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EC number: 814-283-0
CAS number: 42220-47-3
A bacterial reverse mutation assay with the
test item conducted according to OECD 471 and GLP revealed no mutagenic
potential of the test item. A micronucleus test in human lymphocytes in
vitro was negative. The test item did not induce gene mutations at the
HPRT locus in mammalian cells in vitro.
In vitro bacterial reverse mutation
The test substance, waterfree was
tested for its mutagenic potential based on the ability to induce point
mutations in selected loci of several bacterial strains, i.e. Salmonella
typhimurium and Escherichia coli, in a reverse mutation assay.
STRAINS: TA 1535, TA 100, TA 1537, TA
98 and E. coli WP2 uvrA
DOSE RANGE: 33 µg - 5600 µg/plate
(SPT); 33 µg - 5600 µg/plate (PIT)
TEST CONDITIONS: Standard plate test
(SPT) and preincubation test (PIT) both with and without metabolic
activation (liver S9 mix from induced rats).
SOLUBILITY: No precipitation of the
test substance was found with and without S9 mix.
TOXICITY: A weak bacteriotoxic effect
was observed in the standard plate test with tester strain TA 1537 with
S9 mix at concentrations of 2800 and 5600 µg/plate, only.
A biologically relevant increase in
the number of his+ or trp+ revertants (factor > 2: TA 100, TA 98 and
E.coli WP2 uvrA or factor > 3: TA 1535 and TA 1537) was not observed in
the standard plate test or in the preincubation test without S9 mix or
after the addition of a metabolizing system.
Under the experimental conditions of
this study, the test substance , waterfree is not mutagenic in the
Salmonella typhimurium/Escherichia coli reverse mutation assay in the
absence and the presence of metabolic activation.
Micronucleus test in human lymphocytes in
The test item, dissolved in deionised water,
was assessed for its potential to induce micronuclei in human
lymphocytes in vitro in three independent experiments. The following
study design was performed:
Without S9 mix
With S9 mix
Exp. I & IIB
Cytochalasin B exposure
Total culture period
In each experimental group two parallel
cultures were analysed. Per culture at least 1000 binucleated cells were
evaluated for cytogenetic damage.
The highest applied concentration in this
study (4000 µg/mL of the test item) was chosen with regard to the water
content (49.9%) of the test item and with respect to the current OECD
Dose selection of the cytogenetic experiment
was performed considering the toxicity data in accordance with
OECD Guideline 487.
In all experimental parts in the absence and
presence of S9 mix, no cytotoxicity was observed up to the highest
applied and evaluated concentration.
In Experiment I and IIA in the absence of
S9 mix and in Experiment IIB in the presence of S9 mix, no relevant
increase in the number of micronucleated cells was observed after
treatment with the test item. In Experiment I in the presence of S9 mix,
one statistically significant increase in micronucleated cells (1.38 %),
above the historical control data range (95 % control limit:
0.16 – 1.08 %) was observed after treatment with 1306 µg/mL. Since no
dose-dependency was observed, the finding can be regarded as
Appropriate mutagens were used as positive
controls. They induced statistically significant increases in cells with
In conclusion, it can be stated that under
the experimental conditions reported, the test item did not induce
micronuclei as determined by the in vitro micronucleus test in human
Therefore, the test item is considered to be
non-mutagenic in this in vitro micronucleus test, when tested up to the
highest required concentration.
Gene mutation in mammalian cells in vitro
The potential of the test item to induce
gene mutations at the HPRT locus in V79 cells of the Chinese hamster was
investigated. The assay was performed in two independent
experiments, using two parallel cultures each. The first main experiment
was performed with a treatment period of 4 hours with and without
microsomal activation. The second main experiment was performed with a
24 hours treatment period without microsomal activation and a 4 hours
treatment period with microsomal activation.The maximum test item
concentration of the pre-experiment (4000 µg/mL) was chosen with respect
to the current OECD guideline 476 regarding the water content of the
test item. The test item was dissolved in deionised water. No
substantial and reproducible dose dependent increase of the mutation
frequency was observed in 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. In conclusion 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.
Supporting information is available on two
read-across substances 2 -aminoethanol (MEA) and etidronic acid,
the single components of the composition. Furthermore, a QSAR prediction
was performed. Both Ames assays, on 2 -aminoethanol (MEA) and etidronic
acid, yielded negative results. The QSAR prediction with OASIS TIMES was
also negative, for the parent substance as well as for potential
metabolites. The substance is therefore not considered to be mutagenic
in a bacterial reverse mutation assay.
REPORTING FORMAT FOR THE ANALOGUE AND WEIGHT
OF EVIDENCE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The test item is a multi-constituent
substance: etidronic acid composed with 2-ethanolamine. Reliable
experimental data are available for both components, etidronic acid and
2-aminoethanol, which are considered to be suitable read across
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING
INFORMATION ON PURITY AND IMPURITIES)
2-aminoethanol CAS 141-43-5 is already
registered under REACH. The substance purity (analytical grade) for the
Ames test performed was 100%.
Etidronic acid (Phosphonic acid,
P,P'-[1-hydroxyethylidene]bis-, CAS 2809-21-4), the second component is
not yet registered under REACH. An Ames assay (pre-GLP, pre-OECD) was
performed without documentation of the substance purity.
The target substance CAS 42220-47-3 is a
composition of etidronic acid and 2-ethanolamine 1:1.
3. ANALOGUE APPROACH JUSTIFICATION (Read
across and weight of evidence)
Experimental data i.e. Ames assay are
available for 2-aminoethanol and etidronic acid. The Ames test on
2-aminoethanol was performed by the Japanes Ministry of Health and
Environment according to OECD guideline 471. The assay is considered to
be reliable and well documented. The Ames test on etidronic acid,
performed in 1979, is a pre-GLP and pre-OECD study and only briefly
documented. Both compounds, etidronic acid as well as 2-aminoethanol,
are not considered to be mutagenic. The information given on the two
single components is considered to be sufficient to cover the required
endpoint information for the composition thereof. In addition a QSAR
prediction with the target substance was performed (OASIS TIMES,
endpoint: Ames test with metabolic activation). The parent as well as
potential metabolites are not considered to be mutagenic.
Read-across to the single components of the
composition and the QSAR prediction for the target substance together
are supporting the findings of the key studies that the substance has no
Taken together, the three key studies on
gene mutation in bacterial and mammalian cells as well as chromosome
aberration in mammalian cells conducted with the test item did not
indicate any genotoxic potential of the test item. These findings are
further supported by QSAR predictions and a read-across approach to the
single components of the test item.
Labelling, and Packaging Regulation (EC) No 1272/2008
available experimental test data are reliable and suitable for
classification purposes under Regulation (EC) No 1272/2008. Available in
vitro studies were unanimously negative. As a result the substance is
not considered to be classified for genetic toxicity under Regulation
(EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No
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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