Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The evaluation of the potential mutagenicity of cC6O4 ammonium salt is based on four different experimental studies. 

In order to gain an understanding of the potential mutagenicity of the test item cC6O4 Ammomium Salt, an in vitro test battery was implemented:

- A bacterial reverse mutation assay, according to OECD 471

- An In vitromammalian cell gene mutation test, according to OECD 476

- An In vitromammalian chromosome aberration test, according to OECD 473

All studies were performed under GLP and all of them included a preliminary study on cytoxicity of the test item under the specific experimental test conditions.

 

The bacterial reverse mutation assay and the in vitro mammalian cell mutation assay showed clear negative results. Both of the studies identify the substance as non mutagenic.

The in vitro mammalian chromosome aberration test, during the second experiment of the study, showed a positive result at the highest dose level in presence of metabolic activation. Consequently, the substance was identified as mutagenic under that experimental test conditions.

In order to assess the relevance of this positive finding, considering that at the highest dose level signs of toxicity occurred, and considering that metabolic pathways cannot be simulated adequately in vitro, it was deemed appropriate to conduct an in vivo study for chromosome aberration.

The in vivo mammalian bone marrow chromosome aberration test according to OECD 475 guideline was performed on the test item. This study showed a clear negative result since no statistically significant increase in the frequency of aberrant cells occurred at any dose level.

The absorption and the target organ achievement of the substance was confirmed by the available preliminary toxicokinetics study, where after a single oral dose of 26.1 mg/kg b.w. (dry salt) male Wistar rats were sufficiently exposed with a higher absorption of the substance. In the chromosome aberration test, male Wistar rats were treated with higher doses (312.5, 625.0, 1250 mg/kg b.w.) with clear negative results at all doses.

Considering that the results of in vivo test have an higher reliability in respect to the in vitro test, and considering that the positive findings in the in vitro study occurred only at the highest dose in presence of toxicity signs, it can be concluded that the test item cC6O4 ammonium salt does not show potential to induce chromosome aberrations in mammals.

 

Therefore, on the grounds of the available genotoxicity data, it can be concluded that cC6O4 ammonium salt is not characterized by genotoxic properties.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 January 2010 to 14 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
hypoxanthine 1.0×10-4 M aminopterin 2.0×10-7 M thymidine 1.6×10-5 M
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing:
hypoxanthine 1.0×10-4 M thymidine 1.6×10-5 M
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium).

Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated in plastic flasks (Greiner, 72632 Frickenhausen, Germany) in RPMI 1640 complete culture medium. The cells are subcultured two times prior to treatment. The cell cultures are incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix (4 h treatment) : 56.9; 113.8; 227.5; and 455 μg/mL
with S9 mix (4 h treatment) : 56.9; 113.8; 227.5; 455; and 910 μg/mL

Experiment II:
without S9 mix (24 h treatment) : 28.1; 56.3; 112.5; 225; and 337.5 μg/mL
with S9 mix (4 h treatment) : 56.3; 112.5; 225; 450; and 675 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
The test item was dissolved in deionised water on the day of the experiment (immediately before treatment).
The final concentration of deionised water in the culture medium was 10 % v/v.
Concurrent solvent controls (deionised water) were performed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Referred to experiments I and II without metabolic activation Supplier: Sigma, 82024 Taufkirchen, Germany Charge: 76296KJ Expiration Date: May 2010 Purity: > 99% Dissolved in: Nutrient medium Final Concentration: 19.5 μg/mL = 0.18 mM

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Referred to experiments I and II with metabolic activation. Supplier: Aldrich Chemie, 89555 Steinheim, Germany Charge: 097K1311 Expiration Date: March 2011 Purity: ≥ 98 % Dissolved in: 0.9 % saline Final Concentration: 3.0 μg/mL = 10

Details on test system and experimental conditions:

PRE-TEST ON TOXICITY:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.
1x107 cells (3x106 cells at the beginning of 24 h treatment) were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed twice by centrifugation (425 g, 10 min) and resuspended in "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3x105 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector.

DOSE SELECTION
According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiments.
The highest concentration should be 10 mM, but not higher than 5 mg/mL, unless limited by the solubility or toxicity of the test item.
RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20 %.
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 28.4 μg/mL and 3640 μg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the purity (97.5 %) and the molecular weight (357 g/mol) of the test item. Strong toxic effects were observed at 1820 μg/mL and above in the absence of metabolic activation (4 h treatment). In the presence of metabolic activation (4 hours treatment) toxic effects occurred at 455 μg/mL and above. Following continuous treatment (24 hours) strong toxic effects were determined at 227.5 μg/mL and above.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. After 4 hours treatment precipitation of the test item was observed at 910 μg/mL and above in the presence and absence of metabolic activation. Following continuous treatment no precipitation was noted.
There was no relevant shift of the pH value or the osmolarity of the medium even at the maximum concentration of the pre-experiment.
The dose range of the main experiments was limited by the occurrence of toxicity and precipitation. The individual concentrations of the first experiment with and without metabolic activation and the second experiment without metabolic activation were generally spaced by a factor of 2.0. A narrower spacing was used in the upper concentration range to cover the limit of solubility or cytotoxic effects more closely.
Following the expression phase of 48 hours the cultures at the concentrations of 910 and 1365 μg/mL (printed in bold letters) in experiment I without metabolic activation and at 1365 μg/mL with metabolic activation were not analysed due to exceedingly severe toxic effects. In the second experiment the cultures at 450 μg/mL without metabolic activation and 675 μg/mL (culture I) and 900 μg/mL with metabolic activation were not continued for the same reason.

