1-ethylpyrrolidin-2-one

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

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-Identification: 29151288P0
Purity: 99.8%
Expiry date: 22 Feb 2007

Test animals

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

Sexually mature, virgin Himalayan rabbits (Crl:CHBB(HM)) were used. Only animals free from clinical signs of disease were used for the investigations. The breeder had already carried out unique identification of the rabbits by ear tattoo.
During the acclimatization and the study period, the rabbits were housed singly in type stainless steel wire mesh cages. Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dust free embedding, supplied by Ssniff, Soest, Germany).
The animals were accommodated 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%. There were no deviations from these limits. The light cycle rhythm was 12 hours light from 6.00 a.m. to 6.00 p.m. and 12 hours darkness from 6.00 p.m. to 6.00 a.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance was administered to the animals always at approximately the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (doubly distilled water). The volume administered each day was 10 mL/kg body weight. The calculation of the volume administered was based on the most recent individual body weight. On day 29 p.i., blood was taken from all surviving females, which were subsequently sacrificed in randomized order and examined macroscopically (including weight determinations of the liver, the kidneys and the spleen). The fetuses were removed from the uterus and further investigated with different methods.

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 doubly distilled water for a period of at least 7days at room temperature were carried out before the study was initiated. Samples of the test substance solutions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations.
Since the test substance preparations were true solutions, investigations concerning homogeneity were not necessary.

Details on mating procedure:

The animals were supplied at an age of 12 – 20 weeks. After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination. This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe were injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor. This was documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
During the acclimatization period the animals were assigned to the different test groups according to a randomization plan and on the basis of their body weights.
Based on the pregnant animals the body weight on day 0 varied between 1960 - 2870 g.

Duration of treatment / exposure:

day 6 through day 28 post insemination (p.i.)

Frequency of treatment:

daily

Duration of test:

until day 29 p.i.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 60 and 200 mg/kg body weight

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

N-Ethyl-2-pyrrolidone was tested for its prenatal developmental toxicity in Himalayan rabbits. The test substance was administered as an aqueous solution to 25 inseminated female Himalayan rabbits/group by stomach tube at doses of 20; 60 and 200 mg/kg body weight on day 6 through day 28 post insemination (p.i.). A standard dose volume of 10 mL/kg body weight was used for each group. The control group, consisting of 25 females, was dosed with the vehicle only (doubly distilled water). 22 - 23 females/group had implantation sites at terminal sacrifice.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 p.i.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: yes
- Soft tissue examinations: yes
- Skeletal examinations: yes
- Head examinations: yes

Statistics:

