1-methyl-2-pyrrolidone

Carcinogenicity

Description of key information

NMP showed no oncogenic potential in the rat after long-term exposure via inhalation (Lee et al., 1987) or dietary administration (Haskell Laboratories, 1997).

In mice, there was an increased incidence of liver tumors at the highest dose level. However, the liver tumors in mice may be directly related to the observed increase in the cellular proliferation rate, which could likely be due to the observed enzyme induction and weak peroxisome proliferation observed in B6C3F1 mice, which are known to be extremely sensitive to both non-genotoxic effects (BASF, 1999).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March 1995 to 4 April 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.3300 (Carcinogenicity)

Version / remarks:

1994

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material:
Haskell and 20417; BASF and 21699

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature or refrigerated
- Stability under test conditions: stable in diets for up to 14 days at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: NMP was added to the feed and throughly mixed

FORM AS APPLIED IN THE TEST
- mixed within the feed

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. Raleigh, North Carolina, USA
- Age at study initiation: 49 days
- Weight at study initiation: mean body weight at day 0: 251 - 253 g for males and 174 - 175 g for females
- Fasting period before study: no
- Housing: in stainless steel, wire.mesh cages suspended above cage boards
- Diet: ad libitum, PMI Feeds Inc. Irradiated Certified Rodent Diet #5002
- Water: ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 50 ± 10
- Air changes (per hr): not indicated
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 30 March 1995 To: 4 April 1997

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

NMP was added to feed and thoroughly mixed. Control, 1600 ppm and 5000 ppm diests were mixed for 3 minutes; 15000 ppm diets were mixed for 6 minutes. All diets were prepared weekly and refrigerated until use. No animals were presented with diet that had been prepared more than 14 days previously.

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared weekly and refigerated until use
- Mixing appropriate amounts with (Type of food): certified Rodent Diet #5002)
- Storage temperature of food: not indicated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for stability analyses in the diet were stored 0, 7, 14 days at room temperature and 14 days refrigerated. The homogenous distribution in the diet was checked prior to start, after about one year and one and a half year later.  Concentration control analyses were performed on samples of all concentrations prepared on study day 91, 182, 280, 455, 546, 651 and 721. All analyses were performed by means of gas chromatography.

Duration of treatment / exposure:

2 years

Frequency of treatment:

continuously

Post exposure period:

none

Dose / conc.:

1 600 ppm (nominal)

Remarks:

equivalent to 66 mg/kg bw/day in males, 88 mg/kg body weight/day in females

Dose / conc.:

5 000 ppm (nominal)

Remarks:

equivalent to 207 mg/kg bw/day in males, 283 mg/kg body weight/day in females

Dose / conc.:

15 000 ppm (nominal)

Remarks:

equivalent to 678 mg/kg bw/day in males, 939 mg/kg body weight/day in females

No. of animals per sex per dose:

62

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: none
- Dose selection rationale:
The doses for this oncogenicity study were selected according to the results of a 20-day and a 90-day feeding study in rats. In the 28-day feeding study, the NOAEL was 6000 ppm (429 mg/kg bw/day) for males and 18000 ppm (1548 mg/kg bw/day) for females. In the 90-day feeding study, the NOAEL in males and females was 3000 ppm (169 mg/kg bw/day and 217 mg/kg bw/day, respectively).

Positive control:

none

Observations and examinations performed and frequency:

Food consumption and body weights were determined once a week during the first 3 months and once every other week thereafter for the remainder of the study. Clinical signs of toxicity were recorded at least once daily. Ophthalmology was conducted prior to the start and near the end of the study. Blood samples for differential white blood cell counts were collected after about 12, 18 and 24 months.

Sacrifice and pathology:

After about 2 years all surviving rats were sacrificed. The animals were subjected to gross-pathological assessment, followed by comprehensive histopathological examinations in an extent as required by the guideline. Tissues and organs from rats found dead, accidentally killed or sacrificed in extremis were also evaluated.

Statistics:

Statistical analysis: ANOVA (food consumption, body weight, body weight gains, food consumption, food efficiency and clinical pathology). Pairwise comparisons by Dunnett's test. Bartlett's test for homogeneity and of variances (clinical pathology) and, if significant followed by KRUSKAL-WALLIS test and Mann-Whitney U test. Cochran-Armitage trend test (increased incidence in clinical observations, survival, microscopic observations, tumor incidences) Fisher's exact test to compare control and high dose findings with/without Bonferroni correction

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was no toxicologically relevant clinical sign of toxicity in males and females at any dose level. In high and mid dose groups discoloration of urine as indication of systemic availability of the test substance occurred. 

