Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

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 01 May 2012 and 16 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in the UKEMS Recommended Procedures for Basic Mutagenicity Tests (1990).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
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
suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a
viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Cell Culture:
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
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:
Preliminary Toxicity Test:
The dose range of test item used was 4.96, 9.92, 19.84, 39.69, 79.38, 158.75, 317.5, 635, 1270 µg/ml.

Chromosome Aberration Test – Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of N-Isopropylmethacrylamide (NIPMAA) (µg/ml)
4(20)-hour without S9 0*, 39.69, 79.38, 158.75, 317.5*, 635*, 1270*, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 39.69, 79.38, 158.75, 317.5*, 635*, 1270*, CP 5*

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of N-Isopropylmethacrylamide (NIPMAA) (µg/ml)
24-hour without S9 0*, 39.69, 79.38, 158.75, 317.5*, 635*, 1270*, MMC 0.2*
4(20)-hour with S9 (1%) 0*, 39.69, 79.38, 158.75, 317.5*, 635*, 1270*, CP 5*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 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.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide (CP)
Remarks:
In the presence of S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
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. Cells with
chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.


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.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results and 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.


PRELIMINARY TOXICITY TEST (CELL GROWTH INHIBITION TEST)
The mitotic index data are presented in the attached Appendix 1 (5) and (6) (see attached background material - Appendix 1). It can be seen that the test item showed marked evidence of toxicity in the 24-hour exposure group only. No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period in any of the three exposure groups.

Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 1270 µg/ml in all three
exposure groups.

Dose selection for Experiment 1 and Experiment 2 was based on the maximum recommended dose level, the 10 mM concentration which was
1270 µg/ml.

CHROMOSOME ABERRATION TEST - EXPERIMENT 1
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 the maximum test item dose level of 1270 µg/ml in both the presence and absence of metabolic
activation (S9).

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in the attached Form 1, Appendix 2 (attached background material). These data show no mitotic inhibition was demonstrated in the absence or presence of S9 at the maximum recommended dose of 1270 µg/ml.

No precipitate of the test item was observed at the end of the treatment period in either exposure group.

The maximum dose level selected for metaphase analysis was the maximum recommended dose level, the 10 mM concentration dose level
(1270 µg/ml).

The chromosome aberration data are given in the attached Form 1, Appendix 2 (attached background material). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items 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 item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of
metabolic activation (S9).

The polyploid cell frequency data are given in the attached Form 1, Appendix 2 (attached background material). The test item 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 qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of
1270 µg/ml in both the presence and absence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in the attached Form 2, Appendix 2 (attached backgroud material). These data show no growth inhibition was demonstrated in the 4(20)-hour exposure group in the presence of S9 as seen in the previous experiments. In the 24-hour exposure group in the absence of S9 there was a dose related increase in toxicity which achieved 42% and 62% mitotic inhibition at 635 and 1270 µg/ml respectively.

No precipitate of the test item was observed at the end of the treatment period in either exposure group.

The maximum dose level selected for metaphase analysis was the maximum recommended dose level, the 10 mM concentration (1270 µg/ml).

The chromosome aberration data are given in the attached Form 2, Appendix 2 (attached background material). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items 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 item 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 Form 2, Appendix 2. The test item did not induce a 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'.

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

Appendix 1    Report of Results of Chromosomal Aberration Test in Cultured Mammalian Cells

Appendix 2    Results of Chromosome Aberration Test

Appendix 3    Dose Response Curves  

Conclusions:
Interpretation of results: negative.
The test item 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 item 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 (Scott et al, 1990).

The test method used was designed to be compatible with the following internationally accepted guidelines and recommendations:

i)            OECD Guidelines for Testing of Chemicals No. 473 "Genetic Toxicology: Chromosome Aberration Test", Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

ii)           UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in theRecommended Procedures for Basic Mutagenicity Tests (1990).

iii)          US EPA OPPTS 870.5375 Guideline.

The test method was designed to be acceptable to the Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test item, 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, a 4-hour exposure 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-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour 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.

 

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final concentration o fTest Item (µg/ml)

4(20)-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4(20)-hour with S9 (2%)

39.69, 79.38, 158.75, 317.5, 635, 1270

24-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4(20)-hour with S9 (1%)

39.69, 79.38, 158.75, 317.5, 635, 1270

Results.

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

All the positive control items 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 item 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 the maximum recommended dose level.

Conclusion.

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

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 09 July 2012 and 10 September 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
To assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes

- Periodically checked for Mycoplasma contamination:yes

- Periodically checked for karyotype stability: no

- Periodically "cleansed" against high spontaneous background: yes

Cell Line :
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.

