1-butylpyrrolidin-2-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 421 reproductive/developmental screen, no adverse effects were observed for systemic or reproductive toxicity at any dose. The F0 systemic and reproductive NOAEL's for males and females was 500 mg/kg/day, the highest dose tested. The systemic NOAEL for the F1 males and females was 500 mg/kg/day, the highest dose tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study planned

Justification for type of information:

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

- Pre-existing data:
There are no pre-existing data available that would address the pre-natal developmental toxicity study endpoint (GLP or non-GLP). A rat pre-natal developmental toxicity study is not adequate according to the regulation. There are no historical human data that could be used in place of this study.

- (Q)SAR:
There are no accepted QSAR approaches for predicting the outcome of this endpoint. However, there were no Reproductive Toxicity alerts for MiAK.

- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint

- Weight of evidence
WoE is not possible as there are not similar substances that could be used for such a comparison.

- Grouping and read-across
The Registrant has not identified any suitable analogues that could be used to provide data for this endpoint. Other similar substances are quite data poor.

- Substance-tailored exposure driven testing
Based on the use patterns of the registered substance in coatings, exposure-based waiving is not possible.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Justification for study design:

An OECD 443 is required under Annex X. Existing data for this substance suggests no significant developmental toxicity at suitable test article concentations. Also, there is currently (as of August 10, 2021) an OECD 421 ongoing and is in the reporting phase. This study resulted in no reproductive or developmental toxicity to parental animals of either sex or to the offspring. Thus, the lead registrant feels that the base set of the OECD 443 with no additional cohorts or the extension to breed the F2 generation is appropriate. Also, the existing data was primarily conducted by oral gavage. While there was 1 inhalation developmental study, the test substance is both water soluble, has a low vapor pressure and is not volatile. Thus, inhalation would not be considered to be a preferred route of exposure.

Species:

rat

Sex:

male/female

Route of administration:

oral: gavage

Reproductive effects observed:

not specified

Reference 2

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

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:

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

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.

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

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

325 mg/kg bw/day

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

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

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Critical effects observed:

no

Reproductive effects observed:

no

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.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP compliant and is of high quality (Klimish score = 1). The quality of the database is therefore high.

Effects on developmental toxicity

Description of key information

- Inhalation developmental toxicity study (OECD 414): Crl:CD(SD) rats, whole body; conc.: 0, 0.3, 0.6 and 1.2 mg/L (corresponding to 76.2, 152.6, and 315.8 mg/kg/day, respectively); GD 6-19; NOAEC: 0.6 mg/L (152.6 mg/kg bw).

- Dermal developmental study (OECD 414): Crl:CD(SD) rats, dermal daily exposure for approximately 23 hours each day; dose levels: 0, 375, 500 and 750 mg/kg bw; GD: 6-19; NOAEL: 500 mg/kg bw.

- Oral developmental study (OECD 414): Crl:CD(SD) rats, gavage; dose levels: 200, 300, 400 and 500 mg/kg bw; GD: 6-19; NOAEL: 400 mg/kg bw.

- Supporting oral developmental study (OECD 414): CD rats; gavage; dose levels: 0, 5, 50 and 500 mg/kg bw; NOAEL > 500 mg/kg bw for foetal organisms.

- Oral Rabbit developmental toxicity study according to OECD 414. Developmental NOAEL of 300 mg/kg/day, the highest dose tested. No developmental effects found.

Since developmental toxicity is the endpoint of concern by structurally related analogues N-methylpyrrolidone and N-ethylpyrrolidone, NOAELs from developmental toxicity studies will be used for hazard assessment (DNEL derivation). Fertility effects and general toxicity are covered by the developmental NOAELs.

Remark

Additional link to relevant study: ESR contained in section 13 of the IUCLID file

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-19 to 2016-10-24

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

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at room temperature under a nitrogen blanket, and was considered stable under these conditions.
- Stability under test conditions: Documentation regarding the purity and stability of the test substance is on file with the Sponsor and Charles River.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: No

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 75 days old upon receipt; The selected females were approximately 12 weeks old when paired for breeding.
- Weight at study initiation: Body weight values of the selected females ranged from 230 g to 284 g on Gestation Day 0.
- Fasting period before study: no
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH).
- Diet (e.g. ad libitum): ad libitum except during exposure periods (The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River).
- Water (e.g. ad libitum): ad libitum except during exposure periods (Reverse osmosis-purified (on-site) drinking water)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Non-exposure periods: 68°F to 78°F (20°C to 26°C); actual mean daily temperature ranged from 72.3°F to 74.1°F (22.4°C to 23.4°C) during the study. Exposure periods: The mean temperature was between 19°C to 25°C.
- Humidity (%): Non-exposure periods: 30% to 70%; actual mean daily relative humidity ranged from 45.4% to 59.0% during the study. Exposure periods: The mean relative humidity was between 30% to 70%.
- Air changes (per hr): Non-exposure periods: 10. Exposure periods: All chambers were operated under dynamic conditions, at least 12-15 air changes per hour, at a slight negative pressure.
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- Oxygen content was measured during the method development phase and was 20.9% for Groups 2-4.

IN-LIFE DATES: From: 02 Aug 2016 (animal receipt) To: 02 Sep 2016 (last laparohysterectomy).

Route of administration:

inhalation: mixture of vapour and aerosol / mist

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.4 - <= 2.7 µm

Geometric standard deviation (GSD):

1.73

Remarks on MMAD:

The target range for MMAD is 1.0 to 3.0 microns and GSD is 1.5 to 3.0. Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Mean MMAD and GSD were within the target values (table 10). MMAD values were 2.7, 2.4 and 2.4 for the substance-treated groups 2, 3 and 4, respectively. GSD were of 1.81, 1.78 and 1.73 for the substance-treated groups 2, 3 and 4, respectively.

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus: Exposures were conducted using four 1000-L glass and stainless steel whole-body inhalation exposure chambers. One exposure chamber was dedicated for each group for the duration of the study.

- Method of holding animals in test chamber: For each day’s exposure, the animals were transferred to exposure caging in the colony room, transported to the exposure room, exposed for the requisite duration, and returned to their home cages in the animal colony room. The animal cage batteries were rotated on a daily basis between the 3 battery positions within the chamber to help ensure a similar exposure for all animals within each group over the duration of the exposure period.

- Source and rate of air: Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source.

- Method of conditioning air: not reported.

- System of generating particulates/aerosols: An individual generation system was used for each test substance exposure chamber. A mixed liquid aerosol/vapor atmosphere of the test substance was generated as follows. During generation trials as part of the range-finding study, it was noted that the test substance was not compatible with a plastic clinical nebulizer, but it was compatible with the glass jar of a Collison nebulizer. The test substance was aerosolized using 1 or more Collison nebulizers. A 6-jet nebulizer was used for Chamber 2 and Chamber 3, and two 6-jet nebulizers were used for Chamber 4. Using a regulator, breathing quality compressed air from the facility in-house air source was delivered to each nebulizer. Test substance was delivered to the nebulizers through the use of an infusion pump.
For Chamber 4, the resulting liquid aerosol/vapor atmosphere from the 6-jet nebulizers were combined at a 4-way ‘cross’-fitting where it was mixed with dry, compressed air. For Chambers 2 and 3, the resulting liquid aerosol/vapor atmosphere from the respective nebulizer was mixed with additional dry, compressed air at a ‘T’-fitting. The amount of compressed air for Chambers 2, 3, and 4 were controlled using a regulator.
In order to produce an exposure atmosphere with an appropriate particle-size, the resulting liquid aerosol/vapor mixture for all test substance chambers was first directed through a glass cyclone in order to reduce the particle-size to under 3 microns. The output from each cyclone was delivered to the chamber inlet, where it was mixed with supply air to achieve the desired target concentration of the test substance.

- Temperature, humidity, pressure in air chamber: Chamber ventilation rate and negative pressure within the chambers were continually monitored and recorded approximately every 45 minutes during the 6-hour exposure periods. Temperature and relative humidity within each exposure chamber were determined at least 3 times during each period. The mean temperature and mean relative humidity were to be between 19°C to 25°C and 30% to 70%, respectively. Oxygen content was measured during the method development phase and was 20.9% for Groups 2-4.

- Air flow rate: The airflow rates for each whole-body chamber were monitored by measuring the pressure drop between the ports of an orifice plate using a Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge was calibrated for conversion from pressure to airflow in standard liters per minute through the use of a Fox Gas Mass Flowmeter Transmitter (model no. FT2, Fox Thermal Instruments; Marina, CA). Sample flow through the sample train was controlled using a needle valve connected to the facility vacuum source. Prior to each sample collection, the sample flow rate was measured using a Mini-Buck Calibrator (model no. M-5, A.P Buck Inc.; Orlando, FL). An approximate sample flow rate was 2.0 L/min for 8, 2.5, and 1.5 minutes for Chambers 2, 3, and 4, respectively. Chamber ventilation rate (airflow) and negative pressure within each exposure chamber were continually monitored and recorded at approximately 45-minute intervals through the use of the Inhalation Toxicology Data Acquisition System (WINH) and a personal computer.

- Air change rate: All chambers were operated under dynamic conditions, at least 12-15 air changes per hour, at a slight negative pressure.

- Method of particle size determination: Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Aerosol particle-size measurements were conducted using a 7-stage stainless-steel cascade
impactor (model no. 02-100-2L, IN-TOX Products; Moriarty, NM). Pre-weighed, 22-mm stainless-steel collection substrates were used for stage 1 to 7. A pre-weighed, 25-mm glass fiber filter (Type A/E, PALL Corporation) was used as the final collection substrate. Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Samples of the mixed liquid aerosol/vapor atmosphere of test substance were collected at an approximate sample flow rate of 2.0 LPM for 8, 2.5, and 1.5 minutes for Chambers 2, 3, and 4, respectively. Sample flow rates were measured using a Mini-Buck Calibrator (model no. M-5). Following sample collection, the aerosol retained on the filters was determined by re-weighing the filters and the particle-size was calculated based on the impactor stage cut-offs. The particle-size was expressed as the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD).

- Treatment of exhaust air: All test substance atmosphere chamber exhaust passed through an activated-carbon drum prior to passing through the facility exhaust system, consisting of redundant exhaust blowers preceded by activated-charcoal and HEPA-filter units.

TEST ATMOSPHERE
- Brief description of analytical method used: Following each sample collection, aerosol content was measured using standard gravimetric methods and the vapor concentration was determined using analytical techniques (liquid-phase injection gas chromatograph (GC) with flame ionization detection). The filter was re-weighed and the aerosol concentration (mg/L) was calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume.
- Samples taken from breathing zone: yes (the description please see below in "Details on analytical verification of doses or concentrations".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Nominal exposure concentrations were calculated for each test substance exposure chamber from the total amount of test substance used during each generation period and the total volume of air that passed through the chamber during exposure. Test substance usage was determined by weighing pre-filled syringes prior to and at the termination of each generation. Total air volume was calculated by multiplying the daily mean ventilation rate by the duration of generation.

Samples of the exposure atmospheres were collected using a sampling train consisting of a pre-weighed, glass fiber filter in-line with a 30-mL midget impinger at approximately 60-minute intervals. The filter was held in a filter holder and placed in the animal-breathing zone of the chamber. The impinger was filled with approximately 10 mL of isopropanol as the trapping liquid. Prior to each sample collection, the sample flow rate was measured using a calibrator.
Aerosol concentrations
Following each sample collection, aerosol content was measured using standard gravimetric methods and the vapor concentration was determined using analytical techniques. The filter was re-weighed and the aerosol concentration (mg/L) was calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume.
Vapor Concentrations
Analyzed concentrations of the test substance vapor in the exposure atmospheres were determined using a liquid-phase injection gas chromatograph (GC) with flame ionization detection.
Following sample collection, a portion of the impinger liquid was transferred to a 2-mL vial and analyzed using a GC. Liquid sample injection onto the chromatography column occurred via manual injection (approximately 2 μL) into a split/splitless injector, the chromatograph was displayed, and the area under the sample peak was calculated and recorded. Test substance concentration within the impinger liquid, in μg/mL, was calculated using the ln-quadratic equation based on the GC calibration curve. The total amount of vaporized test substance recovered was determined by multiplying the impinger liquid concentration by the impinger solvent volume (10 mL). The vapor concentration was determined by dividing the amount of test substance recovered by the sample volume.
Total Test Substance Concentrations
Total test substance (combined aerosol and vapor) was calculated by addition of the gravimetrically and analytically determined concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused
At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females.
- If cohoused:
- M/F ratio per cage: 1
- Length of cohabitation: not reported.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

6 hours

Frequency of treatment:

once daily

Duration of test:

during Gestation Days 6–19.

Dose / conc.:

0.3 mg/L air (nominal)

Remarks:

Mean analyzed exposure concentrations was 0.29 mg/L and corresponded to mean estimated inhaled (delivered) dosage level 76.2 mg/kg/day.

Dose / conc.:

0.6 mg/L air (nominal)

Remarks:

Mean analyzed exposure concentrations was 0.58 mg/L and corresponded to mean estimated inhaled (delivered) dosage level 152.6 mg/kg/day.

Dose / conc.:

1.2 mg/L air (nominal)

Remarks:

The highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). The mean analyzed exposure concentration was 1.2 mg/L and corresponded to mean estimated inhaled (delivered) dosage levels of 315.8 mg/kg/day.

No. of animals per sex per dose:

24

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: 2-week range-finding study.
In that study, the test substance was administered via whole-body inhalation exposure, 6 hours per day, for 14 consecutive days to 3 groups of 5 female Crl:CD(SD) rats. As part of the generation trials for this study, attempts were made to produce a maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). It was estimated that the highest exposure concentration that met this criteria would be 1.0 mg/L. Therefore, target exposure concentrations were 0.25, 0.5, and 1.0 mg/L for Groups 2, 3, and 4, respectively. Mean exposure concentrations (total test substance; aerosol plus vapor) over the duration of the exposure period were 0.29, 0.64, and 1.23 mg/L for the same respective groups. Minor adjustments to generation settings (nebulizer pressure and/or dilution airflow) allowed the actual achieved concentration to be approximately 1.2 mg/L and consistently maintain a particle size under 3 microns (achieved 2.6 microns).
All animals survived to the scheduled necropsy. There were no test substance-related effects on body weights, food consumption, macroscopic pathology, or lung weights in any of the test substance-exposed groups. Clinical observations noted in the 1.0 mg/L group included partial closure of the right and left eyes, impaired equilibrium, and increased respiration rate. Test substance-related clinical observations of rales were noted in the 0.5 and 1.0 mg/L groups. Observations of rales were noted beginning as early as Study Day 3 in the 1.0 mg/L group and continued through the remainder of the study period. An animal in the 0.5 mg/L group was observed with rales beginning on Study Day 4.
Based on the lack of dose-limiting toxicity observed at up to 1.2 mg/L, exposure concentrations of 0.3, 0.6, and 1.2 mg/L were chosen for this study. The selected route of administration for this study was inhalation exposure because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program, which randomized the animals based on stratification of the Gestation Day 0 body weights in a block design. Animals not assigned to study were transferred to the Charles River rat colony.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily during Gestation Days 0–20 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity at approximately the midpoint of each 6-hour exposure and approximately 1 hour following exposure. The absence or presence of findings was recorded for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on Gestation Days 0 and 6–20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for Gestation Days 6–9, 9–12, 12–15, 15–20, and 6–20.
Gravid uterine weight was collected and net body weight (the Gestation Day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Individual food consumption was recorded on Gestation Days 0 and 6–20 (daily).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. Wet lung weights were recorded for all females at the scheduled necropsy, and the lungs were subsequently placed in 10% neutral-buffered formalin for possible future histopathological examination. Other maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings; representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification.

- External examinations: Yes: all per litter. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.

- Soft tissue examinations: Yes: all per litter. Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development.

- Skeletal examinations: Yes: all per litter. Following fixation in alcohol, each fetus was stained with Alizarin Red S7 and Alcian Blue8. Fetuses were then examined for skeletal malformations and developmental variations.

- Head examinations: Yes: all per litter. Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Statistics:

All statistical tests were performed using WTDMS™. Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group. Each mean was presented with the S.D., S.E., and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, S.D. and S.E. on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, lung weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-exposed groups to the control group.

