1-ethylpyrrolidin-2-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a 90d oral study according to OECD 408 Groups of 10 male and 10 female Wistar rats received doses of 0, 100, 300 and 1000 mg N-Ethyl-2 -pyrrolidone/kg bw/d via their diet (BASF, 2006). Additionally, sperm parameters were determined immediately after necropsy and organ weight determination in this study. Among other effects at the highest dose level (reduced bw and bw gain, centrilobular liver hypertrophy, alpha 2u nephropathy in males, changes in clinical pathology), sperm analysis revealed an increased number of sperms with abnormal heads in males at 1000 mg/kg/d (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). According to the authors of the study, these findings are indicative of disrupted sperm maturation when the test compound is administered at high oral dosages. There were no histopathological changes in the testis and the number of homogenization resistant spermatids, epididymal sperm count and sperm motility was not affected.

In females, there were no effects on the reproductive organs after oral and inhalative subchronic exposure. Due to a possible interference with sperm maturation in males at high oral dosages in repeated dose experiments (NOAEL 300 mg/kg bw/d, LOAEL 1000 mg/kg bw/d), there was a suspicion of a fertility impairing effect.

In the 90d inhalation study, multifocal tubular degeneration in the testes occurred in control and treated males without relation to the concentration resulting in debris and/or oligospermia in the epididymides in some of these males (BASF, 2013). Comparable findings in testes and in the epididymides have been observed frequently in head-nose exposed control animals in past inhalation studies in the same laboratory. Therefore, the occurrence of tubular degeneration as well as of the resulting debris and oligospermia in the epididymides in this inhalative subchronic study was considered to be incidental.

In females, there were no effects on the reproductive organs after oral and inhalative subchronic exposure even at high dose levels (up to 1000 mg/kg bw/d).

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline conforming subchronic toxicity study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Oral, rat

Doses of 0 (distilled water), 50, 250, 500 and 750 mg/kg bw/daywere given toSprague-Dawley rats (19 to 24 pregnant), by gavage, on gestational days (GD) 6–20. Decreased body weight gain and food consumption, demonstrating maternal toxicity was observed in all NEP groups at the beginning of treatment (GD 6–9) (Saillenfait, 2007). The incidence of resorptions, malformed fetuses per litter and the number of litters with malformed fetuses were significantly increased at 500 mg/kg bw/day. Resorption rate reached 83% at 750 mg/kg bw/day. Malformations mainly consisted of edema, anal atresia with absent tail, cardiovascular defects and fused cervical arches. Ossification of skull bones and sternebrae was significantly reduced. at 250 mg/kg bw/day and higher doses the incidence of supernumerary ribs was significantly elevated. In conclusion, NEP administered by gavage is embryotoxic and teratogenic at maternal toxic doses. Beyond this there was transient body weigth gain reduction from day 6-9 GD, day 1-3 of application at concentration 50 mg/kg bw/day onward. This may point to maternal toxicity in the sensitive implantation period.

The no-observed-adverse-effect-level (NOAEL) for maternal toxicity is below 50 mg/kg bw/day and the NOAEL for prenatal developmental toxicity could be fixed at 50 mg/kg bw/day (LOAEL maternal toxicity 50 mg/kg bw/day, LOAEL prenatal developmental toxicity 250 mg/kg bw/d). 

Oral, rabbit

N-Ethyl-2-pyrrolidone (NEP) was tested for its prenatal developmental toxicity in Himalayan rabbits in a GLP guideline study according to OECD 414 (BASF, 2007). NEP was administered as an aqueous solution to 25 inseminated female Himalayan rabbits/group by stomach tube at doses of 0, 20; 60 and 200 mg/kg bw on day 6 through day 28 post insemination. 22 - 23 females/group had implantation sites at terminal sacrifice.

The administration of N-Ethyl-2- pyrrolidone at a dose level of 200 mg/kg bw/day elicited overtsigns of maternal toxicity (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 including the highest dose level (200 mg/kg bw/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 bw/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 bw/day, but induced no substance-related signs of embryo-/fetotoxicity at 20 and 60 mg/kg bw/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 bw/day. Thus, signs of prenatal developmental did only occur at a dose level, which was also clearly toxic to the dams (LOAEL 200 mg/kg bw/d).

There was a supplementary oral developmental toxicity study according to OECD 414 in the same strain of rabbits and a similar study design but with a different dose level (220 mg/kg bw/day) (BASF, 2007). The oral administration of 220 mg N-Ethyl-2 -pyrrolidone/kg bw/day to pregnant Himalayan rabbits during organogenesis caused clear signs of maternal toxicity and unspecific signs of prenatal developmental toxicity similar to those seen in the above mentioned prenatal developmental toxicity study at a dose of 200 mg/kg bw/day. The supplementary study therefore confirms the result of the first study mentioned above.

 

Dermal, rat

NEP was applied dermally (6 hours/day) as an aqueous solution to 25 presumed pregnant female Wistar rats/group another developmental toxicity according to OECD 414 (BASF, 2005). Rats were dosed 200, 400 and 800 mg/kg bw onto the intact shaven dorsal skin using a semi-occlusive dressing on day 6 through day 19 post coitum. NEP elicited clearsigns of maternal toxicity at 800 mg/kg bw/day and less pronounced effects on the 400 mg/kg bw/day mothers (reduced food consumption and impaired absolute and corrected body weight data). No adverse, substance-induced effects on the dams occurred at the low dose level (200 mg/kg bw/day). Dermal application of up to 800 mg/kg bw/day had no influence onthe gestational parameters. Signs of prenatal developmental toxicity (reduced placental and fetal body weights increased rate of skeletal variations, delayed ossification) occurred exclusively at the high dose level (800 mg/kg bw/day) also eliciting clear sings of maternal toxicity. However, no indications for selective teratogenicitywere observed up to and including 800 mg/kg bw/day. No substance-induced signs of embryo-/fetotoxicity at all were observed at 200 or 400 mg/kg bw/day .

Based on the results of this prenatal developmental toxicity study with dermal application, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity is 200 mg/kg bw/day (LOAEL 400 mg/kg bw/d) and the NOAEL for prenatal developmental toxicity could be fixed at 400 mg/kg bw/day (LOAEL 800 mg/kg bw/d). 

