1-butylpyrrolidin-2-one

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-butylpyrrolidin-2-one

EC Number:

222-437-8

EC Name:

1-butylpyrrolidin-2-one

Cas Number:

3470-98-2

Molecular formula:

C8H15NO

IUPAC Name:

1-butylpyrrolidin-2-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): n-butyl pyrrolidone
- Substance type: organic
- Physical state: clear colorless liquid
- Purity test date: 2014-11-28
- Expiration date of the lot/batch: 01 March 2016
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.
- Storage condition of test material: room temperature, in the dark.

Specific details on test material used for the study:

Identification: n-Butyl-Pyrrolidone (TamiSolve® NxG; CAS No. 3470-98-2)
Batch No.: G190180362
Expiration Date: 22 Jan 2022
Physical Description: Clear, colorless liquid
Purity: 99.79 %
Water Content: 0.01 %
Storage Conditions: Kept in a controlled temperature area set to maintain 18 °C to 24 °C, protected from light, under nitrogen
Supplier: Chemical Marketing Concepts

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

SD rats are commonly used in DART studies, and previous testing was done with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing
On arrival, the F0 animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, the F0 animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Following weaning (F1), animals were group housed (2 to 3 animals of the same sex) until euthanasia. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study.
Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dose level, and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions
Target temperatures of 68 °F to 78 °F (20 °C to 26 °C) with a relative target humidity of 30 % to 70 % were maintained. A 12-hour light/12-hour dark cycle was maintained.

Food
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system. Water bottles were provided, if required.
Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
For enrichment, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance and vehicle were administered as a single daily oral gavage dose. F0 males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia. F0 females were dosed for 14 days prior to mating and continuing through Lactation Day 20. Females with no evidence of mating were dosed through the day prior to euthanasia. All animals were dosed at approximately the same time each day.

The offspring of the F0 generation (F1 litters) were potentially exposed to the test substance in utero, as well as via the milk while nursing. Selected F1 pups (1 pup/sex/litter) were directly administered the test substance following weaning from PND 22 through 54 for males and PND 22 through 35 for females.

Details on mating procedure:

After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12 hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses described below were performed by a high performance liquid chromatography method with ultraviolet light absorbance using a validated analytical procedure (Engda, 2019, 00387129).

Concentration Analysis
Duplicate sets of samples for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each concentration was ≤ 10 % and if mean sample concentration results were within or equal to ± 15 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability of test substance formulations was established over the range of concentrations used on this study for at least 24 hours at room temperature and for 13 days of room temperature and refrigerated (2 °C to 8 °C) storage in an analytical extension validation study (Engda, 2019, 00387129). Therefore, stability of test substance formulations was not assessed on this study.

Duration of treatment / exposure:

The test substance and vehicle were administered as a single daily oral gavage dose. F0 males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia. F0 females were dosed for 14 days prior to mating and continuing through Lactation Day 20. Females with no evidence of mating were dosed through the day prior to euthanasia. All animals were dosed at approximately the same time each day.

The offspring of the F0 generation (F1 litters) were potentially exposed to the test substance in utero, as well as via the milk while nursing. Selected F1 pups (1 pup/sex/litter) were directly administered the test substance following weaning from PND 22 through 54 for males and PND 22 through 35 for females

Frequency of treatment:

daily

Details on study schedule:

F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
To reduce variability among the F1 litters, 8 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. The remaining offspring were euthanized by an intraperitoneal injection of sodium pentobarbital and discarded.
For the F1 generation, 1 male and 1 female per litter were selected for the direct dosing phase (for exceptions, see Appendix 1).
The disposition of all animals was documented in the Study Records

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

325 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals: Guideline 421, Reproduction/Development Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12 13 females per group. Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles (e.g., EDDDE), during the pretest period, were excluded from the study, therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
To reduce variability among the F1 litters, 8 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. The remaining offspring were euthanized by an intraperitoneal injection of sodium pentobarbital and discarded.
For the F1 generation, 1 male and 1 female per litter were selected for the direct dosing phase.
The disposition of all animals was documented in the Study Records

The route of administration was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
The dose levels for this study were determined from results of previous studies. In a previous 90 day repeat dose study in rats and an oral prenatal developmental toxicity study in rats, both of which used dose levels up to 500 mg/kg/day, showed that this was suitable as the high-dose level to produce a lowest observed-adverse-effect level (LOAEL) (Wakefield, 2014, 41303953; Tanner, 2016, 00387076). As a result, dose levels of 100, 325, and 500 mg/kg/day were chosen to assess male and female reproduction within the scope of the current screening study.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Viability
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Observations
The animals were removed from the cage, and a detailed clinical observation was performed once daily throughout the study. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded approximately 1 hour postdose.
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

Body Weights
Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 3, 7, 10, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, 14, 17, and 21. A fasted weight was recorded on the day of necropsy. Terminal body weights were not collected from animals found dead or euthanized moribund.

