) and both (>= 50 mg/kg/day) ovaries compared to the control group. Whether or not these changes were related to administration of the test substance is unclear. Left and right ovaries were unable to be determined for histology. Regarding the combined means, without any clear pattern, trend or associated histopathology changes, these increases appear to be due more to natural variability than to relation with the test article. Corpora lutea were present for all animals evaluated in all groups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: viability and growth in the offspring

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1

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

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Dose formulation analyses

The dosing suspensions of the test substance prepared in R.O. deionized water were analyzed following the first and last preparations for the P generation and F1 generation and were found to be within the target concentration and homogeneous under the conditions of this study. 

The first preparation of the dosing suspensions of 10, 30 and 90mg/mL for the P generation were analyzed and found to be 112%, 101% and 95.6% of the target concentrations, respectively. The homogeneity values obtained for the first preparation were 1.3% and 1.6% RSD for the 10 and 90mg/mL formulations, respectively. Although the initial 10 mg/mL concentration was slightly above the protocol-specified ± 10% of the target concentration, this variation from the target concentration was slight and the 5 mg/mL dose preparation was homogeneous and considered to be acceptable for usage on study. The last preparation of the dosing suspensions of 10, 30 and 90mg/mL were analyzed and found to be 106%, 109% and 110% of the target concentrations, respectively. 

The first preparation of the dosing suspensions of 10, 30 and 90mg/mL for the F1 generation were analyzed and found to be 108%, 106% and 104% of the target concentrations, respectively. The homogeneity values obtained for the first preparation were 3.2% and 2.3% RSD for the 10 and 90mg/mL formulations, respectively. The last preparation of the dosing suspensions of 10, 30 and 90mg/mL were analyzed and found to be 110%, 104% and 106% of the target concentrations, respectively. 

Conclusions:

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was considered 150 mg/kg/day for the P generation and F1 generation male and female rats. There was an increased incidence of microscopic findings in the nasal cavity of both the P generation and F1 generation rats at 150 or 450 mg/kg/day that included inflammation, neutrophilic exudates, and squamous metaplasia of the nasopharynx that were considered test substance-related. Luminal hemorrhage was also observed in the nasal cavity of P generation rats at 450 mg/kg/day. There were abnormal breathing sounds and labored breathing observed in the P generation and F1 generation males and females that correlated with these microscopic findings. Mortality was observed in the P generation females and F1 generation males and females. Reductions in body weight and body weight gain were observed in the P generation males and females at 450 mg/kg/day.

The reproductive NOAEL was 450 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. The NOAEL for viability and growth in the offspring was also 450 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Screening study via topical application

In a screening reproductive toxicity study (according to OECD guideline 421), rats were exposed to 3, 10, 30 mg/kg (nominal in water) via topical application (Calvert Laboratories Inc, 2011; key study; Klimisch 1). No systemic effects were observed at the highest dose administered. Therefore, a NOAEL of 30 mg/kg bw/d is derived for male and female reproductive performance. The NOAEL for parental clinical signs was considered to be 10 mg/kg bw/d. There were no treatment-related effects on pregnancy status; gestation, viability and related parameters; fetal weights and fetal morphological observations.

 

Screening study via oral gavage

In a screening reproductive toxicity study (according to OECD guideline 421; Charles River Laboratories Inc., 2021; key; Klimisch 1), rats were exposed to 0, 15, 30, 60, 120 mg/kg/day (in R.O. deionized water) via oral gavage. The following parameters and end points were evaluated in this study: viability, clinical signs, food consumption, body weights, body weight changes, estrous cycle evaluations, reproductive capacity, natural delivery observations, maternal behavior, anogenital distance measurements, nipple presence, hormone analysis, gross necropsy observations, organ weights, and histopathological evaluations.

 

Male rats

Mortality and adverse clinical signs were observed in the male rats at 300 and 600 mg/kg/day. There was 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. There was also an increased incidence of labored breathing and staining on the fur at 600 mg/kg/day. At 600 mg/kg/day, there were decreases or statistically significant decreases in mean body weights beginning on DS 33 and continuing at all intervals for the remainder of the dosing period. Statistically significant decreases in body weight gains were observed at 600 mg/kg/day on DSs 9 to10, 14 to 15 and 34 to 35. 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. At 600 mg/kg/day, there was a statistically significant reduction in the male fertility index and the male pregnancy index. There were no test substance-related gross necropsy observations at doses up to and including 600 mg/kg/day. At 600 mg/kg/day, there was a statistically significant decrease in the terminal body weight. All organ weights (absolute and relative) were unaffected by doses of the test substance up to and including 600 mg/kg/day.
There were no test substance-related differences in T4 thyroid hormone levels and no treatment-related microscopic alterations were observed.

 

Female rats and F1 generation

Mortality and adverse clinical signs were observed in the female rats at 300 and 600 mg/kg/day. There was an increased incidence of abnormal breathing sounds at 300 and 600 mg/kg/day. At 600 mg/kg/day, there was also an increased incidence of excess salivation, labored breathing, erected fur and fur staining. At 600 mg/kg/day, there were decreases or statistically significant decreases in mean body weights beginning on DS 9 and continuing through the pre-mating, gestation and lactation periods. There was a statistically significant body weight loss observed at 600 mg/kg/day on DGs 10 to 11. At 600 mg/kg/day, there were decreases in maternal food consumption observed on DGs 4 to 7, 7 to 11, 11 to 15 and also on DLs 0 to 4. At 600 mg/kg/day, there were decreases in the number of cycles and the number of days in estrous observed during the estrous evaluation performed 14 days prior to the cohabitation period. There were also statistically significant reductions in the female fertility and pregnancy indices. Mating occurred in 10, 10, 10, 9 and 6 females and pregnancy occurred in 10, 8, 9, 9 and 3 females at 0, 75, 150, 300 and 600 mg/kg/day, respectively. At 600 mg/kg/day, there were statistically significant reductions in the number and percentage of pregnant females. No clinical observations in the F1 generation pups were attributable to maternal doses of the test substance as high as 600 mg/kg/day.

 

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. 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. 

 

Extended one-generation reproductive toxicity study

In a key, K1 extended one-generation reproductive toxicity study performed according to OECD guideline 443 and according to GLP requirements, the test substance was administered to rats at dose levels 50, 150, or 450 mg/kg bw/day via oral gavage (Barnett, 2020).

A total of 100 male and 100 female Crl:CD(SD) Sprague-Dawley rats (P generation) were randomly assigned to four dose groups of 25 rats per sex per group. Male rats were given the test substance and/or the control article (Reverse osmosis membrane-processed deionized water [R.O. deionized water]) formulations once daily via oral gavage beginning 70 days before cohabitation, during cohabitation and continuing through the day before euthanasia (a total of 113-116 consecutive doses). Female rats were given the test substance and/or the control substance formulations once daily oral gavage beginning 70 days before cohabitation, during cohabitation and continuing through Postpartum Day 20 (DL 20; rats that delivered a litter not selected for blood sample collection), DL 21 (rats that delivered a litter selected for blood sample collection), or DG 24 (rats that did not deliver a litter). Females not mated after completion of the 14-day cohabitation period were considered to be at DG 0 on the last day of cohabitation. The first day of dosing was designated as Day 1 of study (DS 1). The dose volume was 5 mL/kg based on the most recently recorded body weight, and doses were administered at approximately the same time each day. 

On the day of scheduled euthanasia (10 P generation rats/sex/dose group) or on Day 90 ± 1 postpartum (10 F1 generation rats/sex/dose group), blood samples were collected for clinical pathology and thyroid hormone evaluations.

The following parameters and end points were evaluated in this study for the P generation: viability, clinical signs, body weights, body weight changes, food consumption, mating and fertility, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), thyroid hormone analysis, gross necropsy findings, organ weights, and histopathologic examinations. Male rats were further evaluated for sperm motility, sperm concentration and reproductive organ weights, and female rats were further evaluated for estrous cycling and ovarian and uterine contents.  