Dermal, rabbit

Teratogenic properties of NEP after dermal contact were examinded in the second species as well. NEP was applied as an aqueous preparation to 3 groups of 25 inseminated female Himalayan rabbits each at doses of 100, 300 and 1000 mg/kg bw/day, onto the intact shaven dorsal skin using a semi-occlusive dressing, on gestation day (GD) 6 through GD 28 for 6 hours/day (BASF, 2010). Highly deionized water was used as vehicle. At terminal sacrifice on GD 29, 21-24 females per group had implantation sites.

Statistically significantly reduced mean food consumption (by 42% at maximum) on GD 6-17, reduced mean food consumption during the entire treatment (by 17%) were observe at 1000 mg/kg bw/day. Distinct weight loss at initiation of treatment (-15 grams on GD 6–9) and impaired body weight gain thereafter (61% below control for GD 9–11), recovery from GD 12. In conclusion, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity is 300 mg/kg bw/day based on reduced food consumption and body weight gain. Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. They did not indicate a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. Furthermore, the overall incidences were comparable to the historical control data. For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were without a relation to dosing. One specific skeletal variation, supernumerary 13th rib without cartilage, was statistically significantly increased in the high-dose group compared to the concurrent control. Supernumerary 13th ribs without cartilage are quite common in gestation day 29 rabbit fetuses of this strain, as can be seen from the historical control data. Thus an association of these findings to the treatment was therefore not assumed.The no-observed-adverse-effect-level (NOAEL) for prenatal developmental toxicity was set by the study authors at 1000 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

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

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.

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.

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.

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

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

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.

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

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

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)).

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

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

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: vertebra

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

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.

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.

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch identification: 39522688QO
purity: 99.8%

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Time-mated Wistar rats (CrIGIxBrIHan:Wl) that were free from clinical signs of disease were used for the investigations. The animals were mated by the breeder and supplied on day 0 post coitum (= detection of vaginal plug / sperm). They were assigned to the test groups by random selection from the transport box. After randomization the rats were identified uniquely by ear tattoo. The rats were housed singly from day 0- 20 p .c. in type DK III stainless steel wire mesh cages. The animals were accommodated in fully air-conditioned rooms in which central air conditioning maintained a temperature range of 20 - 24°C and a range of relative humidity of 30 - 70%. There were no deviations from these limits. The day/night rhythm was 12 hours (12 hours light and 12 hours darkness. The food used was ground Kliba maintenance diet mouse-rat (GLP). Food was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy), as was drinking water of tap water quality, from water bottles.

Route of administration:

dermal

Vehicle:

water

Details on exposure:

The 33.3% aqueous test substance solution was applied dermally to the intact shaven dorsal skin of the animals using a semi-occlusive dressing on day 6 through day 19 p.c. The animals of the control group were treated in the same way with the vehicle. Dose volumes of 2.4; 0.6; 1.2 and 2.4 ml/kg body weight/day were used for the test groups 0- 3. The animals were shaved on the dorsal area of the trunk at least 18 hours before the first treatment (day 6 p .c.) and at additional times during the treatment period as necessary. On day 20 p.c., the rats were sacrificed in a randomized order and examined macroscopically. 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 7 days at room temperature were carried out before the study was initiated.

Details on mating procedure:

The animals were paired by the breeder ("time-mated") and supplied on day 0 post coitum (= detection of vaginal plug I sperm).

Duration of treatment / exposure:

day 6 through day 19 post coitum (p.c.)

Frequency of treatment:

6 h/day

Duration of test:

until day 20 p.c.

Remarks:

Doses / Concentrations:
0, 200, 400 and 800 mg/kg body weight

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

A check was made twice a day on working days or once a day. The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited. The application site was examined twice (before and after the 6-hour exposure) (days 6 - 19 p.c.). In case of any observed skin reactions, these would have been scored according to the OECD Guideline for Testing of Chemicals No . 404. With the exception of day 0, the consumption of food was determined on the same days. All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results as was body weight. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day or more oftn when clinical signs of toxicity were elicited (d0-20 p.c.)

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1,3,6,8,10,13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
With the exception of day 0, the consumption of food was determined on the same days as was body weight.

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

Skin examination
The application site was examined twice (before and after the 6-hour exposure) (days 6-19 p.c.). In case of any observed skin reactions, these would have been scored according to the OECD Guideline for Testing of Chemicals No. 404: Acute Dermal
Irritation/Corrosion (adopted July 17, 1992).

POST-MORTEM EXAMINATIONS: Yes
On day 20 p.c., the dams were sacrificed in randomized order by cervical dislocation and the fetuses removed from the uterus. After the dams had been sacrificed, they were necropsied and assessed by grass pathology.
The uterus 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 c1assified as: live fetuses or dead implantations (a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri fram apparently non-pregnant animals
and the 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)

- Organs examined:

Ovaries and uterine content:

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

Fetal examinations:

yes- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [all per litter / half per litter]

Statistics:

yes
DUNNETT-test; Kruskal-Wallis test; Wilcoxon-Test; Fischer´s exact test

Historical control data:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Orange or reddish discolored urine was recorded in all substance-treated females at 400 or 800 mg/kg body weight/day from day 7 p.c. (800 mg/kg) or day 8 p.c. (400 mg/kg) onwards until terminal sacrifice (day 20 p.c.). This urine discoloration mirrors the systemic availability of the test substance, not an adverse, toxie effeel. It is most Iikely due to the exereted test eompound or its metabolites. Furthermore, 3 contral females, 1 low dose female, 7 mid dose females and 4 high dose females showed occasionally vaginal hemorrhage during treatment days 13 - 16 p.c.. This finding, which occurred only on a narraw range of treatment days, showed no clear relation to treatment as several contral dams were also similarly affected. It is very likely, that this finding occurred in association with the dermal, semi-occlusive application of the test substance and the inevitable handling of the pregnant rats and thus is not substance, but treatment-induced. The transient occurrence of vaginal bleeding in some females of all test graups had neither an adverse effect on the mean number of implantation sites (8.7; 9.8; 9.0; 9.0 at 0; 200; 400 or 800 mg/kg body weightlday; historical control range 8.1 - 10.2) nor did it cause an impairment of the mean number of live fetuses (8.2; 9.2; 8.5; 9.0 at 0; 200; 400 or 800 mg/kg body weightlday;