EXPERIMENT PERFORMANCE
In the mutation experiment 1×107 (3x106 during 24 h exposure) cells/flask (80 cm2 flasks) suspended in 10 ml RPMI medium with 3 % horse serum (15 % horse serum during 24 h exposure) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. After 4 h (24 h in the second experiment) the test item was removed by centrifugation (425 x g, 10 min) and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 ml complete culture medium and incubated for an expression and growth period of 48 h.
The cell density was determined each day and adjusted to 3×105 cells/ml, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector.
After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4×103 cells in selective medium (see below) with TFT (Serva, 69042 Heidelberg, Germany). The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37°± 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

SiZE DISTRIBUTION OF THE COLONIES
Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at: 227.5 μg/mL (experiment I), 225 μg/mL (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at: 227.5 μg/mL (experiment I), 225 μg/mL (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item visible to the naked eye was noted at 455 μg/mL and above in the first experiment with metabolic activation and at 450 μg/mL and above in the second experiment with metabolic activation.
Relevant toxic effects indicated by a relative total growth of less than 50 % of relative total growth were observed in experiment I starting at 227.5 μg/mL in the presence and absence of metabolic activation. In the second experiment toxic effects as described above occurred at 225 μg/mL and above with and without metabolic activation. The data generated in the second experiment without metabolic activation at 337.5 μg/mL were not judged as acceptable since the RTG dropped far below the 10% limit in both parallel cultures. The same was true for the data generated in the second experiment with metabolic activation at 675 μg/mL. The RTG of the second culture was only 2.6, the first culture was not analysable at all due to exceedingly severe cytotoxic effects.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments up to the maximum concentration with and without metabolic activation. The mutation frequency exceeded the threshold of 126 above each solvent control in the first culture of the first experiment without metabolic activation at 227.5 and 455.0 μg/mL. In the second culture of the first experiment with metabolic activation the threshold was exceeded at precipitating concentrations of 450 and 910 μg/mL. However, this increase was not reproduced in the parallel culture under identical conditions and consequently judged as artefact. In the second experiment the threshold of 126 above each solvent control count was again exceeded in one of the parallel cultures at a precipitating concentration of 450 μg/mL with metabolic activation. Again, no comparable increase was noted in the parallel culture under identical conditions so this isolated increase was judged as precipitation artefact. Precipitation is a problem in the mouse lymphoma assay since L5178Y cells grow in suspension. The test item is removed by centrifugation at the end of treatment. At precipitating concentrations the test item can not completely be removed at the intended end of treatment since the precipitate sediments together with the cells upon centrifugation. Arbitrary amounts of the test item are carried over to the next steps of the assay leading to irreproducible artefacts.

In this study the range of the solvent controls was from 124 up to 196 mutant colonies per 10E+06 cells; the range of the groups treated with the test item was from 124 up to 764 mutant colonies per 10E+06 cells. The solvent controls exceeded the recommended upper limit of 170 per 10E+06 cells somewhat in the presence of metabolic activation and in one of both cultures following long term treatment for 24 hours. However, the data are judged as acceptable since the IWGT recommendations published in 2003 stated an upper limit of 200 colonies per 10E+06 cells and all solvent controls remained within the historical control range. MMS (19.5 μg/mL in experiment I and 13.0 μg/mL in experiment II) and CPA (3.0 and 4.5 μg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls.

Conclusions:

Interpretation of results: negative

It can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, cC6O4 AMMONIUM SALT is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of cC6O4 AMMONIUM SALT to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. The highest concentration (3640 μg/mL) applied in the pre-experiment was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and precipitation of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, cC6O4 AMMONIUM SALT is considered to be non-mutagenic in this mouse lymphoma assay.

Reference 2

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:

2009-03-09 to 2009-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

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

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimuriumtester strains: histidine requirement
E. coli (WP2 uvrA): tryptophan requirement

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: rfa wall mutation (increasing of permeability to certain classes of chemicals), deficiency in DNA excision repair system (uvrB mutation). The strain is predominantly sensitive to base pair mutagens.

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: rfa wall mutation (increasing of permeability to certain classes of chemicals), deficiency in DNA excision repair system (uvrB mutation). The strain is sensitive to frameshift mutagens.

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: rfa wall mutation (increasing of permeability to certain classes of chemicals), deficiency in DNA excision repair system (uvrB mutation). The strain is sensitive to frameshift mutagens. Presece of pKM101 plasmid.

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: rfa wall mutation (increasing of permeability to certain classes of chemicals), deficiency in DNA excision repair system (uvrB mutation). The strain is predominantly sensitive to base pair mutagens. Presence of pKM101 plasmid.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Tester strain contains an uvrA DNA repair deficiency which enhance its sensitivity to some mutagenic compounds.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : 2 lots of S9 mix obtained from supplier (MOLTOX Molecular Toxicology, Inc.) were used
- method of preparation of S9 mix : rat liver tissue fraction prepared from rat treated with phenobarbital/5,6-Benzoflavone.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 ml in overlay mixture, or 0.5 ml in the pre-incubation solution.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): protein content: 37.6 mg/L and 37.8 mg/L depending on lot.

Test concentrations with justification for top dose:

Toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate.
Assays for reverse mutation I and II: 5000, 2500, 1250, 625 and 313 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used for the test item: Distilled Water.

- Vehicle(s)/solvent(s) used for the positive controls:
Distilled water for Sodium azide, Methylmethanesulphonate (MMS).
DMSO for 9-Aminoacridine, 2-Nitrofluorene, 2-Aminoanthracene.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Preliminary toxicity test
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In these tests a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Main experiments
Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix, and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The first experiment was performed using a plate-incorporation method.
The second experiment was performed using a pre-incubation method.

METHOD OF APPLICATION: in agar (plate incorporation), pre-incubation (in suspension)

DURATION
- Preincubation period: 30 min
- Exposure duration: approximately 72 hours
- Expression time (cells in growth medium): approximately 72 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as: y = a + bx.
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay I , plate incorporation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay I, plate incorporation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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

Remarks:

Main Assay I, plate incorporation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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

Remarks:

Main Assay I, plate incorporation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay I: plate incorporation. Test concentrations: 5000, 2500, 1250, 625 and 313 µg/plate.

