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EC number: 946-788-8
CAS number: -
The test item was examined for mutagenic
activity by assaying for the induction of 5-trifluorothymidine resistant
mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the
absence and presence of S9 metabolic activation, using a fluctuation
method according to the OECD Guideline 490 (2016).
A preliminary solubility trial indicated
that the maximum practicable concentration of the test item in the final
treatment medium was 3400 μg/mL using Minimal Medium A as solvent. On
the basis of this result, a cytotoxicity assay was performed, both in
the absence and presence of S9 metabolic activation, using 9 dose levels
up to 3460 μg/mL.
Based on the results obtained in the
preliminary trial, the first mutation assay was performed using the
following dose levels:
(−S9, 3 h treatment): 830, 692, 576, 480
and 400 µg/mL;
(+S9, 3 h treatment): 865, 721, 601, 501,
417 and 348 µg/mL.
In the absence of S9 metabolic activation,
severe toxicity, reducing Relative Total Growth (RTG) below 10%, was
observed at all concentrations tested, thus the evaluation of mutagenic
effects could not be performed. In the presence of S9 metabolism, severe
toxicity was noted at the three highest concentrations tested, while
marked to moderate toxicity was observed over the three remaining dose
levels, where dose related increases in mutation frequency were noted.
At the highest analysable concentration (501 μg/mL) the RTG was reduced
to 13 % and the observed increase was higher than the Global Evaluation
Factor (GEF). Based on the results obtained, in order to evaluate a
potential mutagenic effect in a sufficient number of concentrations at
adequate levels of cytotoxicity, a second mutation assay was performed,
both in the absence and presence of S9 metabolism, using the short
treatment time and the following lower range of concentrations:
(−S9, 3 h treatment): 346, 288, 240,
200,167, 139 and 116 µg/mL;
(+S9, 3 h treatment): 498, 415, 346, 288,
240 and 200 µg/mL.
Adequate levels of cytotoxicity, covering
a range from the maximum to slight or no toxicity, were observed in both
treatment series. In the presence of S9 metabolism, the induced mutation
frequency was lower than the Global Evaluation Factor (GEF) at all
concentrations tested. However, a statistically significant dose effect
relationship was observed and mutation frequency at the highest
concentration of 498 μg/mL fell out the distribution of the historical
negative control data (95% control limits). In the absence of S9
metabolism, a linear trend was indicated and at the highest dose level,
the observed increase was higher than the GEF and thus was considered a
clear evidence of positive result.
Appropriate negative and positive control
treatments were included in each mutation experiment. The mutant
frequencies in the solvent control cultures fell within the normal
range. Marked increases were obtained with the positive control
treatments indicating the correct functioning of the assay system.
It is concluded that the test item induces
mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the
absence or presence of S9 metabolic activation, under the reported
The test item was assayed for the ability
to induce micronuclei in human lymphocytes, following in vitro
treatment, according to the OECD guideline 487 (2016).
Since the test item gave positive results
in a mouse lymphoma mutation assay without any difference in the absence
and presence of S9 metabolic activation, a direct genotoxic mechanism
This study was intended as a further
investigation to evaluate cytogenetic effects and treatments were
carried out only in the absence of S9 metabolism.
Two treatment series were performed. A
short termtreatment, where the cells were treated for 3 hours and the
harvest time of approximately 32 hours, corresponding to approximately
two cell cycle lengths was used and a long term (continuous), treatment
where the cells were treated until harvest at 31 hours.
Solutions of the test item were prepared
in complete culture medium.
On the basis of the solubility of the test
item and cytotoxicity results obtained in the MLA test, the maximum dose
level of 1250 µg/mL expressed as organic content or 1730 µg/mL expressed
as test item as received, was selected for treatment. Lower dose levels
of 826, 556, 371, 247, 165, 110, 73.2 and 48.8 µg/mL expressed as
organic content or 1140, 769, 513, 342, 228, 152, 101 and 67.5 µg/mL
expressed as test item as received, were chosen for
the short termtreatment. For the
continuous treatment, the additional dose level of 32.5 µg/mL expressed
as organic content or 45.0 µg/mL expressed as test item as received was
The experiment included appropriate
negative and positive controls. Two replicate cell cultures were
prepared at each test point.
The actin polymerisation inhibitor
Cytochalasin B was added prior to the targeted mitosis to allow the
selective analysis of micronucleus frequency in binucleated cells. The
proliferation index CBPI was calculated in
order to evaluate cytotoxicity.
Dose levels for the scoring of micronuclei
were selected with the aim to evaluate the test item concentrations at
adequate levels of cytotoxicity, covering a range from the maximum
(55 ± 5%) to slight or no toxicity. In the
absence of cytotoxicity, the maximum dose level was selected as the
highest dose level for scoring.
Based on the results obtained, the
following concentrations were selected for the scoring of micronuclei:
- short treatment: 1250, 826 and 556 µg/mL
(organic content) with Cytotoxicity 18, 7 and 11 % respectively;
- continuous treatment: 556, 371 and 247
µg/mL (organic content) with Cytotoxicity 47, 18 and 15 % respectively.
One thousand binucleated cells per culture
were scored to assess the frequency of micronucleated cells. Adequate
cell proliferation was observed in negative control cultures and the
appropriate number of doses and cells was analysed. Statistically
significant increases in the incidence of micronucleated cells were
observed following treatments with the positive controlsMitomycin-C and
Colchicine, indicating the correct functioning of the test system. The
study was accepted as valid.
Following treatment with the test item, no
statistically significant increase in the incidence of micronucleated
cells over the concurrent solvent control value was observed at any dose
level, in any treatment series. All incidences were within the
distribution of historical negative control (95% control limits) with
the exception of the result obtained at the intermediate dose level
(short treatment) and high dose level (continuous treatment). These
values slightly exceeded the upper confidence limit, but fell within the
historical control range and no concentration related increase was seen.
Hence, this result was considered without any biological relevance.
It is concluded that the test item does
not induce micronuclei in human lymphocytes after in vitro treatment,
under the reported experimental conditions.
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