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

The skin was free fram any notable findings during days 6 - 19 p.c. before the daily dermal application and after the daily 6-hour exposure time. Moreover, no abnormal skin findings occurred during days 0 - 6 p.c. (pretreatment period) and on day 20 p.c. (day of terminal sacrifice).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of the high dose rats (800 mg/kg body weightlday) were statistically significantly below control values on days 8-13 and 19-20 p.c.. On day 20 p.c., the mean body weight in this test group was about 5% below the mean value of the concurrent control females. In the mid dose group (400 mg/kg body weightlday) the mean body weight was statistically significantly reduced on day 8 p.c.. Mean body weights of test group 1 (200 mg/kg body weightlday) were similar to those of the controls. There was a statistically significantly body weight loss in the dams of test groups 2 and 3 at initiation of treatment (days 6-8 p.c.). On the following treatment days, however, body weight gains of both test groups reached or even exceeded control values (attaining statistical significance on days 8-10 p.c., respectively). Nevertheless, if calculated for the entire treatment period (days 6-19 p.c.) the body weight gains of the mid dose females
were about 10% (without attaining statistical significance) and that of the high dose dams about 22% (with statistical significance) below the respective control values. Moreover, the weight gain of the top dose rats was also statistically significantly impaired during
days 0-20 p.c. (about 15% below the concurrent control value). Body weight gains of the dams of test group 1 (200 mg/kg body weightlday) were similar to those of the concurrent controls. All observable differences in this group in comparison to the controls during the pretreatment and the treatment period are without any biological relevance and reflect the normal variation inherent in the strain of rats used in the present experiment.
The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.) was distinctly and statistically significantly lower in test groups 2 and 3 (400 or 800 mg/kg body weight/day) (about 21% or 43% below the concurrent control value). Furthermore, the carcass weight of the high dose dams was statistically significantly reduced in comparison to the control group (about 5% below controls). These effects are assessed as direct, substance-related signs of maternal toxicity. Mean corrected body weight gain of the dams of test group 1 (200 mg/kg body weight/day) was similar to the corresponding control value and did not show statistically significant or biologically relevant deviations.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the high dose dams (800 mg/kg body weightlday) was statistically significantly reduced on treatment days 6-10 and 17-19 p.c.. If calculated for the entire treatment period (days 6-19 p.c.), it was about 10% below the concurrent contraI value.
In the mid dose graup (400 mg/kg body weightlday) the mean food consumption was statistically significantly decreased on gestation days 6-8 (about 13% below the concurrent control value). Thereafter, food consumption of these rats recovered and was similar to contral values. The statistically significantly increased food consumption valueof the mid dose dams on gestation days 0-1 is, of course, without any biological relevance. The food consumption of the females of test graup 1 (200 mg/kg body weightlday) was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the contrals. The transient reductions in food consumption of the mid dose dams at initiation of treatment and of the high dose dams during the treatment period are considered to be substance- induced; corraborative effects on body weight data and corrected body weight gain of these females did also occur..

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean gravid uterus weight was slightly reduced in test group 3 (800 mg/kg body weightlday) without attaining statistical significance and reached only 94% of the respective control value. This has to be seen in association with the lower placental and fetal body weights in this test group and is considered to be substance-induced. The uterus weights of the animals of test groups 1 and 2 (200 or 400 mg/kg body weightlday) were not influenced by the application of the test substance. The differences between these groups and the control group are without biological relevance.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

The conception rate reached 84% in test group 2 (400 mg/kg body weightlday), 88% in test group 3 (800 mg/kg body weightlday), 92% in test group 1 (200 mg/kg body weightlday) and 96% in test group O. As all presumed pregnant rats had implantation sites at necropsy, a sufficient number of females were available for the purpose of the study (according to the test guidelines listed in chapter 2.3.).
There were no 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 the postimplantation losses, the number of resorptions and viable fetuses.

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

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Clear signs of maternal toxicity at 800 mg/kg body weight/day and less pronounced effects on the 400 mg/kg mothers. The adverse effects on the mid and/or high dose dams were predominantly substantiated by reduced food consumption and impaired absolute and corrected body weight data. No adverse, substance-induced effects on the dams occurred at the low dose level (200 mg/kg body weight/day).

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: maternal toxicity

Key result

Dose descriptor:

LOAEL

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of the high dose fetuses (800 mg/kg body weightlday) were statistically significantly reduced (about 11% below the concurrent control values if both sexes are combined). This was assessed to be test substance-induced. The mean fetal body weights in test groups 1 and 2 (200 and 400 mg/kg body weightlday) were not influenced by the test substance application and were similar to the control values. This includes the marginally, but statistically significantly reduced mean male fetal body weight at 400 mg/kg (3.4 gram versus 3.6 gram in the male contral fetuses).

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

One external malformation was recorded for three fetuses of the control graup. Omphalocele (with ruptured sac) was recorded for 3 male fetuses from one contral dam. The isolated occurrence of this malformation in 3 fetuses of one contral litter is a spontaneous finding. In total, 3 out of 197 control fetuses [=1.5%] (in 1 out of 24 litters [= 4.2%]), none of the 211 low dose fetuses (fram 23 Iitters), none ofthe 178 mid dose fetuses (from 21 litters) and none of the 189 high dose fetuses (from 21 litters) showed external malformations. The mean percentages of affected fetuses/litter with external malformations amounted to 1.4, 0.0, 0.0 and 0.0%, respectively in test groups 0-3 (0, 200, 400 or 800 mg/kg body weightlday) without attaining statistical significance. No external variations and no unclassified external observations were seen in any fetuses of any group.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Malformations of the skeletons occurred in one control, one mid dose and one high dose fetus each, but not in one of the low dose fetuses. A misshapen lumbar vertebra (with present cartilage) was recorded for the affected control and high dose fetuses, whereas the mid dose fetus in question showed several malpositioned and bipartite sternebrae (with unchanged cartilage). In total, one out of 106 control fetuses [= 0.9%] (in one of 24litters [= 4.2%]), none of the 112 low dose fetuses (from 23 litters), one out of 95 mid dose fetuses [= 1.1 %] (in one of 21 litters [= 4.8%]) and one of 100 high dose fetuses [= 1.0%] in one of 21 litters [= 4.8%] showed skeletal malformations. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 1.0, 0.0, 0.8, and 1.0% respectively in test groups 0-3 and thus did not show any relation to dosing.

