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Genetic toxicity: in vitro

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 02 September 2009 and 14 December 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9)
Description : Pale brown viscous liquid
Purity : 99.9%
Batch number : S-001016
Date received : 08 July 2009
Storage conditions: Approximately 4oC in the dark under nitrogen

Copy of Certificate of analysis attached.

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitabilityThe volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) (µg/ml)
4(20)-hour without S9 0*, 1.5, 3, 6*, 9*, 12*, 18, MMC 0.4*
4(20)-hour with S9 0*, 3, 6*, 12*, 24*, 36, 48, CP 5*

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) (µg/ml)
24-hour without S9 0*, 1.5*, 3*, 6*, 12, 18, 24, MMC 0.2*
4(20)-hour with S9 0*, 3*, 6*, 12*, 24, 36, 48, CP 5*
* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl Sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test material was readily soluble in it at the required
concentrations.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl Sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9

Migrated to IUCLID6: (CP)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl Sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9

Migrated to IUCLID6: (MMC)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in medium


DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9.
Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium):
20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells
with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted
according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with
chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results in attached tables.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material suspended in DMSO
- Precipitation:
Premlinary toxictiy test: A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 100 µg/ml in the 4(20)-hour without S9 exposure group and in the continuous exposure group. In the 4(20)-hour with S9 exposure group precipitate was seen at and above 25 µg/ml at the end of exposure. Haemolysis was also seen at the end of exposure at and above 100 µg/ml in the 4(20)-hour
exposure group without S9 and the continuous exposure group. In the 4(20)-hour exposure group in the presence of S9 haemolysis was seen at and
above 200 µg/ml.
Experiment 1: No precipitate of the test material was observed at the end of exposure in either exposure group.
Experiment 2: No precipitate of the test material was observed at the end of exposure in either exposure group.

See attached tables 1 to 8 for results

RESULTS
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 3.13 to 800 µg/ml. The maximum dose selected was 800 µg/ml and was limited by the toxicity
seen in the Mouse Lymphoma Assay on the same test material (Harlan Laboratories Ltd Project No. 0142/0415). A precipitate of the test material was
observed in the parallel blood-free cultures at the end of the exposure, at and above 100 µg/ml in the 4(20)-hour without S9 exposure group and in
the continuous exposure group. In the 4(20)-hour with S9 exposure group precipitate was seen at and above 25 µg/ml at the end of exposure.
Haemolysis was also seen at the end of exposure at and above 100 µg/ml in the 4(20)-hour exposure group without S9 and the continuous exposure
group. In the 4(20)-hour exposure group in the presence of S9 haemolysis was seen at and above 200 µg/ml. Microscopic assessment of the slides
prepared from the exposed cultures showed that metaphase cells were present up to 25 µg/ml in the 4(20)-hour exposure group in the absence of
metabolic activation (S9) and in the continuous exposure group. The maximum dose with metaphases present in the 4(20)-hour exposure group in
the presence of metabolic activation (S9) was 50 µg/ml. The mitotic index data are presented in the attached Table 1. The test material induced dose
related toxicity in all three of the exposure groups.
The selection of the maximum dose level was based on toxicity and was 18 and 48 µg/ml for the 4(20)-hour exposure groups in both the absence and presence of S9 respectively, and was 24 µg/ml for the continuous exposure group used in Experiment 2.

Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) (µg/ml)
4(20)-hour without S9 0*, 1.5, 3, 6*, 9*, 12*, 18, MMC 0.4*
4(20)-hour with S9 0*, 3, 6*, 12*, 24*, 36, 48, CP 5*
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were
metaphases suitable for scoring present at 18 µg/ml and 36 µg/ml in the absence and presence of S9 respectively. No precipitate of the test material
was observed at the end of exposure in either exposure group.
The mitotic index data are given in the attached Table 2. They confirm the qualitative observations in that a dose-related inhibition of mitotic index
was observed, and that 81% and 42% mitotic inhibition was achieved at 18 and 12 µg/ml respectively in the absence of S9. In the presence of S9 73%
and 51% mitotic inhibition was achieved at 36 and 24 µg/ml respectively.
The maximum dose level selected for metaphase analysis was 12 µg/ml and 24 µg/ml in the absence and presence of S9 respectively, selected on the
basis of toxicity.
The chromosome aberration data are given in the attached Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The polyploid cell frequency data are given in the attached Table 8. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) (µg/ml)
24-hour without S9 0*, 1.5*, 3*, 6*, 12, 18, 24, MMC 0.2*
4(20)-hour with S9 0*, 3*, 6*, 12*, 24, 36, 48, CP 5*
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 36 µg/ml in the presence of S9, however due to the obvious toxicity at this dose level it was not selected for a mitotic index or metaphase analysis. In the absence of S9 the maximum test
material dose level with metaphases suitable for scoring was 12 µg/ml. No precipitate of the test material was observed at the end of exposure in
either exposure group.
The mitotic index data are given in the attahed Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 79% and 44% mitotic inhibition was achieved at 24 and 12 µg/ml respectively in the presence of S9. In the continuous exposure
group 64% and 40% mitotic inhibition was achieved at 6 and 3 µg/ml respectively.
The maximum dose level selected for metaphase analysis was based on toxicity and was 6 µg/ml for the continuous exposure group and 12 µg/ml for
the 4(20)-hour exposure group in the presence of S9.
The chromosome aberration data are given in the attached Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with
chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells
with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or
presence of metabolic activation.
The polyploid cell frequency data are given in the attached Table 8. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For the tables and figures of results mentioned above, please refer to the attached background material section for the following tables:

Table 1: Mitotic Index - Preliminary Toxicity Test

Table 2: Mitotic Index - Experiment 1

Table 3: Mitotic Index - Experiment 2

Table 4: Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)

Table 5: Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (S9)

Table 6: Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (S9)

Table 7: Results of Chromosome Aberration Test - Experiment 2 With Metabolic Activation (S9)

Table 8:  Mean Frequency of Polyploid Cells (%)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction. 

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results. 

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition or greater.

Conclusion. 

The test material was considered to be non-clastogenic to human lymphocytes in vitro.