1-methyl-2-pyrrolidone

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: inhalation

Remarks:

developmental neurotoxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

developmental neurotoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: US EPA OPPTS 870.6300

Version / remarks:

not specified, (OPP 83–6 Developmental Neurotoxicity Study (Pesticide Assessment Guidelines, Subdivision F--Hazard Evaluation: Human and Domestic Animals, Addendum 10, EPA report 540/09– 91–123, March 1991)

Deviations:

yes

Remarks:

Only one concentration instead of three concentrations tested. Only tested until day 20 of gestation instead of day-10 postnatally. Only 16 (14 for the control) instead of 20 litters.

GLP compliance:

not specified

Specific details on test material used for the study:

Source: BASF AG, Ludwigshafen, Germany, commercial grade
Purity: at least 99.5%

Species:

rat

Strain:

Wistar

Remarks:

Mol:Wist

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Exposed sex: females
- Nulliparous: yes
- Age at study initiation: not specified
- Weight at study initiation: 100-120 g
- Fasting during study: food and water removed during exposure
- Housing:
- in pairs, wire mesh cages
- after exposure, on post coitum (PC) day 20: housed alone, plastic cages
- Diet: ad libitum; Altromin Standard Diet nr. 1324
- Water: e.g. ad libitum, tap water
- Acclimation period: 1-day adaptation period to the whole-body inhalation chambers

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 (22-25 in the inhalation chambers)
- Humidity (%): 40-60 % (30-50 % in the inhalation chambers)
- Air changes (per hr): 8 (dynamic air flow of 12 air changes per hour during exposure)
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: not specified
- Source and rate of air: not specified
- Method of conditioning air: heated in a glass spiral heated by circulation of warm water
- Temperature in air chamber: ca. 22-25°C
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used:
- infrared gascell spectrophotometer (MIRAN-1A from Foxboro) was used to monitor the concentration of NMP continously; the spectrophotometer was calibrated daily
- air samples from charcoal tubes were regularly analysed by GC (HP5720A with 3380 A integrator, 6 feetx 1/8" SS column with 0.1% SP1000 80/100 mesh Carbopack)

- Samples taken from breathing zone: no

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: from 6 a.m. to 12 p.m.
- Proof of pregnancy:sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 7 through day 20 of gestation

Frequency of treatment:

6 h/d

Duration of test:

21 d

Dose / conc.:

150 ppm (analytical)

Remarks:

equivalent to 0.618 L/min or 620 mg/m3

No. of animals per sex per dose:

not specified, 37 sperm-positive females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: dose level and exposure period were chosen to minimize maternal toxicity and offspring mortality

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily after exposure

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: gestational day (GD 1, 7, 10, 15 and 20)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on postnatal day 22 of weaning females; rest of the dams and sperm-positive females observed right after sacrifice
- Organs examined: not specified, macrospcopical changes examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: Yes: all per litter
- Behavioral test: surface rightening reflex; homing response; auditory startle reflex; air rightening reflex; rotarod test; open field and Morris water maze test for training, learning, retention, transfer, new platform location and operant conditioning
Results are given in table 1.

Statistics:

- litter or one pup per litter = statistical unit
- Mann-Whitney U test and chi-square test: body weights, developmental milestones and reflexes, results from Rotarod test
- Multivariant analyses (MANOVA): Morris maze results, operant behavior test

Historical control data:

Because a pilot study had indicated that fetal viability was not affected by the chosen dose level and exposure period, the size of the litters was not standardized in the present study.

Clinical signs:

no effects observed

Description (incidence and severity):

- no clinical signs were seen

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no effects observed compared to the control group

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- urine of the females in the exposed group was colored bright yellow

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
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): - no effects observed compared to the control group

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

- no effects observed compared to the control group

Dose descriptor:

NOAEC

Effect level:

150 ppm (analytical)

Based on:

test mat.

