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EC number: 261-332-1
CAS number: 58567-11-6
Genetic toxicity in vitro:
Ames test (OECD 471): negative (± S9 mix)
MNT test (OECD 487): negative (± S9 mix)
CA test in Human Lymphocytes (OECD 473): negative (± S9 mix)
HPRT test (OECD 476): negative (± S9 mix)
Table2: Summary of results of the in vitro micronucleus
test in human lymphocytes with AMBERWOOD
Exposure period 4 hrs without S9 mix
Exposure period 20 hrs without S9 mix
* For the positive control groups and the test
item treatment groups the values are related to the solvent controls
** The number of micronucleated cells was
determined in a sample of 2000 binucleated cells
separation occurred at the end of treatment
number of micronucleated cells is statistically significantly higher
than corresponding control values
1 Ethanol 0.5
2 MMC 1.0
3 Demecolcin 150.0
Table 2, cont.: Summary of results of the in
vitro micronucleus test in human lymphocytes with AMBERWOOD
Exposure period 4 hrs with S9 mix
1 Ethanol 0.5
2 CPA 12.5
3 CPA 20.0
The test item, dissolved in ethanol, was assessed for its
potential to induce micronuclei in human lymphocytes in vitro in the
absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the
exposure period was 4 hours with and without S9 mix. In Experiment II,
the exposure periods were 4 hours with S9 mix and 20 hours without
S9 mix. The cells were prepared 40 hours after start of treatment with
the test item.
In each experimental group two parallel cultures were analysed.
1000 binucleate cells per culture were scored for cytogenetic damage on
coded slides. To determine a cytotoxic effect the CBPI was determined in
500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 2500.0 µg/mL
(approx. 10 mM) was chosen with regard to the molecular weight and the
purity (97.7 %) of the test item and with respect to the OECD Guideline
487 for the in vitro mammalian cell micronucleus test.
No precipitation of the test item in the culture medium was
observed. No relevant influence on pH value was observed. Osmolarity was
decreased in Experiment I. Phase separation was observed in Experiment I
at 87.0 µg/mL and above in the absence of S9 mix and at 28.4 µg/mL and
above in the presence of S9 mix. In Experiment II phase separation was
observed at 266.6 µg/mL in the absence of S9 mix and at 28.4 µg/mL and
above in the presence of S9 mix.
In Experiment I and II in the absence of S9
mix and in Experiment II in the presence of S9 mix cytotoxicity was
observed at the highest evaluated concentration (59.4, 77.9 and 54.5 %
cytostasis, respectively). In Experiment I in the presence
of S9 mix no clear cytotoxicity was observed up to the highest applied
In both experiments, in the absence and presence of S9 mix, no
biologically relevant increase in the number of cells carrying
micronuclei was observed. The micronucleus rates of the cells after
treatment with the test item (0.25 –0.95 % micronucleated cells) were
close to the range of the solvent control values (0.25 –0.60 %
micronucleated cells) and within the range of the laboratory historical
In both experiments, either Demecolcin (150.0 ng/mL), MMC
(1.0 µg/mL) or CPA (12.5 or 20.0 µg/mL) were used as positive controls
and showed distinct increases in cells with micronuclei.
In conclusion, it can be stated that under the experimental
conditions reported, the test item did not induce micronuclei in human
lymphocytes in vitro when tested up to cytotoxic or the highest
Reduced background growth was observed in strain TA100 without S9
mix from 333 – 5000 µg/plate in experiment I. No reduction of the
background growth was observed in the remaining strains and in
Toxic effects, evident as a reduction in the number of revertants
(below the indication factor of 0.5), occurred in the test groups at the
following concentrations (µg/plate):
without S9 mix
with S9 mix
333 – 5000
1000 – 5000
2500 – 5000
/ no toxic effects observed
This study was performed to investigate the potential of the substance
to induce gene mutations in the plate incorporation test (experiment I)
and the pre-incubation test (experiment II) using the Salmonella
typhimurium strains TA 1535, TA 1537, TA 98, and
TA 100, and the Escherichia coli strain WP2 uvrA.
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
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000
TA 1535, TA 98, WP2 uvrA: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, TA 1537, TA 100: 1; 3; 10; 33; 100; 333; 1000; 2500; and
Reduced background growth was observed in strain TA 100 without S9 mix
from 333 - 5000 µg/plate in experiment I. No reduction of the background
growth was observed in the remaining strains and in experiment II.
Toxic effects, evident as a reduction in the number of revertants (below
the indication factor of 0.5), were observed in strains TA 1537 and TA
100 with and without S9 mix in experiment I. In experiment II, toxic
effects were observed in strains TA 1535, TA 1537, and TA 100 with S9
No substantial increase in revertant colony numbers of any of the five
tester strains was observed following treatment with the substance 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 mutagens were used as positive controls and showed
a distinct increase of induced revertant colonies.
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 frame shifts in the
genome of the strains used.
Therefore, the substance is considered to be non-mutagenic in this
Salmonella typhimurium and Escherichia coli reverse mutation assay.
