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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The recommendations of the International Workshop on Genotoxicity Tests Workgroup (IWGT).
Deviations:
not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification), containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: R10, basic medium supplemented with 10% v/v heat-inactivated horse serum.
Exposure medium: R5 and R10, basic medium supplemented with 5 or 10% v/v heat-inactivated horse serum (used for the 3- and 24 hour exposure, respectively).
Selective medium: R20, basic medium supplemented with 20% v/v heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
Non-selective medium: R20, basic medium supplemented with 20% v/v heat-inactivated horse serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat (male Sprague Dawley) liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
DOSE RANGE FINDING TEST
33, 100, 333, 1000 and 2500 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period. The highest test concentration corresponds to ca. 0.01 M, the guideline recommended maximum concentration.

MUTAGENICITY TEST
Experiment 1: 3 h exposure in the absence and in the presence of S9-mix (4%; v/v)
Exposure concentrations: 1*, 3, 10, 33, 100, 333, 1000 and 2461 μg/mL exposure medium
*: not used for the evaluation of the mutagenicity in the presence of S9-mix due to infection (1)

Experiment 2: 24 h exposure in the absence of S9-mix
Exposure concentrations: 1°, 3, 10, 33, 100, 333*, 666, 1000, 1400, 1900° and 2461° μg/mL exposure medium
*: not used for the evaluation of the mutagenicity due to infection (1)
°: not used for the mutation experiment. In this part of the study, the highest dose that was tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the negative control.

Experiment 2: 3 h exposure in the presence of S9-mix (8%; v/v):
Exposure concentrations: 1, 3, 10, 33, 100, 333, 1000 and 2461 μg/mL exposure medium


(1): Due to infection the CEday2 could not be determined. The number of mutant colonies of these treatment groups and the mutation frequencies of all other tested dose levels were comparable with the number of colonies of the negative control groups and a clear negative response was observed, thus the testing of only seven dose levels had no effect on the integrity of the study.
Vehicle / solvent:
No vehicle/solvent was used, the test material was sufficiently soluble in the exposure medium.
Untreated negative controls:
yes
Remarks:
exposure medium
Negative solvent / vehicle controls:
no
Remarks:
(the test material was sufficiently soluble in the exposure medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Remarks:
final concentrations: MMS (used in assays without metabolic activation) - 15 and 5 µg/mL for a 3 and 24 hours treatment period, respectively; CP (used in assays with metabolic activation) - 7.5 and 10 µg/mL with 4 and 8% (v/v) S9 fraction, respectively.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

DURATION
- Exposure duration:
Experiment 1: 3 h exposure with and without metabolic activation.
Experiment 2: 3 h exposure with and 24 h exposure without metabolic activation.
- Expression time (cells in growth medium): 2 days.
- Selection time (if incubation with a selection agent): 11-12 days.

SELECTION AGENT (mutation assays): trifluorothymidine (TFT), final concentration 5 µg/mL.
STAIN (for cytogenetic assays): 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for 2 hours, final concentartion 0.5 mg/mL.

NUMBER OF REPLICATIONS :
- Exposure phase: single cultures (duplicates for the negative control).
- Assessment of cloning efficiency : two 96-well microtiter plates per treatment (test material, negative (vehicle) and positive controls.
- Assessment of mutant potential: five 96-well microtiter plates per treatment (test material and negative (vehicle) control); ten 96-well microtiter plates for the positive controls.

NUMBER OF CELLS EVALUATED:
- Assessment of cloning efficiency : 192 cells (one cell per well). In the dose groups of 33 and 666 µg/mL in the second experiment (without S9-mix) a total number of 181 wells/cells was used for the determination of the cloning effeciency due to infection. This loss was only 6% and the CEday2 could be determined, thus it had no effect on the outcome of the study.
- Assessment of mutant potential: 9.6x10E5 cells per concentration (i.e., 2000 cells/well for the test material and the negative controls, 1000 cells/well for the positive controls).

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth, relative total growth, cloning efficiency.

GROWTH CONDITIONS
- ca. 37 °C, ca. 5% CO2 in a humidified incubator (80-100%). Temporary deviations from these values have been observed, caused by openeing and closing of the incubator door. Based on laboratory historical data these deviations were considered not to have effected the study integrity.

OTHER EXAMINATIONS:
- Scoring of small and large colonies
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutant frequency (MF) of more than the sum of the control MF and the Global Evaluation Factor (GEF) in a dose-dependent manner (GEF = 126, International Workshop on Genotoxicity Tests Workgroup (IWGT), see Moore et al. 2006, detailed reference below).

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + GEF.
b) The results are confirmed in an independently repeated test.

