Hexan-4-olide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Dihydro-5-octylfuran-2(3H)-one

EC Number:

218-971-6

EC Name:

Dihydro-5-octylfuran-2(3H)-one

Cas Number:

2305-05-7

Molecular formula:

C12H22O2

IUPAC Name:

Dihydro-5-octylfuran-2(3H)-one

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9, co-factor-supplemented post-mitocondrial fraction prepared from the livers of the rodents treated with enzyme-inducing agents (i.e. Aroclor 1254)

Test concentrations with justification for top dose:

C1 (0,930 µI/ml): 7,5 ml of 1,116 µI/ml were diluted with 1,5 ml of fresh culture medium
C2 (0,776 µI/ml): 7,5 ml of C1 were diluted with 1,5 ml of fresh culture medium
C3 (0,646 µI/ml): 7,5 ml of C2 were diluted with 1,5 ml of fresh culture medium
C4 {0,538 µI/ml): 7,5 ml of C3 were diluted with 1,5 ml of fresh culture medium
C5 (0,448 µI/ml): 7,5 ml of C4 were diluted with 1,5 ml of fresh culture medium
C6 (0,374 µI/ml): 7,5 ml of C5 were diluted with 1,5 ml of fresh culture medium
C7 (0,312 µI/ml): 7,5 ml of C6 were diluted with 1,5 ml of fresh culture medium
C8 (0,260 µI/ml): 7,5 ml of C7 were diluted with 1,5 ml of fresh culture medium

Vehicle / solvent:

fresh culture medium

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

other: Vinblastine

Details on test system and experimental conditions:

Cell line: L5178Y TK+/-
Supplier and cat n°: ATCC CRL-9518
Lymphocytes from peripheral blood
Culture conditions: 37C, 5%C02
’
Culture Medium: Fisher's medium enriched with 10% heat-inactivated Horse serum, 1 mM sodium piruvate, 0.1 % Pluronic F-127, 1 % penicilltn/streptomycin solution.
The cells are stored as frozen stocks in liquid nitrogen.
L5178Y TK+r- cells need to be at first purged to reduce the frequency of spontaneously occurring TK-/- cells.
Cell purging was performed prior to store cells as frozen stocks, applying the procedure described in SOPa-144
A new batch of cells L5178Y TK+'·-purged (NIP 97 batch n° 05/02/2018), stored in liquid nitrogen, was thawed 1 week prior to start with the assay.
Cells were propagated up to 25th passage after thawing (total passage number was 25).
The karyotype, population doubling time (PDT) of the cell batch employed in the assay was controlled together with monitoring mycoplasma absence, as described in SOPa-144. Only cells that respect acceptability criteria for absence of mycoplasma, karotype, PDT are used in the assay.
These acceptability criteria are:
Mycoplasma: absence
Karyotype: 40 chromosomes
PDT:10-12 hours

Evaluation criteria:

Collected data wilI be analyzed with Kaluza Flow Cytometry analysis Software ver. 1.5. The composite of the software saved as “COMPOSITE Sytox EMA MN" will be used.
The composite is a set of histogram and bivariate plots used to discriminate MN from apoptotic chromatin and other spurious events.

Statistics:

Statistical analysis of results will be performed by using mod 399/GxP or statistical analysis software GraphPad.
For all tests statistical significance will be 5%.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In the present study, the test substance “Gamma-Dodecalactone'' batch n° 050170905 is considered as NEGATIVE (i.e. it is unable to induce chromosome breaks and/or gain or loss in the test system employed)