The study was performed to investigate the potential of the substance to
induce gene mutations at the HPRT locus in V79 cells of the Chinese
hamster. The test methods described are designed to be compatible with
the OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian
Cell Gene Mutation Tests" and Method B17 of Commission Regulation (EC)
No 440/2008 of 30 May 2008. The study is comprised of a pre-experiment
and two independent main experiments. In the pre-experiment the cell
cultures were treated with the test item for 4 hours with metabolic
activation and for 4 and 24 hours without metabolic activation. In the
first experiment the treatment period was 4 hours with and without
metabolic activation. The second experiment was performed with a
treatment time of 4 hours with and 24 hours without metabolic
activation. The maximum concentration of 2500 µg/mL used in the
pre-experiment was equal to a molar concentration of about 10 mM. The
concentration range of the main experiments was limited by cytotoxic
effects and phase separation. Ethanol was used as solvent. The maximum
concentration of 2500 µg/mL used in the pre-experiment was equal to a
molar concentration of about 10 mM. The concentration range of the main
experiments was limited by cytotoxic effects and phase separation.
Ethanol was used as solvent. The dose range of the test item was
selected based on the results of a preliminary cytotoxicity test and
were as follows:
Concentration of test item (µg/mL)
The evaluated experimental points and the results are summarised in
Table 1 (see Attached document).
No substantial and reproducible dose dependent increase of the mutation
frequency was observed in either of the main experiments.
Appropriate reference mutagens, used as positive controls, induced a
distinct increase in mutant colonies and thus, showed the sensitivity of
the test system and the activity of the metabolic activation system. It
can be stated that under the experimental conditions reported the test
item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the substance is considered to be non-mutagenic in this HPRT
Table2 Summary of results
Test itemconcentrationin µg/mL
Mitotic indicesin %of control
Aberrant cellsin %
Exposure period 22 hrs without S9 mix
cells carrying exchanges
of 200 metaphases per culture
of 50 metaphases per culture
separation occurred at the end of treatment
frequency statistically significant higher than corresponding control
1 Ethanol 0.5
2 EMS 770.0
3 EMS 550.0
Table 2, cont. Summary of results
2 CPA 15.0
test item Amberwood F, dissolved in ethanol, was assessed for its
potential to induce structural chromosomal aberrations in human
lymphocytesin vitroin four independent experiments. The
following study design was performed:
Without S9 mix
With S9 mix
Exp. IA & IB
Exp. IA, IIA & IIB
each experimental group two parallel cultures were analysed. Per
culture at least 100 metaphases were evaluated for structural
chromosomal aberrations, except for the positive control in Experiment
IIA, in the absence of S9 mix, where only 50 metaphases were evaluated.
highest applied concentration in this study (2425.0 µg/mL of the test
item, approx. 10 mM) was chosen with regard to the molecular weight of
the test item and with respect to the current OECD Guideline 473.
selection of the cytogenetic experiment was performed considering the
toxicity data in accordance with OECD Guideline 473. The rationale for
the dose selection is reported in section3.5.1.
The chosen treatment concentrations are reported inTable
the results are summarised inTable
Experiment IA and IIA in the absence of S9 mix, cytotoxicity was
observed at the highest evaluated concentration. In Experiment IA in
the presence of S9 mix and in Experiment IB in the absence of S9 mix,
concentrations showing clear cytotoxicity were not evaluable for
cytogenetic damage. In Experiment IIB in the presence of S9 mix no
cytotoxicity was observed up to the highest applied concentration.
Experiment IA in the absence of S9 mix, statistically significant
increases in chromosomal aberrations were observed after treatment
with 27.6 and 1385.7 µg/mL (3.0 and 5.5 % aberrant cells, excluding
gaps). The second value exceeded the range of the laboratory
historical solvent control data (0.0 – 3.0 % aberrant cells, excluding
gaps), but no dose-dependency was observed. In Experiment IB this
finding could not be confirmed. In Experiment IIA in the absence of
S9 mix and in Experiment IA and IIB in the presence of S9 mix no
relevant increases in chromosomal aberrations were observed at the
concentrations evaluated for cytogenetic damage.
evidence of an increase in polyploid metaphases was noticed after
treatment with the test item as compared to the control cultures.
mutagens were used as positive controls. They induced statistically
significant increases in cells with structural chromosome aberrations.
Four genetic toxicity in vitro assays were conducted with the substance
covering different modes of action of genetic toxicity. The substance
did not produce significant genetic toxicity in a reliable bacterial
reverse mutation assay with S. typhimurium and E. coli (2012b), in an in
vitro micronucleus assay with human lymphocytes (2013b), in an in vitro
Gene Mutation Assay in Chinese Hamster V79 Cells (V79/HPRT, 2013c) and
in an in vitro chromosome aberration assay in human lymphocytes (2014).
It is therefore concluded that the substance is not genotoxic. Further
testing on the genetic toxicity of the substance is not required.
The substance was not genotoxic in a battery of four reliable in vitro
assays. The substance is considered to be not genotoxic. The substance
does not need to be classified for this endpoint.
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