1-(2-hydroxyethyl)pyrrolidin-2-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL based on a 28 days repeated dose toxicity study with reproductive and developmental screening in rats exposed by oral gavage was determined to be 1000 mg/kg bw/day.

According to the standard information requirements of REACH Annex X an EOGRTS with basic design is proposed.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: Male animals: 332.5 g - 358.3 g; Female animals: 188.4 g - 220.2 g
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Enrichment: wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria)
- Bedding: Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data)
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day before necropsy).
- Water: Drinking water was supplied from water bottles (ad libitum).
- Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24°C and a range
of relative humidity of 30-70%. The air change rate was 15 times per hour. There were no or only minimal deviations from these limits. The day/night
cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h. The animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection) before use. Walls and floor were cleaned once a week with water
containing an appropriate disinfectant.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water and subsequently intensely mixed with a magnetic stirrer until it was completely dissolved.

Details on mating procedure:

In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test substance preparations:
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out prior to the start of the study. Given that test substance was completely miscible with drinking water, solutions were considered to be homogenous without further analysis. Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.
Analytical Method:
The concentrations of the preparations were determined by LC/MS/MS in a GLP compliant study. the following set-up was used:
- HPLC System: Agilent 1100 Series HPLC system (vacuum solvent degasser, binary HPLC pump, column oven), and CTC Analytics HTC-Pal Autosampler.
- HPLC Column: Thermo Aquasil C18 column, 150 mm length, 3.0 mm i.d., 3 μm particle size.
- Injection Volume: 10 μL
- HPLC Method: Solvent A: 0.1 % formic acid in water, Solvent B: 0.1 % formic acid in methanol
Mobile Phase Composition:
Time (min); Flow rate (μL/min); % A; % B
0.00; 400; 90; 10
0.50; 400; 90; 10
0.60; 400; 5; 95
6.00; 400; 5; 95
6.10; 400; 90; 10
9.00; 400; 90; 10
- MS System Applied Biosystems API 3000 triple quadrupole LC-MS/MS system with Turbo IonSpray ESI source.
- Electrospray Ion Source Conditions: Positive ionisation:
Source temperature: 400°C
Curtain gas (CUR): 12
Nebulizer gas (NEB): 14
Ion spray voltage (IS): 5000 V
Collision gas (CAD): 4.00
Entrance potential (EP): 10 V
Resolution Q1 and Q3: Unit
- MS/MS Conditions: MRMs for quantitation of N-(2-Hydroxyethyl)-2-pyrrolidon and for confirmation (not reported): Declustering potential (DP): 26 V:
Q1 Mass; Q3 Mass; CE; CXP; Dwell
Quantitation; 130; 69; 27; 12; 500
Confirmation; 130; 112; 19; 20; 500
The [M-H]+ ion of N-(2-Hydroxyethyl)-2-pyrrolidon at 130 m/z was used as parent ion for MS/MS detection. The MS/MS transition to the daughter ion at 69 m/z was used for quantification of the analyte. For confirmation a 2nd MS/MS transition (130 m/z -> 112 m/z) was monitored.
The nominal concentration of N-(2-Hydroxyethyl)-2-pyrrolidon (PSN 04/0443-2) in water of 10.0, 3.0 and 1.0 g/100 mL were confirmed by deviations in a range of plus 9 % to 0 %.

Duration of treatment / exposure:

Male: 28 days
Female: 49 days

Frequency of treatment:

Daily, females in labor were not treated.

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw /day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: By request of the sponsor
- Rationale for selecting species: The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Parental animals: Observations and examinations:

MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

CAGE SIDE OBSERVATIONS:
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g.inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day

DETAILED CLINICAL OBSERVATIONS (DCO):
Detailed clinical observations were performed in all animals once prior to the first administration and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed: 1. Abnormal behavior in “handling”, 2. Fur, 3. Skin, 4. Posture, 5. Salivation, 6. Respiration, 7. Activity/arousal level, 8. Tremors, 9. Convulsions, 10. Abnormal movements, 11. Gait abnormalities, 12. Lacrimation, 13. Palpebral closure, 14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency), 16. Assessment of the urine discharged during the examination, 17. Pupil size

BODY WEIGHT:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION :
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14 and 14 - 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1- 4.
Food consumption was not determined in females without positive evidence of sperm during the gestation period and in females without litter duringthe lactation period.