In addition, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Reference for GEF:
Moore, M.M., Honma, M., Clements, J., Bolcsfoldi, G., Burlinson, B. Cifone, M., Clarke, J., Delongchamp, R., Durward, R., Fellows, M., Gollapudi, B., Hou, S., Jenkinson, P., Lloyd, M., Majeska, J., Myhr, B., O’Donovan, M, Omori, T, Riach, C., San, R., Stankowski. JR. L.F., Thakur, A.K., Van Goethem, F., Wakuri, S. and Yoshimura, I. (2006). Mouse lymphoma thymidine kinase gene mutation assay: Follow-up meeting of the international workshop on Genotoxicity testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environmental and Molecular Mutagenesis. 47, 1-5.
Statistics:
No statistical analysis was reported.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see 'Additional information on results'
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
No relevant toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 2500 μg/ml (ca. 0.01 M, the maximum concentration recommended in the guideline) compared to the suspension growth of the negative control after 3 hours of treatment both in the absence and presence of S9-mix. After 24 hours of treatment in the absence of S9-mix, the relative suspension growth was 4% at the test substance concentration of 2500 μg/ml compared to the relative suspension growth of the negative control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the control cultures were within the values of the historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First experiment (3 hours exposure with and without metabolic activation): no severe toxicity was observed.
Second experiment (3 hours exposure with metabolic activation and 24 hours exposure without metabolic activation): In the absence of S9-mix, the dose levels of 1 to 100 μg/ml showed no cytotoxicity. At 666, 1000 and 1400 µg/ml the relative total growth was reduced by 55, 52 and 85% compared to the total growth of the negative controls. The dose levels of 1900 and 2461 μg/ml were too toxic for further testing. In the presence of S9-mix, no toxicity was observed.

EXAMINATION OF COLONY SIZE:
The numbers of small and large colonies in the treated cultures were comparable to the numbers of small and large colonies of the negative controls.
For more details, see 'Tables_MLA_T-1063FM.pdf' attached as background material.
Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 March 2003 to 16 May 2003
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)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromatid-type aberrations
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO cells (CHO K-1line), used in the present assay, were obtained from Prof. Dr. A.T. Natarajan, University of Leiden, The Netherlands. This cell line derives from the CHO cells originally isolated from an explant of the ovary of the Chinese hamster (Cricetulus griseus, 2n = 22) by Kao and Puck (1968). The modal chromosome number of these cells is 20-22 (stable aneuploid karyotype). The cellcycle time is 12-14 h. The cells are stored as frozen stock cultures in liquid nitrogen. Subcultures were prepared from these stocks (passage 16) for experimental use. Each passage CHO cells in the liquid nitrogen is checked for mycoplasma contamination and karyotype stability, which were absent and stable, respectively.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
First Test
0, 62.5, 125, 250, 500 μg/ml – in the absence of S9 mix
0, 250, 500, 1000 μg/ml – in the presence of S9 mix

Second Test
0, 50, 75, 100, 300, 500, 750 μg/ml – in the absence of S9 mix
0, 500, 750, 1000, 1500, 2000 μg/ml – in the presence of S9 mix
Vehicle / solvent:
It was decided to use ethanol (100 %) as vehicle for the test substance.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Culture medium for CHO cells: The medium for culturing the CHO cells consisted of: Ham's F-12 medium (with Glutamax-1), supplemented with heat-inactivated (45 min, 56 °C) foetal calf serum (10 %), penicillin (100 IU/ml medium) and streptomycin (100 μg/ml medium).
Metabolic activation system: The S9-mix consisted of a liver homogenate fraction (89) and cofactors as described by Ames et al. (1975) and Maron and Ames (1983). The S9, used in this study, was part of a batch prepared on 25 September 2002. Immediately before use, a S9-mix was prepared by mixing the thawed S9 with a NADPH-generating system. The final concentrations of the various ingredients in the S9-mix were: MgCl2 8 mM; KCl 33 mM; G-6-P 5 mM; NADP 4 mM; sodium phosphate 100 mM (pH 7 .4) and S9 40 % (v/v).
Solubility of the test substance: It was decided to use ethanol (100 %) as vehicle for the test substance. Before use, the test substance was pulverized with a pestle in a mortar. In this condition, the test substance could be suspended in ethanol (100%) up to 200 mg/ml. This stock concentration of 200 mg/ml was stirred for 2 minutes at a magnetic stirrer and appeared as a milky suspension. Thereafter, serial dilutions (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.39 mg/ml) of the stock concentration (200 mg/ml) were prepared in ethanol. Fifty μl of the prepared stock concentrations (ranging from 200 to 0.39 mg/ml) in ethanol was added to 4.95 ml culture medium. This resulted in final concentrations of the test substance in the culture medium of 2000, 1000, 500,250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 μg/ml. The pH of the seven highest final concentrations in the culture medium were measured immediately after preparation. Thereafter, the seven highest final concentrations were incubated for 18 hours at 37 °C in humidified air containing 5% C02. After this incubation period, the pH of the seven highest final concentrations were measured for the second time.
To avoid artefactual chromosome breakage at higher dose levels than 2000 μg/ml, as a result of lower pH values, it was decided to take 2000 μg/ml (final concentration in the culture medium) as highest concentration of the test substance for the first chromosomal aberration test.