Remarks on result:

other: equivalent to 620 mg/m3

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- from birth and throughout preweaning: mean b.w. in litters was significantly lower
- distribution of b.w. was not merely shifted to lower values also more skewed
- difference was not statistically significant after the age of 5 weeks
Results are given in table 2 and table 3.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- no effects observed compared to the control group

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- no effects observed compared to the control group

External malformations:

effects observed, treatment-related

Description (incidence and severity):

- significantly delayed in some recorded developmental milestones

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Behavioral effects:
- exposed pups were significantly delayed in some recorded developmental milestones and in the ontogeny of the surface righting reflex (see Table 4)
- no differences found concerning age at sexual maturation, performance on the Rotarod, or ambulation and percentage of time spent in the center square in open field
- testing for operant behavior indicated no differences in the first training phases concerning outshaping, discrete trials, or visual discrimination
- suggested reduced retention of learned behavior

Dose descriptor:

LOAEC

Effect level:

150 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

external malformations

other: Behavior: impairments of higher cognitive functions (solving difficult tasks)

Remarks on result:

other: equivalent to 620 mg/m3

Abnormalities:

no effects observed

Description (incidence and severity):

insufficient parameter analyzed

Developmental effects observed:

no

Lowest effective dose / conc.:

150 ppm (analytical)

Treatment related:

no

Table 2: Pup body weights.

Body weight after weaning
	Control				NMP
Females	Small*		Medium†		Small*		Medium†
Day 22	41± 1		42 ± 1		37 ± 1§		39 ± 1†
Day 24	45± 1		48 ± 1		41 ± 1§		44 ± †
Day 28 or 29	61 ± 2		65 ± 1		60 ± 2		62 ± 2
7-8 weeks	155± 4		165 ± 5		147 ±3		155 ± 4§
11-12 weeks	197± 5		210 ± 6		190 ± 4		201 ± 4
Males
Day 22	41± 1		43 ± 1		39 ± 1§		40 ± 1‡
Day 24	47 ± 1		48 ± 1		43 ± 1§		44± 1‡
Day 28	65 ± 1		68 ± 1		62 ± 1		66 ± 1
Day 36 (± 1)	108 ± 3		113 ± 3		104 ± 3		105 ± 3‡
7-8 weeks	218 ± 7		221 ± 6		209 ± 6		206 ± 7
11-12 weeks	335 ± 11		340 ± 7		320 ± 6		327 ± 7
14-15 weeks	368 ± 13		375 ± 8		346 ± 8		354 ± 8

*Pup with lowest body weight in litter at weaning;†Pup with median body weight in litter at weaning;‡p<0.05,Mann-Whitney U Test, NMP versus control;§p<0.10, Mann-Whitney U Test, NMP versus control; mean ± SD.

Table 3: Body weight in litters in the preweaning period.

Group	Control	NMP
Number of litters	14	16
Day 1	5.9 ± 0.4	5.5 ± 0.3*
Day 7	12.8 ± 0.9	11.9 ± 1.0*
Day 10	17.6 ± 1.3	16.6 ± 1.6*
Day 14	24.0 ± 1.7	22.6 ± 2.0*
Day 19	32.5 ± 3.2	30.S ± 2.7†
Day 22	42.2 ± 3.7	39.S ± 3.3*

*p<0.05,Mann-Whitney U Test; †p<0.10, Mann­ Whitney U Test; Mean±SD.

Table 4: Results of the developmental milestones and the reflex testing in exposed pups.

