Pentanamide, N-(1-cyanocyclopentyl)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: Detection of clastogenicity and aneugenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 November 2012 to 07 December 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A well documented study according to international guideline but not GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 487 (In vitro Mammalian cells micronucleus test)

Deviations:

no

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N-(1-cyanocyclopentyl)pentanamide

EC Number:

928-489-4

Cas Number:

194984-24-2

Molecular formula:

C11H18N2O

IUPAC Name:

N-(1-cyanocyclopentyl)pentanamide

Constituent 2

Reference substance name:

1-(pentanoylamino)cyclopentanenitrile

IUPAC Name:

1-(pentanoylamino)cyclopentanenitrile

Details on test material:

Test article identification: Valeroil-aminonitrile
Test article batch number: 2U291
Certificate of analysis: Dated 13 September 2012
Conversion factor: 1.082 (calculated taking into account the purity of 92.4%)
Valeroil-aminonitrile was dissolved in Negative control article (DMSO) and diluted in the culture medium (final solvent concentration in the culture medium: 1%). The maximum soluble concentration in the solvent was approximately 500 mg/mL, but the test article precipitated in the culture medium above 250 mg/mL. This concentration was chosen as the top concentration, corresponding to 2500 μg/mL in the culture medium.

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from the liver of Aroclor1254-treated rats (final percentage of S9-mix in the culture medium:1.5%)

Test concentrations with justification for top dose:

From 1 to 2500 µg/ml

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin C (MITC) was used for positive control without metabolic activation and cyclophosphamide (CP) for positive control with metabolic activation.

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;
- 10 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.
In the present study, all acceptance criteria were met.

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;
- 10 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:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Precipitate from 2000 µg/ml. The top concentration retained for scoring 500 μg/mL (56% of cytotoxicity) corresponded to the required cytotoxicity (50 % to 60 %).

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Precipitate from 750 µg/ml. The top concentration retained for scoring 250 μg/mL (51 % of cytotoxicity) corresponded to the required cytotoxicity (50 % to 60 %).

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, Valeroil-aminonitrile was found negative in the exploratory in vitro micronucleus test in mouse lymphoma cells in the presence or absence of metabolic activation.