The first chromosomal aberration test: Exponentially growing cells were seeded in sterile, screw-capped tissue culture flasks (surface area 25 cm2; 120,000 cells per flask) containing 5 ml culture medium and then incubated at 37 °C in humidified air containing 5 % CO2. On the next day, the cells were exposed to the test substance, in both the absence and presence of the S9-mix. Just before use, a stock concentration of the test substance of 200 mg/ml and its serial dilutions (at a twofold interval; ranging from 100 to 0.05 mg/ml) were prepared in ethanol. As positive controls, mitomycin C (in the absence of S9-mix) and cyclophosphamide (in the presence of S9-mix) were used. In all instances duplicate cultures were used. In the absence of S9-mix, the treatment time was 4 hours (pulse treatment) and 18 hours (continuous treatment). In the presence of S9-mix, the treatment time was 4 hours (pulse treatment). In both the absence and presence of S9-mix, the harvesting of the cells was 18 hours after onset of the treatment.
In the absence of the S9-mix, 50 μl of the vehicle (ethanol), 50 μl of each of the dilutions of the test substance and 50 μl of the positive control substance mitomycin C was added directly to 4.95 ml freshly added tissue culture medium in the flasks and the culture medium was checked visually. The cultures were incubated at 37 °C in humidified air containing 5 % C02 and treated for 4 hours (pulse treatment) and 18 hours (continuous treatment). After the 4 hours treatment period (pulse treatment group only), the cells and culture medium of the pulse treatment group were checked again. The medium with the test substance was removed, the cells washed twice with phosphate-buffered saline (pH 7.4) and supplied with 5 ml freshly prepared culture medium. Thereafter, the cells were incubated for an additional 14 hours at 37 °C in humidified air containing 5% CO2. Two hours before the end of the culture period (18 hours), the cells and culture medium were checked visually.
In the presence of the S9-mix, the culture medium with foetal calf serum was replaced by 4.45 ml culture medium with penicillin and streptomycin but without foetal calf serum. Fifty μl of the vehicle (ethanol), 50 μl of each of the dilutions of the test substance and 50 μl of the positive control substance cyclophosphamide was added directly to the tissue culture medium in the flasks and the culture medium was checked visually. Thereafter, 0.5 ml of the S9-mix was added to all the cultures. After the 4 hours treatment period, the cells were checked again. The medium with the test substance was removed, the cells washed twice with phosphate-buffered saline (pH 7.4) and supplied with 5 ml freshly prepared culture medium with foetal calf serum. The cells were incubated for an additional 14 hours at 37 °C in humidified air containing 5 % CO2. Two hours before the end of the culture period (18 hours), the cells and culture medium were checked visually.

The second (independent) chromosomal aberration test: The second (independent) chromosomal aberration test was carried out essentially as the first chromosomal aberration test. In the absence of 89-mix, one group of cultures was treated continuously for 18 hours. A second group of cultures was treated continuously for 32 hours. In the presence of 89-mix, one group of cultures was pulse-treated for 4 hours and harvested 18 hours after onset of the treatment. A second group of cultures was pulse-treated for 4 hours and harvested 32 hours after onset of the treatment. The dose levels, used in the second chromosomal aberration test were based on the results obtained in the first chromosomal aberration test.

