Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
1981-05-12
Deviations:
no
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:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, from continuous production, tank No. 53
- Expiration date of the lot/batch: 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: ensured for the period of the study by the sponsor und specified storage conditions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: supplied to an atomizer for aerosol spraying

FORM AS APPLIED IN THE TEST: aerosol

Test animals

Species:
rat
Strain:
Wistar
Remarks:
SPF-Wistar rats/Chbb :THOM
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-W7950 Biberach, Germany
- Age at study initiation: about 7 weeks on delivery, 9 weeks old at study initiation
- Weight at study initiation: males: 225 g, females: 167 g
- Housing: individually, in Makrolon wire cages (type MD III Becker, Castrop-Rauxel, Germany)
- Diet: Kliba rat/mouse/hamster laboratory diet 24-343-4 10 mm pellets (Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum

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

IN-LIFE DATES: From: 18 March 1991 To: 08 August 1991

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Remarks:
Aerodynamic exposure apparatus: INA 60, BASF AG
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.6 - <= 3.5 µm
Remarks on MMAD:
Particle size analyses for the different test groups
MMAD [µm]
1.6 - 3.5 µm (0.5 mg/L)
2.6 - 2.7 µm (1 mg/L)
2.2 - 2.4 µm (3 mg/L)

GSD
2.1 - 9.6 (0.5 mg/L)
2.8 - 3.3 (1 mg/L)
2.9 - 3.0 (3 mg/L)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: INA 60 (volume about 90 L, BASF AG, Germany)
- Method of holding animals in test chamber: animals restained in exposure tubes (glass tubes); animals conditioned 5 days before experiment with supply air under comparable conditions
- Source and rate of air: The aerosol was sprayed into a mixing stage using compressed air. The liquid aerosol was diluted with conditioned blast air inside the mixing stage.
- System of generating particulates/aerosols: two component atomizer, coupled with a cyclonic seperator
- Temperature, humidity in air chamber: mean temperature 21.2 to 23.0 °C, mean relative humidity 51.9 to 60.8 %
- Method of particle size determination: cascade impactor
- Treatment of exhaust air: exhaust air flow set lower than supply air flow (positive pressure), exhaust air system connected

TEST ATMOSPHERE
- Brief description of analytical method used: Concentration was analysed by gas chromatography after absorption of NMP from measured samples in 2-propanol
- Samples taken from breathing zone: yes

AEROSOL CHARACTERISTICS
The effective aerodynamic cutoff diameter 50 % (EACD 50 %) was 5.5 µm. The mean percentage of respirable aerosol was 82.0, 90.9 and 91.2 % for 0.5, 1 and 3 mg/L, respectively.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
NMP chamber concentrations by gas chromatography (Hewlett-Packard 5880 A with automatic sampler 7672A)
Duration of treatment / exposure:
96 days (65 exposures)
Frequency of treatment:
6 hours/day, 5 times/week
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/L air (analytical)
Remarks:
measured mean concentrations:
2.99 ± 0.143 mg/L
Dose / conc.:
1 mg/L air (analytical)
Remarks:
measured mean concentrations:
1.00 ± 0.060 mg/L
Dose / conc.:
0.5 mg/L air (analytical)
Remarks:
measured mean concentrations:
0.50 ± 0.050 mg/L
No. of animals per sex per dose:
10 rats (control/low/mid/high concentration) for 96 days (main study)
10 rats (control/high concentration) for 4 weeks post-exposure period (recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: information available from earlier studies: no toxic effects in a 2-year study with 100 ppm (0.4 mg/L) but lethality observed after exposure of 28 days of 1 mg/L NMP; a range-finding study to 4, 7 and 10 mg/L also existed
- Rationale for selecting satellite groups: examination to obtain information on the reversibility of possibly occurring toxic effects
- Post-exposure recovery period in satellite groups: 4 weeks for control and high concentration group (recovery)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: on workdays, at least three times on exposure days and, as a rule, once during the preflow period and the post-exposure observation period