Visceral malformations:

no effects observed

Description (incidence and severity):

No soft tissue malformations were seen in any fetuses of any group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

External variations did not occur in any of the fetuses in this study. Soft tissue variations, exclusively in the form of dilated renal pelvis and/or ureters occurred in test groups 0, 200 or 400 mg/kg body weight/day, but not at the top dose and thus did not show any relation to dosing. The overall incidence of skeletal variations, however, was statistically significantly increased at 800 mg/kg body weight/day due to the increased occurrence of delays and minor disturbances in the ossification process of various bones (predominantly skull and sternebrae) and supernumerary 14th ribs in this test group. The observed increased delays in the ossification process of the high dose fetuses have to be seen in conjunction with the distinctly reduced mean fetal body weights in this group. If all variations are summarized, in total 110 of the 197 examined control fetuses [= 56%) in all 24 litters [= 100%), 119 of the 211 examined low dose fetuses [= 56%) in all 23 Iitters [= 100%), 100 out of 178 mid dose fetuses [= 56%) in all 21 litters [= 100%) and 100 out of 189 high dose fetuses [= 53%) in all 21 litters [= 100%) showed variations. The mean percentages of affected fetuses/litter with total variations amounted to 57.0, 56.5, 58.8, and 53.0% at 0, 200,400 or 800 mg/kg body weight/day, respectively. There occurred no external or soft tissue unclassified observations in any of the fetuses. A spontaneous origin is assumed for the few unclassified cartilage observations which were recorded for several fetuses of test groups 0, 1, 2 and 3. Distribution and type of these findings do not suggest any relation to treatment as the mean percentages of affected fetuses/litter with these findings amounted to 61.9, 59.5, 66.1, and 60.8% at 0, 200, 400 or 800 mg/kg body weightlday, respectively.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No substance-induced signs of embryo-/fetotoxicity at all were observed at 200 or 400 mg/kg body weight/day.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: skeletal variations and delayed ossification, supernumerary 14th ribs

Key result

Dose descriptor:

LOAEL

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: increased skelatal variations (delayed ossification of skull and sternebrae), increased number of supernumerary 14th rib

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: skeletal variations (skull, sternebrae, supernumerary ribs)

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day

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

The following substance-related findings were obtained:

Test group 3 (800 mg/kg body weight/day):

- statistically significantly reduced food consumption on days 6-10 and 17-19 p.c. with a 10% reduction in overall food intake if calculated for the entire treatment period (days 6-19 p.c.)

- statistically significantly reduced mean body weights on days 8-13 and 19-20 p.c.; about 5% below controls on day 20 p.c.

- statistically significant body weight loss at initiation of treatment (days 6-8 p.c.) and a statistically significant weight gain reduction if calculated for days 6-19 p.c. (about 22% below controls )

- statistically significantly decreased corrected body weight gain (about 43% below controls)

- statistically significantly lowered carcass weight (about 5% below controls)

- slightly reduced mean gravid uterus weight (about 6% below controls without attaining statistical significance)

- statistically significantly lower mean placental weights (about 17% below controls; both genders combined)

- statistically significantly lower mean fetal body weights (about 11% below controls; both genders combined)

- statistically significantly increased rate of fetuses/litter with skeletal variations (delays/minor disturbances in ossification, predominantly of skull and sternebrae; supernumerary 14th ribs)

Test group 2 (400 mg/kg body weight/day):

statistically significantly reduced food consumption on days 6 - 8 p.c. (about 13% below controls)

- statistically significant body weight loss at initiation of treatment (days 6-8 .c.) and a 10% weight gain reduction if calculated for days 6 - 19 p.c. (without attaining statistical significance )

- statistically significantly decreased corrected body weight gain (about 21 % below controls)

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

Test group 1 (200 mg/kg body weight/day):

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

Under the conditions of this prenatal developmental toxicity study, the dermal application of N-Ethyl-2-pyrrolidone to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (days 6 - 19 p.c.) elicited clear signs of maternal toxicity at 800 mg/kg body weight/day and less pronounced effects on the 400 mg/kg mothers. The adverse effects on the mid and/or high dose dams were predominantly substantiated by reduced food consumption and impaired absolute and corrected body weight data. No adverse, substance-induced effects on the dams occurred at the low dose level (200 mg/kg body weight/day). The dermal application of 200, 400 or 800 mg N-Ethyl-2-pyrrolidone/kg body weight/day had no influence on the gestational parameters. The dermal test substance application evoked signs of prenatal developmental toxicity, which occurred exclusively at the high dose level in the form of significantly reduced placental and fetal body weights and an increased rate of skeletal variations (e.g. delays/ minor disturbances in the ossification of skull and sternebrae as well as supernumerary 14th ribs). However, no indications for selective teratogenicity were observed up to and including 800 mg/kg body weight/day. No substance-induced signs of embryo- /fetotoxicity at all were observed at 200 or 400 mg/kg body weight/day.

Conclusions:

Based on the results of this prenatal developmental toxicity study with dermal application, the no observed adverse effect level (NOAEL) for maternal toxicity is 200 mg/kg body weight/day and the NOAEL for prenatal developmental toxicity could be fixed at 400 mg/kg body weight/day. Thus, signs of prenatal developmental toxicity (with no indications for teratogenicity) occurred only at a dose level that was already clearly toxic to the dams.

Executive summary:

Under the conditions of this prenatal developmental toxicity study, the dermal application of N-Ethyl-2-pyrrolidone to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (days 6 -19 p.c.) elicited clear signs of maternal toxieity at 800 mg/kg body weight/day and less pronounced effects on the 400 mg/kg mothers. The adverse effects on the mid and/or high dose dams were predominantly substantiated by reduced food consumption and impaired absolute and corrected body weight data. No adverse, substance-induced effects on the dams occurred at the low dose level (200 mg/kg body weight/day).