Cell fixation, preparation of slides and scoring: Two hours before the end of the total incubation period, the cells of the remaining cultures were arrested in the metaphase stage of mitosis by the addition of colcemid (final concentration: 0.1 mM medium). At the end of the total incubation period the cells were harvested by trypsinization, treated for 15 min at 37 °C with a hypotonic solution (1 % sodium citrate), fixed with a 3: 1 mixture of methanol:glacial acetic acid (two refreshments of the fixative), and transferred to clean microscope slides. Two slides were prepared from each culture. The slides were stained in a 2 % solution of Giemsa, rinsed in water, dried and embedded with a Tissue-TEK Coverslipper. The slides were coded by a qualified person not involved in scoring the slides, to enable "blind" scoring. At least 1000 nuclei in each culture were examined (500 on each slide) to determine the mitotic index (percentage of cells in mitosis). After the results of the mitotic index scoring and the observations with respect to the quality of the metaphases had been obtained, a selection of the concentrations of the test substance, to be analysed for chromosomal aberrations, was carried out.
In all treatment groups, in both the first and second (independent) chromosomal aberration test, three concentrations of the test substance together with the negative and positive controls were selected for the analysis of chromosomal aberrations. If possible, the highest concentration should reduce the mitotic index with at least 50 % (but not more than 70 %), when compared to the negative control value or exhibit some other clear indication of cytotoxicity. Subsequently, the cultures of the selected concentrations of the test substance, together with the negative and positive control cultures, were analysed for the induction of structural chromosomal aberrations. For each treatment group, 200 well spread metaphases per concentration ( 100 metaphases per culture), each containing 20-22 centromeres, were analysed by microscopic examination for chromatid-type aberrations (gaps, breaks, fragments, interchanges), chromosome-type aberrations (gaps, breaks, minutes, rings, dicentrics) and other anomalies, such as interstitial deletions, endoreduplication, polyploidy and multiple aberrations (>10 aberrations per cell, excluding gaps), according to the criteria recommended by Savage (1975). If heavily damaged or endoreduplicated cells were observed, these cells were recorded but the cells were not counted and included in the 200 analysed cells. The Vernier readings of all aberrant metaphases were recorded
Evaluation criteria:
Data were analysed statistically by Fisher's exact probability test (two-sided) to determine significant differences between treated and control cultures.
Statistics:
The study was considered valid because the positive controls gave the statistically significant increases in the number of aberrant cells and the negative controls were within the historical range.
A response is considered to be positive if a concentration-related increase or a reproducible increase in the number of cells with structural chromosomal aberrations is observed.
A response is considered to be equivocal if the percentage of cells with structural chromosomal aberrations is statistically marginal higher than that of the negative control (0.05A test substance is considered to be clastogenic if a concentration-related increase in the percentage of cells with structural chromosomal aberrations over the concurrent control frequencies is observed, or if a single positive test point is observed in both tests.
A test substance is considered to be negative in the chromosomal aberration test if it produces neither a dose-related increase in the number of structural chromosomal aberrations nor a reproducible positive response at any of the test points.
Gaps (achromatic lesions) are recorded separately and not included in the final assessment of clastogenic activity.
Both statistical significance and biological relevance are considered together in the evaluation of the results.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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:
In both chromosomal aberration tests, the selection of the highest concentration analysed for the induction of chromosomal aberrations was based on toxicity of the test substance to the cells.
In the first chromosomal aberration test, in both pulse treatment groups with and without metabolic activation (S9-mix), the mitotic indices of all the concentrations analysed (1000, 500 and 250 μg/ml) were not reduced, when compared to the mitotic indices of the concurrent controls. In this treatment groups, the test substance did not induce a statistically significant increase in the number of aberrant cells at any of the concentrations analysed.
In the first chromosomal aberration test, in the continuous treatment group without metabolic activation (S9-mix), the mitotic index of the highest concentration analysed (250 μg/ml) was reduced to 45% of that of the concurrent control. The mitotic index of the moderate concentration analysed (125 μg/ml) was slightly reduced to 76% of that of the concurrent control. In this treatment group, the test substance did not induce a statistically significant increase in the number of aberrant cells at any of the concentrations analysed.
In the second chromosomal aberration test, in the continuous treatment group of 18 hours without metabolic activation (S9-mix), the mitotic index of the highest concentration analysed (300 μg/ml) was slightly reduced to 76% of that of the concurrent control. In this treatment group, the test substance did not induce a statistically significant increase in the number of aberrant cells at any of the concentrations analysed.
In the second chromosomal aberration test, in the continuous treatment group of 32 hours without metabolic activation (S9-mix), the mitotic index of the highest concentration analysed (300 μg/ml) was clearly reduced to 31% of that of the concurrent control. The mitotic index of the moderate concentration analysed (100 μg/ml) was slightly reduced to 71% of that of the concurrent control. In this treatment group, the test substance did not induce a statistically significant increase in the number of aberrant cells at any of the concentrations analysed.
In the second chromosomal aberration test, in the pulse treatment group with metabolic activation (S9-mix), at the early sampling time of 18 hours, the mitotic index of the highest concentration analysed (1000 μg/ml) was clearly reduced to 63% of that of the concurrent control. In this treatment group, the test substance did not induce a statistically significant increase in the number of aberrant cells at any of the concentrations analysed.
In the second chromosomal aberration test, in the pulse treatment group with metabolic activation (S9-mix), at the later sampling time of 32 hours, the mitotic indices of all the concentrations analysed (2000, 1500 and 1000 μg/ml) were not reduced, when compared to the mitotic index of the concurrent control. In this treatment group, the test substance did not induce a statistically significant increase in the number of aberrant cells at any of the concentrations analysed.
In both the first and second (independent) chromosomal aberration test, the positive control substances mitomycin C (in the absence of a metabolic activation system) and cyclophosphamide (in the presence of a metabolic activation system), induced the expected statistically significant increases in the incidence of structural chromosomal aberrations.
See 'Tables_CA_T-1063FM.pdf' attached as background material.
Remarks on result:
other: all strains/cell types tested

