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EC number: 248-363-6
CAS number: 27247-96-7
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.
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