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Genetic toxicity in vitro

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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:
2002-08-30 to 2003-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
June 08, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
This in vitro experiment assesses the potential of the test item to induce gene mutations by means of a Thymidine Kinase assay using the mouse lymphoma cell line L5178Y. The Thymidine Kinase (TK) system detects base pair mutations, frameshift mutations, small deletions as well as large, non lethal deletions and rearrangements of the relevant chromosomes.
Cells deficient in the heterozygous TK-Iocus due to the forward mutation TK+/- to TK-/- are resistant to the cytotoxic effects of pyrimidine analogues such as trifluorothymidine (TFT).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 fractions obtained from livers of male Wistar rats which were induced with Phenobarbital (80 mg/kg b.w.) and ß-Naphthoflavone (100 mg/kg b.w.). Final protein concentration of 0.75 mg/mI in the cultures.
Test concentrations with justification for top dose:
The selection of the concentrations was based on data from a pre-experiment.

Experiment I
- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/ml
- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/ml
Experiment II
- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/ml
- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: experiment I short time incubation 4 hours and experiment II long-term incubation 24 hours
- Expression time (cells in growth medium): 72 hours in experiment I and 48 h in experiment II
- Selection time (if incubation with a selection agent): 11 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 17 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: 300000/plate seeded

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, cloning efficiency
Evaluation criteria:
There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency,
- biologically relevant response (at least 2-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) for at least one of the dose groups.
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ratio (< 4), is an indication for potential clastogenic effects and/or chromosomal aberrations.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at
any dose level.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations >/= 16 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The selection of the concentrations was based on data from the pre-experiment. Precipitation of the test item in cell culture medium was observed at concentrations of 50.0 µg/ml and higher. The pH-value detected at a test item concentration of 200 µg/ml was 7.2.

TOXICITY:

Growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 6.85 % for the highest concentration (60.0 µg/ml) evaluated. The highest biologically relevant dose group evaluated with metabolic activation was 40.0 µg/ml with a RTG of 16.19 %. The highest concentration investigated without metabolic activation was 30.0 µg/ml with a RTG of 7.62 %. The highest biologically relevant concentration evaluated without metabolic activation was 20.0 µg/ml with a RTG of 10.19 %.

In experiment II with metabolic activation the relative total growth (RTG) was 13.04 % for the highest concentration (55.0 µg/ml) evaluated. The highest biologically relevant concentration evaluated without metabolic activation was 16.0 g/ml with a RTG of 17.14 %.

MUTAGENICITY:
In experiment I with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the controls.

Mutation frequencies with the negative controls were 66.39 - 100.61 mutants/10exp6 cells. The values of the solvent controls were found to be 64.18 - 90.07 mutants/10exp6 cells and 46.56 - 133.57 mutants/10exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.73 was found at a concentration of 60.0 µg/ml with a RTG of 6.85 %. The mutation factor found at this dose group was considered as not biologically relevant due to the high toxicity found. The highest biologically relevant concentration evaluated was 40.0 µg/ml and showed a mutation factor of 1.07.

In experiment I without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells) except the value (172.70 mutants/10exp6 cells) found at a dose of 25.0 µg/ml. No dose dependency could be observed. Due to the high cytotoxicity of 8.93 % RTG at 25.0 µg/ml the increased mutation factor (above the historical control data) was considered as not biologically relevant. Moreover, if the spontaneous mutant frequency (negative control) is > 100 mutants/10exp6 cell, a doubling of the spontaneous frequency should be required for a definitive positive response. The mutation factor found at a dose of 25.0 µg/ml was 1.45, thus below the threshold value of 2.

Mutation frequencies with the negative controls were 100.99 - 136.72 mutants/10exp6 cells. The values of the solvent controls were found to be 102.49 - 135.07 mutant/10exp6 cells and 65.22 - 172.70 mutants/10exp 6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.45 was found at a concentration of 25.0 µg/ml with a RTG of 8.93 %. The highest biologically relevant dose group
evaluated (20.0 µg/ml) showed a mutation factor of 1.02.

In experiment II with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the controls.

Mutation frequencies with the negative controls were 64.80 - 68.94 mutants/10exp6 cells. The values of the solvent controls were found to be 62.71 - 103.15 mutants/10exp6 cells and 40.30 - 88.61 mutants/10exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.07 was found at a concentration of 15.0 µg/ml with a RTG of 91.69 %.

In experiment II without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation factor of 2, which was found at a dose of 0.50 µg/ml, was considered as not relevant due to the mutant frequency of 136.84 mutants/10exp6 cells, which was within the historical control data.

Mutation frequencies with the negative controls were 76.86 - 111.59 mutants/10exp6 cells. The values of the solvent controls were found to be 64.67 - 72.05 mutants/l0exp6 cells and 49.86 - 136.84 mutants/l0exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 2.00 was found at a concentration of 0.50 µg/ml with a RTG of 83.16 %.

EMS (80 µg/ml and 400 µg/ml) and B[a]P (1.50 µg/ml) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

RELATIONSHIP TO LARGE AND SMALL COLONIES:

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colonies ratio (< 4), is an indication for potential clastogenic effects and/or chromosomal aberrations.

In experiment I, with metabolic activation the quotients of large/small colonies of the negative controls were found to be 14.00 and 73.00. For the
solvent controls the quotients noticed were 7.43 and 10.33. The quotient of the highest dose groups were found to be 17.00 (15.0 µg/ml), 14.50
(20.0 µg/ml), 15.00 (40.0 µg/ml) and 10.38 (60.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in all dose groups evaluated.

In experiment I without metabolic activation the quotients of large/small colonies of the negative controls were found to be 12.60 and 26.00. For the
solvent controls the quotients noticed were 5.91 and 10.43. The quotients of the highest dose groups were found to be 14.50 (18.0 µg/ml), 7.89
(20.0 µg/ml), 4.89 (25.0 µg/mI) and 7.45 (30.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in
all dose groups evaluated.

In experiment II with metabolic activation the quotients of large/small colonies of the negative controls were found to be 7.38 and 11.60. For the
solvent controls the quotients noticed were 7.67 and 9.40. The quotients of the highest dose groups were found to be 18.00 (40.0 µg/ml), 6.43
(45.0 µg/ml), 17.00 (50.0 µg/ml) and 10.60 (55.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in all dose groups evaluated.

