1-methyl-2-pyrrolidone

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

endocrine system modulation

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

1. SOFTWARE
Approach with integrated results from 13 ER related in vitro HTS assays from EPA ToxCast program combined with an ER Interaction Score. The Score described the chance of a chemical being estrogenic. Besides, a quantitative score for ERα/ERβ selectivity and agonist/antagonist activity could be presented.

2. MODEL (incl. version number)
Not specified

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS number, combined with ToxCast chemical library

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The ER Interaction Scores and the in vivo rodent estrogenicity was compared by using data from the OECD guideline and Endocrine Disruptor Screening Program (EDSP) T1S validation studies.
The in vivo data were described as 'active' or 'inactive' for agonist or antagonist activity based on changes in rodent uterine weight.

5. APPLICABILITY DOMAIN
As this model is newly developed an applicability domain is not defined.

Further information are given in Table 1.

Qualifier:

no guideline available

Principles of method if other than guideline:

In this study the results from 13 estrogen receptor (ER) related in vitro high-throughput screening (HTS) assays from the EPA ToxCast program were integrated into an ER Interaction Score that represents the overall liklihood of a chemical being estrogenic.

GLP compliance:

not specified

Type of method:

in vitro

Remarks:

combination of in vitro and in silico

Endpoint addressed:

other: Endocrine property

Specific details on test material used for the study:

Data observed from ToxCast chemical library and ToxCast assay battery data.

Details on study design:

The present study describes a novel approach for incorporating data from multiple in vitro assays into an integrated quantitative score that reflects a chemicals's perturbation of a specific signaling pathway. Experimental data used in the analysis was obtained from a total of 1814 chemicals tested in the 13 assays (Table 1). The panel of in vitro assays interrogated multiple endpoints related to estrogen receptor (ER) signaling, namely binding, agonist, antagonist, and cell growth responses. All assay responses were normalized to 17β-estradiol, except those in the antagonist group, which were normalized to 4 -hydroxytamoxifen.

Details on results:

The test substance did not display indications of interacting with the ER signaling pathway.

Table 1: ER in vitro assay annotation and model grouping

Assay name	Assay information	Group
NVS_NR_hER	Human ER binding assay	binding
NVS_NR_mERa	Murine ERαbinding assay	Binding
NVS_NR_bER	Bovine ER binding assay	binding
OT_ERaERa_1440_agonist	Odyssey Thera ERα-ERαdimerization in agonist mode after 1440 min	agonist
OT_Era_ERb_1440_agonist	Odyssey Thera ERα-ERβdimerization in agonist mode after 1440 min	agonist
OT_ERbERb_1440_agonist	Odyssey Thera ERβ-ERβdimerization in agonist mode after 1440 min	agonist
Tox21_Era_LUC_BG1_Antagonist	ERαluciferase reporter gene assay in human BG-1 ovarian cells in antagonist mode	antagonist
Tox21_Era_BLA_Antagonist_ratio	ERαβ-lactamase reporter gene assay in human HEK-293 cells in antagonist mode	antagonist
Tox21_Era_LUC_BG1_Agonist	ERαluciferase reporter gene assay in human BG-1 ovarian cells in agonist mode	agonist
Tox21_Era_BLA_Agonist_ratio	ERαβ-lactamase reporter gene assay in human HEK-293 cells in agonist mode	agonist
ATG_ERE_CIS	Multiplex ER reporter gene assay using full length receptor in HepG2 cells	agonist
ATG_Era_TRANS	Multiplexed GAL4 reporter construct with human ERαligand-binding domain in HepG2 cells	agonist
ACEA_T47D_80h	Cell growth using real-time cell analysis in T47D cells at 80 h	Cell growth

Conclusions:

The test substance did not display indications of interacting with the ER signaling pathway.

Reference 2

Endpoint:

mechanistic studies

Remarks:

TS-Phase response mouse liver

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of the study was to determine a possible influence of DNA-synthesis/cell proliferation ("S-phase response") in the liver after treatment with N-Methylpyrrolidone for a period of 1 week and 4 weeks.

