1-methyl-2-pyrrolidone

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-05-07 to 1994-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

other: US EPA TSCA, Test guideline 40 CFR 798.2450, US EPA Neurotoxicity Subdivision F

GLP compliance:

yes

Limit test:

no

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

Details on test material:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, from continuous production, tank No. 53
- Analytical purity: 99.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: under N2, to be protected from air
- Stability under test conditions: stability ensured by substance supplier

Specific details on test material used for the study:

- Analytical purity: 99.9 %, including 0.5 % 1,3-, 1,4- and 1,5-dimethylpyrrolidone
- measured concentrations of test substance in stability samples: 90% - 106%
- Lot/batch No.: continuous production from tank 53
- Physical state: colourless liquid
- Stability under test conditions: Stable based upon analyses of samples near the beginning of dietary administration and near the end of dietary administration .

Test animals

Species:

rat

Strain:

other: Crl:CD BR (Sprague-Dawley derived)

Details on species / strain selection:

selected on the bases of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous diseases

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York
- Age at study initiation: about 6 weeks at delivery, about 7 weeks at start of exposure
- Housing: during qurantine 3 / cage; individually housed in stainless steel, wire-mesh cages
- Diet: commercial diet, Purina certified rodent chow # 5002 (Ralston Purina, St. Louis, MO, USA), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina certified rodent chow # 5002
- Storage temperature of food: refrigerated until use (14 days)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification was perfomed by means of gas chromatography. Diet samples collected from the top, middle and bottom of the mixer for each dietary concentration indicate that the test substance was present at the target concentrations and that it was distributed homogenously throughout the diet. The concentrations in the samples analyzed for homogeneity ranged between 92 % and 106 % of nominal values, and the coefficient of variation ranged between 1.3 % and 6.0 % of nominal values.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 000 ppm

Remarks:

equivalent to 169 mg/kg bw/d in male rats and 217 mg/kg bw/d in female rats

Doses / concentrations 2

Dose / conc.:

7 500 ppm

Remarks:

equivalent to 433 mg/kg bw/d in male rats and 565 mg/kg bw/d in female rats

Doses / concentrations 3

Dose / conc.:

18 000 ppm

Remarks:

equivalent to 1057 mg/kg bw/d in male rats and 1344 mg/kg bw/d in female rats

No. of animals per sex per dose:

26 (control and high dose)
20 (low and mid dose)

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: 28 days (control and high dose group)

Examinations

Observations and examinations performed and frequency:

For details on animal allocation, refer to "Any other information on materials and methods incl. tables"

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, at every weighing


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: day -26 and day 87
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 44 and 91 for males, days 45 and 90 for females
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: 10 males and 10 females per dose group
- Parameters examined: number of erythrocytes (RBC), leukocytes (WBC) and platelets (PLAT), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), relative numbers of neutrophils (Neut), band neutrophils (Band), lymphocytes (Lymph), atypical lymphocytes (Alym), monocytes (Mono), eosinophils (Eosin) and basophils (Baso)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 44 and 91 for males, days 45 and 90 for females
- Animals fasted: Yes
- How many animals: 10 males and 10 females per dose group
- Parameters examined: activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and sorbitol dehydrogenase (SDH), concentrations of glucose (GLUCO), urea nitrogen (BUN), calcium (CALC), phosphate (PHOS), bilirubin (BILRN), cholesterol (CHOL), creatinine (CREAT), total protein (TPROT), albumin (ALBMN), sodium (Na), potassium (K), chloride (Cl), serum globulin (GLOBN)


URINALYSIS: Yes
- Time schedule for collection of urine: overnight (16 hours) prior to days 44 and 91 for males and days 45 and 92 for females
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume (VOL), osmolality, urobilinogen concentration (UROBL), pH value, the presence of hemoglobin or occult blood (BLOOD), glucose, protein, bilirubin and ketone (acetoacetic acid), color, transparency, sediment


