1-methyl-2-pyrrolidone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

NMP was not mutagenic in several bacterial and mammalian test systems in vitro covering different genetic endpoints (point mutations, DNA damage and repair):

Negative in two Ames tests: OECD 471, 1986 and 1978

Negative in the HPRT test: OECD 476, 1988

Negative in the Unscheduled DNA Synthesis assay (not recognized as a guideline study since 2014): OECD 486, 1988

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Laboratory identification number 77/585, not further specified

Species / strain / cell type:

S. typhimurium, other: TA98, TA1535, TA100, TA1537

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation (S9 mix from Arochlor 1254 treated male Sprague-Dawley rats)

Test concentrations with justification for top dose:

Experiment 1: 3.15, 10, 31.5, 100, 315, 1000, 3000, 10000, and 30000 µL/plate with S9 mix
Experiment 2: 31.5, 100, 315, 1000, 3000, 10000, and 30000 µL/plate without S9 mix

Vehicle / solvent:

DMSO or water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

benzo(a)pyrene

other: 2-aminoanthracene; N-methyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: Concentrations were tested in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Not indicated.

Species / strain:

S. typhimurium, other: TA98, TA1535, TA100, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No confounding factors were described.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Since a reduction of the his background lawn was not evident even at the highest dose of N-methylpyrrolidone, an overshadowing of a possible mutagenicity by toxicity is unlikely.

Conclusions:

No mutagenic potential was measured in the in vitro gene mutation study in bacteria.

Executive summary:

N-Methylpyrrolidone was tested for mutagenicity with Salmonella typhimurium TA 100, TA 1537 and TA 98. Seven different concentrations from 31.5 to 30,000 nl per plate were used in the experiments for direct mutagenicity. In the experiments with S-9 Mix nine concentrations from 3.15 to 30,000 nl per plate were tested. The test compound was dissolved at all concentrations. No mutagenicity was observed with the test compound under all these conditions. This was also the case when an epoxide hydratase inhibitor was added to the S-9 Mix in order to increase the sensitivity of the test towards compounds which are activated to mutagenic epoxides (this experiment was performed with TA 98). Since a reduction of the his background lawn was not evident even at the highest dose of N-methylpyrrolidone, an overshadowing of a possible mutagenicity by toxicity is unlikely.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-12-21 to 1988-02-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Source: GAF Corp.
- Lot/batch No.: 9084-126A
- Purity: unspecified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's Nutrient Mixture F12 supplemented with l-glutamine, antibiotics and fetal bovine serum (10%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolizing system (S9 fraction) induced with Aroclor 1254

Test concentrations with justification for top dose:

with and without metabolic activation: 0.5; 1.0; 2.0; 3.0; 4.0; and 5.0 mg/mL

Vehicle / solvent:

- Solvent used: Culture medium
- Justification for choice of solvent: NMP was soluble in F12 culture medium up to 50 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

substance was dissolved in culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 5-bromo-2'-deoxyuridine (BrdU)

Remarks:

50 µg/mL without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

5 µg/mL with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium as NMP showed good solubility in the solvent (F12 culture medium) at a concentration of 50.0 mg/mL

DURATION
- Preincubation period: 18 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 to 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 17 days


SELECTION AGENT (mutation assays): 4 µg/mL 6- thioguanine



NUMBER OF REPLICATIONS:
mutagenicity experiment: 3 to 12 replicates
cytotoxicity test: 2 replicates


NUMBER OF CELLS EVALUATED:
treatment mutagenicity test: 4 Mio cells/replicate
treatment cytotoxicity: 200 cells/replicate
expression phase: 1.5 Mio cells/replicate (at subculturing)
selection phase: 20000 cells per replicate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

Mutagenic if a dose-related or toxicity related increase in mutant frequency is observed.

Statistics:

Significant differences to controls were evaluated according to the statistical tables provided by Kastenbaum and Bowman (1970).