GLP compliance:

yes (incl. QA statement)

Type of method:

in vivo

Endpoint addressed:

not applicable

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, from continuous production, tank No. 53
- Analytical purity: 99.9 % (gas chromatography)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature under N2, to be protected from air
- Stability under test conditions: stability ensured by substance supplier (purity proven by reanalysis: 98.8%)

Species:

mouse

Strain:

B6C3F1

Remarks:

86C3F1/Rj IOPS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre d'Elevage R. Janvier, France
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation, group means: males: 27.7 g, females: 22.2 g
- Fasting period before sacrifice: 16-20 hours
- Housing: singly in Makrolon cages (Becker & Co., Castrop-Rauxel, Germany) in a fully air-conditioned room
- Diet: Kliba maintenance diet rat/mouse/hamster, meal (Provimi Kliba, Kaiseraugst, Switzerland), ad libitum
- Water: ad libitum
- Acclimation period: on day of arrival, not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12
Technical failures led to transient, minor deviations from these ranges which had no influence on the results of the study. Documentation of the deviations is retained in the study raw data.

IN-LIFE DATES: From: 2000-05-03 To: 2000-06-07

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: once at the beginng of the study, fodd was changed weekly
- Mixing appropriate amounts with: laboratory feed as used in the study
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Capillary gas chromatograph equipped with autosampler, split injector and flame ionization detector (FID) and adapted to an electronic integrator.

Duration of treatment / exposure:

daily in the diet for 1 and 4 weeks

Frequency of treatment:

daily in the diet

Dose / conc.:

7 200 ppm (nominal)

Remarks:

equivalent to 1,392 mg/kg bw/day in males and 1,906 mg/kg bw/day in females

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

One week prior necropsy, osmotic minipumps containing bromodeoxyuridine (BrdU) were implanted subcutaneously. Bromodeoxyuridine incorporated in the DNA of liver cells was detected by immunohistochemistry and evaluated microscopically.

Examinations:

Food consumption and body weights were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day.

Details on results:

1-week treatment
-         6.9-fold increase of cell proliferation in the liver of males, 3.3-fold increase of cell proliferation in the liver of females
-         mitotic figures increased in the liver of males
-         minimal or slight centrilobular, hepatocellular hypertrophy in males (9/10) and one female (1/10)
-         less pronounced fat storage (males) or loss of fat storage (females) in the liver
-         decrease of liver weights in females

4-week treatment
-         slightly decrease of body weight in males (4.6 % below control on day 28)
-         2.1-fold increase of cell proliferation in the liver of males, 1.7-fold increase of cell proliferation in the liver of males
-         increased number of apoptotic cells in the liver of males
-         minimal or slight centrilobular, hepatocellular hypertrophy in males (7/10) and two females (2/10)
-         less pronounced fat storage (males) or loss of fat storage (females) in the liver An increase of cell proliferation (S-Phase response) in the liver was seen after treatment with NMP for 1 and 4 weeks

There was clear evidence that NMP is able to induce an increase in hepatocellular proliferation after dietary treatment for 1 or 4 weeks.

Conclusions:

According to the results of the study, a clear increase of cell proliferation (S-Phase response) in the liver was seen after treatment with NMP for 1 and 4 weeks.

Executive summary:

N-Methylpyrrolidone (NMP) was administered to groups ot 10 male and 10 temale B6C3F1 mice at a dietary concentration ot 7,200 ppm tor 1 and 4 weeks (mean test substance intake ot 1,392 mg/kg bw/day in males and 1,906 mg/kg bw/day in temales). An untreated group served as control. Food consumption and body weights were determined weekly. The animals were examined for signs ot toxicity or mortality at least once a day. One week prior necropsy, osmotic minipumps containing bromodeoxyuridine (BrdU) were implanted subcutaneously. Bromodeoxyuridine incorporated in the DNA of liver cells was detected by immunohistochemistry and evaluated microscopically.

In conclusion, the following substance-related findings were observed:

1-week treatment:

- 6.9-fold increase of cell proliferation in the liver of males, 3.3-fold increase of cell proliferation in the liver of temales

- mitotic figures increased in the liver ot males

- minimal or slight centrilobular, hepatocellular hypertrophy in males (9/10) and one female (1/10)

- less pronounced tat storage (males) or lass ot tat storage (temales) in the liver

- decrease ot liver weights in temales

4-week treatment:

- slightly decrease of body weight in males (4.6% below control on day 28)

- 2.1-fold increase ot cell proliferation in the liver ot males, 1. 7-fold increase ot cell proliteration in the liver ot males

- increased number of apoptotic cells in the liver of males

- minimal or slight centrilobular, hepatocellular hypertrophy in males (7/10) and two temales (2/10)

- less pronounced tat storage (males) or lass ot tat storage (temales) in the liver

Thus, a clear increase ot cell proliteration (S-Phase response) in the liver was seen after treatment with NMP tor 1 and 4 weeks.

