1-methyl-2-pyrrolidone

Endpoint summary

Description of key information

In the dietary combined subchronic and neurotoxicity study (90-day with 4 week recovery, rat) the NOAEL was 169 and 217 mg/kg bw/day for males and females, respectively (corresponding to 3,000 ppm). This finding was supported by several reliable studies in rats, mice and dogs. No target organ was identified (Haskell Lab., 1995).

The NOAEC for systemic toxicity and local irritation was 500 mg/m³ air (125 ppm) based on the results of a repeated inhalation exposure to a liquid NMP aerosol (90-day with 4-week recovery, rat). At 3,000 mg/m³ air, cellular depletion was observed in the testes at the end of the treatment and recovery periods (BASF SE, 1994).

In the key study for the dermal route of exposure (20-day, rabbit) a NOAEL for systemic toxicity of 826 mg/kg bw/day was derived, while for local skin irritation no NOAEL could be obtained. No target organ for systemic toxicity was identified (Ind. Biol. Res. Lab., 1963).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

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 .

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

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

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)

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.

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.

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

Critical effects observed:

not specified

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

 

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

169 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

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

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

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

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

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)

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

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

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

Critical effects observed:

not specified

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

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 000 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

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

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

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

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

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)

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

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

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

Critical effects observed:

not specified

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

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

500 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1963

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

only 2 animals per dose group and sex instead of 5 as required by guideline

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Methyl pyrrolidone - tested as supplied.

Species:

rabbit

Strain:

other: albino

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: approx. 1.8 - 2.3 kg
- Acclimation period: 2 weeks

Type of coverage:

not specified

Vehicle:

not specified

Details on exposure:

Route of Administration: dermal

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

20 days

Frequency of treatment:

5 days/week

Dose / conc.:

413 mg/kg bw/day (nominal)

Remarks:

cited as 0.4 mL/kg bw/d

Dose / conc.:

826 mg/kg bw/day (nominal)

Remarks:

cited as 0.8 mL/kg bw/d

Dose / conc.:

1 653 mg/kg bw/day (nominal)

Remarks:

cited as 1.6 mL/kg bw/d

No. of animals per sex per dose:

2 rabbits with intact skin
2 rabbits with abraded skin

Control animals:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION:
- No data

FOOD EFFICIENCY:
- No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the beginning and at the end of the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mild local skin irritation was noted after repeated dosing.
At 0.4 and 0.8 mL/kg bw/day (corresponding to 413 and 826 mg/kg bw/day), no systemic signs of toxicity substantiated by clinical examinations.

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Description (incidence):

At 1.6 mL/kg bw/day (corresponding to 1,653 mg/kg bw/day) 1/4 animals died (abraded skin). 

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

At 0.4 and 0.8 mL/kg bw/day (corresponding to 413 and 826 mg/kg bw/day), no systemic signs of toxicity substantiated by hematological examinations.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

At 0.4 and 0.8 mL/kg bw/day (corresponding to 413 and 826 mg/kg bw/day), no systemic signs of toxicity substantiated by histopathological examinations.

Histopathological findings: neoplastic:

not specified

Details on results:

No further findings occurred in the surviving animals.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

826 mg/kg bw/day

Sex:

male

Basis for effect level:

mortality

Dose descriptor:

LOAEL

Effect level:

1 653 mg/kg bw/day

Sex:

male

Basis for effect level:

mortality

Critical effects observed:

no

Conclusions:

The NOAEL for local irritation was <413 mg/kg bw/day (cited as 0.4 mL/kg bw/day) due to mild local skin irritation noted at all dose levels tested.

Executive summary:

The subacute dermal toxicity of NMP was investigated in male albino rabbits. Groups of two rabbits each received concentrations of 0, 413, 826 or 1653 mg/kg bw/day (cited as 0, 0.4, 0.8 or 1.6 mL/kg bw/day) on the intact or abraded skin, applied once a day, 5 days per week, for a total period of 4 weeks. Mild local skin irritation was noted after repeated dosing at 413 mg/kg bw/day and above. Beside the death of one rabbit with abraded skin after one week of treatment out of four in total, which received 1653 mg/kg bw/day, no further signs of systemic toxicity was noted by clinical, hematological and histopathological examinations.

Thus, the NOAEL for systemic toxicity was 826 mg/kg bw/day, while for local irritation no NOAEL could be obtained after repeated application due to missing effects.

This toxicity study in the rabbit is acceptable and satisfies the general guideline requirements for a repeat-dose dermal toxicity study (OECD 410) in rabbits with some restrictions due to the fact that the study was performed prior to the date of guideline publication.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

826 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral: (Haskell, 1995)

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, 3,000, 7,500 or 18,000 ppm (about 0, 169/217, 433/565, 1,057/1,344 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 ≥ 7,500 ppm. At 18,000 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 ≥ 7,500 ppm. A higher incidence in low arousal and slight light palpebral closure, suggestive for a sedative effect, were observed at 18,000 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 18,000 ppm indicating reversibility. The urine showed a yellowish discoloration at ≥ 3,000 ppm as indication for systemic availability. The NOAEL was 3,000 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.

Dermal: (GAF, 1963)

The subacute dermal toxicity of NMP was investigated in male albino rabbits. Groups of two rabbits each received concentrations of 0, 413, 826 or 1,653 mg/kg bw/day (cited as 0, 0.4, 0.8 or 1.6 mL/kg bw/day) on the intact or abraded skin, applied once a day, 5 days per week, for a total period of 4 weeks. Mild local skin irritation was noted after repeated dosing at 413 mg/kg bw/day and above. Beside the death of one rabbit with abraded skin after one week of treatment out of four in total, which received 1653 mg/kg bw/day, no further sign of systemic toxicity was noted by clinical, hematological and histopathological examinations. Thus, the NOAEL for systemic toxicity was 826 mg/kg bw/day, while for local irritation no NOAEL could be obtained after repeated application.

Inhalation: (BASF SE, 1994)

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).

Justification for classification or non-classification

In the key oral study only non-adverse effects, e.g. reduced animal body weights and food consumption were observed. Specific target organ toxicity was not observed. Besides, the NOAEL was out of range for a classication for STOT RE.

Only local effects were monitored in the key inhalative study. The signs of irritation of outer parts of the nose were not supported by findings of irritated airways, hence the existing classification for irritation Cat. 2 for skin and eye as well as the STOT SE 3 for respiratory irritation are sufficiently protective. Besides, the NOAEL was out of range for a classication for STOT RE.

In the key dermal study mild local skin irritation was observed. One out of four animals of the high dose group died but no further systemic effects were registered. The existing classification for irritation Cat. 2 for skin and eye as well as the STOT SE 3 for respiratory irritation are sufficiently protective. Besides, the NOAEL was out of range for a classication for STOT RE.

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.