Developmental milestones and reflexes
		Control	NMP
Number of litters		14	16
Ear unfolding	positive - day 2	2%	0%
	positive - day 3	88%	48%*
Surface righting reflex	negative - day 1	50%	59%
	negative - day 2 negative - day 3	41% 6%	39% 15%†
	negative - day 4	O%	3%
Homing response	positive - day 6	67%	77%
	positive - day 7	95%	95%
Incisor eruption	positive - day 9 positive - day 10	14% 50%	8% †37%
	positive - day 11	91%	77%
	positive - day 12	99%	93%
Auditory startle reflex	positive - day 11	0%	0%
	positive . day 12	11%	5%
	positive- day 13	93%	77%
Eye opening	positive - day 13	6%	9%
	positive - day 14	44%	43%
	positive. day 15	98%	83%*
Air righting reflex	positive- day 14	26%	32%
	positive. day 15	53%	49%
	positive- day 16	98%	99%

Mean of%of litters;*p<5%,Mann-Whitney U Test;†p<5%,x²-test.

Conclusions:

The physical development of the pups was delayed based on retarded body weight gain until weaning and the results of ear unfolding, surface rightening reflex, incisor eruption and eye opening. However, no treatment-related effects were noted on landmarks of physical development and reflexes during the later part of pre-weaning and thereafter and no effects on onset of puberty, open filed parameter and performance on rotarod. The results of the Morris water maze and operant conditioning were contradictory since some parameter and sessions showed an impairment, while other demonstrated no differences.

Executive summary:

Pregnant rats (Mol: WIST) were exposed to 150 ppm N-methylpyrrolidone for 6 hours per day on gestation days 7-20 (Hass et al., 1994). The dose level was selected so as not to induce maternal toxicity or decrease viability of the offspring. In the preweaning period, the exposed offspring had a lower body weight and their physical development was delayed. Neurobehavioral evaluation of the male pups revealed no effects on basal functions of the central nervous system. The animals appeared normal in motor function (rotarod), activity level (open field), and performance in learning tasks with a low grade of complexity were similar in the two groups. However, in more difficult tasks such as the reversal procedure in Morris water maze and operant delayed spatial alternation (Skinner boxes), performance was impaired in exposed offspring.

Data source

Reference

Reference Type:

publication

Title:

Effects of prenatal exposure to N-methylpyrrolidone on postnatal development and behavior in rats

Author:

Hass U., Lund S.P., Elsner J.

Year:

1994

Bibliographic source:

Neurotoxicol. Teratol., 16(3), 241-249

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

Version / remarks:

not specified, (OPP 83–6 Developmental Neurotoxicity Study (Pesticide Assessment Guidelines, Subdivision F--Hazard Evaluation: Human and Domestic Animals, Addendum 10, EPA report 540/09– 91–123, March 1991)

Deviations:

yes

Remarks:

Only one concentration instead of three concentrations tested. Only tested until day 20 of gestation instead of day-10 postnatally. Only 16 (14 for the control) instead of 20 litters. Examinations differ from guideline and are given in table 1.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-2-pyrrolidone

EC Number:

212-828-1

EC Name:

1-methyl-2-pyrrolidone

Cas Number:

872-50-4

Molecular formula:

C5H9NO

IUPAC Name:

1-methylpyrrolidin-2-one

Test material form:

liquid

Specific details on test material used for the study:

Source: BASF AG, Ludwigshafen, Germany, commercial grade
Purity: at least 99.5%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Exposed sex: females
- Nulliparous: yes
- Age at study initiation: not specified
- Weight at study initiation: 100-120 g
- Fasting during study: food and water removed during exposure
- Housing:
- in pairs, wire mesh cages
- after exposure, on post coitum (PC) day 20: housed alone, plastic cages
- Diet: ad libitum; Altromin Standard Diet nr. 1324
- Water: e.g. ad libitum, tap water
- Acclimation period: 1-day adaptation period to the whole-body inhalation chambers

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 (22-25 in the inhalation chambers)
- Humidity (%): 40-60 % (30-50 % in the inhalation chambers)
- Air changes (per hr): 8 (dynamic air flow of 12 air changes per hour during exposure)
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: not specified
- Source and rate of air: not specified
- Method of conditioning air: heated in a glass spiral heated by circulation of warm water
- Temperature in air chamber: ca. 22-25°C
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used:
- infrared gascell spectrophotometer (MIRAN-1A from Foxboro) was used to monitor the concentration of NMP continously; the spectrophotometer was calibrated daily
- air samples from charcoal tubes were regularly analysed by GC (HP5720A with 3380 A integrator, 6 feetx 1/8" SS column with 0.1% SP1000 80/100 mesh Carbopack)