Table 1: Chromosomal aberration test with T-1063FM in the presence of S9-mix: mitotic index (Test 1)

Treatment time:

4h

Harvesting time:

18h

Treatment

Dose (μg/ml)

Number of cells scored

Mitotic index

Percentage of cells in mitosis

Percentage of treated versus negative control value

Selection for chromosomal aberration scoring

Ethanol

0

1000

8.2

100

+

1000

8.7

+

Test substance

1000

1000

9.8

113

+

1000

9.3

+

500

1000

7.8

93

+

1000

8.0

+

250

1000

9.1

107

+

1000

8.9

+

Cyclophosphmide

3.0

1000

4.3

51

+

1000

4.4

+

 

Table 2: Chromosomal aberration test with T-1063FM in the absence of S9-mix: mitotic index (Test 1)

Treatment time:

4h

Harvesting time:

18h

Treatment

Dose (μg/ml)

Number of cells scored

Mitotic index

Percentage of cells in mitosis

Percentage of treated versus negative control value

Selection for chromosomal aberration scoring

Ethanol

0

1000

9.8

100

+

1000

10.5

+

Test substance

1000

1000

9.3

93

+

1000

9.5

+

500

1000

11.4

110

+

1000

10.9

+

250

1000

9.3

95

+

1000

9.9

+

Mitomycin C

0.1

1000

4.2

41

+

1000

4.1

+

 

Table 3: Chromosomal aberration test with T-1063FM in the absence of S9-mix: mitotic index (Test 1)

Treatment time:

18h

Harvesting time:

18h

Treatment

Dose (μg/ml)

Number of cells scored

Mitotic index

Percentage of cells in mitosis

Percentage of treated versus negative control value

Selection for chromosomal aberration scoring

Ethanol

0

1000

9.9

100

+

1000

9.7

+

Test substance

500

1000

3.2

33

-

1000

3.2

-

250

1000

4.2

45

+

1000

4.6

+

125

1000

7.0

76

+

1000

7.9

+

62.5

1000

9.4

95

+

1000

9.2

+

Mitomycin C

0.05

1000

3.6

38

+

1000

3.8

+

-discarded

 

Table 4: Chromosomal aberration test with T-1063FM in the absence of S9-mix: mitotic index (Test 2)

Treatment time:

18h

Harvesting time:

18h

Treatment

Dose (μg/ml)

Number of cells scored

Mitotic index

Percentage of cells in mitosis

Percentage of treated versus negative control value

Selection for chromosomal aberration scoring

Ethanol

0

1000

8.9

100

+

1000

8.3

+

Test substance

750

1000

1.5

19

-

1000

1.7

-

500

1000

1.1

15

-

1000

1.4

-

300

1000

6.5

76

+

1000

6.5

+

100

1000

7.8

95

+

1000

8.5

+

75

1000

8.9

104

+

1000

9.0

+

Mitomycin C

0.05

1000

4.9

55

+

1000

4.6

+

-discarded

 

Table 5: Chromosomal aberration test with T-1063FM in the absence of S9-mix: mitotic index (Test 2)

Treatment time:

32h

Harvesting time:

32h

Treatment

Dose (μg/ml)

Number of cells scored

Mitotic index

Percentage of cells in mitosis

Percentage of treated versus negative control value

Selection for chromosomal aberration scoring

Ethanol

0

1000

8.9

100

+

1000

8.5

+

Test substance

300

1000

2.7

31

+

1000

2.7

+

100

1000

6.3

71

+

1000

6.1

+

75

1000

8.9

101

+

1000

8.7

+

50

1000

8.7

101

-

1000

8.9

-

Mitomycin C

0.025

1000

6.1

68

+

1000

5.7

+

-discarded

 

Table 6: Chromosomal aberration test with T-1063FM in the presence of S9-mix: mitotic index (Test 2)

Treatment time:

4h

Harvesting time:

18h

Treatment

Dose (μg/ml)

Number of cells scored

Mitotic index

Percentage of cells in mitosis

Percentage of treated versus negative control value

Selection for chromosomal aberration scoring

Ethanol

0

1000

10.9

100

+

1000

10.7

+

Test substance

1500

1000

2.7

25

-

1000

2.7

-

1000

1000

7.0

63

+

1000

6.6

+

750

1000

9.4

84

+

1000

8.8

+

500

1000

10.1

90

+

1000

9.3

+

Cyclophosphamide

3.0

1000

4.4

41

+

1000

4.5

+

-discarded

 

Table 7: Chromosomal aberration test with T-1063FM in the presence of S9-mix: mitotic index (Test 2)

Treatment time:

4h

Harvesting time:

32h

Treatment

Dose (μg/ml)

