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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

1) EOGRTS, OECD 443, GLP, rat, oral (drinking water)

NOAEL (systemic, based on body weight and food consumption): F0 male = 4000 ppm, F0 female = 8000 ppm, F1 male = 1500 ppm, F1 female = 4000 ppm

NOAEL (reproduction): F0/F1 male and female > 8000 ppm

2) Reproductive toxicity Screening test; OECD 421; GLP rats, oral (drinking water)

conducted as a Dose-Range finder for the EOGRTS: dosage levels up to 8000 ppm were considered to be acceptable for an extended one-generation reproductive toxicity of 2-pyrrolidone

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Dec 2018 - 03 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Remarks:
Dose-range Finder
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Jun 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals : 10 weeks (as requested by ECHA in the Final decision on a compliance check)
- Basis for dose level selection : Dose level setting shall aim to induce some toxicity at the highest dose level (as requested by ECHA in the Final decision on a compliance check)
- Inclusion/exclusion of extension of Cohort 1B : exclusion of extension of Cohort 1B (not requested by ECHA in the Final decision on a compliance check)
- Termination time for F2
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : yes (as requested by ECHA in the Final decision on a compliance check)
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : no (not requested by ECHA in the Final decision on a compliance check)
- Route of administration : oral via drinking water
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]: not necessary
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Taminco BVBA
- Expiration date of the lot/batch: not specified
- Purity test date: not provided

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 under storage conditions: given (Test substance formulations have been previously shown to be stable over a concentration range of 1 to 80 mg/mL (1000 to 80,000 ppm) for at least 10 days at room temperature. In addition, an extension validation was conducted concurrently to establish stability at a concentration of 500 ppm (0.5 mg/mL) to cover the lowest possible concentration used on the current study. Therefore, stability of test substance drinking water formulations was not assessed on this study.)
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The test substance formulations have been previously shown to be stable over a concentration range of 1 to 80 mg/mL (1000 to 80,000 ppm) for at least 10 days at room temperature. In addition, an extension validation was conducted concurrently to establish stability at a concentration of 500 ppm (0.5 mg/mL) to cover the lowest possible concentration used on the current study. Therefore, stability of test substance drinking water formulations was not assessed on this study, as 2-pyrrolidone expected to be stable in the vehicle (reverse osmosis-purified water)
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any): none
- Preparation of a nanomaterial dispersion (incl. dilution): not applicable
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: not applicable

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

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: (P) 6 wks; (F1) x wks
- Weight at study initiation: the naimals weighted between 130 and 226 g at the initiation of dosing. (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no / not specified
- Housing: On arrival, the F0 animals were group housed (2 to 3 animals of the same sex). During cohabitation, the F0 animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Following weaning (F1), animals were group housed (2 to 3 animals of the same sex) until euthanasia. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis (with test substance added during the treatment period for animals in Groups 2–4) was available ad libitum via amber glass water bottles with metal sipper tubes. Bottles were checked daily for spillage and supplemented as necessary and the occurrence of spillage was documented. Water bottles were changed and sanitized weekly. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
- Acclimation period: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 °F to 78 °F (20 °C to 26 °C)
- Humidity (%): 30 % to 70 %
- Air changes (per hr): Ten or greater air changes per hour with 100 % fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: To:
Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
Reverse osmosis-purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Preparation of Vehicle
The vehicle, reverse osmosis-purified water, was dispensed approximately weekly for administration to Group 1 control animals and preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and were stored at room temperature (18 °C to 24 °C), protected from light until use. Details of the dispensing of the vehicle have been retained in the Study Records.

Preparation of Test Substance
Test substance dosing formulations were prepared based on Sponsor instructions at appropriate concentrations to meet dose level requirements. The dosing formulations 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. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 1500, 4000, and 8000 ppm
- Purity: pure
Details on mating procedure:
During cohabitation, the F0 animals were paired for mating in the home cage of the male. The F0 animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of exposure. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12 hour dark cycle were considered to have been paired for 1 day.
- M/F ratio per cage: 1:1
- Length of cohabitation: maximal 14 days
- Proof of pregnancy: [vaginal plug] referred to as [day 0] of pregnancy
- After successful mating each pregnant female was caged (how): Following the breeding period, animals were individually housed.
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. Analyses were performed by high performance liquid chromatography using ultraviolet absorbance detection using a validated analytical procedure.
Concentration analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded
Homogeneity Analysis: Homogeneity analyses performed on the analytical validation and stability study demonstrated that the test article is soluble in the vehicle when prepared under the same mixing conditions at concentrations bracketing those used in the present study. Solubility data have been retained in the Study Records.
Stability Analysis: Test substance formulations have been previously shown to be stable over a concentration range of 1 to 80 mg/mL (1000 to 80,000 ppm) for at least 10 days at room temperature. In addition, an extension validation was conducted concurrently to establish stability at a concentration of 500 ppm (0.5 mg/mL) to cover the lowest possible concentration used on the current study. Therefore, stability of test substance drinking water formulations was not assessed on this study.
Duration of treatment / exposure:
F0 males: 70 consecutive days prior to mating + continuing until euthanasia (for approximately 19 weeks)
F0 females: 70 consecutive days prior to mating + continuing throughout mating, gestation, and lactation until euthanasia
F1 generation was potentially exposed to the test substance in utero, through nursing during lactation, and through potential direct exposure via drinking water prior to weaning. Following weaning, the offspring selected for the F1 generation were dosed until euthanasia (PND 22 [Cohort 2B], PND 59 [Cohorts 3 and 3A], PND 78 [Cohort 2A], PND 91 [Cohort 1A], and PND 98 [Cohort 1B]).
Frequency of treatment:
The test substance was administered at a constant concentration in the diet (ppm). Drinking water formulations were supplied fresh approximately weekly.
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
1 500 ppm (nominal)
Dose / conc.:
4 000 ppm (nominal)
Dose / conc.:
8 000 ppm (nominal)
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels were determined from results of a previous dose range-finding study with 2 pyrrolidone in rats. In that study, 2-pyrrolidone was administered at concentrations of 3000, 8000, and 12,000 ppm in drinking water to male and female F0 rats beginning 2 weeks prior to mating through euthanasia, and to male and female F1 rats from PND 21 through 35. No test substance related mortality was observed at any concentration level. Lower mean body weights compared to controls were noted for males at 12,000 ppm and for females at 8000 and 12,000 ppm throughout the study. In addition, a lower mean number of F1 pups born, live litter size on PND 0, and postnatal survival (PND 0–1 and from birth to PND 4) were noted at 12,000 ppm. Mean F1 pre-weaning body weights were up to 9.6 % and 15 % lower in the 8000 and 12,000 ppm group males, and up to 9.8 % and 13.1 % lower in the 8000 and 12,000 ppm group females, respectively. Mean body weights for F1 animals during the post-weaning period were up to 13.6 % and 15.3 % lower in males and 10.3 % and 13.8 % lower in females in the 8000 and 12,000 ppm groups, respectively, during PND 22-35. Based on these results, a high concentration level of 8000 ppm was selected for the current study. The low and mid dose levels of 1500 and 4000 ppm, respectively, were selected for the identification of a no observed adverse-effect-level (NOAEL) and for the evaluation of a potential dose response.

- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to blood collection.
- Other: not specified
Positive control:
no
Parental animals: Observations and 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 throughout the study

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 14, and 21. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly until cohabitation. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 14, and 21. Following the breeding period, food consumption for males was quantitatively measured weekly until euthanasia. Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was quantitatively measured weekly until cohabitation. Once evidence of mating was observed, female water consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 14, and 21. Following the breeding period, water consumption for males was quantitatively measured weekly until euthanasia.
- Compound Consumption: The mean amounts of test substance consumed (mg/kg/day) by each sex per dose group were calculated from the mean water consumed (g/kg/day) and the appropriate target concentration of test substance in the water (ppm).

OTHER:
Thyroid Hormone Analysis
- Time schedule for sample collection: Blood samples (Group 1 - 4; sample collected from 10 animals/ sex/group) for thyroid hormone analyses were collected in week 19 (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from the jugular vein into tubes without anticoagulants. Samples were collected prior to fasting for clinical pathology evaluations.
- Sample processing: Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70 °C.
- Sample analysis: Blood samples were analyzed for the following parameters: Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH)

CLINICAL PATHOLOGY:
- Time schedule for sample collection:
- Blood collection for Haematology examinations, coagluation examination and clinical chemistry: Animals were fasted overnight prior to blood collection. Blood samples (Group 1 - 4; sample collected from 10 animals/ sex/group) were collected in week 19. Blood samples for hematology and serum chemistry were collected from the jugular vein and were processed for serum, and the serum was analyzed. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation and were subsequently processed for plasma, and the plasma was analyzed.
- sample treatment: K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants. In addition, blood smears were prepared, stained with Wright Giemsa stain, cover-slipped, and retained for possible future evaluation.
- Urine collection for Urinalysis: Urine was collected overnight using metabolism cages in week 19 from 10 animals/sex/group

- Parameters evaluated (Haematology): Total leukocyte count (WBC); Erythrocyte count (RBC); Hemoglobin (HGB); Hematocrit (HCT); Mean corpuscular volume (MCV); Mean corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin concentration (MCHC); Platelet count (Platelet); Reticulocyte count; Percent (RETIC); Absolute (RETIC Absolute); Differential leukocyte count (- Percent and absolute; -Neutrophil (NEU); -Lymphocyte (LYMPH); -Monocyte (MONO); -Eosinophil (EOS); -Basophil (BASO); -Large unstained cell (LUC)); Red cell distribution width (RDW); Hemoglobin distribution width (HDW); Platelet estimatea; Red cell morphology (RBC Morphology)
- Parameters evaluated (Coagulation): Activated partial thromboplastin time (APTT); Prothrombin time (PT); - Parameters evaluated ( ): Albumin; Total protein, Globulin [by calculation]; Albumin/globulin ratio (A/G Ratio) [by calculation]; Total bilirubin (Total BILI); Urea nitrogen; Creatinine; Alkaline phosphatase (ALP); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST); Gamma glutamyltransferase (GGT); Glucose; Total cholesterol (Cholesterol); Calcium; Chloride; Phosphorus; Potassium; Sodium; Sorbitol dehydrogenase (SDH); Triglycerides (Triglyceride); Bile acids (Bile Acid); Appearance
- Parameters evaluated (Urinalysis): Specific gravity (SG); pH; Urobilinogen (URO); Total volume (TVOL); Color (COL); Clarity (CLA); Protein (PRO); Glucose (GLU); Ketones (KET); Microscopy of sediment; Bilirubin (BIL); Occult blood (BLD); Leukocytes (LEU)
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily (also on day of necropsy). Slides were evaluated microscopically to determine stage of estrous cycle of each F0 female for 14 days prior to cohabitation & until mating or until the end of mating period. Average cycle length was calculated for complete estrous cycles. Estrous cycle length was determined by counting number of days from the first M or D in a cycle to the first M or D in a subsequent cycle.
At the end of the study, the overall pattern of each female was characterized as regularly cycling (RC), irregularly cycling (IC), not cycling (NC), or insufficient data (ID). The following definitions were used for determination of regular cycling, irregular cycling, non-cycling, and insufficient data. Note that for a “complete cycle”, an animal must exhibit at minimum an E from the previous cycle, followed by one cycle (D to E), and a D from the start of the next cycle:
-RC: animal has at least 6 days of data collected & displays 1 complete cycle with no cycles ≥ 5 days in duration, no cycles with 3 or more consecutive days of P and/or E, and no cycles ≤ 4 days in duration.
-IC: animal does not display 1 complete cycle but has at least 1 E and/or P and a partial cycle ≥ 5 days, the animal has at least one E present and either at least 1 cycle (complete or partial) ≥ 5 days in duration or at least 1 cycle with 3 or more consecutive days of P and/or E, at least 1 cycle ≤ 4 days in duration, or 1 irregular cycle and 1 regular cycle.
-NC: no E or P present on any days of estrous cycle determination and at least 5 days of data collected.
- ID: animal does not display at least 1 complete cycle but has at least 1 E and/or P and a partial cycle of ≤ 5 days, no E present on any days of estrous cycle determination and ≤ 4 days of data collected, or at least 1 E or P present and only 1-4 days of data collected.
From the above-mentioned definitions, the percent cycling and percent cycling regularly were calculated.
Sperm parameters (parental animals):
Parameters examined in P/F1 male parental generations: testis weight, epididymis weight, sperm production rate, sperm motility, sperm morphology

Sperm Evaluations
Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37 °C) with 10 mg/mL BSA. After a minimum 10 minute incubation period, a sample of sperm was loaded onto a slide with a 100 µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37 °C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer.
The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.
The right epididymis was then placed in modified Davidson’s solution for microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and cauda epididymis from all males were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20 µm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.
The sperm production rate was calculated.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

Viability: Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Total litter loss: Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia. Litters that were euthanized prior to scheduled euthanasia due to reasons unrelated to test article administration (e.g., mechanical injury, inadvertent removal from room) were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.
Observations: The animals were removed from the cage, and a detailed clinical observation was performed on PND 1, 4, 7, 14, and 21. Any abnormalities in nesting and nursing behavior were recorded.
Sex Determination: Pups were individually sexed on PND 0, 4, 14, and 21.
Body Weights: Pups were weighed individually on PND 1, 4, 7, 14, and 21.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

PREWEANING DEVELOPMENTAL LANDMARKS
Anogenital Distance: The anogenital distance of all pups was measured on PND 1. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.
Assessment of Areolas/Nipple Anlagen: On PND 13, all male pups were evaluated for the presence of thoracic nipples/areola. The number of nipples was recorded.

Thyroid Hormone Analysis
- Time schedule for sample collection: Blood samples for thyroid hormone analysis were collected on Day 4 and 21 (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) via cardiac puncture of animals anesthetized by inhalation of isoflurane (PND 4 culled pups) or via the jugular vein (PND 21 nonselected pups) into tubes without anticoagulants. Samples for Day 4 were collected from culled pups and pooled by litter until a total of 10 samples/dosage level were obtained. To the extent possible, samples from the first 10 litters at each dosage level with sufficient numbers of culled pups were used. Samples of Day 21 were collected from nonselected pups (10/sex/group).