Indices:

Gestation Day 20 Laparohysterectomy
Intrauterine data were summarized using 2 methods of calculation. An example of each method of calculation follows:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group)/ No. Gravid Females/Group

2. Proportional Litter Basis:
Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)/ No. Litters/Group;

Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter)/ No. Implantation Sites/Litter x 100

Fetal Morphological Examination
The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%))/ No. Litters/Group

Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter)/ No. Viable Fetuses/Litter x 100

Historical control data:

Charles River has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat (Number of Datasets in Historical Control: 79-80). This animal model has been proven to be susceptible to the effects of developmental toxicants.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, increased incidences of red material around the nose, mouth, and forelimbs was noted in the 0.6 and 1.2 mg/L groups generally throughout the treatment period; these observations were noted primarily at approximately 1 hour following exposure at 0.6 mg/L and at the daily examinations and approximately 1 hour following exposure at 1.2 m/L. The aforementioned observations were considered adverse at 1.2 mg/L; because these findings were generally resolved by the following daily examination in the 0.6 mg/L group, they were considered nonadverse at this exposure level. In the 1.2 mg/L group, an increased incidence of clear material on the ventral neck was noted primarily during Gestation Days 16–20 at the daily examinations and/or approximately 1 hour following exposure and was considered test substance-related and adverse. No other test substance-related clinical observations were noted at the daily examinations, midpoint of exposure, or approximately 1 hour following exposure at any exposure level. Observations noted in the exposed groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Mortality:

no mortality observed

Description (incidence):

All females in the control, 0.3, 0.6, and 1.2 mg/L groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 1.2 mg/L group, test substance-related mean body weight losses or lower mean body weight gains were noted throughout the exposure period (Gestation Days 6–9, 9–12, 12–15, and 15–20) and resulted in a lower mean body weight gain when the entire exposure period (Gestation Days 6–20) was evaluated compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). In addition, mean body weights in this group were lower (5.4% to 11.5%; significant at p < 0.01) than the control group during Gestation Days 8–20. Lower mean net body weight and net body weight gain were noted in the 1.2 mg/L group compared to the control group; differences were significant (p < 0.01). In addition, a significantly (p < 0.01) lower mean gravid uterine weight was noted in the 1.2 mg/L group compared to the control group. The aforementioned effects noted in the 1.2 mg/L group were considered adverse.
In the 0.3 and 0.6 mg/L groups, significantly (p < 0.01) lower mean body weight gains were noted during Gestation Days 6–9 compared to the control group. Mean body weight gains in these groups were similar to the control group for the remainder of the exposure period (Gestation Days 9–12, 12–15, and 15–20). Due the decrements in mean body weight gain noted at the beginning of the exposure period, a significantly (p < 0.01) lower mean body weight gain was noted in the 0.6 mg/L group compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated; mean body weight gain in the 0.3 mg/L group was similar to the control group for this interval. The lower mean body weight gains noted in the 0.3 and 0.6 mg/L groups were not of sufficient magnitude to affect mean body weights at these exposure levels, and therefore were considered nonadverse. Mean maternal net body weights, net body weight gains, and gravid uterine weights in the 0.3 and 0.6 mg/L groups were unaffected by test substance exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, lower mean maternal food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 1.2 mg/L group throughout the exposure period (Gestation Days 6–9, 9–12, 12–15, and 15–20) and resulted in lower mean food consumption in this group compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated; differences were significant (p < 0.01). The lower mean food consumption in the 1.2 mg/L group correlated with the mean body weight losses and lower mean body weight gains and was considered adverse.
Test substance-related, lower mean food consumption was noted during Gestation Days 6–9 in the 0.3 mg/L group and during Gestation Days 6–9, 9–12, and 12–15 in the 0.6 mg/L group compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). Mean food consumption was similar to the control group during Gestation Days 9–12, 12–15, and 15–20 at 0.3 mg/L and during Gestation Days 15–20 at 0.6 mg/L. As a result of the initial decrements in mean food consumption, significantly (p < 0.05 or p < 0.01) lower mean food consumption was noted in these groups compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated. However, the differences noted in the 0.3 and 0.6 mg/L groups were not of sufficient magnitude to affect mean body weights in these groups, and therefore were considered nonadverse.

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:

no effects observed

Description (incidence and severity):

No test substance-related effects on mean lung weights were noted in the 0.3, 0.6, and 1.2 mg/L groups. Differences from the control group were slight and not statistically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on Gestation Day 20, no test substance-related internal findings were observed at exposure levels of 0.3, 0.6, and 1.2 mg/L. Macroscopic findings observed in the test substance-exposed groups occurred infrequently and/or in a manner that was not exposure-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. Parameters evaluated included postimplantation loss, live litter size, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

see above

Early or late resorptions:

no effects observed

Description (incidence and severity):

see above

Dead fetuses:

no effects observed

Description (incidence and severity):

see above

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

With the exception of 2 and 1 females in the control and 0.6 mg/L groups, respectively, all females were determined to be gravid.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

0.6 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male, female, and combined fetal weights in the 1.2 mg/L group (3.5 g, 3.3 g, and 3.4 g, respectively) were significantly (p < 0.01) lower (approximately 13%) than the control group values (4.0 g, 3.8 g, and 3.9 g, respectively) and were also below the minimum mean values in the Charles River Ashland historical control data (version 2016.03; 3.682 g, 3.458 g, and 3.573 g, respectively). The lower mean fetal weights at 1.2 mg/L correlated with decrements in maternal mean body weight gain and food consumption noted in this group during the fetal period (Gestation Days 15–20), which is the time of greatest fetal growth, and were considered adverse.
Lower (approximately 5%) mean male, female, and combined fetal weights were noted in the 0.3 and 0.6 mg/L groups; differences were generally significant (p < 0.05 or p < 0.01). However, the values at 0.3 and 0.6 mg/L (3.8 g for males, 3.6 g for females, and 3.7 g for combined) were greater than the minimum mean values and only 2% to 3% lower than the mean (3.898 g for males, 3.697 g for females, and 3.801 g for combined) and median (3.883 g for males, 3.696 g for females, and 3.792 g for combined) values within the Charles River Ashland historical control data. The concurrent control group values for fetal body weight (4.0 g for males, 3.8 g for females, and 3.9 g for combined) were 2% to 3% heavier (approximately equal to the 75th quartile; 3.971 g for males, 3.761 g for females, and 3.861 g for combined) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights in the 0.3 and 0.6 mg/L groups were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups. The absence of a dose-response relationship between the 0.3 and 0.6 mg/L group fetuses also suggests a lack of effect at these exposure concentrations. In addition, the magnitude of change in these groups compared to the control group was not as severe as the difference in the 1.2 mg/L group. Therefore, the lower mean fetal weights at 0.3 and 0.6 mg/L were not considered test substance-related.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. Parameters evaluated included postimplantation loss, live litter size, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

see above

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

see above

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 323(22), 358(24), 339(23), and 350(24) in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. Malformations were observed in 2(2), 2(2), 1(1), and 4(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.
No test substance-related external malformations were observed for fetuses at any exposure level. Fetal anasarca was noted for 1 fetus each in the control (No. 6130-16) and 0.3 (No. 6082-07) mg/L groups. In the 0.3 mg/L group, Fetus No. 6094-16 was noted with anophthalmia (bilateral; confirmed skeletally as orbits smaller than normal). The aforementioned malformations were noted in single fetuses, similarly in the control group, and were not observed in an exposure-related manner; therefore, they were not considered test substance-related.
No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related skeletal malformations were noted for fetuses at any exposure level. In the 1.2 mg/L group, 2 fetuses (Nos. 6025-10 and 6113-02) were noted with costal cartilage anomaly (bifurcated or fused costal cartilage) and 1 fetus (No. 6113-07) was noted with 8 cervical vertebrae (with normal presacral vertebrae count). Fetus No. 6065-03 in the 1.2 mg/L group was noted with severely malaligned sternebrae; this finding was also noted for 1 fetus (No. 6089-14) in the control group. Sternoschisis (no. 4 or no. 6 sternal bands not joined) was noted for 1 fetus each in the control (No. 6130-16) and 0.6 (No. 6076-04) mg/L groups. The aforementioned skeletal malformations were observed in single fetuses, similarly in the control group, were not observed in an exposure-related manner, and/or the differences in the mean litter proportions were not statistically significantly different from the concurrent control group and the values were within the ranges of the Charles River Ashland historical control data; therefore, they were not considered test substance-related.
Test substance-related skeletal developmental variations were noted in the 1.2 mg/L group. In the 1.2 mg/L group, a significantly (p < 0.01) lower mean litter proportion of cervical centrum no. 1 ossified was noted compared to the control group (3.4% versus 27.3% per litter). In addition, a higher (not statistically significant) mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified was noted in the 1.2 mg/L group (23.1% per litter) compared to the control group (9.4% per litter); the value at 1.2 mg/L exceeded the maximum mean value in the Charles River Ashland historical control data (19.86% per litter). The aforementioned findings noted at 1.2 mg/L were considered indicators of developmental delay and correlated with the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level.
No other test substance-related skeletal developmental variations were noted at any exposure level. Other findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test substance-related visceral malformations were observed for fetuses at any exposure level. In the 0.3 mg/L group, Fetus No. 6094-16 was noted with an interventricular septal defect (a <1 mm in diameter opening in the anterior portion of the septum); this fetus was also noted with an external malformation. Because this finding was noted in a single fetus and did not occur in an exposure-related manner, this finding was not considered test substance-related. In the control group, Fetus No. 6130-16 was noted with a bulbous aorta (ascending and aortic arches).
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for 2 (Nos. 6020-12 and 6071-13), 1 (No. 6095-14), 1 (No. 6047-06), and 1 (No. 6110-13) fetuses in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred infrequently, at similar frequencies in the control group, and in a manner that was not exposure-related.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 323(22), 358(24), 339(23), and 350(24) in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. Malformations were observed in 2(2), 2(2), 1(1), and 4(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations, when observed in the test substance-exposed groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in an exposure-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations were attributed to the test substance.
Test substance-related fetal skeletal developmental variations were noted in the 1.2 mg/L group. A higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and a lower mean litter proportion of cervical centrum no. 1 ossified were noted in the 1.2 mg/L group. These findings were considered secondary to the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level. No other test substance-related developmental variations were noted at any exposure level.

Key result

Dose descriptor:

NOAEL

Effect level:

0.6 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: Test substance-related fetal skeletal developmental variations

Key result

Abnormalities:

no effects observed

Localisation:

skeletal: sternum

Description (incidence and severity):

Variations: In the 1.2 mg/L group, a test substance-related higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and lower mean litter proportion of cervical centrum no. 1 ossified were noted compared to the control group; these findings were considered indicators of developmental delay and correlated with the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1.2 mg/L air (nominal)

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Table 4. Overall Nominal Exposure Concentrations

Exposure Chamber:	2	3	4
Target Concentration (mg/L):	0.30	0.60	1.2
Nominal Concentration (mg/L):	1.0	1.8	4.9
Standard Deviation:	0.11	0.12	1.13
N:	17	17	17

The overall mean nominal concentrations for each exposure group are presented below. Due to the use of glass cyclones, a large amount of liquid test substance was removed from the generation atmosphere prior to entering the exposure chamber. This resulted in a greater calculated nominal concentration.

The overall mean analyzed exposure concentrations for each group are presented below:

Table 5. Overall Mean Exposure Concentrations

Exposure Chamber:	1	2	3	4
Target Concentration (mg/L):	0	0.3	0.6	1.2
Mean Concentration (mg/L):	0	0.29	0.58	1.2
Standard Deviation:	0.0	0.031	0.035	0.05
N:	5	17	17	17

The overall mean aerosol particle size for each test substance-treated group is presented below:

Table 6. Mean Aerosol Particle-Size

Exposure Chamber:	2	3	4
Mean MMAD (microns):	2.7	2.4	2.4
Mean GSD:	1.81	1.78	1.73
N:	3	3	3

The mean estimated inhaled (delivered) dose and the mean deposited dose for each interval and the overall exposure period are presented below. The deposited dose will be estimated at 10% of the estimated inhaled dose.

Table 7. Mean Estimated Delivered Dosage

Exposure Chamber:	1	2	3	4
Target Concentration (mg/L):	0	0.3	0.6	1.2
Mean Concentration (mg/L):	0	0.29	0.58	1.2
Females (mg/kg/day):	0.0	76.2	152.6	315.8
Deposited Dose (mg/kg/day):	0.0	7.62	15.26	31.58

Conclusions:

Target exposure concentrations for the current study were 0.3, 0.6, and 1.2 mg/L; the highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines).
Maternal toxicity was evidenced by adverse clinical observations and mean body weight losses and lower mean body weight gains with corresponding lower mean food consumption in the 1.2 mg/L group throughout the gestation treatment period. Developmental effects were noted at 1.2 mg/L as evidenced by lower mean fetal weights, which correlated with a lower mean gravid uterine weight and decrements in maternal food consumption and body weight gain during the fetal period (Gestation Days 15-20). Lower mean fetal weights were also noted in the 0.3 and 0.6 mg/L groups; however, mean fetal weight values in these groups were greater than the minimum mean values and only slightly lower than the mean and median values in the Charles River Ashland historical control data. In addition, the concurrent control group values for fetal body weight were 2% to 3% heavier (approximately equal to the 75th quartile) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights at 0.3 and 0.6 mg/L were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups, and were not considered test substance-related. Test substance-related fetal skeletal developmental variations (higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and lower mean litter proportion of cervical centrum no. 1 ossified) were noted in the 1.2 mg/L group, which corresponded with the lower mean fetal weights noted in this group. No adverse maternal or developmental effects were noted at 0.3 and 0.6 mg/L. Based on these results, an exposure level of 0.6 mg/L was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance was administered via whole-body inhalation exposure to bred Crl:CD(SD) rats. Test substance exposure concentrations of 0.3, 0.6, and 1.2 mg/L corresponded to mean estimated inhaled (delivered) dosage levels of 76.2, 152.6, and 315.8 mg/kg/day, respectively.

Executive summary:

The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity. The test substance was administered via whole-body inhalation exposure to 3 groups of 24 bred female Crl:CD(SD) rats for 6 hours per day from Gestation Days 6–19. Based on a range-finding study in nonpregnant female rats, target exposure concentrations for the current study were 0.3, 0.6, and 1.2 mg/L; the highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). Mean analyzed exposure concentrations were 0.29, 0.58, and 1.2 mg/L for the same respective groups. Test substance exposure concentrations of 0.3, 0.6, and 1.2 mg/L corresponded to mean estimated inhaled (delivered) dosage levels of 76.2, 152.6, and 315.8 mg/kg/day, respectively. A concurrent control group composed of 24 bred females were exposed to humidified, filtered air on a comparable regimen. The females were approximately 13 weeks of age at the initiation of exposure. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On Gestation Day 20, a laparohysterectomy was performed on each female and the lungs were weighed. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control, 0.3, 0.6, and 1.2 mg/L groups survived to the scheduled necropsy. Test substance-related, increased incidences of red material around the nose, mouth, and forelimbs were noted in the 0.6 and 1.2 mg/L groups generally throughout the treatment period at the daily examinations and approximately 1 hour following exposure and was considered adverse at 1.2 mg/L; these observations were generally resolved by the following daily examination in the 0.6 mg/L group, and were therefore considered nonadverse at this exposure level. In the 1.2 mg/L group, a test substance-related increased incidence of clear material on the ventral neck was noted during the latter half of gestation at the daily examinations and/or approximately 1 hour following exposure and was considered adverse. No other test substance-related clinical observations were noted at the daily examinations, midpoint of exposure, or approximately 1 hour following exposure at any exposure level. In the 1.2 mg/L group, test substance-related mean body weight losses or lower mean body weight gains with corresponding reductions in mean food consumption were noted throughout the exposure period and resulted in a lower mean body weight gain and food consumption when the entire exposure period (Gestation Days 6–20) was evaluated compared to the control group. In addition, mean body weights in this group were 5.4% to 11.5% lower than the control group during Gestation Days 8-20. Test substance-related lower mean net body weight, net body weight gain, and gravid uterine weight were also noted in the 1.2 mg/L group. The aforementioned body weight effects noted in the 1.2 mg/L group were considered adverse. In the 0.3 and 0.6 mg/L groups, lower mean body weight gains were noted during Gestation Days 6–9 compared to the control group; mean body weight gains in these groups were similar to the control group for the remainder of the exposure period. Lower mean food consumption was noted during Gestation Days 6–9 in the 0.3 mg/L group and during Gestation Days 6–15 in the 0.6 mg/L group compared to the control group and resulted in lower mean food consumption in these groups when the entire treatment period (Gestation Days 6–20) was evaluated. However, the aforementioned differences in body weight gain and food consumption noted at 0.3 and 0.6 mg/L were not of sufficient magnitude to affect mean body weights at these exposure levels, and therefore were considered test substance-related but nonadverse. Mean maternal body weights, net body weights, net body weight gains, and gravid uterine weights in the 0.3 and 0.6 mg/L groups were unaffected by test substance exposure. No test substance-related macroscopic findings or changes in mean lung weights were noted for females at any exposure level at the scheduled necropsy on Gestation Day 20.

Mean fetal weights (male, female, and combined) in the 0.3, 0.6, and 1.2 mg/L groups were up to 5.3%, 5.3%, and 13.2% lower, respectively, than the control group. The differences in mean fetal weights noted in the 1.2 mg/L group correlated with decrements in maternal mean body weight gain and food consumption noted in this group during the fetal period (Gestation Days 15–20), which is the time of greatest fetal growth, and were considered adverse. When compared to the Charles River Ashland historical control data, the mean fetal weight values in the 0.3 and 0.6 mg/L groups were greater than the minimum mean values and only slightly lower than the mean and median values. In addition, the concurrent control group values for fetal body weight were 2% to 3% heavier (approximately equal to the 75th quartile) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights at 0.3 and 0.6 mg/L were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups. Additionally, the magnitude of change in these groups compared to the control group was not as severe as the difference in the 1.2 mg/L group and there was no dose-response relationship between the 0.3 and 0.6 mg/L groups. Therefore, the lower mean fetal weights at 0.3 and 0.6 mg/L were considered unrelated to treatment. Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. A test substance-related, higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and a lower mean litter proportion of cervical centrum no. 1 ossified was noted in the 1.2 mg/L group compared to the control group. The aforementioned findings noted at 1.2 mg/L were considered indicators of developmental delay and secondary to the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level. There were no test substance-related malformations noted at 0.3, 0.6, and 1.2 mg/L or developmental variations noted at 0.3 and 0.6 mg/L.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May 2016 to 10 Aug 2016

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

GLP compliance:

yes

Remarks:

the United States EPA GLP Standards 40 CFR Part 160 and 40 CFR Part 792 (l 6-0ct-1989 and l 8-Sep-1989, respectively) and the OECD Principles of GLP [C(97) 186/Final] (26-Nov-1997).

