2-pyrrolidone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-05 - 2019-02-22 (experimental phase: 2018-06-18 - 2018-08-20)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Remarks:

Dose range-finding study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-04-24 - 2018-11-20 (experimental phase: 2018-04-26 - 2018-05-18)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

A GLP dose-range finding study for OECD 414

Reason / purpose for cross-reference:

reference to other study

Remarks:

main study

Reason / purpose for cross-reference:

reference to other study

Remarks:

tolerability study

Principles of method if other than guideline:

- Principle of test: The objective of this study was to determine dose levels of the test substance to be evaluated in a definitive prenatal developmental toxicity study in rabbits.
- Short description of test conditions: Animals were dosed via oral gavage once daily during Gestation Days 7 – 28 (concentrations: 0, 100, 300, 700, 1000 mg/kg/day)
- Parameters analysed / observed: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, clinical pathology parameters (hematology and serum chemistry), intrauterine growth and survival, gross necropsy findings, organ weights, and external fetal examinations.

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: Kept in a controlled temperature area set to maintain 18 °C to 24 °C, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: stable in dispersion

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA, USA
- Age at study initiation: ~ 6 months on Gestation Day 0
- Weight at study initiation: between 2918 and 3587 g on Gestation Day 0
- Fasting period before study: no
- 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 are accredited by AAALAC International.
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.
- Diet (e.g. ad libitum): 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 (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis 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.
- Acclimation period: After receipt at the Testing Facility (2018-04-20), the New Zealand White rabbits were acclimated prior to the initiation of dosing (2018-04-26).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures of 61 °F to 71°F (16 °C to 22 °C) were maintained.
- Humidity (%): relative target humidity of 30 % to 70 %
- Air changes (per hr): Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained.

IN-LIFE DATES: From: 2018-04-20 To: 2018-05-18 (completion of in-life)

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of Vehicle: The vehicle, deionized water, was dispensed approximately weekly for administration to Group 1 control animals and an adequate amount was dispensed into daily aliquots, which were stored at room temperature (18 °C to 24 °C), protected from light until use.
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, divided into aliquots for daily dispensation and stored at room temperature (18 °C to 24 °C), protected from light, until use.

VEHICLE
- Concentration in vehicle: 0, 20, 60, 140, 200 mg/mL test item
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis
Dose formulation samples were collected for analysis as indicated below:
Table
Dose Formulation Sample Collection Schedule
Interval Concentration Homogeneity Stability
25 Apr 2018 All groups N/A N/A
10 May 2018 All groups N/A N/A
N/A = not applicable.
Samples to be analyzed were transferred to the Analytical Chemistry Department.

Analytical Method
Analyses were performed by high performance liquid chromatography method using ultraviolet absorbance detection and a validated analytical procedure.

Concentration Analysis
Duplicate sets of samples (1.0 mL) for each 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 concentration results were within or equal to ± 10 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Solubility at 85 mg/mL and homogeneity of test substance formulations over the range of concentrations used on this study was established in a separate analytical validation study. Therefore, homogeneity analysis was not conducted on this study.

Stability Analysis
Stability of test substance formulations over the range of concentrations used on this study for 11 days at room temperature (18 °C to 24 °C) was established in a separate analytical validation study. Therefore, stability of test substance formulations was not assessed on this study.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
Time-mated female New Zealand White rabbits were received on Gestation Day 1

Duration of treatment / exposure:

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

Frequency of treatment:

daily

Duration of test:

Gestation day 1 to gestation day 29

Dose / conc.:

0 mg/kg bw/day

Remarks:

control

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

700 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

6 females / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dosage levels were determined from results of a previous tolerability study and were provided by the Sponsor Representative. In that study, nonpregnant rabbits were administered 2- pyrrolidone at dose levels of 100, 300, 700, and 1000 mg/kg/day for 7 days (2). All rabbits survived until scheduled euthanasia. Body weight losses of 6.1 and 9.1 % with correspondingly reduced food consumption (19.6 and 25.9 g/day) and excreta-related findings were noted at 1000 mg/kg/day. At the 700 mg/kg/day dosage level, body weight losses of 3.7 and 7.1 % with lower food consumption (46.1 and 70.7 g/day) was noted during the treatment period. No remarkable effects were noted at 100 and 300 mg/kg/day. Based on these findings, dosage levels of 100, 300, 700, and 1000 mg/kg/day were selected for the current study.
- Rationale for animal assignment (if not random): 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.
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.
- Other:
The route of administration was oral (gavage) as this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
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 an appropriate number to obtain 4 to 5 gravid females/group at termination on this dose range-finding study given the possibility of nongravid animals, unexpected deaths, or treatment-related mortality and/or mortality. This number of animals allowed for meaningful interpretation of the data to adequately assess maternal and developmental toxicological endpoints and the selection of dosage levels for the definitive developmental toxicity study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 2 hours postdose.

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations: Animals were weighed individually on Gestation Days 0 (by supplier), 4, and 7 – 29.
Gravid uterine weight was collected and net body weight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and net 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 AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was quantitatively measured on Gestation Days 4–29.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 29
- Organs examined:
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.
Organ Weights: Kidney, liver, spleen were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to net body weight ratio was calculated.

OTHER:
- Hematology (blood samples via a marginal ear vein):
Differential leukocyte count (percent and absolute): Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Erythrocyte count (RBC), Hemoglobin (HGB), Hemoglobin distribution width (HDW), Hematocrit (HCT), Mean corpuscular hemoglobin (MCH), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (Platelet), Red cell distribution width (RDW), Reticulocyte count, Percent (RETIC), Absolute (RETIC Absolute), Total leukocyte count (WBC), Platelet estimatea, Red cell morphology (RBC Morphology)

- Serum chemistry (blood samples via a marginal ear vein): Alanine aminotransferase (ALT), Albumin, Albumin/globulin ratio (A/G Ratio) [by calculation], Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Calcium, Chloride, Creatinine, Gamma glutamyltransferase (GGT), Globulin [by calculation], Glucose, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase (SDH), Total bilirubin (Total BILI), Total cholesterol (Cholesterol), Total protein, Triglycerides (Triglyceride), Urea nitrogen, Appearance

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
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
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:

Each viable fetus was examined externally, euthanized by an intraperitoneal injection of sodium pentobarbital and discarded. The crownrump 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. The findings were recorded as either developmental variations or malformations.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations 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. Data obtained from nongravid animals were excluded from statistical analyses. 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 by sex. Maternal body weights and body weight changes (absolute and net), food consumption, clinical pathology data, gravid uterine weights, organ weights, numbers of corpora lutea, implantation sites, and viable fetuses were subjected to a parametric one-way ANOVA5 to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test6 was used to compare the test substance-treated groups to the control group. Mean litter proportions of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, and pre- and postimplantation loss) were subjected to the Kruskal-Wallis nonparametric ANOVA test7 to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test8 was used to compare the test substance-treated groups to the control group.