The following parameters and end points were evaluated in this study for the F1 generation: viability, clinical signs, body weights, body weight changes, food consumption, anogenital distance, nipple presence (males), sexual maturation, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), thyroid hormone analysis, gross necropsy findings, organ weights, and histopathologic examinations. Male rats were further evaluated for sperm motility, sperm concentration and reproductive organ weights, and female rats were further evaluated for estrous cycling. 

 

P generation Male Rats

There were no test substance-related mortalities observed in the P generation male rats. One male in the control group was euthanized due to adverse clinical signs on DS 82, a male at 150 mg/kg/day was found dead on DS 3 and a male at 450 mg/kg/day was euthanized due to adverse clinical signs on DS 36. Based on there being single occurrences in each dose group and there being no correlation in the death or moribundity, these deaths were not considered to be test substance related.

An increased incidence of excess salivation and abnormal breathing sounds were observed in the P generation males at 450 mg/kg/day.

At 450 mg/kg/day, there were reductions or statistically significant reductions in mean body weights observed beginning on DS 106 and continuing for the remainder of study. At 450 mg/kg/day, there was a statistically significant reduction in body weight gain on DSs 7 to 8 and a statistically significant body weight loss on DSs 108 to 109. 

Food consumption was unaffected by doses of test substance in the males at all intervals prior to cohabitation at doses up to and including 450 mg/kg/day. 

All P generation male mating and fertility parameters were comparable among all dose levels. 

There were no test substance-related changes in the thyroid hormone levels observed in the P generation male rats. No hematologic, coagulation, clinical chemistry or urinalysis parameter was affected by doses of the test substance as high as 450 mg/kg/day. 

No effect on sperm (motility or count),gross necropsy and organ weights occurred in any dose group.

Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. In two rats at 450 mg/kg/day, segments of the nasopharyngeal epithelium were replaced by squamous metaplasia.

 

P generation Female Rats

The only deaths that were considered to be test substance-related occurred in the 450 mg/kg/day dose group. One female at 50 mg/kg/day and another at 150 mg/kg/day were euthanized due to no surviving pups. There were no test substance related clinical signs or effects on body weights observed in these females. At 450 mg/kg/day, two females were found dead and three females were euthanized due to adverse clinical signs. Common clinical signs in these females included hunched posture, decreased activity and respiratory issues (labored breathing and abnormal breathing signs). The females that were euthanized due to adverse clinical signs also experienced body weight loss prior to euthanasia. 

During the precohabitation, gestation and/or lactation periods, there was an increased incidence of suspected dehydration, labored breathing, abnormal breathing sounds, shallow breathing, hunched posture, thin fur cover erect fur, hyperreactivity and decreased activity at 450 mg/kg/day.

At 450 mg/kg/day, there were statistically significant reductions in mean body weights observed beginning on DS 20 and continuing for the remainder of the pre-mating period. There were reductions or statistically significant reductions in mean body weights at 450 mg/kg/day on DGs 5, 19 and 21. There was a statistically significant body weight loss at 450 mg/kg/day on DSs 9 to 10.

At 450 mg/kg/day, there were decreases or statistically significant decreases in the mean food consumption values observed on DSs 8 to 11, 11 to 15 and at all intervals between 18 to 22 and 46 to 50. 

Estrous cycling, mating, fertility and natural delivery were not affected by doses of the test substance as high as 450 mg/kg/day.

Maternal doses as high as 450 mg/kg/day did not affect the anogenital distance as measured on the day of delivery. No male pup in any group had nipples observed on PND 11. There were no test substance-related adverse clinical signs or effects on pup weights during the pre-weaning period. 

There were no test substance-related changes in the thyroid hormone levels observed in the P generation female rats. No hematologic, coagulation, clinical chemistry or urinalysis parameter was affected by doses of the test substance as high as 450 mg/kg/day. 

No effect on gross necropsy and organ weights occurred in any dose group.

Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. In three females at 450 mg/kg/day, segments of the nasopharyngeal epithelium were replaced by squamous metaplasia. 

 

F1 Generation Male and Female Rats

The only deaths that occurred in the F1 generation rats occurred at 450 mg/kg/day. At 450 mg/kg/day, one male and one female were found dead during the dose period and three males were euthanized due to adverse clinical signs. Common clinical signs in the rats included respiratory issues (labored breathing and abnormal breathing signs) and suspected dehydration. The three males that were euthanized due to adverse clinical signs also experienced body weight loss prior to euthanasia. One male that was euthanized due to adverse clinical signs had necropsy observations that were indicative of an intubation error. 

There was an increased incidence in abnormal breathing sounds observed at 150 and 450 mg/kg/day in the F1 generation female rats and at 450 mg/kg/day in the F1 generation male rats. There was also an increased incidence of suspected dehydration, excess salivation and labored breathing in the F1 generation male rats.

There were no test substance-related differences in body weights, body weight gains and food consumption observed in the F1 generation male or female rats at doses up to and including 450 mg/kg/day.

There were no test substance-related effects on F1 generation male or female sexual maturation at doses as high as 450 mg/kg/day. There were no test substance-related effects on estrous cycling observed in the F1 generation female rats. There were no test substance-related effects on hematology, coagulation, clinical chemistry or urine analysis parameter at termination of the F1 generation male and female adult rats. 

Thyroid hormones were not affected by doses of the test substance as high as 450 mg/kg/day. 

Significant increases in the number of primordial follicles were observed in left (50 and 450 mg/kg/day only), right (>= 50 mg/kg/day) and both (>= 50 mg/kg/day) ovaries. However, there was not a clear pattern, trend, associated histopathology changes, or identifiable orientation of the ovaries; therefore, these increases appear to likely be due to natural variability than to relation to administration of the test substance. Corpora lutea were present for all animals evaluated in all groups.

No test substance-related gross findings or organ weight changes were observed in the F1 generation male and female rats. No effect on sperm (motility or count) occurred in the F1 generation males.

Inflammation was observed within the nasopharynx and multifocally within the nasal cavity of rats at 150 or 450 mg/kg/day. This inflammation was characterized by infiltrates of neutrophils and less frequently mononuclear cells within the epithelium and lamina propria, as well as within the lumen of the nasopharynx/nasal cavity. Segments of epithelium within areas of inflammation were occasionally necrotic/absent. The nasal respiratory epithelium was more frequently affected than the olfactory epithelium. Segments of the nasopharyngeal epithelium were replaced by squamous metaplasia in one F1 generation female at 450 mg/kg/day. 

 

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for general toxicity was 150 mg/kg/day for the P generation and F1 generation male and female rats. There was an increased incidence of microscopic findings in the nasal cavity of both the P generation and F1 generation rats at 450 mg/kg/day that included inflammation, neutrophilic exudates, and squamous metaplasia of the nasopharynx that were considered test substance-related. Luminal hemorrhage was also observed in the nasal cavity of P generation rats at 450 mg/kg/day. There were abnormal breathing sounds and labored breathing observed in the P generation and F1 generation males and females that correlated with these microscopic findings. Mortality was observed in the P generation females and F1 generation males and females. Reductions in body weight and body weight gain were observed in the P generation males and females at 450 mg/kg/day. 

 

The reproductive NOAEL was 450 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. The NOAEL for viability and growth in the offspring was also 450 mg/kg/day.