HAEMATOLOGY:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): 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 (PLT), Differential blood count and Reticulocytes (RET).
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Parameter and method: Prothrombin time (Hepato Quick’s test) (HQT),

CLINICAL CHEMISTRY:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. Parameters:
- Enzymes: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT).
- Blood chemistry parameters: Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids
(TBA).

URINALYSIS:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to semi quantitatively determine urine constituents were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany). Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery was performed in 5 parental male and 5 parental female animals (with litter) per group at the end of the administration period starting at about 10.00h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: 1. Posture, 2. Tremors, 3. Convulsions, 4. Abnormal movements, 5. Impairment of gait, 6. Other findings
- Open field observations: The animals were transferred to a standard arena (50 x 50 cm wide, with side borders which are 25 cm high) and observed for at least 2 minutes. The following parameters were examined: 1. Behavior when removed from cage, 2. Fur, 3. Skin, 4. Salivation, 5. Nose discharge, 6. Lacrimation, 7. Eyes/pupil size, 8. Posture, 9. Palpebral closure, 10. Respiration, 11. Tremors, 12. Convulsions, 13. Abnormal movements/stereotypy, 14. Impairment of gait, 15. Activity/arousal level, 16. Feces excreted within 2 minutes (number of scybala discharged/appearance/consistency), 17. Urine excreted within 2 minutes (amount/color), 18. Number of rearings within 2 minutes
- Sensory motor tests/Reflexes: The animals were removed from the open field and subjected to following sensory motor or reflex tests: 1. Approach response, 2. Touch response, 3. Vision (“visual placing response”), 4. Pupillary reflex, 5. Pinna reflex, 6. Audition (“startle response”), 7. Coordination of movements (“righting response”), 8. Behavior during “handling”, 9. Vocalization, 10. Pain perception (“tail pinch”), 11. Other findings, 12. Grip strength of forelimbs and hindlimbs, 13. Landing foot-splay test
- Motor activity measurement (MA): The MA was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in clean polycarbonate cages for the time of measurement. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.

Sperm parameters (parental animals):

Special attention was given on stages of spermatogenesis in the male gonads.

Litter observations:

PUP NUMBER AND STATUS AT DELIVERY:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUP VIABILITY/MORTALITY:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in section “Necropsy observations”. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

SEX RATIO:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.

PUP CLINICAL OBSERVATIONS:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.

PUP BODYWEIGHT DATA:
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):

GROSS PATHOLOGY:
- Clinical pathology: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using
isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were
carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and
urine was collected overnight. Urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.
- Necropsy: All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
- Weight parameters: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Epididymides and Testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen and Thymus.

HISTOPATHOLOGY:
The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution: 1. All gross lesions, 2. Adrenal glands, 3. Aorta, 4. Bone marrow (femur), 5. Brain, 6. Cecum, 7. Cervix, 8. Coagulating glands, 9. Colon, 10. Duodenum, 11. Eyes with optic nerve, 12. Esophagus, 13. Extraorbital lacrimal glands, 14. Epididymides (modified Davidson’s solution), 15. Femur with knee joint, 16. Heart, 17. Ileum, 18. Jejunum (with Peyer’s patches), 19. Kidneys, 20. Larynx, 21. Liver, 22. Lungs, 23. Lymph nodes (axillary and mesenteric), 24. Mammary gland (male and female), 25. Nose (nasal cavity), 26. Ovaries (modified Davidson’s solution), 27. Oviducts, 28. Pancreas, 29. Parathyroid glands, 30. Pharynx, 31. Pituitary gland, 32. Prostate gland, 33. Rectum, 34. Salivary glands (mandibular and sublingual), 35. Sciatic nerve, 36. Seminal vesicles, 37. Skeletal muscle, 38. Spinal cord (cervical, thoracic and lumbar cord), 39. Spleen, 40. Sternum with marrow, 41. Stomach (fore stomach and glandular stomach), 42. Target organs, 43. Testes (modified Davidson’s solution), 44. Thymus, 45. Thyroid glands, 46. Trachea, 47. Urinary bladder, 48. Uterus, 49. Vagina.
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully with 0.9% NaCl solution. When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing. (SALEWSKI, E.: Färbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte; Naunyn-Schmiedeberg’s Arch. Exp. Pathol. Pharmakol. 247, 367(1964)). Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. Special attention was given on stages of spermatogenesis in the male gonads. A correlation between gross lesions and histopathological findings was attempted.