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay II, pre-incubation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Thinning of the background lawn at 2 highest concentrations, with and without S9

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay II, pre-incubation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Decreased number of revertants (without S9) and thinning of the background lawn (with and without S9), at the 2 highest concentrations (2500, 5000 µg/plate)

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

Remarks:

Main Assay II, pre-incubation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Thinning of the background lawn at the 2 highest concentrations (2500, 5000 µg/plate, with and without S9)

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

Remarks:

Main Assay II, pre-incubation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Decreased number of revertants and thinning of the background lawn at the 3 highest concentrations (1250, 2500, 5000 µg/plate), with and without S9

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Main Assay II, pre-incubation. Test concentrations: 5000, 2500, 1250, 625 and 313 µg/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Thinning of the background lawn at the 2 highest concentrations (2500 and 5000 µg/plate), with and without S9 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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none reported
- Effects of osmolality: none reported
- Evaporation from medium: not reported
- Water solubility: no limitations reported
- Precipitation: No precipitation of the test item was noted at the end of the incubation period at any concentration.
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In these tests a wide range of dose levels of the test item, set at half-log intervals, were used.

COMPARISON WITH HISTORICAL CONTROL DATA: Results show that mean plate counts for untreated and positive control plates fell within the normal historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main Assay I (without pre-incubation): using the plate incorporation method, no toxicity was observed at any dose level with any tester strain both in the absence and presence of S9 metabolic activation.
Main Assay II (with pre-incubation): Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at higher dose levels with all tester strains, both in the absence and presence of S9 metabolic activation.

STERILITY
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Conclusions:

cC6O4 ammonium salt does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item cC6O4 ammonium salt was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item cC6O4 ammonium salt (solution in sterile distilled water)

was assayed in the toxicity test at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No toxicity was observed at any dose level with any tester strain.

In Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels expressed in terms of active ingredient: 5000, 2500, 1250, 625 and 313 µg/plate. No toxicity was observed at any dose level with any tester strain both in the absence and presence of S9 metabolic activation.

As no relevant increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same concentrations employed in Main Assay I. Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at higher dose levels with all tester strains, both in the absence and presence of S9 metabolic activation. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item cC6O4 ammonium salt does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March 2009 to 08 July 2009

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

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:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary cells were obtained from Dr. A.T. Natarajan (State University of Leiden). This cell line derives from the CHO isolate originally described by Kao and Puck (1968).

- Type and identity of media: the medium used for the growth of the cells has the following composition: Ham's F.10=499.0ml, Streptomycin sulphate 50 mg/ml-Penicillin G 50,000 IU/ml = 1.0 ml, Newborn Calf Serum = 88.2 ml.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not reported, presumably yes.

Permanent stocks of CHO cells are stored in liquid nitrogen, and subcultures are prepared from these stocks for experimental use.
All incubation are at 37°C in a 5% carbon dioxide atmosfere (100% humdity nominal).

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

First main experiment (3 hours treatment both in absence and presence of metabolic activation): 5000, 2500, 1250, 625, 313, 156, 78.1, and 39.1 μg/ml.
Second main experiment (3 hours treatment in presence of metabolic activation, 20 hours (continuous) treatment in absence of metabolic activation): 2000, 1330, 889, 593, 395, 263, 176 and 117 μg/ml

Vehicle / solvent:

The solvent used in this study was sterile distilled water of injectable grade, batch no. 08G2803, obtained from Bieffe, Italy.
Solvent controls were performed.

Untreated negative controls:

yes

Remarks:

Untreated cultures

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

In absence of S9 metabolic activation

Positive control substance:

mitomycin C

Remarks:

1st main exp: 0.30 and 0.45 μg/ml for 3 hours treatment; 2nd main exp: 0.10 and 0.15 μg/ml for the continuous treatment

Untreated negative controls:

yes

Remarks:

Untreated cultures

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

In presence of S9 metabolic activation.

Positive control substance:

cyclophosphamide

Remarks:

In both the 1st and 2nd main exp.: 15.0 and 23.0 μg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours in the first main test (with and without metabolic activation), 3 hours in the Second main test with metabolic activation, 20 hours (continuous treatment) in second main test without metabolic activation.
- Expression time (cells in growth medium): 20 hours

NUMBER OF REPLICATIONS: 1.5

NUMBER OF CELLS EVALUATED: 100 eligible metaphase per test culture

DETERMINATION OF CYTOTOXICITY
- reduction of Population Doubling (PD) over the relevant control.

EXAMINATIONS:
-Chromosome exchanges and deletions
-Chromatid exchanges and deletions
-Gaps
-Other anomalies: polyploid cells, endoreduplications, seriously damage cells.


SELECTION OF DOSE LEVELS FOR TREATMENT
The highest dose-level to be used is determined according to the solubility of the test item in the culture medium and solvent vehicle, but will not exceed a maximum concentration of 5mg/ml. Up to two dose-levels at at which precipitation is observed may be included in the treatment series. Seven dose-levels will be used, spaced at approximatly equal intervals.
If an additional experiment is performed, dose-levels will be selected on the basis of the results obtained (e.g. toxicity) in the first experiment.
Duplicated cultures are prepared for each experimental test point.

PREPARATION OF THE TEST CULTURES
Approximatly 20 hours before treatment an appropriate number of flasks for the experiment are perpared from a single pool of cells.
These cell will be in exponential growth phase at the time of treatment.

TREATMENT OF THE TEST CULTURES
Both in the absence or presence of S9 metabolism, the culture are incubated in the treatment media for three hours. The medium is then removed and the flasks are washed twice with Ca/Mg-free Phosphate Buffered Saline (PBS).
Fresh medium is added and the cultures are incubated for a further 17 hours (Recovery period).
Colcemid (0.2 μg/ml final concentration) is added for the last three hours of the recovery period, leading up to harvesting. In this way cultures are prepared for harvesting at 20 hours after treatment commenced.