- Sample processing: Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70 °C.
- Sample analysis: Blood samples were analyzed for the following parameters: Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH)
Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay. Samples to be analyzed for TSH were transferred to the Charles River Ashland ADME/DMPK Department; analyses were performed using a qualified radioimmunoassay.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
F1 DEVELOPMENTAL LANDMARKS, SENSORY FUNCTIONAND NEUROBEHAVIORAL TESTING
Balanopreputial Separation: Each male was observed for balanopreputial separation beginning on PND 35. Examination of the males was continued daily until balanopreputial separation was present, and the age of attainment was recorded. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a partial and complete balanopreputial separation or a persistent thread of tissue between the glans and prepuce was recorded.
Vaginal Patency: Each female was observed for vaginal perforation beginning on PND 25. Examination of the females was continued daily until vaginal patency was present, and the age of attainment was recorded. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a small “pin hole”, a vaginal thread, and complete vaginal opening were recorded.
Auditory Startle Response (Cohort 2A): An auditory startle response test was performed on animals assigned to Cohort 2A on PND 23. Auditory startle response testing was performed in a room equipped with a white-noise generation system. Each test session consisted of a 5 minute acclimation period with a 68 dB to 72 dB broadband background white noise. The startle stimulus for each trial was a 110 dB to 120 dB mixed-frequency noise burst stimulus, approximately 20 ms in duration. Responses were recorded during the first 100 ms following the onset of the startle stimulus for each trial. Each test session consisted of 50 trials, with 8 seconds between each trial. Startle response data were analyzed in 5 blocks of 10 trials each. Startle response measurements obtained were PEAK (peak response amplitude) and Tpeak (latency to PEAK).
FOB Assessments (Cohort 2A): FOB findings were recorded for all Cohort 2A animals on PND 65. The FOB used at Charles River is based on previously developed protocols. Testing was performed by the same trained technicians, when possible, who did not know the animal’s group assignment and was performed at approximately the same time each day. The FOB was performed in a sound attenuated room equipped with a white noise generator. All animals were observed for the following parameters:
- in the home cage: Posture; Convulsions/tremors; Feces consistency; Biting; Palpebral (eyelid) closure
- Handling observations: Ease of removal from cage; Lacrimation/chromodacryorrhea; Piloerection; Palpebral closure; Eye prominence; Red/crusty deposits; Ease of handling animal in hand; Salivation; Fur appearance; Respiratory rate/character; Mucous membranes/eye/skin color; Muscle tone
- Open Field Observations (evaluated over a 2-minute observation period): Mobility; Rearing; Convulsions/tremors; Grooming; Bizarre/stereotypic behavior; Time to first step (seconds); Gait; Arousal; Urination/defecation; Gait score; Backing
- Sensory Observations: Approach response; Startle response; Pupil response; Forelimb extension; Air righting reflex; Touch response; Tail pinch response; Eyeblink response; Hindlimb extension; Olfactory orientation
- Neuromuscular Observations: Hindlimb extensor strength; Hindlimb foot splay; Grip strength hind and forelimb; Rotarod performance
- Physiological Observations: Catalepsy; Body temperature; Body weight
- Motor Activity (Cohort 2A): Motor activity was assessed on animals assigned to Cohort 2A on PND 65 using a series of infrared photobeams to quantify each animal’s motor activity. The motor activity assessment was performed in a sound attenuated room equipped with a white noise generator, and black enclosures were used to decrease the potential for distraction. Data were collected in 5 minute epochs over a period of 60 minutes, and the data were reported in 10 minute subintervals. Total motor activity was defined as a combination of fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of 2 or more consecutive photobeams).

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: All animals in Cohort 3 (10 rats/sex/group) and Cohort 3A (10 rats in the control group, representing as many litters as possible) were administered a single intravenous immunization of sheep red blood cells (sRBC) via a lateral tail vein on PND 54 to elicit a primary antibody response. Each sRBC injection was composed of 0.5 mL of Earle’s Balanced Salt Solution with HEPES buffer solution and contained approximately 2 x 108 sRBC.
Positive control rats in Cohort 3A also received 10 mL/kg/day (25 mg/kg/day) of cyclophosphamide (CPS) by intraperitoneal injection for 5 consecutive days (PND 54–58) prior to and including the day prior to the scheduled necropsy (PND 59). CPS is a known immune suppressant and was prepared in phosphate-buffered saline at a concentration of 2.5 mg/mL, apportioned into at least 5 separate (daily) aliquots and stored at ≤ -10ºC until needed. On the day of injection, the CPS was thawed, stored on ice, and mixed prior to injection.
On PND 59 (approximately 120 hours following immunization with sRBC), all animals were euthanized by carbon dioxide inhalation. Approximately 1 mL to 1.5 mL of blood was collected via the inferior vena cava into tubes without anticoagulant. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Samples were transferred to the Charles River Immunotoxicology Department; t-cell dependent antibody response analysis was conducted using a sRBC IgM ELISA kit.
Following blood collection, a gross necropsy was conducted on each animal. The thymus and spleen were weighed and retained in 10% neutral-buffered formalin for possible future histopathology.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [following the selection of the F1 generation.]
- Maternal animals: All surviving animals [Study Days 132–135]

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

Scheduled Euthanasia
All surviving animals, including females that failed to deliver or with total litter loss, were euthanized by carbon dioxide inhalation following the selection of the F1 generation.

Necropsy
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights
The organs (Adrenal glands; Brain; Epididymidesa (total and cauda); Heart; Kidneys; Levator ani and bulbocavernosus (LABC) muscle group; Liver; Ovaries; Pituitary gland; Prostate gland; Seminal vesicles with coagulating glands (witth accessory fluids); Spleen; Testes; Thyroids with parathyroids; Thymus gland; Uterus with oviducts and cervix) were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise noted.
Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Tissue Collection and Preservation
Representative samples of the tissues (Adrenal glands (2); Aorta; Bone with marrow (sternebrae); Brain; Coagulating glands (2); Eyes with optic nerve (2); Gastrointestinal tract; Esophagus; Stomach; Duodenum; Peyer’s Patches; Jejunum; Ileum; Cecum; Colon; Rectum; Heart; Kidneys (2); Lacrimal/Harderian glands (2); Liver (sections of 2 lobes); Lymph node (axillary [2], mandibular [2], and mesenteric); Lungs (including bronchi, fixed by inflation with fixative); Levator ani and bulbocavernosus (LABC) muscle group; Ovariesc and oviducts (2); Pancreas; Peripheral nerve (sciatic); Pituitary; Prostate; Mandibular salivary gland (2); Seminal vesicles (2); Skeletal muscle (quadriceps); Skin with mammary glandd; pinal cord (cervical); Spleen; Testes with epididymides (2) and vas deferens; Thymus; Thyroids with parathyroids, if present (2); Trachea; Urinary bladder; Uterus with cervix and vagina; All gross lesions (all groups)) were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

Histology
Ovaries were processed at PAI Durham. All other histology procedures were performed at the Testing Facility. Tissues (as specified above) from all animals in the control and high dose groups and from all animals found dead and euthanized in extremis, as well as gross lesions from all animals in all groups, and reproductive organs of all animals suspected of reduced fertility were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Five (5) sections were taken approximately 100 µm apart from the inner third of each ovary from any F0 females suspected of reduced fertility. In addition, a single section was taken from F0 females (with no signs of reduced fertility) for a qualitative bilateral evaluation of each ovary. For females found dead or euthanized in extremis, a single section from each ovary was qualitatively evaluated.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues for microscopic examination (as specified above) were evaluated from all animals in the control and high dose groups and from all animals found dead and euthanized in extremis. Gross lesions were examined from all animals in all groups.
In addition, reproductive organs of all animals suspected of reduced fertility were subjected to a histopathologic evaluation.
Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
Uterine and ovarian histopathology were considered in light of the terminal estrous stage.
Postmortem examinations (offspring):
SACRIFICE
- 1 F1 offspring not selected as parental animals/sex/litter and all F2 offspring were sacrificed at PND4.
- Up to 10 F1 pups/sex/group were sacrificed at PND 21.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained.
Intact offspring that were found dead or euthanized for humane reasons during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead or euthanized for humane reasons after PND 4.

Scheduled Euthanasia
On PND 4, culled pups were euthanized by exsanguination (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital.
On PND 21, nonselected pups were euthanized by carbon dioxide inhalation.

Necropsy
On PND 4, 1 culled pup/sex/litter was subjected to a complete necropsy examination. Pups were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. All remaining culled pups were discarded without examination.
On PND 21, nonselected pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGTHS

Organ Weights
The organs (Brain; Liver; Spleen; Thymus; Thyroid) were weighed at necropsy from up to 10 nonselected F1 pups/sex/group on PND 21. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Tissue Collection and Preservation
Representative specimens with malformations and gross lesions from offspring found dead or euthanized for humane reasons were preserved in 10% neutral buffered formalin.
Representative samples of the tissues (Trachea (with thyroid gland); All gross lesions) were collected 1 culled pup/sex/litter on PND 4 and preserved in 10% neutral buffered formalin.
Representative samples of the tissues (Brain; Liver; Ovaries (2); Skin with Mammary gland; Spleen; Testes (2); Thymus; Thyroid; All gross lesions) were collected from up to 10 nonselected F1 pups/sex/group on PND 21 and preserved in 10% neutral buffered formalin.

F1 - Cohort 1
SACRIFICE
- 20 F1 offsprings/sex/group were sacrificed at PND 91.
- 24 or 25 F1 offsprings/sex/group, respectively, were sacrificed at PND 98.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
Unscheduled Deaths
No animals died during the course of the study.

Scheduled Euthanasia
All surviving animals were euthanized by carbon dioxide inhalation.

Necropsy
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
Sperm Evaluations (Cohort 1A)
Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37 °C) with 10 mg/mL BSA. After a minimum 10 minute incubation period, a sample of sperm was loaded onto a slide with a 100 µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37 °C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.
The right epididymis was then placed in modified Davidson’s solution for microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and cauda epididymis from all males were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20 µm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated.

Organ Weights
The organs (Adrenal glands; Brain; Epididymidesb (totale and cauda); Heart; Kidneys; Levator ani and bulbocavernosus (LABC) muscle group; Liver; Ovaries; Pituitary gland; Prostate gland; Seminal vesicles with coagulating glands (with accessory fluids); Spleen; Testes; Thyroids with parathyroids; Thymus gland; Uterus with oviducts and cervix) were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise noted. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated

Tissue Collection and Preservation
Representative samples of the tissues (Adrenal glands (2); Aorta; Bone with marrow (sternebrae); Brain; Coagulating glands (2); Eyes with optic nerve (2); Gastrointestinal tract; Esophagus; Stomach; Duodenum; Peyer’s Patches; Jejunum; Ileum; Cecum; Colon; Rectum; Heart; Kidneys (2); Lacrimal/Harderian glands (2); Liver (sections of 2 lobes); Lymph node (axillary [2], mandibular [2], and mesenteric); Lungs (including bronchi, fixed by inflation with fixative); Levator ani and bulbocavernosus (LABC) muscle group; Ovariesc and oviducts (2); Pancreas; Peripheral nerve (sciatic); Pituitary; rostate; Mandibular salivary gland (2); Seminal vesicles (2); Skeletal muscle (quadriceps); Skin with mammary gland; Spinal cord (cervical); Spleen; Testes with epididymides (2) and Vass deferens; Thymus; Thyroids with parathyroids, if present (2); Trachea; Urinary bladder; Uterus with cervix and vagina; All gross lesions (all groups)) were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

Histology
Ovaries were processed at PAI Durham. All other histology procedures were performed at the Testing Facility. Tissues identified in Text Table 33 from F1 animals in Cohort 1A in the control and high-dose groups, as well as gross lesions from all animals in all groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Five (5) sections were taken approximately 100 µm apart from the inner third of each ovary from all F1 Cohort 1A females at the scheduled termination. In addition, a single section was taken from all F1 Cohort 1A females per dosage level for a qualitative bilateral evaluation of each ovary.
The coagulating glands, ovaries, pituitary gland, prostate gland, seminal vesicles, testes with epididymides, uterus with cervix and vagina, and gross lesions from Cohort 1B animals were processed to block stage (in paraffin).

Histopathology (Cohort 1A)
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues for microscopic examination (Adrenal glands (2); Aorta; Bone with marrow (sternebrae); Brain; Coagulating glands (2); Eyes with optic nerve (2); Gastrointestinal tract; Esophagus; Stomach; Duodenum; Peyer’s Patches; Jejunum; Ileum; Cecum; Colon; Rectum; Heart; Kidneys (2); Lacrimal/Harderian glands (2); Liver (sections of 2 lobes); Lymph node (axillary [2], mandibular [2], and mesenteric); Lungs (including bronchi, fixed by inflation with fixative); Levator ani and bulbocavernosus (LABC) muscle group; Ovariesc and oviducts (2); Pancreas; Peripheral nerve (sciatic); Pituitary; rostate; Mandibular salivary gland (2); Seminal vesicles (2); Skeletal muscle (quadriceps); Skin with mammary gland; Spinal cord (cervical); Spleen; Testes with epididymides (2) and Vass deferens; Thymus; Thyroids with parathyroids, if present (2); Trachea; Urinary bladder; Uterus with cervix and vagina; All gross lesions (all groups)) were evaluated from all animals in the control and high dose groups. Gross lesions were examined from all animals in all groups.

Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). When possible, sections of the rete testis were examined in the F1 Cohort 1A males.
For all F1 Cohort 1A females in the control and high-dose groups at scheduled termination, a quantitative histopathologic evaluation of multiple sections was conducted. This examination included enumeration of the total number of primordial follicles. Uterine and ovarian histopathology were considered in light of the terminal estrous stage.

Splenic Lymphocyte Immunophenotyping (Cohort 1A)
The spleen was harvested from 10 F1 animals/sex/group in Cohort 1A, weighed, and cut in half. One-half was placed into chilled Hank’s Balanced Buffer Salt solution with 2 % fetal bovine serum; the remaining half was placed in 10 % neutral buffered formalin. Subsequently the following cells were evaluated: Total T lymphocytes (CD45+ CD3+); Helper T lymphocytes (CD45+ CD3+ CD4+); Cytotoxic T lymphocytes (CD45+ CD3+ CD8+); B lymphocytes (CD45+ CD3- CD45RA+); NK cells (CD45+ CD3- CD161a+); NK-T cells (CD45+ CD3+ CD161a+).
Immunophenotyping results were expressed as relative frequency (%) of total spleen leukocytes for each subset. The absolute lymphocyte count (cells/organ) of each subset was calculated by multiplying each leukocyte subset frequency (%) with the total leukocyte count.

