Registration Dossier

Administrative data

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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Species / strain:
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:
water
Negative controls:
yes
Solvent 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 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:
yes
Remarks:
A bacteriotoxic effect was observed in the preincubation test depending an the strain and test conditions at about > 1000 µg/plate.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: A bacteriotoxic effect was observed in the preincubation test depending an the strain and test conditions at about > 1000 ug/plate.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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.

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.