In experiment II without metabolic activation the quotients of large/small colonies of the negative controls were found to be 7.33 and 8.89. For the
solvent controls the quotients noticed were 4.44 and 6.70. The quotients of the highest dose groups were found to be 9.67 (8.00 µg/ml), 7.63 (10.0 µg/ml), 12.25 (12.0 µg/ml) and 5.25 (16.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in
all dose groups evaluated.

CONCLUSION

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Rewoquat V 2815 is considered to be
non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Conclusions:
Interpretation of results:
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item oleic-acid based IQAC, DMS quaternised is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Executive summary:

In a mammalian gene mutation assay, L5178 Y (mouse lympoma thymidine kinase locus) cells cultured in vitro were exposed to the oleic-acid based IQAC; DMS quaternised (98 % a.i.) at the following concentrations:

Experiment I

- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/ml

- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/ml

Experiment II

- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/ml

- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/ml

The oleic-acid based IQAC, DMS quaternised was tested up to cytotoxic concentrations (>/= 16.0 µg/ml without metaboic activation and >/= 40.0 µg/ ml with metabolic activation). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of increased number of induced mutant colonies over background.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2007-10-11 to 2007-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L 1362000, Annex 4A: ”Mutagenicity – In vitro Mammalian Chromosome Aberration Test“, May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chromosome aberration assays detect the induction of chromosome breakage (clastogenesis). Although mutagenic substances produce structural
chromosome aberrations by a variety of mechanisms, the endpoint is a discontinuity in the chromosomal DNA which is left unrejoined, or rejoined
inaccurately, thus producing a mutated chromosome. Chromosome aberrations are generally evaluated in first post treatment mitoses. The majority of chemical mutagens induce aberration of the chromatid type, but chromosome type aberrations also occur.
Species / strain / cell type:
other: human lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy donors not receiving medication.
Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks.
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1; Life Technologies GmbH, 76339 Eggenstein, Germany) containing 10 % FCS (fetal calf serum) provided by PAA Laboratories GmbH (35091 Cölbe, Germany), the antibiotic solution containing 10,000 U/mL penicillin and 10,000 µg/mL streptomycin (SEROMED, D-12247 Berlin). Additionally, the medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), the anticoagulant heparin (25,000 U.S.P.-U/mL, NATTERMANN, 50829 Köln, Germany), and
HEPES (final concentration 10 mM, Serva, 69115 Heidelberg, Germany).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar HanIbm rats.
Test concentrations with justification for top dose:
Experiment I
with metabolic activation: 11.9, 20.8, 36.4, 63.7, 111.4, 195.0, 341.2, 597.1, 1044.9, 1828.6 and 3200 µg/mL
without metabolic activation: 20.8, 36.4, 63.7, 111.4, 195.0, 341.2, 597.1, 1044.9, 1828.6 and 3200 µg/mL

Experiment II
with metabolic activation: 10.0, 20.0, 50.0, 125.0, 250.0, 500.0, 750.0, 1000.0, 1250.0 and 1500.0 µg/mL
without metabolic activation: 8.1, 14.2, 25.9, 43.5, 76.2, 133.3, 233.2, 408.2, 714.3 and 1250 µg/mL
Results from the rangefinding assay were used to determine the dose range to be used in the chromosomal aberrations assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol 0.5%
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Range-finder
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by
the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay.
The pre-test phase was performed with 10 concentrations without S9 mix and 11 concentrations with S9 mix of the test item and a solvent and
positive control. All cell cultures were set up in duplicate. Exposure times were 4 hrs (with and without S9 mix). The preparation interval was 22 hrs
after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 µg/mL) to reassure the replication time of the cultured lymphocytes.

Dose Selection
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests
requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation.
In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate
solvent is possible.
With respect to the ability to formulate a homogeneous suspension of the test item, 3200 µg/mL of test substance
(approx. 4.8 mM) were applied as top concentration for treatment of the cultures in the pre-test. Doses over 3200 µg/mL led to an inhomogeneous
suspension in ethanol that was not applicable. Test item concentrations between 11.9 and 3200 µg/mL, and between 20.8 and 3200 µg/mL (with and without S9 mix, respectively) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed
before start of treatment at 36.4 µg/mL and above in the absence of S9 mix, and at 20.8 µg/mL and above in the presence of S9 mix. Since the
cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, toxic effects of about 50 % of control were observed after 4 hrs treatment
with 597.1 µg/mL and above in the absence and presence of S9 mix. Considering these toxicity data, 1250 µg/mL (without S9 mix) and 1500 µg/mL
(with S9 mix) were chosen as top concentrations in Experiment II.
The cytogenetic evaluation of higher concentrations in the respective preparation interval (with and without S9 mix) was impossible due to strong test item-induced toxic effects (low metaphase numbers, partially paralleled by poor metaphase quality and precipitation on the slides).

Exposure time 4 hours
The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test item.
For the treatment with metabolic activation 50 µL S9 mix per mL medium were used.
Concurrent solvent and positive controls were performed. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The
supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G" solution (NaCl -8000 mg, KCl -400 mg, glucose x H2O -1100 mg, Na2HPO47H2O - 290 mg, KH2PO4 --150 mg). The washing procedure was repeated once as described.
After washing the cells were re-suspended in complete culture medium and cultured until preparation.

Exposure time 22 hours (without S9 mix)
The culture medium was replaced with complete medium (with 10 % FCS) containing the test item without S9 mix. The culture medium at continuous
treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
All cultures were incubated at 37° C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

Preparation of the Cultures
Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by
dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa (MERCK, 64293 Darmstadt, Germany) or according to the Fluorescent plus Giemsa technique, respectively.

Analysis of Metaphase Cells
The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" [4]) using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. At least 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphase plates were scored. Only metaphases with 46 +/-1 centromer regions were included in the analysis. To describe a
cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 250 metaphase cells (% polyploid
metaphases) was scored.
Evaluation criteria:
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 %
aberrant cells, excluding gaps).
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding
gaps) and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be
considered together. If the criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Key result
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
With respect to the ability to formulate a homogeneous suspension of the test item, 3200 µg/mL of the test substance (approx. 4.8 mM) were applied as top concentration for treatment of the cultures in the pre-test. Doses over 3200 µg/mL led to an inhomogeneous suspension in ethanol that was not applicable.
Test item concentrations between 11.9 and 3200 µg/mL, and between 20.8 and 3200 µg/mL (with and without S9 mix, respectively) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed before start of treatment at 36.4 µg/mL and above in the absence of S9 mix, and at 20.8 µg/mL and above in the presence of S9 mix.

Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.
 

Polyploid metaphases:
Polyploid metaphases occured, but in both experiments, no biologically relevant increase in the rate of polyploid metaphases was found
after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.8 %).


POSITIVE CONTROLS
In both experiments, EMS (880 and 770 µg/mL, respectively) and CPA (37.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

 

Summary of results of the chromosomal aberration study with PC-2007-140 (W 575 no solvent)

Exp.

Preparation interval

Test item concentration in µg/mL

Polyploid cells in %

Mitotic indices in % of control

Incl. gaps

Aberrant cells in % excl. gaps

With exchanges

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control

0.8

100.0

2.0

2.0

1.0

 

Positive control

0.2

64.2

10.0

9.0S

0.0

 

20.8

0.2

73.4

3.0

2.0

0.0

 

36.4 P

0.4

82.6

2.5

1.5

0.5

 

341.2 P

0.2

61.3

2.0

1.5

0.0

 

597.1 P

0.4

50.5

0.5

0.5

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control

0.4

100.0

2.0

2.0

0.0

 

Positive# control

0.0

25.3

50.0

49.0S

11.0

 

43.5##

0.2

72.3

5.0

4.5

0.0

 

76.2 P

0.2

67.9

0.0

0.0

0.0

 

133.3 P

0.0

53.6

3.0

3.0

0.0

 

233.2 P

0.0

29.2

2.5

2.0

0.0

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control

0.2

100.0

2.0

1.5

0.0

 

Positive control

0.4

49.7

8.5

 8.5S

2.0

 

11.9

0.0

84.7

1.0

1.0

0.0

 

20.8 P

0.4

88.9

1.5

1.0

0.5

 

341.2 P

0.6

65.3

0.0

0.0

0.0

 

597.1 P

0.4

73.4

2.5

2.0

0.0

II

22 hrs

Solvent control

 

0.0

 

100.0

 

2.0

 

1.0

 

0.0

 

Positive control

 

0.0

 

34.6

 

22.0

   20.0S

 

1.0

 

20.0

0.0

120.1

1.5

1.5

0.0

 

50.0 P##

0.0

111.0

3.5

  3.3S

0.0

 

125.0 P

0.0

110.2

1.5

1.5

0.0

 

P -Test item precipitation was observed

S - Aberration frequency statistically significant higher than corresponding control values

# - Evaluation of 50 metaphases per culture

## - Evaluation of 200 metaphases per culture

Conclusions:

It can be stated that under the experimental conditions reported, the test itempartially unsaturated IQAC, DMS quaternised (no solvent) did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.
Executive summary:

In a mammalian chromosome aberration test performed according to OECD Guideline 473 (1997), human lymphocyte cultures were exposed to partially unsaturated IQAC, DMS quaternised (no solvent), (100% a.i.), suspended in ethanol at concentrations between 11.9 - 3200 µg/mL with metabolic activation and 20.8 - 3200 µg/mL without metabolic activation.

Partially unsaturated IQAC, DMS quaternised (no solvent) was tested up to cytotoxic or precipitating concentrations. 

The following experimental points were microscopically evaluated:11.9, 20.0, 20.8, 50.0, 125.0, 341.2, 597.1 µg/mL with metabolic activation and 20.8, 36.4, 43.5, 76.2, 133.3, 233.2, 341.2, 597.1 µg/mL without metabolic activation.

In the absence of S9 mix, reduced mitotic indices of about or below 50 % of control were observed at the highest evaluated concentrations. In the presence of S9 mix, concentrations showing clear cytotoxic effects were excluded from scoring for the endpoint cytogenicity.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.

 Positive controls induced the appropriate response. There was no evidence of Chromosome Aberration induced over background.

This study is classified as acceptable. It satisfies the requirement for Test Guideline In vitro mammalian Chromosome Aberration test OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2010-07-26 to 2010-08-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and Tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Metabolic activation system was derived from an Aroclor 1254 induced rat liver post mitochondrial fraction (S9), microsomal protein-content: 36.7 mg/mL.
Test concentrations with justification for top dose:
The concentrations tested were 0 µg/plate (control), 62, 185, 556, 1667 and 5000 µg/plate in the first experiment and 0 µg/plate (control), 5, 15, 44, 133 and 400 µg/plate in the second experiment.
Vehicle / solvent:
The test concentrations were prepared by solving the test substance in acetone.
Untreated negative controls:
yes
Remarks:
with and without metabolic activation system
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2- aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: the test was performed by the direct plate incorporation method

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 2

NUMBER OF PLATES EVALUATED: three per dose

DETERMINATION OF CYTOTOXICITY
- Method: The criterion for a biologically significant bacteriotoxic effect is a either a reduction of the survival of the cells to at least 50% or a reduction of the background growth of auxotrophic cells and the number of revertants.



Evaluation criteria:
A test substance producing no biologically relevant positive response in any one of the bacterial strains tested is considered to be non-mutagenic in this system.
A biologically relevant response is described as follows:
If the number of revertants is at least twice (three times in the case of TA 1537) the spontaneous reversion rate in one of the strains and/or if there is a concentration related increasing number of revertants over the range tested.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no biologically relevant increases in revertant colony numbers of any of the five test strains was observed at any concentration level, neither in the presence nor absence of metabolic activation
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity effects started at around 100 µg test substance/plate depending on the test strain and the absence or presence of the metabolic activation system.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
STERILITY CONTROLS
The strain characteristics of the stock cultures (rfa-character, uvrA/uvrB-deletion and resistance to ampicillin and tetracycline) had been confirmed.

NEGATIE CONTROLS
Negative (solvent) and positive control treatments were included for all strains in both experiments. All mean numbers of revertant colonies on negative control plates fell within the ranges of the historical control data and were significantly elevated by positive control treatments.

POSITIVE CONTROLS
The positive control chemicals induced without exceptions large increases in the revertant numbers in the corresponding strains.