Reference 3

Endpoint:

mechanistic studies

Remarks:

Liver Enzyme Induction (Cyt. P450, EROD, PROD, PALCoA, ELMI)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

no guideline available

Principles of method if other than guideline:

The objective of the study was to determine the effect ofNMP on liver enzyme induction or peroxisome proliferation after dietary administration for a period of 2 weeks .

GLP compliance:

yes (incl. QA statement)

Type of method:

in vivo

Endpoint addressed:

not applicable

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, from continuous production, tank No. 53
- Analytical purity: 99.9 % (gas chromatography)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature under N2, to be protected from air
- Stability under test conditions: stability ensured by substance supplier (purity proven by reanalysis: 98.8%)

Species:

mouse

Strain:

B6C3F1

Remarks:

B6C3F1/Rj IOPS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre d'Elevage R. Janvier, France
- Age at study initiation: 12 - 13 weeks old
- Weight at study initiation: group means: male: 27.1 g, female: 22.5 g
- Fasting period before sacrifice: 16-20 hours
- Housing: singly in Makrolon cages in a fully air-conditioned room
- Diet: diet rat/ mouse/ hamster meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: on day of arrival, not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2000-05-16 To: 2000-06-15

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: once at the beginng of the study, fodd was changed weekly
- Mixing appropriate amounts with: laboratory feed as used in the study
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The NMP containing diet was analyzed to verify stability, homogeneity and stability by means of gas chromatography.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily by feeding

Dose / conc.:

7 200 ppm (nominal)

Remarks:

equivalent to 1,364 mg/kg bw/day in males and 1,945 mg/kg bw/day in females

No. of animals per sex per dose:

10 animals for the following enzyme examinations: Cytochrome P450-content (Cyt .P450), Ethoxyresorufin-O-deethylase (EROD), Pentoxyresorufin-O-depentylase (PROD)
5 animals for the following enzyme examinations: Cyanide-insensitive Palmitoyl-CoA-oxidation (PALCoA), Light and electron microscopy of liver regarding changes in structure or amount on the peroxisomes, endoplasmic reticulum or mitochondria (ELMI)

Control animals:

yes, plain diet

Examinations:

The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Furthermore, the animals were examined in detail once a week.
Food consumption was determined weekly over a period of 7 days and calculated as mean food consumption in grams per animal and day.
The water bottles were checked daily for any overt changes in volume.
The body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

The mean test substance intake was 1,364 mg/kg bw/day in males and 1,945 mg/kg bw/day in females.

The following results were obtained:

PALCoA: slightly increased activities in the males

Cyt.P450: no treatment related effects

EROD: no treatment related effects

PROD: no treatment related effects ELMI: slight increase of peroxisomes in two treated males

Conclusions:

NMP led to slightly increased activity in PALCoA in male animals and electron microscopy revealed a slight elevation in peroxisomes in 2/5 males.

Executive summary:

N-Methylpyrrolidone (NMP) was administered to groups of 10 male and 10 female B6C3F1 mice at dietary concentrations of 0 and 7,200 ppm for 2 weeks. Each 2 livers were pooled and used for the following enzyme examinations:

- Cytochrome P450-content (Cyt .P450)

- Ethoxyresorufin-O-deethylase (EROD )

- Pentoxyresorufin-O-depentylase (PROD )

Additional groups of 5 male and 5 female B6C3F1 mice were treated as mentioned above. In these animals, the following examinations were performed :

- Cyanide-insensitive Palmitoyl-CoA-oxidation (PALCoA) ,

- Light and electron microscopy of liver regarding changes in structure or amount on the peroxisomes, endoplasmic reticulum or mitochondria (ELMI)

The mean test substance intake was 1,364 mg/kg bw/day in males and 1,945 mg/kg bw/day in females.

The following results were obtained:

PALCoA : slightly increased activities in the males

Cyt.P450 : no treatment related effects

EROD: no treatment related effects

PROD : no treatment related effects

ELMI : slight increase of peroxisomes in two treated males

Thus, NMP caused minor signs of peroxisome proliferation after treatment for 2 weeks.

Description of key information

In an in silico screening approach the test substance did not display indications of interacting with the ER signaling pathway and thus is not suspected to be an endocrine disruptor (Rotroff et al., Environ. Sci. Technol. 48, 2014).

