Hexan-4-olide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

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

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

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%.

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

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)

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

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

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The highest concentration employed in the assay was 2 µ1/ml.
A stock solution of test substance was prepared by dissolving 300 µI in 1200 µI of DMSO to obtain a 200µl/ml solution. A further 1:10 dilution was obtained by mixing 1,2 ml of the test substance 200 µI/ml concentrated with 10,8 ml of F10 medium, obtaining a test solution 20 µI/ml (final conlcentration of DMSO in the assay < 1%).
This solution resulted to be well dissolved without any sign of precipitation and was used as the first working solution (C1). The final concentrationof 2 µI/ml is reached during the treatment by a further 1 :10 dilution.
Then, 7 dilutions (labeled from C2 to CB) were prepared with the constant dilution factor 1:2 in F10 medium, as follows:
C2 (10µlI/ml): 6 ml of C1 were diluted with 6 ml of F10 medium
C3 (5 µI/ml): 6 ml of C2 were diluted with 6 ml of F10 medium
C4 (2,5 µI/ml): 6 ml of C3 were diluted with 6 mt of F10 medium
CS (1,25 µI/ml): 6 ml of C4 were diluted with 6 ml of F10 medium
C6 (0,625 µI/ml): 6 ml of CS were diluted with 6 ml of F10 medium
C7 (0,313 µI/ml): 6 ml of C6 were diluted with 6 ml of F10 medium
C8 (0, 157 µI/ml): 6 ml of C7 were diluted with 6 ml of F10 medium
Final concentration of each solution is the 1: 10 dilution, due to the dilution that occurs during the test.

Vehicle / solvent:

Test substance was dissolved in DMSO according to the solubility properties of the test substance, keeping the final concentration of DMSO <= 1 %.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

The batch of cells that was employed in this study, batch n° 05/02/2018, was monitored for absence of mycoplasma, karyotype, population doubling time (PDT) and background before starting the study, applying the procedure described in SOPa-121 and in par. 9.4, 9.5 , 9.6 and 9.7.
Acceptability criteria for absence of mycoplasma, Karyotype, PDT and background resulted satisfied.
Cells were maintained at 0.1 x 106 cells/ml in F10 medium and grown in suspension in T75 flasks at 37°C and 5% CO2, under gentle agitation (60-100 rpm). Cells were counted and diluted every 2-3 days to maintain the exponential growth phase.

Evaluation criteria:

Acceptance criteria are summarized in 444-0 GxP data sheet.
The assay is considered valid if the following criteria are met:
The mean suspension growth (SG) of the vehicle control should fall in the range 8-32 following 4 h treatment or between 32 and 180 following 24 h treatment.
The mean mutant frequencies in the vehicle control cultures should fall within the normal ranges (50-170 mutants per 106 viable cells).
The mean cloning efficiency (CEviability) of the vehicle control from the Mutation Experiments should be within the range 65-120%.
At least one positive control should show either an absolute increase in total MF of at least 300 x10-6(note that the MF relative to small colonies should account for at least 40% of that MF), or an increase in small colony mutant frequency of at least 150 x 10-6 above the concurrent vehicle control.
The mean RTG for the positive controls (for at least one tested concentration), vehicle control and test substance in any treatment conditions should be >=0%.
Moreover, the MF for the negative (vehicle) and positive controls must be within the acceptable historical range of the laboratory, evaluated throw the control chart mod. 565/GxP. It was verified that all the acceptability criteria were met. See results reported at par.11.10.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

According to OECD 490: 2016 “tn vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene", negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.
According to OECD 490:2016 “ln vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene", the test substance "Gamma-Dodecalactone” batch n.050170905 is considered not able to induce gene mutations, under the test conditions applied.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: Nitrofurantoine; 4-Nitro-1,2-phenilendiammine

Details on test system and experimental conditions:

In phase A, Salmonella typhimurium TA100 was exposed to 5 different concentrations of test substance (C1-C5), prepared with a √10 dilution factor. As cytotoxicity may vary in the presence of metabolic activation systems, the test was carried out in presence and in absence of S9 (-S9; +S9). The highest concentration (C1) was 5 μl/plate, the highest allowed in the test. This solution resulted completely dissolved.
In phase B, the 5 bacteria strains were exposed to 5 different concentrations of test substance prepared with a √10 dilution factor, in presence and in absence of a metabolic activation system (-S9; +S9). The concentrations were selected according to the preliminary cytotoxicity test, including the first cytotoxic concentration: 0.16 μl/plate (C4) for testing in the absence of metabolic activation system (-S9) and 0.5 μl/plate (C3) for testing in the presence of metabolic activation system (+S9).

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available