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

strain/cell type: CHO-K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: not seen at max. concentration due to solubility
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
A wide range of NMP concentrations was tested for cytotoxicity both with and without S9 metabolic activation. Ten concentrations that spanned a 3-log concentration range (0.005 - 5.0 mg/mL) were used. The maximum concentration applied was 5.0 mg/mL.
The preliminary cytotoxicity test showed that NMP was non-toxic at all dose levels both with and without activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
Pooled negative and solvent controls (from non-activation studies (50 studies and 100 controls):
Mean: 4.3 cells per 1 Mio. cells
Range: 0 to 15.0 cells per 1 Mio. cells

Pooled negative and solvent controls (from activation studies (50 studies and 99 controls):
Mean: 3.8 cells per 1 Mio. cells
Range: 0 to 13.5 cells per 1 Mio. cells

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main experiment, no cytotoxicity was observed up to the highest concentration used.

Conclusions:

The overall result of the Hprt test is a negative outcome.

Executive summary:

NMP was examined for genetic activity in the CHO gene mutation assay in the absence and presence of metabolic activation. Culture medium (HAM's F12) was used as solvent. Point mutation is measured in this test system by examining the induction of 6 -thioguanine (TG) resistance by forward gene mutation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. The test was performed with and without metabolic activation (S9 mix from the liver of Aroclor 1254 induced male Sprague-Dawley rats). One range-finding test and one mutagenicity experiment was carried out. Concentrations of the test substance in the main experiments ranged from:

 

- Main Experiment : 0.5 - 5.0 µg/mL without and with metabolic activation

In the mutagenicity tests, relative growth of the cells was not biologically relevant decreased at any concentration either with or without activation.

Mutant frequencies of all cultures treated with NMP varied randomly with dose within a range acceptable for negative control mutant frequencies. In the S9 metabolic activation mutation assay, one of the six treatment conditions achieved statistical significance. The culture that achieved statistical significance had a mutant frequency within the acceptable range for background mutant frequencies. This effect is considered to be a normal assay variation. Without S9 metabolic activation, none of the six dose levels had mutant frequencies that achieved statistical significance. The sensitivity of the test system was shown since all positive control substances were mutagenic.

Therefore, NMP was considered negative for inducing forward mutation at the HPRT locus in CHO cells under the S) metabolic activation and non-activation conditions of the assay.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-08-22 to 1988-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

not applicable

Species / strain / cell type:

hepatocytes: rat primary culture

Details on mammalian cell type (if applicable):

- Type and identity of media: Williams`Medium E supplemented with 10 % fetal bovine serum, 2mM L-glutamine, 100 µg/mL streptomycin sulfate and 150 µg/mL gentamicin
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Metabolic activation system:

primary hepatocytes are able to metabolize themselves

Test concentrations with justification for top dose:

0; 250; 500; 1000; 2000; 3000; 4000; 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: NMP is soluble in water and culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

NMP was dissolved in culture medium.

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- preincubation period: 1.5 - 2.0 h
- Exposure duration: 18 - 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 19.5 - 22 h

NUMBER OF REPLICATIONS: triplicate cultures for the UDS assay

NUMBER OF CELLS EVALUATED: 50 cells/culture (i.e. 150 cells per dose level)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (two replicates per dose level)

Evaluation criteria:

The test material is considered active in the UDS assay at applied concentrations that cause:
1) An increase in the mean net nuclear grain count to at least six grains per nucleus after subtraction of the concurrent negative control values, and/or
2) An increase in the percent of nuclei having six or more net grains to at least 10 % of the analysed population after subtraction of the concurrent negative control value

Species / strain:

hepatocytes: Primary rat hepatocytes

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 3000 to 5000 µg/mL

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The dose selection procedure was an integral part of the UDS assay in order to select appropriate doses. A range of fifteen concentrations 0.500 - 5000 µg/mL) was applied initially to the cells. A viable cell count (tryphan blue exclusion) was then obtained about 20.9 hours after initiation of the treatments. Six concentrations were chosen for analysis of nuclear labelling, starting with the highest dose that resulted in a sufficient number of survivors with intact morphologies and proceeding to successively lower doses.

COMPARISON WITH HISTORICAL CONTROL DATA:
not applicable

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material was highly toxic at 5000 µg/mL and resulted in cell morphologies unsuitable for analysis. Moderate toxicity was observed at concentrations of 4000 µg/mL and 3000 µg/mL (75.3 % and 88.6 % survival, respectively). Dose levels at and below 2000 µg/mL were non-toxic.