ADDITIONAL EXPERIMENT
If negative or equivocal results both with or without S9 metabolism are obtained, an additional experiment is performed with continuous treatment and a harvest time at approximatly 1.5 cell cycle lenghts.
This experiment is conducted only in absence of S9. The treatment media are added to the tubes and the cultures are incubated for 20 hours. Colcemid (0.2 μg/ml final concentration) is added fro the last three hours of the treatment period, leading up the harvesting. Cells are harvested at twenty hours after beginning of treatment.

Negative results with metabolic activation need to be confirmed on a case-by-case basis.

SELECTION OF THE DOSE-LEVEL FOR SCORING OF CHROMOSOMAL ABERRATIONS
At the time of harvesting, cell counts will be performed for each culture.
The highest dose-level selected for scoring should show a significant reduction in Population Doubling (PD).
PD=[log(N/X0)]/log2
where:
N = final cell count
X0 = starting (baseline) count
In order to evaluate baseline count at the beginning of treatment, two additional control cultures are included in the experimetal scheme.
Ideally the reduction should be approximatly 50% of the control and not greatly exceeding this percentage reduction.

Two lower dose-levels will be alse selected for the scoring of chromosomal aberration. The lowest dose-level for scoring should be on the borderline of cytotoxicity.
The intermediate dose-level will be evenly spaced between the two.

METAPHASE ANALYSIS
Metaphase spreads judged to be of sufficient quality to permit scoring are examined at high magnification. Metaphases that differ from the modal chromosomal complement by more than two centromeres are not scored.
Polyploid and endoreduplicated cells encuntered will be recorded, but not incuded in the count of the eligible metaphases. From 100 eligible metaphases per test culture, the number of chromosomes, the specif types and numbers of aberrations are recordered.
The Vernier readings of aberrant or equivocal metaphases are recordered.

Evaluation criteria:

The evaluation is based on the set of results, which excludes gaps.
According to the protocol of the test, for a test item to be considered clastogenic, four criteria must be met:
(i) Increases over the concurrent controls:
if any dose-leve shows a statistically significant increase in aberration-bearing cells, this will be considered as evidence of a clastogenic effect.
(ii) Increases over the historical controls:
If the increases fall within the range of value normally observed in the negative control culteres, the test item cannot be judged clastogenic. Any significant increases over the concurrent solvent/vehicle controls will therefore be compared with historical values derived from recent studies.
(iii) Reproducibility:
Any increases observed must be present in both replicate cultures.
(iv) Biological significance:
The conclusions must be consistent with the underlying biology of the assay. Specifically:
- it is not required that an increased response is observed at increasing dose-levels, but dose-related activity will be taken as further evidence of clastogenic effect.
- the types of aberrations observed will be taken into consideration.
- an increased incidence of an aberration type with an exceedingly low background frequency will be considered as evidence of a clastogenic effect.

Statistics:

The number of cells bearing aberrations in the control and treated cultures are compared using Fisher's exact test.
The comparison is performed both including and excluding gaps from the aberration counts. The values obtained at all the treatment dose-levels combined are compared with the control values. Since multiple-dose are compared with the negative controls, the problem of Typr I error (change 'positive' results) arises. Accordingly, significance levels for each treatment will also be presented after application of Bonferroni's correction.
The solvent/vehicle controls will be used as the reference point for comparison in the statistical evaluation and the evaluation of the results.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

An increament of endoredulicated cells (no. ER =8) was observed at the highest dose selected for scoring (1250 μg/ml).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Severe toxicity at the higher dose levels of 5000 and 2500 μg/ml (relative PD=0%); moderate tocixity at 1250 μg/ml (relative PD=75%)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

A marked increment of endoreduplicated cells over the control was observed (11 ER cells at 313 μg/ml, 28 ER cells at 625 μg/ml, 26 ER cells at 1250 μg/ml).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Severe toxicity at the higher dose levels of 5000 and 2500 μg/ml (relative PD=0%)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No statistically significant increase in the incidence of cells bearing aberrations gaps was observed. (Cells bearing aberrations: 1% at dose level of 313 μg/ml)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Severe toxicity at 2000 and 1330 μg/ml (relative PD= 0%); toxicity at the dose levels of 889 and 593 μg/ml ( relative PD=31%)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Statistically significant increases in the incidence of cells bearing aberrations (4.5% excluding gaps) at the highest dose level selected for scoring (2000 μg/ml). Marked increases of endoreduplicated cells (22 ER at 1330μg/ml, 30 ER at 2000μg/ml)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Distinct toxicity at 2000 μg/ml (relative PD= 51%). Slight toxicity at the remaining dose range ( relative PD = approximatly 80%).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Following treatment, the pH of the treatment media at the higher dose levels were determined. No relevant effect on pH of the treatment media was observed at any dose level tested
- Effects of osmolality: Following treatment, the osmolality of the treatment media at the higher dose levels were determined. Due to the presence of water in the solvent control cultures, a slight increase in osmolality over the control was observed for cultures treated at the highest dose level selected for treatment. The values however were comparable to those observed in untreated control cultures.
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: During the main experiments, no precipitation or opacity of the media was observed at the beginning or end of treatment.
- Other confounding effects: not reported

COMPARISON WITH HISTORICAL CONTROL DATA: In the second main experiment in presence of metabolic activation, the incidence of aberrant cells exceeded the range of historical values for negative controls when excluding gaps.

Assay for chromosomal aberrations

For the first main assay, dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1 and 39.1 μg/ml were employed both in the absence and presence of S9 metabolism.

Both in the absence and presence of S9 metabolic activation, the treatment time was 3 hours after which the cells were allowed to recover prior to harvesting. The harvest time of 20 hours, corresponding to approximately 1.5 cell cycle, was used.

Since no induction of chromosomal aberrations was obtained in the first main assay, in agreement with the study protocol, a second main experiment was performed where cells were treated with cC6O4 ammonium salt in the absence of S9 metabolism and harvested after 20 hours. A continuous treatment until harvest was used.

For the first main experiment, following treatment with the test item in the presence of S9 metabolism, marked increases in the number of endoreduplicated cells over the control were observed.

