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EC number: 943-080-0
CAS number: -
Main results from experiment I and II (Mean values of both
Relative Total Growth [%]
Mutants per 106Cells
Solvent Control of Test Item
Solvent Control of Positive Control
Test Item 5.00 µL/mL
Test Item 0.63 µL/mL
Test Item 0.31 µL/mL
Test Item 0.16 µL/mL
Test Item 0.08 µL/mL
Test Item 0.04 µL/mL
Test Item 0.02 µL/mL
- Cultures could not be evaluated for mutagenicity because of
* Cultures were not continued since only 4 analysable
concentrations are required by the guideline.
Note: The threshold (number of mutant colonies per 106cells
of the respective solvent control plus 126) was 255 (+S9) and 253 (-S9)
in Experiment I as well as 242 (+S9) and 208 (-S9) in Experiment II.
This study was performed to investigate the potential of the
substance to induce mutations at thethymidine kinase locus(Tk1)
on chromosome 11 and/or structural chromosomal aberrations in mouse
The assay was performed in a pre-test and in two independent main
experiments (experiment I and II) whereby the first experiment I was
invalid and had to be repeated. The results of the invalid experiment
will not be included in this report but will be archived with the raw
data. Therefore, in total one pre-test and three experiments were
The pre-test was done to detect a potential cytotoxic effect of
the test item. Based on the results of this test the concentrations for
the main experiments were determined.
Experiment I was performed with and without metabolic activation
(liver enzyme S9 fraction / “liver S9 mix from male rats, treated with
Aroclor 1254”) and a treatment period of 4 h. Experiment II was
performed with a treatment period of 24 hours in the absence of
metabolic activation and 4 hours in the presence of metabolic activation.
The highest nominal concentration that was used in the experiment
was 5 µL/mL.
Not all tested concentrations could be evaluated for mutagenicity.
Some of them induced a cytotoxic reaction and had to be excluded from
the evaluation of the mutagenicity. However in all analysable
concentrations no substantial and reproducible dose dependent increase
in mutant colony numbers was observed in both experiments. No relevant
shift of the ratio of small versus large colonies was observed up to the
maximal concentration of the test item.
The mean revertant values of the 3 replicates are presented in the
Mean Revertants Experiment 1
Two valid experiments was performed
5 concentrations of the test item, dissolved in DMSO (ranging from
50 to 5000 µg/plate) were used. 5 genetically changed strains ofSalmonella
typhimurium(TA97a, TA98, TA100, TA102 and TA1535) were exposed to
the test item both in the presence and in the absence of a metabolic
activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for
48 h, using the plate incorporation method.
None of the concentrations caused a significant increase in the
number of revertant colonies in the tested strains. The test item did
not show any mutagenic effects in the first experiment.
The test item showed no precipitates on the plates in all tested
No signs of toxicity towards the bacteria could be observed.
The sterility control and the determination of the titre did not
show any inconsistencies. The determined values for the spontaneous
revertants of the negative controls were in the normal range. All
positive controls showed mutagenic effects with and without metabolic
To verify the results of the first experiment, a second experiment
was performed, using 7 concentrations of the test item (ranging from 78
to 5000 µg/plate) and a modification in study performance
The test item did not show mutagenic effects in the second
Experiment I without and with metabolic activation was performed
using an exposure period of 4 h, culture harvest time was 23 h.The
results of the cytotoxicity test of experiment I are presented in the
Results Cytotoxicity Experiment I without S9
Mitotic indices in %
of the solvent control
Experiment I: exposure period 4 h without S9
Positive control EMS 300 µg/mL
Positive control EMS 600 µg/mL
Test item 5 µL/mL
Test item 2.5 µL/mL
Test item 1.25 µL/mL
Test item 0.63 µL/mL
Test item 0.32 µL/mL
Test item 0.16 µL/mL
Test item 0.08 µL/mL
Test item 0.04 µL/mL
Experiment I: exposure period 4 h with S9
Positive control CPA 25 µg/mL
Positive control CPA 35 µg/mL
Experiment III without metabolic activation was performed using an
exposure period of 24 h, culture harvest time was 24 h. The results of
the cytotoxicity test of experiment III without S9 are presented in the
Results Cytotoxicity Experiment III without S9
Experiment III: exposure period 24 h without S9
Test item 0.02 µL/mL
*n.e.= not evaluated because of very strong cytotoxicity
In the experimental part with metabolic activation, 1
concentration of the test item (0.04 µL/mL) showed a statistically
significant (p < 0.05) increase of structural chromosomal aberrations.