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test substance was stored at room temperature under a nitrogen blanket, and was considered stable under these conditions.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance in the vehicle was previously established to be stable and homogeneous following at least 10 days of refrigerated and room temperature storage under a nitrogen blanket at concentrations of 40 to 200 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: preparation of test substance formulations in vehicle.
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: [Crl:CD(SD)]
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 79 days old upon receipt. The selected females were approximately 12 weeks old when paired for breeding.
- Weight at study initiation: The day following receipt, all animals were weighed and clinical observations were recorded. Body weight values ranged from 214 g to 268 g on gestation day 0.
- Fasting period before study: no
- Housing: Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material. All rats were housed throughout the acclimation period and during the study in an environmentally controlled room.
- Diet (e.g. ad libitum): ad libitum; The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River.
- Water (e.g. ad libitum): ad libitum; Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system.
- Acclimation period: 14 days. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behavior. All animals were acclimated to the Elizabethan collars during the acclimation period according to Charles River SOPs.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 73°F ± 5°F (23°C ± 3°C). Actual mean daily temperature ranged from 72.3°F to 72.5°F (22.4°C to 22.5°C) during the study.
- Humidity (%): 50% ± 20%. Actual mean daily relative humidity ranged from 39.5% to 55.8% during the study.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 May 2016 (Experimental starting date (animal receipt)) To: scheduled necropsy on gestation day 20

Route of administration:

dermal

Vehicle:

water

Remarks:

deionized water (prepared on-site)

Details on exposure:

TEST SITE
- Area of exposure: Prior to the first day of dose administration, and as often as needed thereafter, the hair was clipped in a rectangular shape from the dorsal scapular area of each animal; a different set of clippers was used for control and test substance-treated animals to avoid potential cross-contamination. Care was taken not to abrade the skin.
The formulations were applied over the center of the clipped area and distributed to avoid running; the application site was approximately 10% of the total body surface area. The four corners of the application site were marked with indelible ink to allow proper identification of the treated and untreated skin.
- % coverage: 10
- Type of wrap if used: no, the treated animals were outfitted by the Elizabethan collars to restrict access to the dermal application site and avoiding grooming
- Time intervals for shavings or clipplings: Prior to the first day of dose administration, and as often as needed thereafter.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no. Following approximately 23 hours of exposure, the application sites were gently patted with a disposable paper towel to remove residual test substance.
- Time after start of exposure: 23 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
- Concentration (if solution): 75, 100 and 150 mg/mL for dose levels of 375, 500 and 750 mg/kg bw, respectively (see table 2).
- Constant volume or concentration used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (2°C to 8°C), purged with nitrogen. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

The test substance in the vehicle was previously established to be stable and homogeneous following at least 10 days of refrigerated and room temperature storage under a nitrogen blanket at concentrations of 40 to 200 mg/mL.
Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first and last 75, 100, and 150 mg/mL dosing formulations and from the middle stratum of the first and last control dosing formulations. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of an aliquot taken from the first 75 and 150 mg/mL dosing suspensions following refrigerated (2°C to 8°C), purged with nitrogen storage for 9 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored refrigerated (2°C to 8°C) under a nitrogen blanket as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

Details on mating procedure:

- Impregnation procedure: cohoused
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained.
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: not reported
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

single application

Frequency of treatment:

once daily

Duration of test:

from gestation days 6 through 19.

Dose / conc.:

375 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Based on the number of animals available for replacement, the total number of pregnant females assigned to the control, 375, 500, and 750 mg/kg/day groups was 24, 23, 24, and 24, respectively.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of a previous dose range-finding dermal study in rats. In that study, pregnant rats were dosed from gestation days 6-19 at dosage levels of 0, 250, 500, and 750 mg/kg/day. There were no deaths that were considered related to treatment. Lower mean body weight gains were observed in the 750 mg/kg/day group when the overall treatment period was evaluated (gestation days 6-20). In addition, lower mean fetal weights were noted in the 750 mg/kg/day group compared to the control group. As a result, dosage levels of 375, 500, and 750 mg/kg/day were selected for the current study. The high dosage level was expected to produce some evidence of maternal toxicity without resulting in mortality.
- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design.
- Other: Replacement animals were arbitrarily assigned based on body weight. On 13 Jun 2016 (gestation day 6), 23 females were dosed but did not have proper access to food because they were provided the incorrect food jars for use with Elizabethan collars. This resulted in severe body weight loss and reduced food consumption for these females from gestation day 6 to 7. Therefore, these females were removed from study on 14 Jun 2016 and 18 additional females were assigned to the study. Based on the number of animals available for replacement, the total number of pregnant females assigned to the control, 375, 500, and 750 mg/kg/day groups was 24, 23, 24, and 24, respectively.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration and to confirm the Elizabethan collar remained on the animal approximately 3 hours following each dose administration. The absence or presence of findings was recorded for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded.
Wet lung weights were recorded for all females at the scheduled necropsy. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

OTHER: Dermal Observations:
The application site was scored daily (prior to dose administration during the treatment period) for erythema and oedema in accordance with a 4-step grading system of very slight, slight, moderate, and severe. Other remarkable dermal findings, if present, were recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes
Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

Intrauterine data were summarized using 2 methods of calculation. An example of each method of calculation follows:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group))/ No. Gravid Females/Group

2. Proportional Litter Basis:
Summation Per Group (%) = (Sum of Postimplantation Loss/Litter (%)) / No. Litters/Group

Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter) / No. Implantation Sites/Litter x 100

Fetal examinations:

- External examinations: Yes: all per litter. Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the Charles River study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded.
- Soft tissue examinations: Yes: all per litter. Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development.
- Skeletal examinations: Yes: all per litter. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
- Head examinations: Yes: all per litter. Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice.

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the S.D., S.E., and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, S.D., and S.E. on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, organ weights, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Indices:

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%)) / No. Litters/Group
Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter) / (No. Viable Fetuses/Litter) x 100

Historical control data:

WIL Research has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A slightly increased incidence of red material around the eyes was noted for 7, 11, and 12 females in the 375, 500, and 750 mg/kg/day groups, respectively, generally throughout the treatment period at the daily examinations; however, given the low occurrence of this finding (generally 1-4 occurrences per animal) and the inability of the animals to groom while outfitted with Elizabethan collars, this finding was considered nonadverse. No other test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Other findings noted in the treated groups, including red material around the nose and/or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

No dermal observations were noted for females in the control, 375, 500, and 750 mg/kg/day groups.

Mortality:

no mortality observed

Description (incidence):

All females in the control, 375, 500, and 750 mg/kg/day groups survived to the scheduled necropsy on gestation day 20.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight losses were noted in all groups, including the control group, following the initiation of dose administration (gestation day 6-7); these differences were likely due to the placement of the Elizabethan collars. In the 750 mg/kg/day group, a test substance-related mean body weight loss (not statistically significant) was noted during gestation days 6-9 followed by mean body weight gains that were similar to the control group during gestation days 9-12 and 12-15. Mean body weight gains in this group were lower (23.5% to 37.5%) during gestation days 17-19 and resulted in a lower mean body weight gain for the gestation days 15-20 interval compared to the control group; differences were significant (p<0.05 or p<0.01). The decrements in mean body weight gain noted in the 750 mg/kg/day group resulted in a significantly (p<0.01) lower mean body weight gain when the entire treatment period (gestation days 6-20) was evaluated compared to the control group. In addition, mean body weights in the 750 mg/kg/day group were significantly (p<0.05 or p<0.01) lower (up to 7.4%) compared to the control group during gestation days 8-20. The aforementioned body weight effects noted in the 750 mg/kg/day group were considered adverse. A test substance-related lower mean net body weight and net body weight gain were noted in the 750 mg/kg/day group compared to the control group and were considered adverse; differences were significant (p<0.01). In addition, a test substance-related, significantly (p<0.01) lower mean gravid uterine weight was noted in the 750 mg/kg/day group.
In the 375 and 500 mg/kg/day groups, mean body weight gains were generally similar to the control group for the remainder of the treatment period (gestation days 6-9, 9-12, 12-15, 15-20, and 6-20). A significantly (p<0.01) lower mean body weight gain was noted at 500 mg/kg/day compared to the control group on gestation day 17-18; however, this difference was transient and not of sufficient magnitude to affect the overall treatment period interval (gestation days 6-20) in this group, and therefore was not considered test substance-related. Mean body weights in the 500 mg/kg/day group were significantly (p<0.05) lower than the control group during gestation days 18-20; given the small magnitude of change (only 3.8% to 4.0% lower compared to the control group), the lower mean body weights noted at this dosage level were not considered test substance-related. A significantly (p<0.05) lower mean net body weight change was noted at 500 mg/kg/day compared to the control group; however, mean net body weight in this group was similar to the control group, and therefore this change was not considered test substance-related.
Mean maternal body weights, net body weight, and net body weight gain in the 375 mg/kg/day group and gravid uterine weights in the 375 and 500 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, lower mean maternal food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 500 mg/kg/day group during gestation days 6-9 and in the 750 mg/kg/day group during gestation days 6-9 and 15-20 compared to the control group and resulted in lower mean food consumption in these groups when the entire treatment period (gestation days 6-20) was evaluated; differences were generally significant (p<0.05 or p<0.01). The lower mean food consumption in the 750 mg/kg/day group correlated with the lower mean body weights and body weight gains and was considered adverse. The differences noted in the 500 mg/kg/day group were generally not of sufficient magnitude to affect mean body weights and body weight gains in this group, and therefore were considered nonadverse. Mean food consumption at 500 and 750 mg/kg/day was similar to the control group during gestation days 9-12 and 12-15.
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 375 mg/kg/day group was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05) lower mean g/animal/day food consumption values were noted in the 375 mg/kg/day group compared to the control group during gestation days 6-9 and 6-20; however, these differences were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were not considered test substance-related.

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:

no effects observed

Description (incidence and severity):

No test substance-related effects on mean lung weights were noted in the 375, 500, and 750 mg/kg/day groups. Differences from the control group were slight and not statistically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 375, 500, and 750 mg/kg/day. Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related. With the exception of 2 and 1 females in the control and 750 mg/kg/day groups, respectively, all females were determined to be gravid.

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

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Effects on pregnancy duration: no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

With the exception of 2 and 1 females in the control and 750 mg/kg/day groups, respectively, all females were determined to be gravid.

Other effects:

not specified

Details on maternal toxic effects:

Additional parameters evaluated included postimplantation loss, live litter size, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male, female, and combined fetal weights in the 750 mg/kg/day group were lower (5.1%, 7.9%, and 5.3%, respectively) than the control group; differences were significant (p<0.01) and were considered test substance-related. The lower mean fetal weights at 750 mg/kg/day were attributed to the decrements in mean body weight gain and food consumption noted in this group during the fetal period (gestation days 15-20), which is the time of greatest fetal growth. Mean male, female, and combined fetal weights at 375 and 500 mg/kg/day and survival at all dosage levels were unaffected by test substance administration.

Mean male, female, and combined fetal weights at 375 and 500 mg/kg/day were unaffected by test substance administration. The lower mean fetal weights at 750 mg/kg/day were attributed to the decrements in mean maternal body weight gain and food consumption noted in this group during the fetal period (gestation days 15-20), which is the time of greatest fetal growth.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Live litter sizes were similar across all groups. Mean male, female, and combined fetal weights at 375 and 500 mg/kg/day were unaffected by test substance administration. The lower mean fetal weights at 750 mg/kg/day were attributed to the decrements in mean maternal body weight gain and food consumption noted in this group during the fetal period (gestation days 15-20), which is the time of greatest fetal growth.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Fetal survival at all dosage levels was unaffected by test substance administration.

External malformations:

no effects observed

Description (incidence and severity):

No external malformations or developmental variations were observed in fetuses in this study.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The number of fetuses (litters) with skeletal malformations were 1(1), 2(2), and 3(3), and 0(0) in the control, 375, 500, and 750 mg/kg/day groups, respectively. A vertebral anomaly with an associated rib anomaly (fused, misshapen, and/or malpositioned arches; fused malpositioned, absent, and/or malproportioned centra; extra costal cartilage; fused ribs; moderately malaligned sternebrae; 12 left ribs and 11 right ribs present; 25 presacral vertebrae) was noted for 1 fetus each in the 375 (no. 41462-08) and 500 (no. 41525-05) mg/kg/day groups. In the 500 mg/kg/day group, fetus no. 41431-10 was noted with a bent scapula (right) and fetus no. 41459-05 was noted with a costal cartilage anomaly (fused costal cartilage); 1 fetus in the control group (no. 41466-09) was also noted with a costal cartilage anomaly. A rib anomaly (fused and misshapen arches; fused and malpositioned centra; fused ribs; extra costal cartilage; 12 left ribs and 11 right ribs present; moderately malaligned sternebrae; 25 presacral vertebrae) was noted for fetus no. 41498-15 in the 375 mg/kg/day group. The aforementioned skeletal malformations were observed in single fetuses, noted similarly in the control group, did not occur in a doserelated manner, and/or the mean litter proportions were within Charles River Ashland historical control data ranges; therefore, they were not considered test substance-related.
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral malformations were limited to 1 fetus (no. 41522-13) in the 500 mg/kg/day group noted with hydrocephaly (increased cavitation of both the lateral and third ventricles). Because this finding was noted in a single fetus, did not occur in a dose-related manner, and the value was within the range of the Charles River Ashland historical control data, it was not considered test substance-related.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for 2 (nos. 41454-04 and 41466-06), 4 (nos. 41479-06, 41479-11, 41479-14, and 41519-09), and 1 (no. 41502-06) fetuses in the control, 500, and 750 mg/kg/day groups, respectively. Fetus no. 41472-12 in the 500 mg/kg/day group was noted with a white area on the liver. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 305(22), 329(23), 328(24), and 299(23) in the control, 375, 500, and 750 mg/kg/day groups, respectively. Malformations were observed in 1(1), 2(2), 4(4), and 0(0) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

Treatment related:

yes

The analyzed dosing formulations were within Charles River SOP range for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

Results of the analyses of dosing formulations are summarized below.

Table 7. Results of Homogeneity and Concentration Analyses

	Group 2 (75 mg/mL)	Group 3 (100 mg/mL)	Group 4 (150 mg/mL)
Homogeneity and Concentration Assessment of the 08 Jun 2016 Formulations
Mean Concentration (mg/mL)	78.3	106	156
RSD (%)	0.15	0.22	0.46
Mean % of Target	104	106	104
9-Day Refrigerated Storage Resuspension Homogeneity Assessment of the 08 Jun 2016 Formulations
Mean Concentration (mg/mL)	75.8	NA	153
RSD (%)	0.77	NA	0.088
Mean % of Target	101	NA	102
Homogeneity and Concentration Assessment of the 24 Jun 2016 Formulations
Mean Concentration (mg/mL)	79.7	106	155
RSD (%)	0.48	0.21	0.28
Mean % of Target	106	106	103

Conclusions:

Adverse mean body weight losses and lower mean body weight gains with corresponding reduced mean food consumption were noted in the 750 mg/kg/day group, with the greatest magnitude of change occurring during the fetal period, which resulted in lower mean body weights, net body weight, net body weight gain, and gravid uterine weight in this group. The aforementioned decrements in body weight and food consumption parameters at 750 mg/kg/day noted during the period of greatest fetal growth (gestation days 15-20) resulted in lower mean fetal weights in this group. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when n-butyl pyrrolidone was administered by dermal application to bred Crl:CD(SD) rats.

Executive summary:

The objectives of the study were to determine the potential of the test substance, N-butylpyrrolidone, to induce developmental toxicity after maternal dermal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. The test substance, N-butyl pyrrolidone, in the vehicle (deionized water) was administered by dermal application to the dorsal scapular area (approximately 10% of total body surface) to 3 groups of bred female Crl:CD(SD) rats once daily from gestation days 6 through 19; animals were fitted with Elizabethan collars and exposed to the test substance for approximately 23 hours each day. Exposure levels were 375, 500, and 750 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group received the vehicle on a comparable regimen. The females were approximately 13 weeks of age at the initiation of dose administration. In the original study design, each group consisted of 25 bred females. However, the 23 females that were dosed on 13 Jun 2016 (gestation day 6) did not have proper access to food because they were provided the incorrect food jars for use with Elizabethan collars. This resulted in severe body weight loss and reduced food consumption for these females from gestation day 6 to 7. Therefore, these females were removed from study on 14 Jun 2016 and 18 additional females were assigned to the study. Based on the number of animals available for replacement, the total number of pregnant females assigned to the control, 375, 500, and 750 mg/kg/day groups was 24, 23, 24, and 24, respectively.

All animals were observed twice daily for mortality and moribundity. Clinical and dermal observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female and the lungs were weighed. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

Results

All females in the control, 375, 500, and 750 mg/kg/day groups survived to the scheduled necropsy. A slightly increased incidence of red material around the eyes was noted in all test substance-treated groups generally throughout the treatment period at the daily examinations; however, given the low occurrence of this finding (generally 1-4 occurrences per animal) and the inability of the animals to groom while outfitted with the Elizabethan collars, this finding was considered nonadverse. No other test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration in the 375, 500, and 750 mg/kg/day groups. No dermal findings were noted at any dosage level.

Lower mean body weight gains were noted in the 375, 500, and 750 mg/kg/day groups compared to the control group prior to the initiation of dose administration (gestation days 0-6); mean body weights in these groups were similar to the control group during this interval. Mean body weight losses were noted in all groups, including the control group, following the initiation of dose administration (gestation day 6-7); these differences were likely due to the placement of the Elizabethan collars. Test substance-related mean body weight losses or lower mean body weight gains were noted in the 750 mg/kg/day group compared to the control group during gestation days 6-9 and 15-20, and resulted in a lower mean body weight gain when the entire treatment period (gestation days 6-20) was evaluated; mean body weight gains in this group were similar to the control group during gestation days 9-12 and 12-15. In addition, mean body weights in the 750 mg/kg/day group were up to 7.4% lower than the control group during gestation days 8-20. A test substance-related lower mean net body weight and net body weight gain were also noted in the 750 mg/kg/day group. The aforementioned body weight effects noted in the 750 mg/kg/day group were considered adverse. The mean gravid uterine weight in the 750 mg/kg/day group was lower than the control group and was considered test substance-related. Mean body weight gains in the 375 and 500 mg/kg/day groups were generally similar to the control group for the remainder of the treatment period (gestation days 6-9, 9-12, 12-15, 15-20, and 6-20). Mean body weights in the 500 mg/kg/day group were up to 4.0% lower than the control group during gestation days 18-20; given the small magnitude of change and absence of a corresponding effect on mean body weight gain, the lower mean body weights noted at this dosage level were not considered test substance-related. A lower mean net body weight change was noted at 500 mg/kg/day compared to the control group; however, mean net body weight in this group was similar to the control group, and therefore this change was not considered test substance-related. Test substance-related, lower mean maternal food consumption was noted in the 500 and 750 mg/kg/day groups during gestation days 6-9 and/or 15-20 compared to the control group and resulted in lower mean food consumption in these groups when the entire treatment period (gestation days 6-20) was evaluated; mean food consumption in these groups was similar to the control group during gestation days 9-12 and 12-15. The lower mean food consumption in the 750 mg/kg/day group correlated with the lower mean body weights and body weight gains and was considered adverse; the differences noted in the 500 mg/kg/day group were generally not of sufficient magnitude to affect mean body weights and body weight gains, and therefore were considered nonadverse. Mean maternal body weights, net body weight, net body weight gain, and food consumption in the 375 mg/kg/day group and gravid uterine weights in the 375 and 500 mg/kg/day groups were unaffected by test substance administration.