TOXICITY
Toxicity was observed with and without S9 supplementation. Toxicity of the test substance was detected by a reduction in the number of revertant colonies attended by a clearing of the background lawn. The highest concentration level without toxic effects in any of the test strains was 62 µg test substance / plate. Due to the toxicity of the test substance the lower concentrations were chosen for the second experiment.
In the second experiment toxicity was observed with and without S9 supplementation. The toxicity effects started at around 100 µg test substance/plate depending on the test strain and the absence or presence of the metabolic activation system.

Concentration levels of the test substance tested up to the cytotoxic concentration did not show any mutagenic effects neither in the absence nor in the presence of S9 supplementation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The mean values are presented with the underlying individual plate counts recorded in the appendices. Revertant counts higher than 1000 were counted and calculated as 1000.

Conclusions:
Interpretation of results:
negative


No biologically relevant increases in revertant colony numbers of any of the five test strains were observed at any concentration level, neither in the presence nor absence of metabolic activation. Toxic concentration levels of the test substance resulted in a decrease in the number of revertant colonies. Concentration levels of the test substance tested up to the cytotoxic concentration did not show any mutagenic effects neither in the absence nor in the presence of S9 supplementation. Therefore the test substance is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA according to the OECD Guideline No. 471 (1997) were exposed to the oleic-acid based IQAC, DES quaternised, (100 % active matter (99 % solid matter), diluted with acetone) at concentrations of 5, 15, 44, 62, 133, 185, 400, 556, 1667 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

The oleic-acid based IQAC, DES quaternised was tested up to cytotoxic concentrations (400 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of an increased number of induced mutant colonies over background. This study is classified as acceptable.

This study satisfies the requirement for the OECD Guideline No. 471 (1997) for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1988-10-10 to 1988-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US Toxic Substances Control Act (TSCA) Final Rule, EPA (1985)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chromosome aberration assays detect the induction of chromosome breakage (clastogenesis). Although mutagenic substances produce structural chromosome aberrations by a variety of mechanisms, the endpoint is a discontinuity in the chromosomal DNA which is left unrejoined, or rejoined inaccurately, thus producing a mutated chromosome. Chromosome aberrations are generally evaluated in first post treatment mitoses. The majority of chemical mutagens induce aberration of the chromatid type, but chromosome type aberrations also occur.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5a culture medium supplemented with 10% FCS (foetal calf serum) 1% L-glutamine, and 1% penicillin and streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fractions obtained from livers of male Sprague-Dawley rats induced with Aroclor 1254 (commercially purchased)
Test concentrations with justification for top dose:
with metabolic activation: 15.0 to 199 µl/ml
without metabolic activation: 3.74 to 74.8 µl/ml
microscopic analyses:
with metabolic activation: 49.9, 99.7, 150 and 199 µl/ml
without metabolic activation: 24.9, 37.4, 49.9 and 74.8 µl/ml
Results from the range finding assay were used to determine the dose range to be used in the chromosomal aberrations assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Range finding Assays:

In these assays, the cells were cultured for approximately 24 hours prior to treatment by seeding approximately 0.3 x 10exp6 cells per 25 cm² flask into 5 ml of complete McCoy's 5a culture medium. The thymidine analogue, 5-bromo-2'-deoxyuridine (BrUdR), was added at a final concentration of 10 µM approximately two hours after the initial exposure of the cells to the test article.

Range finding Nonactivation Assay:
- The cultures were dosed with the test article for 2 hours when 5-bromo-2'-deoxyuridine (BrUdR) was added at a final concentration of 10 µM. The cultures were then reincubated for approximately 23 hours. Approximately 2.75 hours prior to the harvest of the cells, the test article was washed from the cells with phosphate buffered saline and fresh complete medium with BrUdR (10 µM), and Colcemid® (final concentration 0.1 µg/ml) was added. The cultures were then harvested and were differentially stained for the analysis of cell cycle delay using a modified fluorescence-plus-Giemsa (FPG) technique.

Range finding Assay with Metabolic Activation:
- In this assay, the CHO cells were exposed to the test article for two hours in the presence of a rat liver S9 reaction mixture (S9 15 µl /ml, NADP 1.5 mg/ml, and isocitric acid 2.7 mg/ml). The S9 fraction was derived from the liver of male Sprague-Dawley rats which had been previously treated with Aroclor 1254 to induce the mixed function oxidase enzymes which are capable of metabolizing chemicals to more active forms. The two hour incubation time was used because prolonged exposure to the S9 mixture might be toxic to the cells and the enzyme activity of S9 is lost rapidly at 37°C.
The medium did not have FCS during the exposure period to avoid possible inactivation of short-lived and highly reactive intermediates produced by the S9 enzymes by binding to serum proteins. In this assay, the CHO cells were incubated at 37°C for two hours in the presence of the test article and the S9 reaction mixture in the growth medium without FCS. After the exposure period the cells were washed twice with buffered saline. Complete McCoy's 5a medium with 10 µM BrUdR was added to the cultures which were then incubated for approximately 23 hours. Colcemid® (final concentration of 0.1 µg/ml) was then added for 2.5 hours to collect metaphase cells. The cultures were then harvested, fixed, and slides were prepared and
stained as was described for the nonactivation range finding assay.

Range finding Assay Evaluation:
- The highest three doses with metaphase cells in both trials were analyzed for toxicity (visual observations of monolayer confluence) and cell cycle kinetics. One hundred consecutive metaphase plates, if available, were assessed for the number of cell cycles through which the cells had progressed while in the presence of BrUdR.

Main test:

- Nonactivation Aberrations Assays:
Cultures were initiated by seeding approximately 1.5 x 10exp6 cells per 75 cm² flask into 10 ml of complete McCoy's 5a medium). One day after culture initiation, the CHO cells to be used in the nonactivation trial were treated with the test article at predetermined doses for 7.25 hours. The cultures were then washed with buffered saline and complete McCoy's 5a medium containing 0.1 µg/ml Colcemid® was placed back onto the cells. Two and one half hours later the cells were harvested and air dried slides were made. The slides were then stained in pH 6.8 buffered 5% Giemsa solution for the analysis of chromosomal aberrations.