Cell Culture:
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.

Cell Cleansing:
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by
culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and
grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each
experiment.



Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test
The dose levels of test item used were 4.96, 9.92, 19.84, 39.69, 79.38, 158.75, 317.5, 635 and 1270 µg/ml.

Mutagenicity Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
4-hour without S9 0*, 39.69*, 79.38*, 158.75*, 317.5*, 635*, 1270*, EMS 750*
4-hour with S9 (2%) 0*, 39.69*, 79.38*, 158.75*, 317.5*, 635*, 1270*, *,DMBA 0.5* and 1*

Mutagenicity Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
24-hour without S9 0*, 39.69*, 79.38*, 158.75*, 317.5*, 635*, 1270*, EMS 200* and 300*
4-hour with S9 (1%) 0*, 39.69*, 79.38*, 158.75*, 317.5*, 635*, 1270*, DMBA 0.5* and 1*

* = Dose levels plated for mutant frequency
EMS = Ethyl methane sulphonate
DMBA = Dimethyl benzanthracene

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle:The test material formed a solution with the solvent suitable for dosing at the required concentrations.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethyl benzanthracene (DMBA)
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulphonate (EMS)
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
PRELIMINARY CYTOTOXICITY TEST:
A preliminary cytotoxicity test was performed on cell cultures plated out at 1.5 x 10E6 cells/75 cm2 flask for the 4-hour exposure groups and at 1 x 10E6 cells/75 cm2 flask for the 24-hour exposure group, approximately 48 hours before dosing. On dosing, the growth media was removed and
replaced with serum free media (Hams F12) for the 4 hour exposure groups and Hams F12 with 1% FBS for the 24-hour exposure group. One flask
per dose level was treated with and without S9 metabolic activation, 9 dose levels using halving dilutions and vehicle controls were dosed. The dose
levels of test item used were 4.96, 9.92, 19.84, 39.69, 79.38, 158.75, 317.5, 635 and 1270 µg/ml. Exposure was for 4 hours or 24 hours at 37°C,
after which the cultures were washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in
Hams F12 with 5% FBS, a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 ml of Hams F12 with 5% FBS and incubated for 6 to 7 days at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies.

Results from the preliminary cytotoxicity test were used to select the test item dose levels for the mutagenicity experiments.

MUTAGENICITY TEST:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient
cells for use in the test. For the 4-hour exposure groups of Experiment 1 cells were seeded at 1.5 x 10E6/75 cm2 flask approximately 48 hours
before being exposed to the test or control items. In Experiment 2 cells were seeded approximately 48 hours before being exposed to the test or
control items at 1.0 x 10E6/75 cm2 flask for the 24-hour exposure group and at 1.5 x 10E6/75 cm2 flask for the 4-hour exposure group in the
presence of S9. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with six dose levels of test item, and vehicle and positive controls. Treatment was for 4 hours in serum free media (Ham's F12) or for 24 hours in Hams F12 with 1% serum at 37°C in an
incubator with a humidified atmosphere of 5% CO2 in air.

The dose range of test item was 39.69, 79.38, 158.75, 317.5, 635 and 1270 µg/ml for the 4-hour exposure groups in the absence and presence of S9 and for the 24-hour exposure group of Experiment 2.

At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in Hams F12 with 5% FBS. A sample of
each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10E6 cells/flask in a 225 cm2 flask to allow
growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in
Hams F12 with 5% FBS and incubated at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air.

Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded
to estimate cytotoxicity.

During the 7 Day expression period the cultures were subcultured and maintained at 2 x 10E6 cells/225 cm2 flask on day 3 to maintain logarithmic
growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out
as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 ml of Hams F12 with 5% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage
cloning efficiency for each dose group calculated.
ii) At 2 x 10E5 cells/75 cm2 flask (5 replicates per group) in Hams F12 with 5% FBS, supplemented with 10 µg/ml 6-Thioguanine (6-TG),
to determine mutant frequency. The flasks were incubated for 14 days at 37°C in an incubator with humidified atmosphere of 5% CO2 in air, then
fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.

The percentage of viability and mutation frequency per survivor were calculated for each dose group.

Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with
methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and
without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with
its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance
criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the
scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10E-6. The
background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10E-6 will not be used for the evaluation of a test item.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly
elevated over the concurrent negative control.
iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced
the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test article in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration
that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts.
To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed
duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.
Evaluation criteria:
See assay acceptance criteria
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY CYTOTOXICITY TEST:
A dose range of 4.96, 9.92, 19.84, 39.69, 79.38, 158.75, 317.5, 635 and 1270 µg/ml was used in the preliminary cytotoxicity test. The maximum
dose tested was the maximum recommended dose level, the 10mM concentration.