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during pretest and weeks 4, 8 and 13 (treatment), week 18 (recovery)
- Dose groups that were examined: all dose groups during pretest and treatment, control and high dose group during recovery.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: foot splay

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (for details refer to "Any other information on materials and methods incl. tables")

HISTOPATHOLOGY: Yes (for details refer to "Any other information on materials and methods incl. tables")

Other examinations:

Neuropathological evaluations

Statistics:

One-way ANOVA and Dunnett's test, Bartlett's test for homogeneity of variances, Cochran-Armitage trend test, Shapiro-Wilk's test, Kruskall-Wallis test followed by Dunn's test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treated male and female rats showed bright yellow stained cage board beneath the cage as indicative of urine discoloration in a concentration dependent incidence at all concentrations. This was assessed as indicative for systemic NMP availability (probably caused by metabolite(s)).

Mortality:

mortality observed, treatment-related

Description (incidence):

No substance-induced mortality occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reduced mean body weights and/or body weight gains were recorded in males and females at 7500 and 18000 ppm (see "Any other information on results and tables").

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reduced mean food consumption was noted in males and females at 18000 ppm (see "Any other information on results and tables")

Food efficiency:

not specified

Description (incidence and severity):

For mean substance intake data, refer to "Any other information on results and tables"

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related adverse findings.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related adverse findings.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related adverse findings.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There was no treatment-related effect on forelimb and hindlimb grip strength. Males at 7500 and 18000 ppm displayed increases in foot splay values at 4-, 8- and 13-week examination (see "Any other information on results and tables"). Among all the various tests included in the FOB, only the males at 18000 ppm had a higher incidence of low arousal during the 4-, 8- and 13-week examinations and a higher incidence of slight palpebral closure at 4- and 13-week evaluation in the open field (see "Any other information on results and tables").

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females at 18000 ppm had increased relative liver, kidney and lung weights, while in male rats relative kidney, lung and testes weights were increased but relative brain weights were reduced at this dose level (see "Any other information on results and tables").

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects in neural or muscular tissue.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Females at 18000 ppm displayed an increased incidence of centrilobular hypertrophy in the liver and animals of both sexes showed a minimal increase of a staining pigment (indicative for hemosiderin) within the red pulp of the spleen at this dose level (see "Any other information on results and tables").

Details on results:

DIETARY TEST SUBSTANCE ANALYSIS:
Homogeneity and correctness of concentration was demonstrated (concentration range 92 - 106 %). NMP was stable in the diet as samples after storage of 7 and 10 days at room temperature and 14 days in the refrigerator ranged from 90 - 106 %. Samples for concentration control from feeders on day 35 and 84 ranged between 88 - 100 % and confirmed intended concentration.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: equivalent to 169 mg/kg bw/d in male, 217 mg/kg bw/d in female rats

2

Dose descriptor:

LOAEL

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

Remarks on result:

other: equivalent to 433 mg/kg bw/d in male, 565 mg/kg bw/d in female rats

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mean Body Weight [g]

Day [#]	Dose Group [ppm]
	Male				Female
	0	3000	7500	18000	0	3000	7500	18000
0	275.0	274.6	273.7	273.6	178.8	179.4	179.2	179.6
15	395.0	392.5	382.0	341.2*	227.9	224.9	217.2	207.5*
29	467.1	464.1	452.3	400.1*	252.4	249.0	239.7	229.3*
57	562.0	553.1	541.2	480.8*	284.2	278.2	269.9	256.8*
92	631.9	614.9	608.1	531.2*	302.0	295.6	283.3	272.3*
106	652.6	-	-	567.5*	305.6	-	-	304.6
127	671.1	-	-	618.6	323.1	-	-	312.5
* p<0.05, Dunnett’s test and ANOVA

 

Mean Body Weight Gain [g]

Period	Dose Group [ppm]
Day [#]	Male				Female
	0	3000	7500	18000	0	3000	7500	18000
0-43	240.4	239.1	217.0*	166.0*	96.7	89.1	77.8*	65.0*
43-92	116.4	101.2	117.4	91.6*	26.5	27.1	26.3	27.3
92-127	28.4	-	-	89.6*	25.6	-	-	35.2
* p<0.05, Dunnett’s test and ANOVA

