C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-21 to 2012-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline conform Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

EC Number:

701-479-5

Molecular formula:

C42H82O7

IUPAC Name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Test material form:

other: viscous liquid

Details on test material:

Name: HOSTACERIN DGI
Batch No.: ESD0012304
CAS No.: 67938-21-0
Chemical Name: di(isooctadecanoic)acid, diester with oxydi(propanediol)
Active Components: 100%
Physical State at RT: viscous liquid
pH: 6-8 (20 °C, 5% solution in ethanol/water 1:1)
Colour: colourless to light yellow
Expiry Date: 09 June 2013
Purity: 100%
Storage Conditions: at room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/mL

Experiment I
with and without metabolic activation:
0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/mL

Experiment II
without metabolic activation:
0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/mL

and with metabolic activation:
25, 50, 100, 250, 500, 750, 1250, 2000, 3000 and 5000 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: The test item was suspended in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) and diluted prior to treatment. In higher concentrations oily droplets have been observed in the suspension.

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Migrated to IUCLID6: 300 µg/mL

Controls 2

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Migrated to IUCLID6: 1.0 and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no biologically relevant increase of mutants with and without S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I with S9: ≥ 2500 μg/mL; Experiment II without S9: 5000 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item HOSTACERIN DGI is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus),V79cells culturedin vitrowere exposed to HOSTACERIN DGIsuspended in cell culture medium (MEM)at concentrations of

- 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/mL (without metabolic activation, Experiment I)

- 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/mL (with metabolic activation, Experiment I)

- 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/mL (without metabolic activation, Experiment II)

- 25, 50, 100, 250, 500, 750, 1250, 2000, 3000 and 5000 µg/mL (with metabolic activation, Experiment II).

HOSTACERIN DGI was tested up to cytotoxic concentrations.

No biologically relevant growth inhibition was observed in experiment I without metabolic activation and in experiment II with metabolic activation.

A biologically relevant growth inhibition was observed in experiment I with metabolic activation and in experiment II without metabolic activation.

In experiment I without metabolic activation the relative growth was 115.5% for the highest concentration (5000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 68.4%. In experiment II without metabolic activation the relative growth was 54.7% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 83.6%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.88 was found at a concentration of 2500 µg/mL with a relative growth of 100.7%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.77 was found at a concentration of 2500 µg/mL with a relative growth of 65.3%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.20 was found at a concentration of 10 µg/mL with a relative growth of 98.5%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.14 was found at a concentration of 2000 µg/mL with a relative growth of 88.5%.

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation), No dose-response relationship was observed.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.