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: at the beginning of the pre-flow period, one day before beginning of the exposure period and then, as a rule, once a week. The difference between the body weight on the day of weighing and the body weight on the day before the first exposure was calculated as a group mean. This value was defined as body weight change.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At the beginning of the preflow period (day -10), the eyes of all animals and at the end of the exposure (day 96), the eyes of the animals of the control and high dose group were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 97, day 126/125 (male/female)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all surviving crontol and high dose group animals
- The following parameters were examined: leukocyte count (WBC), erythrocyte count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocyte concentration (RETI) and thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 97, day 126/125 (male/female)
- Animals fasted: No
- How many animals: all surviving control and high dose group animals
- The following parameters were examined: alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), alkaline phosphatase activity (ALP), gamma-glutamyltransferase activity (GGT), sodium concentration (NA), potassium concentration (K), chloride concentration (CL), inorganic phosphate concentration (INP), calcium concentration (CA), urea concentration (UREA), creatinine concentration (CREA), glucose concentration (GLUC), total bilirubin concentration (TBIL), total protein concentration (TPROT), albumin concentration (ALB), globulin concentration (GLOB), triglyceride concentration (TRIG), cholesterol concentration (CHOL) and magnesium concentration (MG).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see "Any other information on materials and methods incl. tables")
HISTOPATHOLOGY: Yes (see "Any other information on materials and methods incl. tables")
Statistics:
Dunnett's test with variance analysis for main groups, Student's T-test for recovery groups

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The animals of all NMP exposed groups showed yellowish-orange discolored urine indicative for systemic availability (probably caused by metabolite(s)). Slight signs of irritation in form of reddish crust formation on nasal edges in males and females at 1 and 3 mg/L. At 3 mg/L impaired general state was noted as indicated by ruffled fur starting about 2 weeks after beginning of exposure up to high stepping gait towards end of exposure.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no substance-induced incidences of death.
One male animal of main group 0 died on test day 96 before exposure.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No impaired body weights or body weight changes were observed in males at 0.5 mg/L and females at any concentration. The isolated occurrence of a slight retarded body weight gain in males at day 5 only was assessed as incidental. The males at 1 mg/L had a statistically significantly retarded mean body weight gain during days 5 - 40 and on day 68, while the mean body weight was not statistically significantly impaired. In the males at 3 mg/L the mean body weight gain was almost the entire exposure statistically significantly retarded in the main and recovery groups, lasting during the 4-week recovery period, while the mean body weight in the main group was statistically significantly reduced during days 19 - 40 and in the recovery group during days 12 - 96 and on post exposure days 103 and 110 (see "Any other information on results incl. tables").
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
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 changes.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no effects in male and female rats at 0.5 and 1 mg/L. At 3 mg/L in males slight increases in erythrocyte concentration, haemoglobin concentration, haematocrit and mean corpuscular volume (MCV) after 3 months of exposure were noted and increased haemoglobin concentration and haematocrit at the end of the 4-week recovery were observed. Females at this concentration revealed increased polymorphonuclear neutrophil counts and decreased lymphocyte counts at the end of the exposure period, totally reversible during the recovery period. Additionally, the females of this group had a prolonged clotting time at the end of exposure only (see "Any other information on results incl. tables").
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 3 mg/L there was an increase in alanine aminotransferase activity in both sexes, reversible during recovery. At 3 mg/L elevated levels for inorganic phosphate, albumin, triglycerides and decreased glucose concentration were noted in males, while females had increased values for inorganic phosphate and triglyceride concentration at the end of exposure only (see "Any other information on results incl. tables").
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related decreased absolute mean testes weight was noted at 3 mg/L (see "Any other information on results incl. tables").
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
One animal of the high dose group (1/10) and two animals of the corresponding recovery group (2/10) exhibited a reduced testes size
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Cellular depletion of the germinal epithelium of a varying degree was noted in 5 animals (control: 0/10, low dose: 1/10, mid dose: 0/10, high dose: 4/10) of the main study and 7 animals (control: 0/10, high dose 7/10) of the recovery group.
In any case no histopathological correlate for the clinically recorded nasal irritation or the findings in hematology or clinical pathology were observed.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
0.5 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Remarks on result:
other: local effects of the upper respiratory tract
Dose descriptor:
LOAEC
Effect level:
1 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Remarks on result:
other: local effects of the upper respiratory tract
Dose descriptor:
NOAEC
Effect level:
1 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: systemic effects
Dose descriptor:
LOAEC
Effect level:
3 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: systemic effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean body weight gain of male and female animals [g]

