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EC number: 248-363-6
CAS number: 27247-96-7
Genotoxic potential of the registered substance has been excluded in a
standard test battery: Ames test (OECD Guideline 471), chromosomal
aberration assay (OECD Guideline 473) and mouse lymphoma assay (OECD
Guideline 476). This was further confirmed in a mammalian cell
transformation assay which was non-guideline and considered to be
the test item did not induce a reduction in the bacterial
background lawns, substantial decreases in TA100 revertant
colony frequency were noted from 500 µg/plate (absence of
S9-mix) and at 5000 µg/plate (presence of S9-mix). No
toxicity was noted to WP2uvrA. The
test item formulation and S9‑mix used in this experiment
were both shown to be sterile.
1: Spontaneous Mutation Rates (Concurrent Negative Control)
Number of revertants (mean number of colonies per plate)
Base-pair substitution type
TABLES OF RESULTS FOR MUTATION TEST: Please see attached
in overall remarks, attachments
B N, McCann J and Yamasaki E (1975), Methods for detecting
carcinogens and mutagens with the Salmonella/mammalian
microsome mutagenicity test, Mutation Research, 31,
D M and Ames B N (1983), Revised Methods for the Salmonella
mutagenicity test, Mutation Research, 113, 173 -
K and Zeiger E (2000), The Ames Salmonella/microsome
mutagenicity assay, Mutation Research, 455, 29-60.
M H L and Muriel W J (1976), Mutagen Testing Using TRP+ Reversion
in Escherichia coli, Mutation Research, 38, 3-
Serres F J and Shelby M D (1979), Recommendations on data
production and analysis using theSalmonella/microsome
mutagenicity assay,Environmental Mutagenesis, 1,
test method was designed to be compatible with the guidelines for
bacterial mutagenicity testing published by the major Japanese
Regulatory Authorities including METI, MHLW and MAFF, the OECD
Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation
Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30
May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.
TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA
were treated with the test item, 2-ethyl-hexyl nitrate,
using both the Ames plate incorporation and pre-incubation methods at
eight dose levels, in triplicate, both with and without the addition of
a rat liver homogenate metabolising system (10% liver S9 in standard
dose range for the first experiment was 1.5 to 5000 µg/plate. The
experiment was repeated on a separate day (pre-incubation method) using
the same dose range, fresh cultures of the bacterial strains and fresh
test item formulations.
dose levels and an expanded dose range were selected in both experiments
in order to achieve four non-toxic dose levels and the toxic limit of
the test item.
vehicle (DMSO) control plates gave counts of revertant colonies within
the normal range. All
of the positive controls used in the test induced marked increases in
the frequency of revertant colonies, both with or without metabolic
the sensitivity of the assay and the efficacy of the S9-mix were
test item caused a visible reduction in the growth of the bacterial
background lawns and/or a substantial reduction in the frequency of
revertant colonies of all of the Salmonella tester strains
(except TA98 dosed in the presence of S9-mix), initially from 50
toxicity was noted to Escherichia coli strain WP2uvrAat
any test item dose level in either the absence or presence of S9-mix. The
sensitivity of the bacterial tester strains to the toxicity of the test
item varied slightly between strain type, exposures with or without
S9‑mix and experimental methodology. These
results were not indicative of toxicity sufficiently severe enough to
prevent the test item being tested up to the maximum recommended dose
level of 5000 µg/plate.
significant increases in the frequency of revertant colonies were
recorded for any of the bacterial strains, with any dose of the test
item, either with or without metabolic activation or exposure method.
test item,2 -ethyl-hexyl nitrate,
was considered to be non-mutagenic under the conditions of this test.
All vehicle controls had frequencies of cells with aberrations within
the range accepted for normal human lymphocytes.
All the positive control materials induces statistically significant
increases in the frequency of cells with aberrations indicating the
satisfactory performance of the test and of the activity of the
The objective of this study was to
evaluate the potential of the test item 2-Ethylhexyl nitrate to
induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse
The study was performed according to
international guidelines (OECD No. 476 and Commission Directive B 17)
and in compliance with the principles of Good Laboratory Practice.
After a preliminary toxicity test,
2-Ethylhexyl nitrate was tested in two independent experiments, with and
without a metabolic activation system
(S9 mix) prepared from a liver
microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.
Cultures of 20 mL at 5 x 105cells/mL
(3-hour treatment) or cultures of 50 mL at 2 x 105cells/mL
(24-hour treatment) were exposed to the test or control items, in the
presence or absence of S9 mix (final concentration of S9 fraction 2%).
During the treatment period, the cells were maintained as suspension
culture in RPMI 1640 culture medium supplemented by heat inactivated
horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a, 5%
CO2humidified incubator. For the 24-hour treatment, flasks
were gently shaken at least once.
Cytotoxicity wasmeasured by assessment
of adjusted relative total growth (Adj. RTG), adjusted relative
suspension growth (Adj. RSG) andcloning efficiency following the
expression time (CE2).
The number of mutant clones
(differentiating small and large colonies) was evaluated after
expression of the mutant phenotype.
The test item was diluted in
The dose-levels for the positive
controls were as follows:
S9 mix: methylmethane sulfonate (MMS), used at a final concentration of
25 µg/mL, (3-hour treatment) or 5 µg/mL (24-hour treatment),
S9 mix: cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.
In this study, the cloning
efficiencies (CE2), the suspension growth and the mutation
frequencies of the vehicle controls as well as the mutation frequencies
of the positive controls were as specified in the acceptance criteria.
The study was therefore considered to be valid.
In the culture medium, at the
dose-level ofno precipitate was observed, and the pH and osmolarity
values were equivalent to those of the vehicle control culture.
Therefore,was selected as the highest dose-level to be tested in the
preliminary toxicity test.
Since the test item was toxic in the
preliminary test, the choice of the highest dose-level for the main test
was based on the level of toxicity, according to the criteria specified
in the international guidelines (decrease in Adj. RTG).
Experiments without S9 mix
Using a treatment volume of 0.5%, the
selected dose-levels were as follows:
0.078, 0.156, 0.313, 0.625 andfor the first experiment (3-hour
0.039, 0.078, 0.156, 0.313,for the second experiment (24‑hour treatment).
No precipitate was noted in culture
medium at the end of the treatment periods (3- or 24-hour treatments).
Following the 3-hour treatment, a
severe toxicity was induced at dose-levels ≥ 0.313 mM, as shown by a
89-96% decrease in Adj. RTG.
Following the 24-hour treatment, a
moderate to severe toxicity was induced at dose‑levels ≥ 0.156 mM, as
shown by a 44-100% decrease in
Following the 3-hour or 24-hour
treatments, no noteworthy increase in the mutation frequency was noted
in comparison to the vehicle control.
Experiments with S9 mix
0.313, 0.625, 1.25, 2.5 andfor the first experiment,
0.156, 0.313, 0.625, 1.25 andfor the second experiment.
In the first experiment, a severe
toxicity was induced at dose-levels ≥ 0.625 mM, as shown by a 82-100%
decrease in Adj. RTG.
In the second experiment, a marked to
severe toxicity was induced at dose-levels ≥ 0.625 mM, as shown by a
65-87% decrease in Adj. RTG.
Nonoteworthy increase in the mutation
frequencywas observed in either of the experiments.
The test item, 2-Ethylhexyl nitrate,
did not show any mutagenic activity in the mouse lymphoma assay, in the
presence or in the absence of a rat metabolizing system.
on results from the available studies, 2 -EHN does not require
classification for genetic toxicity according to Regulation (EC) No
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