F1 Neuropathology (Cohort 2)
Due to the large number of animals to be perfused for neuropathological assessment, perfusions for Cohorts 2A and 2B were performed over several days. The perfusions were performed such that both sexes and all treatment groups were approximately equally represented across each day. The order of perfusions each day was also counterbalanced by sex and treatment group (i.e., 1 male from Group 1, 1 male from Group 2, 1 male from Group 3, 1 male from Group 4, 1 female from Group 1, etc.).

SACRIFICE
- 12 F1 offsprings/sex/group were sacrificed at PND 22.
- 12 F1 offsprings/sex/group, respectively, were sacrificed at PND 78.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY

Scheduled Euthanasia
All animals were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and perfused in situ with fixative (4 % paraformaldehyde solution in 0.1M phosphate buffer).

Necropsy
Central and peripheral nervous system tissues from all animals were dissected. Any abnormal coloration or lesions of the external brain and spinal cord were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS

Organ Weights and Measurements
The whole brains were removed, weighed (including olfactory bulbs), and the dimensions (length [excluding olfactory bulbs] and width) were recorded.

Tissue Collection and Preservation
Representative samples of the tissues (Brain – olfactory bulbs, cerebral cortex (2 levels), hippocampus/dentate gyrus, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, and medulla oblongata (full coronal section was prepared); Spinal cord – at cervical swellings C3 – C7 and at lumbar swellings T13 – L4; Trigeminal ganglion/nerves; Lumbar dorsal root ganglion at T13 – L4*; Lumbar dorsal root fibers at T13 – L4*; Lumbar ventral root fibers at T13 – L4*; Cervical dorsal root ganglion at C3 – C7*; Cervical dorsal root fibers at C3 – C7*; Cervical ventral root fibers at C3 – C7*; Pituitary gland; Caudal nerves (2)+; Sciatic nerves (2)+; Sural nerves (2)+; Tibial nerves (2)+; Peroneal nerves (2)+; Nasal tissue with olfactory epithelium; Optic nerves; Eyes; Skeletal muscle (gastrocnemius)
* Four to 6 tissues were collected at necropsy; 2 tissues were evaluated microscopically.
Other sites: if deemed necessary) were collected from all animals perfused in situ, unless otherwise indicated.

Histology
Tissues were processed at PAI Durham. The brains from all animals perfused in situ on PND 22 (Cohort 2B) were embedded in paraffin. For all animals perfused in situ on PND 78 (Cohort 2A), the central nervous system tissues (please refer to enlisting depicted above) were embedded in paraffin, and the peripheral nervous system tissues were embedded in glycol methacrylate. The brain and central nervous systems tissues from animals in the control and high-dose groups were further sectioned and stained with hematoxylin and eosin for microscopic evaluation and with Luxol fast blue/Cresyl violet for evaluation of myelin. The peripheral nervous system tissues from Cohort 2A animals in the control and high-dose group were sectioned and stained with hematoxylin and eosin. Cross-sections of peripheral nerves from all dose groups were osmicated and resin-embedded, and sections from the control and high-dose group were further sectioned by ultramicrotomy and stained with toluidine blue for evaluation of myelin.

Neuropathology
Neuropathological evaluation was performed by a board-certified veterinary pathologist. For animals perfused in situ on PND 22 (Cohort 2B), sections from all major brain regions, as well as the trigeminal ganglion and nerve and the pituitary gland, were evaluated from all animals in the control and high-exposure groups.
For all animals perfused in situ on PND 78 (Cohort 2A), tissues identified as described above for microscopic examination were evaluated from all animals in the control and high-exposure groups.

Morphometric Analysis (Cohort 2A)
For Cohort 2A rats at PND 78, microscopic morphometric measurements were performed on highly homologous sections of brain from three of the 10 blocks and stained with LFB/CV. The three slides corresponding to these blocks were scanned using a Hamamatsu Nanozoomer whole slide scanner and imported into the Visiopharm software for measurements. Microscopically, 11 linear morphometric measurements, were taken in a blinded random fashion from control and high dose group animals. These 11 linear measurements were as follows:
• Thickness of the frontal cortex bilaterally. This measurement was taken from the dorsal portion of the cerebral cortex within the coronal section passing through the region of the optic chiasm.
• Thickness of the parietal cortex bilaterally. This measurement was taken from the dorsolateral portion of the cerebral cortex within the coronal section taken through the optic chiasm.
• Diagonal width (maximum cross-sectional width) of the caudate-putamen bilaterally. This measurement was performed on the coronal section taken at the level of the optic chiasm.
• Thickness of the corpus callosum bilaterally just lateral to its midpoint at the level of Layer 2 of the overlying cingulate gyrus. This measurement was taken within the section passing through the optic chiasm.
• Thickness of all hippocampal layers combined, bilaterally, extending from the alveus to just lateral to the inferior blade of the dentate gyrus. This measurement was performed within the section taken at the level of the infundibulum.
• Maximum height of the cerebellum at the level of the deep cerebellar nuclei, extending from the roof of the fourth ventricle to the dorsal surface. Note that Lobules 1 or 10 of the cerebellum (which protrude into the fourth ventricle) may be excluded from measurement if inconsistently present on section.

F1 Immunotoxicology (Cohort 3)
All animals in Cohort 3 (10 rats/sex/group) and Cohort 3A (10 rats in the control group, representing as many litters as possible) were administered a single intravenous immunization of sheep red blood cells (sRBC) via a lateral tail vein on PND 54 to elicit a primary antibody response. Each sRBC injection was composed of 0.5 mL of Earle’s Balanced Salt Solution with HEPES buffer solution and contained approximately 2 x 108 sRBC.
Positive control rats in Cohort 3A also received 10 mL/kg/day (25 mg/kg/day) of cyclophosphamide (CPS) by intraperitoneal injection for 5 consecutive days (PND 54–58) prior to and including the day prior to the scheduled necropsy (PND 59). CPS is a known immune suppressant and was prepared in phosphate-buffered saline at a concentration of 2.5 mg/mL, apportioned into at least 5 separate (daily) aliquots and stored at ≤ -10 ºC until needed. On the day of injection, the CPS was thawed, stored on ice, and mixed prior to injection.
On PND 59 (approximately 120 hours following immunization with sRBC), all animals were euthanized by carbon dioxide inhalation. Approximately 1 mL to 1.5 mL of blood was collected via the inferior vena cava into tubes without anticoagulant. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70 °C. Samples were transferred to the Charles River Immunotoxicology Department; t-cell dependent antibody response analysis was conducted using a sRBC IgM ELISA kit.
Following blood collection, a gross necropsy was conducted on each animal. The thymus and spleen were weighed and retained in 10% neutral-buffered formalin for possible future histopathology.
Statistics:
The number of animals or cages (N) used to calculate the mean (+/- Standard Deviaiton (S.D.)). Where applicable, the litter was used as the experimental unit. Due to the use of significant figures & different rounding conventions inherent in the types of software used, the means and S.D. on summary & 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 following the mating period. Statistical analyses were not performed on F0 female weekly body weight data after 1 or more animals had entered the gestation phase or remained in the lactation phase.
All statistical tests for the following parameters were performed using WTDMS™. Analyses were conducted using two tailed tests (except as noted otherwise) for minimum significance levels of 1% & 5%, comparing each test substance exposed group to the control group by sex. Male & female mating, fertility, copulation, & conception indices were analyzed using the Chi square test with Yates’ correction factor.
Clinical signs:
no effects observed
Description (incidence and severity):
No test substance related clinical findings were noted during the generation at the daily examinations. Findings noted in the test substance-exposed groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related effects on survival for F0 parental animals. In the control group, Female No. 6888 was found dead on LD 6; there were no clinical observations noted for this female prior to death. The cause of death was septicemia; this animal had inflammation of the uterus and vagina, and suppurative necrotizing vascular inflammation and thromboses in the lungs. Female No. 6876 in the control group was euthanized in extremis on Study Day 118 due to impaired use of hindlimbs, swollen trunk, hunched posture, and red material around the eyes. The cause of death was necrosis of the mammary gland with coagulative necrosis and dystrophic mineralization with no inflammatory cell component. All other F0 animals survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower mean body weight gains were noted for F0 males at 8000 ppm when the premating (Study Days 0–70) and entire treatment (Study Day 0–131) periods were evaluated; differences from the control group were statistically significant. As a result, mean absolute body weights in this group were up to 10.6 % lower than the control group beginning on Study Day 21 and continuing through euthanasia. These differences were considered test substance-related and adverse.
In the 4000 ppm group F0 males, mean body weight gains were sporadically slightly lower than the control group during entire generation. As a result, mean body weight gains in this group were lower than the control group during the premating period (Study Days 0–70) and when the entire treatment period (Study Days 0–131) was evaluated; the difference was statistically significant for the premating period. Mean absolute body weights for males at 4000 ppm were statistically significantly lower (5.1 % to 9.6 %) than the control group during Study Days 35 - 126. However, gradual recovery was noted beginning on Study Day 105 resulting in an absolute mean body weight that was comparable to the control group value on Study Day 131. Therefore, the body weight effects for males at 4000 ppm were considered test substance-related, but not adverse given the recovery evident during the generation.
There were no test substance-related effects on body weights and body weights gains for F0 males at 1500 or F0 females any exposure level; differences from the control were slight and not statistically significant, with the following exceptions. Statistically significantly lower mean body weight gains were noted for F0 females at 4000 and 8000 ppm compared to the control group when the pre-mating period (Study Days 0–70) was evaluated. These differences did not result in substantial effects on mean absolute body weights in these group, and therefore were not considered test substance-related.

Gestation
Mean maternal body weights and body weight gains were unaffected by test substance exposure during gestation. Differences between the control, 1500, 400, 8000 ppm were slight and not statistically significant, with the following exceptions. Mean maternal body weights for females in the 8000 ppm were 4.5 % lower than the control group on Gestation Day 0; statistical significance was not achieved. Thereafter, slightly lower mean body weight gains were noted for F0 females in the 8000 ppm between Gestation Days 0 and 11 which resulted in statistically significantly lower (5.7 % to 6.4 %) mean absolute body weights for females in this group compared to the control group during Gestation Days 7 through 20. However, because the difference versus controls and versus Gestation Day 0 was marginal, these slightly lower body weights at 8000 ppm were considered incidental and not test substance-related.

Lactation
Mean maternal body weights and body weight gains were unaffected by test substance exposure during lactation. Differences between the control, 1500, 4000, and 8000 ppm groups were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 8000 ppm group F0 males, mean food consumption, evaluated as g/animal/day, and food efficiency were sporadically lower than the control group during the premating (Study Days 0 -70) and postmating (Study Days 84–131) periods. Differences from the control group were frequently statistically significant and corresponded with lower mean body weights and body weight gains in this group, and therefore were considered test substance-related and adverse.
Food consumption, evaluated as g/animal/day, and food efficiency in the 1500 and 4000 ppm group F0 males and females and in the 8000 ppm group F0 females was unaffected by test substance exposure. Any statistically significant differences from the control group were transient, did not occur in a dose-related manner, and therefore were not considered test substance-related.

Gestation
Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance exposure during gestation. Differences between the control, 1500, 4000, and 8000 ppm groups were slight and not statistically significant.

Lactation
Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance exposure during lactation. Differences between the control, 1500, 4000, and 8000 ppm groups were slight and not statistically significant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption, evaluated as g/animal/day, in the 1500, 4000, and 8000 ppm group F0 males and females was unaffected by test substance exposure. Sporadically, statistically significant differences from the control group were achieved but only isolated intervals were affected, and sustained differences were not observed at any exposure level. Therefore, any differences were not considered test substance-related.

Gestation
Mean maternal water consumption, evaluated as g/animal/day, was unaffected by test substance exposure during gestation. Differences between the control, 1500, 4000, and 8000 ppm groups were slight and not statistically significant, with the following exception. Statistically significantly lower mean water consumption was noted in the 8000 ppm group females when the overall gestation period (Gestation Days 0–20) was evaluated. Water consumption in this group was marginally lower, and therefore was not considered test substance-related.

Lactation
Mean maternal water consumption, evaluated as g/animal/day, was unaffected by test substance exposure during lactation. Differences between the control, 1500, 4000, and 8000 ppm groups were slight and not statistically significant. It should be noted that limited water consumption data was available during Lactation Days 14–21 due to water spillage.