The concentration of 5000 µg/mL was not investigated due to high cytotoxicity.

Cell survival (%) was as follows:

NMP concentration (µg/ml)	Relative survival (% of control)
4000	75.3
3000	88.6
2000	96.1
1000	97.6
500	97.2
250	not determined

The minimum criteria for UDS in this assay were a mean net nuclear grain count exceeding 6.66, or at least 16.0 % of the nuclei containing six or more grains. None of the treatments with the test material samples caused nuclear labelling significantly different from the control. Furthermore, no dose-related trend was evident.

NMP did not induce significant changes in nuclear labeling of rat primary hepatocytes at concentrations ranging from 250 - 4000 µg/mL covering a good range of cell survival (75.3 % - 97.6 %).

Conclusions:

The overall result of the UDS assay was a negative outcome.

Executive summary:

In the in vitro Rat Primary Hepatocyte UDS assay, freshly prepared rat hepatocytes were exposed to NMP at concentrations ranging from 0.500 µg/mL to 5000 µg/mL in the presence of 5 µCi/mL 3HTdr (20 Ci/mmole). Treatment at 5000 µg/mL was not analysed for nuclear labeling due to high toxicity. Treatments from 250 µg/mL to 4000 µg/mL, which covered a good range of toxicity (97.6 to 75.3 % survival), were selected for analysis. NMP was soluble in culture medium at all concentrations tested. None of the criteria used to indicate UDS were approached by NMP and no dose-related response was observed.

The sensitivity of the test system was shown since the positive control substance was mutagenic.

NMP did not induce significant changes in the nuclear labeling of rat primary hepatocytes for an applied concentration range of 250 µg/mL to 4000 µg/mL. Therefore, NMP was evaluated as inactive in the Rat Primary Hepatocyte UDS Assay. NMP was therefore evaluated as inactive in the Rat Primary Hepatocyte Assay.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Source: Aldrich
- Analytical purity: 99 %

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

metabolic microsomal enzyme systems (S9 fraction) from Arochlor 1254-induced male Sprague-Dawley rat and male Syrian hamster liver

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333, 10000 µg/plate with and without S9 mix

Vehicle / solvent:

- Vehicle used: distilled water

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix (TA100, TA1535)

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix (TA1537)

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9 mix (TA98)

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

According to Haworth et al. 1983:
1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background (even if less than two-fold)
2) non-mutagenic response: no increase in the number of revertants
3) questionable result: absence of a clear-cut dose-related increase in revertants; when dose-related increases were not reproducible or when the response was of insufficient magnitude to support determination of mutagenicity

Statistics:

none

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
All chemicals were initially tested with strain TA 100 in the presence and absence of S9 mix over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was encountered either by appearance of his- pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning of absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to 10 mg/plate dose level, or to a dose level determined by their solubility. Toxic chemicals were tested up to high dose which exhibited some degree of toxicity.

Results form Experiment Ames test with NMP as described in the tables of NTP (2015), 309283

Strain: TA100
Dose	No Activation		No Activation		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	114	0.7	85	5.5	108	3.7	105	3.5
100	100	9.5	95	4.3	114	5.2	116	3.2
333	126	11.3	90	3.8	125	6.4	112	7.1
1000	123	7.1	91	7.2	122	10.1	115	4.1
3333	111	4.6	88	10.6	98	1.2	124	9.3
10000	111	0.7	88	6.1	106	8.5	115	5
Positive Control	377	7.5	208	16.4	389	29.7	438	5.6
Strain: TA1535
Dose	No Activation		No Activation		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	31	3.8	21	3.3	9	1.7	9	1.9
100	22	4.2	15	1.7	6	0.9	6	1.7
333	25	3.2	15	3.3	8	0.9	5	1.5
1000	21	3.3	10	3.1	4	0.3	7	1.3
3333	17	1.9	13	4	8	3.2	11	2.5
10000	16	1	14	4.8	7	0.7	4	0.6
Positive Control	440	6.9	250	13.5	162	6.1	158	11.5
Strain: TA1537
Dose	No Activation		No Activation		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	3	0.3	4	0.7	10	0.3	3	0.9
100	4	0.7	6	1.2	4	1.3	6	1.5
333	5	1.2	3	0.7	6	0.7	7	0.3
1000	5	0.7	5	1	5	1.2	6	1.5
3333	4	1.5	7	3.1	5	0.7	5	1
10000	3	0.9	5	2.2	4	0	9	2.2
Positive Control	317	31.7	157	28.2	163	25.6	114	5.7
Strain: TA98
Dose	No Activation		No Activation		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	16	1	17	2.2	17	1.5	17	3.6
100	16	1.9	11	1.7	23	4.5	30	8.1
333	14	2.4	15	1.5	24	4	23	3.7
1000	11	1.5	15	4.3	26	0.6	23	3.2
3333	10	2.8	9	2	20	3.5	21	0.6
10000	16	1.2	16	1.7	22	5.4	24	4.3
Positive Control	388	21.7	325	9.7	285	17.7	386	14.6
Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; t = Toxic; c = Contamination