Historical control data:

Historical data on the background incidence of fetal malformations and developmental variations in the New Zealand White rabbit are available

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

An increased incidence of decreased defecation was noted in the 1000 mg/kg/day group at the daily examinations during Gestation Days 11–18 compared to the control group, which corresponded with lower mean food consumption noted in this group. This finding was noted on 3–7 occasions in all animals in this group. Other clinical observations noted at the daily examinations or approximately 2 hours following dose administration, including hair loss or brown material on various body surfaces, small or mucoid feces, decreased urination, and rales, 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

Description (incidence):

All females in the control, 100, 300, 700, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 29.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean body weight losses in the 1000 mg/kg/day group during the Gestation Days 7 - 10 and 10 – 13 intervals (statistically significant to control group)
- Mean body weight gains in this group were comparable to or higher than the control group during Gestation Days 13 – 20 and 20 – 29 (statistically significant differences for the Gestation Day 13 – 20 interval and Gestation Day 26 – 27)
- The initial mean body weight losses in the 1000 mg/kg/day group were of sufficient magnitude to result in a lower mean body weight gain during the entire treatment period (Gestation Days 7 - 29; not statistically significant) and mean absolute body weights that were up to 10.6 % lower compared to the control group during Gestation Days 9 – 26.
- Mean absolute body weight in the 1000 mg/kg/day group was generally comparable to the control group (3.5 % lower; not statistically significant) on Gestation Day 29, suggesting amelioration of the effects.
- Mean body weight gains in the 100, 300, and 700 mg/kg/day groups were generally comparable to the control group throughout the treatment period; the only statistically significant differences were higher mean body weight gains on Gestation Day 20 – 21 (300 and 700 mg/kg/day) and 26 - 27 (100 mg/kg/day).
- Mean body weight gains in these groups were comparable to the control group (when the entire treatment period (Gestation Days 7 – 29) was evaluated)
- Greater mean net body weight losses were noted at 300, 700, and 1000 mg/kg/day compared to the control group (however, not in a non-dose-related manner)
- Mean net body weight change at 100 mg/kg/day and mean net body weights and gravid uterine weights at 100, 300, 700, and 1000 mg/kg/day were compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Statistically significant differences were observed in mean maternal food consumption (evaluated as g/animal/day and g/kg/day): in the 700 and 1000 mg/kg/day groups was lower than the control group beginning at the start of dosing on Gestation Day 7 until Gestation Day 18 (700 mg/kg/day) or 16 (1000 mg/kg/day) and consequently for the Gestation Days 7 – 10, 10 - 13, and/or 13 – 20 cumulative intervals and when the entire treatment period (Gestation Days 7 – 29) was evaluated.
- The lower mean food consumption in the 1000 mg/kg/day group corresponded to body weight losses and decreased defecation in the 1000 mg/kg/day group The decrements in food consumption in the 700 mg/kg/day group were not of sufficient magnitude to result in lower mean body weights during the study.
- Mean food consumption at 700 and 1000 mg/kg/day was compar ble to the control group during Gestation Days 20 – 29.
- Mean maternal food consumption in the 100 and 300 mg/kg/day groups was generally comparable to that in the control group throughout the treatment period. Differences from the control group were slight and not statistically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No effects on hematology were noted at any dosage level. Differences from the control group were not statistically significant and considered to be the result of normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly lower mean creatinine concentration was noted in the 1000 mg/kg/day group. Other differences from the control group were not statistically significant and considered to be 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, treatment-related

Description (incidence and severity):

Mean absolute and relative (to net body weight) liver weight in the 1000 mg/kg/day group were higher (20.8 % for absolute) than the control group; the difference for mean liver weight relative to net body weight was statistically significant. Mean liver weights in the 100, 300, and 700 mg/kg/day groups and mean kidney and spleen weights in the 100, 300, 700, and 1000 mg/kg/day groups were generally comparable to the control group; no statistically significant differences were noted.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the scheduled necropsy on Gestation Day 29, no remarkable internal findings were observed at dosage levels of 100, 300, 700, and 1000 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. One female in the 100 mg/kg/day group was determined to be nongravid.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 100, 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss and mean number and percentage of viable fetuses. Differences from the control group were slight and not statistically significant.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 100, 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss and mean number and percentage of viable fetuses. 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.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 100, 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss and mean number and percentage of viable fetuses. Differences from the control group were slight and not statistically significant.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 100, 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss and mean number and percentage of viable fetuses. Differences from the control group were slight and not statistically significant.

Dead fetuses:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 100, 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss and mean number and percentage of viable fetuses. Differences from the control group were slight and not statistically significant.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 48(6), 39(5), 52(6), 45(6), and 46(6) in the control, 100, 300, 700, and 1000 mg/kg/day groups, respectively. No external malformations or developmental variations were noted at any dosage level.

Changes in sex ratio:

not examined

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 100, 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss and mean number and percentage of viable fetuses. 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:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 100, 300, 700, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss and mean number and percentage of viable fetuses. 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.

External malformations:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 48(6), 39(5), 52(6), 45(6), and 46(6) in the control, 100, 300, 700, and 1000 mg/kg/day groups, respectively. No external malformations or developmental variations were noted at any dosage level.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not specified

Abnormalities:

no effects observed

Conclusions:

Based on the effects on mean body weights and food consumption at 1000 mg/kg/day and no effects on embryo/fetal survival or morphology at dosage levels up to 1000 mg/kg/day, dosage levels of 250, 500, and 1000 mg/kg/day were selected for a prenatal developmental toxicity study of 2-pyrrolidone administered orally by gavage to time-mated New Zealand White rabbits.

Executive summary:

The objective of this study was to determine dose levels of the test substance to be evaluated in a definitive prenatal developmental toxicity study in rabbits. The study design was as follows:

Group Number	Treatment	Dosage Level^a (mg/kg/day)	Concentration (mg/mL)	Dose Volume (mL/kg)	Number of Females
1	Vehicle	0	0	5	6
2	2-Pyrrolidone	100	20	5	6
3	2-Pyrrolidone	300	60	5	6
4	2-Pyrrolidone	700	140	5	6
5	2-Pyrrolidone	1000	200	5	6

a = No correction factor was used.