Food Consumption
Food consumption was quantitatively measured weekly until cohabitation. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 3, 7, 10, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 14, 17, and 21.

Oestrous cyclicity (parental animals):

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed or until the end of the mating period. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

None

Litter observations:

The day parturition was initiated was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups), with the following exception; if a female was completed at the first parturition check of the day, Lactation Day 0 was the day prior. During the period of expected parturition, females were observed 3 times daily (starting at 0800, 1200, and 1600 hours ± 1 hour) for initiation and completion of parturition and for dystocia or other difficulties. All females were allowed to deliver naturally. Beginning on the day parturition was initiated, the number of live pups were recorded. Individual gestation length was calculated using the date delivery was first observed.

Postmortem examinations (parental animals):

Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained.
For females found dead or euthanized during lactation, the number of corpora lutea and former implantation sites were recorded. All pups were euthanized by an intraperitoneal injection of sodium pentobarbital in the scapular region and necropsied.

Scheduled Euthanasia
All surviving animals, including females that failed to deliver, were euthanized by carbon dioxide inhalation.

Necropsy
Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. Uteri of females without macroscopic evidence of implantation were opened and placed in 10 % ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).

Postmortem examinations (offspring):

Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained.
Scheduled Euthanasia
All surviving animals were euthanized by carbon dioxide inhalation.
Necropsy
All animals were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive.

Statistics:

Shown below in "other" section due to a lack of allowed characters here. That should be changed in the next version as there is no reason to restrict character number here!

Reproductive indices:

See results

Offspring viability indices:

See results

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on survival in the F0 generation. Two F0 females in the 500 mg/kg/day group did not survive to the scheduled necropsy. Female No. 4507 in this group was found dead on Lactation Day 16 in the absence of remarkable clinical observations, changes in body weight/food consumption, or gross necropsy findings. Microscopically, hepatocellular hypertrophy (minimal) was noted for this female, which was also observed for 7 of 8 surviving females in this group, and considered a nonadverse, test substance-related effect. Female No. 4508 in the 500 mg/kg/day group was euthanized in extremis on Lactation Day 17 due to the severity of abnormal breathing sounds noted on this day. No remarkable gross findings at necropsy or microscopic findings were noted for this female. The causes of death/moribundity at 500 mg/kg/day were unable to be determined, but were considered unrelated to the test substance due to the absence of evidence of systemic toxicity at this dose level. In the 325 mg/kg/day group, Female No. 3507 was euthanized in extremis on Lactation Day 9 due to poor clinical condition, including limited usage of forepaws and hindpaws, hunched posture, erect fur, skin pallor, and thin body on Lactation Days 8 and 9, and marked body weight loss (25%) with correspondingly lower food consumption (19 to 28 g/day) from Lactation Days 1 7. Necropsy findings for this female consisted of pale foci or discoloration in the heart, liver, and kidney. Microscopically, there was evidence of bacterial sepsis, and therefore this moribundity was considered unrelated to the test substance. All other F0 animals survived to the scheduled necropsy.
Abnormal breathing sounds were limited to 1 F0 male and 5 F0 females in the 500 mg/kg/day group at the daily examinations and/or approximately 1 hour following dose administration. This observation was primarily noted on single occasions, with the exception of 1 F0 female (No. 4505) with 14 occurrences. Salivation-related clinical observations (salivation and wet fur around mouth) were also noted at an increased incidence in the 500 mg/kg/day group F0 females at the daily examinations and/or approximately 1 hour following dose administration compared to the control group. These observations were limited to single animals or sex and occurred in the absence of any effects on parental body weight or food consumption, and therefore were considered test substance-related but not adverse. Other observation noted in the test substance treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males
Mean body weights and body weight gains in the 100, 325, and 500 mg/kg/day group F0 males were unaffected by test substance administration throughout the study (Study Days 0–27). Any statistically significant differences from the control group were transient and did not occur in a dose responsive manner.

Females

Weekly
Mean body weights and body weight gains in the 100, 325, and 500 mg/kg/day group F0 females were unaffected by test substance administration during the premating period (Study Days 0–13). None of the differences from the control group were statistically significant.

