Diphosphoric acid, compd. with piperazine (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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

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

Reference 2

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.

Reference 3

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.