DUNNETT-test; Kruskal-Wallis test; Wilcoxon-Test; Fischer´s exact test;
Statistic of clinical, necropsy and fetal examinations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Orange or reddish discolored urine was recorded in all substance-treated females of test group 3 (200 mg/kg body weight/day) and one dam of test group 2 (60 mg/kg body weight/day) from day 8 p.i. (test group 3) or day 27 p.i. (test group 2) onwards until terminal sacrifice (day 29 p.i.). This urine discoloration mirrors the systemic availability of the test substance, not an adverse, toxic effect. It is most likely due to the excreted test compound or its metabolites. Furthermore, no defecation occurred in mid dose rabbit No. 56 (test group 2, days 27 - 29 p.i.) and is considered as a spontaneous finding due to the scattered occurrence without a relation to dosing. There were no abnormal clinical findings in the other does of the study.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of the females in test groups 1, 2 and 3 (20, 60 and 200 mg/kg body weight/day) did not show any statistically significant differences in comparison to the concurrent control group during the administration period (days 6 – 28 p.i.). At initiation of treatment (days 6 – 9 p.i.), the high dose rabbits showed a considerable and statistically significant weight loss (-20 grams), which is assessed as a substance-induced finding. It is in line with the impaired food consumption of these females during this time period. On the following days until terminal sacrifice the weight gains of the high dose rabbits were sometimes below and sometimes above the concurrent control values. In total, body weight gain of the 200 mg/kg rabbits was about 27% below controls if calculated for the entire treatment phase (days 6 - 28 p.i.) and statistically significantly reduced if calculated for the entire study period (days 0 – 29 p.i.). Body weight gain as presented here, includes both, a maternal and an embryo-/fetal component in general. In the present study, the corrected body weight gain value (reflecting the maternal component only) was lowest in the high dose group animals (see 4.2.1.5.). The respective values of the embryo-/fetal component, i.e. number of resorptions and live fetuses as well as fetal body weights, were not affected by the test substance administration. Thus, the impairments in the high dose dams’ body weight gain, particularly the weight loss at initiation of treatment, are considered as substance–induced, clear signs of maternal toxicity.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The females of the test group 3 (200 mg/kg body weight/day) showed a distinct and statistically significantly lowered food consumption at initiation of treatment (days 6 – 7 p.i.). There were also decreased food uptake values at this dose on the following intervals (attaining statistical significance on days 8 – 17 p.i.). Thereafter, the food consumption of the high dose rabbits was similar to or even exceeded control values. In total, food consumption of the 200 mg/kg rabbits was about 14% below controls if calculated for the entire treatment phase (days 6 - 28 p.i.). This is considered to be substance-related, because body weight gain of these animals was also impaired accordingly (see also 4.2.1.4.). The food consumption of the does of test groups 1 and 2 (20 and 60 mg/kg body weight/day) did not show any substance-related impairments. All differences between these groups and the controls are without any biological relevance and/or not dose related. This includes the statistically significantly higher food consumption in test group 2 (60 mg/kg body weight/day) on days 22 – 23, 24 - 25 and 26 – 27 p.i..

The corrected body weight gain (terminal body weight on day 29 p.i. minus weight of the unopened uterus minus body weight on day 6 p.i.) was slightly lower (about 21%) at 200 mg/kg body weight/day than the concurrent control value. This is, in association with the distinct reductions in food consumption and the body weight loss at initiation of the treatment period, also considered to reflect straight maternal toxicity at 200 mg/kg body weight/day. The corrected body weight gains (terminal body weight on day 29 p.i. minus weight of the unopened uterus minus body weight on day 6 p.i.) of the dams of test groups 1 and 2 (20 and 60 mg/kg body weight/day) revealed no differences of any biological relevance to the corresponding control group. The mean carcass weight (terminal body weight minus mean uterine weight) was unaffected by treatment.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly increased alanine aminotransferase and γ-glutamyltransferase activities were found in the serum of dams given 200 mg/kg bw/day of the test compound. Alanine aminotransferase activities were also higher in the serum of does of the 60 mg/kg-group. No treatment-related changes were seen in the other serum enzymes activities of treated animals. Blood chemistry examinations revealed slightly, but statistically significantly increased inorganic phosphate and calcium concentrations in dams of the 200 mg/kg-group. Increased calcium levels were also found in the serum of does receiving 60 mg/kg bw of the test compound.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Uterus weight
The mean gravid uterus weights of the animals of test groups 1, 2, and 3 (20; 60 or 200 mg/kg body weight/day) did not show any statistically significant differences in comparison tothe control group. The observed differences reflect the usual variation in the strain of rabbits used for this study and thus are without any biological relevance.

Liver weight
The absolute mean liver weights were similar between the controls and test groups 1 and 2 (20 and 60 mg/kg body weight/day) and slightly increased at the top dose (about 13%) without attaining statistical significance. The mean relative liver weights of test group 3 (200 mg/kg body weight/day) were statistically significantly increased (about 16% above the corresponding control value). These weight increases are considered to be substanceinduced, because of corresponding slight increases in serum alanine aminotransferase activity in the high dose group (see 4.2.2.1.2.).The relative mean liver weights of the dams of
test groups 1 and 2 (20 and 60 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant differences.