No test substance-related macroscopic findings or changes in mean lung weights were noted for females at any dosage level at the scheduled necropsy on gestation day 20.

Mean male, female, and combined fetal weights in the 750 mg/kg/day group were 5.1%, 7.9%, and 5.3% lower, respectively, than the control group; the differences were considered test substance-related and attributed to the decrements in mean body weight gain and food consumption noted in this group during the fetal period (gestation days 15-20), which is the time of greatest fetal growth. Intrauterine growth at 375 and 500 mg/kg/day and survival at 375, 500, and 750 mg/kg/day were unaffected by test substance administration. There were no test substance-related external, visceral, and skeletal malformations or developmental variations noted at any dosage level.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-11 to 2016-07-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The actual ESR with UUID b01c04cd-9699-4682-ae06-f1c56743d4ad has been deleted as recommended by ECHA (INC 198666). Due to a false positive virus alert, the dossier got rejected several times. The original content of the study (including the formerly attached Summary Tables) has been reinstalled in a new RSS.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at room temperature under a nitrogen blanket, and was considered stable under these conditions.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Crl:CD(SD) rats (155 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 26-Jan-2016.
- Age at study initiation: 79 days old upon receipt; 13 weeks old when paired for breeding.
- Weight at study initiation: Body weight values ranged from 220 g to 296 g on gestation day 0.
- Fasting period before study: no
- Housing: Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). All rats were housed throughout the acclimation period and during the study in an environmentally controlled room.
- Diet (e.g. ad libitum): ad libitum. The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
- Water (e.g. ad libitum): ad libitum. Reverse osmosis-purified (on-site) drinking water, delivered byan automatic watering system.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71°F ± 5°F (22°C ± 3°C). Actual mean daily temperature ranged from 70.6°F to 70.8°F (21.4°C to 21.6°C) during the study.
- Humidity (%): 50% ± 20%. Actual mean daily relative humidity ranged from 44.1% to 53.5% during the study.
- Air changes (per hr): Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 26-Jan-2016 Experimental starting date (animal receipt)
To: 03-Mar-2016 (Last laparohysterectomy)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated (2°C to 8°C), purged with nitrogen. The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
Dosing formulations were prepared at the test substance concentrations indicated in the table (see below).
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (2°C to 8°C), purged with nitrogen. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
The first test substance dosing formulations were visually inspected by the Study Director’s designee and were found to be visibly homogeneous and acceptable for administration.

VEHICLE
- Concentration in vehicle: 0, 40, 60, 80 and 100 mg/mL for the dosage levels of 0, 200, 300, 400 and 500 mg/kg bw/day, respectively.
- Amount of vehicle (if gavage): The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.
- Purity: The vehicle used in preparation of the test substance formulations and for administration to the control group was deionized water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance in the vehicle was previously established to be stable and homogenous following 11 days of refrigerated and room temperature storage at concentrations of 40 and 100 mg/mL. Samples for homogeneity and/or concentration determination were collected from the top, middle, an d bottom strata of the first 40 and 100 mg/mL dosing formulations and from the middle stratum of the first control, 60, and 80 mg/mL dosing formulations. In addition, samples for resuspension homogeneity determination were collected from the top and bottom strata of an aliquot taken from the first 40 and 100 mg/mL dosing suspensions following room temperature storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum of the last dosing formulations (including the control group) prepared during the study. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored refrigerated (2°C to 8°C) and protected from light as back-ups, and the first dosing formulations were also stored purged with nitrogen. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: not specified.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not specified
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

single exposure

Frequency of treatment:

once daily

Duration of test:

during gestation days 6-19

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Details on study design:

- Dose selection rationale: Dosage levels were determined from results of previous studies (Hansen, 2013; see disregarded study LPT, 2013) and were discussed by the Sponsor Representative and the Study Director.
- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Animals not assigned to study were transferred to the WIL Research colony.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20. When body weights could not be determined for an animal during a given in terval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were designated as “NA” on the individual report tables.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

POST-MORTEM EXAMINATIONS: Yes
A gross necropsy was performed on the female that was found dead during the course of the study. The number and location of implantation sites and corpora lutea were recorded. The female and all products of conception were discarded.
- Sacrifice on gestation day 20.
- Organs examined: Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving rats were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded.

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% am monium sulfide solution for detection of early implantation loss (Salewski, 1964).
Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded (see Deviations from the Protocol). Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). Fetuses were then examined for skeletal malformations and developmental variations.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Statistics:

please see below ("Any other information on materials and methods incl. tables").

Indices:

Gestation Day 20 Laparohysterectomy
Intrauterine data were summarized using 2 methods of calculation. An example of each method of calculation follows:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group)/ No. Gravid Females/Group
2. Proportional Litter Basis:
Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)/ No. Litters/Group;
Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter)/ No. Implantation Sites/Litter x 100
Fetal Morphological Examination
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%))/ No. Litters/Group
Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter)/ No. Viable Fetuses/Litter x 100

Historical control data:

WIL Research has historical control data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical observations were noted in the 500 mg/kg/day group and consisted of rales and clear and red material around the mouth. Rales were noted in the 500 mg/kg/day group at the daily examinations and approximately 1 hour following dose administration generally throughout the treatment period (gestation days 7-20) and were considered adverse. Clear and red material around the mouth were noted for females in the 500 mg/kg/day group approximately 1 hour following dose administration. Because the material findings were noted primarily once per animal, they were not considered adverse. Rales were also noted in the 300 and 400 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration. However, due to the limited incidence of this finding in these groups, rales were considered test substance-related but not adverse. No other test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Findings noted in the treated groups, including hair loss on the forelimbs, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female no. 9857 in the 500 mg/kg/day group was found dead on gestation day 10. No remarkable clinical observations or effects on body weight or food consumption were noted for this female prior to death and the cause of death was not determined at necropsy. Therefore, the death in the 500 mg/kg/day group was not attributed to test substance administration. All other animals survived to the scheduled necropsy on gestation day 20.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test substance-related significant (p<0.01) mean body weight loss was noted in the 500 mg/kg/day group compared to a body weight gain in the control group during gestation days 6-9 due to mean body weight losses noted in this group immediately following the initiation of dose administration (gestation day 6-8, significant [p<0.01] during gestation days 6-7 only). Mean body weight gain in this group was comparable to the control group during the remainder of the treatment period (gestation days 9-12, 12-15 and 15-20). As a result of the initial mean body weight loss noted in the 500 mg/kg/day group, a significantly (p<0.05) lower mean body weight gain was observed for this group compared to the control group when the overall treatment period (gestation days 6-20) was evaluated. In addition, mean body weights in the 500 mg/kg/day group were 5.0% to 6.0% lower than the control group during gestation days 9-15; the differences were significant (p<0.05 or p<0.01). Mean net body weight gain in the 500 mg/kg/day group was also significantly (p<0.01) lower than the control group. Mean net body weight and gravid uterine weight in this group were comparable to the control group.
In the 200, 300, and 400 mg/kg/day groups, test substance-related lower mean body weight gains were noted during gestation days 6-9 compared to the control group due to mean body weight losses noted in these groups immediately following the first dose (gestation day 6-7); the difference was significant (p<0.05) for the 400 mg/kg/day group. Mean body weight gains in these groups were comparable to the control group throughout the remainder of the treatment period (gestation days 9-12, 12-15, and 15-20). Because the initial mean body weight losses observed in the 200, 300, and 400 mg/kg/day groups did not affect the overall mean body weight gain (gestation days 6-20) or mean body weights in these groups, they were not considered adverse. Mean net body weight gains in the 200, 300, and 400 mg/kg/day groups were lower than the control group; the differences at 300 and 400 mg/kg/day were significant (p<0.05 or p<0.01). Mean net body weights and gravid uterine weights in these groups were comparable to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 500 mg/kg/day group during gestation days 6-9 and 9-12; the differences were significant (p<0.01) compared to the control group and corresponded to lower mean body weight gains. Mean food consumption in this group was comparable to the control group during gestation days 12-15 and 15-20. As a result of the lower mean food consumption noted in the 500 mg/kg/day group during the first week of the treatment period, mean food consumption in this group was significantly (p<0.01) lower than the control group when the overall treatment period (gestation days 6-20) was evaluated.
In the 200, 300, and 400 mg/kg/day groups, significantly (p<0.01) lower mean food consumption corresponding to mean body weight losses was noted immediately following the first dose (gestation day 6-7). However, these differences were transient and did not affect the overall treatment period (gestation days 6-20) and therefore were not considered adverse. Other differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

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):

Female no. 9857 in the 500 mg/kg/day group was found dead on gestation day 10. This female had 17 normally developing implantations in utero and no macroscopic findings were noted at necropsy. At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 200, 300, 400, and 500 mg/kg/day. Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose related.
One female each in the control and 200 mg/kg/day groups were nongravid.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

Intrauterine growth in the 200 mg/kg/day group and survival in the 200, 300, 400, and 500 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The mean litter proportions of pre-implantation loss were similar across all groups.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Effects on pregnancy duration: not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

One female each in the control and 200 mg/kg/day groups were nongravid.

Other effects:

no effects observed

Details on maternal toxic effects:

Developmental toxicity was manifested at a dosage level of 500 mg/kg/day. Test substance-related statistically significantly lower (up to 10.0%) mean male, female, and combined fetal body weights were noted in the 500 mg/kg/day group compared to the concurrent control group. There were no test substance-related effects on intrauterine survival or fetal morphology at 500 mg/kg/day. The developmental toxicity noted at 500 mg/kg/day occurred in the presence of maternal toxicity.
At the daily examinations and approximately 1 hour following dose administration, test substancerelated higher incidences of rales were observed at 500 mg/kg/day. In addition, clear and red material around the mouth were noted for females in the 500 mg/kg/day group approximately 1 hour following dose administration. Although these material findings were considered test substance-related, they were not considered adverse because these findings were noted primarily once per animal. A test substance-related mean body weight loss was noted in the 500 mg/kg/day group during gestation days 6-9 which resulted in a lower mean body weight gain in this group when the overall treatment period (gestation days 6-20) was evaluated and mean body weights that were 5.0% to 6.0% lower than the control group during gestation days 9-15. In addition, lower mean food consumption was noted in the 500 mg/kg/day group compared to the control group during gestation days 6-9 and 9 -12 and when the overall treatment period (gestation days 6-20) was evaluated. In the 300 and 400 mg/kg/day groups, rales were noted sporadically throughout the treatment period and therefore were considered test substance-related, but not adverse. Test substance-related lower mean body weight gains and corresponding lower mean food consumption were noted for the 200, 300, and 400 mg/kg/day groups during gestation days 6-9 due to mean body weight losses and lower mean food consumption noted in these groups immediately following the first dose (gestation day 6-7). In the absence of effects on mean body weight gains and food consumption when the overall treatment period was evaluated or mean body weights throughout the study, these transient differences were not considered to be adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male, female, and combined fetal body weights in the 500 mg/kg/day group (3.7 g, 3.6 g, and 3.6 g, respectively) were 7.7% to 10.0% lower than the concurrent control group (4.1 g, 3.9 g, and 4.0 g, respectively) and the values were lower than the mean values in the WIL Research historical control data ranges (3.898 g, 3.697 g, and 3.801 g for male, female, and combined, respectively). The differences in fetal body weights observed in the 500 mg/kg/day group were significant (p<0.01) compared to the concurrent control group (even when the difference in mean number of viable fetuses was incorporated) and considered test substance-related. Significantly (p<0.05 or p<0.01) lower mean fetal body weights (male, female, and combined) were also noted in the 300 and 400 mg/kg/day groups compared to the concurrent control group. The mean fetal body weight values in the 300 mg/kg/day and 400 mg/kg/day groups (3.9 g, 3.7 g, and 3.8 g for male, female, and combined, respectively, for both groups) were comparable to the WIL Research historical control mean values for these parameters (3.9 g, 3.7 g, and 3.8 g, for male, female, and combined, respectively). In addition, the concurrent control group mean values for these parameters (4.1 g, 3.9 g, and 4.0 g for male, female, and combined, respectively) were at the maximum mean values in the WIL Research historical control database comprised of 80 data sets. Furthermore, the magnitude of the differences in fetal weights between the 300 and 400 mg/kg/day groups and the concurrent control group was small (4.9% to 5.1% lower than the concurrent control group). Therefore, the differences in fetal body weights observed in the 300 and 400 mg/kg/day groups were considered to be incidental due to the lower number of fetuses and increased fetal body weights in the concurrent control group and not related to test substance administration.
Fetal/pup body weight changes: not examined

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of viable fetuses in the control group was 4% to 8% less than the test substance treated groups, although this difference was not statistically significant. The mean number of viable fetuses in the control group (13.4 fetuses) was at the lower end of the WIL Research historical control data range of control group values (mean = 14.63, minimum = 12.35, maximum = 16.82, 25th quartile = 14.24, and 75th quartile = 15.01).

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 322(24), 348(24), 352(25), 348(25), and 347(24) in the control, 200, 300, 400, and 500 mg/kg/day groups, respectively. Malformations were observed in 1(1), 1(1), 1(1), 2(2), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. External malformations were observed in 0(0), 1(1), 1(1), 0(0), and 1(1) fetuses (litters) in the control, 200, 300, 400, and 500 mg/kg/day groups, respectively. Fetus no. 9825-18 in the 200 mg/kg/day group and fetus no. 9766-16 in the 500 mg/kg/day group were observed with microphthalmia and anophthalmia, respectively. Skeletally these malformations consisted of orbits smaller than normal.
In the 300 mg/kg/day group, fetus no. 9851-07 had mandibular micrognathia that consisted of small mandibles that were fused along the mandibular symphysis. The external malformations observed in the 200, 300, and 500 mg/kg/day groups were noted in single fetuses, did not occur in a dose-related manner, and/or the mean litter proportions of these findings were not statistically significantly different from the concurrent control group and therefore they were not attributed to the test substance.
There were no external developmental variations noted for fetuses in this study.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were observed in 1(1), 0(0), 0(0), 2(2), and 1(1) fetuses (litters) in the control, 200, 300, 400, and 500 mg/kg/day groups, respectively. One fetus each in the control, 400, and 500 mg/kg/day groups had sternoschisis (sternal band nos. 2 and/or 3 not joined). In addition, fetus no.9738-10 in the 400 mg/kg/day group had bent limb bones (femurs, fibula, tibia, humeri, radii, and ulnae). Because the skeletal malformations were noted for single fetuses, were observed similarly in the concurrent control group, and the mean litter proportions were not statistically significantly different from the concurrent control group and/or were within the WIL Research historical control data, they were not considered to be test substance-related.
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no visceral malformations noted for fetuses in this study.
No test substance-related visceral developmental variations were noted at any dosage level. Findings observed in the test substance-treated groups were noted infrequently, similarly in the concurrent control group, and/or were not observed in a dose-related manner. Furthermore, the differences in the mean litter proportions of these findings were not statistically significant compared to the concurrent control group and/or the values were within the ranges of the WIL Research historical control data. Red discoloration of the eye was observed in fetus no. 9818-08 in the 500 mg/kg/day group and renal papilla(e) not fully developed (Woo and Hoar grade 1) were noted for fetus nos. 9788-01 and 9834-03 in the control and 500 mg/kg/day groups, respectively. These findings were not classified as either malformations or developmental variations, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Other effects:

no effects observed

Description (incidence and severity):

Developmental toxicity was manifested at a dosage level of 500 mg/kg/day. Test substance-related statistically significantly lower (up to 10.0%) mean male, female, and combined fetal body weights were noted in the 500 mg/kg/day group compared to the concurrent control group. There were no test substance-related effects on intrauterine survival or fetal morphology at 500 mg/kg/day. The developmental toxicity noted at 500 mg/kg/day occurred in the presence of maternal toxicity.
Although mean fetal body weights in the 300 and 400 mg/kg/day groups were statistically significantly lower (approximately 5%) than the concurrent control group, the values in these groups were equal to the mean fetal body weight values in the WIL Research historical control database (comprised 80 data sets). In addition, the mean fetal body weight values within the concurrent control group were the maximum values found within the WIL Research historical control database. The very high mean fetal body weight values within the concurrent control group may be due to the lower number of mean viable fetuses within this group. Therefore, the differences in mean fetal body weight in the 300 and 400 mg/kg/day groups were considered incidental due to the lower number of viable fetuses and increased fetal body weights in the concurrent control group and not related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Analyses of Dosing Formulations

The analyzed dosing formulations were within WIL Research SOP range for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).

Results of the analyses of dosing formulations are summarized below.

Text Table 1. Results of Homogeneity Analyses
	Group 2 (40 mg/mL)	Group 5 (100 mg/mL)
Homogeneity Assessment of the 12-Feb-2016 Formulations
Mean Concentration (mg/mL)	40.0	103
RSD (%)	0.57	0.57
Mean % of Target	100	103
10-Day Room Temperature Resuspension Homogeneity Assessment of the 12-Feb-2016 Formulations
Mean Concentration (mg/mL)	40.5	102
RSD (%)	0.53	0.17
Mean % of Target	101	102

Text Table 2. Results of Concentration Analyses
	Mean Concentration, mg/mL (% of Target)
	Group 2	Group 3	Group 4	Group 5
Date of Preparation	(40 mg/mL)	(60 mg/mL)	(80 mg/mL)	(100 mg/mL)
12-Feb-2016	40.0 (100)	61.3 (102)	81.2 (102)	103 (103)
25-Feb-2016	40.6 (102)	61.8 (103)	82.5 (103)	102(102)

The summary tables (S1 - S15) depicted below show the results of the evaluated parameters.