Test substance Consumption:
Mean compound consumptions (mg/kg/day) were based on theoretical water concentrations of the test substance and are presented below.
theroretical Water Concentration (ppm): 1500
=> Mean Test substance Consumption (mg/kg/day) - Males (Prior to Mating/After Mating): 176 / 105 and Females (Prior to Mating/Gestation/Lactation (LD 1-4)/Lactation (LD4-14)): 211 / 191 / 241 / 139
theroretical Water Concentration (ppm): 4000
=> Mean Test substance Consumption (mg/kg/day) - Males (Prior to Mating/After Mating): 514 / 300 and Females (Prior to Mating/Gestation/Lactation (LD 1-4)/Lactation (LD4-14)): 586 / 531 / 672 / 333
theroretical Water Concentration (ppm): 8000
=> Mean Test substance Consumption (mg/kg/day) - Males (Prior to Mating/After Mating): 1012 / 558 and Females (Prior to Mating/Gestation/Lactation 8LD 1-4)/Lactation (LD4-14)): 1203 / 1041 / 1321 / 707
(Limited water consumption data was available during Lactation Days 14–21 due to water spillage; and therefore, this interval was not summarized above).
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on hematology parameters.
There were no test substance-related effects on coagulation parameters. Any differences between the control and 1500, 4000, and 8000 ppm groups were not dose-responsive and were attributed to natural variation.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on serum chemistry parameters. Any differences between the control and 1500, 4000, and 8000 ppm groups were not dose-responsive and were attributed to natural variation.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on urinalysis parameters. Differences between the control, 1500, 4000, and 8000 ppm groups were slight and not statistically significant, with the following exceptions. Statistically significantly lower mean urine volume was noted for F0 males in the 8000 ppm group; while lower volume can be indicative of dehydration, specific gravity for the same animals was noted to be statistically significantly higher than the control group. There were no test substance-related effects on urine volume or specific gravity on F0 females at 8000 ppm or for F1 males and females at the same exposure level on PND 91. Therefore, these differences were considered spurious and not test substance-related.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no 2-pyrrolidone-related histologic changes in the F0 males or females. Any histologic changes were considered to be incidental findings unrelated to administration of 2 pyrrolidone. There was no 2-pyrrolidone related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No test substance related effects were observed on F0 sperm parameters (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage concentration. Differences from the control group were slight and were not statistically significant, with the following exceptions. Statistically significantly lower mean testicular sperm concentrations and sperm production rates were noted in the 1500 and 8000 ppm groups compared to the control group. These differences were not dose responsive, and therefore were not considered test substance-related.
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance related effects on F0 reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test substance exposed groups. Three, 3, 2, and 2 males in the control, 1500, 4000, and 8000 ppm groups, respectively, did not sire a litter. Three, 3, 2, and 2 females in these same respective groups were determined to be nongravid.
The mean numbers of days between pairing and coitus in the test substance-exposed groups were similar to the control group value. The mean lengths of estrous cycles and estrous cycle status were comparable across all groups. None of these differences were statistically significant.
Gestation and Parturition
No test substance related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F0 gestation lengths in the test substance exposed groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 1500, 4000, and 8000 ppm groups were 21.9, 21.9, and 22.0 days, respectively, compared to mean gestation lengths of 22.0 days in the concurrent control group and 21.8 days in the Charles River Ashland historical control data. No signs of dystocia were noted at any exposure level.
Dose descriptor:
NOAEL
Effect level:
4 000 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
other: food consumption
Remarks on result:
other: corresponds to 514 mg/kg bw/day prior to mating and 300 mg/kg bw after mating based on mean compound intake
Dose descriptor:
NOAEL
Effect level:
8 000 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
other: food consumption
Remarks on result:
other: corresponds to 1203 mg/kg bw/day prior to mating, 1041 during gestation, 1321 mg/kg bw during early lactation (LD days 1-4) and 707 mg/kg bw during late lactation (LD day 4-14) based on mean compound intake
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning:
All F1 parental animals in the control, 1500, 4000, and 8000 ppm groups survived to the scheduled necropsy. No test substance related clinical findings were noted during the generation at the daily examinations. Findings noted in the test substance exposed groups, including hair loss and red material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
F1 Generation Following Weaning:
All F1 parental animals in the control, 1500, 4000, and 8000 ppm groups survived to the scheduled necropsy. No test substance related clinical findings were noted during the generation at the daily examinations. Findings noted in the test substance exposed groups, including hair loss and red material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Following Weaning:
Mean body weight gains for F1 males in the 4000 and 8000 ppm groups were lower than the control group throughout the entire generation (PND 21–98); differences were statistically significant. As a result, mean absolute body weights for males in the 4000 and 8000 ppm groups were up to 7.4 % and 11.4 % lower than the control group, respectively, beginning on PND 63 and 28, respectively; differences were statistically significant. The effects on F1 male body weights in these groups were considered test substance-related and adverse.
In the 1500 ppm group F1 males, statistically significantly lower mean body weight gain was noted compared to the control group during PND 21–28. Mean body weight gains in this group were generally similar to the control group for the remainder of the generation (PND 28–98). The initial decrement in mean body weight gain resulted in statistically significantly lower mean body weight gain compared to the control group when PND 21–56 interval was evaluated. However, the transient decrement for males at 1500 ppm did not affect mean body weight gains when PND 21–77 and 21–98 (entire generation) were evaluated, and therefore was not considered test substance related.
Mean body weight gains for F1 females in the 8000 ppm group were sporadically lower than the control group throughout the postweaning period resulting in statistically significantly lower mean body weight gain when the entire generation (PND 21–98) was evaluated. Mean absolute body weights for F1 females in this group were 5.8 % to 8.8 % lower than the control group during PND 28–98; differences were statistically significant. The effects on body weight for F1 females in the 8000 ppm group were considered test substance-related and adverse.
Mean body weights and body weight gains for F1 females at 1500 and 4000 ppm were unaffected by test substance exposure. Any statistically significant differences from the control group were transient and did not affect mean absolute body weight, and therefore was not considered test substance-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Following Weaning:
Food consumption, evaluated as g/animal/day, and food efficiency in the F1 males in the 4000 and 8000 ppm groups were generally lower than the control group throughout the postweaning period (PND 21–98); differences were sporadically statistically significant. The lower mean food consumption occurred in a dose-related manner, corresponded with lower mean body weights for males in this group, and therefore was considered test substance-related and adverse.
Mean food consumption and food efficiency for F1 males at 1500 ppm and for F1 females at 1500, 4000, and 8000 ppm were unaffected by test substance exposure. Any statistically significant differences from the control group were not considered test substance-related because they were transient, did not occur in a dose-related manner, and/or did not correlate with effects on mean absolute body weights.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning:
Water consumption, evaluated as g/animal/day, in the 1500, 4000, and 8000 ppm groups was unaffected by test substance exposure. The values in the test substance exposed groups were generally similar to the control group for the entire generation. Any statistically significant differences from the control group were noted for isolated intervals and sustained differences were not observed at any exposure level.

Test substance Consumption:
Mean compound consumptions (mg/kg/day) were based on theoretical water concentrations of the test substance and are presented ibelow.
theroretical Water Concentration (ppm): 1500
=> Mean Test substance Consumption (mg/kg/day) - Males (PND 21-35/PND 42-98): 134 / 150 and Females (PND 21-35/PND 42-98): 98 / 181
theroretical Water Concentration (ppm): 4000
=> Mean Test substance Consumption (mg/kg/day) - Males (PND 21-35/PND 42-98): 396 / 423 and Females (PND 21-35/PND 42-98): 273 / 530
theroretical Water Concentration (ppm): 8000
=> Mean Test substance Consumption (mg/kg/day) - Males (PND 21-35/PND 42-98): 827 / 853 and Females (PND 21-35/PND 42-98): 561 / 1002
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning (Cohort 1A):
There were no test substance-related effects on hematology nor on coagluation parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning (Cohort 1A):
There were no test substance-related effects on serum chemistry parameters.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning:
Balanopreputial separation:
Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of balanopreputial separation were 44.2, 44.2, and 45.3 days in the 1500, 4000, and 8000 ppm groups, respectively, when compared to 44.3 in the control group. Mean body weights at the age of attainment were 236.0 g, 236.3 g, and 231.5 g in the same respective groups compared to 246.2 g in the control group. Differences from the control group were slight and not statistically significant, with the following exception. The mean body weight at attainment of balanopreputial separation at 8000 ppm was statistically significantly lower than the control group, but within the Charles River Ashland historical control data range. Therefore, this difference was not considered test substance-related.

Vaginal Patency:
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of vaginal patency were 33.4, 33.5, and 35.2 days in the 1500, 4000, and 8000 ppm groups, respectively, when compared to 33.0 days in the control group. Mean body weights at the age of attainment were 124.6 g, 123.0 g, and 129.2 g in the same respective groups compared to 124.4 g in the control group. The mean age of attainment of vaginal patency at 8000 ppm was statistically significantly higher than the concurrent control group and the maximum mean value in the Charles River Ashland historical control data (34.2 days). This difference was considered spurious and not test substance-related.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital Distance
Statistically significantly higher mean anogenital distance was noted for F1male pups at 4000 and 8000 ppm when evaluated as a function of the mean pup body weight (anogenital distance relative to cube root of pup body weight). This finding was considered spurious based on the marginal difference in absolute mean anogenital distances for males in these groups and because the values were within the Charles River Ashland historical control data range. The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 1500 ppm group were similar to the control group values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areola/Nipple Anlagen
Areolae/nipple anlagen in the F1male pups was unaffected by parental administration of the test substance when evaluated on PND 13. No nipples were observed in male pups at any exposure level.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning:

Cohort 1A:
There were no 2-pyrrolidone-related alterations in organ weights in animals of Cohort 1A.
There were several statistically significant changes in organ weights relative to body weight in males and females, yet these changes were considered to reflect lower absolute group mean final body weights in the 1500, 4000, and 8000 ppm group males and females. In addition, there were statistically significant changes in organ weights that were not considered a mere reflection of changes in final body weights but affected the absolute organ weights as well. However, these statistically significant changes were nonetheless considered unrelated to 2-pyrrolidone and to represent biological variability.

Cohort 1B:
There were no 2-pyrrolidone-related alterations in organ weights in animals of Cohort 1B.
There were 2-pyrrolidone-related lower absolute final body weights in 4000 and 8000 ppm group males and in the 8000 ppm group females. Lower final body weight in these males explained several statistically significant organ weight changes. In addition, there were statistically significant changes in organ weights in males that affected absolute organ weights, and therefore were not considered to be a mere reflection of the changes in final body weight value. However, these changes were nonetheless considered to represent biological variability.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning (Cohort 1A and 1B):
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of 2-pyrrolidone.
There were no 2-pyrrolidone-related effects on ovarian follicle counts in F1 females from Cohort 1A. Any variation in counts between those groups was due to normal biological variability.
Histopathological findings:
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning (Cohort 1A):
There were no 2-pyrrolidone-related histologic changes in the F1 males or females of Cohort 1A (PND 91). Any histologic changes were considered to be incidental findings unrelated to administration of 2-pyrrolidone. There was no 2-pyrrolidone-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Description (incidence and severity):
F1 Generation Following Weaning:
Estrous Cycle Data (Cohort 1A)
The mean ages at the first occurrence of estrus in the 1500, 4000, and 8000 ppm groups (35.8, 35.9, and 35.8 days, respectively) were generally comparable to the control group (36.1 days). In addition, the duration from vaginal opening to first estrus in these same respective groups (3.1, 3.1, and 2.6 days) was generally comparable to the control group (3.8 days). None of the differences were statistically significant.
The mean lengths of estrous cycles and cycle status in the test substance-treated groups from PND 75-91 were also similar to the control group value. None of these differences were statistically significant.

Sperm Evaluations (Cohort 1A):
No test substance related effects were observed on F1 sperm parameters (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage concentration. Differences from the control group were slight and were not statistically significant, with the following exception. A statistically significantly lower mean cauda epididymal concentration was noted at 8000 ppm compared to the control group. This difference was not considered test substance-related because it was non dose-responsive and occurred in the absence of any effects on cauda epididymal weight in this group.

Thyroid Hormone Analysis (Cohort 1A):
There were no test substance-related effects on thyroid hormones. Mean TSH levels across all treated groups for males and females appeared higher than the control group, however, these differences were not statistically significant, occurred in a non-dose responsive fashion (females) and were attributed to atypically low control group values (5.2 and 4.0 ng/mL for males and females, respectively) compared to the mean values in the Charles River Ashland historical control data (10.2 and 5.5 ng/mL for males and females, respectively).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
F1 Generation Following Weaning - Sensory Function and Neurobehavioral Testing (Cohort 2A):

Auditory Startle Response (Cohort 2A):
The auditory startle response habituation paradigm was conducted as a longitudinal assessment with selected F1 animals evaluated on PND 23. Administration of 1500, 4000, and 8000 ppm had no significant effect on auditory startle responsiveness. The PEAK and Tpeak values for all 5 epochs (trials 1–10, 11–20, 21–30, 31–40, and 41–50) and the overall 50-trial test session were similar in the F1 males and females among the test substance-exposed and the control groups, with the following exceptions. Statistically significantly lower PEAK values were noted for F1 males at 1500 and 8000 ppm compared to the control group for the overall testing period. In the 4000 ppm group F1 males, a statistically significantly higher overall Tpeak value was noted compared to the control group. The aforementioned differences did not occur in a dose-related manner, and therefore were not considered test substance-related. No effects were noted in the pattern of the habituation response over the entire 50-block test session in adult animals.

F1 Generation Following Weaning - Functional Observational Battery (Cohort 2A):
Home Cage Observations
Home cage parameters were unaffected by test substance administration. Differences between the test substance-exposed F1 males and females and the control group on PND 65 were slight and not statistically significant, with the following exceptions. Statistically significantly higher numbers of F1 females in the 4000 ppm group were asleep, lying on side or curled up and had eyelids completely closed compared to the control group. These findings are considered normal home cage observations and occurred in a non-dose-related manner, and therefore were not considered test substance-related.

Handling Observations
Handling parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-exposed F1 males and females when compared to the control group on PND 65.

Open Field Observations
Home cage parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-exposed F1 males and females when compared to the control group on PND 65.

Sensory Observations
Sensory parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-exposed F1 males and females when compared to the control group on PND 65, with the exception of the aforementioned lower mean body weights for males in the 4000 and 8000 ppm groups and females in the 8000 ppm group.

Neuromuscular Observations
Neuromuscular parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-exposed F1 males and females when compared to the control group on PND 65.

Physiological Observations
Physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-exposed F1 males and females when compared to the control group on PND 65.

F1 Generation Following Weaning - Locomotor Activity (Cohort 2A)
Locomotor activity patterns (total activity as well as ambulatory activity counts) in F1 males and females were unaffected by maternal test substance exposure at all concentrations when evaluated on PND 65. Values obtained from the 6 epochs evaluated (0–10, 11–20, 21–30, 31 40, 41–50, and 51–60 minutes) and the overall 60 minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant, with the following exception. Higher mean total and ambulatory counts were noted in the 1500 ppm group males and females compared to the control group throughout the test session; the difference was statistically significant for the overall test session. However, the values were within the Charles River Ashland historical control data ranges and the difference did not occur in a dose related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance exposed groups when the F1 animals were evaluated at PND 65.
Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Generation Following Weaning - Immunophenotyping (Cohort 1A):
There were no test substance-related effects on mean absolute counts of total T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes, NK cells, and NK-T cells in the F1 generation males and females following exposure to 2-pyrrolidone in utero, through nursing during lactation, or following direct exposure via drinking water following weaning, until the day of scheduled necropsy on PND 91.
Moderately lower mean B lymphocyte values were noted in the 8000 ppm group males (-45.2 %) and females (-37.1 %) when compared to the control group on PND 91. However, these changes were not considered 2-pyyrolidone-related, but most likely due to natural variations because the changes observed when compared to the low-dose group were minimal. In addition, these changes did not correlate with any significant changes in the sRBC-IgM concentrations.
In males, minimal to mild decreases were observed in the 8000 ppm group including a mild decrease (29.9 % to 33.6 % compared to the control control) in the total T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, and NK cells on PND 91. However, these changes were not considered 2-pyrrolidone-related, but rather attributed to unusually low values in these leukocyte subsets observed in 1 of 10 males (No. 6910-05) for the total T lymphocytes, helper T lymphocytes, and cytotoxic T lymphocytes, or 2 of 10 males (Nos. 6895-04 or 6910-05) for the NK cells observed in the 8000 ppm group. In addition, similar magnitude of changes were not noted in the 8000 ppm group females in any of these leukocyte subsets.
F1 Generation Following Weaning - Brain Weights and Measurements (Cohort 2A):
There were no 2-pyrrolidone related changes in the brain weights or measurements in Cohort 2A (PND 78) animals.
There were 2-pyrrolidone-related lower group mean final body weight in Cohort 2A animals, including 4000 and 8000 ppm group males and 8000 ppm group females. These changes in final body weight had no effect on the brain weights or gross brain measurements.