Effects on developmental toxicity

Description of key information

- The NOAEL for embryo-fetal survival, development and growth is set at 115 mg/kg/day when the test substance is administered during organogenesis in the rabbit. A key prenatal development study is performed according to OECD guideline 414 and according to GLP requirements (Renaut, 2016; Klimisch 1)

- As no adverse effects were observed when the test substance is administered during organogenesis in the rat, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was concluded to be 350 mg/kg/day. A key prenatal development study is performed according to OECD guideline 414 and according to GLP requirements (Noble, 2020; Klimisch 1)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-08-25 to 2016-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Principles of method if other than guideline:

The study was also designed to meet the requirements of the following guideline: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): POPDA
- Substance type: yellow liquid
- Physical state: liquid
- Analytical purity: 100%
- Purity test date: no datano data
- Lot/batch No.: DR66700414
- Expiration date of the lot/batch: 2016-12-05
- Stability under test conditions: not specified
- Stability under storage conditions: not specified
- Storage condition of test material: at ambient temperature in the dark
- Action of test substance, intended use: reaction products of propane-1,2-diol, propoxylated by amination of the terminal hydroxyl groups
- Correction factor: none
- Purity/weighing factor: none

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 96 female (sexually mature, virgin) New Zealand White Rabbits, supplied by Charles River (France)
- Age at start of study (day 0 of gestation): 21-22 weeks
- Weight at day 0 (after mating): 3.17 - 4.62 kg
- Fasting period before study: no data
- Housing: One animal per cage (acclimatization and gestation) one stock male and one female per cage during mating; suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least 3 times a week. The cages constituting each group were blocked by group and mounted in batteries.
- Diet (e.g. ad libitum): restricted (initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter), Teklad diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 50 mL of water) was offered, the consumption was recorded. In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly.
- Water (e.g. ad libitum): ad libitum, potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided.
- Acclimation period: at least 19 days. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-20°C
- Humidity (%): 40-70%
- Air changes (per hr): filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 10 hours dark/14 hours light, artificial lighting

IN-LIFE DATES: From: 2016-01-30 To: 2016-02-22 to 2016-02-26

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: The required amount of test item for the highest concentration was mixed with sufficient vehicle to form a solution and mixed with a magnetic stirrer. The solution was made up to the required volume with vehicle and stirred again with a magnetic stirrer. The remaining groups were formulated by serial dilution with further quantities of vehicle.
- Individual dose volume was calculated from the most recently recorded scheduled body weight.
- Frequency of preparation: weekly
- A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.


VEHICLE
- Justification for use and choice of vehicle: not applicable (vehicle is water)
- Concentration in vehicle: 0, 3, 10, 23 mg/mL
- Amount of vehicle (if gavage): 5ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.

The analytical method will involve the dilution of the test formulations with a suitable solvent followed by chromatographic assay.
The formulated samples will be analysed using a method transferred from the sponsor with respect to the determination of the specificity of analysis, limit of
detection, linearity of detector response, repeatability, method accuracy and precision.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: no data
- M/F ratio per cage: 1:1, using identified stock New Zealand White bucks
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: natural mating observed referred to as day 0 of pregnancy
- After mating each female was injected intravenously with 25 i.u. luteinising hormone.
- A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

days 6 to 28 (inclusive) after mating (22 days)

Frequency of treatment:

once daily, at approximately the same time each day

Duration of test:

30 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

115 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females per dose

Control animals:

yes

Details on study design:

- Dose selection rationale: the dose levels were selected by the Sponsor based on a preliminary embryo-fetal study in the rabbit (Envigo Study No. TMR0110), in which the high dose of 150 mg/kg/day elicited extended periods of low food intake in some animals and resulted in one animal being killed for reasons of animal welfare, whereas the intermediate dose of 115 mg/kg/day elicited extended periods of low food consumption in some animals but did not result in premature death. A dose level of 150 mg/kg/day was therefore considered too high for subsequent investigation on a main embryo-fetal study, and therefore the dose level of 115 mg/kg/day was selected as the high dose for the current main embryo-fetal development study.
Intermediate and low dose levels of 50 and 15 mg/kg/day, respectively, were selected to evaluate the response at lower dose levels to identify any dose response. The lowest dose level of 15 mg/kg/day was expected to provide a clear No Observed Effect Level for extended periods of low maternal food intake.
- Route of administration rationale: the oral gavage route of administration was chosen to simulate the conditions of possible human exposure.
- Animal species selection rationale: the rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in the laboratory.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during aclimatization at least once per day, during gestation at least twice daily
- Animals were inspected visually for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the treatment period at the following times in relation to dose administration: 1 to 2 hours after completion of dosing, as late as possible in the working day. - Detailed physical examination on days 0, 6, 12, 18, 24 and 29 after mating
- Detailed physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, on days 0, 3, 6 to 29 after mating.
- All adult animals

FOOD CONSUMPTION: Yes
- Time schedule: daily from day 1 after mating.
- All adult animals
- The weight of food supplied supplied to each animal, that remaining and an estimate of any spilled was recorded
- Consumption of hay and vegetables were monitored qualitatively but not quantitatively. No entry was made in the raw data for the provision of vegetables during the period of 26 Jan 16 – 01 February 2016 and thus during this period only, a twice weekly schedule cannot be verified from the raw data.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: day 29 after mating
- Females that exhibit pregnancy loss were killed on the day of detection.
- All adult animals were killed by intravenous injection of sodium pentobarbitone
- Macroscopic pathology: a full macroscopic examination of the tissues was performed for each animal. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

OTHER:
MORTALITY:
- Time schedule: a viability check was performed near the start and end of each working day.
- Animals were killed for reasons of animal welfare where necessary or if they exhibited pregnancy loss.

Ovaries and uterine content:

For females surviving to term, the following was recorded:
- uterus: gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):
- For each ovary/uterine horn: number of corpora lutea, implantation sites, resorption sites (classified as early or late), fetuses (live and dead).
- Apparently non pregnant animals and for apparently empty uterine horns: the absence or number of uterine implantation sites was confirmed.
- Females exhibiting pregnancy loss: expelled uterine contents were identified and examined, as appropriate.

Fetal examinations:

- External examinations: Yes: [all viable fetuses per litter ]
- Examination of all viable fetuses and placentae: dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, samples as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abnormal cavities and the sex of each fetus was also recorded.
- Soft tissue examinations: No
- Skeletal examinations: Yes [one third per litter ], fixed in Industrial Methylated Spirit and stained with Alizarin Red
- Head examinations: Yes: [appr. 50% of eviscerated fetuses ], fixed in Bouin's fluid
- Remaining fetuses will be eviscerated and fixed in Industrial Methylated Spirit.
- All live fetuses: Internal examination of neck and thoracic and abdominal cavities, sex recorded and individual identification within litter.
- Fetuses were killed by subcutaneous injection of sodium pentobarbitone.
- fetal, litter and placental weights: mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.

Statistics:

Body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
- A parametric analysis if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, Dunnett's test (Dunnett 1955, 1964) was performed instead.
- A non-parametric analysis if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made. For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, Steel's test (Steel 1959) was performed instead.

Litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value:
- Fisher’s exact tests (Fisher 1973).

Pre/post implantation loss and sex ratio: generalized mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991).

Resorptions: exact Wilcoxon rank sum test (Wilcoxon 1945)

Indices:

- Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) / Number of corpora lutea x 100
Where the total number of implantations exceeds corpora lutea observed, pre-implantation loss was assumed to be 0.

