1-ethylpyrrolidin-2-ylmethylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June - 25 July 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study but no informations on GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487 dated 22 July 2010

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1-ethylpyrrolidin-2-ylmethylamine

EC Number:

247-466-3

EC Name:

1-ethylpyrrolidin-2-ylmethylamine

Cas Number:

26116-12-1

Molecular formula:

C7H16N2

IUPAC Name:

1-ethylpyrrolidin-2-ylmethylamine

Test material form:

solid - liquid: suspension

Details on test material:

Test article identification: PE0851 [(1-ethyl 2-pyrrolidinyl) methanamine]
Test article batch number: DS00001
Certificate of analysis: Purity: > 98%

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First test without metabolic activation: 10 to 1500 µg/mL
Second test without metabolic activation: 100 to 600 µg/mL
Test with metabolic activation: 10 to 1500 µg/mL

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide (DMSO)

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide (DMSO)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9-mix

Details on test system and experimental conditions:

L5178Y cells were treated with the test article in 96-well microtiter plates for 3 hours with metabolic activation and for 28 hours without metabolic activation. Without metabolic activation the cells were harvested directly after the treatment time and with metabolic activation 21 hours (recovery time) after the end of the treatment. Cells were then fixed and stained with Giemsa, then scored manually.
The cytotoxicity of the test article was evaluated by the population doubling (PD), expressed as a percentage of the negative control (relative population doubling): PD = (log (Cf /C0 )) / log 2
Cf = final cell count (24 hours after the start of treatment with metabolic activation or 28 hours without metabolic activation)
C0 = initial cell count
The ability of the test article to induce structural/numerical chromosome damage is evaluated by the increase in the number of micronucleated mononucleated cells, scored out of 1000 mononucleated cells.

Evaluation criteria:

Experiment acceptance criteria
The experiment is considered valid when all the following criteria are fulfilled:
• the population doubling of the negative control is higher or equal to 1.5 but not higher than 2.2;
• the number of micronucleated cells per 1000 cells in the concurrent solvent control is lower than or equal to the acceptable upper value for historical solvent control data (99% percentile of our historical negative control data):
- 7 for 28-hour treatment without S9-mix;
- 11 for 3-hour treatment with S9-mix.
• the positive controls induce a marked increase in the number of micronucleated cells;
• at least three analyzable concentrations are scored;
• the highest concentration evaluated corresponds either to 5000 µg/mL (or 10 mM), or to the solubility limit in the solvent, or to the lowest precipitating concentration in the culture medium or to 50% to 60% of cell cytotoxicity.

Test evaluation criteria
A test article is considered to induce a positive response in the in vitro micronucleus test if the two criteria listed below are fulfilled for at least one concentration:
• the test article induces a statistically significant increase in the number of micronucleated cells for a given concentration;
• for this concentration, this value is strictly above positive threshold value (99% percentile of our historical negative control data):
- 7 for 28-hour treatment without S9-mix;
- 11 for 3-hour treatment with S9-mix.
When none of the above criteria are fulfilled, the test article is considered negative.
Results which only partially satisfy one of the above positivity criteria are dealt on a case by case basis, taking into account the concentration relationship, the reproducibility and the biological relevance.

Statistics:

Statistical analysis is performed using the pairwise Fisher exact test with Bonferroni-Holm correction.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

A dose related increase (>7) in the number of micronucleated cells was observed as from 200 µg/mL with a statistical significance at 400 µg/mL.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The top concentration retained for scoring was 400 µg/mL (41% cytotoxicity) as the slides of higher concentrations were unreadable due to damaged cells (indicating severe cytotoxicity). Evaluated concentrations were 100, 200 and 400 µg/ml.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

A statistically significant increase (>7) in the number of micronucleated cells was observed at 500 µg/mL.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The top concentration retained for scoring was 500 µg/mL (35% cytotoxicity) as the slides of higher concentrations were unreadable due to damaged cells (indicating the severe cytotoxicity). Evaluated concentrations were 200, 400 and 500 µg/mL.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Test with metabolic activation: at the beginning of treatment, as the pH of the concurrent negative control was 7.5, the pH of the top dose should not be higher than 8.5 which was obtained at 1000 µg/mL. Evaluated concentrations were 100, 500 and 1000 µg/ml. The population doubling of the negative control was slightly higher than 2.2, but this was considered acceptable.

First test without metabolic activation: binucleated cells and vacuoles in cells were observed from 200 µg/mL.

Second test without metabolic activation: presence of vacuoles in cells as from 200 µg/mL.

Applicant's summary and conclusion

Conclusions:

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

Under the experimental conditions of this study, PE0851 [(1-ethyl 2-pyrrolidinyl) methanamine] was found positive in the exploratory in vitro micronucleus test in mouse lymphoma cells in the absence of metabolic activation and was found negative in the presence of metabolic activation.