 

Results form Experiment Ames test with NMP as described in the tables of NTP (2015), 750004

Strain: TA100
Dose	No Activation		No Activation		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	82	4.6	94	3.9	124	11.2	126	12.1
100	80	4.7	119	12.9	117	6	141	2.5
333	76	2.9	102	4.5	123	3.6	138	23.7
1000	79	2.9	94	2.5	118	4	165	3.4
3333	78	4	118	5.6	119	9.6	142	20.5
10000	77	0.6	98	5	117	6.9	139	20.6
Positive Control	1433	41.8	1571	134.6	2284	41	2640	175.7
Strain: TA1535
Dose	No Activation		No Activation		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	9	1.2	8	0.3	6	1.5	7	0.3
100	6	2.3	5	1.9	9	2.3	13	4.6
333	10	1.9	4	0.9	8	0.3	4	1.5
1000	11	2.4	6	1.7	5	0	10	4.5
3333	9	0.7	6	1.5	8	3.1	6	1.8
10000	4	1.5	5	1.2	5	1.2	3	0.9
Positive Control	769	178.3	1832	75.5	218	73.1	510	48.5
Strain: TA1537
Dose	No Activation		No Activation		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	7	0.3	4	2	9	0.9	12	4.2
100	8	2.3	5	1.7	8	1.5	4	1.2
333	8	0.9	4	1.2	7	1.7	7	1.2
1000	5	0.6	5	0.7	10	2	6	1.3
3333	4	0.9	4	1.3	8	1.8	11	4.4
10000	3	0.3	3	0.9	8	0.9	6	0.9
Positive Control	831	127.3	712	73.5	181	21.9	106	8.8
Strain: TA98
Dose	No Activation		No Activation		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	±SEM	Mean	±SEM	Mean	±SEM	Mean	±SEM
0	18	2.8	19	2.8	23	5.2	18	3.3
100	20	3.5	17	1.2	26	3.1	19	3
333	19	3.5	16	0.7	29	4.6	17	4.4
1000	18	1.2	17	3.1	30	9.7	12	0.6
3333	17	0.9	15	2.2	29	4.2	12	3.4
10000	18	1.2	23	0.9	27	0.6	9	0.3
Positive Control	377	15.5	800	42.8	2216	98.8	1685	133.8
Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; t = Toxic; c = Contamination

Test condition: Comparative study within the comprehensive testing program of the NTP/USA.

 

Conclusions:

No mutagenic potential was measured in the in vitro gene mutation study in bacteria.

Executive summary:

NMP was evaluated for its ability to induce mutations in the histidine operon of Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 1535 and TA 1537. In a preliminary assay, it was shown that doses up to 10000 µg/plate did not reveal toxicity.

In the main assay, doses of 0, 100, 333, 1000, 3333 and 10000 µg/plate were evaluated in triplicate (one concentration/well/plate) in the presence and absence of metabolic activation (from rat liver). Concurrent negative/solvent and positive controls were included.

No relevant increase in the number of histidine (his+) revertants was observed in any of the bacterial strains used either with or without activation by S9 mix. The sensitivity of the test system was shown since all positive control substances were mutagenic.