Postmortem examinations (offspring):

All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Statistics:

- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control
group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically.
Statistics of pathology:
- Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05,
a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Statistics of the clinical examinations:
- Food consumption, body weight and body weight change (parental animals); Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means

Reproductive indices:

For each group, the following calculations were performed:
- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
- Male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100
- Female mating index (%) = (number of females mated / number of females placed with males) x 100
- Female fertility index (%) = (number of pregnant females / number of females mated) x 100
- Gestation index (%) = (number of females with live pups on the day of birth / number of pregnant females) x 100

Offspring viability indices:

For each group, the following calculations were performed:
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
- Postimplantation loss (%) = (number of implantations – number of pups delivered / number of implantations) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100
- Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

CLINICAL SIGNS AND MORTALITY:
There were no test substance-related or spontaneous mortalities in any of the groups. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation andlactation periods. Several male and female animals of dose group 3 (1000 mg/kg bw/d) showed salivation after treatment during premating, mating, gestation and lactation. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. One sperm positive high-dose female (1000 mg/kg bw/d) and four sperm positive mid-dose females (300 mg/kg bw/d) did not become pregnant.

DETAILED CLINICAL OBSERVATIONS:
Male and female animals of all dose groups (1000, 300 and 100 mg/kg bw/d) did not show any abnormalities.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights and mean body weight change of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period. Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3; 100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period.

REPRODUCTIVE PERFORMANCE:
- Males; For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One high-dose male (1000 mg/kg bw/d - No. 38) and four mid-dose males (300 mg/kg bw/d ) did not generate F1 pups. Thus, the male fertility index ranged between 60% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rat of test groups 3 did not show relevant gross lesions.
- Female; The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.4 and 3.7 days without any relation to dosing. All sperm positive rats delivered pups or had implants in utero with the following exceptions: one female in high dose and 4 females in the middle dose. The fertility index varied between 60% in test group 2, 90% in test group 3 and 100% in test group 1 and control. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. None of the non-pregnant females had any relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 22.1 and 22.3 days). The gestation index was 100% in all test groups. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.4 / 13.4 / 11.0 and 12.9 implants/dam in test groups 0-3 (0, 100, 300 and 1000 mg/kg body weight/day)). There were no statistically significant differences in post-implantation loss between the groups (5.7% / 5.7% / 7.8% / 5.5%), and the mean number of F1 pups delivered per dam remained unaffected (11.7 / 12.6 / 10.2 and 12.2 pups/dam at 0, 100, 300 and 1000 mg/kg bw/d). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% (test group 3, 2 and control) and 99.2% (test group 2). Moreover, the number of stillborn pups was comparable between the groups.

ORGAN WEIGHTS:
- Absolute weights: When compared to control group 0 (set to 100%), the mean absolute weight of the brain was significantly changed in test group 1 and 2. The significant changes observed in the brain of males showed no dose-dependency and were therefore regarded as non-treatment related. All other mean absolute weight parameters of treated males and females showed no relevant differences when compared to the control group 0 and are therefore considered to be within the normal range.
- Relative weights: All mean relative organ weights of treated males and females showed no statistically significant differences when compared to the control group 0 and are therefore considered to be within the normal range.

GROSS PATHOLOGY:
All gross findings noted at necropsy are regarded as incidental and spontaneous in nature and are not related to treatment.

HISTOPATHOLOGY:
No treatment-related findings were observed in males and females of test group 3 (1000mg/kg bw/d). All findings noted were either single observations, or were biologically equally distributed between control and treated rats. All of them are considered to be incidental and/or spontaneous in origin.