Gestation
Mean body weights and body weight gains in the 100, 325, and 500 mg/kg/day groups were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

Lactation
Mean body weights and body weight gains in the 100, 325, and 500 mg/kg/day groups were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males
Mean food consumption, evaluated as g/animal/day, in the 100, 325, and 500 mg/kg/day group F0 males was unaffected by test substance administration during the premating period (Study Days 0–13). No statistically significant differences were observed.

Females

Weekly
Mean food consumption, evaluated as g/animal/day, in the 100, 325, and 500 mg/kg/day group F0 females was unaffected by test substance administration during the premating period (Study Days 0–13). None of the differences from the control group were statistically significant.

Gestation
Mean maternal food consumption, evaluated as g/animal/day, in the 100, 325, and 500 mg/kg/day groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

Lactation
Mean maternal food consumption, evaluated as g/animal/day, in the 100, 325, and 500 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology parameters in F0 males at any dose level. Differences from controls were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on serum chemistry parameters in F0 males at any dose level. Any statistically significant differences from the control group did not occur in a clear dose-responsive manner, and therefore, were considered to be the result of normal biological variation.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males at any dose level. Differences from the control group were not statistically significant and were considered the result of normal biological variation.

Urinalysis findings:

not examined

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, non-treatment-related

Description (incidence and severity):

Hepatocellular hypertrophy was characterized by an increase in hepatocyte cytoplasm, particularly in centrilobular regions. It was minimal in severity and was considered an adaptive, nonadverse finding. The hepatocellular hypertrophy correlated to higher mean liver weights and to the gross finding of liver enlargement in a single animal.
Diffuse cortical hypertrophy in the adrenal gland occurred at a notably increased incidence in the 325 and 500 mg/kg/day group F0 females and was characterized by an increase in cortical cell cytoplasm in the zona fasciculata. It was minimal in severity and was considered a nonadverse finding. The diffuse cortical hypertrophy correlated to higher mean adrenal gland weights and to the gross finding of adrenal gland enlargement.
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 test substance administration

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on survival were noted at any dose level. F1 Female Nos. 8506 and 8508 were found dead or euthanized in extremis on PND 27 and 26, respectively, following clinical signs of abnormal breathing sounds and/or labored breathing at the daily examinations and/or approximately 1 hour postdose on the day of death or euthanasia. At necropsy, evidence of dosing error was noted for both animals, including material accumulation in the trachea and/or distended/gas-filled intestines; therefore, the mortality and moribundity were considered unrelated to the test substance. In addition, Male No. 6009 and Female No. 6509 in the 100 mg/kg/day group, selected for the F1 generation direct dosing phase, were inadvertently sent to necropsy on PND 21. All other F1 males and females survived to the scheduled necropsies.
Test substance-related abnormal breathing sounds were observed in 1 and 4 F1 males in the 325 and 500 mg/kg/day groups, respectively, at the daily examinations or approximately 1 hour following dose administration. These observations were limited to a small number of animals and occurred in the absence of effects on body weight and food consumption, and therefore were considered nonadverse. No other remarkable clinical observations were noted for F1 males or females at any dose level.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on survival were noted at any dose level. F1 Female Nos. 8506 and 8508 were found dead or euthanized in extremis on PND 27 and 26, respectively, following clinical signs of abnormal breathing sounds and/or labored breathing at the daily examinations and/or approximately 1 hour postdose on the day of death or euthanasia. At necropsy, evidence of dosing error was noted for both animals, including material accumulation in the trachea and/or distended/gas-filled intestines; therefore, the mortality and moribundity were considered unrelated to the test substance. In addition, Male No. 6009 and Female No. 6509 in the 100 mg/kg/day group, selected for the F1 generation direct dosing phase, were inadvertently sent to necropsy on PND 21. All other F1 males and females survived to the scheduled necropsies

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight gains in the 100, 325, and 500 mg/kg/day group F1 males and females were unaffected by test substance administration throughout the generation (PND 22–55 and PND 22–36, respectively). Any statistically significant differences from the control group were transient and/or did not affect mean absolute body weights.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day, in the 100, 325, and 500 mg/kg/day group F1 males and females was unaffected by test substance administration. No statistically significant differences were observed.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration. The mean ages of attainment of balanopreputial separation were 43.0, 44.7, and 44.6 days in the 100, 325, and 500 mg/kg/day groups, respectively, compared to 43.1 days in the control group. Mean body weights at the age of attainment were 238.6 g, 250.1 g, and 247.0 g in the same respective groups compared to 229.1 g in the control group. None of the differences from the control group were statistically significant.

Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration. The mean ages of attainment of vaginal patency were 32.1, 30.8, and 32.0 days in the 100, 325, and 500 groups, respectively, compared to 32.3 days in the control group. Mean body weights at the age of attainment were 119.7 g, 108.8 g, and 114.6 g in the same respective groups compared to 121.7 g in the control group. None of the differences from the control group were statistically significant.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Test substance-related shorter mean anogenital distances (absolute and normalized for body weight) were noted for male pups in the 325 and 500 mg/kg/day groups compared to the concurrent control group. The differences at 500 mg/kg/day were statistically significant and the absolute values in both groups (3.19 and 2.90 mm, respectively) were below the minimum mean value (3.4 mm) in the Charles River Ashland historical control data. Based on these effects, the study was extended to include developmental landmark evaluations for both F1 males and females assigned to the direct doing phase, to further evaluate possible treatment-related effects on pup development.
Anogenital distances (absolute and normalized for body weight) in the 100 mg/kg/day group males and females were similar to the control group values. Differences were slight and not statistically significant

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. No nipples were retained on PND 13 male pups at any dose level.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid weights in the F1 males and females at any dose level on PND 21. Differences from the control group were not statistically significant and were considered the result of normal biological variation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No internal findings that could be attributed to parental test substance administration were noted at the necropsy of pups euthanized on PND 21. Internal findings included a dilated renal pelvis in Pup No. 2505-11 in the 100 mg/kg/day group. No other internal findings were noted.

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Gross Pathology

	Males				Females^a
Group	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	100	325	500	0	100	325	500
No. Animals per Group	10	10	10	10	10	10	9	8
Liver (No. Examined)	(10)	(10)	(10)	(10)	(10)	(10)	(9)	(8)
Enlargement	0	0	0	0	0	0	0	1
Adrenal gland (No. Examined)	(10)	(10)	(10)	(10)	(10)	(10)	(9)	(8)
Enlargement	0	0	0	0	1	1	1	3
^a  Includes females that failed to deliver.

Histopathology

	Males				Females^a
Group	1	2	3	4	1	2	3	4
Dosage (mg/kg/day)	0	100	325	500	0	100	325	500
No. Animals per Group	10	10	10	10	10	10	9	8
Liver (No. Examined)	-10	-10	-10	-10	-10	-10	-9	-8
Hypertrophy; hepatocellular	0	0	8	10	0	0	0	4
Minimal	-	-	8	10	-	-	-	4
Gland, Adrenal (No. Examined)	-10	-10	-10	-10	-10	-10	-9	-8
Hypertrophy; cortical	0	0	0	0	1	2	6	7
Minimal	-	-	-	-	1	2	6	7
^aIncludes females that failed to deliver.
- = Not applicable.

Reproductive performance

Parameter	Dose Level (mg/kg/day)				CRL HC^a Mean (Range)
	0	100	325	500
Male Mating Index (%)	90.0	100.0	100.0	100.0	98.0 (83.3–100.0)
Female Mating Index (%)	90.0	100.0	100.0	100.0	98.0 (83.3–100.0)
Male Fertility Index (%)^b	100.0	90.0	100.0	100.0	95.7 (80.0–100.0)
Female Fertility Index (%)^c	100.0	90.0	100.0	100.0	95.7 (80.0–100.0)
Male Pregnancy Index (%)^d	90.0	90.0	100.0	100.0	93.9 (80.0–100.0)
Female Pregnancy Index (%)^e	90.0	90.0	100.0	100.0	93.9 (80.0–100.0)
Estrous Cycle Length (days)	4.69	3.95	4.02	3.98	4.2 (3.9–5.2)
Pre-Coital Interval (days)	1.9	1.9	2.3	2.0	2.7 (1.4–4.5)
^a Charles River Ashland historical control data (version 2019.05). ^b  Equivalent to Male Copulation Index in the Charles River Ashland historical control data. ^c  Equivalent to Female Conception Index in the Charles River Ashland historical control data. ^d  Equivalent to Male Fertility Index in the Charles River Ashland historical control data. ^e  Equivalent to Female Fertility Index in the Charles River Ashland historical control data.