NMP was not mutagenic in the Salmonella microsome assay when tested directly or in the presence of a metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo, no clastogenic or aneugenic potential of NMP was reported in the following assays:

Negative in the Micronucleus assay in mice: OECD 474, 1989

Negative in the Chromosome aberration assay in chinese hamster: OECD 475, 1993

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, 88/369
- Analytical purity: 99.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +4°C to +6°C under N2 conditions
- Stability under test conditions: Stability proven by sponsor
- Solubility and stability of the test substance in the solvent/vehicle: Substance was formulated in aqua dest. immediately before administration

FORM AS APPLIED IN THE TEST
- solution

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Wiga, D-8741 Sulzfeld, Germany
- Weight at study initiation: not specified
- Assigned to test groups randomly: yes
- Fasting period before study: not indicated
- Housing:
during acclimation period: in Makrolon cages type III, in groups of 5 per sex
during study period: in Makrolon cages type I, individually
- Diet: ad libitum, standardized pellet (Kliba Haltungsdiät; Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: about one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): fully air conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: aqua dest.
- Justification for choice of solvent/vehicle: NMP is soluble and stable in aqua dest. for > 2 hours
- Concentration of test material in vehicle: 9.5 - 38 g/100 mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test groups 2 were given 3800 mg test substance/kg bw or 10 mL/kg bw of a solution with a concentration of 38 g/100 mL
Test groups 3 were given 1900 mg test substance/kg bw or 10 mL/kg bw of a solution with a concentration of 19 g/100 mL
Test groups 4 were given 950 mg test substance/kg bw or 10 mL/kg bw of a solution with a concentration of 9.5 g/100 mL

Duration of treatment / exposure:

24 h (950 and 1900 mg/kg bw); 16, 24 and 48 h (3,800 mg/kg bw)
24 h (solvent control and positive controls)

Frequency of treatment:

one single test substance administration by gavage

Post exposure period:

not applicable

Dose / conc.:

950 mg/kg bw/day (actual dose received)

Dose / conc.:

1 900 mg/kg bw/day (actual dose received)

Dose / conc.:

3 800 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per sex, dose and sampling interval (for NMP treatment groups and control group)
3 male and 2 female mice for cyclophosphamide treatment group
2 male and 3 female mice for vincristine treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

- Route of administration: gavage
- Doses / concentrations:
cyclophosphamide: 40 mg/kg bw per os (positive agent for clastogenic activity)
vincristine: 0.15 mg/kg intraperitoneal (positive agent for spindle inhibition, aneuploidy)

Tissues and cell types examined:

bone marrow cells
After administration of the test substance, the animals were examined for any evident clinical signs of toxicity.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for determination of the acute oral toxicity, deaths were observed down to a dose of 4640 mg/kg bw. The dose level which all animals survived was 3830 mg/kg bw, but signs of toxicity were observed at this dose level: irregular respiration and abdominal position. In addition, the general state of the animal was poor.
Therefore a dose of 3800 mg/kg bw was selected as the highest dose in this micronucleus test. 1900 mg/kg bw and 950 mg/kg bw were selected as additional dose levels.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All test substance solutions were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animal on the day of the experiment. For control purposes, male and female animals were given the solvent aqua dest. by the same route.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by Schmid, W. (1976 and 1977).
After cutting the epiphyses, the bone marrow ws flushed out of the diaphysis with ca. 2 mL fetal calf serum. The suspension was reduced by centrifugation and bone marrow smears were prepared onto clean microscopic slides. The slides were stained in eosin and methylene blue and after staining with Giemsa, the preparation were embedded in Entellan.

Evaluation criteria:

1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes are also scored. The following parameters are recorded:
- number of polychromatic erythrocytes
- number of polychromatic erythrocytes containing micronuclei
- number of normochromatic erythrocytes
- ratio of polychromatic to normochromatic erythrocytes
- number of small micronuclei (d < D/4) and large micronuclei (d>= D/4)
d= diameter of micronuclei and D = cell diameter
- number of normochromatic erythrocytes containing micronuclei

Statistics:

Statistical analysis: Fisher's exact test and U-test according to Mann-Whitney

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Apart from irregular respiration and abdominal position that lasted from 1h to 1d, depending on the doses, there was no toxicity observed.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Clinical examinations:
Doses of 3800 mg/kg bw and 1900 mg/kg bw induced irregular respiration and abdominal position immediately after test substance administration. These signs indicate the bioavailability of the test substance. 24 hours after treatment, the animals did not show clinical signs any longer.