HAEMATOLOGY:
No treatment-related changes among hematological parameters were observed. In males of test group 3 (1000 mg/kg bw/d) absolute and relative basophil counts were increased. However, mean values were within historical control ranges (absolute basophil counts 0.00-0.07 Giga/L; relative basophil counts 0.0-1.1 %). This, were the only changed clinical pathology parameters in these individuals. Therefore, these alterations were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY:
No treatment-related changes among clinical chemistry parameters were observed. In females of test groups 1 and 2 (100 and 300 mg/kg bw/d), urea levels were higher compared to controls. However, the parameter was not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

URINALYSIS:
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR:
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental.
- Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly increased distance in the landing foot splay test in high-dosed males (test group 3 - 1000 mg/kg bw/d) was considered to be spontaneous in nature and not treatment related.
- Motor activity measurement (MA): No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested.

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested.

VIABILITY/MORTALITY:
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 99.1% (test group 3), 98.6% (test group 2), 100% (test group 1) and 98.6% (control) without showing any association to the treatment.

CLINICAL SIGNS:
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT:
Mean body weights and mean body weight change of the male and female pups in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period. Two female runts were seen in test group 3 (1000 mg/kg bw/d) and two female runts were seen in test group 1 (100 mg/kg bw/d).

SEX RATIO:
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

GROSS PATHOLOGY:
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis and testis discolored (red). These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested.

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP compliant OECD 422 study (BASF SE 2013), the test compound N-(2-Hydroxyethyl)-2-pyrrolidon was administered daily by gavage to treatment groups of 10 male and 10 female Wistar rats at concentrations of 100, 300 and 1000 mg/kg bw/day to test for repeated dose toxicity and screen for potential reproductive and developmental effects. In addition a vehicle control group exposed to the vehicle (water) only was tested as well. Analyses confirmed the overall accuracy of the prepared concentrations and demonstrated the stability of the test substance in drinking water over a period of 7 days at room temperature. No adverse clinical findings were noted. In several male and female high-dose parental animals, salivation was observed after treatment throughout major parts of the treatment period, including the gestation and lactation periods. Since this salivation was transient and occurred directly after dosing, it is probably due to the irritation and/or taste of substance administration, and therefore was not assessed as adverse. No changes in either food consumption or body weight parameters were noted at any dose. Regarding clinical pathology, no treatment-related, adverse effects were observed up to the 1000 mg/kg bw/d dose of the compound. Regarding pathology, no treatment-related organ weight changes, gross lesions or histopathological findings were observed in the parental animals. All organs of the reproduction tract were not affected by treatment. No alterations to parental reproductive performance or fertility were observed at any dose. Thus, under the conditions of this repeated dose and reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for general, systemic toxicity aswell as reproductive performance and fertilityis 1000 mg/kg body weight/day for the F0 parental rats, the highest dose tested.The NOAEL for developmental toxicity was 1000 mg/kg body weight/day, as no developmental toxicity was observed at any dose.

According to the standard information requirements of REACH Annex X an EOGRTS with basic design is proposed by the registrant.

Effects on developmental toxicity

Description of key information

The NOAEL based on a 28 days repeated dose toxicity study with reproductive and developmental screening in rats exposed by oral gavage was determined to be 1000 mg/kg bw/day.

According to the standard information requirements of REACH Annex X a teratogenicity study in a 2nd species is proposed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept. 2017 - June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP conforming guideline study with no limitations

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE, 97949636W0
- Expiration date of the lot/batch: 12 June 2019
- Purity: 99.6 corr. area-% (GC, DB-Wax capillary)
99.8 corr. area-% (GC, CP-Sil 5 CB/MS capillary)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Solubility and stability of the test substance in the solvent/vehicle: The test substance is completely miscible with drinking water. The stability of the test substance in drinking water over a period of 7 days at room tempera-ture was demonstrated before the start of the study in a similar batch.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Time-mated Wistar rats (Crl:WI[Han]) were supplied by Charles River Laboratories, Research Models and Services, Germany GmbH,
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 139.7 – 187.3 g
- Housing: During the study period, the rats were housed individually in Polycarbonate cages (floor area about 800 cm²). Dust-free wooden bedding was used in this study.
- Diet and water: The food used was ground Kliba maintenance diet mouse/rat “GLP” meal. Food and drinking water (potable tap water in water bottles) were available ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The animals were accommodated in fully air-conditioned rooms in which central air condi-tioning maintained a range of temperature of 20-24°C
- Humidity (%): range of relative humidity of 30-70%
- Air changes (per hr): The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.
- Photoperiod (hrs dark / hrs light): The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).