Table S1
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of maternal survival and pregnancy status
Dose group :	1		2		3		4		5
	No.	%	No.	%	No.	%	No.	%	No.	%
Females on study	25		25		25		25		25
Females that aborted or delivered	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
Females that died	0	0.0	0	0.0	0	0.0	0	0.0	1	4.0
Females that aborted	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
nongravid	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
gravid	0	0.0	0	0.0	0	0.0	0	0.0	1	100.0
Females that were euthanized	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
nongravid	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
gravid	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
Females examined at scheduled necropsy	25	100.0	25	100.0	25	100.0	25	100.0	24	96.0
nongravid	1	4.0	1	4.0	0	0.0	0	0.0	0	0.0
gravid	24	96.0	24	96.0	25	100.0	25	100.0	24	100.0
with resorptions only	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
with viable fetuses	24	100.0	24	100.0	25	100.0	25	100.0	24	100.0
Total females gravid	24	96.0	24	96.0	25	100.0	25	100.0	25	100.0
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day

Table S2  (daily examinations)
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of clinical findings: total occurrence/no. of animals
----- f e m a l e -----
Table range:	02 -09 -16 to 03 -03-16
Group:	1	2	3	4	5
normal
-no significant clinical observations	489/25	508/25	505/25	472/25	451/24
disposition
-found dead	0/0	0/0	0/0	0/0	1/1
-scheduled euthanasia; gestation day 20	25/25	25/25	25/25	25/25	24/24
body/integument
-hair loss forelimb(s)	32/ 4	14/2	7/ 2	50/4	44/ 4
-hair loss ventral trunk	0/0	2/1	10/1	0/0	0/0
-hair loss facial area	3/ 1	0/0	0/0	0/0	0/0
cardio-pulmonary
-rales	0/0	0/0	0/0	3/ 3	28/10
eyes/ears/nose
-dried red material around nose	0/0	1/1	1/1	0/0	1/1
body/integ. II
-wet yellow material urogenital area	1/1	2/ 2	2/1	0/0	0/0
-wet yellow material ventral trunk	1/1	0/0	0/0	0/0	0/0
-dried brown material facial area	0/0	1/1	0/0	0/0	0/0
body/integ. II
-dried yellow material dorsal head	0/0	1/1	0/0	0/0	0/0
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day

Table S3
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of post-dose findings: total occurrence/no. of animals
----- f e m a l e -----
Table range:	02-15-16 to 03-02-16
Group:	1	2	3	4	5
normal
1 hour post-dosing
-no significant clinical observations	350/25	348/25	347/25	313/25	294/25
unsched obs (>7s mins)
-no significant clinical observations	0/0	0/0	0/0	24/24	0/0
cardio-pulmonary
1 hour post-dosing
-rales	0/0	0/0	2/2	8/6	28/12
eyes/ears/nose
1 hour post-dosing
-dried red material around nose	0/0	0/0	0/0	0/0	3/3
oral/dental
1 hour post-dosing
-wet clear material around mouth	0/0	1/1	0/0	2/2	8/5
-dried red material around mouth	0/0	1/1	1/1	3/3	6/6
-dried clear material around mouth	0/0	0/0	0/0	0/0	3/3
special ii
time of dose
-undetermined amount of dose expelled	0/0	0/0	0/0	1/1	1/1
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day

Table S4
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of body weights during gestation [g]
group:	0 mg/kg/day	200 mg/kg/day	300 mg/kg/day	400 mg/kg/day	500 mg/kg/day
Day 0
mean	255.	255.	254.	255.	256.
% difference		0.0	-0.4	0.0	0.4
S.D.	13.0	15.4	13.6	15.0	15.7
S.E.	2.6	3.1	2.7	3.0	3.1
n	24	24	25	25	25
Day 6
mean	287.	284.	283.	284.	283.
% difference		-1.0	-1.4	-1.0	-1.4
S.D.	15.5	17.6	14.3	16.3	17.6
S.E.	3.2	3.6	2.9	3.3	3.5
n	24	24	25	25	25
Day 7
mean	289.	283.	281.	282.	279.
% difference		-2.1	-2.8	-2.4	-3.5
S.D.	15.0	17.5	14.3	15.9	18.5
S.E.	3.1	3.6	2.9	3.2	3.7
n	24	24	25	25	25
Day 8
mean	292.	287.	284.	284.	279.
% difference		-1.7	-2.7	-2.7	-4.5
S.D.	16.9	15.8	14.1	15.4	17.9
S.E.	3.5	3.2	2.8	3.1	3.6
n	24	24	25	25	25
Day 9
mean	297.	290.	288.	289.	281.**
% difference		-2.4	-3.0	-2.7	-5.4
S.D.	16.4	16.4	15.1	15.5	19.6
S.E.	3.3	3.4	3.0	3.1	3.9
n	24	24	25	25	25
Day 10
mean	302.	297.	294.	294.	284.**
% difference		-1.7	-2.6	-2.6	-6.0
S.D.	16.1	18.1	13.6	16.3	19.3
S.E.	3.3	3.7	2.7	3.3	3.9
n	24	24	25	25	24
Day 11
mean	307.	302.	299.	299.	289.**
% difference		-1.6	-2.6	-2.6	-5.9
S.D.	16.5	18.3	14.8	17.0	19.0
S.E.	3.4	3.7	3.0	3.4	3.9
n	24	24	25	25	24
Day 12
mean	311.	308.	303.	303.	295.**
% difference		-1.0	-2.6	-2.6	-5.1
S.D.	18.2	18.2	15.3	18.2	20.8
S.E.	3.7	3.7	3.1	3.6	4.2
n	24	24	25	25	24
Day 13
mean	317.	313.	309.	309.	301.*
% difference		-1.3	-2.5	-2.5	-5.0
S.D.	20.0	19.9	15.7	19.2	20.6
S.E.	4.1	4.1	3.1	3.8	4.2
n	24	24	25	25	24
Day 14
mean	324.	318.	314.	314.	305.**
% difference		-1.9	-3.1	-3.1	-5.9
S.D.	18.0	18.9	16.1	18.6	23.0
S.E.	3.7	3.9	3.2	3.7	4.7
n	24	24	25	25	24
Day 15
mean	330.	325.	320.	322.	312.**
% difference		-1.5	-3.0	-2.4	-5.5
S.D.	19.0	19.3	17.3	19.8	24.1
S.E.	3.9	3.9	3.5	4.0	4.9
n	24	24	25	25	24
Day 16
mean	340.	336.	331.	330.	323.
% difference		-1.2	-2.6	-2.9	-5.0
S.D.	20.1	20.3	18.0	22.8	25.0
S.E.	4.1	4.2	3.6	4.6	5.1
n	24	24	25	25	24
Day 17
mean	353.	348.	345.	345.	337.
% difference		-1.4	-2.3	-2.3	-4.5
S.D.	22.7	22.2	19.1	22.6	26.3
S.E.	4.6	4.5	3.8	4.5	5.4
n	24	24	25	25	24
Day 18
mean	369.	366.	362.	360.	353.
% difference		-0.8	-1.9	-2.4	-4.3
S.D.	22.5	22.8	20.7	24.2	27.9
S.E.	4.6	4.7	4.1	4.8	5.7
n	24	24	25	25	24
Day 19
mean	385.	382.	376.	375.	366.
% difference		-0.8	-2.3	-2.6	-4.9
S.D.	25.1	22.6	20.4	25.2	28.5
S.E.	5.1	4.6	4.1	5.0	5.8
n	24	24	25	25	24
Day 20
mean	398.	397.	392.	387.	381.
% difference		-0.3	-1.5	-2.8	-4.3
S.D.	27.5	24.8	22.7	28.3	32.6
S.E.	5.6	5.1	4.5	5.7	6.7
n	24	24	25	25	24
*   = significantly different from the control group at 0.05 using Dunnett's test
** = significantly different from the control group at 0.01 using Dunnett's test
nongravid weight(s) not included in calculation of mean

Table S5
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of body weight changes during gestation [g]
Group:	0 mg/kg/day	200 mg/kg/day	300 mg/kg/day	400 mg/kg/day	500 mg/kg/day
Day     0- 6
mean	33.	28.	28.	29.	27.
S.D.	6.5	5.3	6.7	7.1	6.5
S.E.	1.3	1.1	1.3	1.4	1.3
n	24	24	25	25	25
Day     6- 7
mean	2.	-1.	-2.*	-2.*	-3.**
S.D.	3.3	3.4	3.2	4.8	6.0
S.E.	0.7	0.7	0.6	1.0	1.2
n	24	24	25	25	25
Day     7- 8
mean	3.	5.	3.	2.	-1.
S.D.	4.9	5.3	4.2	5.4	7.1
S.E.	1.0	1.1	0.8	1.1	1.4
n	24	24	25	25	25
Day     8- 9
mean	5.	3.	4.	5.	2.
S.D.	5.7	5.0	4.3	5.8	6.0
S.E.	1.2	1.0	0.9	1.2	1.2
n	24	24	25	25	25
Day     9- 10
mean	5.	7.	5.	5.	4.
S.D.	3.8	5.3	5.8	3.7	6.5
S.E.	0.8	1.1	1.2	0.7	1.3
n	24	24	25	25	24
Day   10- 11
mean	5.	5.	5.	4.	5.
S.D.	4.5	3.9	4.7	4.2	5.6
S.E.	0.9	0.8	0.9	0.8	1.1
n	24	24	25	25	24
Day   11- 12
mean	4.	6.	4.	4.	5.
S.D.	5.0	4.8	6.9	5.3	5.6
S.E.	1.0	1.0	1.4	1.1	1.2
n	24	24	25	25	24
Day   12- 13
mean	6.	5.	6.	6.	6.
S.D.	6.2	6.7	6.8	5.3	5.3
S.E.	1.3	1.4	1.4	1.1	1.1
n	24	24	25	25	24
Day   13- 14
mean	7.	5.	5.	5.	4.
S.D.	5.7	5.7	5.9	5.7	8.1
S.E.	1.2	1.2	1.2	1.1	1.7
n	24	24	25	25	24
Day   14- 15
mean	6.	7.	7.	8.	7.
S.D.	5.2	4.4	3.7	3.8	6.2
S.E.	1.1	0.9	0.7	0.8	1.3
n	24	24	25	25	24
Day   15- 16
mean	10.	11.	10.	8.	11.
S.D.	4.4	6.2	5.3	8.2	5.8
S.E.	0.9	1.3	1.1	1.6	1.2
n	24	24	25	25	24
Day   16- 17
mean	14.	12.	14.	15.	14.
S.D.	6.6	8.5	4.5	5.7	5.8
S.E.	1.4	1.7	0.9	1.1	1.2
n	24	24	25	25	24
Day   17- 18
mean	15.	18.	17.	15.	16.
S.D.	5.7	8.3	6.1	5.1	5.3
S.E.	1.2	1.7	1.2	1.0	1.1
n	24	24	25	25	24
Day   18- 19
mean	16.	15.	15.	15.	13.
S.D.	6.0	4.0	4.1	4.3	5.3
S.E.	1.2	0.8	0.8	0.9	1.1
n	24	24	25	25	24
Day   19- 20
mean	14.	16.	15.	13.	15.
S.D.	5.2	4.5	4.7	7.0	7.3
S.E.	1.1	0.9	0.9	1.4	1.5
n	24	24	25	25	24
Day       6- 9
mean	10.	7.	6.	5.*	-2.**
S.D.	5.2	4.8	4.1	4.0	9.1
S.E.	1.1	1.0	0.8	0.8	1.8
n	24	24	25	25	25
Day     9- 12
mean	14.	17.	15.	14.	15.
S.D.	7.5	6.2	8.0	6.6	9.8
S.E.	1.5	1.3	1.6	1.3	2.0
n	24	24	25	25	24
Day   12- 15
mean	19.	18.	17.	19.	17.
S.D.	6.6	6.1	6.7	5.1	9.0
S.E.	1.4	1.2	1.3	1.0	1.8
n	24	24	25	25	24
Day   15- 20
mean	68.	72.	71.	65.	69.
S.D.	11.4	9.0	10.3	13.6	13.1
S.E.	2.3	1.8	2.1	2.7	2.7
n	24	24	25	25	24
Day     6- 20
mean	111.	113.	109.	103.	99.*
S.D.	16.2	12.2	14.5	19.2	22.9
S.E.	3.3	2.5	2.9	3.8	4.7
n	24	24	25	25	24
*   = significantly different from the control group at 0.05 using Dunnett's test
** = significantly different from the control group at 0.01 using Dunnett's test
mean differences calculated from individual differences
nongravid weight(s) not included in calculation of mean

Table S6
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of gravid uterine wts. and net body wt. changes [g]
Group:	0 mg/kg/day	200 mg/kg/day	300 mg/kg/day	400 mg/kg/day	500 mg/kg/day
initial body wt.
mean	255.	255.	254.	255.	255.
S.D.	13.0	15.4	13.6	15.0	15.8
S.E.	2.6	3.1	2.7	3.0	3.2
n	24	24	25	25	24
terminal body wt.
mean	398.	397.	392.	387.	381.
S.D.	27.5	24.8	22.7	28.3	32.6
S.E.	5.6	5.1	4.5	5.7	6.7
n	24	24	25	25	24
gravid uterine wt.
mean	81.0	84.6	82.9	79.9	80.0
S.D.	13.19	10.15	11.23	13.76	18.87
S.E.	2.69	2.07	2.25	2.75	3.85
n	24	24	25	25	24
net body wt.
mean	317.5	312.6	308.6	307.5	300.8
S.D.	18.87	19.06	19.25	21.19	19.87
S.E.	3.85	3.89	3.85	4.24	4.06
n	24	24	25	25	24
net body wt. change
mean	62.8	57.2	54.3*	52.3**	45.6**
S.D.	13.91	9.92	12.14	12.70	10.38
S.E.	2.84	2.02	2.43	2.54	2.12
n	24	24	25	25	24
*   = significantly different from the control group at 0.05 using Dunnett's test
** = significantly different from the control group at 0.01 using Dunnett's test

Table S7
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of food consumption during gestation [g/animal/day)
Group:	0 mg/kg/day	200 mg/kg/day	300 mg/kg/day	400 mg/kg/day	500 mg/kg/day
Day       0- 0
mean	20.	20.	20.	21.	20.
S.D.	2.0	2.0	1.7	1.6	1.8
S.E.	0.4	0.4	0.3	0.3	0.4
n	24	23	25	25	25
Day       6- 7
mean	21.	17.**	17.**	17.**	14.**
S.D.	2.4	2.0	2.7	4.7	3.4
S.E.	0.5	0.5	0.5	0.9	0.7
n	24	24	25	25	25
Day     7- 8
mean	22.	20.	18.**	21.	15.**
S.D.	2.9	2.7	2.9	2.8	4.5
S.E.	0.8	0.6	0.5	0.5	0.9
n	24	24	25	25	25
Day     8- 9
mean	22.	21.	21.	21.	17.**
S.D.	3.9	2.9	2.3	2.4	4'.5
S.E.	g.b	g.g	g.s	g.s	0.9
n	24	24	25	25	25
Day     9- 10
mean	23.	21.	21.	20.*	17.**
S.D.	2.7	3.5	3.3	3.5	4.2
S.E.	0.5	0.7	0.7	0.7	0.9
n	24	24	25	25	24
Day   10- 11
mean	22.	21.	21.	22.	19.**
S.D.	3.1	3.4	2.6	2.5	4.5
S.E.	0.6	0.7	0.5	0.5	0.9
n	24	24	25	25	24
Day   11- 12
mean	22.	21.	20.	21.	19.*
S.D.	3.2	3.1	3.6	2.8	4.2
S.E.	0.7	0.6	0.7	0.6	0.9
n	24	24	25	25	24
Day   12- 13
mean	22.	24.	24.	23.	21.
S.D.	4.4	4.8	4.8	3.7	3.7
S.E.	0.9	1.0	1.0	0.8	0.8
n	24	24	25	24	24
Day   13- 14
mean	23.	22.	22.	23.	20.
S.D.	4.5	5.3	5.7	5.2	5.3
S.E.	0.9	1.1	1.1	1.0	1.1
n	24	24	25	25	24
Day   14- 15
mean	23.	23.	22.	23.	22.
S.D.	3.5	3.2	5.3	3.0	4.3
S.E.	0.7	0.7	1.1	0.6	0.9
n	24	24	25	25	24
Day   15- 16
mean	24.	25.	24.	24.	22.
S.D.	2.7	3.8	2.6	3.1	3.5
S.E.	0.6	0.8	0.6	0.6	0.7
n	24	24	23	25	24
Day   16- 17
mean	26.	25.	26.	25.	24.
S.D.	3.1	3.5	2.4	3.7	3.3
S.E.	0.6	0.7	0.5	0.7	0.7
n	24	24	25	25	23
Day   17- 18
mean	25.	24.	24.	24.	23.
S.D.	4.6	3.1	4.1	2.9	4.2
S.E.	0.9	0.6	0.8	0.6	0.9
n	24	24	25	25	24
Day   18- 19
mean	26.	25.	25.	24.	24.
S.D.	4.7	4.3	2.9	3.0	4.9
S.E.	1.0	0.9	0.6	0.6	1.0
n	24	24	25	25	23
Day   19- 20
mean	21.	24.	22.	22.	22.
S.D.	6.9	2.8	5.2	5.4	3.0
S.E.	1.4	0.6	1.1	1.1	0.6
n	24	24	24	24	24
Day     6- 9
mean	21.	20.	19.**	20.	16.**
S.D.	2.7	1.8	1.8	2.0	3.3
S.E.	0.5	0.4	0.4	0.4	0.7
n	24	24	25	25	25
Day     9- 12
mean	22.	21.	21.*	21.	18.**
S.D.	2.3	2.3	1.7	2.1	3.5
S.E.	0.5	0.5	0.3	0.4	0.7
n	24	24	25	25	24
Day   12- 15
mean	23.	23.	23.	23.	21.
S.D.	2.5	3.1	3.8	2.8	3.5
S.E.	0.5	0.6	0.8	0.6	0.7
n	24	24	25	25	24
Day   15- 20
mean	24.	25.	24.	24.	23.
S.D.	2.9	2.7	2.2	2.5	2.6
S.E.	0.6	0.6	0.4	0.5	0.5
n	24	24	25	25	24
Day     6- 20
mean	23.	22.	22.	22.	20.**
S.D.	2.2	2.0	1.9	1.7	2.2
S.E.	0.5	0.4	0.4	0.3	0.5
n	24	24	25	25	24
*   = significantly different from the control group at 0.05 using Dunnett's test
** = significantly different from the control group at 0.01 using Dunnett's test
nongravid weight(s) not included in calculation of mean

