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EC number: 271-974-4
CAS number: 68647-86-9
Genetic toxicity in vitro
Ames tests (for coconut shell charcoal)
The study was performed to investigate the
potential of coconut shell charcoal to induce gene mutations according
to the plate incorporation test (experiment I) and the preincubation
test (experiment II) using the Salmonella typhimurium strains TA 1535,
TA 153, TA 98, TA 100 and TA 102.
The assay was performed in two independent
experiments both with and without liver microsomal activation. Each
concentration, including the controls, was tested in triplicate. The
test item was tested at the following concentrations:
Pre-Experiment /Experiment I: 3;
10; 33; 100; 333; 1000; 2500 and 5000 µg/plate
Experiment II: 33;
100; 333; 1000; 2500 and 5000 µg/plate
The plates incubated with the test item
showed normal background growth up to 5000 µg/plate with and without S9
mix in all strains used.
No toxic effects, evident as a reduction in
the number of revertants (below the indication factor of 0.5), occurred
in the test group with and without metabolic activation.
No substantial increase in revertant colony
numbers of any of the five tester strains was observed following
treatment with coconut shell charcoal at any dose level, neither in the
presence nor absence of metabolic activation (S9 mix). There was also no
tendency of higher mutation rates with increasing concentrations in the
range below the generally acknowledged border of biological relevance.
Appropriate reference mutagenes were used as
positive controls and showed a distinct increase of induced revertant
In conclusion, it can be stated that during
the described mutagenicity test and under the experimental conditions
reported, the test item did not induce gene mutations by base pair
changes or frameshifts in the genome of the strains used.
Therefore, coconut shell charcoal is
considered to be non-mutagenic in this Salmonella typhimurium reverse
Ames test (for Read-Across substance
The three tests were performed in the tester
strains TA 97a, TA 98, TA 100, TA 102, and TA 1535 using both the plate
incorporation and the pre-incubation method, both with and without
metabolic activation by rat S9 mix. All three tests were essentially
similar in design and were conducted according to OECD guideline no. 471
and EU method B.13/14.
When tested in amounts corresponding to
nominal test item concentrations of up to 5,000 µg/plate, none of the
extracts did induce an increased rate of revertants in any of the tester
strains, neither in the presence nor in the absence of metabolic
activation. No cytotoxic effects were observed at any of the tested
Concurrently performed negative and solvent
controls as well as appropriate positive controls gave the expected
Accordingly, charcoal was concluded to be
non-mutagenic in the tests.
In vitro gene mutation assay in mammalian
cells (for Read-Across substance charcoal)
In addition, the potential mutagenicity of
Probe 2 was studied in an in vitro mammalian cell gene mutation assay in
mouse lymphoma L5178Y TK+/-cells which was performed
according to OECD guideline no. and EU method B.17.
Based on the results of a preliminary
solubility and toxicity test, concentrations of the extract
corresponding to nominal test item concentrations of 128; 320; 800;
2,000 and 5,000 µg/mL were selected for the two main experiments, assay
1 and 2.
In assay 1, a 3-h treatment, both with and
without metabolic activation was utilised while in assay 2, a 3-h
treatment with metabolic activation and a 24-h treatment without
metabolic activation were used. The relative harmonised survival the
relative total growth of the cells, cell viability, and the potential
mutagenicity (5-trifluorothymidine resistance) were determined.
The extract of the test item was not
cytotoxic up to and including the highest concentration of 5,000 µg/mL.
The mutation frequencies observed in both
assays did not show dose-related tendencies, remained far below the
relevant thresholds for positive results, and were not statistically
significantly different from that of the vehicle control.
Both main assays fulfilled the validity
criteria. Concurrently performed positive and negative controls gave the
expected results thus demonstrating the correct functioning of the assay
In conclusion, the extract of Probe 2 did not
induce gene mutations in cultured mouse lymphoma L5178Y TK+/-cells
under the conditions of this study, neither in the presence nor in the
absence of metabolic activation.
Accordingly, the test item charcoal was
concluded to be non-mutagenic under the conditions of the present study.
In vitro chromosomal aberrations assay
(for Read-Across substance charcoal)
The potential genotoxicity of Probe 2 was
investigated in an in vitro chromosome aberration assay in V79 cells.
The assay was conducted according to OECD guideline no. 473 and EU
method B.10. Based on the results of a pre-test for solubility and
cytotoxicity test, concentrations of 1,250, 2,500, and 5,000μg/mL were
chosen for the main assay. The main assay consisted of two independent
experiments, experiment A (3 h treatment, harvest after 20 h, both with
and without metabolic activation) and experiment B (3 h treatment with
and 20 h treatment without metabolic activation, harvest after 28 h).
In both experiments A and B, the test item
extract did not induce an increase in the number of cells with
aberrations without gaps at any examined concentration, either in the
absence or in the presence of metabolic activation, up to and including
the maximum concentration.
There were no statistical differences between
treatment and concurrent solvent control groups, and no dose-response
relationships were noted.
No statistically significant differences
between treatment and concurrent negative (vehicle) control groups and
no dose-response relationships were noted.
An adequate degree of cytotoxicity was
observed at the highest test concentration in both experiments.
The observed chromosome aberration rates were
within the ranges of historical control data. There were no biologically
relevant increases in the rate of polyploid or endoreduplicated
metaphases in the two experiments, either in the presence or absence of
The validity of the test was shown using
ethyl methanesulphonate (0.4 and 1.0μL/mL) and N-nitrosodimethylamine
(1.0μL/mL) as positive controls.
In conclusion, the test item charcoal was
considered as non-genotoxic in this system.
Taken together, the results of the three Ames
test, the in vitro gene mutation assay in mammalian cells, and the in
vitro chromosomal aberrations assay clearly indicate that charcoal is
non-mutagenic and non-genotoxic.
Genetic toxicity in vivo
on the results obtained in the four tests, the gene mutation assay in
mammalian cells in vitro, and the in vitro chromosomal aberrations
assay, coconut shell charcoal is concluded to be non-genotoxic.
Accordingly, coconut shell charcoal is not classified as a germ cell
mutagen according to the current EU-CLP Regulation. However, results
from specific mutagenicity studies in germ cells are lacking.
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