No precipitate of the test item was observed at the end of exposure in any of the exposure groups.
The results of the individual flask counts and their analysis are presented in the attached Table 1 (attached background material). It can be seen that there was no dose-related reduction in the cloning efficiency (CE) in the 4-hour exposure groups in the presence or absence of S9. In the 24-hour exposure group there was a reduction in the cloning efficiency of 44% in the middle of the dose range at 19.84 µg/ml but since it was not dose related it was considered to be spurious and of no significance. The maximum dose of 1270 µg/ml in the 24-hour exposure group demonstrated only modest toxicity with a 17% reduction in cloning efficiency when compared to the vehicle control

The maximum dose level for the main experiments was the maximum recommended dose level of 1270 µg/ml for all exposure groups in Experiment 1 and Experiment 2.

MUTAGENICITY TEST - EXPERIMENT 1:
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
4-hour without S9 0*, 39.69*, 79.38*, 158.75*, 317.5*, 635*, 1270*, EMS 750*
4-hour with S9 (2%) 0*, 39.69*, 79.38*, 158.75*, 317.5*, 635*, 1270*, *,DMBA 0.5* and 1*

No precipitate of the test item was seen at the end of exposure in either exposure group.

The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in the attached Table 2 and
Table 3 (attached background material). There was no marked reduction in cloning efficiency demonstrated at Day 0 or Day 7 in either of the exposure groups.

The Day 0 vehicle control cloning efficiencies for the 4-hour exposure group in the absence of S9 and the Day 0 and Day 7 cloning efficiencies for the 4-hour exposure group in the presence of S9 did not achieve 70% in all the replicates, however since the value was greater than 50% in all cases this was considered to be acceptable.

The mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3 (attached background material). There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 with or without the presence of S9.

It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

MUTAGENICITY TEST - EXPERIMENT 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/ml)
24-hour without S9 0*, 39.69*, 79.38*, 158.75*, 317.5*, 635*, 1270*, EMS 200* and 300*
4-hour with S9 (1%) 0*, 39.69*, 79.38*, 158.75*, 317.5*, 635*, 1270*, DMBA 0.5* and 1*

No precipitate of the test item was seen at the end of the exposure period in either exposure group.

The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in the attached Tables 4 and 5 (attached background material). It can be seen that, as in Experiment 1, there was no reduction in the Day 0 or Day 7 cloning efficiencies of the 4-hour exposure group in the presence of S9. The 24-hour exposure group demonstrated a dose related increase in toxicity at Day 0 with a 44% reduction in cloning efficiency at 1270 µg/ml when compared to the vehicle controls. This toxicity was greater than that seen in the Preliminary Toxicity Test for this dose group.

The Day 0 vehicle control cloning efficiencies for both exposure groups and the Day 7 cloning efficiencies in the 4-hour exposure group in the
presence of S9 did not achieve 70% cloning efficiency in all the replicates, however since they achieved at least 50% this was considered to be
acceptable.

The mutation frequency counts and mean mutation frequency per survivor per 106 cells values are presented in the attached Tables 4 and 5 (attached background matetial). There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10-6 in either exposure group.
I
t can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6 mutants per viable cell, and that the positive
controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

See attached background material for:

Table 1: Preliminary Cytotoxicity Results

Table 2: Experiment 1 - 4 Hour Exposure Without Metabolic Activation (S9)

Table 3: Experiment 1 - 4 Hour Exposure With Metabolic Activation (S9)

Table 4: Experiment 2 - 24 Hour Exposure Without Metabolic Activation (S9)

Table 5: Experiment 2 - 4 Hour Exposure With Metabolic Activation (S9)

Conclusions:
Interpretation of results: negative.

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.
Executive summary:

Introduction.

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Method B17 of Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the US EPA OPPTS 870.5300 Guideline.. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods.

Chinese hamster ovary (CHO) cells were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4‑hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour 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.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group

Final concentration of test item (µg/ml)

4-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4-hour with S9 (2%)

39.69, 79.38, 158.75, 317.5, 635, 1270

24-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4-hour with S9 (1%)

39.69, 79.38, 158.75, 317.5, 635, 1270

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion. 