- Aberrations Assays with Metabolic Activation:
Cultures were initiated by seeding approximately 1.5 x 10exp cells per 75 cm² flask into 10 ml of complete McCoy's 5a medium. One day after culture initiation. the cultures that were treated under the conditions of metabolic activation were incubated at 37°C for two hours in the presence of the test article and the S9 reaction mixture in McCoy's 5a medium without FCS. After the two hour exposure period the cells were washed twice with buffered saline and the cells were refed with complete McCoy's 5a medium. The cells were incubated for an additional 7.75 hours with 0.1 µg/ml Colcemid®present during the last 2.5 hours of incubation. The metaphase cells were then harvested and prepared for cytogenetic analysis.
NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED:
At least 200 well spread metaphases per concentration and negative/positive controls were scored for cytogenetic damage. The cells scored contained 21 ± 2 centromeres.

HARVEST PROCEDURE AND DETERMINATION OF CYTOTOXICITY
- Prior to the harvest of the cultures visual observations of toxicity were made. These observations included an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic (large rounded cells) or dead cells floating in the medium. The metaphase cells were collected by mitotic shake-off (Terasima and Tolmach, 1961) and were treated with 0.075 M KCl hypotonic solution. This treatment helps to swell the cells and thus disperse the chromosomes. The cultures were then fixed with an absolute methanol: glacial acetic acid (3:1) fixative and were washed several times before air-dried slides were prepared.

SLIDE PREPARATION AND STAINING
-Slides were prepared by dropping the harvested cultures on clean slides. The slides from the range finding assays were differentially stained using a modified fluorescence-plus-Giemsa (FPG) technique (after Perry and Wolff, 1974; Goto, et al., 1978). The slides were stained in Hoechst 33258 stain, exposed to ultraviolet light, and then stained with Giemsa Azure B stain. The slides prepared from the aberrations assay were stained with pH 6.8
buffered 5% Giemsa solution for the analysis of chromosomal aberrations. All slides were then air-dried and coverslipped using Depex@ mounting medium.

ABERRATION ANALYSIS
- Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 (range 19-23) were analyzed. One hundred cells, if possible, from each duplicate culture at four dose levels of the test article and from each of the negative and solvent control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962). At least 25 cells were analyzed for chromosomal aberrations from one of the positive control culture. For control of bias, all slides except for the positive controls were coded prior to analysis. Cells with aberrations, but not gaps, were recorded on the data sheets by the microscope stage location.

ACCEPTABILITY OF THE ASSAY
An assay will be considered acceptable for evaluation of test results only if all of the following criteria are satisfied. Activation and nonactivation sections of the aberrations assay form independent units and will be repeated independently, as needed, to satisfy the acceptance criteria.
- Unsatisfactory Controls
The assay will be repeated if:
a. The negative or the solvent control is more than twice the upper limit of the range of historical control values (upper limit 5% or more of cells with aberrations).
b. The positive control result is not significantly higher (p<0.01)) than the pooled negative and solvent controls. If the positive control result in the test with S9 is adequate in an assay where activation and nonactivation assays are run concurrently but the positive-control in the nonactivation assay fails, the test need not be repeated as the activation positive control demonstrates . the sensitivity of the cells.

- Lack of Toxicity
If the aberration results are negative and there is no significant reduction in confluence or delay in cell cycle progression, the test may be repeated at higher doses if higher doses can be achieved (i.e. not limited by solubility or not greater than 5 mg/ml).

- Excessive Toxicity
The assay will be repeated if results are not available for at least three dose levels. Data will not be evaluated from dose levels with excessive toxicity. Any culture flask with less confluence of the monolayer than observed at the time of dosing will be deemed to have excessive toxicity. Those cultures from which 200 cells were not available for analysis from the duplicate flasks will also be considered to have cytotoxic effects.

- Sporadic Increase
If a significant increase is seen at one or more dose levels but not in consecutive doses and if there is no clear evidence for a positive dose response, the assay will be repeated to verify the significance.

If the assay has to be repeated because of equivocal results, only three dose levels will be analyzed, instead of four, in the second assay.
Evaluation criteria:
The following factors were taken into account in the evaluation of the chromosomal aberrations data:
1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage or cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response.
5. The estimated number of breaks involved in the production of the different types of aberrations which were observed, i.e., complex aberrations may have more significance than simple breaks.

Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test article to induce chromosomal aberrations since they may not represent true chromosomal breaks and may possibly be induced by toxicity.
Statistics:
Statistical analysis employed the Fisher's Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation). Test article significance was established where P
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation: >/= 37.4 µl/ml, with metabolic activation: >/= 99.7 µl/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PREEXPERIMENTS on SOLUBILITY, CELL CYCLE KINETICS, and DOSE DETERMINATION
Solubility was evaluated in water and unpipettable, gelatinous mixtures were obtained at 204, 305, and 500 mg/ml. In an assay that was conducted concurrently, a cloudy white liquid was obtained in water at 52.9 mg/ml. This suspension was considered suitable for use in this assay. Therefore, for this assay a stock concentration of 49.6 mg/ml of the test article was prepared in deionized water. Final concentrations were achieved by a 1:10 dosing of the prepared stock solution or the serial dilutions prepared from this stock. A half-log series of concentrations of 0.165 to 4960 µg/ml was tested in the range finding assays.

- Range finding Assay Without Metabolic Activation
A precipitate was visible at the time of dosing and harvest at 165, 496, 1650, and 4960 µg/ml. Dead cell monolayer with no visible mitotic cells were observed at these concentrations and these cultures were not harvested. An unhealthy cell monolayer, 60% reduction in the monolayer confluence, floating dead cells, and a reduction in visible mitotic cells were observed at 49.6 µg/ml. A slight reduction in visible mitotic cells was still discerned at 16.5 µg/ml. Cell cycle kinetics were evaluated at 4.96, 16.5, and 49.6 µg/ml. No significant cell cycle delay was evident at any of the concentrations analyzed. A 10 hour harvest was selected for testing concentrations of 3.74, 4.99, 12.5, 24.9, 37.4, 49.9, and 74.8 µg/ml in the aberrations assay using a 1:100 dosing volume of the 7.48 mg/ml solution prepared from a 19.9 mg/ml solution of the test article or serial dilutions prepared from the 7.48 mg/ml solution.
- Range finding Assay With Metabolic Activation
A precipitate was visible at the time of dosing and harvest at 496, 1650, and 4960 µg/ml. Floating dead cells, floating debris, no visible mitotic cells, and no visible cell monolayer were observed at these concentrations and the cultures were not harvested. At 165 µg/ml, an unhealthy cell monolayer, slight reduction in visible mitotic cells, and 50% reduction in cell monolayer confluence were discerned. Cell cycle kinetics were evaluated at 16.5, 49.5, and 165 µg/ml. No cell cycle delay was evident at these concentrations. A 10 hour harvest was selected for testing concentrations of 15.0, 19.9, 49.9, 99.7, 150, and 199 µg/ml using a 1:100 dosing volume of the 19.9 mg/ml stock solution or the serial dilutions prepared from the stock solution.