- Induction of micronuclei (for Micronucleus assay):
No increased frequency of polychromatic erythrocytes containing either small or large micronuclei (see also table under "Any other information on results incl. tables").
- Ratio of PCE/NCE (for Micronucleus assay): Inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at 3800 mg/kg  bw (approx. 80 % of LD50) after 24 h sacrifice only.

- Statistical evaluation:
There was no statistically significant increase in the rate of polychromatic cells with micronuclei in any animal treated with NMP
The positive controls cyclophosphamide and vincristine induced significant increases in the number of micronucleated polychromatic erythrocytes

Results at 24 h:

 

Dose level (mg/kg bw)	MN/1000 PCE	PCE examined	NCE (found)
0	22 (2.2%)	10000	3423
NMP          950	16 (1.6)	10000	3198
NMP         1900	22 (2.2)	10000	2952
NMP        3800	21 (2.1)	10000	6605
CPP             40	90 (18.0)	5000	2292
VC                 0.15	516 (103)	5000	4132

MN = cells with micronuclei per 100 polychromatic erythrocytes

PCE = poylchromatic erythrocytes

NCE = normochromatic erythrocytes

CP = cyclophosphamide

VC = vincristine

Conclusions:

The overall result of the in vivo micronucleus assay was a negative outcome.

Executive summary:

NMP was tested for clastogenicity and for the ability to have spindle poison effects in the mouse micronucleus test in NMRI mice. NMP dissolved in aqua dest., was administered as a single dose orally to 5 males and 5 females/test group at dose levels of 3800, 1900 and 950 mg/kg bw in a volume of 10 mL/kg body weight. Concurrent negative control test groups (5 male and 5 female mice) were administered merely the solvent aqua.dest by the same route. As positive control for clastogenicity, 40 mg of cyclophosphamide/kg bw, dissolved in aqua dest., was administered orally to 3 male and 3 female animals. As positive control for spindel poison effects, 0.15 mg of vincristine/kg bw, dissolved in aqua dest., was administered intraperitoneally to 2 male and 2 female animals. The femora of the animals in the respective groups were prepared 16, 24 and 48 hours after test substance administration in the highest dose group of 3800 mg/kg bw. In the test groups of 1900 and 95 mg/kg bw, the negative control group and in the positive control groups, the 24 -hour interval was investigated, only. After staining of the preparations, 1000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normochromatic erythrocytes were also registered. After administration of the test substance, the animals were examined for any evident clinical signs of toxicity.

Doses of 3800 mg/kg bw and 1900 mg/kg bw induced irregular respiration and abdominal position immediately after test substance administration. These signs indicate the bioavailability of the test substance. 24 hours after treatment, the animals did not show clinical signs any longer.

NMP did not increase the frequency of poylchromatic erythrocytes containing either small or large micronuclei.

The positive control substances cyclophosphamide/vincristine sulfate increased clearly the numbers of micronucleated polychromatic erythrocytes indicating the sensitivity of the test system.

These results indicate that NMP has neither a clastogenic effect nor a spindle poison effect in vivo.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Remarks:

study conducted in a GLP compliant laboratory

Type of assay:

chromosome aberration assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG
- Analytical purity: >99.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +4°C to +6°C under N2 conditions

FORM AS APPLIED IN THE TEST
- solution

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Knoll AG, Ludwigshafen/Rh, Germany
- Age at study initiation: not indicated
- Weight at study initiation: 25.9 g (mean weight)
- Assigned to test groups randomly: yes

Route of administration:

oral: gavage

Vehicle:

NMP was dissolved in aqua dest.