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, clinical pathology parameters (hematology and serum chemistry), intrauterine growth and survival, gross necropsy findings, organ weights, and external fetal examinations. All females survived to the scheduled necropsy on Gestation Day 29. A higher incidence of decreased defecation was noted in the 1000 mg/kg/day group at the daily examinations, which corresponded with lower mean food consumption noted in this group; no other noteworthy clinical observations were noted. Mean body weight losses during Gestation Days 7 – 13 and lower mean food consumption from Gestation Day 7 – 16 were noted in the 1000 mg/kg/day group when compared to the control group. As a result, mean body weights in this group were up to 10.6% lower than the control group during Gestation Days 9 – 26. Mean absolute body weight in the 1000 mg/kg/day group was comparable to the control group (3.5 % lower) on Gestation Day 29, suggesting amelioration of the effects. Mean food consumption in the 700 mg/kg/day group was lower than the control group during Gestation Days 7 - 18. Mean food consumption in the 100 and 300 mg/kg/day groups, mean body weights and body weight gains in the 100, 300, and 700 mg/kg/day groups, and mean net body weights, net body weight changes, and gravid uterine weights in the 100, 300, 700, and 1000 mg/kg/day groups were generally comparable to the control group. Hematology parameters in the test substance-treated groups were generally comparable to the control group. A lower mean creatinine concentration was noted in the 1000 mg/kg/day; there were no other noteworthy changes in serum chemistry parameters at any dosage level. Maternal macroscopic findings in the test substance-treated groups occurred infrequently, in a manner that was not dose-related, and/or similarly in the control group. Mean absolute and relative (to net body weight) liver weights in the 1000 mg/kg/day group were higher than the control group. Mean liver weights in the 100, 300, and 700 mg/kg/day groups and mean kidney and spleen weights in the 100, 300, 700, and 1000 mg/kg/day groups were generally comparable to the control group. Intrauterine survival in the 100, 300, 700, and 1000 mg/kg/day groups were comparable to the control group. There were no external malformations or developmental variations noted in fetuses at any dosage level. Based on the effects on mean body weights and food consumption at 1000 mg/kg/day and no effects on embryo/fetal survival or morphology at dosage levels up to 1000 mg/kg/day, dosage levels of 250, 500, and 1000 mg/kg/day were selected for a prenatal developmental toxicity study of 2 pyrrolidone administered orally by gavage to time-mated New Zealand White rabbits.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Remarks:

tolerability study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-03-01 - 2018-06-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Tolerability study for DRF for OECD 414

Remarks:

The design of this study was based on the study objective(s) and the overall product development strategy for the test substance.

Reason / purpose for cross-reference:

reference to other study

Remarks:

Dose-range finding study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Main study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The objective of this study was to determine the tolerability of the test substance in nonpregnant female rabbits following oral administration for up to 7 days. The results of this study allowed dose selection for a subsequent oral range-finding embryo/fetal development toxicity study in rabbits.
- Short description of test conditions: Animals were dosed via oral gavage once daily during Study Days 0–6 (concentrations: 100, 300, 700, 1000 mg/kg /day).
- Parameters analysed / observed: clinical signs, body weights, body weight gains, and food consumption.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area set to maintain 18°C to 24°C, protected from light

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under storage conditions: The test substance is considered to be stable under the storage conditions provided by the Sponsor.
- Stability under test conditions and solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Analyses to determine homogeneity, stability, and concentration of test substance formulations were not conducted on this non-GLP study.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Species:

rabbit

Strain:

New Zealand White

Remarks:

(Hra:[NZW]SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA, USA
- Age at study initiation: ~7 months
- Weight at study initiation: 3056 and 3380 g (initiation of dosing)
- Fasting period before study: no
- Housing: Animals were individually housed in stainless steel perforated floor cages suspended above appropriate bedding equipped with an automatic watering valve. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal number, 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.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322 was provided ad libitum (throughout the study)
- Water (e.g. ad libitum):Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system
- Acclimation period:After receipt at the Testing Facility, the New Zealand White rabbits were acclimated prior to the initiation of dosing (not specified)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 22
- Humidity (%): 30 to 70
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light):12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 3 days prior to Study To: Study day 7

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The vehicle, deionized water, was dispensed daily for preparation of the test substance formulations.
- Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and stored at room temperature (18 °C to 24 °C), protected from light, until use.

VEHICLE
- Concentration in vehicle: 20, 60, 140, 200 mg test item /mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Analyses to determine homogeneity, stability, and concentration of test substance formulations were not conducted on this non-GLP study.

Details on mating procedure:

-use of non pregants rabbits

Duration of treatment / exposure:

From Study Day 0 through 6, inclusively

Frequency of treatment:

The test substance was administered as a single daily oral gavage dose. All animals were dosed at approximately the same time each day.

Duration of test:

From 3 days prior to Study to Study Day 7 (scheduled euthanasia, the day following the last day of dosing)

Dose / conc.:

100 mg/kg bw/day

Remarks:

No correction factor was used.

Dose / conc.:

300 mg/kg bw/day

Remarks:

No correction factor was used.

Dose / conc.:

700 mg/kg bw/day

Remarks:

No correction factor was used.

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

No correction factor was used.

No. of animals per sex per dose:

2 females / dose

Control animals:

other: Comparison to pretreatment values or to examinations on Study day 0

Details on study design:

- Dose selection rationale:
The dosage levels were provided by the Sponsor Representative and are considered standard doses for tolerability studies. A high dose of 1000 mg/kg/day was selected to be the limit dose because there is limited or no previous data for this test substance in rabbits. The subsequent doses were selected to provide a potential dose response, and aid in the selection of dosage levels for a subsequent range-finding study in rabbits.
- Rationale for animal assignment (if not random): 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.
- Other: Rationale for route: The route of administration was oral (gavage) because this is the potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Maternal examinations:

VIABILITY: Yes
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were removed from the cage, and a detailed clinical observation was performed once daily. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded approximately 2 hours postdose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually daily from 3 days prior to Study Day 0 (the first day of dose administration) until euthanasia.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was quantitatively measured daily from 3 days prior to Study Day 0 (the first day of dose administration) until euthanasia.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Due to the limited group sizes (N = 2), individual animal data were presented; no comparative statistical analysis was conducted.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg/day group, Female No. 4624 had findings of soft feces, decreased defecation and brown material on the anogenital area, urogenital area, and hindlimbs at the daily examinations as early as Study Day 3. Female No. 4620 in the 1000 mg/kg/day group had findings of rales on Study Days 6 and 7 and decreased defecation on Study Days 5 and 6.In the 700 mg/kg/day group, Female No. 4621 had findings of brown material on the anogenital area on Study Days 1, 3, and 6 and Female No. 4616 had decreased defecation on Study Day 6. No other remarkable clinical observations were noted for any animals at the daily examinations. No clinical findings were noted approximately 2 hours following dose administration for any of the females.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females survived to the scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

All females in the 700 and 1000 mg/kg/day groups lost weight over the course of the treatment period (Study Days 0 – 7). Body weight losses were 227 g and 99 g (700 mg/kg/day group) or 322 g and 189 g (1000 mg/kg/day group) when the entire treatment period was evaluated. As a result, body weights for these females were 7.1 % and 3.1 % lower (700 mg/kg/day group) or 9.5 % and 6.1 % lower (1000 mg/kg/day group) on Study Day 7 when compared to Study Day 0. Body weights and body weight gains in the 100 and 300 mg/kg/day groups were generally unaffected over the course of the treatment period. Body weight gains for females in these groups ranged from 12 g to 47 g when the entire treatment period was evaluated and were generally comparable to pretreatment period values.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for females in the 700 and 1000 mg/kg/day groups during the treatment period was generally lower compared to the pretreatment period. Food consumption (g/animal/day) values for these females were 46 g/day and 71 g/day (700 mg/kg/day group) or 20 g/day and 26 g/day (1000 mg/kg/day group) when the entire treatment period (Study Days 0 - 7) was evaluated. These results corresponded to the body weight losses noted for females in these groups during the treatment period. Food consumption for females in the 100 and 300 mg/kg/day groups was generally comparable during the treatment and pretreatment period for all animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Conclusions:

When 2-pyrrolidone was administered by oral (gavage) to non-pregnant New Zealand White rabbits for 7 consecutive days, rales was noted for 1 female at 1000 mg/kg/day and excreta-related findings were noted for both females at 1000 mg/kg/day; limited occurrences of excreta-related findings were also noted at 700 mg/kg/day. In addition, body weight losses and reduced food consumption (compared to pretreatment values) were generally noted in a dose-related manner for all females in the 700 and 1000 mg/kg/day groups. Based on these results, dosage levels of 100, 300, 700, and 1000 mg/kg/day were selected for a dose range-finding embryo/fetal development study in rabbits.