In a total of 4 cultures chromatid exchanges were observed. This can be
assessed as a strong hint, that the test item has mutagenic properties,
because this type of aberration is practically never seen in untreated
The results of experiment I are presented in the following table:
Results Genotoxicity Experiment I
Aberrant cells in %
Solvent control serum free medium RPMI 1640
Solvent control DMSO
Positive control EMS 300 µg/ml
Solvent control for the positive control NaCl 0.9 %
Positive control CPA 25 µg/ml
*Inclusive cells carrying exchanges
SAberration frequency statistically
significant higher than the corresponding control values
1)chromosome exchange (e.g. translocation)2)chromatid
Note: in this table, the mean values are stated. Data for the
individual cultures are listed in the annex, see chapter14.2, page31.
The 3 highest evaluated concentrations of the test item showed
statistically relevant increases of structural chromosomal aberrations.
Again, chromatid exchanges could be found in 2 cultures.
The results of experiment III are presented in the following table:
Results Genotoxicity Experiment II
Positive control EMS 300mg/mL
3 experiments were performed. The 1stand the 3rdexperiment
were valid.The 2ndexperiment was not valid, because the
positive control caused complete cytotoxicity.This experiment is not
reported in this report, the raw data are kept in the test facility in
the GLP- archive.
This study was performed to assess the mutagenic potential of the
substance to induce structural chromosomal aberrations in human
lymphocytes culturedin vitroin the absence and the presence of an
exogenous metabolic activation system (liver S9 mix from male rats,
treated with Aroclor 1254).
The test item (whitish waxy pastilles) was melted at 50 °C and the
series of dilutions was prepared with pre-heated DMSO (50 °C). 50 µl of
the pure test item as well as 50 µL of the resp. solution to be tested
was added to the cultures, corresponding to a maximum concentration of 5
µL/mL in the test.
Human lymphocytes, in whole blood culture, were stimulated to
divide by addition of phytohaemagglutinin and exposed to medium control,
solvent control, test item and positive control both in the presence and
the absence of S9 mix derived from rat livers. 3 h before the end of
cultivation, cell division was arrested using colcemid, the cells were
harvested and slides were prepared. Then, the metaphase cells were
examined for chromosomal damage.
The following schedule was observed:
without S9 mix
with S9 mix
Expression time in growth medium
Culture harvest time
Concentrations selected for metaphase analysis
5, 0.16, 0.08 and 0.04 mL/mL
5, 0.32, 0.08
* performed because the results of experiment I were equivocal and
experiment II was invalid
In both experiments, all cell cultures were set up in duplicates.
In order to assess the toxicity of the test solution to cultured human
lymphocytes; the mitotic index was calculated for all cultures treated
with medium control, solvent control, test item and positive control. On
the basis of the data from the mitotic index, the concentrations (see
table 3-a, page7) were selected for metaphase analysis.
In both experiments, toxicity was detected in the intermediate
concentrations of the test item. In the 1stexperiment with
metabolic activation, a statistically significant increase of structural
chromosomal aberrations was detected in the concentration 0.04 µL/mL. In
the 3rdexperiment, a statistically significant increase of
structural chromosomal aberrations was observed in 3 tested
concentrations (5 µL/mL, 0.32 µL/mL and 0.08 µL/mL). Furthermore,
biologically relevant types of aberrations were detected at several
evaluated concentrations in all independent experiments.
All positive control compounds caused large, statistically
significant increases in the proportion of aberrant cells, demonstrating
the sensitivity of the test system.
In conclusion, under the experimental conditions reported, the
substance has the ability to induce structural chromosomal aberrations
in human lymphocytesin vitro.
The test item is considered as “mutagenic under the conditions of
Micronucleus test in bone marrow cells of the mouse with Reaction
product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids.