Details on exposure:

applied by gavage in a volume of 10 mL/kg bw

Duration of treatment / exposure:

24 h (1900 mg/kg bw); 24 and 48 h (3800 mg/kg bw)

Frequency of treatment:

once

Dose / conc.:

3 800 mg/kg bw/day (actual dose received)

Dose / conc.:

1 900 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per sex, dose and sampling interval (for NMP and cyclophosphamide treatment groups)
3 animals per sex, dose and sampling interval (for vincristine and benomyl treatment groups)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Route of administration: oral
- Doses / concentrations:
cyclophosphamide: 40 mg/kg bw per os (positive agent for clastogenic activity)
vincristine: 2 and 3 mg/kg intraperitoneal (positive agent for spindle inhibition, aneuploidy)
benomyl: 2500 and 3000 mg/kg bw per os (positive agent for spindle inhibition, aneuploidy)

Tissues and cell types examined:

bone marrow;
bone marrow cells in mitosis

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
2-3 hours before sacrifice, Chinese hamsters were intraperitoneally injected with 3.3 mg/kg Colcemid in order to arrest mitosis in the metaphase stage. The bone marrow chromosomes were prepared according to a modified method of Schmidt and Staiger 1969; the slides were stained using Giemsa.

METHOD OF ANALYSIS:
100 well-spread mitosis of each animal were analysed for numerical chromosoe aberrations (hyperploidies and polyploidies) and structural chromosomal aberrations according to Evans and O'Riordan (1975) and Savage (1975)
Cells were analysed for gaps, for exchanges, multiple aberrabt cells (< 5 aberrations/cell) and for polyploidy and aneuploidy.

Evaluation criteria:

statistical significance if p<=0.05 or p<=0.01

Statistics:

Fisher's exact test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

NMP treatment led to signs of toxicity including irregular respiration, abdominal position and poor general state and discoloration of urine indicating systemic availability.

NMP caused no statistically significant increase in the number of cells containing structural chromosomal alterations or numerical chromosomal aberrations. 
The vehicle control (distilled water) showed an appropriate result.
The positive control substances cyclophosphamide/vincristine sulfate induced chromosomal aberrations and vincristine/benomyl enhanced the number of polyploid and aneuploid cells indicating the sensitivity of the test system for these types of mutagenic endpoints.

Conclusions:

The treatment led to signs of toxicity including irregular respiration, abdominal position and poor general state and discoloration of urine indicating systemic availability.
There was no increase in either the number of mitoses containing structural chromosomal alterations or numerical chromosomal aberrations after the administration of NMP. Thus, NMP did not reveal any clastogenic or aneugenic activity.

Executive summary:

NMP was investigated in the Chinese hamster bone marrow test for structural and numerical aberrations. This test can detect both types of mutations as demonstrated by appropriate positive control substances (cyclophosphamide, vincristine sulfate and benomyl).

NMP was dissolved in aqua dest. and given once orally in a volume of 10 mL/kg bw to 5 male and 5 female animals per test group and sacrifice interval. The dose levels were 1900 and 3800 mg/kg bw. Bone marrow was sampled after 24 hours in the lower dose group or after 24 and 48 hours in the higher dose group. For the positive controls on aneuploidy, vincristine and benomyl, and cyclophosphamide for clastogenicity, 3 male and 3 female animals were used at sacrifice interval of 24 hours. Concurrent vehicle control (aqua dest. only) animals (5 males and 5 females) were sacrificed at 24 hours after administration. After preparation of bone marrow cells, frequencies of aberrant cells inclusive and exclusive gaps and polyploid or aneuploid cells were evaluated (each 100 cells/animal).

NMP treatment led to signs of toxicity including irregular respiration, abdominal position and poor general state and discoloration of urine indicating systemic availability.

NMP at single oral doses of 1900 and 3800 mg/kg bodyweight (approximately 80% of LD50) did not lead to an increase either in structural or numerical aberrations when bone marrow was sampled 24 and 48 hours after treatment for cytogenetic analysis.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

NMP was evaluated for its ability to induce mutations in the histidine operon of Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 1535 and TA 1537. In a preliminary assay, it was shown that doses up to 10000 µg/plate did not reveal any toxicity. In the main assay, doses of 0, 100, 333, 1000, 3333 and 10000 µg/plate were evaluated in triplicate (one concentration/well/plate) in the presence and absence of metabolic activation (from rat liver). Concurrent negative/solvent and positive controls were included. No relevant increase in the number of histidine (his+) revertants was observed in any of the bacterial strains used either with or without activation by S9 mix. The sensitivity of the test system was shown since all positive control substances were mutagenic. NMP was not mutagenic in the Salmonella microsome assay when tested directly or in the presence of a metabolic activation system (Mortelmans et al., 1986). These negative results were also confirmed by data from other bacterial test systems (BASF SE, 1978).