- Post implantation loss (%) = (Number of implantations – Number of live fetuses) / Number of implantations x 100

Historical control data:

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Female number 82 receiving 115 mg/kg/day was despatched to necropsy for reason of animal welfare on day 23 of gestation following a prolonged period of marked inappetance from day 9 of gestation and subsequent body weight loss of 590 g from day 6 of gestation. At necropsy this female showed 8 normal implantations, and there were no remarkable macroscopic observations. This prolonged period of reduced food intake is likely to be related to treatment.
- Female number 83 receiving 115 mg/kg/day was despatched to necropsy following evidence of abortion. This female had shown body weight loss of 310 g from day 10 of gestation, and food consumption had been very low from day 11 of gestation. Fetal material was observed in the cage under tray on day 20 of gestation. At necropsy this female showed a pale liver, and the animal was confirmed as pregnant with 12 implantations, 11 of which were live young and one was an aborted implant. Due to its isolated nature, a relationship between treatment and abortion is considered unproven.
- Female number 58 receiving 50 mg/kg/day was despatched to necropsy for reason of animal welfare following the observation of a small amount of clotted blood, and subsequently further blood, in the cage under tray, and signs of piloerection and dark eyes on day 15 of gestation. This female had progressively lost bodyweight (to a total of 260 g) with the most marked loss occurring from day 13 of gestation, and food consumption was very low during days 13 and 14. At necropsy there were no macroscopic changes, but the animal was confirmed as pregnant with 11 implantations, which were all noted to be early resorptions. This rabbit therefore showed total litter resorption. Our preliminary evaluation is that this is not related to treatment.
- Female number 65 receiving 50 mg/kg/day was despatched to necropsy for reason of animal welfare on day 23 of gestation following the observation of a swelling on the ventral abdomen (suspected hernia) on days 22-23 of gestation. This female also showed signs of little hay/water consumed, underactive behaviour, reduced body temperature (assessed by touch), deep and irregular breathing, thin build, few faeces, reduced urine output and dark eyes on day 23 of gestation. Body weight loss of 330 g was evident from day 15 of gestation, and food consumption was markedly low from day 14 of gestation. At necropsy the hernia was confirmed (jejunum protruded through the abdomen wall), stomach contents were dark and liquefied, the stomach was distended with dark areas on the glandular mucosa, the jejunum and duodenum were distended and the caecum had firm contents. This female was pregnant with 10 implantations, all of which were late resorptions. This rabbit therefore also showed total litter resorption. Our preliminary evaluation is that this is not related to treatment.
- In animals surviving to scheduled termination there was an increased incidence of females showing clinical signs indicative of inappetance at 115 mg/kg/day. These signs included thin build, little hay eaten, reduced urine output and few, small and pale faeces. Three females receiving 50 mg/kg/day were also noted to have thin build.
- There were no other test article related clinical signs, nor any dosing signs, observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

- Two females receiving 115 mg/kg/day, and two females receiving 50 mg/kg/day, were killed for reasons of animal welfare.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 115 mg/kg/day mean body weight gain during days 6-14 of gestation was similar to Controls, however, during days 14-28 of gestation mean bodyweight loss of approximately 120 g was evident, compared to body weight gain (ca. 90 g) in the Control group. When mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, overall maternal mean body weight loss during days 6-29 of gestation was greater than Controls at 115 mg/kg/day, confirming that the maternal portion of gestational body weight gain was affected by treatment at this high dose level.
- Body weight performance at 15 or 50 mg/kg/day was unaffected by treatment with mean body weight gain during days 6-14 and 14-28 being similar to Control values. When adjusted for the contribution of the gravid uterus, adjusted body weight change values at 50 or 15 mg/kg/day were similar to Controls indicating that maternal weight gain was unaffected by treatment at these dose levels.
More details are available in data tables attached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Mean values of food consumption at 115 mg/kg/day were lower than Controls from day 15 of gestation to termination on day 29 of gestation. Resulting overall mean food consumption values for the treatment period (days 6-28) were 79 % of Controls.
- Food consumption at 50 or 15 mg/kg/day was similar to Controls throughout gestation and unaffected by treatment.

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:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no maternal findings observed at macroscopic examination that were considered to relate to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

In addition to the premature deaths observed, there was also one female in each treatment group that was observed to be not pregnant at necropsy on Day 29 after mating. The following assessment of litter data is therefore based on a total of 22, 21, 19 and 19 pregnant females reaching full term in groups 1-4, respectively.
More details are available in data tables attached.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Mean post-implantation loss at 115 mg/kg/day was marginally higher than Control values, resulting in a slight increase in the numbers of early and late, and therefore total, resorptions; and subsequently slightly fewer live young when compared with Control values, however, no statistical significance was attained.
Embryo-fetal survival was unaffected by treatment at 15 and 50 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and postimplantation loss being similar to Control values.
More details are available in data tables attached.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Mean post-implantation loss at 115 mg/kg/day was marginally higher than Control values, resulting in a slight increase in the numbers of early and late, and therefore total, resorptions; and subsequently slightly fewer live young when compared with Control values, however, no statistical significance was attained.
Embryo-fetal survival was unaffected by treatment at 15 and 50 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and postimplantation loss being similar to Control values.
More details are available in data tables attached.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Mean post-implantation loss at 115 mg/kg/day was marginally higher than Control values, resulting in a slight increase in the numbers of early and late, and therefore total, resorptions; and subsequently slightly fewer live young when compared with Control values, however, no statistical significance was attained. Embryo-fetal survival was unaffected by treatment at 15 and 50 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and postimplantation loss being similar to Control values.
More details are available in data tables attached.

Dead fetuses:

no effects observed

Description (incidence and severity):

Embryo-fetal survival was unaffected by treatment at 15 and 50 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and postimplantation loss being similar to Control values.
More details are available in data tables attached.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean placental, litter and fetal weights at 115 mg/kg/day were marginally lower than Controls, with statistical significance attained for litter weight, and male and overall fetal weights.
Mean placental, litter, and male, female and overall fetal weights at 15 and 50 mg/kg/day were similar to Controls and unaffected by treatment.
More details are available in data tables attached.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Mean post-implantation loss at 115 mg/kg/day was marginally higher than Control values, resulting in a slight increase in the numbers of early and late, and therefore total, resorptions; and subsequently slightly fewer live young when compared with Control values, however, no statistical significance was attained.
Embryo-fetal survival was unaffected by treatment at 15 and 50 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and postimplantation loss being similar to Control values.
More details are available in data tables attached.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Embryo-fetal survival was unaffected by treatment at 15 and 50 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and postimplantation loss being similar to Control values.
More details are available in data tables attached.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Mean placental, litter and fetal weights at 115 mg/kg/day were marginally lower than Controls, with statistical significance attained for litter weight, and male and overall fetal weights.
Mean placental, litter, and male, female and overall fetal weights at 15 and 50 mg/kg/day were similar to Controls and unaffected by treatment.
More details are available in data tables attached.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- At 115 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of large anterior fontanelle, delayed/incomplete ossification/unossified sternebrae, pubes, epiphyses, talus, metacarpals/metatarsals/phalanges compared to the concurrent Control group, and which were outside of historical control data (HCD) ranges. This delay in ossification is a transient stage in fetal development that may be attributed to the slight decrease in mean fetal weight observed and as such this is not considered to be adverse.
- in addition, at 115 mg/kg/day there was also a slightly increased incidence of the minor abnormality of fused/partially fused/misaligned sternebral hemicentres, with associated misaligned costal cartilage when compared to concurrent Control, and these were also slightly outside of HCD ranges. As these incidences were small (max 4 fetuses from 3 litters) a relationship to treatment is considered not proven.
- at 15 and 50 mg/kg/day there was a high incidence of the minor abnormality of delayed/incomplete ossification/unossified sternebrae compared to concurrent control and outside HCD ranges. As at 115 mg/kg/day this delay in ossification is a transient stage in fetal development and as such this is not considered to be adverse.
More details are available in data tables attached.

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter data:
- In addition to the premature deaths observed, there was also one female in each treatment group that was observed to be not pregnant at necropsy on day 29 after mating. The following assessment of litter data is therefore based on a total of 22, 21, 19 and 19 pregnant females reaching full term in Groups 1-4, respectively.
- Mean post-implantation loss at 115 mg/kg/day was marginally higher than Control values, resulting in a slight increase in the numbers of early and late, and therefore total, resorptions; and subsequently slightly fewer live young when compared with Control values.
- Embryo-fetal survival was unaffected by treatment at 15 or 50 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre and post-implantation loss being similar to Control values.