Mortality:

mortality observed, treatment-related

Description (incidence):

There was a test substance-related decrease in survival associated with chronic progressive nephropathy/uremia in the 15000 ppm males. No further substance-related effect on survival was noted in males or females. The lower percentages in males at 5000 ppm and females at 1600 and 15000 ppm were related to slightly higher incidences of spontaneous tumors (skin and/or pituitary tumors).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weights and retarded body weight gains as well as lower food consumption and reduced food efficiency were recorded in both sexes at the high dose only.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption were recorded in both sexes at the high dose group only.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Reduced food efficiency was recorded in both sexes at the high dose group only.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No substance-related effects were observed.

Haematological findings:

no effects observed

Description (incidence and severity):

No substance-related effects were observed.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Only the high dose males revealed treatment-related macroscopic findings consisting of an increased incidence of large kidneys, kidneys diagnosed with chronic nephropathy, fluid in the pleural cavity and small testes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was no increased incidence in treatment-related benign or malignant tumors in male or female rats at any dose level.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Predominant non-neoplastic lesions were the increased severities of chronic progressive nephropathy and pigment accumulation in the spleen of both sexes at 15000 ppm only. Some other lesions were also increased -mainly in males - but these were secondary to renal failure associated with debilitated health condition.

Dose descriptor:

NOAEL

Effect level:

ca. 5 000 ppm (nominal)

Based on:

test mat.

Remarks:

equal to 207/283 mg/kg bw in male/female animals

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

food efficiency

mortality

Dose descriptor:

LOAEL

Effect level:

15 000 ppm (nominal)

Based on:

test mat.

Remarks:

equal to 678/939 mg/kg bw in males/females

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

food efficiency

mortality

Dose descriptor:

NOEL

Effect level:

>= 15 000 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other:

Critical effects observed:

no

Dietary test substance analysis:
The stability was demonstrated for storage up to 14 day at room temperature and for 14 day refrigerated (recovery
range: 91.0 - 106.3%). The homogeneity analyses revealed the homogeneous distribution (mean top, middle and bottom recovery range: 95.3 - 103.3%) and the concentration control analysis showed mean recovery rates between 89.2 - 112.5 % of nominal as indication for the correctness of the concentrations.

Mortality:
 The survival incidences on day 729 are as  follows:

Test group	Dose level (ppm)	% survival males	% survival females
0	0	32	32
1	1600	48	24
2	5000	21	34
3	15000	24*	23

* p<0.05 Cochran Armitage

Clinical signs:
Incidence of urine discoloration:

Dose level (ppm)	0	1600	5000	15000
Males	0/62	0/62	43/62*	41/62*
Females	0/62	0/62	36/62*	41/62*

* p<0.05 Cochran Armitage

Body weight data, food consumption/efficiency:

Dose level (ppm)	0	1600	5000	15000
Males
Body weight (g) at day 729	629	678	687	518*
Body weight gain (g) days 0 - 729	448	428	437	269*
Food consumption (g/day) at day 729	26.1	26.7	25.9	24.6
Food efficiency (g food/bw gain/d) days 0 - 729	0.023	0.022	0.023	0.015
Females
Body weight (g) at day 729	504	500	461	326*
Body weight gain (g) days 0 - 729	335	329	386	153*
Food consumption (g/day) at day 729	21.8	22.4	21.6	20.5
Food efficiency (g food/bw gain/d) days 0 - 729	0.021	0.021	0.018	0.010

* p< 0.05 ANOVA and Dunnett

Mean substance intake:

Dose level (ppm)	1600	5000	15000
Males (mg/kg bw)	66	207	678
Females (mg/kg bw)	88	283	939

 

Gross pathology: observations in males

Dose level (ppm)	0	1,600	5,000	15,000
Large kidney	1/62	4/62	1/62	9/62
Nephropathy	4/62	6/62	8/62	16/62
Pleural fluid	1/62	0/62	1/62	7/62
Small testes	6/62	6/62	8/62	14/62

Neoplastic lesions:

Dose level (ppm)	0	1600	5000	15000
Males
Liver neoplasm	2/62	6/62	3/62	4/62
Renal tubular neoplasm	1/62	0/62	3/62	2/62
Total animals with benign tumors	52/62	46/62	50/62	52/62
Total animals with malignant tumors	13/62	22/62	17/62	12/62
Females
Liver neoplasm	0/62	0/62	1/62	0/62
Mammary neoplasm	29/62	23/56	23/53	28/62
Total animals with benign tumors	59/62	57/62	60/62	53/62
Total animals with malignant tumors	26/62	21/62	23/62	27/62

 

Non-neoplastic lesions:

Dose level (ppm)	0	1600	5000	15000
Males
Kidney Severe chronic progressive nephropathy	7/62	10/62	12/62	51/62*
Spleen Moderate accumulation pigment containing macrophage	18/62	14/62	16/62	28/62*
Females
Spleen Moderate accumulation pigment containing macrophage	38/62	35/62	36/62	51/62*

* p>0.05 by Cochran-Armitage trend test

Conclusions:

NMP was not oncogenic at dietary levels of up to 15000 ppm in male and female rats (up to approximately: 678/939 mg/kg bw in males/females).
The NOAEL was 5000 ppm (approximately 207/283 mg/kg bw in males/females) based on effects on body weight/body weight gain, food consumption/efficiency, renal failure and reduced survival at the high dose.

Executive summary:

NMP was examined for its chronic toxicity and carcinogenic potential in groups of each 62 male and 62 female Sprague-Dawley CD rats at dietary concentrations of 0, 1600, 5000 or 15000 ppm (about 66/88, 207/283, 678/939 mg/kg bw/day, males/females) for two years. Food consumption and body weights were determined at regular intervals. Clinical signs of toxicity were recorded at least once daily. Ophthalmology was conducted prior to the start and near the end of the study. Blood samples for differential white blood cell counts were collected after about 12, 18 and 24 months. After about 2 years all surviving rats were sacrificed. The animals were subjected to gross-pathological assessment, followed by comprehensive histopathological examinations. Tissues and organs from rats found dead, accidentally killed or sacrificed in extremis were also evaluated.

The survival of the female animals was not affected. The survival of the males in the high dose group was lower due to an increase in severe chronic-progressive nephropathy as a typical finding in aging male rats. There was a reduction in body weights and retarded body weight gain with a corresponding reduction in food consumption and efficiency at 15000 ppm. The incidence of benign or malignant tumors was not increased among male or female rats, indicating no oncogenic potential up to a dietary concentration of 15000 ppm in male and female rats (approximately: 678/939 mg/kg bw/day in males/females). Only the high dose males revealed treatment-related macroscopic findings consisting of an increased incidence of large kidneys, kidneys diagnosed with chronic nephropathy, fluid in the pleural cavity and small testes. There was an increased incidence in spleenic hemosiderin as an indication of a higher turn-over of red blood cells at 15000 ppm. In high and mid dose groups discoloration of urine as indication of systemic availability of the test substance occurred.

NMP was not oncogenic at dietary levels of up to 15000 ppm in male and female rats (up to approximately: 678/939 mg/kg bw in males/females). No specific target organ was detected as the chronic progressive nephropathy with related changes (uremia, reduced survival) were considered as secondary to aging. The increased incidence in pigment containing (hemosiderin) macrophages in the spleen is interpreted as a higher turn-over of red blood cells at 15000 ppm. The NOAEL was 5000 ppm (approximately 207/283 mg/kg bw in males/females) based on effects on body weight/body weight gain, food consumption/efficiency, renal failure and reduced survival at the high dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

678 mg/kg bw/day

Study duration:

chronic

Species:

rat

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

- colorless, light yellow
- mild amine vapor
- '100% pure NMP'

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA, USA) 
- Age at study initiation: 32 days
- Weight at study initiation: not indicated
- Fasting period before study: not indicated
- Housing: during pre-test and non-exposure four per cage, stainless-steel, wire-mesh cages  during exposure: stainless steel and glass chambers, chambers operated in a one-pass, flow through mode with an air-flow rate of about 3500 L/min.
- Diet: commercial diet (Purina Rodent Laboratory Chow #5001); ad libitum
- Water: ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 3
- Humidity (%): 50 ± 10
- Air changes (per hr): not indicated during non-exposure period; during exposure period: 3500 L/min
- Photoperiod (hrs dark / hrs light): Lightening: 12 hours light/12 hours dark


IN-LIFE DATES: From: 20 September 1976 To: 25 September 1978

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Whole-body exposure system, NMP vapors were generated by bubbling pre-heated nitrogen gas (80 or 130 °C) through liquid NMP. The aerosol fraction was minimized by an inline segregation vessel, but was not further quantified because only trace amounts were detected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

NMP chamber concentrations were determined by gas chromatography during about the first 4 months and by Miran spectrometry during the remainder study. Early analysis was with a flame ionization detector, followed by equipment with a nitrogen-phosphorus detector. Chamber atmospheres were measured daily at about 30 min intervals with time-weighed average concentrations for each chamber calculated.