Number of cells scored

Mitotic index

Percentage of cells in mitosis

Percentage of treated versus negative control value

Selection for chromosomal aberration scoring

Ethanol

0

1000

9.4

100

+

1000

9.8

+

Test substance

2000

1000

8.8

95

+

1000

9.5

+

1500

1000

9.0

100

+

1000

10.2

+

1000

1000

9.7

99

+

1000

9.4

+

750

1000

9.3

99

-

1000

9.8

-

Cyclophosphamide

3.0

1000

8.5

86

+

1000

8.1

+

-discarded

Conclusions:
Interpretation of results:
negative with and without metabolic activation

These data, obtained in two (independent) chromosomal aberration tests, support the conclusion that, under the conditions used in this study, the test substance T-1063FM was not clastogenic for CHO cells.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 February 2003 to 17 May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine or tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate
Test concentrations with justification for top dose:
0, 62, 185, 556, 1667 and 5000 μg/plate
Vehicle / solvent:
Ethanol was chosen as vehicle (instead of DMSO), because the test substance solution could not be suspended in DMSO. For the first and second test just before use, the test substance was suspended in the vehicle at 50 mg/ml, assumed purity of 100%. A white milky suspension was obtained. Serial3-fold dilutions in ethanol were prepared, under frequently stirring and in total five concentrations were tested, ranging from 62 to 5000 μg/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: N-ethyl-N-nitrosourea, 2-aminoanthracene
Details on test system and experimental conditions:
Metabolic activation system (S9-mix): On the day of use, aliquots of S9 liver homogenate were thawed and mixed with a NADPH generating system. The final concentrations of the various ingredients in the S9-mix were: MgCl2 8 mM; KCl 33 mM; G-6-P 5 mM; NADP 4 mM; sodium phosphate 100 mM (pH 7.4), 46 mM NaCl, and S9 10 %. The S9-mix was kept on ice until use.

Mutagenicity assay: The plate-incorporation method with the histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98 and TA 100 and the tryptophan-requiring Escherichia coli mutant WP2 uvrA as indicator strains were applied. The assay has been described in detail by Ames et al. (1975) and by Maron and Ames (1983). A preliminary test to assess the toxicity of the test substance was not performed. Therefore the toxicity test was incorporated in the first mutagenicity assay.
The actual concentrations of the test substance in the test solutions were not determined. Therefore, the concentrations quoted in this report are nominal concentrations.
The background spontaneous reversion rate observed in the Salmonella typhimurium T A98 was outside the acceptable ranges for negative control data. Therefore the assay was repeated with Salmonella typhimurium T A98.
On the request of the sponsor a second independent test was performed.
Briefly, the mutagenicity assay was carried out as follows. To 2 ml molten top agar (containing 0.6% agar, 0.5% NaCl and 0.05 mM L-histidine.HCI/0.05 mM biotin for the S. typhimurium strains, and supplemented with 0.05 mM tryptophane for the E. coli WP2 uvrA strain), maintained at ca. 46 °C, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain, 0.1 ml of the appropriate test substance solution, or of the negative or positive control substance solution, and 0.5 ml S9-mix for with metabolic activation or 0.5 ml sodium phosphate 100 mM (pH 7.4) for without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2% glucose). All determinations were made in triplicate. The plates were incubated at ca. 37 °C for 48-72 hours. Subsequently, the his+ and trp+ revertants were counted. The background lawn of bacterial growth was examined microscopically to determine any growth-diminishing or growth enhancing effects by the test substance, if a two-fold or greater increase in the mean number of his+ or trp+ revertants was observed. Cytotoxicity is defined as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.
Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5 % of the plates are lost through contamination or other unforeseen events.
A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.
A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.
Omission of the second assay under these conditions is acceptable as a single assay does not or hardly results in false negative conclusions (TNO historical data and Kirkland and Dean, 1994).
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.
Both numerical significance and biological relevance are considered together in the evaluation.
Statistics:
No statistical analysis was performed.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test substance was suspended in ethanol. Two bacterial reverse mutation tests were performed with all strains in the absence and the presence of S9-mix with five different concentrations of the test substance, ranging from 62 - 5000 μg/plate. Negative controls (ethanol) and positive controls were run simultaneously with the test substance.
In the first test T-1063FM was slightly toxic to strain TA 1537 at the highest dose level, as was evidenced by the presence of a decrease in the mean number of revertant colonies compared to the negative controls. No toxicity was observed in the second test. A dose related precipitation of the test substance was observed in the top agar, but not on the plates.
In the first test, the background spontaneous reversion rate observed in the Salmonella typhimurium TA98 was outside the acceptable ranges for the negative control data. Therefore the assay was repeated with Salmonella typhimurium TA98. On the request of the sponsor a second independent test was performed with all strains.
In both the absence and the presence of S9-mix and in all strains, T-1063FM did not cause a more than two-fold increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control.
The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.
See 'Tables_Ames_T-1063FM.pdf' attached as background material.

