(Z)-non-6-enal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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: BlueScreen HC Assay

GLP compliance:

no

Remarks:

The BlueScreen HC Assay is a fairly newly developed assay designed to provide predictive screening, and thus GLP standards are not yet applied to this assay.

Type of assay:

other: BlueScreen HC Assay

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-non-6-enal

EC Number:

218-900-9

EC Name:

(Z)-non-6-enal

Cas Number:

2277-19-2

Molecular formula:

C9H16O

IUPAC Name:

(Z)-non-6-enal

Test material form:

liquid

Method

Target gene:

GADD45a

Species / strain

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: cultured human lymphoblastoid TK6 cells
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors:
- Whether whole blood or separated lymphocytes were used:
- Whether blood from different donors were pooled or not:
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: 5% CO2, 95% humidity, 37 degrees C

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Aroclor-1254 induced rat liver S9 fraction mix
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium: 1%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

78.1, 156.3, 312.5, 625, 1250, 2500, 5000, 10000 uM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : at least duplicate
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Evaluation criteria:

A test substance was considered positive for cytotoxicity if cell density was reduced to less than 80%. A test substance was considered genotoxic without metabolic activation if the gene repair activity was increased 80% over vehicle controls, and genotoxic with metabolic activation if gene repair activity was increased 50% or more over vehicle controls.

Results and discussion

Test results

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The cell density was reduced to less than 80%, indicating cytotoxicity of the test substance.

Applicant's summary and conclusion

Conclusions:

The test substance was not genotoxic with or without metabolic activation.

Executive summary:

A BlueScreen HC Assay was used to determine the genotoxicity of the test substance. The test is performed on a strain of genetically modified human lymphoblastoid TK6 cells. The cells are modified for increased expression of GLuc when exposed to genotoxic chemicals. Coelenterazine substrate is added to the test media. The coelenterazine reacts with GLuc, which causes luminescence which can be detected to determine the amount of GLuc present, and thus the genotoxicity. The test was done both with and without metabolic activation with S9. The test substance was not genotoxic with or without metabolic activation. However, the test substance was cytotoxic.