Pyrrolidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Substance was only tested without metabolic activation, replicates were included although number of replicates unclear, limited number of concentrations with limited range (2 concentrations; 426.7 - 633 mg/L) were tested. However, substances tested up to cytotoxic concentrations.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1988

Report date:

1987

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effect of pyrrolidine was tested on the diploid strain D61.M of Saccharamyces cerevisiae.

GLP compliance:

no

Type of assay:

yeast cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Pyrrolidine

EC Number:

204-648-7

EC Name:

Pyrrolidine

Cas Number:

123-75-1

Molecular formula:

C4H9N

IUPAC Name:

pyrrolidine

Details on test material:

- Name of test material (as cited in study report): pyrrolidine
- Cas no.: 123-75-1
- Supplier: Sigma Chemical Co., St. Louis, USA
- Analytical purity: no data
- Lot/ batch No.: no data

Method

Target gene:

cyh2 (cycloheximide resistance); ade6 (white-adenine requirement); leul (leucine requirement)

Species / strain

Species / strain / cell type:

Saccharomyces cerevisiae

Details on mammalian cell type (if applicable):

Diploid strain D61.M

Metabolic activation:

without

Test concentrations with justification for top dose:

6.0, 8.9, 11.9 and 14.8 mM (corresponds to 426.7, 633, 846.3, 1052.6 mg/L)

Vehicle / solvent:

Not described

Controls

Untreated negative controls:

yes

Remarks:

No additions

Positive controls:

yes

Remarks:

1-methyl-2-pyrrolidinone at 77.6 and 103.2 mM

Details on test system and experimental conditions:

Preperations of cultures:
Cultures selected to have a low spontaneous frequency of cycloheximide resistance (typically <1 x 10-6) were diluted 1:10 into fresh YEPD medium (1% yeast extract, 2% peptone, and 2% glucose) and incubated at 28°C for 4 hr to bring the cells into exponential growth phase before addition of the test chemical.

Treatment procedure:
The exponential phase culture was adjusted to 5x106 cell/ml in YEPD medium. Treatments were carried out in 2-ml aliquots in glass test tubes. The growing yeast cells were treated in a shaker water bath at approximately 200 rpm at 28°C for 4 hr; then the cultures were refrigerated at 4°C in a water bath for 16 hr. The cold holding period was followed by a second 4-hr incubation at 28°C before the cultures were diluted and plated on the appropriate media. When necessary, cultures were diluted to approximately 1-2 x 107 cells/ml, and 0.1 ml aliquots were plated directly onto the selective cycloheximide-YEPD medium to determine the resistant population. Appropriate dilutions were plated onto YEPD medium to determine the surviving population. Plates were incubated for 5-7 days , and colonies were enumerated.
On selective medium the resistant colonies were either red or white. The red colonies resulted from the occurrence of genetic events such as gene conversion or mutation affecting the CYH2 locus only and not from chromosome malsegregation. The cycloheximide-resistant white colonies are presumably due to chromosome loss because the recessive cyh2 and the recessive ade6 alleles are being simultaneously expressed.
To confirm that the white resistant colonies are really monosomic for chromosome VII, each colony to be tested was streaked onto YEPD master plates, which were incubated overnight at 28°C, and then replicas were plated onto both a synthetic complete medium and onto the same medium lacking leucine. White (ade6) and cycloheximide-resistant (cyh2) colonies must also require leucine (leu1) to be considered monosomic.

Evaluation criteria:

Reference given: Zimmermann, FK (1984): Basic principles and methods of genotoxicity testing in the yeast Saccharamyces cerevisiae. In Kilbey BJ, Legator M, Nichols W, Ramel C (eds): " Handbook of Mutagenicity Test procedures", 2nd Ed., Amsterdam: Elsevier, pp 215-238.

Results and discussion

Test results

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 11.9 mM (846.3 mg/L) and above

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No other information provided.

Remarks on result:

other: strain/cell type: diploid strain D61.M

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

Pyrrolidine was shown not to induce aneuploidy or other genetic effects in the diploid strain D61.M of Saccharamyces cerevisiae.

Executive summary:

The effect of pyrrolidine was tested on the diploid strain D61.M of Saccharamyces cerevisiae. Cytotoxicity was found at concentrations of 11.9 mM (which corresponds with 846.3 mg/L) and above. Pyrrolidine was shown not to induce aneuploidy or other genetic effects without metabolic activation.