Table S8
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of food consumption during gestation [g/kg/day]
Group:	0 mg/kg/day	200 mg/kg/day	300 mg/kg/day	400 mg/kg/day	500 mg/kg/day
Day     0- 6
mean	75.	73.	74.	77.	73.
S.D.	6.0	6.2	6.6	4.8	5.2
S.E.	1.2	1.3	1.3	1.0	1.0
n	24	23	25	25	25
Day     6- 7
mean	72.	61.**	59.**	60.**	50.**
S.D.	7.4	8.6	9.1	15.9	11.4
S.E.	1.5	1.8	1.8	3.2	2.3
n	24	24	25	25	25
Day     7- 8
mean	74.	70.	65.*	73.	55.**
S.D.	7.7	8.8	10.1	11.4	15.7
S.E.	1.6	1.8	2.0	2.3	3.1
n	24	24	25	25	25
Day     8- 9
mean	73.	74.	72.	75.	61.**
S.D.	11.3	10.0	7.1	9.3	14.6
S.E.	2.3	2.0	1.4	1.9	2.9
n	24	24	25	25	25
Day     9- 10
mean	77.	70.	71.	68.*	61.**
S.D.	8.5	10.8	11.9	10.4	13.7
S.E.	1.7	2.2	2.4	2.1	2.8
n	24	24	25	25	24
Day   10- 11
mean	73.	72.	70.	74.	65.*
S.D.	8.7	12.1	9.1	8.2	15.5
S.E.	1.8	2.5	1.8	1.6	3.2
n	24	24	25	25	24
Day   11- 12
mean	70.	70.	67.	71.	64.
S.D.	9.6	8.5	11.4	9.5	13.0
S.E.	2.0	1.7	2.3	1.9	2.7
n	24	24	25	25	24
Day   12- 13
mean	70.	77.	79.	75.	69.
S.D.	12.9	16.1	14.7	11.2	10.2
S.E.	2.6	3.3	2.9	2.3	2.1
n	24	24	25	24	24
Day   13- 14
mean	71.	70.	70.	73.	65.
S.D.	13.2	14.9	16.6	13.8	16.1
S.E.	2.7	3.0	3.3	2.8	3.3
n	24	24	25	25	24
Day   14- 15
mean	70.	72.	68.	72.	70.
S.D.	10.4	8.6	15.7	7.5	10.7
S.E.	2.1	1.8	3.1	1.5	2.2
n	24	24	25	25	24
Day   15- 16
mean	72.	75.	73.	74.	70.
S.D.	7.5	8.7	7.0	7.5	8.6
S.E.	1.5	1.8	1.5	1.5	1.7
n	24	24	23	25	24
Day   16- 17
mean	74.	71.	76.	74.	74.
S.D.	7.2	8.2	5.0	9.7	8.1
S.E.	1.5	1.7	1.0	1.9	1.7
n	24	24	25	25	23
Day   17- 18
mean	69.	68.	68.	69.	67.
S.D.	11.9	7.0	11.0	6.0	12.4
S.E.	2.4	1.4	2.2	1.2	2.5
n	24	24	25	25	24
Day   18- 19
mean	70.	67.	67.	67.	67.
S.D.	11.6	11.1	8.2	8.9	11.4
S.E.	2.4	2.3	1.6	1.8	2.4
n	24	24	25	25	23
Day   19- 20
mean	53.	61.	57.	57.	60.
S.D.	17.1	5.6	13.0	13.5	9.0
S.E.	3.5	1.2	2.7	2.8	1.8
n	24	24	24	24	24
Day     6- 9
mean	73.	68.	66.**	69.	55.**
S.D.	7.1	5.9	5.6	7.4	10.9
S.E.	1.4	1.2	1.1	1.5	2.2
n	24	24	25	25	25
Day     9- 12
mean	73.	70.	69.	72.	63.**
S.D.	6.3	6.8	5.7	6.5	11.2
S.E.	1.3	1.4	1.1	1.3	2.3
n	24	24	25	25	24
Day   12- 15
mean	70.	73.	72.	73.	68.
S.D.	6.6	8.9	10.7	6.0	8.3
S.E.	1.3	1.8	2.1	1.2	1.7
n	24	24	25	25	24
Day   15- 20
mean	67.	68.	68.	68.	67.
S.D.	6.8	5.4	5.6	5.2	6.4
S.E.	1.4	1.1	1.1	1.0	1.3
n	24	24	25	25	24
Day     6- 20
mean	70.	69.	68.	69.	64.**
S.D.	5.2	4.4	4.8	3.5	5.0
S.E.	1.1	0.9	1.0	0.7	1.0
n	24	24	25	25	24
*   = significantly different from the control group at 0.05 using Dunnett's test
** = significantly different from the control group at 0.01 using Dunnett's test
nongravid weight(s) not included in calculation of mean

Table S9
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of maternal macroscopic findings
Group:	1	2	3	4	5
number examined	25	25	25	25	25
no significant changes observed	23	23	23	25	23
nongravid -- ammonium sulfide negative	1	1	0	0	0
found dead	0	0	0	0	1
vagina: contents, brown	1	0	0	0	0
uterus: contents, dark red	0	0	0	0	1
spleen: area(s), white	0	1	0	0	0
placentae: pale	0	0	1	0	0
uterus: contents, brown fluid	0	0	1	0	0
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day

Table S10
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of fetal data at scheduled necropsy
Group:		sex		viable fetuses	dead	resorptions		post implantation loss	implantation sites	corpora lutea	pre implantation loss	fetal weights in grams	no. of gravid females
		m	f		fetuses	early	late
1	total	176	146	322	0	21	0	21	343	372	29	na	24
	mean	7.3	6.1	13.4	0.0	0.9	0.0	0.9	14.3	15.5	1.2	4.0
	S.D.	1.93	2.21	2.38	0.00	0.85	0.00	0.85	2.16	2.72	1.82	0.27
	S.E.	0.39	0.45	0.48	0.00	0.17	0.00	0.17	0.44	0.55	0.37	0.05
2	total	160	188	348	0	22	1	23	371	387	16	na	24
	mean	6.7	7.8	14.5	0.0	0.9	0.0	1.0	15.5	16.1	0.7	3.9
	S.D.	1.97	1.71	1.93	0.00	0.88	0.20	0.91	1.72	2.25	1.27	0.26
	S.E.	0.40	0.35	0.39	0.00	0.18	0.04	0.19	0.35	0.46	0.26	0.05
3	total	175	177	352	0	19	4	23	375	406	31	na	25
	mean	7.0	7.1	14.1	0.0	0.8	0.2	0.9	15.0	16.2	1.2	3.8*
	S.D.	2.16	1.98	2.18	0.00	0.97	0.80	1.41	1.83	2.28	1.90	0.21
	S.E.	0.43	0.40	0.44	0.00	0.19	0.16	0.28	0.37	0.46	0.38	0.04
4	total	171	177	348	0	18	0	18	366	406	40	na	25
	mean	6.8	7.1	13.9	0.0	0.7	0.0	0.7	14.6	16.2	1.6	3.8**
	S.D.	1.93	1.98	2.48	0.00	0.79	0.00	0.79	2.55	2.30	2.68	0.22
	S.E.	0.39	0.40	0.50	0.00	0.16	0.00	0.16	0.51	0.46	0.54	0.04
5	total	167	180	347	0	8	0	8	355	368	13	na	24
	mean	7.0	7.5	14.5	0.0	0.3	0.0	0.3	14.8	15.3	0.5	3.6**
	S.D.	2.44	2.55	3.44	0.00	0.56	0.00	0.56	3.49	3.67	0.78	0.20
	S.E.	0.50	0.52	0.70	0.00	0.12	0.00	0.12	0.71	0.75	0.16	0.04
*   = significantly different from the control group at 0.05
** = significantly different from the control group at 0.01
na = not applicable
mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea, fetal weights compared using dunnett's test
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day

Table S11
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor: EASTMAN CHEMICAL Co.	Summary of fetal data at scheduled necropsy [% per litter]
Group:	0 mg/kg/day	200 mg/kg/day	300 mg/kg/day	400 mg/kg/day	500 mg/kg/day
corpora lutea
mean	15.5	16.1	16.2	16.2	15.3
S.D.	2.72	2.25	2.28	2.30	3.67
S.E.	0.55	0.46	0.46	0.46	0.75
n	24	24	25	25	24
implantation sites
mean	14.3	15.5	15.0	14.6	14.8
S.D.	2.16	1.72	1.83	2.55	3.49
S.E.	0.44	0.35	0.37	0.51	0.71
n	24	24	25	25	24
viable fetuses (%)
mean	93.7	93.8	93.9	95.2	97.8
S.D.	6.44	6.07	9.05	5.17	3.68
S.E.	1.32	1.24	1.81	1.03	0.75
n	24	24	25	25	24
dead fetuses (%)
mean	0.0	0.0	0.0	0.0	0.0
S.D.	0.00	0.00	0.00	0.00	0.00
S.E.	0.00	0.00	0.00	0.00	0.00
n	24	24	25	25	24
early resorptions (%)
mean	6.3	6.0	5.0	4.8	2.2
S.D.	6.44	5.89	5.92	5.18	3.68
S.E.	1.32	1.20	1.18	1.04	0.75
n	24	24	25	25	24
late resorptions (%)
mean	0.0	0.3	1.1	0.0	0.0
S.D.	0.00	1.37	5.34	0.00	0.00
S.E.	0.00	0.28	1.07	0.00	0.00
n	24	24	25	25	24
total resorptions (%)
mean	6.3	6.3	6.1	4.8	2.2
S.D.	6.44	6.07	9.05	5.18	3.68
S.E.	1.32	1.24	1.81	1.04	0.75
n	24	24	25	25	24
pre-implantation loss (%)
mean	6.9	3.7	6.9	9.1	3.3
S.D.	9.48	5.89	9.96	14.43	4.69
S.E.	1.94	1.20	1.99	2.89	0.96
n	24	24	25	25	24
post-implantation loss (%)
mean	6.3	6.3	6.1	4.8	2.2
S.D.	6.44	6.07	9.05	5.18	3.68
S.E.	1.32	1.24	1.81	1.04	0.75
n	24	24	25	25	24
males (%)
mean	55.1	45.6	49.6	49.2	50.1
S.D.	13.13	10.93	13.09	13.26	15.80
S.E.	2.68	2.23	2.62	2.65	3.22
n	24	24	25	25	24
females (%)
mean	44.9	54.4	50.6	50.8	49.9
S.D.	13.13	10.93	13.09	13.26	15.80
S.E.	2.68	2.23	2.62	2.65	3.22
n	24	24	25	25	24
male fetal weights (g)
mean	4.1	3.9	3.9*	3.9**	3.7**
% difference		-4.9	-4.9	-4.9	-9.8
S.D.	0.26	0.26	0.22	0.24	0.24
S.E.	0.05	0.05	0.04	0.05	0.05
n	24	24	25	25	24
female fetal weights (g)
mean	3.9	3.8	3.7*	3.7*	3.6**
% difference		-2.g	-5.1	-5.1	-7.7
S.D.	0.26	0.27	0.18	0.23	0.19
S.E.	0.05	0.06	0.04	0.05	0.04
n	24	24	25	25	23
combined fetal weights (g)
mean	4.0	3.9	3.8*	3.8**	3.6**
% difference		- 2.5	- 5.0	- 5.0	- 10.0
S.D.	0.27	0.26	0.21	0.22	0.20
S.E.	0.05	0.05	0.04	0.04	0.04
n	24	24	24	25	24
proportional (%) data compared using Dunn's test
corpora lutea and implantation sites compared using Dunnett's test
fetal weights compared using Dunnett's test
modified statistics used.
* indicates parametric analysis
+ indicates non-parametric analysis.
*   = significantly different from the control group at 0.05
** = significantly different from the control group at 0.01

Table S12
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor:EASTMAN CHEMICAL Co.	summary of fetuses and litters with malformations  [absolute no.]
Day 20
	fetuses					litters
Dose group:	1	2	3	4	5	1	2	3	4	5
number examined externally	322	348	352	348	347	24	24	25	25	24
microphthalmia and/or anophthalmia	0	1	0	0	1	0	1	0	0	1
mandibular micrognathia	0	0	1	0	0	0	0	1	0	0
number examined viscerally	322	348	352	348	347	24	24	25	25	24
number with findings	0	0	0	0	0	0	0	0	0	0
number examined skeletally	322	348	352	348	347	24	24	25	25	24
sternoschisis	1	0	0	1	1	1	0	0	1	1
bent limb bone(s)	0	0	0	1	0	0	0	0	1	0
total number with malformations
external :	0	1	1	0	1	0	1	1	0	1
soft tissue :	0	0	0	0	0	0	0	0	0	0
skeletal :	1	0	0	2	1	1	0	0	2	1
combined :	1	1	1	2	1	1	1	1	2	1
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day

Table S13
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor:EASTMAN CHEMICAL Co.	Summary of litter proportions of malformations % per litter
day 20
Dose group:		1	2	3	4	5
number of litters examined externally		24	24	25	25	24
microphthalmia and/or anophthalmia	mean	0.0	0.2	0.0	0.0	0.3
	S.D.	0.00	1.20	0.00	0.00	1.46
	S.E.	0.00	0.25	0.00	0.00	0.30
mandibular micrognathia	mean	0.0	0.0	0.4	0.0	0.0
	S.D.	0.00	0.00	2.00	0.00	0.00
	S.E.	0.00	0.00	0.40	0.00	0.00
number of litters examined viscerally		24	24	25	25	24
number of litters with findings		0	0	0	0	0
number of litters examined skeletally		24	24	25	25	24
sternoschisis	mean	0.3	0.0	0.0	0.3	0.3
	S.D.	1.28	0.00	0.00	1.54	1.46
	S.E.	0.26	0.00	0.00	0.31	0.30
bent limb bone(s)	mean	0.0	0.0	0.0	0.4	0.0
	S.D.	0.00	0.00	0.00	2.22	0.00
	S.E.	0.00	0.00	0.00	0.44	0.00
total malformations
percent per litter with external malformations	mean	0.0	0.2	0.4	0.0	0.3
	S.D.	0.00	1.20	2.00	0.00	1.46
	S.E.	0.00	0.25	0.40	0.00	0.30
percent per litter with soft tissue malformations	mean	0.0	0.0	0.0	0.0	0.0
	S.D.	0.00	0.00	0.00	0.00	0.00
	S.E.	0.00	0.00	0.00	0.00	0.00
percent per litter with skeletal malformations	mean	0.3	0.0	0.0	0.8	0.3
	S.D.	1.28	0.00	0.00	2.65	1.46
	S.E.	0.26	0.00	0.00	0.53	0.30
total percent per litter with malformations	mean	0.3	0.2	0.4	0.8	0.3
	S.D.	1.28	1.20	2.00	2.65	1.46
	S.E.	0.26	0.25	0.40	0.53	0.30
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day
modified statistics used.
none significantly different from control group

Table S14
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor:EASTMAN CHEMICAL Co.	Summary of fetuses and litters with variations [absolute no.]
Day 20
	Dose group:	fetuses					litters
Dose group:		1	2	3	4	5	1	2	3	4	5
number examined externally		322	348	352	348	347	24	24	25	25	24
number with findings		0	0	0	0	0	0	0	0	0	0
number examined viscerally		322	348	352	348	347	24	24	25	25	24
liver- accessory lobule(s)		0	2	4	0	1	0	2	2	0	1
hemorrhagic iris		0	0	1	0	0	0	0	1	0	0
major blood vessel variation		2	2	1	0	0	2	2	1	0	0
haemorrhagic ring around the iris		0	0	0	2	0	0	0	0	1	0
renal papilla(e) not developed and/or distended ureter(s)		2	0	1	0	2	1	0	1	0	2
number examined skeletally		322	348	352	348	347	24	24	25	25	24
sternebra(e)  #5 and/or #6 unossified		23	44	41	39	66	12	13	15	15	16
bent rib(s)		4	3	4	8	7	4	1	3	5	3
14th rudimentary rib(s)		28	22	40	50	64	10	13	17	15	18
reduced ossification of the skull		3	2	3	1	6	2	1	3	1	3
cervical centrum #1 ossified		51	68	53	53	49	19	20	20	17	13
sternebra(e) malaligned(slight or moderate)		3	2	5	1	1	3	2	5	1	1
pubis unossified		0	0	0	1	0	0	0	0	1	0
7th cervical rib(s)		2	4	2	2	9	2	4	2	2	4
sternebra(e)  #1,#2,#3 and/or #4 unossified		0	0	2	0	1	0	0	2	0	1
reduced ossification of the vertebral arches		2	0	1	1	1	2	0	1	1	1
reduced ossification of the 13th rib(s)		0	2	4	0	0	0	2	3	0	0
hyoid unossified		1	0	0	2	0	1	0	0	2	0
2 7 presacral vertebrae		0	0	0	4	1	0	0	0	1	1
bent scapula(e)		0	0	0	1	1	0	0	0	1	1
entire sternum unossified		0	0	0	1	1	0	0	0	1	1
14th full rib(s)		0	0	0	5	0	0	0	0	1	0
vertebral centra unossified		0	0	0	1	0	0	0	0	1	0
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day