The dermal application of 200, 400 or 800 mg N-Ethyl-2-pyrrolidone/kg body weight/day had no influence on the gestational parameters. The treatment evoked signs of prenatal developmental toxicity, which occurred exclusively at the high dose level in the form of significantly reduced placental and fetal body weights and an increased rate of skeletal variations (e.g. delays/minor disturbances in the ossification of skull and sternebrae as weil as supernumerary 14th ribs). However, no indications for selective teratogenicity were observed up to and including 800 mg/kg body weight/day. No substance-induced signs of embryo/fetotoxicity at all were observed at 200 or 400 mg/kg body weight/day.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: only publication available; guideline not reported, not reported whether study was conducted according to GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-surl’Arbresle, France)
- Weight at study initiation: 180–200 g
- Housing: single in polycarbonate cages and corn cob granules as bedding.
- Diet (e.g. ad libitum): food pellets(UAR Alimentation Villemoisson, France) ad libitum
- Water (e.g. ad libitum): filtered tap water ad libitum
- Acclimation period: 1 to 2 weeks of acclimatisation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C,
- Humidity (%): 50 ± 5%,
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were formulated in distilled water as vehicle.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Females were housed overnight with adult males from the same strain and supplier. The day sperm was detected in the vaginal smear was considered to be day 0 of gestation (GD). The dosing volume was 5 ml kg−1. Initial doses were based on the GD 6 weight and adjusted every 3 days throughout the treatment period. A control group received the vehicle under the same conditions.

Duration of treatment / exposure:

Pregnant females were given daily doses of NEP, at approximately the same time eachday, by gastric intubation on GD 6–20.

Frequency of treatment:

daily

Duration of test:

GD0 -20

Remarks:

Doses / Concentrations:
0 (distilled water), 50, 250, 500 and 750 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group size ranged from 24 to 25 time-mated rats (19 to 24 pregnant).

Control animals:

yes, concurrent vehicle

Details on study design:

The dosage levels of the definitive study were based on the findings of a dose-range finding study

Maternal examinations:

CAGE SIDE OBSERVATIONS: No
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
Maternal body weights were recorded on GD 0, 6, 9, 12, 15, 18 and 21.
FOOD CONSUMPTION: Yes
Food consumption was measured at 3-day intervals starting on GD 6.
POST-MORTEM EXAMINATIONS: No

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:

Live fetuses were weighed, sexed and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were
examined for visceral changes. The others were used for subsequent skeletal examination.

Statistics:

values are given in mean ± SD.
The number of corpora lutea, implantation sites and live fetuses and various body weights were analysed by oneway analysis of variance, followed by Dunnett’s test if differences were found. The frequency of post-implantation loss, dead fetuses, resorptions and alterations among litters was evaluated by using the Kruskal-Wallis test followed by the Mann-Whitney test where appropriate.
Rates of pregnancy and of litters with dead fetuses or resorptions, and incidences of fetal alterations per dose were analysed using Fisher’s test. Where applicable, least-squares analysis was carried out. The reported level of statistical significance was P < 0.05. The litter was used as the basis for the analysis of fetal variables.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Significant reduced food consumption at 750 mg/kg/day and reduced body weightwere observed. Beyond this there was transient body weigth gain reduction from day 6-9 GD, day 1-3 of application at concentration 50 mg/kg onward. This may point to maternal toxicity in the sensitive implantation period.

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:

Reduced fetal bw and supranumerary ribs discoverd at 250 mg/kg/day. Administration of 500 mg/kg/day caused delayed ossification and resorptions. Resorption rate at 750 mg/kg/ day was 83%. Malformations described after administration of 750 mg/kg/day included edema, anal atresia with absent tail, cardiovascular defects and fused cervical arches.

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Basis for effect level:

fetal/pup body weight changes

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

effects observed, treatment-related

Localisation:

other: visceral, skeletal and external malformations

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

NEP administered by gavage was embryotoxic and teratogenic at maternal toxic doses (NOAEL 50 mg/kg bw/day).

Executive summary:

N-ethyl-2-pyrrolidone (NEP) was studied for oral teratogenicity in a second species in a developmantal toxicity study similar to OECD 414 (val.2). Doses of 0 (distilled water), 50, 250, 500 and 750 mg/kg/daywere given to Sprague-Dawley rats (19 to 24 pregnant), by gavage, on gestational days (GD) 6–20. Decreased body weight gain and food consumption, demonstrating maternal toxicity was observed in all NEP groups at the beginning of treatment (GD 6–9). The incidence of resorptions, malformed fetuses per litter and the number of litters with malformed fetuses were significantly increased at 500 mg/kg^/day. Resorption rate reached 83% at 750 mg/kg/day. Malformations mainly consisted of edema, anal atresia with absent tail, cardiovascular defects and fused cervical arches. Ossification of skull bones and sternebrae was significantly reduced. At 250 mg/kg/day and higher doses the incidence of supernumerary ribs was significantly elevated. In conclusion, NEP administered by gavage is embryotoxic and teratogenic at maternal toxic doses. Beyond this there was transient body weigth gain reduction from day 6-9 GD, day 1-3 of application at concentration 50 mg/kg onward. This may point to maternal toxicity in the sensitive implantation period. Maternal toxicity however may not explain teratogenic effects.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 414 Guideline study according to GLP Full study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

dermal

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

dermal

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

dermal

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch Id. : 29151288P0
Purity: 99.8 corr. peak area-%

Species:

rabbit

Strain:

Himalayan

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: 17-20 weeks
- body weight: 2077-2681 g (GD0)
- Housing: singly in type 12.2395, C stainless steel wire mesh cages (floor area about 3000 cm2). For enrichment, wooden gnawing blocks (Typ KNH E-041)
- Diet (e.g. ad libitum): Kliba maintenance diet for rabbits & guinea pigs, GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland (ad libitum)
- Water (e.g. ad libitum): Tap water (ad libitum)
- Acclimation period: 1 - 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): range of temperature of 20–24°C
- Humidity (%): 30 70%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12h / 12 h

Route of administration:

dermal

Vehicle:

water

Details on exposure:

TEST SITE AND REMOVAL OF TEST SUBSTANCE
The test substance preparation was supplied on on the intact shaven dorsal skin using one-way syringes (e.g. Omnifix®, B. Braun, Melsungen AG, Melsungen, Germany) and covered by a 8-layer gauze patch, measured about 10 x 20 cm. The patch will be fixed (semi-occlusive) by means of a stretchable bandage (e.g. Fixomull®, Beiersdorf AG, Hamburg, Germany). After the daily 6-hour treatment period, the dressing will be removed and the application site will be washed off with luke-warm water and dried.