Placental, litter and fetal weight
- Mean placental, litter and fetal weights at 115 mg/kg/day were marginally lower than Controls.
- Mean placental, litter, and male, female and overall fetal weights at 15 or 50 mg/kg/day were similar to Controls and unaffected by treatment.

Macropathology:
- At 115 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of large anterior fontanelle, delayed/incomplete ossification/ unossified sternebrae, pubes, epiphyses, talus, metacarpals/metatarsals/phalanges compared to the concurrent Control group, and which were outside of Historical Control Data (HCD) ranges (See Annex 3). This delay in ossification is a transient stage in fetal development that may be attributed to the slight decrease in mean fetal weight observed and as such this is not considered to be adverse.
- In addition, at 115 mg/kg/day there was also a slightly increased incidence of the minor abnormality of fused/partially fused/misaligned sternebral hemicentres, with associated misaligned costal cartilage when compared to concurrent Control, and these were also slightly outside of HCD ranges. As these incidences were small (max 4 fetuses from 3 litters) a relationship to treatment is considered not proven.
- At 15 and 50 mg/kg/day there was a high incidence of the minor abnormality of delayed/incomplete ossification/ unossified sternebrae compared to concurrent control and outside of HCD ranges. As at 115 mg/kg/day this delay in ossification is a transient stage in fetal development and as such this is not considered to be adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

115 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Formulation analysis:

The mean concentrations of the test substance in test formulations were within applied limits +/- 10%, confirming the accuracy of formulation. Difference from mean values were within 2% confirming precise analysis.

Conclusions:

Based on the results of this prenatal developmental toxicity study, it was concluded that the No-Adverse-Effect-Level (NOAEL) of the test substance for maternal toxicity was 50 mg/kg/day as the higher dose of 115 mg/kg/day elicited low food consumption that directly resulted in a single mortality, and elicited clinical signs relating to inappetance and overall body weight loss during gestation compared with body weight gain in all other groups.
The NOAEL for embryo-fetal survival, development and growth is set at 115 mg/kg/day when the test substance is administered during organogenesis in the rabbit.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015-10-13 to 2016-01-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD guideline 407

Principles of method if other than guideline:

The study design was based on OECD guideline 407.

GLP compliance:

no

Remarks:

the work performed generally followed GLP principles

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): POPDA
- Substance type: yellow liquid
- Physical state: liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: DR66700414
- Expiration date of the lot/batch:2016-12-05
- Stability under test conditions: refrigerated (nominally 4 – 8°C) or ambient (nominally 21°C) storage for 15 days
- Storage condition of test material: at ambient temperature in the dark
- Action of test substance: reaction products of propane-1,2-diol, propoxylated by amination of the terminal hydroxyl groups
- purity/weighing factor: none
- total correction factor: none

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 36 female (sexually mature, virgin) New Zealand White rabbits, supplied by Charles River (France)
- Age at start of the study: 18-19 weeks (pilot phase); 24 to 25 weeks (preliminary phase)
- Weight range at start of the study: 3.15 - 4.26 kg (pilot phase); 3.87 - 4.54 kg (preliminary phase)
- Fasting period before study: no data
- Housing: individual housing during acclimatization and gestation, one stock male and one female during mating; suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least 3 times a week. Cages were also fitted with a plastic resting platform. The cages constituting each group were blocked by group and mounted in batteries.
- Diet (e.g. ad libitum): restricted, initially 150 g/rabbit/day during acclimatisation up to one week prior to the onset of mating and 200 g/animal/day thereafter; Harlan Teklad 2930, pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50g pelleted diet moistened with 50 ml of water) was offered, the consumption was recorded. In addition, a small supplement of autoclaved hay was given on a daily basis to promote gastric mobility and a small amount of chopped fresh vegetables were given twice weekly.
- Water (e.g. ad libitum): ad libitum, potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided.
- Acclimation period: 15 days (pilot phase), 55 days (preliminary phase)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-20 °C
- Humidity (%): 40-70%
- Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were considered not to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr): filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
- Pilot phase: from 2015-11-03 to: 2015-11-26
- Preliminary phase: from 2015-12-13 to 2016-01-05

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The required amount of test item for the highest concentration was mixed with sufficient vehicle to form a solution and mixed with a magnetic stirrer. The solution was made up to the required volume with vehicle and stirred again with a magnetic stirrer. The remaining group was formulated by serial dilution with further quantities of vehicle.
- Frequency of preparation: weekly, batches may be prepared in advance but will be used within documented stability limits.
- Storage of formulations: at ambient temperature (21°C)
- Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

VEHICLE
- Concentration in vehicle:
- pilot phase: 15 mg/ml (75 mg/kg) and 30 mg/ml (150 mg/kg);
- preliminary phase: 23 mg/ml (115 mg/kg) and 30 mg/ml (150 mg/kg)
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: no data
- M/F ratio per cage: 1:1 using identified stock New Zealand White bucks
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes, a colony of stock males of the same strain was maintained specifically for the purpose of mating; these animals are not part of the study and were maintained as stock animals.
- Proof of pregnancy: natural mating observed referred to as day 0 of pregnancy
- checks: natural mating observed
- after mating: each female was injected intravenously with 25 i.u. luteinising hormone
- day 0 of gestation = day of mating

Duration of treatment / exposure:

days 6 to 28, after mating

Frequency of treatment:

once daily, at approximately the same time each day

Duration of test:

22 days (day 6 to day 28 after mating)

No. of animals per sex per dose:

6 females per dose (pilot and preliminary phase), except control group (3 in pilot phase and 3 in preliminary phase)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Pilot phase: the dose levels for the Pilot phase were selected in conjunction with the Sponsor based on a previous Preliminary Toxicity study in the New Zealand White Rabbit (Study number TMR0109). In that study, a single dose of 600 mg/kg/day was not tolerated, and was associated with marked macroscopic findings relating to depressions or perforation on the stomach, and other gastro-intestinal findings including dark areas on the jejunum and thickened omental adipose tissue.
Repeat daily dosing at a lower dose of 300 mg/kg/day was initially tolerated for a period of 11 days, but did elicit a marked reduction in food consumption for two out of three females. After receiving 11 doses one female was found dead and at macroscopic examination showed multiple depressions on the stomach. Repeat daily dosing at 300 mg/kg/day was subsequently suspended due to the similarity of the stomach findings in the decedent to those observed at the higher dose of 600 mg/kg/day. At necropsy a total of two out of three females receiving a daily dose of 300 mg/kg/day showed a marked reduction in food intake and macroscopic findings in the stomach and GI tract whilst the third animal was not similarly affected.
It is considered that daily dosing at 300 mg/kg/day for a period of 11 days or more is clearly unsuitable for any further investigation. Due to the marked nature of the findings observed in the stomach and GI tract the starting high dose level for the pilot phase of this preliminary embryo-fetal study in the New Zealand White Rabbit is set at 150 mg/kg/day, with the low dose set at 75 mg/kg/day.
Preliminary phase: Results from the pilot phase of the current study suggested potential effects in individual animals in terms of food consumption, and the possibility of marginally low fetal weights, at 150 mg/kg/day. The high dose level of 150 mg/kg/day was therefore repeated on the preliminary phase in order to substantiate these findings. A lower dose level of 115 mg/kg/day was also implemented on the preliminary phase to investigate the response between 75 and 150 mg/kg/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (at least once per day during the acclimatisation period)
- Animals visually inspected for evidence of reaction to treatment or ill-health. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the treatment period at the following times in relation to dose administration: pre-dose observation, 1 to 2 hours after dosing, as late as possible in the working day; detailed physical examination was performed on days 0, 6, 12, 18, 23 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatisation, on day of mating (day 0) and days 3 and 6-29 after mating
- all adult animals

FOOD CONSUMPTION: Yes
- Time schedule: daily from day 1 after mating
- all adult animals
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded.