Table S15
Project No.:WIL-387076	Rat oral prenatal developmental tox study of n-butyl-pyrrolidone
Sponsor:EASTMAN CHEMICAL Co.	Summary of litter proportions of variations % per litter
Day 20
dose group:			1	2	3	4	5
number of litters examined externally			24	24	25	25	24
number of litters with findings			0	0	0	0	0
number of litters examined viscerally			24	24	25	25	24
liver- accessory lobule(s)		mean	0.0	0.7	1.0	0.0	0.3
		S.D.	0.00	2.26	3.93	0.00	1.28
		S.E.	0.00	0.46	0.79	0.00	0.26
hemorrhagic iris		mean	0.0	0.0	0.3	0.0	0.0
		S.D.	0.00	0.00	1.25	0.00	0.00
		S.E.	0.00	0.00	0.25	0.00	0.00
major blood vessel variation		mean	0.6	0.5	0.4	0.0	0.0
		S.D.	2.03	1.85	2.00	0.00	0.00
		S.E.	0.41	0.38	0.40	0.00	0.00
hemorrhagic ring around the iris		mean	0.0	0.0	0.0	0.6	0.0
		S.D.	0.00	0.00	0.00	2.86	0.00
		S.E.	0.00	0.00	0.00	0.57	0.00
renal papilla(e) not developed and/or distended ureter(s)		mean	0.8	0.0	0.3	0.0	0.6
		S.D.	4.08	0.00	1.54	0.00	1.90
		S.E.	0.83	0.00	0.31	0.00	0.39
sternebra(e)  #5 and/or #6 unossified		mean	6.7	12.0	11.6	11.3	18.2
		S.D.	8.14	15.49	14.34	12.68	21.35
		S.E.	1.66	3.16	2.87	2.54	4.36
bent rib(s)		mean	1.4	0.8	1.2	2.6	2.2
		S.D.	3.26	4.08	3.76	6.48	6.45
		S.E.	0.67	0.83	0.75	1.30	1.32
14th rudimentary rib(s)		mean	9.1	6.3	11.4	14.5	17.4
		S.D.	12.38	8.29	12.84	19.57	19.92
		S.E.	2.53	1.69	2.57	3.91	4.07
reduced ossification of the skull		mean	1.1	0.6	0.8	0.3	1.7
		S.D.	3.93	2.72	2.29	1.67	4.61
		S.E.	0.80	0.56	0.46	0.33	0.94
cervical centrum #1 ossified		mean	15.5	19.9	15.1	14.9	13.4
		S.D.	13.02	20.84	12.82	15.72	21.49
		S.E.	2.66	4.25	2.56	3.14	4.39
sternebra(e) malaligned(slight or moderate)		mean	1.1	0.5	1.6	0.5	0.3
		S.D.	3.10	1.82	3.39	2.50	1.28
		S.E.	0.63	0.37	0.68	0.50	0.26
pubis unossified		mean	0.0	0.0	0.0	0.3	0.0
		S.D.	0.00	0.00	0.00	1.25	0.00
		S.E.	0.00	0.00	0.00	0.25	0.00
7th cervical rib(s)		mean	0.6	1.1	0.5	0.6	2.2
		S.D.	2.13	2.57	1.70	2.15	6.72
		S.E.	0.44	0.53	0.34	0.43	1.37
sternebra(e)  #1,#2,#3 and/or #4 unossified		mean	0.0	0.0	0.5	0.0	0.3
		S.D.	0.00	0.00	1.85	0.00	1.46
		S.E.	0.00	0.00	0.37	0.00	0.30
reduced ossification of the vertebral arches		mean	0.7	0.0	0.3	0.4	0.3
		S.D.	2.25	0.00	1.33	2.22	1.70
		S.E.	0.46	0.00	0.27	0.44	0.35
reduced ossification of the 13th rib(s)		mean	0.0	0.6	1.1	0.0	0.0
		S.D.	0.00	2.04	3.13	0.00	0.00
		S.E.	0.00	0.42	0.63	0.00	0.00
hyoid unossified		mean	0.4	0.0	0.0	0.7	0.0
		S.D.	2.04	0.00	0.00	2.54	0.00
		S.E.	0.42	0.00	0.00	0.51	0.00
27 presacral vertebrae		mean	0.0	0.0	0.0	1.1	0.2
		S.D.	0.00	0.00	0.00	5.33	1.20
		S.E.	0.00	0.00	0.00	1.07	0.25
bent scapula(e)		S.E.	0.0	0.0	0.0	0.4	0.3
		S.D.	0.00	0.00	0.00	2.22	1.70
		S.E.	0.00	0.00	0.00	0.44	0.35
entire sternum unossified		mean	0.0	0.0	0.0	0.4	0.3
		S.D.	0.00	0.00	0.00	2.22	1.70
		S.E.	0.00	0.00	0.00	0.44	0.35
14th full rib(s)		mean	g.g	g.g	g.g	1.3	g.g
		S.D.	g.gg	g.gg	g.gg	g.g7	g.gg
		S.E.	g.gg	g.gg	g.gg	1.33	g.gg
vertebral centra unossified		mean	g.g	g.g	g.g	g.4	g.g
		S.D.	g.gg	g.gg	g.gg	2.22	g.gg
		S.E.	g.gg	g.gg	g.gg	g.44	g.gg
total variations
percent per litter with external variations		mean	0.0	0.0	0.0	0.0	0.0
		S.D.	0.00	0.00	0.00	0.00	0.00
		S.E.	0.00	0.00	0.00	0.00	0.00
percent per litter with soft tissue variations		mean	1.4	1.2	2.0	0.6	0.8
		S.D.	4.44	2.79	5.52	2.86	2.22
		S.E.	0.91	0.57	1.10	0.57	0.45
percent per litter with skeletal variations		mean	32.3	35.5	37.3	41.7	45.1
		S.D.	16.86	21.01	21.02	21.49	25.95
		S.E.	3.44	4.29	4.20	4.30	5.30
total percent per litter with variations		mean	33.3	36.5	38.6	42.3	45.3
		S.D.	16.75	21.09	20.91	21.12	25.57
		S.E.	3.42	4.31	4.18	4.22	5.22
1 - 0 mg/kg/day
2 - 200 mg/kg/day
3 - 300 mg/kg/day
4 - 400 mg/kg/day
5 - 500 mg/kg/day
modified statistics used.
none significantly different from control group

Conclusions:

Maternal toxicity was evidenced at 500 mg/kg/day by higher incidences of rales and lower mean body weight gains, body weights, and food consumption during the treatment period. Although test substance-related effects on body weight gains and food consumption were noted at 300 and 400 mg/kg/day and rales were noted at 300 and 400 mg/kg/day, these effects were transient and not considered adverse. Developmental toxicity was noted at 500 mg/kg/day group as evidenced by lower mean fetal body weights. No evidence of developmental toxicity was observed at 200, 300, or 400 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day was considered to be the noobserved-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when n-butyl pyrrolidone was administered orally by gavage to bred Crl:CD(SD) rats.

Executive summary:

The objectives of the study were to determine the potential of the test substance, N-Butylpyrrolidone, to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a noobserved-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity.

The test substance, N-butyl pyrrolidone, in the vehicle (deionized water) was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 200, 300, 400, and 500 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Results

One female in the 500 mg/kg/day group was found dead on gestation day 10. There were no remarkable clinical observations or effects on body weight or food consumption noted for this female prior to death and no remarkable macroscopic findings were noted at necropsy. Therefore, the single death in the 500 mg/kg/day group was not attributed to test substance administration. All other females survived to the scheduled necropsy on gestation day 20. Rales were noted in a dose-related manner in the 300, 400, and 500 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration; however, based on the incidence and frequency of this finding, rales were only considered adverse at 500 mg/kg/day. Clear and red material around the mouth were noted for females in the 500 mg/kg/day group approximately 1 hour following dose administration. These findings were generally noted once per animal and therefore were considered test substance-related, but not adverse. There were no other test substance-related clinical observations noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

A test substance-related lower mean body weight gain and lower mean food consumption were noted for the 500 mg/kg/day group when the overall treatment period (gestation days 6-20) was evaluated due to a mean body weight loss during gestation days 6-9 and lower mean food consumption during the first week of dosing (gestation days 6-12). As a result, mean body weights in the 500 mg/kg/day group were lower (5.0% to 6.0%) than the control group during gestation days 9-15 and a lower mean net body weight gain was observed compared to the control group. Mean net body weight and gravid uterine weight in the 500 mg/kg/day group was comparable to the control group.

Test substance related mean body weight losses and lower mean food consumption were noted in the 200, 300, and 400 mg/kg/day groups immediately following the first dose (gestation day 6-7). Mean body weight gains and food consumption in these groups were comparable to the control group throughout the remainder of the treatment period. Furthermore, mean body weights in the 200, 300, and 400 mg/kg/day groups were unaffected by test substance administration. Therefore, the initial decrements in body weight and food consumption observed in these groups were not considered adverse. Mean net body weight gains in the 300 and 400 mg/kg/day groups were lower than the control group. Mean net body weight gain in the 200 mg/kg/day group and mean net body weights and gravid uterine weights in the 200, 300, and 400 mg/kg/day groups were unaffected by test substance administration.

There were no test substance-related macroscopic findings noted at any dosage level. Although the control group had approximately 4% to 8% fewer mean live fetuses than the test substance-treated groups, these differences were not statistically significant. The mean number of viable fetuses for the concurrent control group (13.4 fetuses) was at the lower end of the WIL Research historical control data, containing data from 80 data sets. Correspondingly, the concurrent control group had the highest mean fetal body weights (4.1 g, 3.9 g, and 4.0 g, for male, female and combined, respectively) compared to the WIL Research historical control data. Test substance-related lower (up to 10%) mean fetal body weights were observed at 500 mg/kg/day. Slight decrements (5%) in mean fetal body weights in the 300 and 400 mg/kg/day groups were considered due to the lower number of fetuses per litter and increased fetal body weights in the concurrent control group. Mean fetal body weights of the 300 and 400 mg/kg/day groups were equal to the mean values in the WIL Research historical control. Therefore, intrauterine growth and survival at 400 mg/kg/day and below were considered unaffected by test substance administration. No test substance fetal malformations or developmental variations were noted at 200, 300, 400, and 500 mg/kg/day.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was conducted as part of the product development and commercialization strategy for the United States by a US company. The data is being updated in the REACH dossier to satisfy the data notification requirements, even though this study was not performed for REACH or for entities in the EU.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification : n-Butyl Pyrrolidone (also known as TamiSolve® NxG; CAS No. 3470 98-2)
Batch No.: G180445430
Receipt Date: 14 May 2019
Expiration Date: 19 Apr 2020
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 (US)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test System
Receipt
On 11 Oct 2019, time-mated female New Zealand White rabbits were received from Envigo Global Services, Inc., Denver, PA on Gestation Day 2, 3, or 4. The animals were approximately 6 months old at receipt and weighed between 2956 and 4426 g on Gestation Day 0.

Justification for Test System and Number of Animals
The New Zealand White rabbit is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical data on the background incidence of fetal malformations and developmental variations in the New Zealand White rabbit. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The number of animals selected for this study was based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, August 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, January 2001, which recommend evaluation of approximately 20 females/group with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, 24 females/group was an appropriate number of animals to obtain a sample size of 20 females/group at termination.

Animal Identification
Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system).

Quarantine
After receipt at the Testing Facility, the New Zealand White rabbits were acclimated prior to the initiation of dosing.

Selection, Assignment, and Disposition of Animals
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Animals at extremes of body weight range were not assigned to groups.
The disposition of all animals was documented in the Study Records.

Husbandry
Housing
Animals were individually housed in stainless steel perforated floor cages suspended above appropriate bedding and equipped with an automatic watering valve.
Each cage was clearly labeled with a color-coded cage card indicating study, group, animal number, dosage 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. The animal facilities at Charles River Ashland are accredited by AAALAC International.
Environmental Conditions
Target temperatures of 61 °F to 71 °F (16 °C to 22 °C) with a relative target humidity of 30 % to 70 % were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms.
Food
PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322 was provided ad libitum throughout the study, except during the acclimation period when food was provided according to Testing Facility SOPs.
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
Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment or to aid in maintaining the animals’ oral or gastrointestinal health.
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.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 7 through 28. All animals were dosed at approximately the same time each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulation and Analysis
Preparation of Vehicle

The vehicle, deionized water, was dispensed daily for administration to Group 1 control animals and weekly for preparation of the test substance formulations. The vehicle was stirred continuously during dosing. Details of the dispensing of the vehicle have been retained in the Study Records.

Preparation of Test Substance
Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (target of 5 °C), protected from light, under nitrogen, until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

Sample Collection and Analysis
Dose formulation samples were collected for analysis.

Analytical Method
Analyses were performed by a high performance liquid chromatography method using ultraviolet absorbance detection using a validated analytical procedure (Engda E. Analytical Validation and Stability Extension Study of n-Butyl Pyrrolidone in Aqueous Formulations (Study No. 00387129). Charles River, Ashland, OH, 2019.)

Concentration Analysis
Duplicate sets of samples (1.0 mL) 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 (1.0 mL) 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 sampling location 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. Therefore, stability of test substance formulations was not assessed on this study.

Details on mating procedure:

N/A, animals were purchased time-mated

Duration of treatment / exposure:

21 days

Frequency of treatment:

Daily from Gestation 7 to 28

Duration of test:

21 days

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were dosed via oral gavage once daily during Gestation Days 7–28.
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, gross necropsy findings, intrauterine growth and survival, and fetal morphology.

Maternal 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, beginning on the day of receipt and lasting through euthanasia. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded approximately 1 hour.

Body Weights
Animals were weighed individually on Gestation Days 0 (by supplier), 4, and 7–29.
Gravid uterine weight was collected and corrected body weight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and corrected body weight change (the Gestation Day 0–29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Food Consumption
Food consumption was quantitatively measured on Gestation Days 4–29.

Ovaries and uterine content:

Necropsy
Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

Tissue Collection and Preservation
Gross lesions were collected and preserved in 10 % neutral buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.

Ovarian and Uterine Examinations
The uterus was weighed, and the ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. The placentae were also examined. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:

Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.). Representative photographs of all malformations, as appropriate, were included in the Study Records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus, or normal littermate, were also included in the Study Records as needed and as appropriate for comparison, when possible.
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis.

External
Each viable fetus was examined in detail, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

Visceral (Internal)
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination. The heads from all fetuses were examined by a mid coronal slice.
All carcasses were eviscerated, skinned, and fixed in 100% ethyl alcohol for subsequent examination of skeletons.

Skeletal
Each eviscerated fetus, following fixation in alcohol, was stained with Alizarin Red S and Alcian Blue. The skeletal examination was made following this procedure.

Statistics:

All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1 % and 5 % levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated by sex and occasion or by litter. Descriptive statistics number, mean, standard deviation, percentage, and/or incidence were reported whenever possible. Calculated values on the Provantis tables may not be reproducible from the individual values presented because all calculations are conducted using non-rounded values. Inferential statistics were performed according to the matrix below when possible, but excluded semi quantitative data, and any group with less than 3 observations. Data obtained from nongravid animals were excluded from statistical analysis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Dose-dependent, increased incidences of decreased fecal output were observed for 2 and 5 females (1 to 8 occasions each) in the 150 and 300 mg/kg/day groups, respectively, at the daily examinations sporadically during Gestation Days 12–29; a single occurrence of absent fecal output was also noted for 1 of the females in the 300 mg/kg/day group. The excreta-related observations noted in the 300 mg/kg/day group corresponded to the reduced mean food consumption noted in this group. No other test substance related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level. Abnormal breathing sounds were noted for 1, 5, 3, and 3 females in the control, 50, 150, and 300 mg/kg/day groups, respectively, at the daily examinations and/or postdosing observations sporadically during Gestation Days 10-29. Due to the absence of a dose response and the occurrence in the control group, this observation was attributed to the dosing procedures and not the test substance. Other observations noted in the test substance-treated groups, including fur staining on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
With the exception of Female Nos. 1502, 2523, and 3507 in the control, 50, and 150 mg/kg/day groups, respectively, all females were determined to be gravid.

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Description (incidence):

All females in the control, 50, 150, and 300 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 29, including Female No. 2524 in the 50 mg/kg/day group that delivered 1 fetus following euthanasia on Gestation Day 29.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant mean body weight loss was noted in the 300 mg/kg/day group during Gestation Days 7–10 (2.58 % loss) compared to the control group (0.97 % gain). Lower (not statistically significant) mean body weight gains were noted in this group during Gestation Days 10–13 (0.72 % gain compared to 1.54 % gain in the control group) and 13–20 (2.64 % gain compared to 3.78 % gain in the control group), with a statistically significantly higher body weight gain noted during Gestation Days 20–29 (5.69 % gain compared to 2.36 % gain in the control group). The higher mean body weight gain noted at the end of the dosing period was not of sufficient magnitude to negate the early decrements, thus resulting in a lower mean body weight gain (6.45 % gain compared to 8.91 % gain in the control group) when the entire treatment period (Gestation Days 7–29) was evaluated. Although these decrements were not of sufficient magnitude to result in statistically significantly lower mean absolute body weights during the treatment period (0.3 % to 5.6 % lower than the control group), a lower (45.4 %, not statistically significant) corrected body weight gain was noted in this group compared to the control group. Therefore, the body weight effects noted in the 300 mg/kg/day group were considered test substance-related and adverse.

Mean corrected body weight and gravid uterine weight in the 300 mg/kg/day group were generally comparable to the control group; no statistically significant differences were noted.

Lower (not statistically significant) mean body weight gains were noted in the 50 and 150 mg/kg/day groups following the initiation of dosing (Gestation Days 7–10), which were considered test substance-related but not adverse. Mean absolute body weights, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 150 mg/kg/day groups were generally comparable to the control group; no statistically significant differences were noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day, in the 300 mg/kg/day group was statistically significantly lower than the control group during Gestation Days 7–10 (48.82 %), 10 13 (47.45 %), and 13–20 (34.79 %), as well as when the entire treatment period (Gestation Days 7–29; 26.44 %) was evaluated. These decrements corresponded to the aforementioned body weight effects.
Mean maternal food consumption in the 50 and 150 mg/kg/day groups were unaffected by test substance administration and was generally comparable to the control group throughout the study. Any statistically significant differences from the control group were transient, had no corresponding effect on absolute body weight/body weight gain, and did not occur in a dose-related manner.

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:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on Gestation Day 29, no test substance-related internal findings were observed at dosage levels of 50, 150, and 300 mg/kg/day. Macroscopic findings observed 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 50, 150, and 300 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not part of this study design as there was not parturition.