Day [#]

Dose Group [mg/L]

 

Male

Female

 

0

0.5

1.0

3.0

0

0.5

1.0

3.0

Main Group

  -1

272.8

274.3

274.8

273.1

186.8

186.4

184.9

189.7

 12

307.2

303.5

299.6

289.7

203.5

204.2

199.4

203.2

 33

362.0

354.2

344.8

329.4**

225.1

227.6

222.7

227.3

 61

404.2

395.2

387.8

373.4

240.2

244.9

239.4

244.2

 96

427.8

420.8

416.7

395.9

251.1

258.3

250.0

253.8

Recovery Group

  -1

284.0

-

-

278.6

185.2

-

-

186.1

 12

319.4

-

-

293.1#

199.8

-

-

197.9

 33

376.5

-

-

336.6#

225.2

-

-

223.2

  61

423.5

-

-

375.9#

241.3

-

-

237.7

 96

467.8

-

-

399.8##

245.8

-

-

246.2

110

490.8

-

-

445.7#

260.8

-

-

267.8

124

515.8

-

-

472.5

276.9

-

-

276.8

** p<0.01, Dunnett’s test and ANOVA
#p<0.05,##p<0.01 Student’s T-test

 

 

Table 2: Haematology parameter observed in the study

Endpoint

Dose Group [mg/L]

 

Male

 

0

0.5

1.0

3.0

Main Group (Day 97)

  RBC [1012/L]

7.95

8.40

8.12

8.02

  HGB [mmol/L]

8.82

9.30

9.22

9.14

  HCT [L/L]

0.411

0.439

0.432

0.423

  MCV [10-15L]

51.68

52.22

53.17

52.65

Recovery Group (Day 97)

  RBC [1012/L]

7.75

-

-

8.25*

  HGB [mmol/L]

8.75

-

-

9.43**

  HCT [L/L]

0.403

-

-

0.441**

  MCV [10-15L]

51.91

-

-

53.35*

Recovery Group (Day 126)

  RBC [1012/L]

8.30

-

-

8.47

  HGB [mmol/L]

9.01

-

-

9.29*

  HCT [L/L]

0.433

-

-

0.451*

  MCV [10-15L]

52.12

-

-

53.13

* p<0.05, ** p<0.01 Student’s T-test

 

 

 

 

Endpoint

Dose Group [mg/L]

 

Female

 

0

0.5

1.0

3.0

Main Group (Day 97)

  HQT [s]

23.7

23.8

25.3

25.9

  Neutro [109/L]

0.40

0.38

0.44

1.25

  Lympho [109/L]

3.27

3.14

2.82

2.42

Recovery Group (Day 97)

  HQT [s]

24.6

-

-

26.5*

  Neutro [109/L]

0.48

-

-

0.95

  Lympho [109/L]

2.82

-

-

2.45

Recovery Group (Day 125)

  HQT [s]

25.5

-

-

24.7

  Neutro [109/L]

0.58

-

-

0.53

  Lympho [109/L]

2.77

-

-

2.52

* p<0.05, ** p<0.01 Student’s T-test

 

 

 

Table 3: Selected clinical chemistry results from the study

Endpoint

Dose Group [mg/L]

 

Male

 

0

0.5

1.0

3.0

Main Group (Day 97)

  ALT [µkat/L]

1.19

1.32

1.21

1.52*

  INP [mmol/L]

2.07

2.52

2.26

2.39

  ALB [g/L]

31.98

34.42*

33.71

34.31*

  TRIG [mmol/L]

2.09

2.83

2.64

3.67**

  GLUC [mmol/L]

6.81

6.50

6.51

6.65

Recovery Group (Day 97)

  ALT [µkat/L]

1.26

-

-

1.41

  INP [mmol/L]

2.20

-

-

2.63#

  ALB [g/L]

32.55

-

-

34.85#

  TRIG [mmol/L]

2.79

-

-

3.07

  GLUC [mmol/L]