Executive summary:

Summary

The objective of this study was to determine the tolerability of the test substance in nonpregnant female rabbits following oral administration for up to 7 days. The results of this study allowed dose selection for a subsequent oral range-finding embryo/fetal development toxicity study in rabbits.

The study design was as follows:

Group Number	Treatment	Dosage Level (mg/kg/day)*	Dose Concentration (mg/mL)	Dose Volume (mL/kg)	Number of Females
1	2-pyrrolidone	100	20	5	2
2	2-pyrrolidone	300	60	5	2
3	2-pyrrolidone	700	140	5	2
4	2-pyrrolidone	1000	200	5	2

* No correction factor was used.

Animals were dosed via oral gavage once daily during Study Days 0 – 6. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, and food consumption. All females survived to the scheduled euthanasia. In the 1000 mg/kg/day group, clinical observations were noted for both females at the daily examinations and included rales, soft feces, decreased defecation, and/or brown material on the anogenital area, urogenital area, and hindlimbs. In the 700 mg/kg/day group, 1 female had findings of brown material on the anogenital area and the other female had a single occurrence of decreased defecation at the daily examinations. No remarkable clinical observations were noted for any other females at the daily examinations. No clinical findings were noted approximately 2 hours following dose administration for any of the females. Body weight losses and reduced food consumption (compared to pretreatment values) were generally noted in a dose-related manner for all females in the 700 and 1000 mg/kg/day groups. As a result, body weights for these females on Study Day 7 were 7.1 % and 3.1 % lower (700 mg/kg/day group) or 9.5 % and 6.1 % lower (1000 mg/kg/day group) when compared to Study Day 0. Body weight gains and food consumption for females in the 100 and 300 mg/kg/day groups were generally comparable to pretreatment values over the course of the treatment period. When 2-pyrrolidone was administered by oral (gavage) to nonpregnant New Zealand White rabbits for 7 consecutive days, rales was noted for 1 female at 1000 mg/kg/day and excreta-related findings were noted for both females at 1000 mg/kg/day; limited occurrences of excreta-related findings were also noted at 700 mg/kg/day. In addition, body weight losses and reduced food consumption (compared to pretreatment values) were generally noted in a dose-related manner for all females in the 700 and 1000 mg/kg/day groups. Based on these results, dosage levels of 100, 300, 700, and 1000 mg/kg/day were selected for a dose range-finding embryo/fetal development study in rabbits.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-pyrrolidone

EC Number:

210-483-1

EC Name:

2-pyrrolidone

Cas Number:

616-45-5

Molecular formula:

C4H7NO

IUPAC Name:

pyrrolidin-2-one

Test material form:

liquid

Remarks:

clear, colorless

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area set to maintain 18 °C to 24 °C, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: stable in dispersion

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA, USA
- Age at study initiation: 6-7 months on Gestation Day 0
- Weight at study initiation: between 2934 and 3893 g on Gestation Day 0
- Fasting period before study: no
- 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 are accredited by AAALAC International.
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.
- Diet (e.g. ad libitum): 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 (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis 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.
- Acclimation period: After receipt at the Testing Facility (2018-06-15), the New Zealand White rabbits were acclimated prior to the initiation of dosing (2018-06-18).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures of 61 °F to 71 °F (16 °C to 22 °C) were maintained.
- Humidity (%): relative target humidity of 30 % to 70 %
- Air changes (per hr): Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained.

IN-LIFE DATES: From: 2018-06-15 To: 2018-07-12 (completion of in-life)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of Vehicle: The vehicle, deionized water, was dispensed approximately weekly for administration to Group 1 control animals and an adequate amount was dispensed into daily aliquots, which were stored at room temperature (18 °C to 24 °C), protected from light until use.
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 at room temperature (18 °C to 24 °C), protected from light, until use.

VEHICLE
- Concentration in vehicle: 0, 50, 100, 200 mg/mL test item
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis
Dose formulation samples were collected for analysis as indicated below:
Table
Dose Formulation Sample Collection Schedule
Interval Concentration Homogeneity Stability
15 Jun 2018 All groups N/A N/A
05 Jul 2018 All groups N/A N/A
N/A = not applicable.
Samples to be analyzed were transferred to the Analytical Chemistry Department.

Analytical Method
Analyses were performed by high performance liquid chromatography method using ultraviolet absorbance detection and a validated analytical procedure.

Concentration Analysis
Duplicate sets of samples (1.0 mL) for each time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility in reserve. Concentration results were considered acceptable if mean concentration results were within or equal to ± 10 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Solubility at 85 mg/mL and homogeneity of test substance formulations over the range of concentrations used on this study was established in a separate analytical validation study. Therefore, homogeneity analysis was not conducted on this study.

Stability Analysis
Stability of test substance formulations over the range of concentrations used on this study for 11 days at room temperature (18 °C to 24 °C) was established in a separate analytical validation study. Therefore, stability of test substance formulations was not assessed on this study.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
Time-mated female New Zealand White rabbits were received on Gestation Day 2, 3, or 4.

Duration of treatment / 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.

Frequency of treatment:

daily

Duration of test:

Gestation day 2-4 to gestation day 29

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 females / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dosage levels were determined from results of a previous range-finding study. In that study, pregnant rabbits were administered 2-pyrrolidone at dosage levels of 100, 300, 700, and 1000 mg/kg/day in deionized water. An overall mean body weight loss was noted at 1000 mg/kg/day from Gestation Days 7–13 resulting in a mean body weight that was approximately 10.6 % lower than controls on Gestation Day 13; correspondingly lower mean food consumption was noted during Gestation Days 7–13. Mean body weight gain was higher than or comparable to the control group during Gestation Days 13–20 and 20–29, and absolute mean body weight was unaffected at the end of the treatment period on Gestation Day 29. Slightly lower mean net body weight change was also observed for these females in the absence of any effect on gravid uterine weight or net body weight. Based on these results, dosage levels of 250, 500, and 1000 mg/kg/day were selected for the current study to identify a NOAEL and for the evaluation of a potential dose-response.
- Rationale for animal assignment (if not random): Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Animals in poor health or at extremes of body weight range 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.
- Other:
The route of administration was oral (gavage) as this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
Justification for Test System and Number of Animals: The New Zealand White rabbit is recognized as appropriate for developmental toxicity studies. The laboratory 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 with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, 24 rabbits/group was an appropriate number of animals to obtain a sample size of 20 females at termination.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 2 hours postdose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Gestation Days 0 (by supplier), 5, and 7–29.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured on Gestation Days 5–29.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
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.
Organ Weights: The liver was weighed at necropsy for all scheduled euthanasia animals. Organ to net body weight ratio was calculated.
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.