External malformations:

no effects observed

Description (incidence and severity):

There were no test substance-related external malformations observed in fetuses in this study. External malformations were observed in 1 fetus each in the control and 150 mg/kg/day groups. Fetus No. 3503-05 in the 150 mg/kg/day group was noted with an inwardly rotated right hindlimb. This finding was not considered test substance-related because it occurred in a single fetus in the mid dosage group. Fetus No. 1508-09 in the control group was noted with cleft lower mandible (medial), cleft upper lip (medial), short snout, cleft palate (entire length), and split tongue.
No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related skeletal malformations observed in fetuses in this study. Skeletal malformations were observed in 1(1), 0(0), 3(2), and 1(1) fetuses (litters) in the control, 50, 150, and 300 mg/kg/day groups, respectively.
Fetus No. 4503-01 in the 300 mg/kg/day group had 8 lumbar vertebrae, fused lumbar centra, and a lumbar hemivertebra. In the 150 mg/kg/day group, Fetus No. 3506-03 was noted with 8 lumbar vertebrae, Fetus No. 3523-04 was noted with 6 lumbar vertebrae, and Fetus No. 3523 06 was noted with branched ribs, 8 cervical vertebrae, 11 thoracic vertebrae, and a thoracic hemivertebra. These findings were not considered test substance-related due to the absence of a dose response and the mean litter proportions of the individual findings were not statistically significantly different from the control group. In the control group, Fetus No. 1508 09 was noted with misshapen right mandible and premaxilla bones and split palatine bones, which correlated to the cleft malformations or short snout observed externally; fused ventral arch atlas, cervical centra, and thoracic centra were also noted for this fetus.
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance treated groups were noted infrequently, similarly in the control group, and/or were not observed in a dose-related manner.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no test substance-related visceral malformations observed in fetuses in this study. Visceral malformations were observed in 3(3), 0(0), 1(1), and 2(2) fetuses (litters) in the control, 50, 150, and 300 mg/kg/day groups, respectively.
In the 300 mg/kg/day group, Fetus No. 4506-01 had an absent right thyroid gland and Fetus No. 4508-03 was noted with a retroesophageal right subclavian artery. Fetus No. 3518-08 in the 150 mg/kg/day group was also noted with a retroesophageal right subclavian artery. These findings were not considered test substance-related due to the absence of a dose response and the finding was noted similarly in the control group (Fetus No. 1519-04). Fetus No. 1508 09 in the control group was noted with a diaphragmatic hernia; this fetus also had multiple external malformations. Also, in the control group, Fetus No. 1521 05 was noted with a caudally malpositioned right kidney.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance treated groups were noted infrequently, similarly in the control group, and/or were not observed in a dose-related manner.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 210(23), 194(23), 211(23), and 216(24) in the control, 50, 150, and 300 mg/kg/day groups, respectively. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Remarks on result:

other:

Remarks:

No adverse effects found

Abnormalities:

no effects observed

Developmental effects observed:

no

Summary data tables are attached in the "background material" section as a single .PDF.

Conclusions:

Based on adverse maternal effects, including body weight loss, reduced food consumption, and corresponding excreta-related clinical observations at 300 mg/kg/day, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when N-Butylpyrrolidone was administered orally by gavage to time-mated New Zealand White rabbits. Intrauterine growth and survival and fetal morphology were unaffected by test substance exposure at all dosage levels. Therefore, 300 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity.

Executive summary:

The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

All females survived to the scheduled necropsy on Gestion Day 29, including 1 female in the 50 mg/kg/day group that delivered on Gestation Day 29. Test substance-related, dose-dependent incidences of decreased fecal output were observed in the 150 and 300 mg/kg/day groups at the daily examinations; this observation corresponded to the reduced mean food consumption noted in the 300 mg/kg/day group. No other test substance-related clinical observations were noted at any dosage level at the daily examinations or approximately 1-hour following dose administration.

Test substance-related mean body weight losses following the initiation of treatment (Gestation Days 7–10) and lower mean body weight gains during Gestation Days 10-13 and 13-20 were noted in the 300 mg/kg/day group, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated. Mean food consumption in this group was also lower than the control group throughout the treatment period. Although the decrements in this group were not of sufficient magnitude to result in noteworthy changes in mean absolute body weights throughout the treatment period, a lower corrected body weight gain was noted in this group compared to the control group; therefore, the body weight effects noted in the 300 mg/kg/day group were considered adverse. Mean corrected body weights and gravid uterine weights in this group were comparable to the control group. In the 50 and 150 mg/kg/day groups, test substance-related lower mean body weight gains, compared to the control group, were also noted following the initiation of treatment (Gestation Days 7–10). Mean body weight gains in these groups were comparable to the control group for the remainder of the treatment period, as well as when the overall treatment period was evaluated. In addition, mean absolute body weights, food consumption, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 150 mg/kg/day groups were comparable to the control group. Therefore, the initial decrements in these groups were considered nonadverse.

No test substance-related internal findings were observed at any dosage level at the scheduled necropsy on Gestation Day 29.

Intrauterine growth and survival were unaffected by test substance administration at all dosage levels. There were also no test substance-related effects on fetal morphology in the 50, 150, and 300 mg/kg/day groups.

In conclusion, based on adverse maternal effects, including body weight loss, reduced food consumption, and corresponding excreta-related clinical observations at 300 mg/kg/day, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when N-Butylpyrrolidone was administered orally by gavage to time-mated New Zealand White rabbits. Intrauterine growth and survival and fetal morphology were unaffected by test substance exposure at all dosage levels. Therefore, 300 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Species:

rat

Quality of whole database:

The key study is GLP compliant and is of high quality (Klimish score = 1). The quality of the database is therefore high.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

600 mg/m³

Species:

rat

Quality of whole database:

The key study is GLP compliant and is of high quality (Klimish score = 1). The quality of the database is therefore high.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Species:

rat

Quality of whole database:

The key study is GLP compliant and is of high quality (Klimish score = 1). The quality of the database is therefore high.

Additional information

There are four reliable developmental toxicity studies available for inhalation ( 1 in rats), oral (1 in rats, 1 in rabbits)and dermal routes of exposure (1 in rats).

 

In an inhalation developmental toxicity study (Charles River Laboratories, 2016c; Report No. WIL – 387081), the test substance was administered via whole-body inhalation exposure to 3 groups of 24 bred female Crl:CD(SD) rats for 6 hours per day from Gestation Days 6–19. Based on a range-finding study in nonpregnant female rats, target exposure concentrations for the current study were 0.3, 0.6, and 1.2 mg/L; the highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). Maternal toxicity was evidenced by adverse clinical observations and mean body weight losses and lower mean body weight gains with corresponding lower mean food consumption in the 1.2 mg/L group throughout the gestation treatment period. Developmental effects were noted at 1.2 mg/L as evidenced by lower mean fetal weights, which correlated with a lower mean gravid uterine weight and decrements in maternal food consumption and body weight gain during the fetal period (Gestation Days 15-20). Lower mean fetal weights were also noted in the 0.3 and 0.6 mg/L groups; however, mean fetal weight values in these groups were greater than the minimum mean values and only slightly lower than the mean and median values in the test facility historical control data. In addition, the concurrent control group values for fetal body weight were 2% to 3% heavier (approximately equal to the 75th quartile) than the mean values in the test facility historical control data. Therefore, the lower mean fetal body weights at 0.3 and 0.6 mg/L were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups, and were not considered test substance-related. Test substance-related fetal skeletal developmental variations (higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and lower mean litter proportion of cervical centrum no. 1 ossified) were noted in the 1.2 mg/L group, which corresponded with the lower mean fetal weights noted in this group. No adverse maternal or developmental effects were noted at 0.3 and 0.6 mg/L. Based on these results, an exposure level of 0.6 mg/L was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance was administered via whole-body inhalation exposure to bred Crl:CD(SD) rats. Test substance exposure concentrations of 0.3, 0.6, and 1.2 mg/L corresponded to mean estimated inhaled (delivered) dosage levels of 76.2, 152.6, and 315.8 mg/kg/day, respectively.

 

In an oral developmental toxicity study (OECD 414; Charles River Laboratories, 2016a; Report No. WIL - 387076), the test substance was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 200, 300, 400, and 500 mg/kg/day. Maternal toxicity was evidenced at 500 mg/kg/day by higher incidences of rales and lower mean body weight gains, body weights, and food consumption during the treatment period. Although test substance-related effects on body weight gains and food consumption were noted at 300 and 400 mg/kg/day and rales were noted at 300 and 400 mg/kg/day, these effects were transient and not considered adverse. Developmental toxicity was noted at 500 mg/kg/day group as evidenced by lower mean fetal body weights. No evidence of developmental toxicity was observed at 200, 300, or 400 mg/kg/day. Test substance-related lower (up to 10%) mean fetal body weights were observed at 500 mg/kg/day. Slight decrements (5%) in mean fetal body weights in the 300 and 400 mg/kg/day groups were considered due to the lower number of fetuses per litter and increased fetal body weights in the concurrent control group. Mean fetal body weights of the 300 and 400 mg/kg/day groups were equal to the mean values in the WIL Research historical control. Therefore, intrauterine growth and survival at 400 mg/kg/day and below were considered unaffected by test substance administration. No test substance fetal malformations or developmental variations were noted at 200, 300, 400, and 500 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when N-butyl pyrrolidone was administered orally by gavage to bred Crl:CD(SD) rats.

 

In an oral developmental toxicity study (OECD 414, CHarles River Laboratories; 2020; Report No. WIL-), the test substance was administered orally by gavage to rabbits to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. All females survived to the scheduled necropsy on Gestion Day 29. Test substance-related, dose-dependent incidences of decreased fecal output were observed in the 150 and 300 mg/kg/day groups at the daily examinations; this observation corresponded to the reduced mean food consumption noted in the 300 mg/kg/day group. No other test substance-related clinical observations were noted at any dosage level at the daily examinations or approximately 1-hour following dose administration.

Test substance-related mean body weight losses following the initiation of treatment (Gestation Days 7–10) and lower mean body weight gains during Gestation Days 10-13 and 13-20 were noted in the 300 mg/kg/day group, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated. Mean food consumption in this group was also lower than the control group throughout the treatment period. Although the decrements in this group were not of sufficient magnitude to result in noteworthy changes in mean absolute body weights throughout the treatment period, a lower corrected body weight gain was noted in this group compared to the control group; therefore, the body weight effects noted in the 300 mg/kg/day group were considered adverse. Mean corrected body weights and gravid uterine weights in this group were comparable to the control group. In the 50 and 150 mg/kg/day groups, test substance-related lower mean body weight gains, compared to the control group, were also noted following the initiation of treatment (Gestation Days 7–10). Mean body weight gains in these groups were comparable to the control group for the remainder of the treatment period, as well as when the overall treatment period was evaluated. In addition, mean absolute body weights, food consumption, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 150 mg/kg/day groups were comparable to the control group. Therefore, the initial decrements in these groups were considered nonadverse.

No test substance-related internal findings were observed at any dosage level at the scheduled necropsy on Gestation Day 29.

Intrauterine growth and survival were unaffected by test substance administration at all dosage levels. There were also no test substance-related effects on fetal morphology in the 50, 150, and 300 mg/kg/day groups.

In conclusion, based on adverse maternal effects, including body weight loss, reduced food consumption, and corresponding excreta-related clinical observations at 300 mg/kg/day, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity when N-Butylpyrrolidone was administered orally by gavage to time-mated New Zealand White rabbits. Intrauterine growth and survival and fetal morphology were unaffected by test substance exposure at all dosage levels. Therefore, 300 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity.

 

In a dermal developmental toxicity study (OECD 414; Charles River Laboratories, 2016b; Report No. WIL - 387079), the test substance was administered by dermal application to 3 groups of bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Animals were exposed to the test substance for approximately 23 hours each day. Exposure levels were 375, 500, and 750 mg/kg/day. All females in the control, 375, 500, and 750 mg/kg/day groups survived to the scheduled necropsy. A slightly increased incidence of red material around the eyes was noted in all test substance-treated groups generally throughout the treatment period at the daily examinations; however, given the low occurrence of this finding (generally 1-4 occurrences per animal) and the inability of the animals to groom while outfitted with the Elizabethan collars, this finding was considered nonadverse. No other test substance-related clinical findings were noted at the daily examinations or approximately 1 hour following dose administration in the 375, 500, and 750 mg/kg/day groups. No dermal findings were noted at any dosage level. Adverse mean body weight losses and lower mean body weight gains with corresponding reduced mean food consumption were noted in the 750 mg/kg/day group, with the greatest magnitude of change occurring during the fetal period, which resulted in lower mean body weights, net body weight, net body weight gain, and gravid uterine weight in this group. The decrements in body weight and food consumption parameters at 750 mg/kg/day noted during the period of greatest fetal growth (gestation days 15-20) resulted in lower mean fetal weights in this group. Intrauterine growth at 375 and 500 mg/kg/day and survival at 375, 500, and 750 mg/kg/day were unaffected by test substance administration. There were no test substance-related external, visceral, and skeletal malformations or developmental variations noted at any dosage level. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when N-butyl pyrrolidone was administered by dermal application to bred Crl:CD(SD) rats.

 

In a supporting oral developmental toxicity study the doses were determined in a range-finding study (LPT, 2013d; Report No. 29177). Dose levels of 5, 50 or 500 mg/kg bw/day had been administered to female rats, by gavage from the 6th to 19th day of pregnancy. No test item-related influences were noted at the low and intermediate dose level (5 and 50 mg/kg bw/day) concerning the behavioural toxicity, body weight, food and water consumption, uterus and carcass weights, the examination at autopsy and the parameters of reproduction (number of corpora lutea, implantation sites, resorptions, percentage of pre- and post implantation loss). At the high dose level (500 mg/kg b.w./day) piloerection was noted in 2 of 3 dams and laboured breathing in one dam. Starting of treatment resulted in a distinct reduction in food consumption and a decline in body weight. A total loss of implantation sites was noted in 1 of the 3 high dose dams, whereas the other 2 showed no influence on the parameters of reproduction. The foetuses from the dams of the low and intermediate dose group (5 and 50 mg/kg bw/day) showed no reduction in body weight and no malformations or variations were found during the external examination. In the high dose group (500 mg /kg bw/day), the foetuses from the 2 dams showed a slightly reduced body weight. Again, no malformations or variations were found during the external examination. Therefore, the dose levels for the main study were 5, 50 and 500 mg/kg bw.

 

In the supporting main study (OECD 414; LPT, 2013c; Report No. 29197), the test substance was administered to female CD® / Crl:CD (SD) rats also at dose levels of 5, 50 or 500 mg/kg bw/day (administration volume: 5 mL/kg b.w./day), orally by gavage from the 6th to 19th day of pregnancy. For dams, the NOAEL was determined as 50 mg/kg b.w./day. At the high dose group (500 mg/kg bw/day), the following observations were made: a slight reduction in body weight and a more distinct reduction in the net and absolute body weight change were noted during the treatment period. After the start of treatment a reduction in food consumption was noted in the high dose group for 2 days. Thereafter, normal level of food intake was reached. In addition, slight reductions of the gravid uterus weight and the carcass weight were noted. No test substance-related influence was noted on the reproductive parameters of the dams. For foetal organisms, the NOAEL was set to > 500 mg/kg b.w./day. The external / internal, skeletal and soft tissue examinations revealed no malformations. No test substance-related increase was noted in the incidence of external / internal, skeletal and soft tissue variations or the skeletal retardations. No dead foetuses and no test substance-related differences in the placental and foetal weights, the number of corpora lutea, implantation sites, resorptions and in the number of runts were noted in the treatment groups. The percentages of pre- and post-implantation loss were also not influenced by the test item.

Even though the study itself is considered reliable and valid, the results of the study are considered not to allow a meaningful interpretation of the results due to the selection of dose levels based upon differences of a factor of ten (log order). The dose range finder had already shown only minimal changes at 500 mg/kg/day in both the fetus and the dams, which suggested that the NOAEL would be around 500 mg/kg/day. Therefore, a different dose level spacing would have been indicated. A further reason to consider the results to be inappropriate for interpretation, is the absence of external, internal or visceral malformations in any of the rats, while the strain of Sprague Dawley rats has a background malformation rate of around 0.5 - 0.8 %. Finally, although there was a decrease in placental weights in male, female and combined conceptuses and a decrease in female fetal body weight; the authors interpreted these changes as not related to treatment (non-adverse) based upon comparison to historical control values. The authors argument is that although the 11.3% reduction in placental weight (p ≤ 0.01) and 8.8% reduction in female fetal weight (p ≤ 0.01) in the 500 mg/kg bw/day group were present, the values were within the historical control (HC) range for the laboratory. Problems with this argument include 1) the concurrent control group is the appropriate value for analysis and unless a problem can be demonstrated with the concurrent control group value, the comparison to HC values should be of minor consideration and 2) where within the HC range of values the treated group falls should be considered. In this instance, the concurrent control values pretty much fall almost exactly mid-range of the HC values, indicating that the concurrent control is producing values that are normal for this strain or rat in this laboratory. In addition, the value for the female fetal body weight (3.1 grams) is at the extreme low end of the HC values. The same can be considered for the male fetal body weights and the combined fetal body weights for the 500 mg/kg bw/day group, although these values were not statistically significantly different from the concurrent control. All of these factors appear to discredit the reasoning for considering these findings as “not related to treatment”. Therefore, a NOAEL of 50 mg/kg bw has to be established for general toxicity because of reduced body weight and body weight gains of the dams and reduced food consumption and gravid uterine weights in the highest dose group.

In conclusion, even though, the data itself is valid, the poor choice of doses made a meaningful interpretation impossible.

The results of the dose range-finding study (OECD 414; LPT, 2013d; Report No. 29177) are also considered not to allow a meaningful interpretation due to the same reasons. The low value of 50 mg/kg bw are overestimating greatly the actual NOAEL, which would have been identified using a more appropriate dose spacing regime.

Justification for classification or non-classification

The fertility and developmental parameters were not affected by test substance administration at all dose levels tested in the OECD 414 developmental toxicity studies in rats and rabbits. NOAEL for maternal toxicity was established at the same dose levels of general systemic toxicity and developmental toxicity. Therefore, reproductive toxicity observed at the highest dose levels is considered to represent a secondary non-specific consequence of other toxic effects of the test substance administration. Additionally, in the 90-day study, no adverse effects were detected during the oestrous cycle assessment and semen parameters assessment. There were no abnormalities detected in reproductive organs, their weights and histopathology. Based on the results of the 90-day study, the highest dose level of 500 mg/kg bw may be regarded as NOAEL for reproductive function. Based on these data, N-Butylpyrrolidone does not need to be classified and labelled as reproductive or developmental toxicant according to Regulation (EC) 1272/2008.