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

POST-MORTEM EXAMINATIONS:
- Necropsy: all adult animals were subject to a detailed necropsy. Animals surviving until the end of the scheduled study period were killed at day 29 after mating; All adult animals were killed by intravenous injection of sodium pentobarbitone. Fetuses were killed by subcutaneous injection of sodium pentobarbitone.
- Macroscopic pathology: after a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

MORTALITY:
- A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

Ovaries and uterine content:

- The uterine content was examined after termination only for females exhibiting pregnancy loss.
- For females surviving to full term (day 29 after mating), the following was recorded: gravid uterine weight (including cervix and ovaries)
- The following was recorded for all animals (including those prematurely sacrificed, where possible): for each ovary/uterine horn: number of: corpora lutea, implantation sites, intrauterine deaths (assessed as early or late resorptions), fetuses (live and dead); apparently non-pregnant animals and for apparently empty uterine horns: the absence or number of uterine implantation sites was confirmed.
- Any photographs of unusual findings were taken at the discretion of the necropsy supervisor.
- Fetuses and placentae were dissected from the uterus and weighed individually.

Fetal examinations:

- External examinations: Yes, each placenta and fetus was externally examined.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Internal examination of neck and thoracic and abdominal cavities was performed and any abnormalities were recorded, sampled as appropriate and retained in appropriate fixative.
- The sex of each fetus was recorded.
- Grossly normal fetuses were discarded.

Statistics:

No statistical analysis performed.

Indices:

- Prenatal losses are seperated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant.
Pre-implantation loss (%) = (Number corpora lutea – Number implantations)/Number corpora lutea x 100
Where the total number of implantations exceeds corpora lutea observed, preimplantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
- Post implantation loss (%) = (Number implantations – Number live fetuses)/Number implantations x 100
- All group values and SD (as appropriate) were calculated from the individual litter values.

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality and clinical signs:
One female (number 15) receiving 150 mg/kg/day in the pilot phase was despatched to necropsy for reasons of animal welfare following an extended period of inappetance and clinical signs indicative of inappetance (little diet/water consumed, small and reduced faeces) and underactive behaviour. Upon veterinary examination it was deemed necessary to despatch this female to necropsy. Macroscopic examination revealed no findings, in particular no findings related to abnormalities of the gastro-intestinal tract. This mortality was considered to be related to inappetance, which is likely to be caused by the test material.There were no premature deaths at 150 mg/kg/day in the prelim phase.
There were no premature mortalities at 115 or 75 mg/kg/day.
In animals surviving to scheduled termination there were no consistent clinical signs, nor were there any dosing signs, observed that were considered to be related to treatment at any of the dose levels investigated.
Signs considered to be related to treatment related effects upon food consumption included two animals at 115 mg/kg/day, and one at 150 mg/kg/day, on the prelim phase that were observed to be thin, and pale and few faeces, which were observed in 4 animals at 115 mg/kg/day and few faeces in two animals receiving 150 mg/kg/day.

Body weight and gravid uterine weight:
Body weight was not clearly affected by treatment.
On the pilot phase, group mean overall body weight gain during Days 6-29 of gestation was lower than controls amongst females receiving 75 or 150 mg/kg/day; however, when mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was similar to controls at both dose levels investigated. This suggested that the reduced gravid uterine weight observed was the influencing factor in treated animals not gaining as much weight as controls and that the maternal portion of gestational body weight gain was unaffected by treatment.
On the prelim phase, slight group mean overall bodyweight loss was evident at 115 mg/kg/day and overall bodyweight gain lower than that of Controls was evident at 150 mg/kg/day. When mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was slightly greater than controls at both dose levels.
At 115 mg/kg/day overall body weight change was heavily influenced by two females (numbers 23 and 24) showing marked weight loss of 0.40 – 0.41 kg, however, all other females at this dose level showed lower weight gain than in controls.
At 150 mg/kg/day on the prelim phase overall body weight change was heavily influenced by a single female (number 28) that showed weight loss of 0.20 kg, however, all other females at this dose level (i.e. 5/6 females) showed overall weight gain that was similar to controls.
Individual values of weight change following adjustment for the contribution of the gravid uterus indicated 4 / 6 females at 115 mg/kg/day, and two females at 150 mg/kg/day, with greater adjusted body weight loss than controls. Control values were also influenced by one female (number 6) showing overall body weight gain after adjustment for the contribution of the gravid uterus, due to a very low litter size (3 implantations).

Food consumption:
At 150 mg/kg/day, four out of a total of 12 animals at this dose level showed periods of low food intake. Females 15 (pilot phase) and 28 (prelim phase) showed low food from day 15 after mating, female 16 showed low food during days 15-20 after mating and female 18 showed low food intake from day 22 after mating.
At 115 mg/kg/day three out of six females showed periods of low food intake. Female 23 showed low food from day 13 after mating, female 24 showed low food intake from day 14 after mating and female 19 showed low food from day 24 after mating.
These incidences were compared to no similar cases in the control or 75 mg/kg/day groups.

Macropathology
There were no maternal findings observed at macroscopic examination of adult females receiving 75, 115 or 150 mg/kg/day that were considered to relate to treatment.

Key result

Dose descriptor:

dose level: other

Remarks:

no remarks

Effect level:

ca. 115 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

no remarks

Basis for effect level:

mortality

Remarks on result:

other: 115 mg/kg considered appropriate high dose in main OECD414 study due to mortality observed at 150 mg/kg as a direct result of sustained period of very low food intake.

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter data:
All females on the pilot phase were pregnant. One Group 3 female receiving 150 mg/kg/day was killed for welfare reasons therefore the assessment of litter data is based on a total of 6, 6 and 5 pregnant females reaching full term in Groups 1-3, respectively.
One female on the preliminary phase was found to be not pregnant at necropsy, female 27 receiving 150 mg/kg/day. The assessment of litter data on the preliminary phase is therefore based on a total of 6, 6 and 5 pregnant females reaching full term in Groups 1, 4 and 5, respectively.
Embryo-fetal survival was unaffected by treatment at 75, 115 or 150 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and post-implantation loss being similar to control values.

Placenta, litter and fetal weight:
Mean placental weight at 150 mg/kg/day was marginally lower than controls in the pilot phase but similar to Controls in the preliminary phase, therefore considered unrelated to treatment.
On the pilot phase mean litter weight was lower than controls in both treated groups, despite mean litter size being considered essentially similar across all groups. In the preliminary phase, mean litter weight was similar to Controls at 150 mg/kg/day but marginally lower than controls at 115 mg/kg/day.
Mean male, female and overall fetal weights at 75, 115 or 150 mg/kg/day were marginally lower than controls, with evidence of a slight dose response apparent. This was replicated on both the pilot and prelim phases at 150 mg/kg/day.

Macropathology:
There were no consistent fetal abnormalities observed at macroscopic examination of adult females receiving 75, 115 or 150 mg/kg/day.
There was one fetus in a litter of an adult female receiving 75 mg/kg/day that showed cardiovascular abnormalities including dilated aortic arch, narrow pulmonary trunk and ventricular septal defect.
At 115 mg/kg/day there were three fetuses from a litter of an adult female receiving 115 mg/kg/day with dark thyroids, two of these fetuses also showed enlarged thyroids.
There was one fetus in a litter of an adult female receiving 150 mg/kg/day on the pilot phase that showed the cardiovascular abnormality of retroesophageal subclavian artery, and further findings of small lungs and bilateral forepaw flexure, and a single fetus at 150 mg/kg/day on the preliminary phase that showed unilateral forepaw extension and flexure.
In isolation these findings are currently of uncertain relationship to treatment.