TEST MATERIAL AND VEHICLE
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the period of established stability. For the preparation of the test substance, one part of test substance and two parts of highly deionized water were given in a volumetric flask and intensely mixed.

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 7 days at room temperature were carried out before the study was initiated (in a similar batch).

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.

A synthetic hormone (0.2 mL) which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male Himalayan rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as 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 (NIJENHUIS and WILF) and on the basis of their body weights.

The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.

Duration of treatment / exposure:

GD 6-28

Frequency of treatment:

once daily 6 h dermal semi occlusive exposure

Duration of test:

GD 0-29

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

N-Ethyl-2-pyrrolidone was tested for its prenatal developmental toxicity in Himalayan rabbits after maternal exposure during the critical period of organogenesis. The test substance was applied dermally (6 hours/day) as an aqueous preparation to 3 groups of 25 inseminated female Himalayan rabbits each at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d), onto the intact shaven dorsal skin using a semi-occlusive dressing, on gestation day (GD) 6 through GD 28. Different volumes (i.e. 0.3, 0.9 and 3.0 mL N-Ethyl-2-pyrrolidone/kg body weight) of a 33.3% aqueous solution were dermally applied to the rabbits of test groups 1, 2 or 3. The control group, consisting of 25 females, was dosed with the vehicle (highly deionized water at a volume of 3.0 mL/kg bw/d) in parallel. At terminal sacrifice on GD 29, 21-24 females per group had implantation sites.

Maternal examinations:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0 29). A cageside clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).

BODY WEIGHT: Yes
All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The food consumption was determined daily on GD 1–29.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Consumption was determined daily on GD 1–29.

POST-MORTEM EXAMINATIONS: Yes
Animals were necropsied and assessed by gross pathology in randomized order

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:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
Each fetus was weighed and examined macroscopically for any external findings. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded.

- Soft tissue examinations: Yes: all per litter includes sex determination by examination of the gonads in situ.
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

see below

Historical control data:

yes

Description (incidence and severity):

Reddish discolored urine was recorded in all substance-treated females of test group 3 (1000 mg/kg bw/d) from GD 8 onwards until sacrifice on GD 29. 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. Reduced defecation occurred in one control rabbit (GD 21-26). No defecation was noted in one mid-dose rabbit (GD 28-29) before it aborted on GD 29. There were no abnormal clinical findings in the other does.

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal each from the low- and the mid-dose group either died intercurrently or was sacrificed after abortion on GD 29, without showing any preterminal signs of illness or impaired well-being. Spontaneous abortions or deaths in single does are not uncommon findings in rabbits.This and the lacking dose response do not suggest a relationship to the treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

After initiation of treatment (GD 6-9), the high-dose rabbits showed a marked and statistically significantly decreased body weight gain, the animals actually lost weight (approx. -15 grams). On the following treatment days (GD 9-11) the body weight gain was still considerably reduced (by 61%) although not statistically significant. Afterwards the weight gain of the high dose group recovered to normal, thus the average body weight gain of the high-dose rabbits was not statistically significantly reduced when looked at the whole treatment period (GD 6-28). The average high-dose body weights were below the control from GD 6 onwards until the end of the study, the difference was, however, not statistically significant. The mean body weights and body weight gain of the low- and mid-dose rabbits (100 and 300 mg/kg bw/d) were comparable to the concurrent control. The observable, insignificant differences between the test substance-treated groups and the controls were biologically not relevant. Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all test groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption in the high-dose females (1000 mg/kg bw/d) was distinctly and statistically significantly reduced (by 42% at maximum) on GD 6-17. This was considered to be treatment-related. During the last third of gestation (from GD 22 onwards) food consumption of the high-dose rabbits recovered slightly and was occasionally higher than the concurrent control (statistically significant GD 27/28, +20%). The average food consumption during the treatment (GD 6-28) was about 17% below control in the high-dose rabbits. The food consumption of the low- and mid-dose does (100 or 300 mg/kg bw/d) did not show test substance-related changes.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of test groups 1, 2, and 3 (100, 300 or 1000 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One animal each from the mid-dose group either died intercurrently was sacrificed after abortion on GD 29 (No. 61), without showing any preterminal signs of illness or impaired well-being. Spontaneous abortions or deaths in single does are
not uncommon findings in rabbits.This and the lacking dose response do not suggest a relationship to the treatment.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The conception rate reached 88% in test group 2 (300 mg/kg bw/d), 92% in the control group and in test group 3 (1000 mg/kg bw/d) and 100% in test group 1 (100 mg/kg bw/d). Importantly, a sufficient number of pregnant females was available for the purpose of the study (according to test guidelines listed in section 2.3.), as 21-24 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice. There were no test substance-related and/or biologically relevant differences between the control and all dosed groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.

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:
Statistically significantly reduced mean food consumption (by 42% at maximum) on GD 6-17, reduced mean food consumption during the entire treatment (by 17%) were observe at 100 mg/kg. Distinct weight loss at initiation of treatment (-15 grams on GD 6–9) and impaired body weight gain thereafter (61% below control for GD 9–11), recovery from GD 12. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300
mg/kg bw/d based on reduced food consumption and body weight gain.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

One sole external malformation (cleft palate) occurred in one fetus of the high dose group. This was an isolated finding which may occur spontaneously in rabbits. Thus, an association of this finding to treatment was therefore not assumed.

see table 1 below

Skeletal malformations:

no effects observed

Description (incidence and severity):

Malformations of the fetal skeletons were noted in single fetuses of all test groups including control (0, 100, 300 and 1000 mg/kg bw/d). Neither statistically significant differences between treated groups and the control were calculated nor was a dose-response relationship observed. Most of the individual malformations are present in the historical control data. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.

see table 2 below

Visceral malformations:

no effects observed

Description (incidence and severity):

The examination of the soft tissues revealed a number of malformations in litters of all test groups including control (0, 100, 300, and 1000 mg/kg bw/d). No statistically significant differences between the test groups and the control were observed. Neither a dose response-relationship nor a malformation pattern was evident. Most of the individual soft tissue malformations were present in the historical control data at comparable frequencies, some (like absent gallbladder) are quite common in this rabbit strain. Thus, an association of these findings to the treatment was therefore not assumed.

ses table 3 below

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal skeletal variations
For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were without a relation to dosing. One specific skeletal variation, supernumerary 13th rib without cartilage, was statistically significantly increased in the high-dose group compared to the concurrent control. Supernumerary 13th ribs without cartilage are quite common in gestation day 29 rabbit fetuses of this strain, as can be seen from the historical control data. Thus an association of these findings to the treatment was therefore not assumed.