Table 1: Bacterial reverse mutation test with T-1063FM (first test)

 

TA 1535

TA 1537

TA 98

TA 100

E. Coli

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0μg/plate

31

28

18

25

80

79

181

217

43

43

25

20

16

22

86

91

193

143

36

43

32

31

14

19

216

84

119

167

43

43

Mean

29

26

16

22

127

85

164

176

41

43

StDev

4

6

2

3

77

6

40

38

4

0

62μg/plate

22

22

11

22

79

103

177

250

25

32

23

30

14

19

58

90

129

190

43

29

28

32

18

20

60

79

98

155

40

42

Mean

24

28

14

20

65

91

135

198

36

34

StDev

3

5

4

2

11

12

40

48

10

7

185μg/plate

24

23

16

36

49

86

165

138

36

48

26

23

14

16

44

86

137

145

60

44

28

34

19

28

43

76

150

145

48

53

Mean

26

27

16

27

45

83

151

143

48

48

StDev

2

6

3

10

3

6

14

4

12

5

556μg/plate

29

32

7

20

44

106

156

172

42

42

28

31

13

22

54

96

133

147

50

47

26

20

12

14

68

***

***

160

42

44

Mean

28

28

11

19

55

101

145

160

45

44

StDev

2

7

3

4

12

7

16

13

5

3

1667μg/plate

20

25

10

24

38

98

189

***

40

49

23

26

14

8

71

83

137

155

36

44

32

24

14

20

32

77

135

133

46

53

Mean

25

25

13

17

47

86

154

144

41

49

StDev

6

1

2

8

21

11

31

16

5

5

5000μg/plate

19

24

12

14

47

71

126

169

46

43

24

20

12

17

46

71

160

137

36

55

24

32

12

13

44

90

139

175

53

37

Mean

22

25

12

15

46

77

142

160

45

45

StDev

3

6

0

2

2

11

17

20

9

9

Pos. Control

556

772

4332

253

1878

1811

718

3260

219

1537

586

1016

5441

275

2364

1880

644

3394

148

1690

555

908

5506

350

2378

1835

730

3352

191

1702

Mean

569

899

5093

293

2207

1842

697

3335

186

1643

StDev

16

122

660

51

285

35

47

69

36

92

Mean = Average number of revertants per plate

StDev = Standard deviation

S9 = Liver homogenate from rats treated with aroclor

*** = Plate not counted due to contamination

Pos. Control = Positive control

 

Table 2: Bacterial reverse mutation test with T-1063FM (repeated first test)

 

TA 98

-S9

+S9

0μg/plate

44

59

48

64

41

77

Mean

44

67

StDev

4

9

62μg/plate

38

53

34

71

35

59

Mean

36

61

StDev

2

9

185μg/plate

44

60

41

58

30

59

Mean

38

59

StDev

7

1

556μg/plate

61

76

42

74

31

64

Mean

45

71

StDev

15

6

1667μg/plate

42

53

48

71

41

66

Mean

44

63

StDev

4

9

5000μg/plate

60

68

42

73

43

73

Mean

48

71

StDev

10

3

Pos. Control

1656

1614

1581

1444

1786

1217

Mean

1674

1425

StDev

104

199

Mean = Average number of revertants per plate

StDev = Standard deviation

S9 = Liver homogenate from rats treated with aroclor

Pos. Control = Positive control

Conclusions:
Interpretation of results:
negative with and without metabolic activation