Text Table 25
Summary of Organ Weight Data – Terminal Euthanasia (F0)^a
	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	100	325	500	0	100	325	500
No. Animals per Group	10	10	10	10	9	9	9	8
Liver (No. Weighed)^b	-10	-10	-10	-10	-9	-9	-9	-8
Absolute value	15.32	16.10 (5.09)	19.25** (25.67)	19.92** (30.01)	16.44	17.44 (6.06)	12.04** (15.42)	24.84** (31.68)
Relative to body weight	3.43	3.70* (7.88)	4.29** (25.09)	4.47** (30.07)	4.85	4.94 (1.81)	5.50** (13.31)	5.92** (22.00)
Relative to brain weight	722.2	770.6 (6.70)	904.5** (25.24)	931.7** (29.00)	816.8	880.4 (7.78)	958.3** (17.32)	1084.2** (32.74)
Adrenal gland (No. Weighed)^b	-10	-10	-10	-10	-9	-9	-9	-8
Absolute value	0.07	0.07 (‑2.58)	0.08 (1.15)	0.08 (1.55)	0.09	0.09 (5.98)	0.11* (23.30)	0.12** (33.23)
Relative to body weight	0.017	0.017 (0.307)	0.017 (1.078)	0.017 (2.00)	0.026	0.026 (2.23)	0.031* (21.04)	0.032** (23.72)
Relative to brain weight	3.52	3.48 (‑1.08)	3.55 (0.98)	3.55 (0.89)	4.36	4.72 (8.25)	5.49* (25.79)	5.87** (34.43)
Kidney (No. Weighed)^b	-10	-10	-10	-10	-9	-9	-9	-8
Absolute value	3.22	3.20 (‑0.67)	3.56* (10.62)	3.48 (7.94)	2.41	2.57 (6.61)	2.70* (12.04)	3.01** (24.84)
Relative to body weight	0.72	0.74 (2.17)	0.80* (10.32)	0.78* (8.14)	0.71	0.73 (2.36)	0.78* (10.04)	0.82** (15.59)
Relative to brain weight	151.7	152.8 (0.74)	167.6* (10.47)	162.7 (7.23)	119.6	129.6 (8.33)	136.1** (13.84)	150.4** (25.72)
^aOrgan weight values rounded to the appropriate decimal place to account for the size of the organ (values presented to all decimal places in Provantis tables).
^bFor all groups, means are shown. For test substance-treated groups, percent differences from control are shown in parentheses. Statistical significance is based on actual data (not on the percent differences).
* = Statistically significantly different from the control group at ≤ 0.05 using Dunnett’s test.
** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s test.
Bold= Values considered to be test substance-related.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dose level. Therefore, a dose level of 500 mg/kg/day (the highest dose level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of n Butyl Pyrrolidone when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical observations of effects on mean body weights, body weight changes, and food consumption noted at any dose level. Furthermore, there were no adverse effects on organ weights, thyroid hormones, or macroscopic and/or microscopic alterations in F0 and F1 males and females at any dose level. Therefore, the NOAEL for systemic toxicity was considered to be 500 mg/kg/day. The NOAEL for postnatal toxicity was 500 mg/kg/day based on the absence of effects on F1 offspring at all dose levels.

Executive summary:

The objective of this study was to provide information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through weaning on Day 21 of postnatal life, and subsequent direct dosing of the offspring until Day 35 (females) or 54 (males).

Animals were dosed via oral gavage once daily. F₀males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia (Study Day 27). F₀females were dosed for 14 days prior to mating and continuing through Lactation Day 20.F₁animals were potentially exposed to the test substance in utero and via maternal milk from birth until Postnatal Day (PND) 21. Following weaning, F₁male and female pups selected to constitute the F₁generation were dosed once dailybeginning at weaning and continuing until 1 day prior to euthanasia on PND 36 (females) or 55 (males).

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, pre-and postweaning developmental landmarks, thyroid hormones, clinical pathology, macroscopic findings, organ weights, and microscopic examinations.

The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous.

There were no test substance-related effects on survival in the F₀generation. In the 500 mg/kg/day group, 2 females did not survive to the scheduled necropsy. One female in this group was found dead on Lactation Day 16 without remarkable antemortem findings; upon microscopic examination, hepatocellular hypertrophy (minimal) was noted for this female, which was also observed for 7 of 8 surviving females in this group, and considered test substance‑related, but nonadverse. An additional female in the 500 mg/kg/day group was euthanized in extremis on Lactation Day 17 due to the severity of abnormal breathing sounds noted on this day. No remarkable gross findings at necropsy or microscopic findings were noted for this female. The causes of death/moribundity at 500 mg/kg/day were unable to be determined, but considered unrelated to the test substance due to the absence of any other evidence of systemic toxicity at this dose level. In the 325 mg/kg/day group, 1 female was euthanized in extremis on Lactation Day 9 due to poor clinical condition and marked body weight loss with correspondingly lower food consumption from Lactation Days 1‑7. Microscopically, there was evidence of bacterial sepsis, and therefore the moribund condition of this female was considered unrelated to test substance administration. All other F₀females and all F₀males survived to the scheduled necropsy.