- Samples taken from breathing zone: no

Duration of treatment / exposure:

day 7 through day 20 of gestation

Frequency of treatment:

6 h/d

Doses / concentrations

Dose / conc.:

150 ppm (analytical)

Remarks:

equivalent to 0.618 L/min or 620 mg/m3

No. of animals per sex per dose:

not specified, 37 sperm-positive females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: dose level and exposure period were chosen to minimize maternal toxicity and offspring mortality

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily after exposure

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: gestational day (GD 1, 7, 10, 15 and 20)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on postnatal day 22 of weaning females; rest of the dams and sperm-positive females observed right after sacrifice
- Organs examined: not specified, macrospcopical changes examined

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

Details of the examinations/behavioral determinations and the number of animals used in the test are given in table 1.

Behavioral Tests
- testing performed between 0800 h and 1400 h

Surface righting reflex
- the time taken by the pup to right itself on all four feet after it had been placed in a supine position
- maximum time allowed was 15 s; number of pups per litter to fulfill criterion was recorded daily, one trial per day on PND 1 and the following days until all pups in the litter met the criterion

Homing response (nest-seeking response mediated by the olfactory system)
- conducted on PNDs 6 and 7
- littermates and dam were removed during testing
- pup placed longitudinally on a "bridge" of plastic (8 x 20 cm) connecting the homecage and a cage with clean bedding
- pups choosing the home bedding site by crossing the edge of the "bridge" with the front paws and head were recorded as having fulfilled the criterion in the test

Auditory startle reflex
- presence or absence of starle reflex was assessed by visual observation following the delivery of a click from a BIC-pencil held at a distance of 1 cm from each ear

Air righting reflex
- pups were held in a supine position and dropped from a height of 40 cm on a 2-3 cm thick layer of home bedding
- criterion fullfilled by landing on all four feet

Rotarod
- two males per litter tested as described by Ladefoged (Ladefoged et al., Pharmacol. Toxixol. 68:384-390, 1991) with minor modifications
- speed: 10 rpm, 10 rpm, and 12 rpm on PNDs 24, 25, and 26, respectively, and the maximum time was 30 s
- rats scored for the amount of time on the rod, the percentage of animals in each group reaching the 30 s criterion

Open field
- on days 27 and 28, 2 males per litter were recorded on videotape for 3 min in a square open field (75 cm)
- videotapes analysed by means of a computer with a frame-grabber (DT2853, Data Translation), a subroutine library for image processing (OT-IRIS, Data Translation) and a lab specific program
- calculation of the total distance the animals moved in the field and the percentage time spent in the center square of the field when dividing the field into 9 squares of equal size