CLASTOGENICITY

- Chromosomal Aberrations Assay Without Metabolic Activation
No precipitate was visible after dosing at any of the concentrations. A precipitate was visible at the time of harvest at 49.9 and 74.8 µg/ml, the two highest concentrations evaluated in this test. An unhealthy cell monolayer, reductions in the cell monolayer confluence (30% at 49.9 and 40% at 74.8 µg/ml), and a severe reduction in visible mitotic cells were observed at 49.9 and 74.8 µg/ml. A slightly unhealthy cell monolayer, 30% reduction in the cell monolayer confluency, and reduction in visible mitotic cells were observed at 37.4 µg/ml. Results were evaluated at 24.9, 37.4, 49.9, and 74.8 µg/ml.
A significant increase in chromosomally aberrant cells was not observed at the concentrations analyzed.
The test article is considered negative for inducing chromosomal aberrations under nonactivation conditions.

- Chromosomal Aberrations Assay With Metabolic Activation
No precipitate was visible after dosing at any of the concentrations. A precipitate was visible at the time of harvest at 49.9, 99.7, 150, and 199 µg/ml. Floating dead cells, floating debris, unhealthy cell monolayer, severe reduction in visible mitotic cells, and reductions in the cell monolayer confluency (30% at 99.7 and 150 µg/ml, and 40% at 199 µg/ml) were observed at 99.7, 150, and 199 µg/ml. A slightly unhealthy cell monolayer, floating debris, and a slight reduction in visible mitotic cells were observed at 49.9 µg/ml. Results were analyzed at 49.9, 99.7, 150, and 199 µg/ml.
There was not a significant increase in chromosomally aberrant cells at the doses analyzed.
The successful activation of the metabolic system is illustrated by the increased incidence of chromosomally aberrant cells in the cultures induced with cyclophosphamide, the positive control agent.
The test article is considered negative for inducing chromosomal aberrations under conditions of metabolic activation.

POSITIVE CONTROLS
Mitomycin C for the nonactivation series and cyclophosphamide in the metabolic activation series were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberration.
Conclusions:
Interpretation of results:
negative

It can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item partially unsaturated IQAC, DMS quaternised (75 %) did not induce structural chromosomal aberrations in the CHO Chinese hamster cell line.

Therefore, the test item Varisoft 475 (methyl-1-tallow amido ethyl-2-tallow imidazolium-methyl sulfate, 75 %) is considered to be non-clastogenic.
Executive summary:

In a mammalian cell cytogenetics assay according to OECD Guideline 473, 1983 and US EPA-TSCA Guidelines, CHO cell cultures were exposed to the patially unsaturated IQAC, DMS quaternised (75 %) at concentrations of 15.0 to 199 µl/ml with metabolic activation and at 3.74 to 74.8 µl/ml without metabolic activation. Evaluation for aberrant cells was performed at for aberrant cells at 49.9, 99.7, 150 and 199 µl/ml with metabolic activation and at 24.9, 37.4, 49.9 and 74.8 µl/ml without metabolic activation.

Cytotoxic effects of the test item were observed with and without metabolic activation; at the highest evaluated concentrations reduction in the cell monolayer confluency was 40 % in both experiments, without and with metabolic activation.

Neither without metabolic activation nor with metabolic activation a significant increase in chromosomally aberrant cells was observed at the concentrations analyzed.

As positive controls reference mutagens Mitomycin C and cyclophosphamide were tested in parallel to the test item. They induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

There was no evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement of the mentioned guidelines for in vitro mammalian

cytogenetics assays.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998-06-19 to 1998-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Dec. 29 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Tryptophan and Histidine locus
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The 9000 g supernatant of rat-liver homogenate (phenobarbital/ß-naphtoflavone incuded) purchased by CCR GmbH & Co. KG, D-64380 Roßdorf , was used in assays with metabolic activation. The lot number was 30797, microsomal protein-content was 24.7 mg/ml.
Test concentrations with justification for top dose:
Range finding test: Concentration µg/plate: 19.53, 39.06, 78.13, 156.25, 312.50, 625, 1250, 2500, 5000
Main test: In two independent mutation experiments, cells were exposed to concentrations of 125, 250, 500, 1000, 2000 µg/plate (1st assay) and 156.25, 312,50, 625, 1250, 2500 µg/plate (2nd assay) in presence and absence of S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Aqua dest.
- Justification for choice of solvent/vehicle: 50 mg of test substance could be dissolved in 1 ml Aqua dest.
Untreated negative controls:
yes
Remarks:
for all strains with Aqua dest., with and without metabolic activation system
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h


SELECTION AGENT (mutation assays): histidine


NUMBER OF REPLICATIONS: 2


NUMBER OF PLATES EVALUATED: three per dose


DETERMINATION OF CYTOTOXICITY
- Method: The criterion for a biologically significant bacteriotoxic effect is a either a reduction of the survival of the cells to at least 50% or a reduction of the background growth of auxotrophic cells and the number of revertants.



Evaluation criteria:
Selection of the Test Concentrations
The highest concentration used in the test must either reduce the survival of the cells to at least 50% or reduce the background growth of auxotrophic cells and the number of revertants.
If no toxicity is observed, the survey is carried out with the highest soluble concentration of the test article, however, not exceeding 5 mg/plate for solids and 10 mM of liquids or 200 µl of extracts/plate. The concentration intervals between the test doses have a factor of 5.