OTHER: Unscheduled Deaths
A necropsy was conducted for animals that aborted on study, and specified tissues were saved. The number and location of former implantation sites, corpora lutea, and viable fetuses were recorded. Recognizable fetuses were examined externally, euthanized (if necessary), and preserved in 10 % neutral buffered formalin. Recognizable fetuses and kits aborted on Gestation Day 29 were examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
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
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 developmental variations or malformations. 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 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

- External examinations: Yes
Each viable fetus was examined in detail, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Nonviable fetuses (the degree of autolysis is minimal or absent) were examined, crown-rump length measured, weighed, sexed and tagged individually. 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.
- Soft tissue examinations: Yes
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 examinations: Yes:
Each eviscerated fetus, following fixation in alcohol, was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue. The skeletal examination was made following this procedure.
- Head examinations: Yes
The heads from all fetuses were examined by a mid-coronal slice.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations 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. Data obtained from nongravid animals were excluded from statistical analyses.
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 by sex.
Maternal body weights and body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, fetal body weights (separately by sex and combined), and liver weights 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 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, 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.

Historical control data:

Available, based on 91 datasets.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased incidences of decreased defecation were noted in the 500 and 1000 mg/kg/day groups at the daily examinations compared to the control group. Decreased defecation was noted during Gestation Days 12–26 (19 animals) and 10–26 (22 animals) in the same respective groups and corresponded to the lower food consumption noted at these dosage levels. Other clinical observations noted in the test substance-treated groups at the daily examinations or approximately 2 hours following dose administration, including thin body condition, small/soft feces, brown material on various body surfaces, and rales, 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:

mortality observed, treatment-related

Description (incidence):

In the 1000 mg/kg/day group, 1 and 2 females aborted or were euthanized in extremis, respectively, prior to the scheduled necropsy. Female No. 5768 in this group aborted on Gestation Day 27 following a body weight loss of 17 % and lower food consumption (0 to 67 g/day) from Gestation Days 19 to 26 (with corresponding decreased defecation on most days during this period); this female aborted 7 dead fetuses (with no apparent malformations for those that were not cannibalized) and 1 late resorption and no internal findings were noted at necropsy. Female Nos. 5723 and 5749 in the same group were euthanized in extremis on Gestation Days 28 and 15, respectively. Female No. 5723 was noted with red material on the urogenital and anogenital areas and red material in the cage pan on Gestation Day 28 prior to euthanasia. There were no remarkable effects on body weight or food consumption for this animal. At necropsy, this female was noted with 8 viable fetuses and 1 late resorption in utero (with no apparent malformations) and a prolapsed vagina. Female No. 5749 was noted with hypoactivity on the day of euthanasia, a body weight loss of 11 %, lower food consumption (0 to 29 g/day) from Gestation Days 7 to 15 and corresponding decreased defecation during Gestation Days 10–15. At necropsy, this female was noted with 7 normally developing implantations in utero. The abortion and moribundity at a dosage level of 1000 mg/kg/day were considered test substance-related and adverse.
In the 250 mg/kg/day group, Female No. 5754 was euthanized in extremis on Gestation Day 19. This animal had a body weight loss of 9.8% from Gestation Days 13 to 19 and corresponding lower food consumption (0 to 28 g/day) from Gestation Days 14 to 19. At necropsy, this female had 2 early resorptions and 6 normally developing implantations in utero. This moribundity was not considered test substance-related as no adverse effects were noted at this dosage level and no mortality/moribundity was noted at the 500 mg/kg/day dosage level.
In the control group, Female No. 5732 aborted on Gestation Day 29; this female lost 7.8 % of its body weight during Gestation Days 23–29 with corresponding low food consumption (0–1 g/day) during Gestation Days 24–29.
All other females survived to the scheduled necropsy on Gestation Day 29.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Days 7–10 and 10–13 when compared to the control group (statistically significant). Mean body weight gains in this group throughout the remainder of treatment (Gestation Days 13–20 and 20–29) were slightly higher than the control group values (not statistically significant). The initial body weight losses in the 1000 mg/kg/day group resulted in a lower mean body weight gain when the overall treatment period (Gestation Days 7–29) was evaluated and mean absolute body weights that were up to 10.9 % lower than the control group values during Gestation Days 10–29; all of these differences were statistically significant. Lower mean net body weight and greater mean net body weight loss were noted in the 1000 mg/kg/day group when compared to the control group (statistically significant), while mean gravid uterine weight in this group was similar to the control group value. The effects on maternal body weight and body weight change in the 1000 mg/kg/day group were considered test substance-related and adverse.
Mean maternal body weight losses were noted in the 500 mg/kg/day group during Gestation Days 7–10 and 10–13 when compared to the control group (statistically significant for Gestation Days 10–13). Slightly lower mean body weight gains were noted in this group during Gestation Days 13–20 and 20–29 (not statistically significant). The initial body weight losses in the 500 mg/kg/day group resulted in a statistically significantly lower mean body weight gain for the overall treatment period (Gestation Days 7–29) and mean absolute body weights that were up to 5.8 % lower than the control group values during Gestation Days 22–29 (generally statistically significant). A greater mean net body weight loss was noted in the 500 mg/kg/day group compared to the control group (statistically significant), while mean net body weight and gravid uterine weight in this group were similar to the control group values. The effects on maternal body weight and body weight change in the 500 mg/kg/day group were considered test substance-related and adverse.
Mean maternal body weights, body weight gains, net body weight, net body weight gain, and gravid uterine weight in the 250 mg/kg/day group 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):

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 500 and 1000 mg/kg/day groups was lower than the control group values during the first 2 weeks of treatment (Gestation Days 7–10, 10–13, and 13–20) and when the overall treatment period (Gestation Days 7-29) was evaluated; the differences were statistically significant. The lower food consumption generally corresponded to the body weight deficits and decreased defecation noted in these groups. During Gestation Days 20-29, mean food consumption (g/animal/day and g/kg/day) in the 500 and 1000 mg/kg/day groups was similar to the control group values.
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 250 mg/kg/day group was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

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):

Mean liver weights (absolute and relative to net body weight) were unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day. 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 29, no test substance-related internal findings were observed at dosage levels of 250, 500, and 1000 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

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios. Differences from the control group were slight and not statistically significant.
A single occurrence of abortion on Gestation Day 27 was noted in the high-dose group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, 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.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, and fetal sex ratios. Differences from the control group were slight and not statistically significant.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number and percentage of viable fetuses, 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

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

250 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

gross pathology

mortality

organ weights and organ / body weight ratios

2

Dose descriptor:

LOAEL

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

gross pathology

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

dead fetuses

early or late resorptions

effects on pregnancy duration

number of abortions

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects on intrauterine growth were noted at dosage levels of 500 and 1000 mg/kg/day. Mean fetal body weights (male, female, and combined) in the 1000 mg/kg/day group were 19.6 %, 19.8 %, and 20.0 % lower, respectively, than the control group values (statistically significant) and below the range of the historical control data. At the 500 mg/kg/day dosage level, mean fetal body weights (male, female, and combined) were 14.5 %, 11.6 %, and 14.1 % lower, respectively, than the control group values (statistically significant) and below the range of the historical control data. The lower mean fetal body weights in the 500 and 1000 mg/kg/day groups were considered adverse. Mean fetal body weights in the 250 mg/kg/day group were unaffected by test substance administration; differences from the control group were slight and not statistically significant.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day. Differences from the control group were slight and not statistically significant.
The numbers of fetuses (litters) available for morphological evaluation were 195(23), 193(22), 200(24), and 179(20) in the control, 250, 500, and 1000 mg/kg/day groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day. Parameters evaluated included fetal sex ratios.
Differences from the control group were slight and not statistically significant.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weights (male, female, and combined) in the 1000 mg/kg/day group were 19.6 %, 19.8 %, and 20.0 % lower, respectively, than the control group values (statistically significant) and below the range of the historical control data. At the 500 mg/kg/day dosage level, mean fetal body weights (male, female, and combined) were 14.5 %, 11.6 %, and 14.1 % lower, respectively, than the control group values (statistically significant) and below the range of the historical control data. The lower mean fetal body weights in the 500 and 1000 mg/kg/day groups were considered adverse. Mean fetal body weights in the 250 mg/kg/day group were unaffected by test substance administration; differences from the control group were slight and not statistically significant.
Intrauterine survival was unaffected by test substance administration at dosage levels of 250, 500, and 1000 mg/kg/day.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were observed in 1(1), 0(0), 0(0), and 3(2) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Spina bifida (the vertebral column was open in the sacral region and/or covered in skin) was noted for Fetus Nos. 5769-05 and 5769-08 in the 1000 mg/kg/day group. Skeletally, the finding consisted of the dorsal aspects of lumbar, sacral, and/or caudal arches that were reflected more laterally than normal and/or fused sacral and/or caudal arches. Short tails (2–4 mm in length) were also noted for these same two fetuses; skeletally, the finding consisted of approximately 7–9 caudal vertebrae present and small, fused, misshapen, and/or malpositioned sacral and/or caudal vertebrae. Fetus No. 5769-05 was also noted with localized fetal edema (in the ventral neck region). Spina bifida has not been observed previously in the current historical control data, and the mean litter proportions for localized fetal edema and short tail were above the range or near the upper end of the range, respectively, of the historical control data. In addition, 1 late resorption in the same litter (No. 5769-06) was also noted with a short tail (4 mm in length). The occurrences of spina bifida, short tail, and localized fetal edema at a dosage level of 1000 mg/kg/day were not considered test substance-related as they affected a single litter. Omphalocele (a few loops of the intestine protruded through an opening in the umbilicus, with remnants of a membranous sac) was noted for Fetus Nos. 5673-01 and 5712-06 in the control and 1000 mg/kg/day groups, respectively. Fetus No. 5673-01 in the control group was also noted with open eyelids. Based on the occurrence of omphalocele in the control group and the limited occurrence in 1 fetus at the 1000 mg/kg/day dosage level, this finding was not attributed to test substance administration.
No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal malformations were observed in 2(2), 0(0), 2(2), and 6(4) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Costal cartilage anomalies (bifurcated, malpositioned and/or fused costal cartilage and/or misshapen sternebra) were noted for 1, 0, 2, and 2 fetuses in the same respective groups. Vertebral anomalies (extra, fused, malpositioned, and/or absent ribs, arches, centra or costal cartilage) were noted for Fetus Nos. 5719-09 and 5769-03 in the 500 and 1000 mg/kg/day groups, respectively; Fetus No. 5769-03 also had small heart ventricles (bilateral) (see below). Also in the 1000 mg/kg/day group, Fetus No. 5769-05 was noted with a rib anomaly (extra, fused, and/or malpositioned ribs and costal cartilage) and a skull anomaly (small interparietal bone) (this fetus also had spina bifida, see above); Fetus No. 5769-08 was observed with 14th full ribs and a vertebral centra anomaly (fused or malpositioned thoracic centra) (this fetus also had spina bifida, above); and Fetus No. 5684-02 had sternoschisis (sternal bands not joined at the No. 6 position). Fetus No. 5674-01 in the 500 mg/kg/day group had 28 presacral vertebrae (8 cervical vertebrae). The skeletal malformations observed in the test substance-treated groups were noted similarly in the control group, were not observed in a dose-related manner, were noted in single fetuses, were not statistically significant (mean litter proportions) compared to the concurrent control group, and/or the values were within the ranges of the historical control data; therefore, these skeletal malformations were not attributed to test substance administration. One fetus (No. 5717-02) in the control group had fused sternebrae (Nos. 4 and 5).
Increased incidences of the skeletal variations 13th full rib(s) and 27 presacral vertebrae were noted in the 250, 500, and 1000 mg/kg/day groups in a dose-responsive manner; the mean litter proportions of these findings (as well as the total percent per litter with skeletal developmental variations in this group) were generally statistically significant compared to the concurrent control group and above the ranges of the historical control data; therefore, these findings were considered test substance-related but nonadverse. Other skeletal variations observed in the test substance treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the mean litter proportions were not statistically significantly different from the concurrent control group, and/or the values were within the ranges of the historical control data.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Visceral malformations were observed in 4(4), 1(1), 6(3), and 11(6) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Interventricular septal defect was noted for 1, 1, 0, and 2 fetuses in the same respective groups. This finding consisted of an opening in the anterior portion of the septum for the affected fetuses in the control and 250 mg/kg/day groups and Fetus No. 5742-15 in the 1000 mg/kg/day group, whereas the entire septum was absent for Fetus No. 5729-04 in the 1000 mg/kg/day group. Bulbous aorta (ascending and/or aortic arch) was noted for Fetus Nos. 5682-07 and 5729-04 in the 1000 mg/kg/day group. The mean litter proportions for interventricular septal defect and bulbous aorta were above the range or near the upper end of the range, respectively, of the historical control data. Therefore, the occurrences of interventricular septal defect and bulbous aorta at a dosage level of 1000 mg/kg/day were considered test substance-related and adverse. A small heart ventricle (right or bilateral) was noted for 3 fetuses in the 1000 mg/kg/day group (Fetus Nos. 5712-02, 5712-10, and 5769-03). Although a small heart ventricle (right) was also noted in the control group (Fetus No. 5686-09), the mean litter proportion of this finding at 1000 mg/kg/day (2.1 % per litter) was above the maximum mean value in the historical control data (0.91 % per litter). Therefore, the occurrences of small heart ventricle at a dosage level of 1000mg/kg/day were considered test substance-related and adverse. Lobular agenesis of the lungs (absent right accessory lobe) was observed in 1, 4, and 5 fetuses in the control, 500, and 1000 mg/kg/day groups, respectively. Persistent truncus arteriosus was observed in 2, 1, and 1 fetuses in the same respective groups; the finding consisted of pulmonary arteries that arose from the truncus arteriosus or the ductus arteriosus, a right pulmonary artery that coursed retroesophageal, and/or a retroesophageal right subclavian artery, along with an interventricular septal defect (an opening in the anterior portion of the septum or the entire septum and tricuspid valve were absent). A malpositioned kidney (the left kidney was located more posterior than normal) was noted for Fetus No. 5716-03 in the 500 mg/kg/day group. The findings of lobular agenesis of the lungs, persistent truncus arteriosus, and malpositioned kidney in the test substance-treated groups were noted similarly in the control group, were not observed in a dose-related manner, were not statistically significant (mean litter proportions) compared to the concurrent control group, and/or the values were within the ranges of the historical control data; therefore, these visceral malformations were not attributed to test substance administration.
No test substance-related visceral developmental variations were noted. Visceral variations observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the mean litter proportions were not statistically significantly different from the concurrent control group, and/or the values were within the ranges of the historical control data.
Other visceral findings noted in the 1000 mg/kg/day group included a distended abdomen, clear fluid contents in the abdominal cavity, and a mottled liver (all lobes) for Fetus No. 5769-05.
White precipitate contents were noted in the right ureter and kidney of Fetus No. 5677-01 in the control group and both kidneys of Fetus No. 5719-08 in the 500 mg/kg/day group. In the 250 mg/kg/day group, white fluid contents in the abdominal cavity were noted for Fetus Nos. 5692-02 and 5692-03 (also noted with white fluid contents in the thoracic cavity and dark red discoloration of the lungs). Fetus No. 5692-10 in the 250 mg/kg/day group was also noted with dark red discoloration of the lungs. A cystic oviduct was noted for Fetus Nos. 5736-06 and 5736-11 in the 250 mg/kg/day group. In the control group, a dark red area in the right cornea was noted for Fetus No. 5673-01 and a renal papilla that was not fully developed (Woo and Hoar Grade 1) was noted for Fetus No. 5762-11. 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:

not specified

Details on embryotoxic / teratogenic effects:

Summary of External, Visceral, and Skeletal Examinations
The numbers of fetuses (litters) available for morphological evaluation were 195(23), 193(22), 200(24), and 179(20) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Malformations were observed in 7(6), 1(1), 8(5), and 15(8) fetuses (litters) in these same respective dose groups. Test substance-related, adverse occurrences of external malformations (spina bifida, short tail, and localized fetal edema) and visceral malformations (interventricular septal defect, small heart ventricle, and bulbous aorta) were noted at a dosage level of 1000 mg/kg/day. Test substance-related, nonadverse increases in the mean litter proportions of skeletal developmental variations (13th full rib[s], 27 presacral vertebrae, and total skeletal variations) were also noted in the 250, 500, and 1000 mg/kg/day groups. The mean litter proportions of total malformations in the 1000 mg/kg/day group and total developmental variations in the 250 and 1000 mg/kg/day groups were slightly higher than the concurrent control group (statistically significant for variations in both groups). However, the values were within (malformations) or only slightly above (variations) the ranges of the historical control data; therefore, these differences were not considered test substance-related. Other 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 historical control data ranges, and therefore were not attributed to the test substance.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Remarks:

overall

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

2

Dose descriptor:

LOAEL

Remarks:

overall

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

3

Dose descriptor:

NOAEL

Remarks:

classification

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

visceral malformations

4

Dose descriptor:

LOAEL

Remarks:

classification

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

visceral malformations

Fetal abnormalities

1

Abnormalities:

effects observed, treatment-related

Localisation:

visceral/soft tissue: cardiovascular

Description (incidence and severity):

Substance-related visceral malformations (interventricular septal defect, small heart ventricle, and bulbous aorta) were noted at a dosage level of 1000 mg/kg/day.

2

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: tail

external: trunk

visceral/soft tissue: integumentary

Description (incidence and severity):

Spina bifida (the vertebral column was open in the sacral region and/or covered in skin) Short tails (2–4 mm in length)
Localized fetal edema (in the ventral neck region)

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 414, OPPTS 870.3700 and European Commission Annex V, B.31 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Clear NOAELs and LOAELs could be identified for the relevant developmental toxic effects. Hence, the results can be considered as reliable to assess the potential of 2-Pyrrolidone to induce developmental toxicity after maternal exposure.

Developmental toxicity was manifested at dosage levels of 500 and 1000mg/kg/day, including lower mean fetal body weights at 500 and 1000 mg/kg/day and visceral malformations of interventricular septal defect, small heart ventricle, and bulbous aorta in 2, 3, and 2 fetuses, respectively at a dosage level of 1000 mg/kg/day. There was no correlation between these malformations and the individual fetal weights to attribute the occurrence of the malformations to general growth retardation. Two of the fetuses with these findings had body weights that were lower than the mean of that litter; the remaining affected fetuses had body weights that were higher than the mean of that litter.
Two fetuses from a single litter at 1000 mg/kg/day were noted with external malformations of spina bifida, short tail, and/or edema. Because these findings affected just one litter, they were not considered test substance-related. There were no maternal effects to explain the appearance of these malformations. Test substance-related, non-adverse increases in the mean litter proportions of skeletal developmental variations (13th full rib[s], 27 presacral vertebrae, and total skeletal variations) were also noted in the 250, 500, and 1000 mg/kg/day groups.
The developmental effects at 500 and 1000 mg/kg/day were noted in the presence of maternal toxicity, as evidenced by effects on food consumption and body weights. Test substance-related, adverse occurrences of moribundity (2 females on Gestation Days 15 or 28) resulting in euthanasia (due to reduced food consumption, body weight loss, hypoactivity, and/or red material in the cage pan) were noted at the 1000 mg/kg/day dosage level. A single additional occurrence of abortion on Gestation Day 27 was also noted at this dosage level following reduced food and body weight loss. In addition, test substance-related mean body weight losses were noted in the 500 and 1000 mg/kg/day groups during the first 1 or 2 weeks of treatment resulting in adverse lower mean body weights that were up to 5.8% and 10.9% lower, respectively, compared to the control group. Corresponding lower food consumption during the first 2 weeks of treatment and observations of decreased defecation were noted in these groups.
The effect on maternal food consumption and body weight gain during GD 7 - 20 occurred at dose levels with lower fetal body weights at termination. However, the maternal food consumption and body weight gain during GD 20 - 29 was comparable to control normal and since this is the period of time when fetal body mass is accumulated, the decreases in fetal body weight was likely unrelated to decreases in maternal food consumption.
In conclusion, based on moribundity at 1000 mg/kg/day, maternal body weight deficits and lower food consumption, with corresponding decreased defecation, at 500 and 1000 mg/kg/day, lower fetal body weights at 500 and1000 mg/kg/day, and fetal malformations (bulbous aorta, small heart ventricle, and interventricular septal defect) at 1000 mg/kg/day, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal and prenatal developmental toxicity when 2-pyrrolidone was administered orally by gavage to time-mated New Zealand White rabbits.

