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EC number: 269-950-3
CAS number: 68391-42-4
Gene mutation assay in bacteria (AMES
test) is in progress and expected to be negative.
Gene mutation assays in mammalian cells
(HPRT test): negative.
A study was conducted to determine the
genetic toxicity in vitro of test substance according to OECD Guideline
476 and EU method B.17.
In an in vitro mammalian cell gene
mutation test (forward gene mutation in HPRT), Chinese hamster V79 cells
were exposed to the test substance at concentrations ranging from 2.34
to 300.0 µg/ml with or without metabolic activation (S9 mix).
Positive and negative controls were valid.
In Main Assay I, without S9 mix, moderate reduction in relative survival
(RS = 18 - 19 %) was noted at the two highest dose levels (150.0 and
75.0 µg/ml), treatment at 37.5 µg/ml yielded a reduction of relative
survival to 36 % of negative control, while no relevant toxicity was
noted over the remaining concentrations tested. With S9 mix, slight
reduction in relative survival (RS = 70 %) was noted at the highest dose
level (300 µg/ml), while no relevant toxicity was observed over the
remaining concentrations tested. In Main Assay II, without S9 mix,
treatment at the highest dose (120 µg/ml) level yielded a reduction of
relative survival to 11 % of negative control, moderate toxicity (RS =
55 %) was noted at the next lower concentration of 60.0 µg/ml, while no
relevant toxicity was observed over the remaining concentrations tested.
With S9 mix, moderate toxicity (RS = 55 %) was noted at the highest dose
level (300 µg/ml), while no relevant toxicity was observed over the
remaining concentrations tested. Opacity of the treatment medium was
noted starting from 30.0 µg/ml, without S9 mix. Opacity and slight
precipitation were observed at the highest dose level, both with and
without S9 mix. No ﬁve-fold or greater increase in mutant frequency,
compared with negative control, was observed at the highest dose level
or at two consecutive doses of test substance, both with and without S9
mix. No reproducible evidence of a dose eﬀect relationship was noticed.
Under the study conditions, test substance did not induce gene mutation
in Chinese hamster V79 cells both with and without S9 metabolic
An in vitro mammalian cell gene mutation
test (forward gene mutation HPRT) was run according to OECD guideline
476. Chinese hamster V79 cells were exposed to test substance at
concentrations ranging from 0.5 to 50.0 µg/plate with or without
metabolic activation (S9 mix). The assay was performed in two
independent experiments with and without liver microsomal activation.
Treatment duration was 4 h in the first experiment and in the second
experiment with metabolic activation. In the second experiment without
metabolic activation, treatment duration was extended to 24 h. Positive
and negative controls were valid.
Strong cytotoxic effects occurred in the
first experiment in both cultures at the maximal concentration without
metabolic activation. Only minor toxic effects were observed up to the
maximal concentration with metabolic activation.
In the second experiment strong cytotoxic
effects occurred only in one out of two parallel cultures at the maximal
concentration without metabolic activation. No relevant and reproducible
increase in mutant colony numbers was observed up to the highest
investigated concentration, neither in the presence nor in the absence
of metabolic activation. Under study conditions, test substance did not
induce gene mutations at the HPRT locus in V79 cells and thus was not
considered as genotoxic.
Micronucleus assay in bone marrow cells of
Evaluation of available information on
genotoxicity was done as reported in ECHA Guidance Chapter R.7a:
Endpoint specific guidance, Version 5.0 – December 2016.
A preliminary assessment should normally
include data from a gene mutation test in bacteria unless existing data
for analogous substances indicates this would be inappropriate.
When the result of the bacterial test is
positive, it is important to consider the possibility of the substance
being genotoxic in mammalian cells.
In order to ensure the necessary minimum
level of information is provided, at least a further test is required in
addition to the gene mutation test in bacteria. This should be an in
vitro mammalian cell test capable of detecting both structural and
numerical chromosome aberrations. Suitable options are in vitro
chromosome aberration test (OECD guideline 473), i.e. a cytogenetic
assay for structural chromosome aberrations, or in vitro
micronucleus test (OECD guideline 487), i.e. a cytogenetic assay to
detect not only structural chromosomal aberrations but also aneuploidy.
It is possible to present data from an in vivo cytogenetic test
as an alternative to the first in vitro mammalian cell test. For
instance, if an adequately performed in vivo micronucleus test is
available, it may be presented as an alternative.
An in vitro gene mutation study in
mammalian cells (OECD guideline 476) is taken into account to complete
In general, substances that are:
- positive in the gene mutation test in
- negative in in vitro or in vivo
tests to measure chromosomal damage, e.g. chromosome aberration or
- negative in in vitro or in vivo
gene mutation tests, e.g. HPRT assay
may be considered as non-genotoxic.