It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that T-1063FM was not mutagenic under the conditions employed in this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 February 2004 to 15 March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
female
Details on test animals or test system and environmental conditions:
Animals, housing and care: For the micronucleus test (28 females), young adult swiss mice, were obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany. The animals arrived on 18 February 2004. The mice were taken in their unopened shipping containers directly to room number 5.2.08. After routine serological examination, carried out on the day of arrival, the animals stayed in the same room. The results of this serological examination were satisfactory. The animals were housed in sterilised Macrolon cages (type IT), fitted with a grid cover of stainless steel and with a bedding of wood shavings (Espen E001). During the quarantine and acclimatization period the animals were observed daily for overt signs of ill health and anomalies. Housing conditions were conventional. For safety reasons, the animals of the positive control group were housed in a laminar down-flow cabinet, just prior to administration and until sacrifice. The animal rooms were ventilated with about 10 air changes per hour and were maintained at a temperature of 22 +1-3 °C and a relative humidity of at least 30% and not exceeding 70% other than during room cleaning. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Feed and drinking water: With the exception of the fasting period prior to dosing, feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study. Fresh pellet diet was provided once weekly.
The animals received a commercial rodent diet (Rat and Mouse No. 3 Breeding Diet, RM3). Batch 3272 (expiry date 12 May 2004) was used for this micronucleus test. Each batch of this diet is analysed by the supplier (SDS Special Diets Services, Witham, England) for nutrients and contaminants. The certificates of analysis pertaining to batch 3272 are stored in the archives of TNO Nutrition and Food Research.
The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water for human consumption (quality guidelines according to Dutch legislation based on the EEC Council Directive 98/83/EEC), was supplied by N.V. Hydron Midden-Nederland. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to TNO Nutrition and Food Research. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of TNO in Zeist for a limited number of variables. The results of the samples taken during or close to the conduct of the study are stored in the archives of TNO Nutrition and Food Research.
Route of administration:
oral: gavage
Vehicle:
physiological saline
Details on exposure:
Prior to dosing, the test substance was suspended in physiological saline, to yield a milky aqueous suspension with a concentration of 100 mg/ml.
Duration of treatment / exposure:
24 & 48 hours
Frequency of treatment:
Single dose
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per dose/per scheduled sacrifice time
Control animals:
yes
Positive control(s):
5 mice treated with the positive control substance mitomycin C
Tissues and cell types examined:
polychromatic and normochromatic erythrocytes
Details of tissue and slide preparation:
From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glass drawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grtinwald Giemsa solution. The other smear was stored as reserve slide.
Microscopic examination of bone marrow smears: The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear. The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.
Evaluation criteria:
The study is considered valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range.
A test substance is considered to cause chromosomal damage and/or damage to the mitotic apparatus, if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.
A test substance is considered to be negative in the micronucleus test if it produces no positive response at any of the time points analysed.
The test substance or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test substance statistically reduce the mean number of PE/E or causes systemic toxicity.
Both statistical significance and biological relevance are considered together in the evaluation.
Statistics:
Data on MPE and PE were subjected to a Two Way Anova with factor group (A and B) and time point (24 hours and 48 hours). If one of the factors and/or interactions yielded a significant effect (p<0.05), it was followed by pooled error variance t-tests or, if variances were not homogeneous, separate variance t-tests. These t-tests were applied to the negative control group A versus treatment group B per time point. Furthermore, the positive control group C and the negative control group A at time point 24 hours were compared using t-tests.

All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Clinical signs in the micronucleus test: No clinical signs, as a result of the treatment with the test substance T -1063FM, could be demonstrated during the performance of the micronucleus test.
Statistical analysis of the micronucleus test results: At both sacrifice times of 24 hours and 48 hours after treatment, the Two Way Anova did not yield a statistically significant effect for MPE and PE. This indicates that treatment with the test substance T-1063FM, at the limit dose level of 2000 mg/kg-bw, did not result in genotoxicity or clastogenicity to the bone marrow target cells.
At the sacrifice time of 24 hours, in the positive control group, the incidence of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly different (P<0.001) from the negative control A. This demonstrates the validity of the test system.
The results of this micronucleus test did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in female mice, treated orally with the test substance T-1063FM.
See 'Tables_MN_T-1063FM.pdf' attached as background material.

Table 1: Micronucleated Polychromatic Erythrocytes (MPE) in the mice of the micronucleus test

The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE)

Group:

A

Vehicle control (physiological saline)

B

T-1063FM (dose level)

C

Pos. control mitomycin C (dose level)

Sex

† (h)

 

(2000 mg/kg-bw)

(0.75 mg/kg-bw)

Female

24

1.6±0.5

1.4±0.5

45.4±9.8***

Female

48

1.6±0.9

2.8±1.1

-

Means and standard deviations: ***P<0.001 (t-tests); group size: 5

The positive control group C, at time point 24 hours, differed significantly from the negative control A (P<0.001).

 

Table 2: Polychromatic Erythrocytes (PE) in the mice of the micronucleus test

The group mean numbers of polychromatic erythrocytes (PE) per 200 erythrocytes (E)

Group:

A

Vehicle control (physiological saline)

B

T-1063FM (dose level)

C

Pos. control mitomycin C (dose level)

Sex

† (h)

 

(2000 mg/kg-bw)

(0.75 mg/kg-bw)

Female

24

96.4±9.5

97.8±16.0

89.4±22.2

Female

48

92.4±10.1

89.4±10.7

-

 

Conclusions:
Interpretation of results: negative.
These data support the conclusion that, under the conditions used in this study, the test substance T-1063FM did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Short description of key information:

In vitro Ames Assay: Negative with and without metabolic activation.

In vitro Chromosome Aberration Test:  Negative with and without metabolic activation.

In vitro Mammalian Cell Gene Mutation Test: Negative with and without metabolic activation.

In vivo Micronucleus Test: Negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative results attained in the in vitro and in vivo genotoxicity studies T-1063FM is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to REGULATION (EC) 1272/2008.