1-ethylpyrrolidin-2-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-ethylpyrrolidin-2-one

EC Number:

220-250-6

EC Name:

1-ethylpyrrolidin-2-one

Cas Number:

2687-91-4

Molecular formula:

C6H11NO

IUPAC Name:

1-ethylpyrrolidin-2-one

Details on test material:

purity: 99.8%

Specific details on test material used for the study:

Batch-Id. 29151288P0
Purity 99.8%
Expiry date: Feb 22, 2007

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

35 +/- 1 day males/females were used. The rats were identified clearly by ear tattoo. The rats were housed singly in type DK III stainless steel wire mesh cages. Underneath the cages, waste trays were fixed containing absorbent material. Motor activity measurements were conducted in Polycarbonate cages with wire covers and small amounts of absorbent material. The animals were housed in a fully air-conditioned room. Central airconditioning guaranteed a range of 20 - 24°C for temperature and of 30 - 70% for relative humidity. The day/night cycle was 12 hours light and 12 hours dark. Deviations from these ranges did not occur. The animal room was completely disinfected prior to the study. The floor and the walls were cleaned once a week. The food used was ground Kliba maintenance diet mouse/rat and drinking water (from water bottles) were available ad libitum.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

For each concentration, the test substance was weighed out and mixed with a small amount of food. Then corresponding amounts of food, depending on dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dietary concentrations of N-Ethyl-2-pyrrolidon for each group and sex were adjusted weekly, based on body weight and food consumption measurements from the previous week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out. The stability of the test substance in the diet over a period of 49 days was proven prior to the study. Homogeneity and concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period.

Duration of treatment / exposure:

3 months

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day (groups 0, 1, 2, 3)
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery (FOB) and measurement of motor activity was carried out after 85 resp. 89 days of treatment in males and 86 resp. 90 days of treatment in females. Vaginal smears for estrus cycle determination of all female animals were prepared and evaluated each day during the last 4 weeks of the study. Clinicochemical, hematological examinations and urinalyses were performed towards the end of the administration period. Ophthalmological examinations were performed before and towards the end of the administration period. All animals were assessed by gross pathology, followed by histopathological examinations. Additionally, sperm parameters were determined immediately after necropsy and organ weight determination.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
FOOD EFFICIENCY: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
HAEMATOLOGY: Yes
CLINICAL CHEMISTRY: Yes
URINALYSIS: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes

Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy
The test animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Weight parameters
Weight assessment was carried out on all animals sacrificed at scheduled dates. The weights of the following organs were determined:
Anesthetised animals, Liver, Kidneys, Adrenals, Testes, Epididymides, Ovaries, Uterus, Spleen, Brain, Heart, Thymus, Thyroids,,

Organ / Tissue preservation list
The left testis, left epididymis and both ovaries of all animals sacrificed at scheduled dates were fixed in Bouin’s solution. The right testis and right epididymis were used for sperm and spermatide examination. The following organs or tissues were preserved in neutral buffered 4% formaldehyde:
All gross lesions, Salivary glands (mandibular and sublingual glands), Esophagus, Stomach (forestomach and glandular stomach), Duodenum, jejunum and ileum, Cecum, colon and rectum, Liver, Pancreas, Brain, Pituitary, Sciatic nerve, Spinal cord (cervical, thoracic and lumbar cords), Eyes, Adrenals, Thyroids, Parathyroids, Trachea, Lungs, Pharynx, Larynx, Nose (nasal cavity), Aorta, Heart, Bone marrow (femur), Lymph nodes (mesenteric and mandibular lymph nodes, Spleen, Thymus, Kidneys, Urinary bladder, Oviducts, uterus and vagina, Prostate and seminal vesicle, Female mammary gland, Skin, Skeletal muscle, Sternum with marrow, Femur with knee joint, Extraorbital lacrimal glands

The following organ samples were processed histotechnically
All gross lesions, Salivary glands (mandibular andsublingual glands), Esophagus, Stomach (forestomach and glandular stomach), Duodenum and ileum, Jejunum (with Peyer’s patches), Cecum, colon and rectum, Liver, Pancreas, Brain, Pituitary, Sciatic nerve, Spinal cord (cervical, thoracic and lumbar cords), Eyes, Adrenals (cortex and medulla), Thyroids, Parathyroids, Trachea, Lungs, Pharynx, Larynx, Nasal cavity (level III), Aorta, Heart, Bone marrow (femur), Lymph nodes (mesenteric and mandibular lymph nodes), Spleen, Thymus

Other examinations:

A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 a.m. 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.