Since no adequate toxicity was observed at any dose level for the evaluation of genotoxic effects, following the approval of a protocol amendment, for the second main experiment an additional treatment in the presence of S9 metabolism was performed in order to confirm the observed results. For this experiment a modified dose range was adopted in order to investigate more closely those doses most likely to exhibit endoreduplication.

For the second main experiment, the dose levels selected for treatment were 2000, 1330, 889, 593, 395, 263, 176 and 117 microgram/ml.

Appropriate negative and positive control cultures were included in the experiment. Positive control treated cultures received Mitomycin-C 0.30 and 0.45 μg/ml for 3 hours treatment or 0.10 and 0.15 μg/ml for the continuous treatment in the absence of S9 metabolism or Cyclophosphamide 15.0 and 23.0 μg/ml in the presence of S9.

Two cultures were prepared at each test point. Air-dried slides were prepared from each culture and stained with 3% Giemsa.

Following treatment, the pH and osmolality of the treatment media at the higher dose levels were determined. No relevant effect on pH of the treatment media was observed at any dose level tested.

Due to the presence of water in the solvent control cultures, a slight increase in osmolality over the control was observed for cultures treated at the highest dose level selected for treatment. The values however were comparable to those observed in untreated control cultures.

During the main experiments, no precipitation or opacity of the media was observed at the beginning or end of treatment.

Selection of dose levels for scoring.

In the first main experiment, following treatment in the absence or presence of S9 metabolism, a severe toxicity was observed at the higher dose levels of 5000 and 2500 microgramm/ml, where no cells or few cells where recovered at harvesting.

In the absence of S9, moderate toxicity was observed at the next lower dose level of 1250 microgramm/ml where PD was 75% of the control. No cytotoxicity was observed over the remaining dose range.

In the presence of S9 metabolism, no remarkable toxicity was observed over the remaining dose range.

In the second main experiment following treatment in the absence of S9, a severe toxicity was observed at the higher dose levels of 2000 and 1330 microgram/ml, where few cells were recovered at harvesting.

Marked toxicity was also observed at the dose levels of 889 and 593 microgram/ml where the PD was 31% of the control. No remarkable toxicity was observed over the remaining dose range.

In the presence of S9 metabolism distinct toxicity was observed at the highest dose level of 2000 microgramm/ml where the PD was 51% of the control. Slight toxicity reducing the PD at approximately 80% of the control was observed over the remaining dose range.

The highest dose level selected for the scoring of aberrations should be a concentration causing moderate toxicity (ideally the reduction of PD should be approximately 50%).

If no toxicity is observed then the highest practicable dose level should be selected.

On the basis of the above described results the dose levels selected for scoring were the following:

Experiment No. S9 Treatment time (hours) Harvest time (hours) Dose level (microgram/ml) 1 + - 3 20 1250, 625 and 313 2 + - 3 20 20 2000, 1330 and 889 889, 593 and 39

The dose levels of 0.30 and 0.10 μg/ml were selected for the scoring of cultures treated with Mitomycin-C (first and second main experiment respectively) and the dose level of 15.0 μg/ml was selected for the scoring of cultures treated with Cyclophosphamide.

Assay results

One hundred metaphase spreads were scored for chromosomal aberrations from each culture, with the exception of one replicate culture treated with Cyclophsphamide (first main experiment), where 75 metaphases were examined. In addition, for the first main experiment, due to the high frequency of aberrant cells (excluding gaps), found for replicate cultures treated with Mitomycin-C, scoring was terminated at 50 metaphases.

The numbers and types of aberrations are recordered, together with the total number of aberrations (chromatid type and chromosome type) including and excluding gaps. The total number of aberrant metaphases including and excluding gaps is also recordered.

For the first experiment, following treatment with the test item cC6O4 ammonium salt in the absence or presence of S9 metabolism, no remarkable increase over the control values in the number of chromosome aberrations including or excluding gaps was observed.

Following treatment in the presence of S9 metabolism, marked increases in the number of endoreduplicated cells over the control were observe. An increase in the number of endoreduplicated cells was also observed in the absence of S9 metabolism at the highest dose selected for scoring.

For the second experiment, following treatment with the test item in the absence of S9 metabolism, slight increases in the incidence of cells bearing aberrations excluding gaps, over the control values were observed at all dose levels selected for scoring. More remarkable increases of aberrant cells were observed when including gaps.

In the presence of S9 metabolism, increases in the incidence of aberrant cells including and excluding gaps, were observed at the highest dose selected for scoring (2000 μg/ml). The incidences exceeded the range of our historical values for negative controls when excluding gaps. Additionally the presence of a heavily damaged cell, bearing more than five aberrations was also noted. In this experiment the induction of endoreduplicated cells was confirmed since marked increases were seen at the intermediate and high dose levels selected for scoring.

Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substances, indicating the correct functioning of the assay system.

The modal number of chromosomes observed in the cells of untreated cultures (without S9 metabolism) was 21 (64.25%). The frequency of cells containing 20, 22 and 23 chromosomes was 0.75%, 34.75% and 0.25%. No metaphases were found with 19 chromosomes.

Statistical analysis

For the statistical analysis, Fisher's Exact Test is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis is performed using sets of data either including or excluding gaps.

Following treatment with the test item in the absence of S9 metabolism, no statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps was observed.

In the presence of S9 metabolism, statistically significant increases in the incidence of cells bearing aberrations, including and excluding gaps, exceeding the range of our historical controls, were observed at the highest dose level selected for scoring.

TABLES

The following tables show, for each test culture used in the main assay and by treatment group totals, the types and numbers of aberrations, identified as follows:

Gaps - This refers to either chromatid or chromosome gaps.

Del - Interstitial or terminal deletions.

Exch - Exchanges:

a) Chromatid exchanges

Includes symmetrical and asymmetrical exchanges; intra- and inter-chromosome exchanges.

b) Chromosome exchanges

Includes both dicentric and ring types.