The in vivo liver cell proliferation assay in mice showed a clear increase of cell proliferation in the liver after treatment with NMP for 1 and 4 week (BASF, 99C0225/93070, 2002).

In the in vivo liver enzyme induction assay in mice after a 2 week feeding period of 7,200 ppm, NMP caused minor signs of peroxisome proliferation and a slightly increased activity in PALCoA in male animals (BASF, 99C0225/93071, 2002).

Additional information

In silico assay (Rotroff et al., Environ. Sci. Technol. 48, 2014):

In this study the results from 13 estrogen receptor (ER) related in vitro high-throughput screening (HTS) assays from the EPA ToxCast program were integrated into an ER Interaction Score that represents the overall likelyhood of a chemical being estrogenic. Experimental data used in the analysis was obtained from a total of 1814 chemicals tested in the 13 assays. 1-methyl-2-pyrrolidone was one of the substances tested. The panel of in vitro assays interrogated multiple endpoints related to estrogen receptor (ER) signaling, namely binding, agonist, antagonist, and cell growth responses. All assay responses were normalized to 17β-estradiol, except those in the antagonist group, which were normalized to 4 -hydroxytamoxifen. 1-methyl-2-pyrrolidone did not display indications of interacting with the ER signaling pathway.

In vivo liver cell proliferation assay (BASF, 99C0225/93070, 2002):

N-Methylpyrrolidone (NMP) was administered to groups ot 10 male and 10 temale B6C3F1 mice at a dietary concentration ot 7,200 ppm tor 1 and 4 weeks (mean test substance intake ot 1,392 mg/kg bw/day in males and 1,906 mg/kg bw/day in temales). An untreated group served as control. Food consumption and body weights were determined weekly. The animals were examined for signs ot toxicity or mortality at least once a day. One week prior necropsy, osmotic minipumps containing bromodeoxyuridine (BrdU) were implanted subcutaneously. Bromodeoxyuridine incorporated in the DNA of liver cells was detected by immunohistochemistry and evaluated microscopically.

In conclusion, the following substance-related findings were observed:

1-week treatment:

- 6.9-fold increase of cell proliferation in the liver of males, 3.3-fold increase of cell proliferation in the liver of temales

- mitotic figures increased in the liver ot males

- minimal or slight centrilobular, hepatocellular hypertrophy in males (9/10) and one female (1/10)

- less pronounced tat storage (males) or lass ot tat storage (temales) in the liver

- decrease ot liver weights in temales

4-week treatment:

- slightly decrease of body weight in males (4.6% below control on day 28)

- 2.1-fold increase ot cell proliferation in the liver ot males, 1. 7-fold increase ot cell proliteration in the liver ot males

- increased number of apoptotic cells in the liver of males

- minimal or slight centrilobular, hepatocellular hypertrophy in males (7/10) and two temales (2/10)

- less pronounced tat storage (males) or lass ot tat storage (temales) in the liver

Thus, a clear increase ot cell proliteration (S-Phase response) in the liver was seen after treatment with NMP tor 1 and 4 weeks.

In vivo liver enzyme induction assay (BASF, 99C0225/93071, 2002):

N-Methylpyrrolidone (NMP) was administered to groups of 10 male and 10 female B6C3F1 mice at dietary concentrations of 0 and 7,200 ppm for 2 weeks. Each 2 livers were pooled and used for the following enzyme examinations:

- Cytochrome P450-content (Cyt .P450)

- Ethoxyresorufin-O-deethylase (EROD )

- Pentoxyresorufin-O-depentylase (PROD )

Additional groups of 5 male and 5 female B6C3F1 mice were treated as mentioned above. In these animals, the following examinations were performed :

- Cyanide-insensitive Palmitoyl-CoA-oxidation (PALCoA) ,

- Light and electron microscopy of liver regarding changes in structure or amount on the peroxisomes, endoplasmic reticulum or mitochondria (ELMI)

The mean test substance intake was 1,364 mg/kg bw/day in males and 1,945 mg/kg bw/day in females.

The following results were obtained:

PALCoA : slightly increased activities in the males

Cyt.P450 : no treatment related effects

EROD: no treatment related effects

PROD : no treatment related effects

ELMI : slight increase of peroxisomes in two treated males

Thus, NMP caused minor signs of peroxisome proliferation after treatment for 2 weeks.