F1 Generation Following Weaning - Neurohistopathological Examination (Cohorts 2A and 2B):
There were no 2-pyrrolidone-related microscopic findings in any of the tissues evaluated in the neurohistopathology Cohorts 2A (PND 78) or 2B (PND 22) animals.

F1 Generation Following Weaning - Neuropathology (Cohorts 2A and 2B:
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of 2-pyrrolidone.

F1 Generation Following Weaning - Brain Weights and Measurements (Cohort 2B):
There were no 2-pyrrolidone related changes in the brain weights or measurements in Cohort 2B (PND 22) animals.

F1 Generation Following Weaning - Morphometric Analysis (Cohort 2A):
There were no 2-pyrrolidone-related effects on the morphometry measurements in F1 males or females of Cohort 2A. There were statistically significant changes in several group mean measurements, but these were considered incidental. Included in these changes were higher S1 (thickness of frontal cortex) in 4000 and 8000 ppm females, higher S2 and S3 (thickness of parietal cortex and width of caudate-putamen, respectively) in 1500 and 4000 ppm females, lower S5 (thickness of hippocampus) in 4000 ppm males, and lower S6 (height of cerebellum) in 1500 and 8000 ppm males. The higher measurements were in a direction opposite to that normally associated with developmental toxicity, and none of the changes (higher or lower) followed a dose response profile. Therefore, all statistically significant changes in measurements were considered incidental and represented biological variability.

F1 Generation Following Weaning - T-Cell Dependent Antibody Response (TDAR) Assay (Cohort 3):
Subsequent to immunization with sRBC, the CPS (a known immunosuppressant)-treated positive control group males and females (Cohort 3A) had statistically significant, markedly lower mean sRBC IgM concentrations (-98.0 % and -97.3 %, respectively) when compared to the control group. Immunosuppressive changes noted in the positive control group were indicative of the pharmacodynamic effect of cyclophosphamide and demonstrated the acceptability of the sRBC IgM ELISA assay to detect a decrease in the T cell-mediated response to an antigen.
However, compared to the control group, there were no significant changes in the sRBC-IgM concentration in any of the test substance-treated groups. Even though mild changes were noted in the 8000 ppm group males and females, these were considered due to natural variations because the results were variable and contrary between both sexes. In males, a mild increase was observed, while in females, a mild decrease was observed.
Based on comparison of the data from the CPS positive control group, which demonstrated a suppression of immune response as indicated by almost a complete ablation of the sRBC-IgM responses and based on the lack of 2-pyrrolidone-related findings in the immunophenotypic parameters, it can be concluded that there was no effect related to test substance exposure on the humoral immune system.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 500 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
other: food consumption
Remarks on result:
other: corresponds to 134 mg/kg bw/d for PND 21–35 and 150 mg/kg bw for PND 42–98 based on mean compound intake
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
4 000 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
other: food consumption
Remarks on result:
other: corresponds to 273 mg/kg bw/d for PND 21–35 and 530 mg/kg bw for PND 42–98 based on mean compound intake
Critical effects observed:
no
Reproductive effects observed:
no

F1 Generation Prior to Weaning

PND Litter data and postnatal survival

The mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre‑selection), 4 (post-selection)–7, 7–14, 14–21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection)–21 were unaffected by the test substance at all dosage concentrations. Differences from the control group were slight, were not statistically significant, and/or did not occur in a dose‑related manner.

             

Observations

The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance exposure. Twenty-one (13), 10(8), 17 (11), and 10(8) pups (litters) in the control, 1500, 4000, and 8000 ppm groups, respectively, were found dead or euthanized in extremis. Eight (6), 7(7), 3(2), and 4(4) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. There were no remarkable clinical observations noted during the preweaning period, with the exception that a marginally higher number of pups in the 8000 ppm group were noted with small stature compared to the control group. These findings correlated with the lower body weights for pups in this group.

Offspring body weights

Mean male and female pup birth weights (PND 1) were slightly lower (2.9 % to 4.3 %) than the control group for the 4000 and 8000 ppm groups; differences were not statistically significant. These differences were accompanied by statistically significantly lower mean body weight gains for male and female pups during PND 1–4. As a result, mean absolute male and female body weights in the 4000 and 8000 ppm groups were 7.6 % to 8.0 % lower than the control group on PND 4; differences were statistically significant for males in both groups. Thereafter, mean body weight gains for pups in these groups were generally slightly lower or comparable to the control group and an overall slow recovery was noted in absolute mean body weights; mean pup body weights were within approximately 5% of the control means on PND 21. The initial decrements in mean pup body weight gains for males and females at 4000 and 8000 ppm were considered test substance-related, but nonadverse due to the transient nature of the effects.

Mean body weights and body weight changes in the 1500 ppm group were unaffected by test substance exposure throughout the postnatal period. No statistically significant differences from the control group were noted.

Developmental Landmarks

Anogenital Distance

Statistically significantly higher mean anogenital distance was noted for F1male pups at 4000 and 8000 ppm when evaluated as a function of the mean pup body weight (anogenital distance relative to cube root of pup body weight). This finding was considered spurious based on the marginal difference in absolute mean anogenital distances for males in these groups and because the values were within the Charles River Ashland historical control data range. The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 1500 ppm group were similar to the control group values.

Areola/Nipple Anlagen

Areolae/nipple anlagen in the F1male pups was unaffected by parental administration of the test substance when evaluated on PND 13. No nipples were observed in male pups at any exposure level.

Thyroid hormone analysis

PND 4 culled pups

There were no test substance-related effects on mean serum concentrations of T4 and TSH for PND 4 culled pups at any exposure level. Differences from the control group were slight and not dose-responsive.

PND 21 Pups selected for hormone analysis

There were no test substance-related effects on mean serum concentrations of T4 and TSH for nonselected male and female pups on PND 21 at any exposure level. Differences from the control group were slight and not dose-responsive.

Gross pathology

Unscheduled deaths

Twenty-one (13), 10(8), 17 (11), and 10(8) pups (litters) in the control, 1500, 4000, and 8000 ppm groups, respectively, were found dead or euthanized in extremis from PND 0 through the selection of the F1generation. No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead or euthanized in extremis. Aside from the presence or absence of milk in the stomach, internal findings included distended intestines (duodenum, jejunum, and ileum) in Pup No. 6898-09 in the 4000 ppm group, a dilated renal pelvis in Pup No. 6931-15 in the 1500 ppm group, and a malformation consisting of absent intestines (duodenum, jejunum, ileum, and cecum) in Pup No. 6879-01 in the control group. These findings were noted infrequently and in a manner that was not dose-related, and therefore were not considered test substance-related.

PND 4 Culled pups

No internal findings were noted at the necropsy of culled pups euthanized on PND 4.

PND 21 Nonselected pups

No internal findings that could be attributed to parental exposure with the test substance were noted at the necropsy of pups euthanized on PND 21. Internal findings included dilated renal pelvis for Pup No. 6881-04 in the 1500 ppm group.

PND 21 Selected Pups for Hormone Analysis

No internal findings were noted at the PND 21 necropsy of F1weanlings selected for organ weights at any exposure level.

Organ weights

No test substance‑related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for F1pups on PND 21 at any dosage level when the test substance‑exposed groups were compared to the control group. Any statistically significant differences from the control group were not dose responsive, and therefore were not considered test substance-related.

Discussion

The objective of this study was to evaluate the potential adverse effects of the test substance on reproduction in an extended one-generation study. This included the evaluation of life stages not covered by other types of toxicity studies and test for effects that may occur as a result of pre- and postnatal chemical exposure.

In the F0generation, lower mean body weight gains with corresponding sporadically lower mean food consumption values were noted for F0males at 8000 ppm throughout the entire generation (Study Days 0‑131) and mean absolute body weights in this group were up to 10.6 % lower than the control group during the exposure period; these differences were considered test article related and adverse. In the 4000 ppm group F0males, mean body weight gains were sporadically lower than the control group resulting in lower mean body weight gains for the premating period (Study Days 0–70) and lower (5.1 % to 9.6 %) mean absolute body weights during Study Days 35‑126. However, gradual recovery of body weights was noted in this group beginning on Study Day 105 resulting in an absolute group mean body weight that was comparable to the control group value at the end of the generation (Study Day 131). Therefore, given the evidence of recover during the generation, the body weight effects noted for F0males in the 4000 ppm were considered test substance-related, but not adverse. 

In the F1generation, lower mean body weight gains with corresponding sporadically lower mean food consumption were noted for F1males in the 4000 and 8000 ppm groups compared to the control group throughout the entire generation (PND 21–98). As a result, mean absolute body weights for F1males in these groups were lower (up to 7.4 % and 11.4 %, respectively) than the control group beginning on PND 63 and 28, respectively. In F1females in the 8000 ppm group, sporadically lower mean body weight gains were noted throughout the postweaning period (PND 21‑98) resulting in lower mean body weight gains and lower (5.8 % to 8.8 %) mean absolute body weights during PND 28‑98. These effects in F1male and female body weights and F1male food consumption were considered test substance-related and adverse.

In F1generation males and females (Cohort 3),there were no 2-pyrrolidone-related effects on immunophenotyping parameters. In addition, there were no 2‑pyrrolidone-related effects on the humoral immune system as indicated by lack of any changes in the sRBC-IgM responses in the T-cell Dependent Antibody Response (TDAR) Assay (Cohort 1A).

With the exception of the abovementioned effects on F0male body weights at 8000 ppm, F1male body weights at 4000 and 8000 ppm, F1female body weights at 8000 ppm, and lower B lymphocytecounts[WF1] in F1males and females, there were no other adverse effects noted on this study. Reproductive endpoints in the F0 and F1 generations and neurotoxicity endpoints in the F1 generation were unaffected by administration of 2-pyrrolidone orally (via drinking water) to Crl:CD(SD) rats at 1500, 4000, and 8000 ppm.

Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, as described in the OECD Guidance Document 117, a second generation was not assessed on this study.

CONCLUSIONS

In conclusion, there was no test substance-related mortality or moribundity noted at any dosage level. Adverse effects on body weights, body weight gain, and food consumption were noted for F0males at 8000 ppm. Therefore, exposure levels of 4000 ppm and 8000 ppm were considered the no-observed-adverse-effect levels (NOAEL) for F0male and female systemic toxicity, respectively, when 2-pyrrolidone was administered orally (via drinking water) to Crl:CD(SD) rats. During the F1generation, lower mean body weights and body weight gains with corresponding decrements in food consumption were noted for F1males at 4000 and 8000 ppm and for F1female at 8000 ppm when the entire exposure period (PND 21–98) was evaluated. Therefore 1500 ppm and 4000 ppm, respectively, were considered the NOAEL for F1male and female systemic toxicity.

There was no evidence of reproductive toxicity at any exposure level based on evaluation of reproductive performance in the F0generation and sperm measurement and estrous cyclicity in the F0and F1generations. Therefore, the NOAEL for F0and F1reproductive toxicity was considered to be 8000 ppm. Due to the lack of adverse effects noted for F1litters, an exposure level of 8000 ppm was considered the NOAEL for neonatal toxicity.

There were no effects on F1behavioral development, brain weights and linear measurements, macroscopic and microscopic findings, and brain morphometry. Based on these results, the NOAEL for F1neurotoxicity was considered to be 8000 ppm. There were no test substance‑related effects on F1 splenic lymphocyte immunophenotyping or the humoral immune system, and therefore 8000 ppm was considered the NOAEL for F1 immunotoxicity when 2‑pyrrolidone was administered orally (via drinking water) to Crl:CD(SD) rats.

Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, as described in the OECD Guidance Document 117, a second generation was not assessed on this study.

Conclusions:
In conclusion, there was no test substance-related mortality or moribundity noted at any dosage level. Adverse effects on body weights, body weight gain, and food consumption were noted for F0 males at 8000 ppm. Therefore, exposure levels of 4000 ppm and 8000 ppm were considered the no-observed-adverse-effect levels (NOAEL) for F0 male and female systemic toxicity, respectively, when 2-pyrrolidone was administered orally (via drinking water) to Crl:CD(SD) rats. During the F1 generation, lower mean body weights and body weight gains with corresponding decrements in food consumption were noted for F1 males at 4000 and 8000 ppm and for F1 female at 8000 ppm when the entire exposure period (PND 21–98) was evaluated. Therefore 1500 ppm and 4000 ppm, respectively, were considered the NOAEL for F1 male and female systemic toxicity.
There was no evidence of reproductive toxicity at any exposure level based on evaluation of reproductive performance in the F0 generation and sperm measurement and estrous cyclicity in the F0 and F1generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 8000 ppm. Due to the lack of adverse effects noted for F1 litters, an exposure level of 8000 ppm was considered the NOAEL for neonatal toxicity.
There were no effects on F1 behavioral development, brain weights and linear measurements, macroscopic and microscopic findings, and brain morphometry. Based on these results, the NOAEL for F1 neurotoxicity was considered to be 8000 ppm. There were no test substance‑related effects on F1 splenic lymphocyte immunophenotyping or the humoral immune system, and therefore 8000 ppm was considered the NOAEL for F1 immunotoxicity when 2‑pyrrolidone was administered orally (via drinking water) to Crl:CD(SD) rats.
Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, as described in the OECD Guidance Document 117, a second generation was not assessed on this study.
Executive summary:

The objective of this study was to evaluate the potential adverse effects of the test substance 2 -pyrrolidone on reproduction in an extended one-generation study. This included the evaluation of life stages not covered by other types of toxicity studies and test for effects that may occur as a result of pre- and postnatal chemical exposure.

The study design was as follows:

Table1 - Experimental Design

GroupNumber

Treatment

Target DosageLevela,b(ppm)

Number of Animals

Males

Females

1

Vehicle

0

30

30

2

2-pyrrolidone

1500

30

30

3

2-pyrrolidone

4000

30

30

4

2-pyrrolidone

8000

30

30

 

a  No correction factor was used.

b  Dosage levels for F0females that delivered were reduced by 50 % (750, 2000, and 4000 ppm) beginning on Lactation Day (LD) 4 and continuing until euthanasia to account for approximately 2X higher maternal water consumption (and consequently higher test substance consumption) which is normally associated with the higher caloric demands of milk production. During the first 2 weeks of the postweaning period (Postnatal Day [PND] 21–35), the dosage levels for F1 pups were also reduced by 50 % to account for higher pup growth rates and food efficiency (body weight gained as a percentage of food consumed) which can also result in higher than expected test substance consumption. Beginning on PND 36, all F1 pups received drinking water formulations at target dosage levels of 1500, 4000, and 8000 ppm.