A modified AMES test with nitroreductase deficient
strains is in progress on the target substance. The genotoxic potential
of target substance was assessed also based on data on Similar Substance
01. Available studies, namely in vitro HPRT assay and in vivo
micronucleus assay, examined the potential of test substance to induce in
vitro gene mutations, both in bacteria and in mammalian cells, as
well as in vivo chromosomal aberrations. Available data was
considered as sufficient to draw a conclusion on genotoxicity of target
assays were conducted on Similar Substance 01 following OECD guideline
the first assay (1998), a pre-test on cytotoxicity was run to select the
concentrations. Two experiments were run in the main assay using V79
cells of Chinese hamster. In particular:
exp. I with and without metabolic activation, 4 h exposure to
concentrations of 3, 5, 10, 25 and 50 µg/ml;
exp. II with metabolic activation, 4 h exposure to concentrations of 3,
5, 10 and 50 µg/ml; without metabolic activation, 24 h exposure at
concentrations of 1, 3, 5 and 10 µg/ml.
was evaluated in terms of cloning efficiency. Strong toxic effects were
noted in exp. I without metabolic activation at the highest
concentration adopted. Only minor toxic effects were seen up to the
maximal concentration in the presence of metabolic activation. In exp.
II, toxic effects were seen in one out of two parallell cultures at the
without metabolic activation. Mutagenicity
was evaluated by considering the number of mutant colonies.
the second assay (2016), dose levels were selected based on
precipitation and toxic effects recorded in a preliminary cytotoxicity
assay. Two assays were run in duplicate using chinese hamster lung
main assay I, with and without metabolic activation, 3 h exposure to
dose levels of 2.34, 4.69, 9.38, 18.8, 37.5, 75, 150, 300 and 600 µg/ml.
Precipitation and opacity were noted, depending on the concentration.
main assay II, 3 h exposure to concentrations of 120 or 150 and 300
µg/ml without and with metabolic activation.
and negative (solvent DMSO) controls were used.
effects were noted at 300 µg/ml with metabolic activation and starting
at 37.5 µg/ml without metabolic activation. Mutagenic effects, in terms
of increase in mutant frequency, were not noted.
A micronucleus assay in bone marrow cells
of mouse was conducted using Similar Substance 01 according to OECD
Test substance was administered orally by
gavage. Males were dosed for 2 consecutive weeks prior to pairing,
through the mating period up to the day before necropsy (day 38 - 39);
females were doses 2 consecutive weeks prior to pairing, during pairing,
up to day 3 post partum. Doses of 62.5, 250, 500 and 1000/500 mg/kgt
bw/d were used. Vehicle control and positive controls were used.
Bone marrow cells of the mouse were
collected after 24 h and 48 h for micronucleus analysis. Ten animals
(5/sex) per test group were evaluated. For each animal, 4 slides were
prepared. 4000 polychromatic erythrocytes (PCE) per animal were scored
for micronuclei; number of normal and normochromatic erythrocytes (NCEs)
was also recorded. Cytotoxicity was estimated by the ratio of
polychromatic (PCE) and normochromatic erythrocytes (NCE) in the same
sample; a reduction of the ratio is indicative of inhibition of cell
division. The incidence of micronucleated PCEs provides an index of
induced genetic damage.
Under the study conditions, no relevant
inhibitory effect of erythropoietic cell division was seen at any dose
level. Therefore, test substance is considered to be non-genotoxic in
According to the CLP Regulation (EC
1272/2008), Annex I, Part 3, substances which cause concern for humans
owing to the possibility that they may induce heritable mutations in the
germ cells of humans are classified in Category 2. This classification
is based on positive evidence obtained in:
— somatic cell mutagenicity tests in
vivo, in mammals; or
— other in vivo somatic cell
genotoxicity tests which are supported by positive results from in vitro
Note: substances which are positive in in
vitro mammalian mutagenicity assays, and which also show chemical
structure activity relationship to known germ cell mutagens, shall be
considered for classification as Category 2 mutagens.
In vitro mutagenicity tests are the
— in vitro mammalian
chromosome aberration test;
— in vitro mammalian cell
gene mutation test;
— bacterial reverse mutation tests.
The overall assessement on the genotoxic
potential was based on: expected negative outcome in bacterial reverse
mutation assay (AMES test), negative outcomes in in vitro HPRT
assays and negative result in in vivo micronucleus test in bone
marrow cells of mouse.
All studies were conducted according to
OECD guidelines and have a high reliability.
Based on these results, target susbstance
was considered as non genotoxic and it was not classified within the CLP
Regulation (EC 1272/2008).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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