Analysis of Results
In the Ames assay a test compound is considered as mutagenic if there is a dose dependent increase in the number of revertant colonies of one or more strains. For the highest non-toxic concentration an increase should be by a factor of about 2 and deemed to be biologically relevant. Any evidence of mutagenic activity must be reproducible in an independent experiment. In addition statistical evaluation may aid data interpretation.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
<= 2500 µg/plate in preliminary tests on TA 100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
<= 2500 µg/plate in preliminary tests on TA 100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
<= 2500 µg/plate in preliminary tests on TA 100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
<= 2500 µg/plate in preliminary tests on TA 100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
37% at 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test compound reduced the survival of the strain TA 100 at a concentration of 2500 µg per plate to 0.6 % of the control value.
The number of the his--cells of the strain TA 100 at the concentration of 5000 µg/plate was reduced to 15.9 % of the control value. In addition the background growth is reduced at concentrations from 1250 to 5000 µg/plate.
Precipitation of the test compound was observed from concentrations for 1250 to 5000 µg/plate. Therefore the mutagenicity assays were performed in a concentration range from 125 to 2500 µg per plate.


MAIN TEST
Concentrations between 125 and 2500 µg/plate were applied to the bacterial strains TA 1535, TA 1537, TA 98 and TA 100.

No evidence of biologically significant mutagenic activity of test substance was found. The results of the 1st and the 2nd assay indicate, that in the tested concentration range with and without metabolic activation no significant increase in the numbers of his+- or trp+-revertants over the spontaneous values could be detected with the Salmonella tester strains TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvrA pkm (101).

Slight bacteriotoxicity was observed for E. coli WP2 uvr A pKM 101 at the highest concentration tested (2500 µg/plate in the second experiment.

STERILITY CONTROLS
The plates for the sterility control of top agar, S9-mix and test compound showed no growth.

NEGATIE CONTROLS
In all experiments the control plates without mutagen showed a normal number of spontaneous revertants

POSITIVE CONTROLS
The positive controls demonstrated the sensitivity of the indicator strains and the activity of the metabolizing syste.
Conclusions:
Interpretation of results:
negative

In summary, the results indicate that partially unsaturated IQAC, DMS quaternised caused no mutagenic effects at concentrations ranging from 125 to 2500 µg/plate in thefive tested strains.
Significant bacteriotoxic effects were not observed in the main study up to and including the highest concentration tested of 2500 µg/plate. In the range finding study partially unsaturated IQAC, DMS quaternised reduced the survival at a concentration of 2500 µg per plate to 0.6 % of the control value.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and WP2 uvr A pkM 101 of E. coli were exposed to partially unsaturated IQAC, DMS quaternised (a.i 76.9 %) diluted in water at concentrations of 125 to 2500 µg per plate in the presence and absence of mammalian metabolic activation (co-incubation).

Significant bacteriotoxic effects were not observed in the main study up to and including the highest concentration tested of 2500 µl/plate. In the range finding study partially unsaturated IQAC, DMS quaternised reduced the survival at a concentration of 2500 µg per plate to 0.6 % of the control value. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 and 472, as well as EU Method B.13/14, 29th December 1992 for in vitro mutagenicity (bacterial reverse gene mutation) data.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutagenicity test

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1535, TA 1537, TA 98 and TA 100 ofS. typhimuriumand WP2 uvr A pkM 101 ofE. coliwere exposed to the read-across substance partially unsaturated IQAC, DMS quaternised (a.i 76.9 %) diluted in water at concentrations of 125 to 2500 µg per plate in the presence and absence of mammalian metabolic activation (co-incubation).

Significant bacteriotoxic effects were not observed in the main study up to and including the highest concentration tested of 2500 µl/plate. In the range finding study partially unsaturated IQAC, DMS quaternised reduced the survival at a concentration of 2500 µg per plate to 0.6 % of the control value.There was no evidence of induced mutant colonies over backgroundin both independently performed experiments.

 

Supporting information is available from a reverse gene mutation assay in bacteria according to the OECD Guideline No. 471 (1997). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA were exposed to the oleic-acid based IQAC, DES quaternised, (100 % active matter (99 % solid matter), diluted with acetone) at concentrations of 5, 15, 44, 62, 133, 185, 400, 556, 1667 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

The oleic-acid based IQAC, DES quaternised was tested up to cytotoxic concentrations (400 µg/plate).The positive control treatments in both the non-activation and S9 activation assays induced large increases in the revertant numbers with all the indicator strains, which demonstrated the effectiveness of the S9 activation system and the ability of the test system to detect known mutagens.

No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments.

 

Mouse Lymphoma Assay for mutagenic activity in mammalian cells

In a mammalian gene mutation assay,according to OECD guideline 476, (1997) and EU method B.17 (2000), L5178 Y (mouse lymphoma thymidine kinase locus) cells cultured in vitro were exposed to oleic-acid based IQAC, DMS quaternised (98 % a.i.) at the following concentrations:

Experiment I

- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/ml

- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/ml

Experiment II

- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/ml

- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/ml

Oleic-acid based IQAC, DMS quaternised was tested up to cytotoxic concentrations (≥16.0 µg/ml without metabolic activation and ≥ 40.0 µg/ ml with metabolic activation). The positive controls induced the appropriate responses. There was no evidence of induced mutant colonies over background. Furthermore there were no indications of clastogenic effects and/or chromosomal aberrations in the study, based on evaluation of colony sizing (ratio of small vs. large colonies).

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item oleic-acid based IQAC, DMS quaternised is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

 

Chromosomal aberration test in mammalian cells:

In a mammalian cell cytogenetics assay according to OECD Guideline 473 (1983) and US EPA-TSCA Guidelines, CHO cell cultures were exposed with partially unsaturated IQAC,DMS quaternised(tallow fatty acids), 75 %) at concentrations of 15.0 to 199 µl/ml with metabolic activation and with 3.74 to 74.8 µl/ml without metabolic activation. Microscopic evaluation of aberrant cells was performed for the concentrations of 49.9, 99.7, 150 and 199 µl/ml (with metabolic activation) and 24.9, 37.4, 49.9, and 74.8 µl/ml, respectively, without metabolic activation.

Cytotoxic effects of the test item were observed with and without metabolic activation; at the highest evaluated concentrations as evident by reduction of the cell monolayer confluency to 40 % in both experiments, without and with metabolic activation.