Duration of treatment / exposure:

2 years

Frequency of treatment:

6 h/d; 5 d/week

Dose / conc.:

0.04 mg/L air (nominal)

Remarks:

equivalent to 10 ppm

Dose / conc.:

0.4 mg/L air (nominal)

Remarks:

equivalent to 100 ppm

No. of animals per sex per dose:

120 rats/sex/group; 3 groups per sex

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: none
- Dose selection rationale: The dose levels were selected according to the results of a repeated inhalation toxicity study in rats (up to 4 weeks): The NOAEC of NMP in the study was < 0.088 mg/L.

Observations and examinations performed and frequency:

Body weights were determined individually once a week during the first 8 weeks, during weeks 10 - 95 all cage mates were group weighed once every other week and during weeks 97 - 105 all surviving rats were weighed individually once every other week. All rats were observed twice daily for abnormal behavior and clinical signs of toxicity. Ten male and 10 female rats per group were subjected after 1, 3, 6, 12 and 18 months to hematological, clinical chemical and urinalysis. 

Sacrifice and pathology:

Ten rats per sex and group were sacrificed and subjected to gross pathology and histopathology after 3, 12 and 18 months. All dead and moribund rats (when found) were examined macroscopically and microscopically. After 24 months all surviving rats were sacrificed and macroscopically examined. Of these rats, all high concentration rats and all control rats were completely subjected to comprehensive histopathology.

Statistics:

One way ANOVA, Dunnett's test, least significant difference test (Steel and Torie, 1960)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No specific signs of intoxication. Only increased incidence in animals with wet and/or stained perinea.

Mortality:

mortality observed, treatment-related

Description (incidence):

No treatment-related effect on survival. Animals that died within the first 18 months suffered from chronic progressive nephropathy (8/23 vs. 4/19 of the control).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight of high-exposed males was significantly reduced by about 6 %.

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:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No substance-related findings.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No substance-related findings.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No substance-related findings.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not specified

Description (incidence and severity):

No substance-related findings.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No substance-related findings.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No substance-related findings.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There was no increased incidence in treatment-related benign or malignent tumours in male or female ats at any concentration.

Relevance of carcinogenic effects / potential:

There was no increased incidence in treatment-related benign or malignant tumors in male or female rats at any concentration.

Dose descriptor:

NOAEC

Effect level:

>= 0.4 mg/L air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects observed

Dose descriptor:

NOAEC

Effect level:

ca. 0.04 mg/L air

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEC

Effect level:

ca. 0.4 mg/L air

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

NMP concentrations: Mean low and high weekly time-weighed average concentrations were 97.4 % and 99.3 % (overall means=10.3 ppm 99.3 ppm).

Primary (benign and malignant) tumor incidence

	LIVER (18 Months)			LUNG (18 Months)
Dose level	0 ppm	10 ppm	100 ppm	0 ppm	10 ppm	100 ppm
Males	0/15	0/14	0/13	0/15	0/14	0/13
Females	0/14	1/15	0/15	0/14	0/15	0/15
	LIVER (24 Months)			LUNG (24 Months)
Dose level	0 ppm	10 ppm	100 ppm	0 ppm	10 ppm	100 ppm
Males	2/82	ND	5/85*	0/82	ND	0/85
Females	0/84	ND	3/82	0/83	ND	0/82

ND Not determined

* Not Significant (Report doesn't provide significance level – assumed to be p < 0.05)

 

 

 

Tumor summary for all organs examined at 24 months

Sex	Males		Females
Dose level	0 ppm	100 ppm	0 ppm	100 ppm
No. primary tumors	124	128	189	138
No. tumor-bearing rats	71/84	66/85	80/84	69/84
No. benign tumors	84	85	115	115
No. malignant tumors	40	43	23	23

Conclusions:

There was no treatment-related effect on survival and no increased incidence in treatment-related benign or malignant tumors in male or female rats at any concentration. Thus, NMP was not oncogenic at the investigated inhalative concentrations of 0.04 or 0.4 mg/L (10, 100 ppm).