See table 4

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect on prenatal development of offspring at any of the dose level

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect on prenatal development of offspring at any of the dose level

Developmental effects observed:

no

The following test substance-related adverse effects/findings were noted:

Test group 3 (1000 mg/kg bw/d):

• Statistically significantly reduced mean food consumption (by 42% at maximum) on GD 6-17, reduced mean food consumption during the entire treatment (by 17%)

• Distinct weight loss at initiation of treatment (-15 grams on GD 6–9) and impaired body weight gain thereafter (61% below control for GD 9–11), recovery from GD 12

• Lower average body weights GD 6-29, not statistically significant

Test group 2 (300 mg/kg bw/d):

• No test substance-related adverse effects on does, gestational parameters or fetuses

Test group 1 (100 mg/kg bw/d):

• No test substance-related adverse effects on does, gestational parameters or fetuses

Table 1 Total fetal external 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	23 155	24 155	21 134	23 144
Fetal incidence	N	0.0	0.0	0.0	1 (0.7%)
Litter incidence	N	0.0	0.0	0.0	1 (4.3%)
Affected fetuses/litter	Mean%	0.0	0.0	0.0	0.7

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

Table 2. Individual fetal skeletal malformations:

 

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	12-07, M	Absent lumbar vertebra
	14-05, F	Branched rib-unilateral
1 (100 mg/kg bw/d)	29-05, F	Sternebrae severely fused (bony plate)
	36-01, M	Absent lumbar vertebra
	48-01, F	Absent acromion-unilateral
	50-08, F	Absent lumbar vertebra
2 (300 mg/kg bw/d)	63-04, F	Sternebrae severely fused (bony plate)
3 (1000 mg/kg bw/d)	77-04, F	Sternebrae severely fused (bony plate)

mg/kg bw/d = milligram per kilogram body weight per day; M = male; F = female

Table 3 Individual fetal soft tissue malformations:

 

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	1-08, F	Absent kidney and ureter-unilateral
1 (100 mg/kg bw/d)	28-08, M	Absent kidney and ureter-unilateral
	37-01, F	Absent gallbladder
	37-07, F	Absent gallbladder
	37-11, F	Absent gallbladder
	38-01, F	Membranous ventricular septum defect of the heart
	38-02, F	Aortic arch atresia
2 (300 mg/kg bw/d)	53-04, M	Absent gallbladder
	65-01, F	Misshapen spleen
	68-02, M	Absent gallbladder
	68-05, M	Asplenia, absent pancreas
	68-07, M	Absent gallbladder
	74-04, M	Absent subclavian-unilateral
3 (1000 mg/kg bw/d)	82-02, F	Absent subclavian-unilateral, membranous ventricular septum defect of the heart
	86-02, F	Membranous ventricular septum defect of the heart
	91-08, M	Dextrocardia, supernumerary lung lobe
	91-09, M	Dextrocardia
	91-10, M	Dextrocardia

mg/kg bw/d = milligram per kilogram body weight per day; M = male; F = female

 

 

Table 4 Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

 

Finding Supernumerary 13thrib, cartilage notpresent	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	Hist Control Data Mean % (range)
Fetal incidence
N	12	1	16	23	6.9
%	7.7	0.6	12	16	2.5-13.9
Litter incidence
N	6 Fi	1	8	13*	28.3
%	26	4.2	38	57	8.0-52.2
Affected fetuses per litter mean%	8.2 Wi	0.6	14.3	15.2*	7.3 (2.1-17.5)

mg/kg bw/d = mg/kg body weight/day; HCD = Historical control data;

Fi = Fisher’s exact test [one-sided]; Wi = Wilcoxon-Test [one-sided]; * = p =< 0.05

Conclusions:

N-Ethyl-2-pyrrolidone was administered to pregnant Himalayan rabbits dermally (6 hours/day onto the intact shaven dorsal skin using a semi-occlusive dressing) from implantation to one day prior to the expected day of parturition (GD 6-28).The no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg bw/d based on reduced food consumption and body weight gain in the does at 1000 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity was according to the study authors 1000 mg/kg bw/d.

Executive summary:

NEP was applied as an aqueous preparation to 3 groups of 25 inseminated female Himalayan rabbits each at doses of 100, 300 and 1000 mg/kg bw/day onto the intact shaven dorsal skin using a semi-occlusive dressing on gestation day (GD) 6 through GD 28 for 6 hours/day. Highly deionized water was used as vehicle. At terminal sacrifice on GD 29, 21-24 females per group had implantation sites.

Statistically significantly reduced mean food consumption (by 42% at maximum) on GD 6-17, reduced mean food consumption during the entire treatment (by 17%) were observed at 100 mg/kg. Distinct weight loss at initiation of treatment (-15 grams on GD 6–9) and impaired body weight gain thereafter (61% below control for GD 9–11), recovery from GD 12. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg bw/d based on reduced food consumption and body weight gain. Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. They showed neither a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. Furthermore, the overall incidences were comparable to the historical control data. For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were without a relation to dosing. One specific skeletal variation, supernumerary 13th rib without cartilage, was statistically significantly increased in the high-dose group compared to the concurrent control. Supernumerary 13th ribs without cartilage are quite common in gestation day 29 rabbit fetuses of this strain, as can be seen from the historical control data. Thus an association of these findings to the treatment was therefore not assumed.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity was according to the study authors set at 1000 mg/kg bw/d.

