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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4rd April 2018 - 9th April 2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Titanium 2,2',2''-nitrilotrisethanolate
EC Number:
240-015-1
EC Name:
Titanium 2,2',2''-nitrilotrisethanolate
Cas Number:
15879-01-3
Molecular formula:
C6H15NO3.xTi
IUPAC Name:
titanium 2,2',2''-nitrilotrisethanolate
Test material form:
liquid
Details on test material:
Test Item Name Tyzor TE
IUPAC Name titanium 2,2',2''-nitrilotrisethanolate
CAS No. 15879-01-3
EC No. 240-015-1
Appearance Light yellow liquid
manufactured by Dorf Ketal Speciality Catalyst Pvt Limited




Specific details on test material used for the study:
Test Item Name Tyzor TE
IUPAC Name titanium 2,2',2''-nitrilotrisethanolate
CAS No. 15879-01-3
EC No. 240-015-1
Appearance Light yellow liquid
manufactured by Dorf Ketal Speciality Catalyst Pvt Limited

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Cytokinesis block (if used):
Colchicine
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 homogentate. prepared from male Wistar rats induced with a single IP injection of Aroclor 1254 (0.7 mL/rat), 5 days before sacrifice.
Test concentrations with justification for top dose:
Preliliminary cytotoxicity test:
31.25, 62.5, 125, 250, 500, 1000, 2000 ug/mL
Following concentrations of the test item were used in the preliminary cytotoxicity test:
Experiment 1 and 2: 222, 667, 2000 ug/ml
Experiment 3: 62.5, 250, 1000 ug/ml

Justification for the top dose; percipitation and cytotoxicity


Vehicle / solvent:
Ethanol- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item precipitated when mixed with sterile water at 200mg/mL and the test item was soluble in DMSO at 200 mg/mL

- Justification for percentage of solvent in the final culture medium:
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Preparation of Target Cells

Exponentially growing CHO-K1 cells were plated at a density of approximately 5 x 106 cells in 25 cm2 flask in duplicate and incubated for approximately 24 hours at 37 ± 1oC in a humidified atmosphere of 5 ± 0.2 % CO2 in air.
At the time of preparation of target cells, two parallel cultures were kept along with the vehicle control and treatment groups. Cell counts were made from these cultures at the 0-hour treatment to obtain the baseline cell count for estimation of RICC.

Exposure of Target Cells to Treatment

After the incubation period, the medium from the test flasks was removed by aspiration and replaced with 13.5 mL and 15 mL F-12 FBS5, in Experiments 1 and 2 and with 15 mL F-12 FBS10 in Experiment 3.
For the experiment in the presence of metabolic activation, 1.5 mL S9 mix was added to the appropriate test flasks to achieve a final concentration of
10 % (v/v) in the test medium.

• For Experiments 1 and 3, the target cells in duplicate were exposed to the vehicle control, positive control and the appropriate concentrations of the test item for 3-hours in the presence and for 21-hours in the absence of metabolic activation, respectively.

• For Experiment 2, the target cells in duplicate were exposed to the vehicle control and the appropriate concentrations of the test item for 3-hours in the absence of metabolic activation.

After the treatment period, the cultures from Experiments 1 and 2 were drained, washed twice with phosphate buffered saline, re-suspended in fresh medium and incubated for approximately another 18 hours.

Mitotic Arrest

Approximately at 19 hours after the start of the treatment, 300 L Colchicine (0.2 µg/mL) was added to the flasks, mixed and further incubated.

Cytotoxicity Assessment and Chromosome Preparation

Each culture from the vehicle control, positive control and treatment groups was harvested approximately at 21 hours after the beginning of the treatment and processed separately for the preparation of chromosomes.

At the end of the incubation period, mitotic cells were suspended in F-12 FBS5 after trypsinization. Two hundred microlitres (200 µL) of mitotic cells of each group from individual replicates were pooled into respective test tubes, mixed well and the cell counts were made separately using a hemocytometer for the assessment of cytotoxicity.