Animals were administered the test substance continuously in the drinking water for at least 70 consecutive days prior to mating and continuing through the day of euthanasia. The test substance was administered to the offspring selected to become the F1parental generation beginning at weaning and continuing until euthanasia.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food and water consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, developmental landmarks, neurobehavior, thyroid hormones, clinical pathology, gross necropsy findings, sperm parameters, organ weights and measurements, histopathologic examinations, and neuropathologic and brain morphometric examinations.

F1animals were further subdivided into cohorts following weaning and specifically evaluated for the following: Cohort 1 for reproductive/developmental toxicity (including estrous cycles and sperm evaluation), Cohort 2 for developmental neurotoxicity (including neurobehavior, neuropathology, and brain morphometry), Cohort 3 for immunotoxicity testing.

The average quantities of test substance consumed (mg/kg/day) during the F0and F1generations are presented below:

Table2 - Mean Test Substance Consumption

Theoretical Water Concentration (ppm)

Mean Test Substance Consumption (mg/kg/day)

F0Males

F0Females

Prior to Mating

After Mating

Prior to Mating

Gestation

Lactation
(LD 1–4)

Lactation
(LD 1–14)a
 

1500

176

105

211

191

241

139

4000

514

300

586

531

672

333 

8000

1012

558

1203

1041

1321

707 

F1Generation

F1Males

F1Females 

 

PND 21–35

PND 42–98

PND 21–35

PND 42 -98

1500

134

150

98

181

4000

396

423

273

530

8000

827

853

561

1002 

a Limited water consumption data was available during Lactation Days 14–21 due to water spillage and therefore, this interval was not summarized above

There were no test substance-related effects on survival for F0 and F1 animals at any exposure level. In the control group, 1 female was found dead on Lactation Day (LD) 6 and 1 female was euthanized on Study Day 118 due to impaired use of hindlimbs, swollen trunk, red material around the nose, and hunched posture. All other F0 and F1 animals survived to the scheduled necropsy. There were no test substance-related clinical observations noted for F0 and F1 animals at any exposure level.

Lower mean body weight gains with corresponding sporadically lower mean food consumption values were noted for F0 males at 8000 ppm throughout the entire generation (Study Days 0‑131). As a result, mean absolute body weights in this group were up to 10.6 % lower than the control group beginning on Study Day 21 and continuing through euthanasia; these differences were considered test article related and adverse. 

In the 4000 ppm group F0 males, mean body weight gains were sporadically lower than the control group during the entire generation resulting in lower mean body weight gain for the premating period (Study Days 0–70) and lower (5.1 % to 9.6 %) mean absolute body weights during Study Days 35‑126. However, gradual recovery was noted beginning on Study Day 105 resulting in an absolute group mean body weight that was comparable to the control group value at the end of the generation (Study Day 131). Therefore, the body weight effects for F0 males at 4000 ppm were considered test substance-related, but not adverse given the recovery evident during the generation. F0 mean body weights and body weight gains for males at 1500 ppm and F0 females at 1500, 4000, and 8000 ppm and food consumption and food efficiency for males at 1500 and 4000 ppm and females at 1500, 4000, and 8000 ppm were unaffected by test substance exposure throughout the treatment period, including during gestation and lactation for females. Water consumption for F0 males and females at all exposure levels was comparable to the control group.

No test substance-related effects were noted on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0 or F1 estrous cycling or spermatogenic parameters (testicular and epididymal sperm concentration, sperm production rate, sperm motility, and sperm morphology) at any exposure level.

There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance, or developmental landmarks (areolae/nipple anlagen, vaginal patency, and balanopreputial separation).

Mean male and female F1 pup birth weights (Postnatal Day [PND] 1) at 4000 and 8000 ppm were slightly lower than the control group and were followed by lower mean body weight gains during PND 1–4. As a result, mean absolute male and female body weights in the 4000 and 8000 ppm groups were lower (7.6 % to 8.0 %) than the control group on PND 4. Thereafter, mean body weight gains for pups in these groups were generally slightly lower or comparable to the control group and an overall slow recovery was noted in absolute mean body weights; mean pup body weights were within approximately 5 % of the control means on PND 21. Therefore, the initial decrements in mean pup body weight gains for males and females at 4000 and 8000 ppm were considered test substance-related, but nonadverse due to the transient nature of the effects. Mean F1pup body weights and body weight changes in the 1500 ppm group were unaffected by test substance exposure throughout the postnatal period.

During the postweaning period (PND 21–98), lower mean body weight gains with corresponding sporadically lower mean food consumption were noted for F1males in the 4000 and 8000 ppm groups compared to the control group throughout the entire generation (PND 21–98). As a result, mean absolute body weights for F1males at 4000 and 8000 ppm were lower (up to 7.4 % and 11.4 %, respectively) than the control group beginning on PND 63 and 28, respectively. In the 8000 ppm group F1 females, sporadically lower mean body weight gains were noted throughout the postweaning period resulting in lower mean body weight gains for the entire generation (PND 21‑98) and lower (5.6 % to 8.8 %) mean absolute body weights during PND 28‑98. The aforementioned effects were considered test substance-related and adverse. Mean body weights and body weight gains for F1 males at 1500 ppm and F1 females at 1500 and 4000 ppm and food consumption for F1 males at 1500 ppm and F1 females at all exposure levels were unaffected by test substance exposure.

In F1 generation males and females (Cohort 1), there were no test substance-related effects on mean estrous status, estrous cycle lengths, or spermatogenic parameters at any exposure level.

Also, in F1 generation males and females (Cohort 2), there were no test substance-related effects on auditory startle response, functional observational battery, or locomotor activity at any exposure level. At scheduled termination, there were no test substance-related alterations in brain weights and measurements, or any neurohistopathological findings, or alterations in brain morphometry measurements (PND 78) and any exposure level.

In F1 generation males and females (Cohort 3), there were no 2-pyrrolidone-related effects on immunophenotyping parameters. In addition, there were no 2‑pyrrolidone-related effects on the humoral immune system as indicated by lack of any changes in the sRBC-IgM responses in the T-cell Dependent Antibody Response (TDAR) Assay (Cohort1A).

No test substance-related effects on mean T4 and TSH levels or on serum chemistry, hematology, coagulation, and urinalysis parameters were noted in the F0 or F1 males and females at any exposure level. There were also no test substance-related effects on gross or microscopic pathology findings, or organ weight changes for F0 or F1 animals at any exposure level.

In conclusion, there was no test substance-related mortality or moribundity noted at any dosage level. Adverse effects on body weights, body weight gain, and food consumption were noted for F0 males at 8000 ppm. Therefore, exposure levels of 4000 ppm and 8000 ppm were considered the no-observed-adverse-effect levels (NOAEL) for F0 male and female systemic toxicity, respectively, when 2-pyrrolidone was administered orally (via drinking water) to Crl:CD(SD) rats. During the F1 generation, lower mean body weights and body weight gains with corresponding decrements in food consumption were noted for F1 males at 4000 and 8000 ppm and for F1 female at 8000 ppm when the entire exposure period (PND 21–98) was evaluated. Therefore 1500 ppm and 4000 ppm, respectively, were considered the NOAEL for F1 male and female systemic toxicity.

There was no evidence of reproductive toxicity at any exposure level based on evaluation of reproductive performance in the F0 generation and sperm measurement and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 8000 ppm. Due to the lack of adverse effects noted for F1 litters, an exposure level of 8000 ppm was considered the NOAEL for neonatal toxicity.

There were no effects on F1 behavioral development, brain weights and linear measurements, macroscopic and microscopic findings, and brain morphometry. Based on these results, the NOAEL for F1 neurotoxicity was considered to be 8000 ppm. There were no test substance‑related effects on F1splenic lymphocyte immunophenotyping or the humoral immune system, and therefore 8000 ppm was considered the NOAEL for F1 immunotoxicity when 2‑pyrrolidone was administered orally (via drinking water) to Crl:CD(SD) rats.

Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, as described in the OECD Guidance Document 117, a second generation was not assessed on this study.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 012 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There is an GLP-compliant EOGRTS according to OECD Guideline in rat available, rated with Klimisch 1, so the database is of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 443, GLP

The objective of this study was to evaluate the potential adverse effects of the test substance 2 -pyrrolidone on reproduction in an extended one-generation study. This included the evaluation of life stages not covered by other types of toxicity studies and test for effects that may occur as a result of pre- and postnatal chemical exposure.

The study design was as follows:

Table1 -Experimental Design

GroupNumber

Treatment

Target DosageLevela,b(ppm)

Number of Animals

Males

Females

1

Vehicle

0

30

30

2

2-pyrrolidone

1500

30

30

3

2-pyrrolidone

4000

30

30

4

2-pyrrolidone

8000

30

30

 

a  No correction factor was used.

b  Dosage levels for F0females that delivered were reduced by 50 % (750, 2000, and 4000 ppm) beginning on Lactation Day (LD) 4 and continuing until euthanasia to account for approximately 2X higher maternal water consumption (and consequently higher test substance consumption) which is normally associated with the higher caloric demands of milk production. During the first 2 weeks of the postweaning period (Postnatal Day [PND] 21–35), the dosage levels for F1pups were also reduced by 50 % to account for higher pup growth rates and food efficiency (body weight gained as a percentage of food consumed) which can also result in higher than expected test substance consumption. Beginning on PND 36, all F1pups received drinking water formulations at target dosage levels of 1500, 4000, and 8000 ppm.

Animals were administered the test substance continuously in the drinking water for at least 70 consecutive days prior to mating and continuing through the day of euthanasia. The test substance was administered to the offspring selected to become the F1parental generation beginning at weaning and continuing until euthanasia.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food and water consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, developmental landmarks, neurobehavior, thyroid hormones, clinical pathology, gross necropsy findings, sperm parameters, organ weights and measurements, histopathologic examinations, and neuropathologic and brain morphometric examinations.

F1animals were further subdivided into cohorts following weaning and specifically evaluated for the following: Cohort 1 for reproductive/developmental toxicity (including estrous cycles and sperm evaluation), Cohort 2 for developmental neurotoxicity (including neurobehavior, neuropathology, and brain morphometry), Cohort 3 for immunotoxicity testing.

The average quantities of test substance consumed (mg/kg/day) during the F0and F1generations are presented below:

Table2 -Mean Test Substance Consumption

Theoretical Water Concentration (ppm)

Mean Test Substance Consumption (mg/kg/day)

F0Males

F0Females

Prior to Mating

After Mating

Prior to Mating

Gestation

Lactation
(LD 1–4)

Lactation
(LD 1–14)a
 

1500

176

105

211

191

241

139

4000

514

300

586

531

672

333 

8000

1012

558

1203

1041

1321

707 

F1Generation

F1Males

F1Females 

 

PND 21–35

PND 42–98

PND 21–35

PND 42 -98

1500

134

150

98

181

4000

396

423

273

530

8000

827

853

561

1002 

a Limited water consumption data was available during Lactation Days 14–21 due to water spillage and therefore, this interval was not summarized above

There were no test substance-related effects on survival for F0and F1animals at any exposure level. In the control group, 1 female was found dead on Lactation Day (LD) 6 and 1 female was euthanized on Study Day 118 due to impaired use of hindlimbs, swollen trunk, red material around the nose, and hunched posture. All other F0and F1animals survived to the scheduled necropsy. There were no test substance-related clinical observations noted for F0and F1animals at any exposure level.

Lower mean body weight gains with corresponding sporadically lower mean food consumption values were noted for F0males at 8000 ppm throughout the entire generation (Study Days 0‑131). As a result, mean absolute body weights in this group were up to 10.6 % lower than the control group beginning on Study Day 21 and continuing through euthanasia; these differences were considered test article related and adverse. 

In the 4000 ppm group F0males, mean body weight gains were sporadically lower than the control group during the entire generation resulting in lower mean body weight gain for the premating period (Study Days 0–70) and lower (5.1 % to 9.6 %) mean absolute body weights during Study Days 35‑126. However, gradual recovery was noted beginning on Study Day 105 resulting in an absolute group mean body weight that was comparable to the control group value at the end of the generation (Study Day 131). Therefore, the body weight effects for F0males at 4000 ppm were considered test substance-related, but not adverse given the recovery evident during the generation. F0mean body weights and body weight gains for males at 1500 ppm and F0females at 1500, 4000, and 8000 ppm and food consumption and food efficiency for males at 1500 and 4000 ppm and females at 1500, 4000, and 8000 ppm were unaffected by test substance exposure throughout the treatment period, including during gestation and lactation for females. Water consumption for F0males and females at all exposure levels was comparable to the control group.

No test substance-related effects were noted on F0reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0or F1estrous cycling or spermatogenic parameters (testicular and epididymal sperm concentration, sperm production rate, sperm motility, and sperm morphology) at any exposure level.

There were no test substance-related effects on the number of F1pups born, live litter size, percentage of males at birth, F1postnatal survival, clinical observations, anogenital distance, or developmental landmarks (areolae/nipple anlagen, vaginal patency, and balanopreputial separation).

Mean male and female F1pup birth weights (Postnatal Day [PND] 1) at 4000 and 8000 ppm were slightly lower than the control group and were followed by lower mean body weight gains during PND 1–4. As a result, mean absolute male and female body weights in the 4000 and 8000 ppm groups were lower (7.6 % to 8.0 %) than the control group on PND 4. Thereafter, mean body weight gains for pups in these groups were generally slightly lower or comparable to the control group and an overall slow recovery was noted in absolute mean body weights; mean pup body weights were within approximately 5 % of the control means on PND 21. Therefore, the initial decrements in mean pup body weight gains for males and females at 4000 and 8000 ppm were considered test substance-related, but nonadverse due to the transient nature of the effects. Mean F1pup body weights and body weight changes in the 1500 ppm group were unaffected by test substance exposure throughout the postnatal period.