Other - These include

H : Heavily damaged cells

(more than 5 aberrations/cell)

ER: Endoreduplicated cells

PP: Polyploid cells

Isolocus - Includes isochromatid and isolocus breaks when these cannot be distinguished.

Tot.abs - Total number of aberrations observed.

Cells with abs. - Cells with aberrations.

MAIN ASSAY: 1 - without metabolic activation

SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE

TREATMENT TIME: 3 hours

SAMPLING TIME: 20 hours

Treatment	Dose level (μg/ml)	Culture no.	Cells scored	Gaps	Chromatid		Chromosome		Isolocus	Other	Tot. Abs (+gaps)	Tot. Abs (-gaps)	Cells with abs (+gaps)	Cell with abs (-gaps)
					Del	Exch	Del	Exch
Untreated		1	100	0	0	0	0	0	0	0	0	0	0	0
		2	100	0	0	0	0	0	0	0	0	0	0	0
			200	0	0	0	0	0	0	0	0	0	0	0
Solvent	10%	3	100	3	0	0	0	0	0	0	3	0	3	0
		4	100	1	0	0	0	0	0	0	1	0	1	0
			200	4	0	0	0	0	0	0	4	0	4	0
Test Item	313	13	100	2	0	0	0	0	0	0	2	0	2	0
		14	100	2	2	0	1	0	0	0	5	3	3	1
			200	4	2	0	1	0	0	0	7	3	5	1
Test Item	625	11	100	0	0	0	0	0	0	0	0	0	0	0
		12	100	4	0	0	0	0	0	0	4	0	4	0
			200	4	0	0	0	0	0	0	4	0	4	0
Test Item	1250	9	100	0	0	0	0	0	0	3ER	0	0	0	0
		10	100	1	0	0	0	0	0	5ER	1	0	1	0
			200	1	0	0	0	0	0	8ER	1	0	1	0
Mitomycin-C	0.3	21	75	1	4	51	3	1	10	0	70	69	41	40
		22	100	0	0	40	8	2	2	2H	52	52	33	33
			175	1	4	91	11	3	12	2H	122	121	74	73

MAIN ASSAY: 1 - with metabolic activation

SOLVENT: STERILE DISTILLED WATER OF INJECTABLE GRADE

TREATMENT TIME: 3 hours

SAMPLING TIME: 20 hours

Treatment	Dose level (μg/ml)	Culture no.	Cells scored	Gaps	Chromatid		Chromosome		Isolocus	Other	Tot. Abs (+gaps)	Tot. Abs (-gaps)	Cells with abs (+gaps)	Cell with abs (-gaps)
					Del	Exch	Del	Exch
Untreated		25	100	4	0	0	0	0	0	1ER,1PP	4	0	4	0
		26	100	4	2	0	0	0	0	0	6	2	5	2
			200	8	2	0	0	0	0	1ER, 1PP	10	2	9	2
Solvent	10%	27	100	1	0	0	0	0	0	0	1	0	1	0
		28	100	2	0	0	0	0	0	0	2	0	2	0
			200	3	0	0	0	0	0	0	3	0	3	0
Test Item	313	37	100	2	1	0	0	0	0	4ER	3	1	3	1
		38	100	2	0	0	0	1	0	7ER	3	1	3	1
			200	4	1	0	0	1	0	11ER	6	2	6	2
Test Item	625	35	100	4	0	0	0	0	0	18ER	4	0	4	0
		36	100	0	0	0	0	0	0	10ER	0	0	0	0
			200	4	0	0	0	0	0	28ER	4	0	4	0
Test Item	1250	33	100	4	0	0	0	0	0	14ER	4	0	4	0
		34	100	0	0	0	0	0	0	12ER	0	0	0	0
			200	4	0	0	0	0	0	26ER	4	0	4	0
Cyclophosphamide	15.0	45	50	5	13	31	0	1	3	1H	53	48	35	35
		46	50	4	20	25	0	0	1	1H	50	46	34	33
			100	9	33	56	0	1	4	2H	103	94	69	68

Conclusions:

Interpretation of results: positive with metabolic activation

Based on the positive results obtained in the presence of S9 metabolism, together with increases in the numbers of endoreduplicated cells and in accordance with the criteria for the outcome of the study, the test item was considered to induce chromosomal aberrations and inhibition of cell cycle progression in Chinese hamster ovary cells in vitro.
Statistically significant increases in aberrant cells compared with the relevant control values were seen in cultures treated with the positive controls Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

Executive summary:

The test item cC6O4 ammonium salt was assayed for the ability to cause chromosomal damage in Chinese hamster ovary cells, following in vitro treatment in the absence and presence of S9 metabolic activation. Two main experiments for chromosomal damage were performed.

 

In the first experiment, the cells were treated for 3 hours with cC6O4 ammonium salt in the presence and absence of S9 metabolism. The cells were then harvested after 20 hours, corresponding to approximately 1.5 cell cycle. Dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1, and 39.1 microgram/ml were used in the absence and presence of S9 metabolism.

No statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps was observed in the absence or presence of S9 metabolism. In the presence of S9 metabolism, marked increases in the number of endoreduplicated cells over the control were observed. An increase in the number of endoreduplicated cells was also observed in the absence of S9 metabolism at the highest dose selected for scoring.

A second main experiment was performed where cells were treated with cC6O4 ammonium salt in the absence of S9 metabolism and harvested after 20 hours. A continuous treatment until harvest was used.

For the second experiment, following treatment with the test item in the absence of S9 metabolism, slight increases but not statistically significant in the incidence of cells bearing aberrations, excluding gaps, over the control values were observed at all dose levels selected for scoring. More remarkable increases of aberrant cells were observed when including gaps. In the presence of S9 metabolism, statistically significant increases in the incidence of aberrant cells including and excluding gaps were observed at the highest dose selected for scoring (2000 μg/ml). The incidences exceeded the range of our historical values for negative controls when excluding gaps. Marked increases in the number of endoreduplicated cells over the controls were seen at the intermediate and high dose levels selected for scoring.