IN-LIFE DATES: From 20 Sep 2017 to 05 Oct 2017

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with drinking water in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.
Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in drinking water over a period of 7 days at room temperature had been verified prior to the start of the study in a similar batch. Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations. Given that the test substance was completely miscible with drinking water, solutions were considered to be homogenous without further analysis. All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to orig-inal samples after agreement by the Study Director.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).

Duration of treatment / exposure:

The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control group were treated with the vehicle (drinking water) in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

Frequency of treatment:

Daily

Duration of test:

On GD 20, the females were sacrificed in a randomized order and examined macroscopically.

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25 female animals per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results on preceding reproductive and developmental toxicity screening study in rats accord. to OECD TG 422.
- Rationale for animal assignment (if not random): Rats are considered to be a suitable species for the detection of a developmental toxicological hazard.
- The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Maternal examinations:

Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20)
Clinical symptoms: A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.
Food consumption: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.
Body weight data: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.
Corrected (net) body weight gain: Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

Ovaries and uterine content:

Terminal examinations of the dams:
Necropsy: On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.
After the dams had been sacrificed, they were necropsied and assessed for gross pathology. The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
- Live fetuses
- Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and th e empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters (except of gross pathology including organ weights) were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
These data were used to calculate conception rate and pre- and postimplantation losses.

Fetal examinations:

Examinations of the fetuses after dissection from the uterus:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were ex-amined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses:
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Statistics:

Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight:
Simultaneous comparison of all dose groups with the con-trol group using the DUNNETT-test (two-sided) for the hypothesis of equal means

Female mortality, females preg-nant at terminal sacrifice, number of litters with fetal findings:
Pairwise comparison of each dose group with the control group using FISHER'S
EXACT test (one-sided) for the hypothesis of equal proportions

Proportions of fetuses with malformations, variations and/or unclassified observations in each litter:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hy-pothesis of equal medians

Clinical signs:

no effects observed

Description (incidence and severity):

Some (7 out of 25) females of the high-dose group (1000 mg/kg bw/d) showed occasional salivation during the treatment period. Salivation occurred in the respective animals only shortly, i.e. within 0-2h, after treatment and was observed on GD 7 and 11. No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic toxicity. No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 100, 300 or 1000 mg/kg bw/d).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. The corrected body weight gain of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group (bw change from day 6: 22 g, 25 g, 24 g and 25 g for controls, 100, 300 and 1000 mg/kg bw/d, respectively). Moreover, mean carcass weights of all test groups remained unaffected by the treatment (240.5 g, 235.4 g, 243.0 g and 234.6 g for controls, 100, 300 and 1000 mg/kg bw/d, respectively).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the high-, mid- and low-dose dams (1000, 300 and 100 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period. The statistically significantly increased food consumption value in the low- and mid-dose dams during GD 19-20 was not considered biologically relevant due to the lack of dose re-sponse relationship.

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):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

One spontaneous finding was noted in two individual females of the control group, i.e. dilated renal pelvis.
No further necropsy findings which could be attributed to the test substance were seen in any dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test substance related and/or biologically relevant differences between the different test groups in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age. See table 1 for summary of data.
The statistically significantly decreased number of implantation sites in test group 1 was assessed as an incidental finding and biologically not relevant, since all mean values (mean number 11.4/10.0*/10.6/11.0 [* = p ≤ 0.05 Dunnett-test]) were within the historical control range (implantation sites mean: 10.8 [9.9 - 11.8]). In addition, there was neither an effect on pre-implantation loss, nor a relation to dose. Therefore, this finding was not assessed as treatment-related and adverse.
Solely as a consequence of the lower number of implants, a statistically significantly decreased number of live fetuses (10.9/9.4*/9.7/10.1 [* = p ≤ 0.05 Dunnett-test]), including live female fetuses (mean number 6.0/4.2**/4.9/5.4 [** = p ≤ 0.01 Dunnett-test]), was observed in test group 1. There was neither an effect on post-implantation loss nor a relation to dose. In addition, the mean value of live fetuses was within the range of the historical control data (HCD: viable fetuses, mean: 10.0 (9.3 - 11.2)). Therefore, this finding was not assessed as treatment-related and adverse.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate was 88% in the control group (0 mg/kg bw/d), 96% in the mid-dose group (300 mg/kg bw/d) and 100% in the low- and high-dose groups (100 and 1000 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance related and/or biologically relevant differences between the different test groups in conception rate. See table 1.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean placental weights were comparable between the control and test groups 1 and 2 (0, 100 and 300 mg/kg bw/d). In test group 3 (1000 mg/kg bw/d), a statistically significantly lower mean placental weight (0.44 g) of female fetuses and of all viable fetuses (0.46 g) was observed. Both mean values were within the historical control range (female fetuses - mean 0.46 g; range 0.32 - 1.10 g; all viable fetuses - mean 0.47 g (0.35 - 1.13 g) and there was no effect on fetal weights. Therefore, these decreases were not assessed as treatment-related and adverse. The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): See table 2

