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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 11 to May 25, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was not planned under REACH strategy. It was performed for other regulatory purposes.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test

Test material

Constituent 1
Reference substance name:
2-[(2-hydroxyethyl)(octadecyl)amino]ethan-1-ol; 2-[(2-hydroxyethyl)(octadecyl)amino]ethyl hexadecanoate; 2-[(2-hydroxyethyl)(octadecyl)amino]ethyl octadecanoate; 2-{[2-(hexadecanoyloxy)ethyl](octadecyl)amino}ethyl hexadecanoate; 2-{[2-(hexadecanoyloxy)ethyl](octadecyl)amino}ethyl octadecanoate; 2-{[2-(octadecanoyloxy)ethyl](octadecyl)amino}ethyl octadecanoate
EC Number:
943-080-0
Molecular formula:
Not applicable, UVCB
IUPAC Name:
2-[(2-hydroxyethyl)(octadecyl)amino]ethan-1-ol; 2-[(2-hydroxyethyl)(octadecyl)amino]ethyl hexadecanoate; 2-[(2-hydroxyethyl)(octadecyl)amino]ethyl octadecanoate; 2-{[2-(hexadecanoyloxy)ethyl](octadecyl)amino}ethyl hexadecanoate; 2-{[2-(hexadecanoyloxy)ethyl](octadecyl)amino}ethyl octadecanoate; 2-{[2-(octadecanoyloxy)ethyl](octadecyl)amino}ethyl octadecanoate
Test material form:
solid
Details on test material:
Appearance Slightly yellow pastilles
Batch 20089403
Test item storage At room temperature
Stable under storage conditions until 20 January 2017 (expiry date)
Purity/composition correction factor No correction factor required
Test item handling No specific handling conditions required
Stability at higher temperatures Stable
Chemical name (IUPAC), synonym or
trade name
Reaction product of: stearyl-diethanol-amine with C16-C18
saturated fatty acids
SABOSTAT A300
CAS Number 52497-24-2 (main component)
Molecular structure UVCB
Molecular formula Not indicated
Molecular weight 668 g/mol calculated on overage UVCB composition

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
NMRI mice (SPF) were used as the test system. These mice are recommended by international
guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided
by Charles River, Sulzfeld, Germany.
Young adult animals were selected (6 weeks old at the start of treatment). The total number of animals
used in the dose range finding study was 3 and in the main study 25. In the micronucleus main study
5 male mice were treated per sampling time in each treatment group.
The body weights of the mice at the start of the treatment in the main study were within 20% of the sex
mean. The mean body weights were 34.3 ± 1.3 g and the range was 32 - 37 g. The mice were
identified by a unique number on the tail written with a marker pen. The animals were allocated at
random to the treatment groups.
The acclimatisation period was at least 6 days before the start of treatment under laboratory
conditions.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected
animals were in a good state of health.
The animals were housed in room number A0.18.
Conditions
A controlled environment was maintained in the room with optimal conditions of approximately 10 air
changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 21.4 - 22.2°C), a relative humidity of
40 - 70% (actual range: 39 - 56%) and a 12 hour light/12 hour dark cycle. Due to e.g. cleaning
procedures, temporary deviations from the light/dark cycle (with a maximum of 4 hours) and the
minimum\maximum level for humidity (with max. 13%) occurred. Based on laboratory historical data
these deviations are considered not to affect the study integrity.
Accommodation
The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages
(type MIII height 180 mm, length 380 mm and width 220 mm) containing sterilised sawdust as bedding
material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). A
shelter (disposable paper corner home, MCORN 404, Datesand Ltd, USA) and paper bedding (Envirodri,
Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment.
Diet
The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH,
Soest, Germany).
Water
The animals had free access to tap-water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was
performed according to facility standard procedures. There were no findings that could interfere with
the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The test item was suspended in propylene glycol (Merck, Darmstadt, Germany). The specific gravity of
propylene glycol is 1.036 g/ml.
Details on exposure:
Test item concentrations were treated with ultra-sonic waves and
heating to approximately 40 - 55ºC to obtain a homogeneous suspension. Test item concentrations
were dosed within 4 hours after preparation.
Frequency of treatment:
Two treatments were performed, administered at a 24-hour interval. The test item was administered as
a split dose, i.e., two treatments on the same day separated by no more than 2-3 hours, to facilitate
administering a large volume necessary due to limited solubility of the test item.
Post exposure period:
48 h
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males for each dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0;
Baxter B.V., Utrecht, The Netherlands) dissolved in physiological saline (Eurovet Animal Health,
Bladel, the Netherlands) dosed as a single oral intubation of 40 mg/kg body weight.
The route of administration of the positive control was the same as that of the test item.

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
Bone marrow was sampled 48 hours after the first dosing. The animals were sacrificed by cervical
dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were
shortened until a small opening to the marrow canal became visible. The bone was flushed with
approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell
suspension was collected and centrifuged at 216 g for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells
in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was
placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v)
ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were
marked with the study identification number and the animal number. The drop was spread by moving a
clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of
bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck)
and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer
(Hematek 3000, Siemens Healthcare, Den Haag, the Netherlands). This staining is based on Giemsa.
The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The
Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica
Microsystems B.V., Rijswijk, The Netherlands).
To prevent bias, all slides were randomly coded before examination. An adhesive label with the study
identification number and code was stuck over the marked slide. At first the slides were screened at a
magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread,
undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of
micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes
(with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was
determined by counting and differentiating at least the first 1000 erythrocytes at the same time.
Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were
calculated.
Evaluation criteria:
A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase
in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent
negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the
frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative
control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The mean number of micronucleated polychromatic
erythrocytes scored in Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids
treated groups were compared with the corresponding vehicle control group.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the
bone marrow of test item treated animals compared to the vehicle treated animals.
All animals treated with Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty
acids exhibited both group mean and individual MN PCE which were comparable with both the
concurrent vehicle control and the laboratory’s historical vehicle control data.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control
animals was within the 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 animals of the groups which were treated with the test item showed no decrease in the ratio of
polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item
on the erythropoiesis. The animals of the group treated with cyclophosphamide showed an expected
decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on
erythropoiesis.
The concentrations analysed in the formulations 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 the above, the formulations were found to be stable during storage at room temperature
under normal laboratory light conditions for at least 4 hours.

Applicant's summary and conclusion

Conclusions:
It is concluded that this test is valid and 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 conditions described.
Executive summary:

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 conditions described.