According to Regulation (EC) 1272/2008, “Reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.” In the current study, the latter was evaluated. “For pragmatic purposes of classification, developmental toxicity essentially means adverse effects induced during pregnancy, or as a result of parental exposure. These effects can be manifested at any point in the life span of the organism. The major manifestations of developmental toxicity include (1) death of the developing organism, (2) structural abnormality, (3) altered growth, and (4) functional deficiency.” Further, “for the purpose of classification for reproductive toxicity, substances are allocated to one of two categories”. Category 1B is defined as follows: “Presumed human reproductive toxicant: The classification of a substance in Category 1B is largely based on data from animal studies. Such data shall provide clear evidence of an adverse effect on sexual function and fertility or on development in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of other toxic effects. However, when there is mechanistic information that raises doubt about the relevance of the effect for humans, classification in Category 2 may be more appropriate.”

The findings in the fetuses in the present study are considered relevant for humans, but there is no indication that rats and rabbits might be considerably less sensitive to these effects of 2-pyrrolidone than humans.
The trigger findings for classification of 2-pyrrolidone as a category 1B reproductive toxin is the slightly increased incidences in malformations at the top dose levels (1000 mg/kg bw/d). Although the toxic effects in the dams might have contributed to the observed fetal toxicity, a substance specific developmental toxic hazard cannot be ruled out. The oral NOAEL for developmental toxicity relevant for classification were 500 mg/kg bw/d for rabbits and the corresponding LOAELs relevant for classification were 1000 mg/kg bw/d in rabbits.
In consequence, 2-pyrrolidone should be classified as reproductive toxicant Cat. 1B according to Regulation (EC) 1272/2008.

Further, as outlined in more detail in section 13, Regulation (EC) 1272/2008 (Article 10(1)) foresees the possibility to assign a specific concentration limit (SCL) for a substance under certain conditions based on the intrinsic potency for the specific hazard. Assigning SCLs for reproductive toxicity is described in the ECHA guidance document on the application of the CLP criteria (ECHA, 2017; chapter 3.7.2.6) and was followed there. In brief, according to this method, a preliminary assessment should be conducted whether the substance shows high, medium or low potency based on an ED10 value which is defined as the lowest dose inducing reproductive effects with an incidence or magnitude of 10 % after correction for the spontaneous incidence. The ED10 trigger values of for Category 1A and 1B substances group are < 4 mg/kg bw/day (high potency, SCL 0.03%), ≥ 4 < 400 mg/kg bw day (medium potency, GCL 0.3 %) and ≥ 400 mg/kg bw/day (low potency, SCL 3 %). The ED10 value was based on %fetuses per litter with any malformations (lowest effect level) by interpolation between NOAEL (4.4 % at 500 mg/kg bw/d) and LOAEL (9.3 % at 1000 mg/kg bw/d), which leads to an ED10 of 1489 mg/kg bw/d.
The derived ED10 values clearly classify 2-pyrrolidone as a low potency reproductive toxin (ED10 ≥ 400 mg/kg bw/day). For this group (Cat. 1B) showing low developmental potency, a Specific Concentration Limit (SCL) for mixtures of 3 % is proposed in the ECHA Guidance which is applied for 2-pyrrolidone as SCL for reproductive toxicity.

Executive summary:

The objective of this study according to OPPTS 870.3700, OECD Test Guideline 414, and European Commission Annex V, B.31, under GLP, was to determine the potential of 2-pyrrolidone 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.

The study design was as follows:

 

Table Experimental Design

Group Number	Treatment	Dosage Level (mg/kg/day)^a	Dose Concentration (mg/mL)	Dose Volume (mL/kg)	Number of Females
1	Vehicle Control	0	0		24
2	2-pyrrolidone	250	50		24
3	2-pyrrolidone	500	100		24
4	2-pyrrolidone	1000	200		24

^aNo correction factor was used.

 

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, organ weights, intrauterine growth and survival, and fetal examinations.

Test substance-related, adverse occurrences of moribundity (2 females on Gestation Days 15 or 28) resulting in euthanasia were noted at the 1000 mg/kg/day dosage level. A single additional occurrence of abortion on Gestation Day 27 was also noted at this dosage level. Body weight losses with corresponding lower food consumption and decreased defecation were observed for the female that aborted and 1 of the moribund females (also noted as hypoactive) prior to euthanasia. Red material on the urogenital and anogenital areas and red material in the cage pan, without any remarkable effects on body weight or food consumption were noted for the other female euthanized in extremis in this group. One female in the 250 mg/kg/day group was euthanized in extremis on Gestation Day 19 following a body weight loss of 9.8 % from Gestation Days 13 to 19 and corresponding lower food consumption (0 to 28 g/day) from Gestation Days 14 to 19. Based on the lack of mortality or moribundity at the 500 mg/kg/day dosage level, this moribundity was not considered test substance-related. One control group female aborted on Gestation Day 29 following a body weight loss of 7.8 % during Gestation Days 23-29 with corresponding low food consumption(0-1 g/day) during Gestation Days 24-29. All other females survived to the scheduled necropsy on Gestation Day 29.

Test substance-related mean maternal body weight losses were noted in the 500 and 1000 mg/kg/day groups during the first 2 weeks of treatment, resulting in lower mean body weight gains during the overall treatment period (Gestation Days 7-29) and mean body weights that were up to 5.8 % and 10.9 % lower, respectively, compared to the control group. Corresponding lower food consumption and observations of decreased defecation were noted in these groups. These body weight deficits were considered adverse at both dosage levels. Mean maternal body weights, body weight changes, and food consumption at a dosage level of 250 mg/kg/day were unaffected by test substance administration.

No test substance-related macroscopic findings or effects on liver weights were noted at any dosage level.

Test substance-related lower mean fetal body weights were observed in the 500 and 1000 mg/kg/day groups compared to the control group and were considered adverse. No test substance-related effects were noted for mean fetal body weights at a dosage level of 250 mg/kg/day or intrauterine survival at any dosage level tested.

Test substance-related, adverse occurrences of visceral malformations (interventricular septal defect, small heart ventricle, and bulbous aorta) were noted at a dosage level of 1000 mg/kg/day. Test substance-related, nonadverse increases in the mean litter proportions of skeletal developmental variations (13th full rib[s], 27 presacral vertebrae, and total skeletal variations) were also noted in the 250, 500, and 1000 mg/kg/day group. No test substance-related fetal malformations were noted at dosage levels of 250 and 500 mg/kg/day.

Based on moribundity at 1000 mg/kg/day, maternal body weight deficits and lower food consumption, with corresponding decreased defecation, at 500 and 1000 mg/kg/day, lower fetal body weights at 500 and 1000 mg/kg/day, and fetal malformations (bulbous aorta, small heart ventricle, and interventricular septal defect) at 1000 mg/kg/day, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal and prenatal developmental toxicity when 2-pyrrolidone was administered orally by gavage to time-mated New Zealand White rabbits.