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EC number: 232-077-3
CAS number: 7785-26-4
In a reverse gene mutation assay in
bacteria, performed according to OECD Guideline 471 and in compliance
with GLP, histidine-dependent auxotrophic mutants of Salmonella
typhimurium, strains TA1535, TA1537, TA98 and TA100, and a
tryptophan-dependent mutant of Escherichia coli, strain WP2uvrA
(pKM101), were exposed to (-)-alpha-pinene diluted in Acetone using both
the Ames plate incorporation and pre-incubation methods at up to eleven
dose levels, in triplicate, both with and without the addition of a rat
liver homogenate metabolizing system (10% liver S9 in standard
The dose range for Experiment 1 (plate
incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate.
The experiment was repeated on a separate day (pre-incubation method)
using fresh cultures of the bacterial strains and fresh test item
formulations which exhibited excessive toxicity employing the
pre-incubation method. Thereby, a repeat test was performed for all of
the strains dosed in the absence of S9-mix employing the toxic limit of
the test item as the maximum concentration.
First Test (Plate incorporation
method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and
Second Test (Pre-incubation method):
1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with S9-mix
Repeated Second Test (Pre-incubation
method): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate, without
The vehicle (acetone) control plates
gave counts of revertant colonies within the normal range. All of the
positive control chemicals used in the test induced marked increases in
the frequency of revertant colonies, both with and without metabolic
activation. Thus, the sensitivity of the assay and the efficacy of the
S9-mix were validated.
There was no visible reduction in the
growth of the bacterial background lawn at any dose level, either in the
presence or absence of metabolic activation (S9-mix), in the first
mutation test (plate incorporation method).
Results from the second mutation test
(employing the pre-incubation modification) showed that the test item
induced a toxic response as weakened bacterial background lawns and
substantial reductions in revertant colony frequency in the absence of
S9-mix from 15 μg/plate. In the presence of S9-mix, weakened bacterial
background lawns were initially noted from 150 μg/plate (TA1535), 500
μg/plate (TA100) and 1500 μg/plate (TA98 and TA1537). No toxicity was
noted to Escherichia coli strain WP2uvrA at any test item dose level in
the 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.
No test item precipitate was observed
on the plates at any of the doses tested in either the presence or
absence of metabolic activation (S9 -mix) in Experiments 1 and 2.
There were no significant increases in
the frequency of revertant colonies recorded for any of the bacterial
strains, with any dose of the test item, either with or without
metabolic activation (S9-mix) in Experiment 1 (plate incorporation
Similarly, no 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 (S9-mix) in Experiment 2 (pre-incubation
Therefore, (-)-alpha-pinene was
considered to be non-mutagenic under the conditions of this test.
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