Ophthalmoscopy
Prior to the start of the administration period the eyes of all animals, and on day 91 the eyes of the control and high dose animals were examined for any changes using an ophthalmoscope after administration of a mydriaticum.

Vaginal smears for cycle determination were prepared in the morning and evaluated.

Sperm parameters
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
The following parameters were determined:
• Sperm motility (%, microscopic evaluation)
• Sperm morphology (%, vital staining with eosin, microscopic evaluation)
• Sperm head count (sperm heads x 10E6/ g cauda epididymis) (microscopic evaluaion with MAKLER chamber after homogenization)
• Sperm head count (sperm heads x 10E6 g testis, microscopic evaluaion with MAKLER chamber after homogenization)

Statistics:

yes

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

“Anogenital region, stained, slight” was observed in 7/10 males and 4/10 females of group 3 and 3/10 males of group 2 from day 63 till the end of the study; “anogenital region, stained, moderate” was observed in 1/10 males of group 3 from day 63 till day 92. “Urine discolored, orange” was observed in 10/10 males and females of groups 3 and 2 and 10/10 females of group 1 from day 2 till the end of the study. “Urine, discolored, dark yellow” was observed in 10/10 males of group 1 from day 7 till day 92. These findings were assessed as substance-related but caused by the physical property of the test article (yellow) and do not represent real toxicity. “High stepping gait, slight” was observed in 1 female of group 3 as well as in 1 female of dose group 1, from day 70 till day 83 and 76, respectively. This finding was assessed as not related to the test article due to the absence of a dose-response relationship. Findings like “alopecia, body, flank, left” in 1 female of group 3 from day 84 till day 93 and “aggressiveness, moderate” in 1 male of group 3 from day 35 till day 55 were assessed as spontaneous in nature and therefore not substance-related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight in both sexes of group 3 was statistically significantly decreased from day 7 till day 91, up to -17.6% in males and -18.3% in females on day 91. Body weight was also decreased in both sexes of group 2, statistically significantly in males on days 28 and 91 and in females on day 21 and from day 49 till day 91, up to -8.7% in males on day 91 and in females on day 70. Body weight change in both sexes of group 3 and 2 was statistically significantly decreased during the whole study period in group 3, resp. the main part of the study in group 2, up to -39.1% in males and -52.6% in females of group 3 and -18.8% in males and -23.5% in females of group 2 on day 7 of the study, each. Due to a clear dose-response relationship, these findings were clearly substance-related. The increase in body weight change of both sexes in group 1 animals on day 7 of the study with +8.9% in males and +6.9% in females was assessed as being incidental, biologically not relevant as well as not substance-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in both sexes of group 3 was statistically significantly decreased during the whole study period, up to -21.1% in males and -23.3% in females on day 7. Food consumption in both sexes of group 2 was decreased during the study, statistically significantly on days 7, 21, 28, 63, 70 and 91 in males, up to -13.2% on day 7 and on days 7, not statistically significantly, and 70 in females, up to -7.5% on day 7, also. Due to a clear dose-response relationship, the influence on food consumption was assessed to be substance-related.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency was decreased on days 7 and 14 in males and on days 7 and 70 in females of group 3. This finding was assessed as being incidental and biologically not relevant.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

All findings were incidental in nature, due to the occurrence in single animals, only, and/or the
lack of a dose-response relationship.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period significantly increased platelet counts were found in the peripheral blood of males at 1000 mg/kg bw and in all treatment groups of females. Moreover, clotting analysis revealed significantly shortened prothrombin times in rats of either sex at 1000 mg/kg. No treatment-related changes were noted in the other hematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Marginally, but significantly decreased alanine aminotransferase and aspartate aminotransferase activities were found in the serum of male and female rats given 1000 mg/kg bw and reduced alanine aminotransferase activities were also seen in female animals at 100 mg/kg and 300 mg/kg. No treatment-related changes were observed in the other serum enzymes examined. Blood chemistry investigations revealed significantly increased inorganic phosphate, calcium, triglyceride and cholesterol concentrations and decreased creatinine levels in the serum of both sexes at 1000 mg/kg. The increase in triglycerides in males and the decrease in creatinine in females, however, were not significantly different to the respective control, but were seen only as a trend toward higher concentrations. In addition, decreased chloride, glucose and total bilirubin values were noted in males at 1000 mg/kg and total bilirubin was also reduced in males at 300 mg/kg. In the serum of females given 300 mg/kg bw and 1000 mg/kg bw significantly lower total protein and albumin concentrations were noted. No treatment-related changes were found in the other blood chemistry parameters.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males given 100 mg/kg bw and animals of either sex receiving 300 mg/kg bw and 1000 mg/kg bw of the test compound excreted urine, which was discolored from dark yellow to orange. Furthermore, microscopic examination of the urine sediments revealed increased numbers of transitional epithelial cells, granular casts and epithelial cell casts in all treated males at the end of the administration period. Most of the transitional epithelial cells found in the sediments were degenerated. No test article-related changes were observed in the other urine parameters of both sexes.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Home cage observations: No substance related findings were observed.