 

The potential mutagenic effect of NMP on mammalian cells was examined by assaying Chinese hamster ovary cells (HGPRT assay). The concentrations ranged from 0.5 to 5.0 mg/mL (with and without S9 mix). The test substance was dissolved in F12 culture medium, which was also used as solvent control. Positive control substances (5-bromo-2'-deoxyuridine (BrdU) and 3-methylcholanthrene) were additionally investigated. NMP showed no cytotoxicity and did not increase the mutation rate (GAF Chemical Corp., 1988).

 

The ability of NMP to interact with DNA was investigated in vitro in primary hepatocyte cultures from the liver of an untreated male F-344 rat. The concentrations ranged from 250 - 5000 µg/mL. The cell cultures were maintained in Williams' Medium E plus 1 % serum. 2-acetylaminofluorene was examined as positive control substance. Unscheduled DNA synthesis was quantified by net nuclear increase of black silver grains for 50 cells per slide. NMP was soluble including the highest concentration, but was shown to be slightly cytotoxic at concentrations ≥ 4000 µg/mL. NMP did not induce significant changes in nuclear labeling of rat primary hepatocytes at concentrations ranging from 500 - 5000 µg/mL covering a good range of cell survival (53.2 - 98.6 %); (Vetline Inc., 1988).

 

In vivo studies

NMP was investigated for its clastogenic/genotoxic potential in vivo in the Chinese hamster cytogenic assay. The test substance was dissolved in distilled water and administered to groups of 5 male and 5 female Chinese hamsters once daily by gavage in doses of 1900 and 3800 mg/kg bw/day using an application volume of 10 mg/kg bw/day. A vehicle control (distilled water) and three positive controls (cyclophosphamide, vincristine sulfate, benomyl) were also tested. Following dosing, the animals were examined for mortality or clinical signs. The animals were sacrificed 24 h (1900 mg/kg bw/day) or 24 and 48 h (3800 mg/kg bw/day) after treatment. Animals received 3.3 mg/kg of colcemid i.p. prior to sacrifice. Bone marrow chromosomes were prepared and slides were stained with Giemsa. 100 mitotic cells per animal were analyzed for numerical and structural chromosomal aberrations. NMP treatment led to signs of systemic toxicity. The vehicle control showed an appropriate result and no increase in the number of mitosis containing structural chromosomal alterations or numerical chromosomal aberrations was observed after treatment with NMP. The positive control substances cyclophosphamide/vincristine sulfate induced chromosomal aberrations and benomyl enhanced the number of polyploid and aneuploid cells indicating the sensitivity of the test system (Engelhardt and Fleig, 1993).

NMP was also investigated in the mouse bone marrow micronucleus test, dissolved in distilled water and administered to groups of 5 male and 5 female NMRI mice once daily by gavage in doses of 950, 1900 and 3800 mg/kg bw/day. A vehicle control and two positive controls (cyclophosphamide, vincristine sulfate) were also tested. Following dosing, the animals were examined for mortality or clinical signs. Bone marrow for micronuclei examination was prepared and 1000 polychromatic erythrocytes were evaluated per animal and investigated for small and large micronuclei. NMP treatment led to clinical signs of toxicity including irregular respiration, abdominal position and poor general state. NMP did not induce micronuclei in the polychromatic erythrocytes of mice treated up to a dose showing clinical signs of toxicity and bone marrow toxicity in form of inhibition of erythropoiesis determined by the ratio of polychromatic to normochromatic erythrocytes. No indication of a spindle poisoning effect was detected (BASF SE, 1989).

Justification for classification or non-classification

NMP was found to be non-mutagenic in vitro and in vivo. The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.