The cell suspension was centrifuged at 2000 rpm for 10 minutes and suspended in warm 0.56 % KCl solution and incubated for 10 minutes at room temperature. After incubation, the cell suspensions were centrifuged at 2000 rpm for 10 minutes. The supernatant was removed and to each tube, freshly prepared cold methanol: acetic acid fixative (3:1) was added drop-wise while shaking the tube gently to re-suspend the cells. The tubes were incubated for 10 minutes at room temperature, centrifuged at approximately at 2000 rpm for 10 minutes and the supernatant discarded.

Once again, fixative was added drop wise and the tubes allowed to stand in the refrigerator for at least 1 hour. After refrigeration, the cell suspension was centrifuged at 2000 rpm for 10 minutes, the supernatant discarded, the cell button re-suspended in fixative and the tubes incubated at room temperature for 10 minutes.The above procedure was repeated, the cell button re-suspended in required quantity of fixative and the cell suspension incubated at room temperature for at least 10 minutes prior to preparing the slides.

Slide Preparation

The cell suspension was dropped onto a clean chilled slide, flame dried and dried on a slide warmer maintained at approximately 35 to 40 °C. The slides were marked with the study number, treatment group, activation, experiment number, replicate number and the slide number with a diamond point marker. Five slides were prepared per replicate.

Staining

The slides were stained with freshly prepared Giemsa stain in water, for 120 minutes, washed in water, air dried, immersed in xylene and mounted with DPX. The slides were then coded by an individual not involved in scoring process before evaluation.


Rationale for test conditions:
In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, CHO-K1 cells exposed to the test item, did not exhibit the required level of cytotoxicity as RICC even at the highest tested concentration of 2000 µg/mL compared to the DMSO control, either in the presence or in the absence of metabolic activation with 3 -hour exposure. However, in the absence of metabolic activation with 21 -hour exposure, required level of cytotoxicity was observed between 1000 and 2000 µg/mL.
The test item did not precipitate in the test medium and did not cause any appreciable changes in the pH and osmolality of the test medium. Based on these observations, a maximum of 2000 µg/mL in Experiments 1 & 2 and a maximum of 1000 µg/mL in Experiment 3 were tested in the chromosomal aberration assay.

Evaluation criteria:
a. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

• At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
• The increase is dose-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

b. A test chemical is considered to be clearly negative if, in all experimental conditions examined:

• None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

c. The results will be considered equivocal if they do not meet the criteria specified for a positive or negative response. Additional experimental work may be required to clarify such results.
Statistics:
Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
There was no evidence of induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation.The study indicated that the test item, titanium 2,2',2''-nitrilotrisethanolate, Tyzor ET is not clastogenic at the concentrations tested and under the conditions of testing.
Executive summary:

The clastogenic potential of the test item, titanium 2,2',2''-nitrilotrisethanolate, Tyzor ET to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, CHO-K1 cells exposed to the test item, did not exhibit the required level of cytotoxicity as RICC even at the highest tested concentration of 2000 µg/mL compared to the DMSO control, either in the presence or in the absence of metabolic activation with 3 -hour exposure. However, in the absence of metabolic activation with 21 -hour exposure, required level of cytotoxicity was observed between 1000 and 2000 µg/mL.

The test item did not precipitate in the test medium and did not cause any appreciable changes in the pH and osmolality of the test medium. Based on these observations, a maximum of 2000 µg/mL in Experiments 1 & 2 and a maximum of 1000 µg/mL in Experiment 3 were tested in the chromosomal aberration assay.

In the definitive chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate for at the concentrations of 222, 667 and 2000 mg/mL in Experiments 1 and 2 and at 62.5, 250 and 1000 µg/mL in Experiment 3 of the chromosomal aberration assay. Concurrent vehicle (DMSO) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

At the respective highest concentrations tested, the reduction in cell growth as RICC was 55, 56 and 50 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.  

There was no evidence of induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

The study indicated that the test item, titanium 2,2',2''-nitrilotrisethanolate, Tyzor ET was not clastogenic at the concentrations tested and under the conditions of testing.