Test substance-related clinical findings of abnormal breathing sounds and salivation-related observations (salivation and wet fur around mouth) were observed at higher incidences for F₀ males and females in the 500 mg/kg/day group at the daily examinations and/or approximately 1 hour following dose administration; these findings were not observed in the control group but were considered nonadverse based on the transient nature of the findings.

Mean body weights, body weight gains, food consumption, reproductive performance, gestation length, parturition, clinical pathology parameters (hematology, coagulation, and serum chemistry; assessed for males only), and thyroid hormone values (analyzed for males only) were unaffected by test substance administration in F₀ males and females at any dose level.

Administration of n-Butyl Pyrrolidone resulted in nonadverse findings in the liver, adrenal glands, and kidney. Minimal hepatocellular hypertrophy was noted in the 325 mg/kg/day group F₀males and in the 500 mg/kg/day group F₀males and females, and correlated to higher mean liver weights and to the gross finding of liver enlargement. Minimal diffuse adrenal cortical hypertrophy occurred at an increased incidence in the 325 and 500 mg/kg/day group F₀females and correlated to higher mean adrenal gland weights and to the gross finding of adrenal gland enlargement. Higher mean kidney weights were noted in the 325 and 500 mg/kg/day groups, but did not have a microscopic correlate. There were no test substance related macroscopic or microscopic findings or alterations in organ weights for F₀males and females at 100 mg/kg/day.

The mean number of pups born, live litter size, percentage of males at birth, postnatal survival in the 100, 325, and 500 mg/kg/day groups were unaffected by test substance administration.

F₁preweaning clinical observations, mean body weights, body weight gains, areolae/nipple anlagen, and thyroid hormone levels were unaffected by F₀administration of the test substance. Test substance-related lower mean anogenital distances (absolute and normalized for body weight) on PND 1 were noted for F₁male pups in the 325 and 500 mg/kg/day groups compared to the control group. Based on these effects, and to further evaluate possible treatment-related effects on pup development, the study was extended to include postweaning developmental landmarks evaluated for both F₁males and females assigned to the direct dosing phase. There were no test substance‑related effects on the age or weight at attainment for balanopreputial separation or vaginal patency for F₁males and females at any dose level. Therefore, the shorter anogenital distances observed for male pups at 325 and 500 mg/kg/day on PND 1 were considered nonadverse. No internal findings or effects on thyroid weights that could be attributed to F₀parental test substance administration were noted for pups euthanized on PND 21.

In the F₁generation, no test substance-related effects on survival were noted at any dose level. Two females in the 500 mg/kg/day group were found dead or euthanized in extremis on PND 26 or 27; at necropsy,evidence of dosing error was noted for both animals, including material accumulation in the trachea and/or distended/gas-filled intestines. In addition, 2 animals (1/sex) in the 100 mg/kg/day group selected for the F₁generation were inadvertently send to necropsy on PND 21. Test substance-related abnormal breathing sounds was noted in F₁males in the 325 and 500 mg/kg/day groups at the daily examinations and/or approximately 1 hour postdose, and not observed in the control group. These observations were limited to a small number of animals and occurred in the absence of other signs of systemic toxicity, and therefore were considered nonadverse.

Mean body weights, body weight gains, food consumption, macroscopic findings, and organ weights in the F₁males and females were unaffected by test substance administration at all dose levels.

Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dose level. Therefore, a dose level of 500 mg/kg/day (the highest dose level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of n‑Butyl‑Pyrrolidone when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical observations of effects on mean body weights, body weight changes, and food consumption noted at any dose level. Furthermore, there were no adverse effects on organ weights, thyroid hormones, or macroscopic and/or microscopic alterations in F₀and F₁males and females at any dose level. Therefore, the NOAEL for systemic toxicity was considered to be 500 mg/kg/day. The NOAEL for postnatal toxicity was 500 mg/kg/day based on the absence of effects on F₁offspring at all dose levels.