Open field observations:
“Discolored fur” was observed in 7 males and 4 females of group 3 and 2 males of group 2, caused by discolored urine. “Discoloration of urine, orange” was observed in 4 males and females of group 3, 2 males and 8 females of group 2 and 4 females of group 1; “discoloration of urine, dark yellow” was observed in 8 males of group 1. These findings were assessed as substance-related but caused by the physical property of the test article (yellow) and do not represent real toxicity. “Alopecia” was observed in 1 female of group 3. This was assessed as spontaneous in nature and therefore not substance-related.

Sensorimotor tests/reflexes:
“Very frequent vocalizations when touched” was observed in 2 males and 1 female of group 3, 2 males and females of group 2, 3 males and 2 females of group 1 and 1 male and females of group 0. This was assessed as spontaneous in nature and therefore not substance-related. The value of the “grip strength forelimbs” was statistically significantly decreased in male animals of group 3 (-40.1%) and 2 (-33.0%). These findings were assessed as being related to the test article caused by systemic toxicity and reduced body weight, respectively.

Regarding the overall motor activity a statistically significantly decreased deviation was seen in females of group 3. This finding was assessed as being related to the test article caused by systemic toxicity and reduced body weight, respectively. Comparing the single intervals with the control groups, no substance-related findings were observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-dependent decrease of the terminal body weights in dose groups 2 and 3. The significant increase of liver weights in dose group 3 is considered to be a treatmentrelated effect. The significant decrease of brain, heart, spleen, and thymus weights in dose groups 2 and 3 is regarded to be secondary to the decreased terminal body weights.
There was a dose-dependent decrease of the terminal body weights in dose groups 2 and 3. The significant decrease of adrenal glands, brain, heart, spleen, and thymus weights in dose group 3 is regarded to be secondary to the decreased terminal body weights. The significant weight increase of the thymus weights in dose group 1 is considered to be incidental and rather represents the wide, physiological biological range of thymus weights of animals of this age than a treatment-related effect.

The significant weight increase of the adrenal glands, brain, epididymides, and testes is considered to be secondary to the significant decrease of terminal body weights in the dose groups 2 and 3. In parts this is also true for the significant increase of the kidney and liver weights but histological examination also detected a direct compound-related effect that may be additionally responsible for the significant weight increase in dose groups 1 to 3.

All significant weight changes in dose groups 2 and 3 were regarded to be secondary to the significant weight decrease of the terminal body weights and do not represent a direct compound-related effect. The liver weight increase of dose group 3 animals is considered to be partly due to direct effect as treatment-related histological findings are present. The significant increase of the thymus weights in dose groups 1 and 2 represent rather the wide biological range of thymus weights of animals in this age than a treatment-related effect.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All gross lesions are considered to be spontaneous lesions in origin and are not related to treatment.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Regarding pathology, the liver and the kidneys of male test animals are found to be target organs. In the liver, a centrilobular hypertrophy of hepatocytes is found in all dose groups. In males, centrilobular hypertrophy of hepatocytes is found in all dose groups. The gradings
indicate a dose-dependent increase after treatment. In females, only the 1000 mg/kg bw/day dose group animals show centrilobular hypertrophy of hepatocytes. In the kidneys of male test animals, morphological indications for the development of an alpha 2u globulin nephropathy are found in all dose groups.
For each of the dose groups, one male kidney was immunhistochemically stained for the proof of alpha 2u globulin. In all cases, the hyaline droplets that showed positive staining in the Mallory Heidenhain stain, were shown to represent specifically alpha 2u globulin. All groups show an increased severity of basophilic tubules and accumulation of hyaline droplets in the proximal tubules, when compared to the control. All other findings noted are considered to be spontaneous or incidental in origin and not related to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sperm examination showed an increased number of sperms with abnormal heads in males receiving 1000 mg/kg bw of the test compound (2.0, 2.2, 2.8 and 11.4% in controls, low, mid and high dose, respectively; males with >4% abnormal sperm: 0, 1, 2, 8 in controls, low, mid and high dose, respectively).. The most common sperm head anomalies were abnormal hooks and amorphous heads. Test substance administration did not affect number of homogenization resistant spermatids, epididymal sperm count and sperm motility.

