Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable publication that meets basic scientific principles, read-across.
Justification for type of information:
Bis (2-ethylhexyl) adipate (DEHA) is appropriate for read-across because it is considered as a similar substance, based on similar structure, toxicokinetic and toxicological profile useful for this interpolation (please see also read-across justification).

Data source

Reference Type:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
not specified
GLP compliance:
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) adipate
EC Number:
EC Name:
Bis(2-ethylhexyl) adipate
Cas Number:
Molecular formula:
bis(2-ethylhexyl) adipate
Details on test material:
Supplied from the National Toxicology Program Chemical Repository, Radian Corporation, Austin, TX 78766.


Target gene:
Thymidine kinase locus.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Tested for absence of mycoplasma.
Periodically "cleansed" against high spontaneous background: yes.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenates.
Test concentrations with justification for top dose:
312.5, 625, 1000, 1250, 1800, 2000, 2500, 2600, 3000, 4000, 4200, 5000 µg/ml.
Vehicle / solvent:
Solvents used: medium without serum, distilled water or DMSO.
Controlsopen allclose all
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Migrated to IUCLID6: positive control for activation assay.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Migrated to IUCLID6: positive control for nonactivation asay.
Details on test system and experimental conditions:
Experimental design:
Each experiment, other than the initial toxicity test, normally consisted of the
following groups: vehicle control, four cultures; positive control, two cultures; at
least five test compound concentrations, two cultures per concentration . With any
chemical, the first experiment was a toxicity test in which cell population expansion
was measured . Ten-fold differences in test compound concentrations were used in the
toxicity test, the highest being 5 mg/ml unless a much lower concentration was
indicated by the poor solubility of a compound.This test was followed by at least two
experiments in the absence of S9 mix. If no clear positive response was observed,
then two experiments were performed in the presence of induced S9 (RLI) . Sometimes, insignificant
responses with these two activation conditions were investigated by tests
involving uninduced S9 (RLN) . Test compound concentrations were primarily two-fold dilutions
from the highest testable concentration, as estimated from the toxicity test.

Mutation experiments:
Each exposed culture consisted of 6 X 106 cells in a final volume
of 10 ml F5p in a 30-ml screw-cap plastic tube (Sterilin Ltd .) . This tube was incubated
for 4 hr on a horizontal axis roller drum rotating at 10 rpm. At the end of the
incubation time, the cells were sedimented by centrifugation at 500 g .av . for 10 min,
washed, and finally resuspended in 20 ml F10p . These cell suspensions (3 X 105
cells/ml) were incubated for a 2-day expression period, the cell population density
being adjusted back to 20 ml of 3 X 105 cells/ml after 24 hr . After 48 hr, the cell
population densities were estimated and culture volumes containing 3 X 106 cells
adjusted to 15 ml with F10p, giving a cell population density of 2 X 105 cells/ml.

Cloning efficiency:
A 0.1-ml sample of the cell suspension was withdrawn
and diluted 1 :100. Three 0 .1-ml samples (200 cells) of the diluted cultures were
transferred to 30-ml tubes, mixed with 25-ml cloning medium (Fischer's medium
containing 20% heat-inactivated horse serum, i .e ., F20p) containing 0 .35% Noble
agar and poured into 90-mm Petri plates.
Mutant selection:
Three aliquots (each containing 106 cells) of the remaining
culture were distributed to 30-ml tubes, mixed with 20-ml cloning medium to give
final concentrations of 0.35% Noble agar and 3 µg trifluorothymidine/ml, then
poured into 90-mm Petri plates.
The agar was gelled at 4 ºC for 5-10 min, then the plates were
incubated for 11-14 days in 5% C02:95% air at 37ºC.
Colony counting:
Colonies were counted using an Artek 880 Automated
Colony Counter, with the colony size discriminator control in the "off" position .
Evaluation criteria:
According to OECD guideline 476.
Toxicity was expressed as either a reduction of cell population
growth in suspension during the expression period or a reduction in cloning
efficiency . A measure of the overall toxicity was the relative total growth (RTG),
which is defined as
RTG = (total suspension growth X cloning efficiency) in dosed culture
(total suspension growth X cloning efficiency) in control culture
Mutant fraction (MF) was calculated as follows :
MF = 200 X mutant clones per plate (usually a mean of 3)
total clones per plate (usually a mean of 3)
= mutants/106 clonable cells.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
Precipitation of di(2-ethylhexyl)adipate was observed at 1000 µg/ml, but testing was continued up 5000 µg/ml. In the absence of S9 mix there was no indication of a mutagenic response in two experiments. In the presence of S9 mix, one experiment similarly failed to show any mutagenic effect, while significant increases in mutant fraction occured in a second experiment. The LOED in this experiment was 2,000 µg/ml, higher than the precipitation concentration. SIgnificant toxicity was observed in all experiments. It was concluded that di(2-ethylhexyl) adipate was not mutagenic in the system.

Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Interpretation of results (migrated information):
Executive summary:

The read-across substance diethylhexyl adipate was negative in the mouse lymphoma test.