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

See table 1

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. See table 1

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No external malformations were recorded. No external variations were recorded. One unclassified external observation, i.e. placentae fused, was recorded in one litter of the mid-dose group (300 mg/kg bw/d). This finding was not considered as treatment-related and adverse since it was not related to dose and the mean value of affected fetuses per litter was within the historical control range (affected fetuses per litter, mean 0.0, range: 0.0 – 0.4%).
See table 3.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were noted in one fetus, each, in test groups 0, 2 and 3 (0, 300 and 1000 mg/kg bw/d. No statistically significant differences between the substance-treated groups and the control were noted and no dose-response relationship was observed. The isolated finding in test groups 2 and 3 each were single cases and the mean values of affected fetuses per litter of both findings were within the range of the historical control data. Therefore, these findings were not considered to be treatment-related. See tables 7 and 8.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data The increased incidences of skeletal variations were not related to the dose and they were inside the historical control range. Therefore, they are not considered as treatment-related. See table 9.

Visceral malformations:

no effects observed

Description (incidence and severity):

One fetus of test group 3 (1000 mg/kg bw/d) had two soft tissue malformations – right-sided aortic arch and abnormal lung lobation. Abnormal lung lobation can be found in the historical control data at a comparable incidence (litter incidence, mean 0.1%, range: 0.0 - 4.0%). However, these were single events in one fetus and no cluster of any of these individual malformations were seen in the other offspring of this or the other treated groups. Thus, no association to the treatment was assumed.
The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data. See table 5.
Four soft tissue variations were detected, i.e. misaligned palatal rugae, short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they were not as-sessed as treatment-related. See table 6.

Other effects:

no effects observed

Description (incidence and severity):

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing. See table 11.

Details on embryotoxic / teratogenic effects:

Assessment of all fetal external, soft tissue and skeletal observations

External malformations did not occur in any of the fetuses in this study. There were noted soft tissue and skeletal malformations in test groups 0, 2 and 3 (0, 300 and 1000 mg/kg bw/d). Two fetuses frome one litter carried more than one malformation. Female high-dose fetus No. 81-02 (1000 mg/kg bw/d) had a right-sided aortic arch and abnormal lung lobation. Female high-dose fetus No. 81-03 of the same litter had multiple malformations affecting the vertebral column, for example fused cervical arch, misshapen thoracic vertebra and absent lumbar vertebra. Further malformations, i.e. malpositioned and bipartite sternebra and cleft sternum were observed in individual fetuses, unrelated to the dose. All these findings were single cases, all of them can (except of right-sided aortic arch) be found in the historical control data. No ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influ-ence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment. See table 12

External variations did not occur in any of the fetuses in this study. Four soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the total incidences showed a relation to dose. The individual variations were equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency. See table 13.

No unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified external and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. See tables 3 and 11.

Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest dose tested (1000 mg/kg bw/d).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1

Summary of reproduction data

	0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Females mated (N)	25	25	25	25
Pregnant (N) Conception rate (%)	22 88	25 100	24 96	25 100
Aborted (N)	0	0	0	0
Premature births (N)	0	0	0	0
Dams with viable fetuses (N)	22	25	24	25
Dams with all resorptions (N)	0	0	0	0
Female mortality (N)	0	0	0	0
Corpora lutea Mean SD Total	11.8 D 1.63 259	10.9 1.89 273	11.5 1.67 277	11.7 1.44 292
Implantation sites Mean SD Total	11.4 D 1.68 250	10.0 * 1.68 250	10.6 2.24 254	11.0 1.17 276
Preimplantation loss Mean% SD	3.5 D 5.66	7.7 10.45	9.4 12.90	5.0 7.26
Postimplantation loss Mean% SD	4.1 D 5.45	6.3 10.57	9.1 13.21	8.0 10.56
Resorptions Total Mean SD Total Mean% SD   Resorptions Early Mean SD Total Mean% SD   Resorptions Late Mean SD Total Mean% SD	0.5 D 0.67 11 4.1 D 5.45     0.5 D 0.67 5.45 4.1 5.45     0.0 D 0.0 0 0.0 D 0.0	0.6 1.19 16 6.3 10.57     0.5 0.92 12 4.8 8.91     0.2 0.47 4 1.5 4.19	0.9 0.99 21 9.1 13.21     0.8 0.9 18 6.6 7.67     0.1 0.61 3 2.5 12.25	0.9 1.26 23 8.0 10.56     0.8 1.04 20 7.0 8.63     0.1 0.44 3 1.1 3.94
Dead fetuses (N)	0	0	0	0
Dams with viable fetuses	22	25	24	25
Live fetuses Mean SD Total	10.9 D 1.55 239 95.9 D 5.45	9.4* 1.85 234 93.7 10.57	9.7 2.40 233 90.9 13.21	10.1 1.39 253 92.0 10.56
Females Mean SD Total Mean % SD	6.0 D 2.07 132 52.7 16.04	4.2 ** 1.42 106 42.8 13.29	4.9 2.21 118 45.7 15.26	5.4 1.50 134 49.0 14.11
Males Mean SD Total Mean %	4.9 D 1.83 107 43.1 D 15.89	5.1 2.11 128 50.9 17.24	4.8 1.93 115 45.2 18.45	4.8 1.54 119 43.0 12.65
Per cent live females	55.2	45.3	50.6	53.0
Per cent live males	44.8	54.7	49.4	47.0

Statistics: D : Dunnett-test (two-sided), * : p <= 0.05, ** : p < = 0.01

Table 2

Mean Fetal body weights

 

Fetal weights (g)	0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
All viable fetuses Mean SD N	3.5 D 0.29 22	3.7 0.23 25	3.5 0.25 24	3.5 0.25 25
Male fetuses Mean SD N	3.7 D 0.26 22	3.7 0.25 25	3.7 0.27 23	3.6 0.26 25
Female fetuses Mean SD N	3.5 0.31 22	3.6 0.23 25	3.5 0.25 24	3.4 0.22 25

 

D: Dunnett-test (two-sided), * : p <= 0.05, ** : P <= 0.01

Table 3

Total external unclassified observations

 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 239	25 234	24 233	25 253
Fetal incidence	N (%)	0.0	0.0	1 (0.4)	0.0
Litter incidence	N (%)	0.0	0.0	1 (4.2)	0.0
Affected fetuses/litter	Mean%	0.0	0.0	0.3	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 4

Individual fetal soft tissue malformations

Test group	Dam No.-, Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	81-02 F	right-sided aortic arch, abnormal lung lobation

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Table 5

Total soft tissue malformations

 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 114	25 111	24 109	25 121
Fetal incidence	N (%)	0.0	0.0	0.0	1 (0.8)
Litter incidence	N (%)	0.0	0.0	0.0	1 (4.0)
Affected fetuses/litter	Mean%	0.0	0.0	0.0	1.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 6

Total soft tissue variations

 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 114	25 111	24 109	25 121
Fetal incidence	N (%)	1 (0.9)	3 (2.7)	3 (2.8)	3 (2.5)
Litter incidence	N (%)	1 (4.5)	3 (12)	3 (13)	3 (12)
Affected fetuses/litter	Mean%	0.9	2.8	2.7	2.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 7

Individual fetal skeletal malformations

 

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	19-01 M	cleft sternum
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	54-07 M	malpositioned and bipartite sternebra
3 (1000 mg/kg bw/d)	81-03 F	severely malformed vertebral column

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

 