Executive summary:

In a 2-year inhalation study, CD rats (120 per sex per dose level) were exposed (whole body) to NMP vapor concentrations of 0, 10 and 100 ppm (0.04 and 0.4 mg/L) for 6 h/day, 5 days/week. Ten rats per sex were subjected to hematology and blood and urine chemistry analysis after 1, 3, 6, 12, and 18 months of exposure. Ten rats per sex were sacrificed after 3, 12, and 18 months. All surviving rats were killed at the end of 24 months and examined macroscopically and by comprehensive histopathology. There was no treatment-related effect on survival. Animals that died within the first 18 months suffered from chronic progressive nephropathy. Beside an increased incidence in animals with wet and/or stained perineal fur as an indication of systemic NMP availability, no specific signs of intoxication were observed clinically. Body weight of males exposed to the high concentration was significantly reduced by about 6 %. Hematology, clinical chemistry, urinalysis as well as gross pathology and histopathology revealed no substance-related findings. Especially, there was no increased incidence in treatment-related benign or malignant tumors in male or female rats at any concentration. Thus, NMP was not oncogenic at the investigated inhalative concentrations of 0.04 or 0.4 mg/L (10, 100 ppm). The NOAEC was 0.04 mg/L (10 ppm) in males due to body weight gain reductions at the high concentration. The NOAEC in females was 0.4 mg/L (100 ppm).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

400 mg/m³

Study duration:

chronic

Species:

rat

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

NMP shall not be classified for carcinogenicity according to Regulation 1272/2008/EC, amended for the tenth time in Regulation (EU) No 2017/776, as no oncogenic potential was found in the rat after long-term exposure via inhalation or dietary administration. The liver tumors in mice may be directly related to the observed increase in the cellular proliferation rate, which could likely be due to the observed enzyme induction and weak peroxisome proliferation observed in B6C3F1 mice, which are known to be extremely sensitive to both non-genotoxic effects.

Additional information

Inhalation study

In a 2-year inhalation study, CD rats (120 per sex per dose level) were exposed (whole body) to NMP vapor concentrations of 0.10 and 100 ppm (0.04 and 0.4 mg/L) for 6 h/day, 5 days/week. Ten rats per sex were subjected to hematology and blood and urine chemistry analysis after 1, 3, 6, 12, and 18 months of exposure. Ten rats per sex were sacrificed after 3, 12, and 18 months. All surviving rats were killed at the end of 24 months and examined macroscopically and by comprehensive histopathology.

There was no treatment-related effect on survival. Animals that died within the first 18 months suffered from chronic progressive nephropathy. Beside an increased incidence in animals with wet and/or stained perineal fur as an indication of systemic NMP availability, no specific signs of intoxication were observed clinically. Body weight of males exposed to the high concentration was significantly reduced by about 6 %. Hematology, clinical chemistry, urinalysis as well as gross pathology and histopathology revealed no substance-related findings. Especially, there was no increased incidence in treatment-related benign or malignant tumors in male or female rats at any concentration. Thus, NMP was not oncogenic at the investigated inhalative concentrations of 0.04 or 0.4 mg/L (10, 100 ppm).

The NOAEC was 0.04 mg/L (10 ppm) in males due to body weight gain reductions at the high concentration and 0.4 mg/L (100 ppm) in females (Lee et al., 1987)

 

Oral feeding studies

Rat

NMP was examined for its chronic toxicity and carcinogenic potential in groups of each 62 male and 62 female Sprague-Dawley CD rats at dietary concentrations of 0, 1600, 5000 or 15000 ppm (about 66/88, 207/283, 678/939 mg/kg bw/day, males/females) for two years. Food consumption and body weights were determined at regular intervals. Clinical signs of toxicity were recorded at least once daily. Ophthalmology was conducted prior to the start and near the end of the study. Blood samples for differential white blood cell counts were collected after about 12, 18 and 24 months. After about 2 years all surviving rats were sacrificed. The animals were subjected to gross-pathological assessment, followed by comprehensive histopathological examinations. Tissues and organs from rats found dead, accidentally killed or sacrificed in extremis were also evaluated.