Morris water maze
- testing of spatial learning and retention in Morris water maze performed in a common manner
- 13 to 15 male rats used, representing 6 to 7 litters from each group
- black, circular plastic pool with a diameter of 220 cm filled to a depth of 30 cm with tap water at room temperature (20°C)
- four points on the rim of the pool, i.e., N, W, E, and S, used as starting points and for dividing the pool into four arbitrary quadrants
- circular platform (diameter 10 cm) situated on a solid support equidistant from the side and the center of the pool
- latency to reach the platform measured by stopwatch and the maximum trial latency: 60 s
- animals tested in blocks of four trials using the four starting points assigned in a pseudo-random sequence
- two cages with 4 animals placed near the pool, one animal after the other started at the first point, tested and then returned to the cage
- same procedure was repeated for the other three starting points
- when the rat swam to and climbed onto the platform, the trial was ended
- if the animal failed to locate the platform within 60 s, it was placed on the platform for 15 s and then returned to the cage
SCHEME USED
- Training: with the platform situated 1 cm above the surface of the water in the center of SW quadrant, the animals were trained for 3 blocks of 4 trials (Trials 1-12), one block per day on consecutive days
- Learning: with the platform situated 1 cm below the surface of the water at the same location in the pool, the animals were trained until a stable performance was established, i.e., 5 blocks of 4 trials (Trials 13-32) twice a day on consecutive days
- Retention: one month later, animals were tested again with the platform in the same location in the pool, 5 blocks of 4 trials (Trials 1-20), two blocks per day on consecutive days
- Transfer test: immediately after two more trials (Trials 21 and 22), the platform was removed from the pool, each animal had one trial from the same starting point, swim path was followed for 60 s, time spent in the target quadrant, expressed as a percentage of the total time, was calculated by the image analysing program
- New platform location: animals tested in a reversal procedure with the platform located in the center of the NE quadrant opposite the original location used earlier, animals were tested for 6 blocks of 4 trials on 3 consecutive days (Trials 24-47)
- Operant conditioning (delayed spatial alternation): 12 male rats (from 12 litters, 11 with median weight and 1 with low weight at weaning) from each group used, animals were transported by air-freight from Copenhagen to Zurich (total travel time of approximately 8 h) at the age of 4 months, rats were housed by four in Makrolon IV cages and the environmental conditions were: 12L: 12D cycle (light on at 6 a.m.), temperature 23 ± 1°C and relative humidity 48 ± 5%

Sacrifice and (histo)pathology:

- Time point of sacrifice: postnatal day (PND) 80 for female pups and PND 100 for male pups selected, PND 22 for rest of the pups, the dams and the sperm-positive females who had not given birth
- Number of animals sacrificed: not specified
- Brain weight: not measured

Other examinations:

- offspring weighed in the preweaning period on PNDs 1, 2, 3, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 19, 22; weighed once every week
- sexual maturation assessed by observation of vaginal opening in females and cleavage of the balano-prepubital skinfold in the males

Statistics:

- litter or one pup per litter = statistical unit
- Mann-Whitney U test and chi-square test: body weights, developmental milestones and reflexes, results from Rotarod test
- Multivariant analyses (MANOVA): Morris maze results, operant behavior test

Results and discussion

Results of examinations

Clinical signs:

not specified

Description (incidence and severity):

Maternal animals:
- no clinical signs were seen

Pups:
- significantly delayed in some recorded developmental milestones (as described in table 4)

Mortality:

not specified

Description (incidence):

Maternal animals:
- not specified

Pups:
- no effects observed compared to the control group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pups:
- from birth and throughout preweaning: mean b.w. in litters was significantly lower
- distribution of b.w. was not merely shifted to lower values also more skewed
- difference was not statistically significant after the age of 5 weeks
Results are given in table 2 and table 3.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- urine of the females in the exposed group was colored bright yellow

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Behavioral effects:
- exposed pups were significantly delayed in some recorded developmental milestones and in the ontogeny of the surface righting reflex (see Table 4)
- no differences found concerning age at sexual maturation, performance on the Rotarod, or ambulation and percentage of time spent in the center square in open field
- testing for operant behavior indicated no differences in the first training phases concerning outshaping, discrete trials, or visual discrimination
- suggested reduced retention of learned behavior

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

LOAEC

Effect level:

150 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 2: Pup body weights.

