Hexane-1,3,6-tricarbonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022.01.10-2022.07.06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Hexane-1,3,6-tricarbonitrile

EC Number:

217-199-7

EC Name:

Hexane-1,3,6-tricarbonitrile

Cas Number:

1772-25-4

Molecular formula:

C9H11N3

IUPAC Name:

hexane-1,3,6-tricarbonitrile

Method

Target gene:

mouse lymphoma thymidine kinase locus (tk+/-)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

An appropriate quantity of S9 fraction was thawed and mixed with a co-factor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. In 100 mM sodium phosphate buffer pH 7.4, the following concentrations were achieved: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP. During the experiment the S9 mix was stored on ice.

Test concentrations with justification for top dose:

Criteria to determine the highest concentration are cytotoxicity, solubility in the test system, and changes of pH or osmolality. Cytotoxicity was determined with and without metabolic activation.
The test item was investigated at the following concentrations:
STE(-): 1.25, 2.5, 5.0, 7.5, 10 mM
STE(+): 1.25, 2.5, 5.0, 7.5, 10 mM
Solvent and negative controls were tested in duplicate.

Vehicle / solvent:

DSMO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

other: ethylmethanesulfonate

Details on test system and experimental conditions:

accordant with the OECD TG 490

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Growth inhibition was observed in STE(-). The relative total growth (RTG) was 61% for highest concentration evaluated (10 mM) . No growth inhibition was observed in STE(+)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, in this in vitro Mammalian Cell Gene Mutation Assay (Thymidine Kinase Locus/tk+/-) in L5178Y Mouse Lymphoma Cells, under the experimental conditions reported, the test item Hexane-1,3,6-tricarbonitrile is considered to be non-mutagenic.

Executive summary:

The test item Hexane-1,3,6-tricarbonitrile was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus (tk^+/-) using the L5178Y cell line.

For the short-term experiments (STE), the test item was dissolved in DMSO and incubated with cells for 4 hours. The STEs were performed independently: with (STE⁽⁺⁾) and without (STE^(-)) metabolic activation.

The test item was investigated at the following concentrations:

STE^(-):    1.25, 2.5, 5.0, 7.5, 10 mM

STE⁽⁺⁾     1.25, 2.5, 5.0, 7.5, 10 mM

No precipitation of the dissolved test item was noted in any experiment.

Growth inhibition was observed in STE^(-). The relative total growth (RTG) was 61% for highest concentration evaluated (10 mM) (Table 3).

No growth inhibition was observed in STE⁽⁺⁾ (Table 6).

No biologically relevant increase of mutants was found in any experiments. The global evaluation factor (GEF) was not exceeded by the induced mutant frequency.

Ethyl methanesulfonate (EMS), Methyl methanesulfonate (MMS), and Benzo[a]pyrene (B[a]P) were used as positive controls and demonstrated distinct and biologically relevant responses in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potentially clastogenic effects.

In conclusion, in this in vitro Mammalian Cell Gene Mutation Assay (Thymidine Kinase Locus/tk^+/-) in L5178Y Mouse Lymphoma Cells, under the experimental conditions reported, the test item Hexane-1,3,6-tricarbonitrile is considered to be non-mutagenic.