During the postweaning period (PND 21–98), lower mean body weight gains with corresponding sporadically lower mean food consumption were noted for F1males in the 4000 and 8000 ppm groups compared to the control group throughout the entire generation (PND 21–98). As a result, mean absolute body weights for F1males at 4000 and 8000 ppm were lower (up to 7.4 % and 11.4 %, respectively) than the control group beginning on PND 63 and 28, respectively. In the 8000 ppm group F1females, sporadically lower mean body weight gains were noted throughout the postweaning period resulting in lower mean body weight gains for the entire generation (PND 21‑98) and lower (5.6 % to 8.8 %) mean absolute body weights during PND 28‑98. The aforementioned effects were considered test substance-related and adverse. Mean body weights and body weight gains for F1males at 1500 ppm and F1females at 1500 and 4000 ppm and food consumption for F1males at 1500 ppm and F1females at all exposure levels were unaffected by test substance exposure.

In F1generation males and females (Cohort 1), there were no test substance-related effects on mean estrous status, estrous cycle lengths, or spermatogenic parameters at any exposure level.

Also, in F1generation males and females (Cohort 2), there were no test substance-related effects on auditory startle response, functional observational battery, or locomotor activity at any exposure level. At scheduled termination, there were no test substance-related alterations in brain weights and measurements, or any neurohistopathological findings, or alterations in brain morphometry measurements (PND 78) and any exposure level.

In F1 generation males and females (Cohort 3), there were no 2-pyrrolidone-related effects on immunophenotyping parameters. In addition, there were no 2‑pyrrolidone-related effects on the humoral immune system as indicated by lack of any changes in the sRBC-IgM responses in the T-cell Dependent Antibody Response (TDAR) Assay (Cohort1A).

No test substance-related effects on mean T4and TSH levels or on serum chemistry, hematology, coagulation, and urinalysis parameters were noted in the F0or F1males and females at any exposure level. There were also no test substance-related effects on gross or microscopic pathology findings, or organ weight changes for F0or F1animals at any exposure level.

In conclusion, there was no test substance-related mortality or moribundity noted at any dosage level. Adverse effects on body weights, body weight gain, and food consumption were noted for F0males at 8000 ppm. Therefore, exposure levels of 4000 ppm and 8000 ppm were considered the no-observed-adverse-effect levels (NOAEL) for F0male and female systemic toxicity, respectively, when 2-pyrrolidone was administered orally (via drinking water) to Crl:CD(SD) rats. During the F1generation, lower mean body weights and body weight gains with corresponding decrements in food consumption were noted for F1males at 4000 and 8000 ppm and for F1female at 8000 ppm when the entire exposure period (PND 21–98) was evaluated. Therefore 1500 ppm and 4000 ppm, respectively, were considered the NOAEL for F1male and female systemic toxicity.

There was no evidence of reproductive toxicity at any exposure level based on evaluation of reproductive performance in the F0 generation and sperm measurement and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 8000 ppm. Due to the lack of adverse effects noted for F1 litters, an exposure level of 8000 ppm was considered the NOAEL for neonatal toxicity.

There were no effects on F1 behavioral development, brain weights and linear measurements, macroscopic and microscopic findings, and brain morphometry. Based on these results, the NOAEL for F1 neurotoxicity was considered to be 8000 ppm. There were no test substance‑related effects on F1 splenic lymphocyte immunophenotyping or the humoral immune system, and therefore 8000 ppm was considered the NOAEL for F1 immunotoxicity when 2‑pyrrolidone was administered orally (via drinking water) to Crl:CD(SD) rats.

Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, as described in the OECD Guidance Document 117, a second generation was not assessed on this study.

Furthermore, there is an OECD 421 available for 2.pyrrolidone. The objective of this study was to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through weaning and following direct dosing from postnatal day (PND) 21 through PND 35. The study design was as follows:

4 Dose groups (control group, 3000 ppm, 8000 ppm, 12 000 ppm) consisting each of 10 male and 10 female animals.

Animals were administered the test substance continuously in the drinking water. F0 males were exposed for 13 days prior to mating and continuing until the day of euthanasia. F0 females were exposed for 13 days prior to mating, and continuing through Lactation Day 21. F1 male and female weanlings (10/sex/group) were administered the vehicle or test substance drinking water formulations beginning on PND 21 through euthanasia (PND 35).

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, water consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, thyroid hormones, gross necropsy findings, organ weights, and histopathologic examinations.

Mean F0 test substance consumption was 333, 590, and 1251 mg/kg/day in males and 488, 1082, and 1094 mg/kg/day in females during Study Days 0-7; 321, 834, and 953 mg/kg/day during gestation; and 604, 1820, and 2313 mg/kg/day during lactation in the 3000, 8000, and 12,000 ppm groups, respectively. Mean F1 test substance consumption was 595, 1590, and 2594 mg/kg/day in males and 588, 1716, and 2593 mg/kg/day in females in the 3000, 8000, and 12,000 ppm groups during PND 21–35.

There was no F0 mortality and no remarkable clinical observations noted at any exposure level during the study. A single female in the 12,000 ppm group had a total litter loss on LD 1 and was euthanized.

Lower mean absolute F0 body weights in the 12,000 ppm group and lower mean body weight gains (16.3 % and 17.0 % for the overall Study Day 0–28 interval) in the 8000 and 12,000 ppm group males, respectively, were noted compared to the control group throughout the study, with no corresponding effect on food or water consumption during Study Days 0-7. Lower mean body weight gains were noted in the 8000 and 12,000 ppm group females during Study Days 0–7 (46.7 % and 80 %, respectively) and 7–14 compared to the control group, with corresponding effects on food and water consumption (11.1 % and 26.7 %, respectively) in the 12,000 ppm group females during Study Days 0-7. Lower mean body weight gains and food and water consumption (19.5 %, 15.4 %, and 8.1 %, respectively, at 8000 ppm and 31.7 %, 19.2 %, and 32.4 %, respectively, at12,000 ppm) continued through gestation and lower mean food and water consumption in the 12,000 ppm group females continued during lactation when compared to the control group. Mean body weight gains in the 8000 and 12,000 ppm group females and mean food and water consumption in the 8000 ppm group females were comparable to the control group during lactation. However, due to the decrements in body weight gain noted during the premating period and gestation, mean absolute body weights in the 8000 and 12,000 ppm group females were lower than the control group beginning on Study Day 14 (11.2 % and 9.8 %, respectively) and continuing throughout the gestation and lactation periods (up to 11.0 % and 11.6 %, respectively, at 8000 ppm and up to 15.7 % and 16.2 %, respectively, at 12,000 ppm). Mean absolute body weights in the 8000 ppm group males, and mean absolute body weights, body weight gains, and food and water consumption in the 3000 ppm group males and females were comparable to the control group throughout the study.

In the F0 males, slight exposure-dependent increases in mean T3 and TSH levels were noted at 3000, 8000, and 12,000 ppm groups compared to the control group. Mean T4 levels were unaffected at all dosage levels. There were no effects on F0 reproductive performance (mating, fertility, conception, and copulation indices), estrous cyclicity, precoital interval, gestation length, or the process of parturition. A lower mean number of implantation sites was noted at 12,000 ppm. The mean numbers of implantation sites at 3000 and 8000 ppm and the mean number of unaccounted-for sites at 3000, 8000, and 12,000 ppm were comparable to the control group.

In the F0 females, test substance-related lower spleen and thymus weights (absolute and relative to final body and brain weights) were seen at ≥ 3000 ppm; of uncertain relationship to test substance administration were lower ovaries/oviducts weights (absolute and relative to final body and brain weights) and pituitary gland weights (absolute and relative to brain weight) at 12,000 ppm. Mean organ weights in the 3000, 8000, and 12,000 ppm group males were generally comparable to the control group. Small thymus was noted at necropsy in 2 females from the 12,000 ppm group, and microscopic examination of those 2 tissues revealed moderate lymphoid depletion; those were the only gross and histopathological findings attributed to 2-pyrrolidone administration. No noteworthy macroscopic or microscopic findings were observed in the 3000 and 8000 ppm group males and females or the 12,000 ppm group males.

A lower mean number of F1 pups born, live litter size on PND 0, and postnatal survival (PND 0-1 and from birth to PND 4) were noted at 12,000 ppm. Mean numbers of pups born, live litter size on PND 0, and postnatal survival at 3000 and 8000 ppm and the mean litter proportions of males at birth at 3000, 8000, and 12,000 ppm were comparable to the control group. Lower mean offspring body weights and body weight gains were noted in the 8000 and 12,000 ppm males and females during the preweaning period (beginning on PND 10). Mean body weights were up to 9.6 and 15 % lower in the 8000 and 12,000 ppm group males, and up to 9.8 and 13.1 % lower in the 8000 and 12,000 pp group females, respectively. Mean F1 body weights and body weight gains at 3000 ppm were comparable to the control group.

Anogenital distance and areolae/nipple retention at 3000, 8000, and 12,000 ppm were comparable to the control group. No noteworthy clinical observations were noted in the F1 pups at any exposure level during the preweaning or postweaning periods (PND 0–21 and 21–35, respectively).

The lower mean absolute body weights in the 8000 and 12,000 ppm group males and females continued into the postweaning period. As a result, mean body weights were up to 13.6 % and 15.3 % lower in males and 10.3 % and 13.8 % lower in females in the 8000 and 12,000 ppm groups, respectively, during PND 22-35. Mean F1 body weights and body weight gains at 3000 ppm were comparable to the control group. Mean food and water consumption in the 3000, 8000, and 12,000 ppm group F1 males and females were generally comparable to the control group throughout the postweaning period.

Mean T3 levels at 8000 and 12,000 ppm and TSH levels at 3000, 8000, and 12,000 ppm were lower than the control group for F1 pups on PND 4. Mean thyroid hormone levels on PND 21 were comparable to the control group across exposure levels; therefore, the findings observed on PND 4 were not considered test substance-related. There were no noteworthy macroscopic findings in the F1 males and females at any exposure level on PND 21 or 35.

Mean F1 spleen weights (absolute and relative to final body and brain weights) in the 3000, 8000, and 12,000 ppm group males and females were lower than the control group on PND 21. Lower mean liver and thymus weights (absolute and relative to final body and/or brain weights) were also noted in the 12,000 ppm group males (liver) and females (liver and thymus) on PND 21; the effects on liver weights were attributed to lower final body weights. Lower mean thymus weights (absolute and relative to final body and brain weights) were noted in the 12,000 ppm group males and lower mean spleen and thymus weights (absolute and relative to final body and brain weights) were noted in the 12,000 ppm females on PND 35. Mean organ weights in the 3000 and 8000 ppm group males and females were comparable to the control group on PND 35.

 

Based on the results of this study, dosage levels up to 8000 ppm were considered to be acceptable for an extended one-generation reproductive toxicity of 2-pyrrolidone when administered orally via drinking water to Crl:CD(SD) rats.

Effects on developmental toxicity

Description of key information

1. Bio-Research Laboratories, 1990. An Oral Teratology Study of 2-Pyrrolidone in the Rat. Report No. 83880. GLP, comparable to the OECD guideline 414, rats, gavage, gestation day 6-15, conc.tested: 0, 190, 600 and 1900 mg/kg bw/day
2. Charles River, 2019: An Oral (Gavage) Prenatal Developmental Toxicity study of 2 -Pyrrolidone in Rabbits. Report No.00738024. OPPTS 870.3700, OECD 414, EU method B.31. Rabbits, NewZealand White, oral:gavage, 0, 250, 500, 1000 mg/kg/d on gestation days 7 -28. NOAEL(maternal and prenatal developmental toxicity) = 250 mg/kg/d

3. BASF AG, 1971. Report on the testing of pyrrolidone for any teratogenic effect in the rat after oral administration. Report No. XIX/421. FDA BASF Test. Rats, Sprague Dawley, oral, gavage, 1875 mg/kg bw/day.
4. BASF AG, 1970. Report on the testing of pyrrolidone for any teratogenic effect in the mouse. Report No. XIX/421. FDA BASF Test. Mouse NMRI, oral, gavage, conc. tested: 0, 1279, 3199 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose for cross-reference:
reference to other study
Remarks:
Dose range-finding study
Reason / purpose for cross-reference:
reference to other study
Remarks:
tolerability study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
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: 6-7 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)
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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
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.
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.
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
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
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
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
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
Abnormalities:
no effects observed
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.
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
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
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
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
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.
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)
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
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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study (GLP)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York, U.S.A.
- Age at study initiation: 80-93 d
- Mean weight at study initiation: 231-320 g
- Acclimation period: 14 d
- Diet: Certified Purina Rodent Laboratory Chow No. 5002
- Water: fresh municipal tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: until positive identification of spemtatozoa in the daily vaginal lavage referred to as day 0 of pregnancy
- Verification of same strain and source of both sexes: yes
Duration of treatment / exposure:
gestation day 6 - 15
Frequency of treatment:
daily
Duration of test:
until gestation day 20
Dose / conc.:
190 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
1 900 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes
Maternal examinations:
Animals were checked twice daily for mortality and clinical signs. Pregnant females were examined prior to and following dosing for reactions to treatment, indications of poor health and abnormal behavior from day 6 to day 15 of gestation. Animals were weighed once each week during the acclimatization period and on days 0, 6, 9, 12, 15, 18 and 20 of gestation. Food intake was assessed for all animals on days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 of gestation. On day 20 of gestation, female rats were killed by carbon dioxide asphyxiation followed by exsanguination from the abdominal aorta, each was given a complete gross pathological examination.
Ovaries and uterine content:
The reproductive tract of each female was dissected out, the ovaries removed and the corpora lutea counted. The uterus was weighed. The uterine contents were examined and the number and position of live fetuses, dead fetuses, early (endometrial gland with or without some placental tissue), middle (discernible placental and fetal tissue present) and late (fetal structure apparent) resorptions were recorded. The fetuses were then removed from the uterus for examination. The uterus of any animal judged to be nonpregnant was stained with 10 % aqueous (v/v) ammonium sulphide solution and was then examined for implantation sites.
Fetal examinations:
Each fetus was weighed, given a detailed external examination with external sex being recorded and then killed. A detailed internal examination using a dissecting microscope was performed on approximately one half of the fetuses, selected randomly from each litter, which were then eviscerated. The heads of these fetuses were removed and placed in Bouin’s fluid for examination by the technique of Wilson. The remaining fetuses in each litter were eviscerated; these and the bodies of those fetuses examined internally were placed in 85 % ethanol/15 % methanol for subsequent staining with Alizarin Red S using a modified Dawson technique for skeletal examination. Abnormalities were classified as major malformations, minor visceral or skeletal anomalies or common skeletal variants.
Statistics:
The group mean body weights and body weight gains of animals with live fetuses were calculated. The group mean corrected body weights for day 20 of gestation (body weight on day 20 minus gravid uterus weight) and the corrected body weight gains from day 6 to 20 (corrected body weight day 20 minus body weight day 6) were calculated (Data for non-pregnant animals were not included). These parameters were analyzed using one-way analysis of variance, and where the F value was found to be of significance (P < 0.05), intergroup differences between control and treated groups were examined using Dunnett’s t-test. The group mean live litter size, corpora lutea count, number of implants and number of resorptions were calculated. The individual and group litter mean for the sex ratio and pre-and post-implantation losses were calculated. Statistical analyses were performed using the Kruskal-Wallis test and where the H value was significant (P < 0.05) the Mann-Whitney U-test was used to analyze for differences between control and test groups. The litter mean fetal weights and group mean fetal weights were calculated and statistical analysis was performed using an analysis of variance (one-way classification) and Dunnett’s t-test. The incidences of major malformations and minor anomalies were reported as the number of litters with abnormalities in each group and the number fetuses affected. Statistical analyses comparing the number of litters (containing major malformations) in each test group with the control values were performed using either the chi-square test (with Yates correction factor) or Fisher’s exact probability test. The incidence of minor anomalies was analyzed in the same manner. The incidence of common skeletal variants was reported as the litter mean percentage of fetuses affected. Statistical analyses were performed by comparing the litter mean percentage incidences of each test group with the u-tests.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were seen. The incidence of alopecia showed marked intergroup variation but did not show a dose-related effect.
Mortality:
no mortality observed
Description (incidence):
No animals died during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Between day 6 and day 9 of gestation, the 1900 mg/kg/day group lost weight (P < 0.01) while the body weight gains were significantly (P < 0.01) reduced in the 600 mg/kg/day group. There were significantly (P < 0.01) reduced body weight gains over the day 9 to 12 interval in the 1900 mg/kg/day group. These reduced body weight gains resulted in significantly reduced (P < 0.05 or P < 0.01) body weights from day 9 to 20 of gestation in both the 600 and 1900 mg/kg/day groups. The corrected body weights were significantly (P < 0.01) decreased in the 600 and 1900 mg/kg/day groups by 5 and 8.5 %, respectively, and the corrected body weight gain was decreased significantly (P < 0.01) in the 1900 mg/kg/day group.
Description (incidence and severity):
Over days 6 to 9 and 9 to 12 of gestation, food consumption in both the 600 and 1900 mg/kg/day groups was significantly (P < 0.01) reduced by maximum 7.8 and 17.8 %, respectively. Food consumption continued to be significantly (P < 0.01) reduced over days 12 to 15 of gestation in the 1900 mg/kg/day group only (-11.2 %).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathological examinations revealed no abnormalities related to treatment. There were few incidental findings among animals on the study. These included dilatation of the renal pelvis(es), which was observed in 2 animals of the 190 mg/kg/day group and in one animal of the 600 mg/kg/day group. There were no gross pathological findings in any of the animals of the 1900 mg/kg/day group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
Details on results:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL FINDINGS
No animals died during the study. No treatment-related clinical signs were seen. The incidence of alopecia showed marked intergroup variation but did not show a dose-related effect.