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phase of this study was performed between 14 October 2007 and 09 November 2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 1500 and 5000 µg/plate (with and without metabolic activation for TA100).
Mutation Test - Experiment 1: 50, 150, 500, 1500 and 5000 µg/plate. (with and without metabolic activation for all test strains).
Mutation Test - Experiment 2: 50, 150, 500, 1500 and 5000 µg/plate. (with and without metabolic activation for all test strains).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water at 50 mg/ml but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates for TA100 and TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
ENNG used without S9 (3 µg/plate for TA100 and 5 µg/plate for TA1535)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates for TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9AA used without S9 mix (80 µg/plate for TA1537)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates for TA102
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
MMC used without S9 mix (0.5 µg/plate for TA102)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates for TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4NQO used without S9 mix (0.2 µg/plate for TA98)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates for TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
Used with S9 mix (1 µg/plate)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates for TA1535 and TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
Used with S9 mix (2 µg/plate)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates for TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
BP used with S9 mix (5 µg/plate)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates for TA102
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone (DAN)
Remarks:
DAN used with S9 mix (10 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 X 10E9 bacteria per ml.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to the maximum recommended dose level of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST:
The test material was non-toxic to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.
The number of revertant colonies for the toxicity assay were shown in the table in 'any other information on results incl. tables' section.

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (attached background material) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 to Table 5 (see attached background material).

A history profile of vehicle and positive control values is presented in Appendix 1 (see attached background material).

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.








Preliminary Toxicity Test:

The number of revertant colonies for the toxicity assay were:

 

With (+) or without (-) metabolic activation

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

113

75

86

97

86

72

73

81

83

82

79

+

TA100

84

76

95

77

79

92

92

65

65

83

74

Conclusions:
Interpretation of results: negative.

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (l0% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation Test "Ames Test":

Introduction.

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (l0% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

Chromosome Aberration Test in Human Lymphocytes in vitro

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 (Scott et al, 1990).

The test method used was designed to be compatible with the following internationally accepted guidelines and recommendations:

i)            OECD Guidelines for Testing of Chemicals No. 473 "Genetic Toxicology: Chromosome Aberration Test", Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

ii)           UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in theRecommended Procedures for Basic Mutagenicity Tests (1990).

iii)          US EPA OPPTS 870.5375 Guideline.

The test method was designed to be acceptable to the Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test item, 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, a 4-hour exposure 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-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour 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.

 

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final concentration of Test Item (µg/ml)

4(20)-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4(20)-hour with S9 (2%)

39.69, 79.38, 158.75, 317.5, 635, 1270

24-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4(20)-hour with S9 (1%)

39.69, 79.38, 158.75, 317.5, 635, 1270

Results.

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

All the positive control items 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 item 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 the maximum recommended dose level.

Conclusion.

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

CHO HPRT Forward Mutation Assay

Introduction.

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476' In Vitro Mammalian Cell Gene Mutation Tests', Method B17 of Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Coleet al, 1990) and the US EPA OPPTS 870.5300 Guideline.. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods.

Chinese hamster ovary (CHO) cells were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4‑hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour 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.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group

Final concentration of test item (µg/ml)

4-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4-hour with S9 (2%)

39.69, 79.38, 158.75, 317.5, 635, 1270

24-hour without S9

39.69, 79.38, 158.75, 317.5, 635, 1270

4-hour with S9 (1%)

39.69, 79.38, 158.75, 317.5, 635, 1270

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion. 

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.


Justification for selection of genetic toxicity endpoint
Three separate in vitro genetic toxicity studies have been conducted on the test item as follows:

OECD Guideline 471 Bacterial Reverse Mutation Test "Ames Test"
OECD Guideline 473 Chromosome Aberration Test
OECD 476 In Vitro Mammalian Cell Gene Mutation Tests

All 3 studies have been conducted according to OECD Guidelines and GLP and are adequately reported. All studies have been assigned a reliability 1.

Short description of key information:
OECD Guideline 471 Bacterial Reverse Mutation Test "Ames Test":
The test material was considered to be non-mutagenic under the conditions of this test.

OECD Guideline 473 Chromosome Aberration Test in Human Lymphocytes in vitro:
The test item was considered to be non-clastogenic to human lymphocytes in vitro.

OECD 476 In Vitro Mammalian Cell Gene Mutation Tests (CHO HPRT Forward Mutation Assay):
The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on negative results in the three following in-vitro studies, the substance is not classified for mutagencity.

 

- Reverse mutation assay 'Ames Test'

The test item was considered to be non-mutagenic under the conditions of this test.

- Chromosome aberration test in human lymphocytes:

The test item is considered to be non-clastogenic to human lymphocytes in vitro

- CHO HPRT forward mutation assay:

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.