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Repeated dose toxicity: oral

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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

Constituent 1
Chemical structure
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 / concentrationsopen allclose all
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
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
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

open allclose all
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
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.