Spleen weight
Absolute and relative mean spleen weights of the dams of test groups 1, 2, and 3 (20, 60 and 200 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant difference

Kidney weights
The absolute mean kidney weights were similar between the controls and the substancetreated groups. The mean relative kidney weights of test group 3 (200 mg/kg body weight/day) were marginally, but statistically significantly increased (about 7% above the corresponding control value). The relative mean kidney weights of the dams of test groups 1 and 2 (20 and 60 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant differences. The marginal, but statistically significantly increased relative kidney weights at the top dose are probably by chance findings, because similar effects did not occur in a supplementary prenatal developmental toxicity study in rabbits, which was performed under comparable study conditions, but with a dose of 220 mg/kg body weight/day. Moreover, a corresponding morphological correlate was missing of 220 mg/kg body weight/day was missing.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One low dose female was sacrificed after abortion before schedule. Spontaneous abortions or deaths in single does are not uncommon findings in the strain of rabbits used for this study.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

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

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Overt signs of maternal toxicity were observed at the top dose, i.e. 200 mg/kg body weight/day.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

organ weights and organ / body weight ratios

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of all test groups were not influenced by the test substance administration and were close to or even exceeded the corresponding control values. It is very likely, that some of the observed differences in fetal body weights were caused by the spontaneous fluctuations in the mean number of live fetuses/doe between the groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

The scattered occurrence of external (Tab. IC-003) and/or soft tissue malformations throughout all test groups including the controls without a consistent pattern, without a clear dose-response relationship and/or at incidences, which were generally similar to historical control rates, does not suggest any substance-induced origin of these findings. The external malformations affected the neural tube; the visceral malformations were related to heart, gallbladder, urinary and genital tract.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal examinations revealed a slight, statistically significant increase of skeletal malformations in the high dose group (200 mg/kg body weight/day. The most salient of these malformations affected the skull (severely malformed skull bones), the vertebral column (e.g. misshapen cervical or lumbar vertebra, absent lumbar vertebra, cervical hemivertebra, small/fused cervical or splayed lumbar arch), the sternum (sternebrae severely fused) and the ribs (branched rib). Since a number of different skeletal components of different ontogenic origin was affected, these findings do not form a distinct malformation pattern.

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

If all different types of malformations are summarized, in total 4 of the 154 examined control fetuses [= 2.6%] in 4 out of 23 litters [= 17%], 4 of the 135 examined low dose fetuses [= 3.0%] in 4 out of 22 litters [= 18%], 2 out of 138 examined mid dose fetuses [= 1.4%] in 2 out of 22 litters [= 9.1%] and 12 out of 126 examined high dose fetuses [= 9.5%] in 11* out of 23 litters [= 48%] showed malformations. The mean percentages of affected fetuses/litter with total malformations amounted to 2.9, 3.9, 1.5 and 11.8*% at 0; 20; 60 or 200 mg/kg body weight/day, respectively (* = p≤ 0.05 Wilcoxon-test (one-sided)).

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

2

Key result

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: vertebra

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

The following substance-related, adverse effects/findings occurred:

Test group 3 (200 mg/kg body weight/day):

- statistically significantly reduced food consumption (up to 46% below the corresponding control value on the first treatment days, 14% if calculated for the entire treatment phase)

- distinct body weight loss at initiation of treatment (-20 grams on days 6 - 9 p.i.) and impaired weight gains thereafter (27% below controls if calculated for days 6 - 28 p.i.)

- corrected body weight change 21% below the corresponding control value

- slightly increased absolute (+ 13%) and statistically significantly increased relative (+ 16%) liver weights

- statistically significant increases of alanine aminotransferase, inorganic phosphate and calcium values

- slight, statistically significant increase in the rate of fetuses/litter with skeletal malformations (8.1%* versus 1.3% in the controls) affecting skull, vertebral column, sternum and/or ribs and consequently slight, statistically significant increase in the rate of fetuses/litter with total malformations (11.8%* versus 2.9% in the controls) * = p< 0.05, ** = p≤< 0.01 Wilcoxon-test (one-sided)

