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EC number: 700-748-4
CAS number: 1226911-69-8
- Ames Test (OECD 471, GLP, K, rel. 1): non
mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA
1537, TA 98, TA 100 & E.coli WP2uvrA.
- Chromosome aberration test (OECD 473, GLP,
K, rel. 1): non clastogenic up to cytotoxic concentrations in human
The test item formulation and S9-mix used in
this experiment were both shown to be sterile.
Prior to use, the master strains were
checked for characteristics, viability and spontaneous reversion rate
(all were found to be satisfactory). The amino acid supplemented top
agar and the S9-mix used in both experiments was shown to be sterile.
Tables: cf Attached background material.
a reverse gene mutation assay performed according to the OECD test
guideline No. 471 and in compliance with GLP, Salmonella
TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA
were treated with the test item, ST 01 C 11, using both the Ames
plate incorporation and pre-incubation methods at seven dose levels, in
triplicate, both with and without the addition of a rat liver homogenate
metabolising system (10% liver S9 in standard co-factors). The dose
range for the range-finding test was determined in a preliminary
toxicity assay and ranged between 0.15 and 500 μg/plate, depending on
bacterial strain type and presence or absence of S9-mix. The experiment
was repeated on a separate day (pre-incubation method) using the same
dose range, fresh cultures of the bacterial strains and test item
formulations. Additional dose levels and an expanded dose range were
selected in each experiment in order to achieve both four non-toxic dose
levels and the toxic limit of the test item. Negative,
vehicle (dimethyl sulphoxide) and positive control groups were also
included in mutagenicity tests.
vehicle (dimethyl sulphoxide) control plates gave counts of revertant
colonies within the normal range. All of the positive control chemicals
used in the test induced marked increases in the frequency of revertant
colonies, both with or without metabolic activation. Thus, the
sensitivity of the assay and the efficacy of the S9-mix were validated.
item caused a visible reduction in the growth of the bacterial
background lawns of all of the tester strains, initially from 150
μg/plate after employing plate incorporation methodology (range-finding
test) and from 15 μg/plate following the pre-incubation method (main
test). The sensitivity of the bacterial tester strains to the toxicity
of the test item varied slightly between strain type, exposures with or
without S9-mix and experimental methodology. The test item was tested up
to the toxic limit. A test item precipitate (light and oily in
appearance) was observed at 5000 μg/plate. The test item was tested up
to the toxic limit in the range-finding and main tests, therefore, this
observation was only noted in the preliminary toxicity test.
significant increases in the frequency of revertant colonies were
recorded for any of the bacterial strains, with any dose of the test
item, either with or without metabolic activation or exposure method.
the test condition, test item is not mutagenic with and without
metabolic activation in S. typhimurium (strains TA1535, TA1537,
TA98 and TA100) and E. coli WP2 uvrA-.
study is considered as acceptable and satisfies the requirement for
reverse gene mutation endpoint.
an in vitro chromosome aberration test performed according to
OECD Guideline 473 and in compliance with GLP, cultured human
lymphocytes were exposed to test item at the following concentrations:
7.34, 14.69, 29.38, 58.75, 117.5, 235, 470, 940 and 1880 μg/mL; 4 h
exposure time with and without metabolic activation followed by a 20 h
recovery period, and a continuous exposure of 24 h without metabolic
without S9: 0, 3.75, 7.5, 15, 30, 60 and 120 μg/mL;
with S9 (2%): 0, 15, 30, 60, 80, 100 and 120 μg/mL
without S9: 0, 3.75, 7.5, 15, 30, 40 and 60 μg/mL;
with S9 (1%): 0, 7.5, 15, 30, 60, 80, 100 μg/mL
activity was arrested by addition of colcemid at 0.1 μg/mL for each
culture, two hours before the harvest. The cells were then treated with
a hypotonic solution, fixed, stained and examined for mitotic indices
and chromosomal aberrations. Vehicle and positive controls were also
included in this test.
vehicle (solvent) controls had frequencies of cells with aberrations
within the range expected for normal human lymphocytes. All the positive
control items induced statistically significant increases in the frequency
of cells with aberrations indicating the satisfactory performance of the
test and of the activity of the metabolising system.
test item did not induce any statistically significant increases in the
frequency of cells with aberrations, in either the absence or presence
of S9, in two separate experiments.
the test conditions, the test item was considered to be non-clastogenic
to human lymphocytes in vitro.
7.6/1: Summary of genotoxicity tests
Test / Guideline
K, rel. 1
E. coli WP2
Up to cytotoxic concentration
-S9 : non mutagenic
+S9 : non mutagenic
HL/CAT (OECD 473)
-S9 : non clastogenic
+S9 : non clastogenic
Gene mutation Assays (Test n°1):
A Bacterial Reverse mutation Assay (Ames
test) was performed according to OECD guideline No. 471 with the
substance (See Table 7.6/1). A test item precipitate (light and oily in
appearance) was observed at 5000 μg/plate in the preliminary test. The
test substance was tested up to 150 µg/plate in the main test for all
Salmonella strains -S9 and up to 500 µg/plate for E.coli +S9 and -S9,
and for all Salmonella strains +S9. No significant increases in the
frequency of revertant colonies were recorded for any of the bacterial
strains under the test condition, with any dose of the substance, either
in the presence or absence of metabolic activation. The substance does
not induce gene mutations in bacteria whereas all positive control
chemicals (with and without metabolic activation) induced significant
increase of colonies.The substance is therefore considered as
non-mutagenic according to the Ames test.
Chromosomal aberration test (Test n°2)
The clastogenic potential of the substance
was determined using an in vitro chromosome aberration test in human
lymphocytes (OECD guideline No. 473), which measures the potential of a
substance to increase the incidence of structural chromosome aberrations
in cultured human lymphocytes.
None of the dose levels up to the
cytotoxicity limit with the substance, either in the presence or absence
of metabolic activation, induced significant increases in the frequency
of cells with aberrations in either of two separate experiments. The
substance does not induce structural aberrations in the chromosomes of
human lymphocytes under activation and non-activation conditionsusing a
dose range that included a dose level that induced approximately 50%
mitotic inhibition, whereas all the positive control items induced
significant increases in the frequency of aberrant cells indicating that
the sensitivity of the assay and the efficacy of the S9-mix were
validated. The substance is therefore considered as negative for
inducing chromosomal mutations in human lymphocyte cells under
activation and non-activation conditions used in this assay.
The substance has no harmonized
classification for human health according to the Regulation (EC) No.
Based on the available data, no additional
classification is proposed regarding genetic toxicity according to the
Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
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