1-methylpyrrolidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test substance was not mutagenic in the Ames test either with or without metabolic activation. Aneuploidy induction in yeast: The test substance did not cause aneuploidy in Saccharomyces cerevisiae D61.M.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

20, 100, 500, 2500, 5000 µg/plate (standard plate test)
125, 250, 500, 1000, 1500 µg/plate (preincubation test)

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S9-mix: 2-aminoanthracene (all strains); without S9-mix: 2-aminoanthracene (all strains), N-methyl-N'-nitro-N-nitrosoguanidine (TA1535, 100), 4-nitro-o-phenylenediamine (TA98), 9-aminoacridine (TA1537) and N-ethyl-N'-nitro-N-nitrosoguanidine (E.coli)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Expression time (cells in growth medium): 48-72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducable increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

The arithmetic mean of the counted colonies per concentration was calculated.

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:

cytotoxicity

Remarks:

A bacteriotoxic effect was observed in the preincubation test depending an the strain and test conditions at about > 1000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed in the preincubation test depending an the strain and test conditions at about > 1000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Number of revertants per plate (mean of three plates), test 1

	strain TA98		strain TA100		strain TA1535		strain TA1537
conc. [µg/mL]	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0 (water)	27	46	159	158	20	20	12
20	27	46	157	158	20	22	10
100	26	37	157	159	19	20	11
500	22	29	127	157	20	16	10
2500		8		83		8
5000
4-nitro-o-phenyleridiamine 10 µg	1266
2-Aminoanthracene 2.5 µg		1215		1536		135		147
N-methyl-N' -nitro-N-nitrosoguanidine 5 µg			2239		1424
9-aminoacridine 100 µg							472

 

 

 

Table 2: Number of revertants per plate (mean of three plates), test 2

	strain TA98		strain TA100		strain TA1535		strain TA1537		E. coli WP2 uvrA
conc. [µg/mL]	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0 (water)	30	48	137	151	13	12	10	11	33	38
125	28	39	141	154	13	15	13	11	32	39
250	35	46	136	152	16	12	9	10	33	35
500	35	46	158	139	12	15	9	12	35	41
1000	28	46	104	119	10	15	6	9	22	37
1500	34	38	108	135	-	12	1	12	17	27
4-nitro-o-phenyleridiamine 10 µg	1113
2-Aminoanthracene 2.5 µg*		786		662		88		129		112
N-methyl-N' -nitro-N-nitrosoguanidine 5 µg			1297		1083
9-aminoacridine 100 µg							346
N-ethylN-nitro-N-nitrosoguanidine 10 µg									772

  *except for E.coli: 60 µg

 

 

 

 

 

 

 

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames Test

The substance 1-Methylpyrrolidin was tested for mutagenicity in the Salmonella typhimurium/ Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing System (S-9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The tested concentrations were in the range of 20-5000 µg/plate. A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his or trp revertants, reduction in the titer was observed in the preincubation test depending on the strain and test conditions at about > 1,000 µg/plate. No test substance precipitation was found. According to the results of the present study, the test substance did not lead to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in several experiments carried out independently of each other (standard plate test and preincubation assay). Thus, under the experimental conditions chosen, it is concluded that 1-Methylpyrrolidin is not a mutagenic agent in a bacterial reverse mutation test in vitro.

Aneuploidy induction in yeast

Two publications are available describing anauploidy induction studies in yeast. The first assay was conducted on the diploid strain D61.M of Saccharomyces cerevisiae (according to Zimmermann, F.K., In: Kilbey, B.J. et al.: Handbook of Mutagenicity Test Procedures, 2nd Ed., Elsevier, Amsterdam, pp. 215-238) to assess the potential of several substances to induce aneuploidy in yeast. 11 substances were tested including the test substance 1-methylpyrrolidine, which was tested in the following nominal concentrations: 12.8, 13.3, 13.8, 14.2, 14.7, 15.2 mM (1089.92, 1132.5, 1175.07, 1209.13, 1251.71, 1294.28 mg/L). On YEPD medium containing cycloheximide, colonies resulting from the loss of chromosome VII were white and colonies containing gene mutations had a red colour. White colonies were confirmed to be monosomic if they were leucine auxotroph and thus not growing on medium without leucine. The test results gave no indications for an aneuploidy-causing property of the test substance. 1-methylpyrrolidine was found to be cytotoxic in concentrations from 10 mM onwards. This result conforms with results summarized in another publication (Liu et al. 1997), where 1 -methylpyrrolidine was also concluded to be non aneuploidy-causing in Saccharomyces cerevisiae D61.M. But, as basic details are not given in this publication, it was considered to be not reliable.

Justification for selection of genetic toxicity endpoint
only one bacterial reverse mutation test available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available Ames Test is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genetic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

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