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EC number: 242-582-0
CAS number: 18794-84-8
See "Attached background material" section below
Method Synopsis: Prior to the cytotoxic
screen, solubility of the test article was checked in Dimethyl sulfoxide
(DMSO). The test article was freely soluble at a concentration of 50
μl/ml in DMSO. Therefore, DMSO was chosen as the vehicle in the main
assay. A cytotoxicity screen was conducted in the TA100
tester strain using eight concentrations
(0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 μl) of the test article,
two plates per dose, on the bacterial tester strain Salmonella
typhimurium (S. typh.)TA100. The test article was combined with the
bacteria and top agar in the presence and absence of a metabolic
activation buffer containing S9 and overlaid onto minimal glucose agar
plates. A DMSO vehicle control was run concurrently, with S9.
Based on the cytotoxicity results, five
concentrations (0.05, 0.16, 0.5, 1.6 and 5 μl/plate, 2 plates per dose)
of the test article were tested in each of five bacterial tester strains
(Escherichia coli (E. coli) WP2 uvrA, and S. typhimurium strains TA97a,
TA1535, TA98, and TA100). Vehicle controls and positive controls
specific to each bacterial strain were treated in the same manner as the
test article concentrations. The plates were incubated at 37ºC ±1ºC for
48-72 hours. Revertant colony growth was determined by counting the
colonies per plate using an AlphaImager® imaging system. The number of
revertants of the test article treatment plates and positive control
plates was divided by the number of revertants of the vehicle plates. In
a positive result is determined by a 2-fold
increase above the vehicle control.
Due to negative results in the main assay
and cytotoxicity found in the entire dose range in strains TA97a, and
TA100, an independent repeat assay was conducted in all tester strains.
Test article concentrations of 0.05, 0.16, 0.5, 1.6 and 5 μl/plate for
strains WP2, TA98, TA1535 (all both with and without S9), and TA97A and
TA100 (both without S9 only). Concentrations of 0.005, 0.016, 0.05,
0.16, 0.5 μl/plate were used for strains TA97A and TA100 (both with S9
Results summary: In the screen, bacterial
background lawn checks and colony counts indicated that the test article
at 5 and 1 μl/plate was slightly cytotoxic to TA100. In the main assay,
slight cytotoxicity of the test article, based on the bacterial
background lawn checks, was observed in strains TA97a (1.6 μl/plate with
S9 and 5 μl/plate without S9), TA100 (5 μl/plate with and without S9),
and TA 1535 (5 μl/plate with S9 only). No positive response or
dose-related increased response of the test article in any strain was
found in the assay.
However, a dose-related decrease of
revertant frequencies found in most concentrations in strains TA97a and
TA100, with S9, raised a concern that these concentrations were
cytotoxic. In the independent repeat assay, no obvious cytotoxicity,
based on the bacterial background lawn check, was observed in any of the
strains. As in the main assay, no positive response or
concentration-related increase response in any strain was found.
Conclusion: Under test conditions for both
main and independent repeat assays, the test article, trans-ß-farnesene,
CAS#18794-84-8, was negative for mutagenicity in the Bacterial Reverse
Mutation Assay (Ames Assay).
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