Statistically significant increases in the number of cells bearing aberrations (including and excluding gaps) were observed following treatments with the positive controls Cyclophosphamide (with metabolic activation) and Mitomycin-C (without metabolic activation), indicating the correct functioning of the test system.

 

On the basis of these results it is concluded that cC6O4 ammonium salt induces chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment under the reported experimental conditions.

It is also concluded that the test item inhibits cell cycle progression and chromosome segregation under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The in vivo mammalian bone marrow chromosome aberration test according to OECD 475 guideline was performed on the test item. This study showed a clear negative result since no statistically significant increase in the frequency of aberrant cells occurred at any dose level.

The absorption and the target organ achievement of the substance was confirmed by the available preliminary toxicokinetics study, where after a single oral dose of 26.1 mg/kg b.w. (dry salt) male Wistar rats were sufficiently exposed with absorption of the substance and systemic distribution. In the chromosome aberration test, male Wistar rats were treated with higher doses (312.5, 625.0, 1250 mg/kg b.w.) with clear negative results at all doses.

 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 December 2009 to 13 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 180.2 ± 5.5 g
- Assigned to test groups randomly: yes. The animals are identified by their cage number and tail tags.
- Fasting period before study: not reported
- Housing: In group, Cage Type: Makrolon Type IV, with wire mesh top; Bedding: granulated soft wood bedding.
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories GmbH, 33178 Borchen, Germany)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, 64380 Roßdorf, Germany)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 33 - 65 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12 hours - (artificial light: 6.00 a.m. - 6.00 p.m.)


IN-LIFE DATES: The animals were sacrifed 24 h after treatment. Animals of one of the two groups tested at high dose were sacrifed 48 h after treatment .

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sterile water (Supplier: B. Braun Melsungen AG; Catalogue no.: 6724092.00.00)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): All animals received a single standard volume of 10 ml/kg b.w. orally

The vehicle of the test item was used as vehicle control.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was formulated in sterile water.
All animals received a single standard volume of 10 ml/kg b.w. orally.

DIET PREPARATION:
Pelleted standard diet. Supplied by Harlan Laboratories GmbH, 33178 Borchen, Germany.

Duration of treatment / exposure:

The animals received the test item by gavage.

Frequency of treatment:

The animals received the test item once.

Post exposure period:

24 hours. For the highest dose level an additional sample was taken at 48 hours after treatment.

Dose / conc.:

312.5 mg/kg bw/day (nominal)

Remarks:

Sampled at 24 hours post treatment

Dose / conc.:

625 mg/kg bw/day (nominal)

Remarks:

Sampled at 24 hours post treatment

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

Sampled at 24 hours post treatment

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

Sampled at 48 hours post treatment

No. of animals per sex per dose:

7 males are assigned to each test group.
One group were tested at low dose (Sampled at 24 hours post treatment).
One group were tested at medium dose (Sampled at 24 hours post treatment).
Two groups were tested at the highe dose. (Sampled at 24 and 48 hours post treatment).

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

Name: CPA; Cyclophosphamide (Supplier: Sigma-Aldrich Vertriebs-GmbH 82041 Deisenhofen, Germany; Catalogue no.: C 0768)
Dissolved in: sterile water
Dosing: 15 mg/kg b.w.
Route and Frequency of Administration: orally, once
Volume Administered: 10 ml/kg b.w.
Solution prepared on day of administration.

The stability of CPA at room temperature is good. At 25°C only 3.5 % of its potency is lost after 24 hours.

Tissues and cell types examined:

Bone marrow cells from the femoral epiphyses.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with two animals per sex and test group each under identical conditions as in the mutagenicity study concerning. (Doses: 1000, 1250, 1750, 2000 mg/kg b.w)
The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of 1 h, 2-4h, 6 h, 24h, 30 h (except for the first pre-experiment), and 48 h after each administration of the test item.
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose levels were determined to be the doses that caused toxic reactions without having effects on survival within 48 hours after the treatment. On the basis of the preliminary study, 1250 mg/kg b.w. was estimated to be suitable as the highest dose for the main study.
In the main study three adequately spaced dose levels, spaced by a factor of 2, were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test item once. Seven males were treated per dose group. The animals were examined for acute toxic symptoms around 1 h, 2-4 h, 6 h, 24 h and 48 h after treatment.
Prior (2.5 to 3 hours) to sacrifice, animals were injected intraperitoneally with the spindle inhibitor colcemid (2.0 mg/kg b.w.), to arrest cells in metaphase.

DETAILS OF SLIDE PREPARATION: The femora were removed, the epiphyses were cut off and the marrow was flushed out with approximately 5 ml hypotonic potassium chloride solution (0.56 % w/v, prewarmed to 37 °C). The hypotonic cell suspension was then incubated for 20 min at 37 °C. The cells were sedimented by a brief centrifugation (1000 rpm), the hypotonic supernatant was discarded and the cell pellet was fixed with 3+1 absolute methanol+glacial acetic acid fixative for 60 min. Then the cell pellet was gently resuspended with fixative and stored overnight at 4°C. Prior to making
slides the fixative was changed and enough fixative was added to make a relatively thin cell suspension. The fixative-cell suspension was spread by flame-drying and stained with Giemsa. Cover slips were mounted with EUKITT (KINDLER, 79110 Freiburg, Germany).
One or more slides were made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. At least 100 well spread metaphases per animal were scored for cytogenetic damage on coded slides. The number of chromosome aberrations per metaphase was determined. Only metaphases with the characteristic chromosome number of 42 ± 2 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis; 1000 cells are scored) was determined.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase in the number of structural chromosomal aberrations and a reproducible statistically significant positive response for at least one of the test points.
A test item producing neither a dose-related increase in the number of structural chromosomal aberration nor a statistically significant and reproducible positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the non-parametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

The test is acceptable since at least 5 evaluable animals per group were available, the positive control shows a statistically significant response and the aberration rate of the vehicle control (excl. gaps) is below 2 %.

