Distilled acetalization product between glucose and C12, C14, C18, C20 ,C22 alcohol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2010-08-17 and 2011-02-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study perfomed according GLP standards and include analytical assessement

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

922-178-7

EC Number:

922-178-7

IUPAC Name:

922-178-7

Details on test material:

Designation :LCE10036
Batch no. T93611
Receipt no. 45742
Date of receipt :August 2, 2010
Characteristics: Pearls, solid
Storage conditions :At room temperature in a well ventilated place
Stability (re-test date) :August 31, 2011

For further information see Appendix 2:‘Certificate of Analysis’.
Retention sample :Stored at of the test item LPT Laboratory of Pharmacology
and Toxicology GmbH & Co. KG
Archive 11
Redderweg 8
21147 Hamburg
Germany

Method

Target gene:

detection of
structural chromosomal aberrations in human peripheral lymphocytes

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral lymphocyte

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venipuncture from healthy donors known to
be without any medication and collected in heparinised vessels. Small innocula of
whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture
medium (see Appendix 1). The tubes were sealed and incubated at 37°C with occasional
shaking to prevent clumping.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Concentrations of 1.0, 2.5, 10, 25, 100, 125 and 250 μg
LCE10036/mL medium were employed in an experiment without and with metabolic
activation.

Vehicle / solvent:

The test item was completely dissolved in ethanol1 by using an ultrasonic bath for
3 minutes at 37°C shortly before use. 250 μg/mL medium was the maximum
concentration that could be achieved because of the limit of solubility. The final
solvent concentration in the medium was 1%.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

ethanol

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

in absence of metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

ethanol

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in presence of metabolic activation

Details on test system and experimental conditions:

A preliminary cytotoxicity study was conducted to establish the top concentration for
the main cytogenetic test. Concentrations of 1.0, 2.5, 10, 25, 100, 125 and 250 μg
LCE10036/mL medium were employed in an experiment without and with metabolic
activation. At least three analysable concentrations have to be used in the main
experiment. Where cytotoxicity occurs, these concentrations cover a range from the
maximum to little or no toxicity; the concentrations are separated by no more than a
factor between 2 and √10. At the time of harvesting, the highest concentration would
show a significant reduction in the mitotic index (greater than 50%). For relatively noncytotoxic
compounds the maximum concentration would be 5 μL/mL or 5 mg/mL, or
0.01 M, whichever is lowest.
For relatively insoluble test items that are not cytotoxic at concentrations lower than the
insoluble concentration, the highest dose used would be a concentration above the limit
of solubility in the final culture medium at the end of the treatment period. One
concentration with visible precipitation would be tested.
The solubility was assessed at the beginning and the end of treatment, as solubility
could change during the course of exposure in the test system due to presence of cells,
S9, serum etc. Insolubility would be detected by using the unaided eye. The precipitate
should not interfere with the scoring.
Each treatment was tested in the absence and in the presence of S9 mix.
Samples (50 μL) of the test item solutions were added 48 hours after culture
establishment and cultures were incubated for a further 24 hours at 37°C .
Cultures were harvested and 1 slide per culture was prepared. 1000
lymphocytes per culture were examined at a magnification of x 400; the mitotic index
was calculated as the percentage of lymphocytes examined which were in mitosis
(metaphase). Slides were coded before analysis.
In this preliminary experiment cytotoxicity in form of haemolysis was noted at a
concentration of 250 μg/mL in the experiments without and with metabolic activation.
In addition, cytotoxicity was noted at a concentration of 125 μg/mL in the experiment
without metabolic activation (24-h exposure).
Hence, the highest concentrations employed in the main study were 250 μg
LCE10036/mL in the experiments without and with metabolic activation (4-h
exposure) and 125 μg/mL in the second experiment without S9 mix (24-h exposure).
After 48 hours of culture in complete medium the tubes were centrifuged, and the cell
pellet resuspended to 4.5 mL (for S9 mix addition) or 5.0 mL with treatment medium
(see Appendix 1) including LCE10036 at the final concentrations. Treatments have
been added at a volume of 50 μL.
S9 mix (0.5 mL) was added to the appropriate cultures. During
treatment the tubes were incubated for 4 hours in a shaking water bath at 37°C.
After this period, the tubes were centrifuged and the cells were washed with 5 mL
treatment medium to remove the test item and S9 mix. After a further centrifugation
the cell pellet was resuspended in 5 mL of complete medium and returned to the
incubator for a further 20 hours .
The incubation procedure took place in the dark.
In a second set of the experiment a continuous treatment of 24 hours without
metabolic activation was carried out and the 4-hour treatment with metabolic
activation was repeated.

Results and discussion

Test results

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the precipitating concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the main study cytotoxicity in form of haemolysis was noted at a concentration of
250 μg/mL in the experiments without and with metabolic activation (4-h exposure).
In the second experiment without S9 mix (24-h exposure) cytotoxicity was noted at
the top concentration of 125 μg/mL.

Remarks on result:

other: other: lymphocytes extracted from peripheric blood

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, the test item tested up to cytotoxic concentrations in the absence and in the presence of metabiolic acitivationemploying two exposures times(without S9) and one exposure time (with S9) revealed no indicatioon of mutagenic properties with respect to chromosal or chromatid damage. In the same test, mytomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

Executive summary:

Test samples of LCE10036 were assayed in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate. LCE10036 was completely dissolved in ethanol. 250 μg/mL medium was the maximum concentration that could be achieved because of the limit of solubility. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment cytotoxicity in form of haemolysis was noted at a concentration of 250 μg/mL in the experiments without and with metabolic activation. In addition, cytotoxicity was noted at a concentration of 125 μg/mL in the experiment without metabolic activation (24-h exposure). Hence, the highest concentrations employed in the main study were 250 μg LCE10036/mL in the experiments without and with metabolic activation (4-h exposure) and 125 μg/mL in the second experiment without S9 mix (24-h exposure). In the main study cytotoxicity in form of haemolysis was noted at a concentration of 250 μg/mL in the experiments without and with metabolic activation (4-h exposure). In the second experiment without S9 mix (24-h exposure) cytotoxicity was noted at the top concentration of 125 μg/mL. Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively. Tests without metabolic activation (4- and 24-hour exposure) The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with LCE10036 at concentrations from 31.3 to 250 or 15.63 to 125 μg/mL medium (4 h and 24-h exposure, respectively), in the absence of metabolic activation ranged from 0.6% to 3.4%. The results obtained are within the normal range of the solvent control where a mean incidence of chromosomal aberrations (excluding gaps) of 1.5% or 1.0% was observed after a 4-hour and 24-hour exposure, respectively (historical range: 0 – 4%).

Test with metabolic activation (4-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated

with LCE10036 at concentrations from 31.3 to 250 μg/mL medium in the presence of

metabolic activation in the first and second experiment, respectively, ranged from

0.5% to 3.6%.

The results obtained are within the normal range of the solvent control where a mean

incidence of chromosomal aberrations (excluding gaps) of 1.5% was observed after a

4-hour exposure in the first and second experiment (historical range: 0 – 4%).

No test item-related polyploidy or endoreduplication were noted in the experiments

without or with metabolic activation.

Under the test conditions, the test item tested up to cytotoxic concentrations in the absence and in the presence of metabiolic acitivationemploying two exposures times(without S9) and one exposure time (with S9) revealed no indicatioon of mutagenic properties with respect to chromosal or chromatid damage. In the same test, mytomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.