 

Mean Food Consumption [g]

Period	Dose Group [ppm]
Day [#]	Male				Female
	0	3000	7500	18000	0	3000	7500	18000
0-43	28.9	28.4	27.8	25.2*	20.2	19.4	19.2	18.2*
43-92	28.4	27.6	27.8	26.0*	19.2	18.6	18.9	17.7*
92-127	29.4	-	-	31.1	21.9	-	-	20.0
* p<0.05, Dunnett’s test and ANOVA

 

Mean Substance Intake [mg/kg bw]

Period	Dose Group [ppm]
Day [#]	Male			Female
	3000	7500	18000	3000	7500	18000
0-92	169	433	1057	217	565	1344

Mean Foot Splay [cm] 

Week [#]	Dose Group [ppm]
	Male				Female
	0	3000	7500	18000	0	3000	7500	18000
4	7.9	8.9	9.6*	9.8*	7.8	7.3	8.3	8.2
8	7.8	9.4	9.7*	9.8*	7.1	7.3	8.2	8.2
13	7.7	7.4	8.9	9.1	8.0	7.2	8.5	8.4
18	6.0	-	-	8.3*	7.1	-	-	6.2
* p<0.05, Bartlett’s test, Dunnett’s test and ANOVA

 

Functional Observation Battery [number affected/total number] 

Endpoint	Dose Group [ppm]
	Male
	0	3000	7500	18000
Low arousal
Week  4	1/16	1/10	2/10	5/16*
Week  8	2/16	0/10	3/10	5/16
Week 13	4/16	3/10	3/10	9/16
Week 18	1/10	-	-	2/10
Palpebral closure
Week  4	0/16	0/10	1/10	3/16*
Week  8	2/16	0/10	2/10	2/16
Week 13	2/16	2/10	0/10	7/16*
Week 18	1/10	-	-	1/10
* p<0.05, Cochran-Armitage trend test or Fisher’s Exact test

 

 

Applicant's summary and conclusion

Conclusions:

The NOAEL was 3000 ppm for both sexes (about 169 mg/kg bw/day in males, 217 mg/kg bw/day in females). A specific target organ for compound-related adverse systemic toxicity was not identified.

Executive summary:

The subchronic toxicity of NMP was investigated in a combined subchronic and neurotoxicity study. Groups of 20 to 26 male and female Sprague-Dawley rats received dietary NMP concentrations of 0, 3000, 7500 or 18000 ppm (about 0, 169/217, 433/565, 1057/1344 mg/kg bw/day, males/females) for 3 months. Ten animals per sex from the control and high dose group were observed for recovery for 1 month after treatment. Functional and morphological evaluations of neurotoxicity were performed as part of the study. Decrements in body weight, food consumption and efficiency were observed at ≥7500 ppm. At 18000 ppm the liver weights in females were increased (increased incidence of centrilobular hypertrophy) and the kidney weights of males and females were increased without corresponding histopathological findings. In addition, animals of both sexes showed a minimal increase in splenic hemosiderin at this dose. In males, only 3 of 36 neurotoxicity parameters were affected by NMP exposure; females were unaffected. Males exhibited an increase in foot splay at ≥7500 ppm. A higher incidence in low arousal and slight light palpebral closure, suggestive for a sedative effect, were observed at 18000 ppm. After 1 month of recovery the effect in foot splay turned to normal and no difference to controls was observed for low arousal and palpebral closure at the investigated concentration of 18000 ppm indicating reversibility. The urine showed a yellowish discoloration at ≥3000 ppm as indication for systemic availability.

The NOAEL was 3000 ppm for both sexes (about 169 mg/kg bw/day in males, 217 mg/kg bw/day in females). A specific target organ for compound-related adverse systemic toxicity was not identified.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rats.