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EC number: 242-582-0
CAS number: 18794-84-8
Three in-vitro genotoxicity studies are available for farnesene - Ames
Assay, chromosomal aberration study and a mouse lymphoma assay. All
three studies were carried out in accordance with OECD guidelines. None
of the studies showed any evidence of genotoxic activity. Hence
Farnesene is not considered to be genotoxic and does not need to be
classified according to the CLP Regulation (EC) N° (1272-2008).
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).
Additional information from genetic toxicity in vitro:
Three guideline genotoxicity studies are available for Farnesene.
Details are summarised as follows:
Ames study: In a screening study, 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 an 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 it is concluded that farnesene
was not mutagenic.
Lymphocyte Chromosomal Aberration Assay:
In a study conducted to OECD guideline 473, human lymphocytes were
exposed to farnesene dissolved in DMSO, at concentrations of 6.25, 12.5,
25, 31.25, 50 and 75 μg/mL in the presence of S-9 mix. Incubations in
the absence of S-9 mix were carried out at 3.125, 6.25, 12,5,31.25 and
50 μg/mL. Positive control materials tested (mitomycin C and
cyclophosphamide) gave acceptable responses, showing a valid test system
Positive controls (mitomycin C without S9,
cyclophosphamide with S9) induced the appropriate
response. Vehicle controls were also valid. Farnesene
was moderately toxic but did not induce any
statistically significant increases in the frequency
of cells with aberrations, using a dose range that
included dose level that approached the 50% mitotic
inhibition limit. It was concluded that farnesene was
not clastogenic to human lymphocytes in-vitro.
Mouse Lymphoma Assay
The study was conducted
using mouse lymphoma L5178Y cells and according to
OECD method 476. Two independent experiments were
performed. In Experiment 1, L5178Y
TK +/- 3.7.2c mouse lymphoma cells (heterozygous at
the thymidine kinase locus) were treated with
farnesene at eight dose levels, in duplicate, together
with vehicle (solvent) and positive controls using
4-hour exposure groups both in the absence and
presence of metabolic activation (2% S9 final
concentration). In Experiment 2, the
cells were treated with farnesene at eight dose levels
using a 4‑hour exposure group in the presence of
metabolic activation (2% S9 final concentration) and a
24‑hour exposure group in the absence of metabolic
activation. The dose range of test item was selected
following the results of a preliminary toxicity test
and for Experiment 1 was 0.25 to 12 µg/ml in the
absence of metabolic activation, and 1 to 64 µg/ml in
the presence of metabolic activation. In
Experiment 2 the dose range was 0.5 to 20 µg/ml in the
absence of metabolic activation, and 2 to 48 µg/ml in
the presence of metabolic activation.
The maximum dose levels
used in the assay were limited by test item-induced
toxicity. Precipitate of the test
item was not observed at any of the dose levels in the
Mutagenicity Test. The vehicle
(solvent) controls had mutant frequency values that
were considered acceptable for the L5178Y cell line at
the TK +/- locus. The positive
control items induced marked increases in the mutant
frequency indicating the satisfactory performance of
the test and of the activity of the metabolising
Farnesene did not induce
any toxicologically significant dose-related increases
in the mutant frequency at any dose level, either with
or without metabolic activation, in either the first
or second experiment. It was concluded that Farnesene
was not mutagenic to L5178Y cells under the conditions
of the test.
Justification for selection of genetic toxicity endpoint
One of 3, Klimisch 1, guideline studies carried out on Farnesene.
All showed negative results
Farnesene showed no evidence of genotoxicity in three
in-vitro assay systems and does not need to be
classified according to the CLP Regulation (EC)
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