Table 8

Total skeletal malformations

 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 125	25 123	24 124	25 132
Fetal incidence	N (%)	1 (0.8)	0.0	1 (0.8)	1 (0.8)
Litter incidence	N (%)	1 (4.5)	0.0	1 (4.2)	1 (4.0)
Affected fetuses/litter	Mean%	0.8	0.0	0.8	0.8

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 9

Total fetal skeletal variations

 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 125	25 123	24 124	25 132
Fetal incidence	N (%)	123 (98)	121 (98)	123 (99)	127 (96)
Litter incidence	N (%)	22 (100)	25 (100)	24 (100)	25 (100)
Affected fetuses/litter	Mean%	98.5	98.0	99.0	96.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 10

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter). All incidences were expressed on a fetus per litter basis.

 

Finding	Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of skull; unchanged cartilage	3.9	8.1	18.0*	7.5	8.1 (0.8 – 21.4)
Supernumerary thoracic vertebra	2.4	2.2	7.5*	0.7	4.3 (0.8 – 11.0)
Supernumerary rib (14^th); cartilage present	4.9	6.4	13.7*	3.6	7.4 (1.9 – 14.7)
Wavy rib	0.8	7.5*	7.2*	4.0	5.1 (1.4 – 13.3)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided]), ** = p <= 0.01 (Wilcoxon-test [one-sided])

Table  11

Total unclassified cartilage observations

 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 125	25 123	24 124	25 132
Fetal incidence	N (%)	99 (79)	108 (88)	97 (78)	101 (77)
Litter incidence	N (%)	22 (100)	24 (96)	24 (100)	24 (96)
Affected fetuses/litter	Mean%	79.4	85.7	79.4	76.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 12

Total fetal malformations

 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 239	25 234	24 233	25 253
Fetal incidence	N (%)	1 (0.4)	0.0	1 (0.4)	2 (0.8)
Litter incidence	N (%)	1 (4.5)	0.0	1 (4.2)	1 (4.0)
Affected fetuses/litter	Mean%	0.4	0.0	0.4	0.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 13

Total fetal variations

 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	22 239	25 234	24 233	25 253
Fetal incidence	N (%)	124 (52)	124 (53)	126 (54)	130 (51)
Litter incidence	N (%)	22 (100)	25 (100)	24 (100)	25 (100)
Affected fetuses/litter	Mean%	52.1	52.9	53.9	51.6

mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

Conclusions:

Under the conditions of a prenatal developmental toxicity study conducted according to OECD TG 414, the oral administration of N-(2-Hydroxyethyl)-2 -pyrrolidonto pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/day caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/day.

Executive summary:

N-(2-Hydroxyethyl)-2-pyrrolidon was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg bw/day on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water) in parallel. At terminal sacrifice on GD 20, 22-25 females per group had implantation sites. Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. The stability of the test substance preparations over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown.

No test substance-related adverse effects on dams, gestational parameters or fetuses were observed at any dose level (0, 100, 300, 1000 mg/kg bw/day). Under the conditions of this prenatal developmental toxicity study, the oral administration of N-(2-Hydroxyethyl)-2 -pyrrolidonto pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/day caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

N-(2-Hydroxyethyl)-2-pyrrolidon was tested for its prenatal developmental toxicity in Wistar rats (BASF SE, 2018). The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg bw/day on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water) in parallel. At terminal sacrifice on GD 20, 22-25 females per group had implantation sites. Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. The stability of the test substance preparations over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown.

No test substance-related adverse effects on dams, gestational parameters or fetuseswere observed at any dose level (0, 100, 300, 1000 mg/kg bw/day).Under the conditions of this prenatal developmental toxicity study, the oral administration of N-(2-Hydroxyethyl)-2 -pyrrolidonto pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/day caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/day.

Supportingly, no adverse effects on pre- and periimplanation losses were oberved after treatment of pregnant rats with 90 mg/kg/d hydroxyethylpyrrolidone by gavage on GD0 -5 (Menegola et al., 1998).

According to the standard information requirements of REACH Annex X a teratogenicity study in a 2nd species is proposed by the registrant.

Mode of Action Analysis / Human Relevance Framework

no further data available

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study with reproductive and developmental screening and a prenatal developmental toxicity study in rats, the test substance does not need to be classified for toxicity to reproduction according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.