Statistics:

Mann-Whitney test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Toxic reactions in the main test: Ruffled fur at 312.5 mg/kg b.w. Ruffled fur and reduction of spontaneous activity at 625 and 1250 mg/kg b.w.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000. 1250. 1750, 2000 mg/kg b.w.
- Clinical signs of toxicity in test animals: Ruffled fur at 1000 mg/kg b.w. Ruffled fur, reduction of spontaneous activity and hunchback at 1250 mg/kg. Ruffled fur, reduction of spontaneous activity, hunchback, salivation and death at 1750 mg/kg. Ruffled fur, reduction of spontaneous activity, hunchback and death at 2000 mg/kg.
- Evidence of cytotoxicity in tissue analyzed: Tissue were not analyzed during the preliminary study. However the mitotic indices calculated in the main test were not relevantly reduced after treatment with cC6O4 AMMONIUM SALT, indicating that the test item did not have cytotoxic effects in the bone marrow.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels:
reported in TABLE 2

- Appropriateness of dose levels and route:
Dose levels and route are appropriate.

- Statistical evaluation:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
No statistically significant or biologically relevant enhancement of the aberration frequencies occurred (aberration rates of the test item treated animals ranged between 0.0-1.1%) as compared to the vehicle control value (0.4%). The mean aberration frequencies observed after treatment with cC6O4 AMMONIUM SALT were consistently below 2 % aberrant cells exclusive gaps (given as the upper limit of a tolerable vehicle
control value; see acceptance criteria). There were also no relevant dose dependent increases in the aberration rates of the treated animals.
The study was acceptable as the positive control showed a statistically significant response (aberration rate excluding gaps was 7.3 %) and the aberration frequency exclusive gaps of the vehicle control was below 2 %.

The mitotic indices were not relevantly reduced after treatment with cC6O4 AMMONIUM SALT, indicating that the test item did not have cytotoxic effects in the bone marrow.

Table 1: Summary of Results

Experimental group	dose mg/kg b.w.	preparation hours post administration	number of cells scored	% aberrant cells		mean mitotic index (%)
				incl. gaps	excl. gaps
1: sterile water	0	24	1000	0.4	0.4	4.67
2: cC604 ammonium salt	312.5	24	1000	0.0	0.0	5.27
3: cC604 ammonium salt	625	24	1000	0.7	0.7	3.06
4: cC604 ammonium salt	1250	24	1000	0.7	0.7	3.94
5: cyclophosphamide	15	24	1000	7.3	7.3	3.59
6: cC604 ammonium salt	1250	48	1000	1.1	1.1	5.19

 

 

Table 2: Analysis of aberration types

Group	gap	isogap	break	isobreak	fragment	iso-fragment	deletion	multiple aberration a	exchange	chrom. Disintegration b
1	0	0	0	0	2	1	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	2	0	2	3	0	0	0	0
4	0	0	1	0	4	1	0	0	0	0
5	0	0	39	0	9	4	0	4	18	0
6	0	0	1	0	3	4	0	0	0	0

 

a More than 5 aberrations excluding gaps in one cell; exchanges (but no other aberrations) were recorded separately

b Pulverization

Conclusions:

During the mutagenicity test described and under the experimental conditions reported, the test item did not induce chromosome damages as determined by the chromosome aberration test with bone marrow cells of the rat.
Therefore, cC6O4 AMMONIUM SALT is considered to be non-mutagenic in this chromosome aberration assay in vivo.

Executive summary:

This study was performed to investigate the potential of cC6O4 AMMONIUM SALT to induce chromosome aberrations in bone marrow cells of the rat.

The test item was formulated in sterile water. The volume administered orally was 10 ml/kg body weight (b.w.). The animals were treated once. 24 h and 48 h (only the high dose group) after the treatment the bone marrow cells were collected for chromosome

aberration analysis. 7 males per test group were evaluated for the occurrence of cytogenetic damage. Per animal 100 well spread metaphases were scored for gaps, breaks, fragments, deletions, multiple aberrations, exchanges, and chromosomal disintegrations.

The test item was investigated in this cytogenetic assay at the following doses:

Males: 312.5, 625, and 1250 mg/kg b.w.

The maximum doses for the cytogenetic assay were determined in a pre-experiment for toxicity. 1250 mg/kg b.w. was estimated as to be close to the maximum tolerated doses. The animals treated with this dose showed clinical signs like a reduction in spontaneous activity, ruffled fur and hunchback posture indicating bioavailability of the test item.

No relevant reduction of the mitotic indices could be observed after treatment with the test item, indicating that the test item at the indicated concentrations was not cytotoxic in the bone marrow.

No statistically significant increase in the frequency of aberrant cells occurred after treatment with the test item as compared to the vehicle control.

An appropriate reference mutagen (cyclophosphamide) was used as positive control and showed a distinct and statistically significant increase of induced aberration frequency.

In conclusion, it can be stated that during the mutagenicity test described and under the experimental conditions reported, the test item did not induce chromosome mutations as determined by the chromosome aberration test with bone marrow cells of the rat.

Therefore, cC6O4 AMMONIUM SALT is considered to be non-mutagenic in this chromosome aberration assay in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Considering that the results of in vivo test have an higher reliability in respect to the in vitro test, and considering that the positive findings in the in vitro study occurred only at the highest dose in presence of toxicity signs, it can be concluded that the test item cC6O4 ammonium salt does not show potential to induce chromosome aberrations in mammals.

 

Therefore, on the grounds of the available genotoxicity data, it can be concluded that cC6O4 ammonium salt is not characterized by genotoxic properties.

Justification for classification or non-classification

The evaluation of the potential mutagenicity of the test item is based on four different experimental studies.
Basing on the results of the studies, it was concluded that the test item cC6O4 ammonium salt does not show genotoxic hazard.

Basing on the results of the reported studies, according to the criteria reported in the REGULATION (EC) No 1272/2008, the test item cC6O4 Ammonium salt would indicate the following:

Classification: not required

Signal word: none indicated

Hazard statement: none indicated