Test group 2 (60 mg/kg body weight/day):

- no substance-related adverse effects on dams, gestational parameters or fetuses Test group 1 (20 mg/kg body weight/day):

- no substance-related adverse effects on dams, gestational parameters or fetuses

Thus, under the conditions of this full-scale study, the administration of N-Ethyl-2- pyrrolidone to pregnant female Himalayan rabbits by stomach tube at a dose level of 200 mg/kg body weight/day from implantation to one day prior to the expected day of parturition (days 6 - 28 p.i.) elicited overt signs of maternal toxicity. Maternal toxicity was substantiated by e.g. reduced food consumption, impairments in absolute and corrected body weight gain and indications of a mild liver damage. There were no substance-related influences on the gestational parameters up to and including the highest dose level (200 mg/kg body weight/day). Conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or the values calculated for the pre- and postimplantation losses were unaffected by the treatment. No substance-related differences to the control were recorded for placental and fetal body weights in any of the substance-treated groups. External and soft tissue examinations of the low, mid and high dose fetuses did not suggest any substance-induced background for the observed findings. The examination of the fetal skeletons, however, revealed signs of substance-induced prenatal developmental toxicity at the top dose (200 mg/kg body weight/day), which were substantiated by statistically significantly increased rates of skeletal and total malformations. A number of skeletal components of different ontogenic origin was affected (skull, vertebral column and ribs), without forming a distinct malformation pattern. Thus, the oral administration of N-Ethyl-2-pyrrolidone to pregnant Himalayan rabbits during organogenesis caused unspecific, borderline effects on fetal morphology at a dose of 200 mg/kg body weight/day, but induced no substance-related signs of embryo-/fetotoxicity at 20 and 60 mg/kg body weight/day.

Applicant's summary and conclusion

Conclusions:

The oral administration of N-Ethyl-2-pyrrolidone to pregnant Himalayan rabbits during organogenesis caused unspecific, borderline effects on fetal morphology at a dose of 200 mg/kg body weight/day, but induced no substance-related signs of embryo-/fetotoxicity at 20 and 60 mg/kg body weight/day. Based on the results of this prenatal developmental toxicity study, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 60 mg/kg body weight/day. Thus, signs of prenatal developmental toxicity did only occur at a dose level, which was also clearly toxic to the dams.

Executive summary:

The administration of N-Ethyl-2 -pyrrolidone to pregnant female Himalayan rabbits by stomach tube at a dose level of 200 mg/kg body weight/day from implantation to one day prior to the expected day of parturition (days 6 - 28 p.i.) elicited overt signs of maternal toxicity. Maternal toxicity was substantiated by e.g. reduced food consumption, impairments in absolute and corrected body weight gain and indications of a mild liver damage. There were no substance-related influences on the gestational parameters up to and including the highest dose level (200 mg/kg body weight/day). Conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or the values calculated for the pre- and postimplantation losses were unaffected by the treatment. No substance-related differences to the control were recorded for placental and fetal body weights in any of the substance-treated groups. External and soft tissue examinations of the low, mid and high dose fetuses did not suggest any substance-induced background for the observed findings. The examination of the fetal skeletons, however, revealed signs of substance-induced prenatal developmental toxicity at the top dose (200 mg/kg body weight/day), which were substantiated by statistically significantly increased rates of skeletal and total malformations. A number of skeletal components of different ontogenic origin was affected (skull, vertebral column and ribs), without forming a distinct malformation pattern. Thus, the oral administration of N-Ethyl-2-pyrrolidone to pregnant Himalayan rabbits during organogenesis caused unspecific, borderline effects on fetal morphology at a dose of 200 mg/kg body weight/day, but induced no substance-related signs of embryo-/fetotoxicity at 20 and 60 mg/kg body weight/day. Based on the results of this prenatal developmental toxicity study, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 60 mg/kg body weight/day. Thus, signs of prenatal developmental toxicity did only occur at a dose level, which was also clearly toxic to the dams.