Reaction mass of 5,8,11,14-Tetraoxaoctadecane and 5,8,11,14,17-Pentaoxaheneicosane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2011-08-10 to 2011-12-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Scientifically well performed study; Guideline study; GLP study Rational: the components of the registration substance and the read-across supporting substance form "chain length category", whereby the number of unit CH2CH2O varies in the center of the moluecule; n = 2 for the read-across supporting substance and n = 3, 4 for the components of the registration substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

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:

Bis(2-butoxyethyl) ether

EC Number:

204-001-9

EC Name:

Bis(2-butoxyethyl) ether

Cas Number:

112-73-2

Molecular formula:

C12H26O3

IUPAC Name:

1,1'-[oxybis(ethane-2,1-diyloxy)]dibutane

Constituent 2

Reference substance name:

DEGDBE

IUPAC Name:

DEGDBE

Details on test material:

Name: Diethyleneglycoldibutylether
CAS No.: 112-73-2
Chemical Name: Bis(2-butoxyethyl)ether
Molecular Weight: 218.33
Active Components: 100%
Physical State: liquid
pH: 6.5-7.5 (25°C, 100 g/L i-propanol/water 4:1)
Colour: colourless
Storage Conditions: at room temperature

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):
3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 10.0 mM

Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mM

Experiment I
without metabolic activation:
0.05, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0 and 3.5 mM
and with metabolic activation:
0.5, 1.0, 2.0, 2.25, 2.5, 2.75, 3.0 and 3.5 mM

Experiment II
without metabolic activation:
0.2, 0.5, 1.0, 2.0, 2.15, 2.30, 2.45 and 2.6 mM
and with metabolic activation:
0.03, 0.07, 0.15, 0.3, 0.7, 1.4, 1.8, 2.1 and 2.4 mM

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: Ethylmethanesulfonate (300µg/mL), with metabolic activation: 7,12-Dimethylbenz(a)anthracene (1 and 1.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved 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; cloning efficiency

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

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

yes (Experiment I without S9: ≥ 3.0 mM; experiment I with S9: ≥ 2.75 mM; Experiment II without S9: ≥ 2.00 mM; Experiment II with S9:≥ 2.10 mM

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In a mammalian cell gene mutation assay (HPRT locus),V79 cells cultured in vitro were exposed to diethylene glycol dibutyl ether (DEGDBE) dissolved in MEM (+ 0% FBS (4h treatment) + 10% FBS (20h treatment)) at concentrations of

- 0.05, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0 and 3.5 mM (without metabolic activation, Experiment I)

- 0.5, 1.0, 2.0, 2.25, 2.5, 2.75, 3.0 and 3.5 mM (with metabolic activation, Experiment I)

- 0.2, 0.5, 1.0, 2.0, 2.15, 2.30, 2.45 and 2.6 mM (without metabolic activation, Experiment II)

- 0.03, 0.07, 0.15, 0.3, 0.7, 1.4, 1.8, 2.1 and 2.4 mM (with metabolic activation, Experiment II).

DEGDBE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I the highest biologically relevant concentration evaluated without and with metabolic activation was 3.5 mM with a relative growth of 16.5% (without metabolic activation) and 9.0% (with metabolic activation).In experiment II without metabolic activation the relative growth was 12.2% for the highest concentration (2.6 mM) evaluated. The highest concentrations evaluated with metabolic activation were 2.1 and 2.4 mM with a relative growth of 42.8% and 6.7%, respectively.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 2.62 was found at a concentration of 0.05 mM with a relative growth of 101.2%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.52 was found at a concentration of 3.0 mM with a relative growth of 45.7%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative controls values) of 1.30 was found at a concentration of 2.15 mM with a relative growth of 52.7%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative controls values) of 2.95 was found at a concentration of 2.4 mM with a relative growth of 6.7%. At this concentration an increased number of mutant colonies (70.12 mutants per 106cells) was observed, exceeding the historical data range. Due to the fact that this concentration was accompanied with a strong cytotoxic effect, the observed increased number of mutants/106cells is considered to be not biologically relevant..

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 forin vitromutagenicity (mammalian forward gene mutation) data

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The mutagenicity property of the registration substance is derived based on the read-across from diethyleneglycoldibutylether (DEGDBE). DEGDBE was investigated for its mutagenicity in bacteria according to the OECD Guideline 471 (Ames test). DEGDBE was not mutagenic with and without metabolic activation in two independent experiments. No mutagenicitiy could be concluded for DEGDBE. Likewise no mutagenicity in bacteria can be derived for the registration substance.

Executive summary:

The mutagenicity property of the registration substance is derived based on the read-across from diethyleneglycoldibutylether (DEGDBE). DEGDBE was investigated for its mutagenicity potential in HPRT. DEGDBE was not mutagenic with and without metabolic activation in two independent experiments. No mutagenicitiy could be concluded for DEGDBE. Likewise no mutagenicity in mammalian cells can be derived for the registration substance.