BODY WEIGHT
Between day 6 and day 9 of gestation, the 1900 mg/kg/day group lost weight (P < 0.01) while the body weight gains were significantly (P < 0.01) reduced in the 600 mg/kg/day group. There were significantly (P < 0.01) reduced body weight gains over the day 9 to 12 interval in the 1900 mg/kg/day group. These reduced body weight gains resulted in significantly reduced (P < 0.05 or P < 0.01) body weights from day 9 to 20 of gestation in both the 600 and 1900 mg/kg/day groups. The corrected body weights were significantly (P < 0.01) decreased in the 600 and 1900 mg/kg/day groups by 5 and 8.5 %, respectively, and the corrected body weight gain was decreased significantly (P < 0.01) in the 1900 mg/kg/day group.

FOOD CONSUMPTION
Over days 6 to 9 and 9 to 12 of gestation, food consumption in both the 600 and 1900 mg/kg/day groups was significantly (P < 0.01) reduced by maximum 7.8 and 17.8 %, respectively. Food consumption continued to be significantly (P < 0.01) reduced over days 12 to 15 of gestation in the 1900 mg/kg/day group only (-11.2 %).

GROSS PATHOLOGICAL FINDINGS
Gross pathological examinations revealed no abnormalities related to treatment. There were few incidental findings among animals on the study. These included dilatation of the renal pelvis(es), which was observed in 2 animals of the 190 mg/kg/day group and in one animal of the 600 mg/kg/day group. There were no gross pathological findings in any of the animals of the 1900 mg/kg/day group.
Number of abortions:
no effects observed
Description (incidence and severity):
The pregnancy rate was at least 88.0 % in all groups. Ammonium sulphide staining revealed no other pregnancies.
There were no significant differences between control and treated groups for the following ovarian and uterine parameters: total corpora lutea, total implantation sites, numbers of male and female fetuses, sex ratio, number of live fetuses, number of dead fetuses, early, middle and late resorptions, total resorptions and pre- and post-implantation losses.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no significant differences between control and treated groups for the following ovarian and uterine parameters: total corpora lutea, total implantation sites, numbers of male and female fetuses, sex ratio, number of live fetuses, number of dead fetuses, early, middle and late resorptions, total resorptions and pre- and post-implantation losses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no significant differences between control and treated groups for the following ovarian and uterine parameters: total corpora lutea, total implantation sites, numbers of male and female fetuses, sex ratio, number of live fetuses, number of dead fetuses, early, middle and late resorptions, total resorptions and pre- and post-implantation losses.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no significant differences between control and treated groups for the following ovarian and uterine parameters: total corpora lutea, total implantation sites, numbers of male and female fetuses, sex ratio, number of live fetuses, number of dead fetuses, early, middle and late resorptions, total resorptions and pre- and post-implantation losses.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no significant differences between control and treated groups for the following ovarian and uterine parameters: total corpora lutea, total implantation sites, numbers of male and female fetuses, sex ratio, number of live fetuses, number of dead fetuses, early, middle and late resorptions, total resorptions and pre- and post-implantation losses.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The pregnancy rate was at least 88.0 % in all groups. Ammonium sulphide staining revealed no other pregnancies.
Other effects:
no effects observed
Description (incidence and severity):
No further observations were made.
Details on maternal toxic effects:
The pregnancy rate was at least 88.0 % in all groups. Ammonium sulphide staining revealed no other pregnancies. Gravid uterus weights were significantly (P < 0.01) reduced in rats of the 1900 mg/kg/day group by 13.5 %. There were no significant differences between control and treated groups for the following ovarian and uterine parameters: total corpora lutea, total implantation sites, numbers of male and female fetuses, sex ratio, number of live fetuses, number of dead fetuses, early, middle and late resorptions, total resorptions and pre- and post-implantation losses.
Dose descriptor:
NOAEL
Effect level:
190 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Description (incidence and severity):
The fetal weights were significantly (P < 0.01) reduced for males, females and totals in the 1900 mg/kg/day group only (by 10.4 - 10.5 %).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
effects observed, treatment-related
Description (incidence and severity):
In the 1900 mg/kg/day group there was a significant (P < 0.05) increase in the incidence of litters and fetuses with major malformations. All 5 affected fetuses had acaudia or microcaudia and anal atresia. In addition one of these fetuses had absence of some thoracic and all lumbar, sacral and caudal vertebrae and absence of 9 pairs of ribs. The incidence of major malformations in the 190 and 600 mg/kg/day groups was unaffected.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
The overall incidence of fetuses with minor skeletal anomalies was significantly (P < 0.001) increased in the 1900 mg/kg/day group (140 affected fetuses vs. 82 in the control). This increase was primarily the result of significantly increased incidences of several findings which included reduced ossification of frontal bones (P < 0.05; 26 vs. 10 in the control), irregular ossification of supraoccipital bones (P < 0.01; 33 vs. 12 in the control), reduced number of pre-sacral vertebrae (P <0.01; 26 vs. 7 in the control) and ossification centers on the seventh cervical vertebra (P < 0.001; 52 vs. 4 in the control). In the 190 or 600 mg/kg/day groups, statistically significant differences (P < 0.05) in the incidences of reduced ossification of the interparietal bone, ossification centers on the first lumbar vertebra, reduced ossification of the pubic bones, reduced ossification of the ischial bones or absent ribs were attributed to intergroup variation. The percentage of fetuses with thoracic centrum variants was significantly (P < 0.01) decreased in the 1900 mg/kg/day group (11.3 vs. 30.8 % in the control). There was a statistically significant (P < 0.05) reduction in the percentage of fetuses with sternebral (5 or xiphisternum) variants in the 190 mg/kg/day group which was attributed to intergroup variation.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
There was no effect upon the overall incidence of litters with minor visceral anomalies, but the incidence of fetuses affected was significantly increased (P < 0.05) in the 1900 mg/kg/day group.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
UTERINE FINDINGS
The pregnancy rate was at least 88.0 % in all groups. Ammonium sulphide staining revealed no other pregnancies. Gravid uterus weights were significantly (P < 0.01) reduced in rats of the 1900 mg/kg/day group by 13.5 %. There were no significant differences between control and treated groups for the following ovarian and uterine parameters: total corpora lutea, total implantation sites, numbers of male and female fetuses, sex ratio, number of live fetuses, number of dead fetuses, early, middle and late resorptions, total resorptions and pre- and post-implantation losses.

FETAL FINDINGS
The fetal weights were significantly (P < 0.01) reduced for males, females and totals in the 1900 mg/kg/day group only (by 10.4 - 10.5 %). In the 1900 mg/kg/day group there was a significant (P < 0.05) increase in the incidence of litters and fetuses with major malformations. All 5 affected fetuses had acaudia or microcaudia and anal atresia. In addition one of these fetuses had absence of some thoracic and all lumbar, sacral and caudal vertebrae and absence of 9 pairs of ribs. The incidence of major malformations in the 190 and 600 mg/kg/day groups was unaffected. There was no effect upon the overall incidence of litters with minor visceral anomalies, but the incidence of fetuses affected was significantly increased (P < 0.05) in the 1900 mg/kg/day group. The overall incidence of fetuses with minor skeletal anomalies was significantly (P < 0.001) increased in the 1900 mg/kg/day group (140 affected fetuses vs. 82 in the control). This increase was primarily the result of significantly increased incidences of several findings which included reduced ossification of frontal bones (P < 0.05; 26 vs. 10 in the control), irregular ossification of supraoccipital bones (P < 0.01; 33 vs. 12 in the control), reduced number of pre-sacral vertebrae (P <0.01; 26 vs. 7 in the control) and ossification centers on the seventh cervical vertebra (P < 0.001; 52 vs. 4 in the control). In the 190 or 600 mg/kg/day groups, statistically significant differences (P < 0.05) in the incidences of reduced ossification of the interparietal bone, ossification centers on the first lumbar vertebra, reduced ossification of the pubic bones, reduced ossification of the ischial bones or absent ribs were attributed to intergroup variation. The percentage of fetuses with thoracic centrum variants was significantly (P < 0.01) decreased in the 1900 mg/kg/day group (11.3 vs. 30.8 % in the control). There was a statistically significant (P < 0.05) reduction in the percentage of fetuses with sternebral (5 or xiphisternum) variants in the 190 mg/kg/day group which was attributed to intergroup variation.
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
2-pyrrolidone displays its embryotoxicity/teratogenicity effects if administered oral at high doses during pregnancy.
Executive summary:

The GLP-compliant oral teratology study of 2 -pyrrolidone in rats was conducted by a method that is similar with the OECD Guideline 414.

2 -pyrrolidone at dose levels of 0, 190, 600 and 1900 mg/kg/bw was administered oral to rats from day 6 to 15 of gestation. Maternal toxic effects occured at 600 and 1900 mg/kg/ bw. Increased numbers of malformations were observed only at the highest dose level. The overall NOAELs for maternal toxicity and embryotoxicity/teratogenicity is considered to be 190 and 600 mg/kg/bw, respectively.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Species:
rabbit
Quality of whole database:
There are two consistent prenatal developmental toxicity studies in two species (rat, rabbit, rated with Klimisch 1) available, so the database is of high quality.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP conform developmental toxicity study according to OECD 414, groups of pregnant female Sprague-Dawley rats (25/group) received 190, 600 or 1900 mg/kg bw. per gavage during gestation days 6 -15 (Bio-Research Laboratories 1990).

Body weights of rats of the 600 and 1900 mg/kg bw. dose groups were significantly reduced by 5 and 8.5 %, respectively. In correlation to this result, food consumption was significantly lower temporarily (600 mg/kg bw.) or for the whole administration period (1900 mg/kg bw.). No abnormalities related to treatment were found in dams at necropsy. At the 1900 mg/kg/day level there were increased incidences of major malformations, minor visceral and skeletal anomalies and decreased fetal weights. Anal atresia combined with acaudia or microacaudia (5 pups of 5 litters), absent inominate artery (2 pups of 2 litters), dilatation of lateral ventricles (4 pups of 2 litters) were major malformations observed. No effect upon postimplantation loss was observed. The incidence of major malformations in the 190 and 600 mg/kg bw. groups was unaffected. Therefore, the developmental NOAEL for rats is considered to be 600 mg/kg.

In a GLP conform developmental toxicity study according to OECD 414, groups of 24 pregnant New Zealand White rabbits were dosed with 0, 250, 500, or 1000 mg/kg/d 2 -pyrrolidone via oral gavage once daily during Gestation Days 7 -28.

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.

In an older limit test performed according to FDA requirements, 20 pregnant Sprague-Dawley were treated orally with 1875 mg 2-Pyrrolidone per kg bw. on gd 6-15 (BASF AG 1971). No signs of maternal or developmental toxicity were found. The malformations observed in fetuses were characteristic for the strain tested and considered to be not related to treatment. Subsequently, the NOAEL for both maternal toxicity and developmental toxicity is considered to be >= 1875 mg/kg bw.

A study in mice was performed following standardized, internal test conditions (BASF 1970). 12 pregnant NMRI mice per treatment were administered 1279 or 3199 mg/kg bw. on gestation day 11-15. No signs of maternal toxicity were found. Slightly increased numbers of malformations were found in the low dose (4.9 %) and the high dose group (3.3 %) if compared with the control data. Authors considered this finding as probably in the range of physiological fluctuations and therefore not relevant for the NOAEL assessment. In this study, the NOAEL for both, the maternal and developmental toxicity is >= 3199 mg/kg bw.

Justification for classification or non-classification

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 available OECD 414 rabbit study was considered the most sensitive one. 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.

The findings in the fetuses in this 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 are 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.

Additional information