Key result

Dose descriptor:

dose level:

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

Remarks on result:

not determinable

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Based on the results of this study it is concluded that a dose level of 150 mg/kg/day would be too high for investigation on a main embryo-fetal study in the pregnant New Zealand White Rabbit, due to the mortality observed as a direct result of a sustained period of very low food intake.
A dose level of 115 mg/kg/day would therefore be considered appropriate for use as the high dose on a main embryo-fetal study in the pregnant New Zealand White Rabbit. This dose level could be expected to elicit slight maternal toxicity in terms of prolonged periods of lower food intake in some females, but did not result in any premature mortality on this preliminary study.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-09 to final report date

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): POPDA
- Substance type: Colorless to pale yellow liquid
- Physical state: liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: DRW2000305
- Expiration date of the lot/batch: 07 April 2022
- Action of test substance: reaction products of propane-1,2-diol, propoxylated by amination of the terminal hydroxyl groups - purity/weighing factor: none
- total correction factor: none

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15 to 25°C), protected from air,
light and humidity.
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: the stability of formulations in the concentration range 3 to 200 mg/mL was determined as one day at ambient temperature (15 to 25°C) and 15 days refrigerated (2 to 8°C).


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

Species:

rat

Strain:

other: Crl:CD(SD) rat

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 88 females Crl:CD(SD) rats Charles River (UK) Ltd.
- Age at study initiation: 10 to 11 weeks old
- Weight at study initiation: 0.235 to 0.300 kg
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. The cages constituting each group were blocked by group and mounted in batteries. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage : during acclimatization up to four animals, during pairing: one (stock) male and one female, during gestation: one female.
- Diet: ad libitum, SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: ad libitum, Potable water from the public supply via polycarbonate
bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 6 days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: from 2020-10-14 to: 2020-11-13

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: Starting with the highest concentration, the required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer for a minimum of twenty minutes. The remaining concentrations were prepared by serial dilution with further quantities of vehicle and stirred using a magnetic stirrer.
- Frequency of preparation: weekly
- Storage of formulation: Refrigerated (2 to 8°C).
- Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts as checked before the formulations were dispen sed.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

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

- The pH of the formulations was high (alkaline). Therefore, following filling of the dose equipment for each animal, the catheter was dipped in tap water, wiped dry, then dipped in tap water again before administration, to prevent any unwanted animal response during the dosing procedure. A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

VEHICLE
- Justification for use and choice of vehicle: not applicable (vehicle is water)
- Concentration in vehicle: 0, 8, 25, 70 mg/ml
- Amount of vehicle (if gavage): 5ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of formulations in the concentration range 3 to 200 mg/ml was determined as one day at ambient temperature (15 to 25°C) and 15 days refrigerated (2 to 8 °C).
Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.

The analytical method will involve the dilution of the test formulations with a suitable solvent followed by chromatographic assay.
The formulated samples were analysed using a method transferred from the sponsor with respect to the determination of the specificity of analysis, limit of
detection, linearity of detector response, repeatability, method accuracy and precision.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: 6 days
- M/F ratio per cage: 1:1, one (stock) male and one female
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
- Any other deviations from standard protocol: Not applicable

Duration of treatment / exposure:

days 6 to 19 after mating

Frequency of treatment:

once daily, at approximately the same time each day

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: the dose levels for investigation were selected in conjunction with the Sponsor and based on the results of a preliminary embryo-fetal development study (Covance Study No. 8442209). In that study dose levels of 125, 250 and 500 mg/kg/day were investigated.
At 500 mg/kg/day there were two unexplained premature deaths. In addition, body weight
gain and food intake were reduced and a slight body weight loss was observed when adjusted
for the contribution of the gravid uterine weight. There were no findings at macroscopic examination of the adult females and no treatment related findings at external examination of the fetuses at any dose level investigated.
Due to the premature deaths of 2/6 females (33%) and adjusted maternal body weight loss at 500 mg/kg/day this dose level was above the maximum tolerated dose. The high dose on the current main OECD 414 study was set at 350 mg/kg/day, with intermediate and low dose levels set at 125 and 40 mg/kg/day, respectively, in order to fulfill the two-fold to four-fold dosing interval as specified in the test guideline.

Route of administration rationale: The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Animal species selection rationale: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily during acclimatization at least once per day, at least twice daily for reaction to treatment, daily amongst occupants.
- Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during treatment period in relation to dose administration; on days 0, 5, 12, 18 and 20 after mating (to monitor general health). Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration: Pre-dose observation, one to two hours after completion of dosing and as late as possible in the working day.
- A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20
after mating to monitor general health.


BODY WEIGHT: Yes
- The weight of each adult was recorded on Days 0, 3 and 6 to 20 after mating.

FOOD CONSUMPTION: Yes
-The weight of food supplied to each adult, that remaining and an estimate of any spilled was
recorded for the periods Days 0-3, 3-6, 6-10, 10-14, 14-18 and 18-20 after mating inclusive.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: on day 20 after mating.
- Females that exhibit pregnancy loss were killed on the day of detection.
- Macroscopic pathology: a full macroscopic examination of the tissues was performed for each animal. All external features and orifices were examined visually.

- Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
o Organ weights: For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals euthanised at scheduled intervals. Organs: kidney, liver, thyroid (weighed after partial fixation), gravid uterus (with cervix).
o tissue fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin. Organs: kidney, liver, thyroid
o histology: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with hematoxylin and eosin. Organs: thyroid
o microscopic examination: Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings. Organs: thyroid

OTHER:
MORTALITY:
- Animals were killed for reasons of animal welfare where necessary or if they exhibited pregnancy loss.

Ovaries and uterine content:

For females surviving to term, the following was recorded:
- uterus: gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):
- For each ovary/uterine horn: number of corpora lutea, implantation sites, resorption sites (classified as early or late), fetuses (live and dead).
- Apparently non pregnant animals and for apparently empty uterine horns: the absence or number of uterine implantation sites was confirmed.
- Females exhibiting pregnancy loss: expelled uterine contents were identified and examined, as appropriate.

Fetal examinations:

- External examinations: Yes: [all viable fetuses per litter ]
- Soft tissue examinations: No
- Skeletal examinations: Yes [one third per litter ], fixed in Industrial Methylated Spirit and stained with Alizarin Red
- Head examinations: No
- Remaining fetuses will be eviscerated and fixed in Bouin’s fluid.
- All live fetuses: Internal examination of neck and thoracic and abdominal cavities, sex recorded and individual identification within litter.
- Fetuses were killed by chilling on a cool plate (approximately 0°C).
- Fetal, litter and placental weight: Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter. Group mean values and SD were calculated using individual litter mean values.
- Ano-genital distance: Ano-genital distance were presented both as absolute/unadjusted and adjusted for fetal body weight, using the weight recorded at necropsy.

Statistics:

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

Indices:

- Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) / Number of corpora lutea x 100
Where the total number of implantations exceeds corpora lutea observed, pre-implantation loss was assumed to be 0.

- Post implantation loss (%) = (Number of implantations – Number of live fetuses) / Number of implantations x 100

Historical control data:

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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During routine physical examination, there were no signs that were attributed to treatment with the test substance. Transient signs of dry rales were observed post-dose in two females that received 125 mg/kg/day (on Day 7/8 or Day 17 of gestation) and one female that received 350 mg/kg/day (on Day 12 of gestation). Transient signs of wet rales were also observed in Covance Study Number 8442199 - 30 - one female that received 125 mg/kg/day (on Day 15 and 17 of gestation) and two females that received 350 mg/kg/day (on Day 13 or 14 of gestation).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No test item-related mortality occurred. All animals survived to their scheduled sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 350 mg/kg/day was associated with mean body weight stasis between Days 7-8
of gestation followed by minor mean weight loss of 2 g between Days 8-9 of gestation.
Thereafter, mean body weight gains were generally similar to control until Day 18 of
gestation, then mean body weight gain during Days 18-20 of gestation was lower than in
Controls (20 g v 30 g in Controls). These effects resulted in mean overall weight gain being
27% lower than in Controls. Body weight gain was unaffected by treatment at 125 or
40 mg/kg/day.
More details are available in data tables attached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced throughout the treatment period for females that received
350 mg/kg/day such that the overall mean intake between Day 6 to 20 of gestation was 20%
lower than Control. Overall group mean food intake was also marginally lower than Control
at 125 mg/kg/day. There was no effect of treatment on food intake at 40 mg/kg/day.