7.08

-

-

5.96###

Recovery Group (Day 126)

  ALT [µkat/L]

1.08

-

-

1.17

  INP [mmol/L]

2.00

-

-

2.13

  ALB [g/L]

31.74

-

-

32.62

  TRIG [mmol/L]

4.13

-

-

3.13

  GLUC [mmol/L]

7.38

-

-

7.04

* p<0.05, ** p<0.01, ANOVA plus Dunnett’s test
#p<0.05,###p<0.001, Student’s T-test

 

 

 

 

Endpoint

Dose Group [mg/L]

 

Female

 

0

0.5

1.0

3.0

Main Group (Day 97)

  ALT [µkat/L]

1.05

1.15

1.05

1.23*

  INP [mmol/L]

1.99

2.02

2.01

2.21

  TRIG [mmol/L]

2.18

2.35

2.44

3.48*

Recovery Group (Day 97)

  ALT [µkat/L]

1.24

-

-

1.31

  INP [mmol/L]

1.93

-

-

2.25#

  TRIG [mmol/L]

1.94

-

-

2.17

Recovery Group (Day 125)

  ALT [µkat/L]

1.03

-

-

1.06

  INP [mmol/L]

1.60

-

-

1.69

  TRIG [mmol/L]

1.81

-

-

2.07

* p<0.05, ANOVA plus Dunnett’s test
#p<0.05, Student’s T-test

 

 

 

Table 4: Selected organ weights resulted from NMP exposure

Organ

Dose Group [mg/L]

 

Male

 

0

0.5

1.0

3.0

Main Group

  Testes, absolute [g]

3.546

3.541

3.519

3.003*

  Testes, bw relative [%]

0.829

0.863

0.856

0.774

Recovery Group

  Testes, absolute [g]

3.626

-

-

3.220

  Testes, bw relative [%]

0.710

-

-

0.694

* p<0.05, Dunnett’s test

 

 

 

Applicant's summary and conclusion

Conclusions:
High concentrations (3 mg/L) of NMP aerosol inhaled for 6 hours per working day for 3 months caused clinical symptoms of upper respiratory tract irritation, general unspecific systemic toxicity, clinicochemical signs of slight liver injury and in some of the male animals
cellular depletion of the germinal epithelium of testes. Whereas the symptoms of general toxicity nearly vanished during a 4-week recovery-period, testes damage was still resent to a comparable extent.
The No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity was 1.0 mg/l under the conditions of this test.
Because of slight clinical signs of nasal irritation at 1.0 mg/l without any pathological correlate the NOAEC for local effects to the upper respiratory tract is judged to be 0.5 mg/L .
Executive summary:

The subchronic toxicity of NMP after inhalative exposure was investigated in male and female Wistar rats using head-nose exposure. Ten rats per sex and group were exposed to 0, 0.5, 1.0 or 3.0 mg/L (0, 125, 250, 750 ppm) for 6 hours daily, 5 days/week for 13 weeks.

These groups were sacrificed and examined at the end of exposure. A satellite group of 10 rats per sex was exposed to 0 or 3.0 mg/L for 13 weeks followed by a 4 week recovery period. The NMP atmospheres consisted of a large proportion (82 – 92 %) of respirable aerosol particles (MMAD: 1.6 – 3.5 μm). Discoloration of the urine was observed at all concentrations as indication of systemic availability. Nasal irritation as shown by crust formation on nasal edges was observed at ≥1.0 mg/L. At 1.0 mg/L the male rats showed a retardation of the body weight gain, while at 3.0 mg/L in male rats, body weight/body weight gain was significantly decreased and testicular finding in form of cellular depletion was recorded. Impaired red blood cell parameters in males and females, an increase in polymorphonuclear neutrophils and a decrease in lymphocytes were observed. Clinical chemistry revealed an indication of impaired liver function. Examination of the satellite group after recovery showed a significant lower body weight gain in males and cellular depletion in the testes. All other findings nearly disappeared during recovery. The NOAEC for systemic toxicity and for local irritation was 0.5 mg/L (125 ppm). This subchronic inhalation study in the rat is acceptable and satisfies the guideline requirement for a subchronic inhalation study (OECD 413) in the rat.

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