Estrous cycle determination:
No substance-related findings were observed.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

3

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

1000 mg/kg bw/day

- Food consumption in both sexes was statistically significantly decreased during the whole study period, up to -21.1% in males and -23.3% in females

- Body weight in both sexes was statistically significantly decreased from day 7 till day 91, up to -17.6% in males and -18.3% in females

- Body weight change in both sexes was statistically significantly decreased during the whole study period, up to -39.1% in males and -52.6% in females

- Grip strength forelimbs in males was statistically significantly decreased of -40.1% during functional observational battery

- Motor activity (summation of intervals) in females was statistically significantly decreased

- Platelets, inorganic phosphate, calcium, cholesterol and triglycerides were increased in both sexes

- Number of sperms with abnormal heads was increased in males

- Prothrombin time was shortened and creatinine was decreased in both sexes

- Chloride, glucose and total bilirubin were decreased in males

- Total protein and albumin were decreased in females

- Terminal body weight decrease in both sexes

- Liver weight increase in both sexes (+53% in males, + 29% in females)

- Centrilobular hypertrophy of hepatocytes in both sexes

- rel. kidney weight increase in males and females (+32% and + 22% respectively)

- Increase of basophilic tubules and accumulation of hyaline droplets (alpha 2u globulin) in males

300 mg/kg bw/day

- Food consumption in both sexes was decreased during the whole study period, statistically significantly on several days

- Body weight in both sexes was decreased from day 7 till day 91, statistically significantly in males on days 28 and 91 and in females on day 21 and from day 49 till day 91, up to -8.7%, each

- Body weight change in both sexes was statistically significantly decreased during the main part of the study, up to -18.8% in males and -23.5% in females

- Grip strength forelimbs in males was statistically significantly decreased of -33.0% during functional observational battery

- Total bilirubin in males and total protein and albumin in females was decreased

- Terminal body weight decrease in both sexes

- rel. liver weight increase in both sexes (+13% in males, + 9% in females)

- Centrilobular hypertrophy of hepatocytes in males

- rel. kidney weight increase in males (+14%)

- Increase of basophilic tubules and accumulation of hyaline droplets (alpha 2u globulin) in males

100 mg/kg bw/day

- rel. Liver weight increase in males (+7%)

- Centrilobular hypertrophy of hepatocytes in males

- rel. kidney weight increase in males (+9%)

- Increase of basophilic tubules and accumulation of hyaline droplets (alpha 2u globulin) in males

- No effects in females at 100 mg/kw/bw

Additional comments to be considered

The kidney effects observed in males at 100 mg/kg bw/day onwards are not relevant to humans (alpha-2 µ-globulin).

The centrilobular hypertrophy of hepatocytes is indicative for an enzyme induction of the cytochrome P450 system and is considered to be a detoxifying, adaptive effect (no signs for cytotoxicity recorded). In males, a centrilobular hypertrophy was also noted for the mid (300 mg/kg bw/day) and low (100 mg/kg bw/day) dose groups, although dose-dependently less pronounced. In females, no centrilobular hypertrophy was noted in the mid and low dose groups.

Sperm analysis revealed an increased number of sperms with abnormal heads in males at 1000 mg/kg/day abnormal sperm: (2.0, 2.2 , 2.8 and 11.4% in controls, low, mid and high dose, respectively; males with >4% abnormal sperm: 0, 1, 2, 8 for

in controls, low, mid and high dose, respectively). The summary of sperm evaluations can be found in the attached table. The most common sperm head anomalies were abnormal hooks and amorphous heads. These results are indicative of disrupted sperm maturation, when the test compound is administered at extremely high dosages. It should be noted that this effect was not associated with any weight changes or (histo)pathological changes in the testis. Beyond this, administration did not affect the number of homogenization resistant spermatids, epididymal sperm count and sperm motility (see table). The functional relevance of this effect is unclear as fertility was not examined in this study.

Applicant's summary and conclusion

Conclusions:

The target organs were liver and kidneys.