Neither without metabolic activation nor with metabolic activation a significant increase in chromosomally aberrant cells was observed at the concentrations analysed.

Positive control references Mitomycin C and cyclophosphamide were tested in parallel to the testitem. They induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

There was no evidence of chromosome aberration induced over background in the CHO Chinese hamster cell line by partially unsaturated IQAC,DMS quaternised(tallow fatty acids). Therefore, the test item partially unsaturated IQAC,DMS quaternised(tallow fatty acids), 75 %) is considered to be non-clastogenic.

 

Further information is available from a mammalian chromosome aberration test performed according to OECD Guideline 473 (1997). Human lymphocyte cultures were exposed to partially unsaturated IQAC, DMS quaternised (no solvent), (100% a.i.), suspended in ethanol at concentrations between 11.9 - 3200 µg/mL with metabolic activation and 20.8 - 3200 µg/mL without metabolic activation.

Partially unsaturated IQAC, DMS quaternised (no solvent) was tested up to cytotoxic or precipitating concentrations. 

The following experimental points were microscopically evaluated: 11.9, 20.0, 20.8, 50.0, 125.0, 341.2, 597.1 µg/mL with metabolic activation and 20.8, 36.4, 43.5, 76.2, 133.3, 233.2, 341.2, 597.1 µg/mL without metabolic activation.

In the absence of S9 mix, reduced mitotic indices of about or below 50 % of control were observed at the highest evaluated concentrations. In the presence of S9 mix, concentrations showing clear cytotoxic effects were excluded from scoring for the endpoint cytogenicity.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.  Positive controls induced the appropriate response. 

There was no evidence of Chromosome Aberration induced over background.

As there were no indications of genotoxicity in the three in-vitro test systems mandatory for Annex IX substances and reported above, no in-vivo genotoxicity tests have been conducted or proposed.

 

Read-across

The toxicological profile for systemic endpoints is expected to be similar for target substance oleic-acid based IQAC, DMS quaternised when compared to the source substance partially unsaturated IQAC, DMS quaternised. Read-across is expected to be reliable.

This read-across is based on the hypothesis that source and target substances, both Imidazolium quaternary ammonium compounds (IQACs)have similar toxicological properties, based on the followingunderlying identical generic structure. There are only minor differences in carbon chain length (C16 and C18) and degree of saturation of the fatty acid moiety present in the structure of source and target substance.

This prediction is supported by toxicological data on the substances themselves. Associated Robust study summaries are provided in the Technical Dossier.

 

Detailed information on substance identity of source and target substance can be found in the “Justification for read-across - toxicological information”, provided in a separate document in chapter 13 of Technical Dossier and attached to the endpoint summary.

Read-across within the endpoint genetic toxicity is considered an appropriate adaptation to meet the standard information requirements according to REACH Regulation. 

 

Overview on available genotoxicity data:

 

 

Endpoint

Source substance

 

Partially unsaturated IQAC, DMS quaternised

Supporting source substance

Oleic-acid based IQAC, DES quaternised

Target substance

 

Oleic-acid based IQAC, DMS quaternised

In vitrogene mutation study in bacteria

negative

in S. typhimurium and E. coliwith and without metabolic activation

negative

inS. typhimuriumandE. coliwith and without metabolic activation

 

Mutagenicity;In vitrogene mutation study in mammalian cells

 

 

negative

inMouse lymphoma L5178Ycells with and without metabolic activation

no indicationof clastogenic effects and/or chromosomal aberrations based on colony sizing.

In vitrocytogenicity study in mammalian cells

negative

inChinese hamster Ovary (CHO)with and without metabolic activation

 

 

negative

inHuman lymphocyteswith and without metabolic activation

 

 

  

The structural similarities between the source and the target substances are the basis for the read-across hypothesis. Adequate, reliable and available scientific information indicates that the source and target substances have similar physicochemical properties and toxicity profiles and thus support the read-across hypothesis. For detailed information refer to the separate document “Justification for read-across - toxicological information”, provided in chapter 13 of Technical Dossier.

Moreover for bacterial reverse mutation testing additional supporting information is available from oleic-acid based IQAC, DES quaternised. This substance is quaternised with diethylamin instead of dimethlyamin which is used for source and target substance. In essence the structure is nearly identical to source and target substance and therefore the toxicological profiles are expected to be comparable.

This assumption is proven by the same low oral toxicity for the oleic-acid based IQAC, DES quaternised, as shown for source and target substance. The oral LD50 in rats was determined to be > 2000 mg/kg bw.

 

Mammalian gene mutation was evaluated in L5178 Y (mouse lymphoma thymidine kinase locus) cells with the target substance (oleic-acid based IQAC, DMS quaternised). The substance was considered to be non-mutagenic in this study. Furthermore there were no indications of clastogenic effects and/or chromosomal aberrations in the study, based on evaluation of colony sizing (ratio of small/large colonies).

 

This negative genotoxic profile is proven by further genetic toxicity studies conducted with source substance (partially unsaturated IQAC, DMS quaternised) and supported by data from oleic-acid based IQAC, DES quaternised, evaluating point mutations in bacteria and chromosomal aberrations inChinese hamster Ovary (CHO) and human lymphocytes.

 

In conclusion there were no indications of genotoxic properties in the three in-vitro test systems and thus there is no evidence for genotoxicity of the evaluated group of IQACs.

 

 

 


Justification for selection of genetic toxicity endpoint
No single key study has been selected since all available studies were negative.

Short description of key information:
In vitro data from a bacterial reverse mutation assay (Ames Test) are available with partially unsaturated IQAC, DMS quaternised and oleic-acid based IQAC, DES quaternised as a read-across substance. In addition data are available from a mammalian cell gene mutation assay (L 5178Y/ TK Mouse Lymphoma assay) with oleic-acid based IQAC, DMS quaternised and a chromosome aberration test in Chinese Hamster Ovary Cells based on the read-across partially unsaturated IQAC, DMS quaternised.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The oleic-acid based IQAC, DMS quaternised is considered to have no genotoxic properties as shown in the Ames-Tests, mouse lymphoma assay and chromosome aberration study.

Therefore, the oleic-acid based IQAC, DMS quaternised does not need to be classified as “genotoxic” according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008. No labelling is required.