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

Description (incidence and severity):

Absolute and terminal body weight adjusted liver weights showed a dose-dependent decrease
with statistical significance attained for all treated groups. There was no effect of treatment on kidney or thyroid weights.
There was no effect of treatment on gravid uterine weight. The group mean terminal body
weight (adjusted for the gravid uterine weight) was statistically significantly low, when
compared to controls, for females that received 350 mg/kg/day and the adjusted body weight
change revealed a small group mean body weight loss (-4 g) for females that received
350 mg/kg/day. A review of the individual data revealed particularly large adjusted body
weight losses (-46 g and -34 g) in 2 females at 350 mg/kg/day.
More details are available in data tables attached.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no maternal findings observed at macroscopic examination that were considered
to be related to treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related findings observed at histopathological examination of the
thyroid.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect of treatment on litter data as assessed by pre- or post-implantation loss.
More details are available in data tables attached.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no effect of treatment on litter data as assessed by the number of implantations,
resorptions (early, late and total).
More details are available in data tables attached.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females were found to be pregnant.
More details are available in data tables attached.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male, female and overall fetal weights were marginally lower than Control at 350 mg/kg/day
(5% lower) with statistical significance attained for female and overall fetal weights.
More details are available in data tables attached.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of treatment on number of live young.
More details are available in data tables attached.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of treatment on sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of treatment on placental or litter weights at any dose level investigated.
More details are available in data tables attached.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants showed no relationship
to treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants showed no relationship
to treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Formulation Analysis 

Rework was performed for all sample extracts due to standard verification failure during the initial analysis.

The mean concentrations were within 8% of the nominal concentration for Groups 3 and 4, confirming the accuracy of formulation.  The difference from mean remained within 2% for Groups 3 and 4, confirming precise analysis.

Group 2 formulations for the first and last preparations had high variability in the rework analysis and upon examination of the chromatography, there was found to be baseline interference causing variability in the responses in the Group 2 samples.  The initial analysis results were examined and found to have unusual variability in the internal standard response, which is thought to have contributed to the failure of the standard verification check.

Overall, the results from the initial analysis of Group 2 samples were considered more reliable than the rework results, although both fell outside the acceptance criterion of ±10% of nominal concentration.  The samples for both preparations were within ±20% of nominal concentration in the initial analysis; these results are reported for information only.

Conclusions:

As no adverse effects were observed, the No-Observed-Adverse-Effect-Level (NOAEL) for
maternal toxicity and embryo-fetal survival and development was concluded to be
350 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

115 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

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

Screening study via topical application

No adverse effects on development were demonstrated in a reproductive/developmental toxicity screening study (OECD 421), with the test substance administered up to 30 mg/kg/day via dermal application.

 

Prenatal development toxicity study in rabbit

A combined pilot and preliminary embryo-fetal development study was performed in rabbits with daily administration from day 6 to 28 after mating of the test substance at 75 or 150 mg/kg/day (pilot phase) or 115 or 150 mg/kg/day (preliminary phase) (Renaut, 2016; Klimisch 1, OECD guideline 414). Mortality was observed as a direct result of a sustained period of very low food intake, hence 150 mg/kg/day is considered too high to investigate in the main embryo-fetal development study. The dose level of 115 mg/kg/day is considered appropriate as high dose, as no premature mortality in this study is observed.

 

Three groups of 22 females received the test substance at doses of 15, 50 or 115 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle only, purified water, at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight, and food consumption are recorded. Adult females were examined microscopically at necropsy on Day 29 after mating; the gravid uterine weight was recorded, and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Treatment of pregnant female New Zealand White rabbits with the test substance daily from Day 6 to Day 28 after mating, inclusive, at 115 mg/kg/day was associated with a prolonged period of low food intake in one female to an extent that necessitated premature euthanasia for welfare reasons. There was also one female killed for welfare reasons following abortion at 115 mg/kg/day; however, in isolation this was considered unlikely to be a result of treatment.

At 50 mg/kg/day there were no mortalities that were considered to be related to treatment.

At 115 mg/kg/day there was an increased incidence, when compared to Controls, of females showing clinical signs indicative of inappetence including thin build, little hay eaten, reduced urine output and few, small and pale faeces. These signs were not observed at the same magnitude at 15 or 50 mg/kg/day.

Mean maternal body weight loss during Days 14 -29 of gestation, and markedly low food intake during Days 15 -28 of gestation, were observed at 115 mg/kg/day only. Body weight and food consumption at 15 or 50 mg/kg/day was similar to the control group and unaffected by treatment.

There were no maternal findings observed at macroscopic examination of adult females receiving 15, 50 or 115 mg/kg/day that were considered to relate to treatment.

Embryo-fetal survival was considered unaffected by treatment at dose levels up to 115 mg/kg/day.

There were no effects of treatment upon embryo-fetal growth or development at any of the dose levels investigated that were considered to be adverse. At 115 mg/kg/day mean fetal weight was slightly lower than in the control group and this, and the higher incidence of a number of minor fetal skeletal abnormalities (primarily delayed ossification), may reflect the reduced maternal food consumption observed at this dose level. At 15 or 50 mg/kg/day there was a higher incidence of delayed ossification of the sternebrae but this was considered no to represent an adverse effect of treatment.

Based on the results of this prenatal development toxicity study, it was concluded that the No-Adverse-Effect-Level (NOAEL) of the test substance for maternal toxicity was 50 mg/kg/day as the higher dose of 115 mg/kg/day elicited low food consumption that directly resulted in a single mortality, and elicited clinical signs relating to inappetence and overall body weight loss during gestation compared with body weight gain in all other groups. The NOAEL for embryo-fetal survival, development and growth is set at 115 mg/kg/day when the test substance is administered during organogenesis in the rabbit.

 

Prenatal development toxicity study in rat

The purpose of this study was to assess the influence of maternal exposure to the test substance on embryo-fetal survival and development in Sprague Dawley rats when administered by oral gavage at doses of 40, 125 or 350 mg/kg/day throughout the organogenesis and fetal growth phases of pregnancy (Day 6 to 19 of gestation).

There was no adverse effect of treatment on embryo-fetal survival or development at any dose level investigated.  A dose dependent decrease in mean T3 and T4 concentrations was observed in all groups of treated maternal females, however, there was considered to be no conclusive evidence that thyroid function was affected by treatment because the group mean T3 and T4 values were within the Historical Control Data (HCD) range, as were a majority of the individual values, there was no effect on TSH concentrations at any dose level, there were no associated thyroid weight changes and no macroscopic or microscopic changes detected in the thyroids at any dose level investigated.  Male, female and overall fetal weights were marginally lower (5% lower) than Control at 350 mg/kg/day with statistical significance attained for female and overall fetal weights, however, the extent of these changes were not considered adverse.

At 350 mg/kg/day maternal body weight gain and food intake were reduced with a slight body weight loss observed when adjusted for the contribution of the gravid uterine weight.  A review of the individual data revealed large adjusted body weight losses in 2 females at 350 mg/kg/day, however, there were no associated changes in maternal clinical condition and no associated effect of embryo-fetal survival or development.  Therefore, the extent of these maternal body weight and food intake changes were not considered adverse.  The extent of the decrease in liver weights at 350 mg/kg/day was not considered adverse.  The extent of the effects on body weight body weight performance and food intake at 125 mg/kg/day were not considered adverse.

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-fetal survival and development administered during organogenesis in the rat, was concluded to be 350 mg/kg/day.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, the substance should not be classified for toxicity to reproduction.