Body weight after weaning
	Control				NMP
Females	Small*		Medium†		Small*		Medium†
Day 22	41± 1		42 ± 1		37 ± 1§		39 ± 1†
Day 24	45± 1		48 ± 1		41 ± 1§		44 ± †
Day 28 or 29	61 ± 2		65 ± 1		60 ± 2		62 ± 2
7-8 weeks	155± 4		165 ± 5		147 ±3		155 ± 4§
11-12 weeks	197± 5		210 ± 6		190 ± 4		201 ± 4
Males
Day 22	41± 1		43 ± 1		39 ± 1§		40 ± 1‡
Day 24	47 ± 1		48 ± 1		43 ± 1§		44± 1‡
Day 28	65 ± 1		68 ± 1		62 ± 1		66 ± 1
Day 36 (± 1)	108 ± 3		113 ± 3		104 ± 3		105 ± 3‡
7-8 weeks	218 ± 7		221 ± 6		209 ± 6		206 ± 7
11-12 weeks	335 ± 11		340 ± 7		320 ± 6		327 ± 7
14-15 weeks	368 ± 13		375 ± 8		346 ± 8		354 ± 8

*Pup with lowest body weight in litter at weaning;†Pup with median body weight in litter at weaning;‡p<0.05,Mann-Whitney U Test, NMP versus control;§p<0.10, Mann-Whitney U Test, NMP versus control; mean ± SD.

Table 3: Body weight in litters in the preweaning period.

Group	Control	NMP
Number of litters	14	16
Day 1	5.9 ± 0.4	5.5 ± 0.3*
Day 7	12.8 ± 0.9	11.9 ± 1.0*
Day 10	17.6 ± 1.3	16.6 ± 1.6*
Day 14	24.0 ± 1.7	22.6 ± 2.0*
Day 19	32.5 ± 3.2	30.S ± 2.7†
Day 22	42.2 ± 3.7	39.S ± 3.3*

*p<0.05,Mann-WhitneyU Test;†p<0.10, Mann­ Whitney U Test; Mean±SD.

Table 4: Results of the developmental milestones and the reflex testing in exposed pups.

Developmental milestones and reflexes
		Control	NMP
Number of litters		14	16
Ear unfolding	positive - day 2	2%	0%
	positive - day 3	88%	48%*
Surface righting reflex	negative - day 1	50%	59%
	negative - day 2 negative - day 3	41% 6%	39% 15%†
	negative - day 4	O%	3%
Homing response	positive - day 6	67%	77%
	positive - day 7	95%	95%
Incisor eruption	positive - day 9 positive - day 10	14% 50%	8% †37%
	positive - day 11	91%	77%
	positive - day 12	99%	93%
Auditory startle reflex	positive - day 11	0%	0%
	positive . day 12	11%	5%
	positive- day 13	93%	77%
Eye opening	positive - day 13	6%	9%
	positive - day 14	44%	43%
	positive. day 15	98%	83%*
Air righting reflex	positive- day 14	26%	32%
	positive. day 15	53%	49%
	positive- day 16	98%	99%

Mean of%of litters;*p<5%,Mann-Whitney U Test;†p<5%,x²-test.

Applicant's summary and conclusion

Conclusions:

The physical development of the pups was delayed based on retarded body weight gain until weaning and the results of ear unfolding, surface rightening reflex, incisor eruption and eye opening.

However, no treatment-related effects were noted on landmarks of physical development and reflexes during the later part of pre-weaning and thereafter and no effects on onset of puberty, open filed parameter and performance on rotarod.

The results of the Morris water maze and operant conditioning were contradictory since some parameter and sessions showed an impairment, while other demonstrated no differences.

Executive summary:

Pregnant rats (Mol: WIST) were exposed to 150 ppm N-methylpyrrolidone for 6 hours per day on gestation days 7-20. The dose level was selected so as not to induce maternal toxicity or decrease viability of the offspring. In the preweaning period, the exposed offspring had a lower body weight and their physical development was delayed. Neurobehavioral evaluation of the male pups revealed no effects on basal functions of the central nervous system. The animals appeared normal in motor function (rotarod), activity level (open field), and performance in learning tasks with a low grade of complexity were similar in the two groups. However, in more difficult tasks such as the reversal procedure in Morris water maze and operant delayed spatial alternation (Skinner boxes), performance was impaired in exposed offspring.