Reaction product of: stearyl-diethanol-amine with C16-C18 saturated
fatty acids was tested in the Micronucleus Test in mice, to evaluate its
genotoxic effect in developing erythrocytes (polychromatic erythrocytes)
in the bone marrow.
Reaction product of: stearyl-diethanol-amine with C16-C18 saturated
fatty acids consisted of slightly yellow pastilles. The test item was
suspended in propylene glycol. The concentrations analysed in the
formulations of were in agreement with target concentrations (i.e. mean
accuracies between 85% and 115%). No test item was detected in the
vehicle control samples. The formulations were homogeneous (i.e.
coefficient of variation ≤ 10%). Analysis of formulations after storage
yielded a relative difference of ≤ 10%. Based on this, the formulations
were found to be stable during storage at room temperature under normal
laboratory light conditions for at least 4 hours. In the dose range
finding study males were dosed once via oral gavage 2000 mg Reaction
product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids
per kg body weight. The animals showed no treatment related clinical
signs or mortality after dosing. In the main study male animals were
dosed twice via oral gavage with vehicle or with 2000 and 1000 mg
Reaction product of: stearyl-diethanol-amine with C16-C18 saturated
fatty acids per kg body weight. A positive control group was dosed once
via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In
total 5 treatment groups were used, each consisting of 5 animals. No
treatment related clinical signs or mortality were noted in any animal
treated with the test item or control animals receiving vehicle or
cyclophosphamide. Bone marrow was sampled 48 hours after the first
dosing. No increase in the mean frequency of micronucleated
polychromatic erythrocytes was observed in the bone marrow of animals
treated with the test item compared to the vehicle treated animals. The
incidence of micronucleated polychromatic erythrocytes in the bone
marrow of all negative control animals was within the 95% control limits
of the distribution of the historical negative control database.
Cyclophosphamide, the positive control item, induced a statistically
significant increase in the number of micronucleated polychromatic
erythrocytes. In addition, the number of micronucleated polychromatic
erythrocytes found in the positive control animals was within the 95%
control limits of the distribution of the historical positive control
database. Hence, all criteria for an acceptable assay were met. The
groups that were treated with the test item showed no decrease in the
ratio of polychromatic to normochromatic erythrocytes compared to the
concurrent vehicle control group, indicating a lack of toxic effects of
this test item on erythropoiesis. The group that was treated with
cyclophosphamide showed an expected decrease in the ratio of
polychromatic to normochromatic erythrocytes compared to the vehicle
control, demonstrating toxic effects on erythropoiesis. It is concluded
that Reaction product of: stearyl-diethanol-amine with C16-C18 saturated
fatty acids is not clastogenic or aneugenic in the bone marrow of male
mice up to a dose of 2000 mg/kg (the maximum recommended dose in
accordance with current regulatory guidelines) under the experimental
In vitro testing of the target substance has produced the following
- OECD 471: gene mutation test in bacteria (Ames test): negative
- OECD 476: gene mutation assay in mammalian cells: negative
- OECD 473: in vitro chromosome aberration test: positive
According to ECHA’s Guidance on the Application of the CLP Criteria
(Version 4.1), “in vitro results can only lead to a Category 2 mutagen
classification in a case where there is support by chemical structure
activity relationship to known germ cell mutagens.” The target substance
is a UVBC with the main component
2-[(2-hydroxyethyl)octadecylamino]ethyl stearate and has the following
- Acids (C16/C18);
- ethoxylated amines (C16/C18);
- Monoester of the ethoxylated amines (C16/C18); and
- Diester of the ethoxylated amines (C16/C18).
There are no known chemical structure activity relationships to known
germ cell mutagens for these components.
While there is one positive in vitro result there are no known chemical
structure activity relationships to known germ cell mutagens. Therefore,
the target substance is not classified for germ cell mutagenicity
according to the criteria of Regulation (EC) No. 1272/2008 (as amended).
This conclusion is supported by an in vivo study performed for different
regulatory purposes. The negative result coming from the mammalian
erythrocytes micronucleous test (no effects on mice after oral gavage at
2000 mg/kg) confirms that the substance should not be classified as
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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