N-(2-chloroethyl)pyrrolidine hydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation toxicity study was performed to determine the mutagenic nature of Pyrrolidinoethyl chloride. The study was performed using the modified Ames assay using eight histidine auxotrophs of Salmonella typhimurium (G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, and TA98) and two tryptophan auxotraphs of Escherichia coli (WP2 and WP2 uvrA-) with and without S9 metabolic activation system. The inocula of ten bacterial tester strains were streaked across square agar plates containing a concentration gradient of test compound and mutagenicity was scored by noting the number of tester strains showing mutant colonies over a given concentration range. The test compound was incorporated into four gradient plates to give a tenfold concentration range per plate, thus providing a 10,000-fold concentration range for the test. Pyrrolidinoethyl chloride induced gene mutation in Salmonella typhimurium strains G46, TA1535, TA100 and E. coli strain WP2 uvrA without metabolic activation system at a minimum mutagenic concentration of 0.6 nanomoles/mL. It however failed to induce gene mutation in Salmonella typhimurium (C3076, TA1537, D3052, TA1538, and TA98) and Escherichia coli (WP2) in the presence and absence of S9 metabolic activation system.

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:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviwed journal

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro gene toxicity study was performed by the gradient plate test to determine the mutagenic nature of Pyrrolidinoethyl chloride in Salmonella typhimurium and E coli

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Specific details on test material used for the study:

- Name of test material: Pyrrolidinoethyl chloride
- IUPAC name: 1-(2-chloroethyl)pyrrolidine hydrochloride
- Molecular formula: C8H9Cl3O
- Molecular weight: 227.517 g/mol
- Substance type: organic
- Physical state: No data available
- Purity :No data available
- Impurities (identity and concentrations): No data available

Target gene:

Histidine for Salmonella typhimurium strains and tryptophan for E. coli strains

Species / strain / cell type:

S. typhimurium, other: G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, and TA98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Histidine auxotrophs

Species / strain / cell type:

E. coli, other: WP2 and WP2uvrA-

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Histidine auxotrophs

Cytokinesis block (if used):

No data

Metabolic activation:

without

Metabolic activation system:

Liver activating system

Test concentrations with justification for top dose:

The highest compound concentration tested was 1,000 mcg/ml of agar (Strain G46: 500-1000, TA1535: 100-1000, TA100: 1-1000, WP2: 0.1-300, WP2uvrA-: 0.1-50 mcg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: The chemical was soluble in sterile distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The chemical Pyrrolidinoethyl chloride was screened for its ability to produce an increase in revertants in any of the strains used for the study

Statistics:

No data available

Species / strain:

S. typhimurium, other: C3076, TA1537, D3052, TA1538, and TA98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium, other: G46, TA1535, TA100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

E. coli, other: WP2 and WP2uvrA-

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Conclusions:

Pyrrolidinoethyl chloride did not induce an increase in the number of revertants in Salmonella typhimurium strains C3076, TA1537, D3052, TA1538, and TA98 in the absence of liver activating enzymes. It however induced an increase in the number of revertants in Salmonella typhimurium strains G46, TA1535, TA100 and E. coli strains WP2 and WP2uvrA- in the absence of liver activating enzymes.




in the concentration of 1 to 1,000 mcg/ml /plate and 0.1 to 300.0 mcg/ml show bacterial cell mutation toxicity when Salmonella typhimurium (G46, TA1535, TA100) and E coli (WP2 and WP2uvrA-) cells were exposed to the test chemical.

Executive summary:

In vitro gene toxicity study was performed to determine the mutagenic nature of Pyrrolidinoethyl chloride using Salmonella typhimurium strains G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, and TA98 and E. coli strains WP2 and WP2uvrA-. Bacterial mutagenesis was performed by the gradient plate test. Pyrrolidinoethyl chloride was incorporated into the top layer of agar on four 10- X 10-cm square plates to give a tenfold concentration gradient per plate, thus providing a 10,000-fold concentration range. The highest compound concentration tested was 1,000 mcg/ml of agar ( Strain G46: 500-1000, TA1535: 100-1000, TA100: 1-1000, WP2: 0.1-300, WP2uvrA-: 0.1-50 mcg/mL). Since Pyrrolidinoethyl chloride is a direct acting alkylating agent, the assay was performed in the absence of liver activating system. The chemical was screened for its ability to produce an increase in revertants in any of the strains used for the study. Pyrrolidinoethyl chloride did not induce an increase in the number of revertants in Salmonella typhimurium strains C3076, TA1537, D3052, TA1538, and TA98 in the absence of liver activating enzymes. It however induced an increase in the number of revertants in Salmonella typhimurium strains G46 (500 -1000 mcg/mL), TA1535 (100 -1000 mcg/mL), TA100 (1 -1000 mcg/mL) and E. coli strains WP2 (1.0 -300.0 mcg/mL) and WP2uvrA- (0.1- 50.0 mcg/mL) in the absence of liver activating enzymes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Variuos peer reviewed publications were reviewed to determine the mutagenic nature of Pyrrolidinoethyl chloride. The studies are as mentioned below:

Gene mutation toxicity study was performed by Probst et al ( Environmental Mutagenesis, 1981) to determine the mutagenic nature of Pyrrolidinoethyl chloride. The study was performed using the modified Ames assay using eight histidine auxotrophs of Salmonella typhimurium (G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, and TA98) and two tryptophan auxotraphs of Escherichia coli (WP2 and WP2 uvrA-) with and without S9 metabolic activation system. The inocula of ten bacterial tester strains were streaked across square agar plates containing a concentration gradient of test compound and mutagenicity was scored by noting the number of tester strains showing mutant colonies over a given concentration range. The test compound was incorporated into four gradient plates to give a tenfold concentration range per plate, thus providing a 10,000-fold concentration range for the test. Pyrrolidinoethyl chloride induced gene mutation in Salmonella typhimurium strains G46, TA1535, TA100 and E. coli strain WP2 uvrA without metabolic activation system at a minimum mutagenic concentration of 0.6 nanomoles/mL. It however failed to induce gene mutation in Salmonella typhimurium (C3076, TA1537, D3052, TA1538, and TA98) and Escherichia coli (WP2) in the presence and absence of S9 metabolic activation system.

Thompson et al (Environmental mutagenesis, 1981) performed in vitro gene toxicity study to determine the mutagenic nature of Pyrrolidinoethyl chloride using Salmonella typhimurium strains G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, and TA98 and E. coli strains WP2 and WP2uvrA-. Bacterial mutagenesis was performed by the gradient plate test. Pyrrolidinoethyl chloride was incorporated into the top layer of agar on four 10- X 10-cm square plates to give a tenfold concentration gradient per plate, thus providing a 10,000-fold concentration range. The highest compound concentration tested was 1,000 mcg/ml of agar ( Strain G46: 500-1000, TA1535: 100-1000, TA100: 1-1000, WP2: 0.1-300, WP2uvrA-: 0.1-50 mcg/mL). Since Pyrrolidinoethyl chloride is a direct acting alkylating agent, the assay was performed in the absence of liver activating system. The chemical was screened for its ability to produce an increase in revertants in any of the strains used for the study. Pyrrolidinoethyl chloride did not induce an increase in the number of revertants in Salmonella typhimurium strains C3076, TA1537, D3052, TA1538, and TA98 in the absence of liver activating enzymes. It however induced an increase in the number of revertants in Salmonella typhimurium strains G46 (500 -1000 mcg/mL), TA1535 (100 -1000 mcg/mL), TA100 (1 -1000 mcg/mL) and E. coli strains WP2 (1.0 -300.0 mcg/mL) and WP2uvrA- (0.1- 50.0 mcg/mL) in the absence of liver activating enzymes.

In the same study mentioned by Thompson et al, In vitro gene toxicity study was performed by Ames assay to determine the mutagenic nature of Pyrrolidinoethyl chloride in Salmonella typhimurium and E coli. The study was performed using Salmenella typhimurium strains TA1535 and TA100 and E. coli strains WP2 and WP2 uvrA-. The highest concentration for the study was 1800 µM /plate. The chemical was screened for its ability to produce an increase in revertants in any of the strains used for the study. Pyrrolidinoethyl chloride induced an increase in the number of revertants in Salmonella typhimurium strains TA1535, TA100 and E. coli strains WP2 and WP2uvrA- and hence is likely to be mutagenic in vitro.

Another study was also mentioned by Thomson et al, In vitro gene toxicity study was performed for Pyrrolidinoethyl chloride in mouse lymphoma cells using Mouse Lymphoma Cell Forward Mutation Assay. The assay procedure used was patterned as that mentioned by Clive et al and employed the modifications described by Amacher et al. Pyrrolidinoethyl chloride was evaluated for its potential to increase the rate of mutation at the thymidine kinase locus scored as trifluorothymidine (TFT) resistance. The test chemical was dissolved using sterile distilled water and used at a concentration from 0,11, 22, 44, 88, 176 nanomoles/mL. Pyrrolidinoethyl chloride induced an increase in the rate of mutation at the thymidine kinase locus when L5178Y Mouse Lymphoma Cell  were exposed to the test chemical and hence is positive for gene mutation in vitro.

Based on the available data for the target chemical, Pyrrolidinoethyl chloride (CAS no 7250 -67 -1) is not likely to exhibit gene mutation in vitro. Though positive effect has been shown by the test chemical as discussed in the some of the studies but the clarity on the use of exogeneous metabolic activation system is unknown. Due to lack of clarity of the studies, the positive effect is not taken into consideretion for classification. Hence, on the basis of the key study and its supporting data, the chemical is not likely to be classified as a gene mutant in vitro.

Justification for classification or non-classification

Based on the available data for the target chemical, Pyrrolidinoethyl chloride (CAS no 7250 -67 -1) is not likely to exhibit gene mutation in vitro. Hence the chemical is not likely to be classified as a gene mutant in vitro.