Reference 5

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance was administered 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 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 solution were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentration. Since the test substance preparation was a true solution, investigations concerning homogeneity were not necessary.

Details on mating procedure:

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 2295 - 2943 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.

Remarks:

Doses / Concentrations:
0, 220 mg/kg body weight
Basis:

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

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:

clinical, necropsy and fetal examinations; clinical pathology

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Substance-related, overt signs of maternal toxicity were observed at 220 mg/kg b.w.

Key result

Dose descriptor:

LOAEL

Effect level:

220 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

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

Key result

Dose descriptor:

LOAEL

Effect level:

220 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in litter size and weights

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: various external, skeletal and visceral malformations

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

220 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:

not specified

Relevant for humans:

not specified

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

Test group 1 (220 mg/kg body weight/day):

- statistically significantly reduced food consumption (up to 47% below the corresponding control value on the first treatment days, 19% if calculated for the entire treatment phase)

- distinct body weight loss at initiation of treatment (-38 grams on days 6 - 9 p.i.) and impaired weight gains thereafter (38% below controls if calculated for days 6 - 28 p.i.).

- corrected body weight change 23% below the corresponding control value

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

- statistically significant increase of alanine aminotransferase

- statistically significantly lower placental and fetal body weights

- statistically significantly increased rates of fetuses/litter with soft tissue (13.2%** versus 5.1% in the controls), skeletal (8.8%* versus 2.6% in the controls) and total malformations (16.9%** versus 7.2% in the controls)

- statistically significantly increased rates of fetuses/litter with skeletal (82.0%** versus 61.7% in the controls) and consequently total variations (82.7%** versus 63.0% in the controls) * = p < 0.05; ** = p < 0.01 Wilcoxon-test (one-sided)

Thus, under the conditions of this supplementary study, the administration of N-Ethyl-2-pyrrolidone to pregnant female Himalayan rabbits by stomach tube at a dose level of 220 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. 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. Indications for substance-induced prenatal developmental toxicity consisted of reduced placental and fetal body weights, statistically significantly increased rates of soft tissue, skeletal and total malformations and of statistically significantly increased rates of skeletal and total variations. The observed broad, non selective spectrum of malformations of different ontogenic origin and the spontaneous occurrence of several of these malformations in actual and/or historical control groups does not indicate a distinct malformation pattern. The increased variation rate has to be seen in association with the statistically significantly reduced mean fetal weights and the frank maternal toxicity.

Conclusions:

The oral administration of 220 mg N-Ethyl-2-pyrrolidone/kg body weight/day to pregnant Himalayan rabbits during organogenesis caused clear signs of maternal toxicity and unspecific signs of prenatal developmental toxicity similar to those seen in the previous prenatal developmental toxicity study (BASF AG 2007; Project No. 40R0033/04058) at a dose of 200 mg/kg body weight/day. Based on the results of the previous (BASF AG 2007; Project No. 40R0033/04058) and the present, supplementary prenatal developmental toxicity study, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 60 mg/kg body weight/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Species:

rabbit

Quality of whole database:

Klimisch 1 study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Species:

rat

Quality of whole database:

Klmisch 1 study

Justification for classification or non-classification

Fertility

In a 90 -day oral study according to OECD 408, sperm analysis revealed an increased number of sperms with abnormal heads in males at 1000 mg/kg among other toxic effects of 1-Ethyl-pyrrolidin-2 -one (NEP) at this dose level. There were no histopathological changes in the testis and the number of homogenization resistant spermatids, epididymal sperm count and sperm motility was not affected. Fertility was not examined in this study. According to the authors of the study, these findings are indicative of disrupted sperm maturation when the test compound is administered at high oral dosages (NOAEL 300 mg/kg bw/d, LOAEL 1000 mg/kg bw/d). In females, there were no effects on the reproductive organs after oral and inhalative subchronic exposure. In addition, in inhalation studies with exposure up to 13 weeks, effects on sperm were not observed.

According to the CLP regulation, substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects. 

In the case of NEP, effects on sperm morphology were observed in an oral 90d oral study at the limit dose of 1000 mg/kg bw//d. An adverse effect on sperm maturation after high oral exposures therefore cannot be completely excluded. A classification Category 2 according to CLP criteria for possible fertility impairing effects seems to be justified based on a weight of evidence approach using the observations in the oral subchronic study.

 

Developmantal toxicity

1-Ethyl-pyrrolidin-2 -one (NEP) is not teratogenic via the dermal route in two species (rat; rabbit). Developmental effects were observed in presence of maternal toxicity. N-Ethyl-2 -pyrrolidone was tested for its prenatal developmental toxicity in Himalayan rabbits in a GLP guideline study according to OECD 414 by gavage. The examination of the fetal skeletons, 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. The same oral dose level of 200 mg/kg body weight/day elicited overt signs of maternal toxicity (reduced food consumption (-46%), impairments in absolute and corrected body weight gain (-27%, -21 %) and indications of a mild liver damage). Liver damage was visible by slightly increased absolute (+13%) and statistically significantly increased relative (+16%) liver weights as well as statistically significant increases of alanine aminotransferase and y-glutamyltransferase.

 

NEP was studied for oral teratogenicity (gavage) in a second species in a study similar to OECD 414. Decreased body weight gain and food consumption, demonstrating maternal toxicity was observed in all NEP groups. The incidence of resorptions, malformed fetuses per litter and the number of litters with malformed fetuses were significantly increased at 500 mg/kg^/day. Resorption rate reached 83% at 750 mg/kg/day. Malformations mainly consisted of edema, anal atresia with absent tail, cardiovascular defects and fused cervical arches. Ossification of skull bones and sternebrae was significantly reduced. at 250 mg/kg/day and higher doses the incidence of supernumerary ribs was significantly elevated. In conclusion, NEP administered by gavage is embryotoxic and teratogenic at maternal toxic doses.

Based on the above described results in the studies with oral and dermal administration a category 1B for developmental toxicity according to Regulation (EC) No. 1272/2008 is warranted. This classification for developmental toxicity was included in the 5th ATP to CLP became mandatory by 01 January 2015.