The survival of the female animals was not affected. The survival of the males in the high dose group was lower due an increase in severe chronic-progressive nephropathy as a typical finding in aging male rats. There was a reduction in body weights and retarded body weight gain with a corresponding reduction in food consumption and efficiency at 15000 ppm. The incidence of benign or malignant tumors was not increased among male or female rats, indicating no oncogenic potential up to a dietary concentration of 15000 ppm in male and female rats (approximately: 678/939 mg/kg bw/day in males/females). Only the high dose males revealed treatment-related macroscopic findings consisting of an increased incidence of large kidneys, kidneys diagnosed with chronic nephropathy, fluid in the pleural cavity and small testes. There was an increased incidence in spleenic hemosiderin as an indication ofa higher turn-over of red blood cells at 15000 ppm. In high and mid dose groups discoloration of urine as indication of systemic availability of the test substance occurred.

The NOAEL was 5000 ppm (approximately 207/283 mg/kg bw/day in males/females; Haskell et al., 1997).

 

Mouse

The oncogenic potential of NMP in the mouse was investigated in groups of each 50 male and 50 female B6C3F1 receiving dietary concentrations of 0, 600, 1,200 and 7,200 ppm (about 89/115, 173/221, 1089/1399 mg/kg bw/day, males/females) in an 18-month study. Food consumption and body weights were determined at regular intervals. A check of the general state of health of the animals was made at least daily and the animals were palpated once a week. Blood smears were prepared after 12 months and 18 months, and from all animals killed in extremis. After 18 months of treatment all surviving mice were sacrificed and liver, kidney, adrenal glands, testes, ovaries and brain were weighed. The animals were subjected to gross-pathological assessment, followed by comprehensive histopathological examinations in an extent as required by the guidelines.

There was no effect on survival in male or female mice. NMP caused substance-related effects at 1,200 and 7,200 ppm. Target organ was the liver with respect to increased metabolic activity. Increased liver weights, an increase in the incidence of foci of cellular alteration in the liver and of liver adenoma were noted at 7,200 ppm in both sexes. Among the 7,200 ppm males, the incidence of liver carcinomas was also increased, while the incidence in females was within the historical control range. Increased liver weights were also observed among the 1,200 ppm group males and 3/50 of these animals showed a centrilobular liver cell hypertrophy. Furthermore, discoloration of urine was observed at 7,200 and 1,200 ppm as a sign of systemic availability of the test substance.

The NOAEL was 600 ppm (89 mg/kg body weight/day) in males and 1200 ppm (221 mg/kg body weight/day) in females (BASF, 1999).

 

Mechanistic studies (Please refer to the Specific investigations)

NMP was investigated in male and female B6C3F1 mice for its potential to induce liver enzymes or peroxisome proliferation at the dose level of 7200 ppm (1364/1945 mg/kg bw/day in males/females) which was shown to cause an increase in liver tumors. The livers of each 10 animals per sex were examined for the cytochrome P450-content, ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD). In addition, each 5 male and 5 female mice were investigated for cyanide-insensitive Palmitoyl-CoA-oxidation (PALCoA) and by light or electron microscopy regarding changes in structure or amount on the peroxisomes, endoplasmic reticulum or mitochondria. NMP led to a slight increase in the activity of PALCoA in male animals and electron microscopy revealed a slight elevation in peroxisomes in 2/5 males (BASF, 2002).

The effect of NMP on cell proliferation in the liver (S-phase response) after dietary administration for 1 and 4 weeks of 7200 ppm (1392/1906 mg/kg bw/day in males/females) was also investigated in this strain of mice. Beside general signs of toxicity, the incorporation of bromodeoxyuridine (BrdU) in the liver DNA was examined microscopically. The cell proliferation rate in the livers increased 6.9-fold in the treated males and 3.3-fold in the treated females compared to the untreated control animals. An increase in the mitotic rate was observed in males only. Especially male animals (9/10) showed a minimal or slight centrilobular, hepatocellular hypertrophy compared to an incidence of 1/10 in the females. Fat storage in the liver was less pronounced in males, while a loss of fat storage accompanied with a decrease in the liver weight occurred in the females only. The cell proliferation rate in the livers showed an increase of 2.1-fold in males and 1.7-fold in females, respectively. The incidence of apoptotic cells in the liver was increased in males only. Minimal or slight centrilobular hypertrophy in the liver was recorded in 7/10 male and in 2/10 female mice, respectively. The effect on the fat storage in the liver was comparable to the findings after treatment for 1 week. Finally, there was clear evidence that NMP is able to induce an increase in hepatocellular proliferation after dietary treatment for 1 or 4 weeks (BASF, 2002).