Titanium 2,2',2''-nitrilotrisethanolate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): non-mutagenic with or without metabolic activation

In vivo cytogenicity assay (OECD 473): negative with or without metabolic activation

In vitro gene mutation assay (OECD 476): negative with or without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 2, 1977 - April 18, 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

TA 102 bacterial strain was not used and negative results were not confirmed

Principles of method if other than guideline:

Suspension of Salmonella Typhimurium cells were exposed to the test substance in the presence and in the absence of an exogenous metbolic activation system. These suspensions were mixed with with an overlay agar and plated immediately onto minimal medium. After two days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates. Study was performed according to reference Ames, B. N., McCann, J. and Yamasaki, E. (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutat. Res. 31, 347-364.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus in selected strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

rat-liver homogenate activation system (S-9)

Test concentrations with justification for top dose:

1500, 3000, 4500, 6000 and 7500 µg/plate with and without metabolic activation

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Remarks:

plate without S-9 activators

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

used for assay with metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

used for assay without metabolic activation

Details on test system and experimental conditions:

Five histidine-requiring strains of Salmonella typhimurium were used in the mutagen assays.
Strains TA 1535 and TA 100 were used to detect base-pair substitution mutations, whereas strains TA 1537, T 1538 and TA 98 were used to detect frame-shift mutations.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Titanic acid, tetra(2,2',2''-Nitrilotriethanol)ester Tyzor TE was not mutagenic in this bacterial reverse mutagenetic test. Thus this substance is concluded to have no genotoxic properties.

Executive summary:

Titanic acid, tetra(2,2',2''-Nitrilotriethanol)ester Tyzor TE

was tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in concentrations up to 7500 µg per plate. The compound was not mutagenic in the microbial assays either in the presence or absence of a liver microsomal system, i.e., it did not induce a significant increase over the spontaneous mutation frequency.

This study was regarded reliable with restriction since the study does not include full range of recommended strains and there is no confirmation of negative results. However the study method is comparable to OECD 471 guideline. Thus, the result of this study is used as a key value in hazard assessment.

Reference 2

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)

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

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

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:

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.

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

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4rd April -16th May 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1, (ATCC CCL-61, Lot 4765275)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate

Test concentrations with justification for top dose:

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, Tyzor TE did not show evidence of sgnificant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) at any of the tested concentrations, either in the presence or in the absence of metabolic activation. Based on these observations the highest recommended test concentration of 2000 µg/mL was tested in the gene mutation assay both in the presence and absence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
The test item precipitated on the sides of the tube when mixed with sterile water at 200 mg/mL. The test item was soluble in DMSO at 200 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

DURATION
- Preincubation period:
- Exposure duration: 3h
- Expression time (cells in growth medium): 24h
- Selection time (if incubation with a selection agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 6-Thioguanine (6-TG)

NUMBER OF REPLICATIONS: two

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were stained with 0.5 % methylene blue and counted for both cloning efficiency and mutant selection after 10 days of incubation.


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

The assay will be considered valid if the following criteria are met:

a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b) The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.

When all the validity criteria are fulfilled:

1. 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 compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

2. 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 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

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed using the formula:

Y = (X + A) B

where,
Y = transformed mutant frequency
X = observed mutant frequency
and A, B = constants.

Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

Cytotoxicity is evaluated by relative survival, i.e., Cloning Efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in vehicle controls (assigned a survival of 100%).

Adjusted CE for a culture treated by a test item is calculated as:


Adjusted CE (ACE) = CE x ( No. of cells at the end of treatment / No. of cells at the beginning of treatment)
No. of colonies
CE = ------------------------
No. of cells plated

Relative Survival (RS) for a culture treated by a test item is calculated as:

Adjusted CE in treated culture
RS = --------------------------------------------- x 100
Adjusted CE in the vehicle control
Mutant Frequency (MF) is the cloning efficiency of mutant colonies in selective medium divided by the cloning efficiency in the non-selective medium measured for the same culture at the time of selection.

CE of mutant colonies in selective medium
MF = -------------------------------------------------------
CE in non-selective medium

MF is usually expressed as mutants per 106 clonable cells.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

There was no evidence of excessive cytotoxicity (i.e., <10 % RS) at any of the tested concentrations either in the presence or absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the end of 3-hour exposure, the pH of the test medium, in the presence of metabolic activation, ranged between 7.03 and 7.13 with 7.04 in the DMSO control. Similarly, in the absence of metabolic activation, the pH of the test medium ranged between 7.03 and 7.12 with 7.03 in the Ethanol control.
- Effects of osmolality: At the end of 3-hour exposure, in the presence of metabolic activation, osmolality of the test medium at the highest test item treatment level (2000 ug/mL) was 0.418 OSMOL/kg; and 0.389 OSMOL/kg in the presence and absence of metabolic activation, respectively.
The corresponding osmolality in the DMSO control was 0.435 and 0.432 OSMOL/kg in the presence and absence of metabolic activation, respectively.

- Precipitation: At the end of 3-hour exposure, the test item did not precipitate in the test medium at any concentration tested.

RANGE-FINDING/SCREENING STUDIES: The test item did not show evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) at any of the tested concentrations, either in the presence and absence of metabolic activation.

Based on these observations it is decided to test up to the highest recommended concentration of of 2000 µg/mL in the gene mutation assay both in the presence and absence of metabolic activation..

HISTORICAL CONTROL DATA
- Positive historical control data: the positive control 3-methylcholanthrene (3-MCA) induced statistically significant increases in the mutant frequency as compared with the vehicle control.
- Negative (solvent/vehicle) historical control data: The frequency of mutants in the DMSO control was within the range of the in-house historical control data

Conclusions:

It is concluded that the test item, TYZOR TE does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The mutagenic potential of the test item TYZOR TE to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

TYZOR TE was soluble in DMSO at 200 mg/mL and was found stable in DMSO at room temperature for 24 hours at the concentrations of 50 and 200,000 µg/mL.

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, Tyzor TE did not show evidence of sgnificant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) at any of the tested concentrations, either in the presence or in the absence of metabolic activation. Based on these observations the highest recommended test concentration of 2000 µg/mL was tested in the gene mutation assay both in the presence and absence of metabolic activation.

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 74, 222, 667 and 2000 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

The results of the forward gene mutation test at the hprt locus with TYZOR TE indicated that the test item was non-mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames (equivalent to OECD471)

Taken together, the results indicated that TYZOR TE does not have the potential to induce gene mutation either in the presence or in the absence of metabolic activation at the doses tested. Tyzor TE

was tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in concentrations up to 7500 µg per plate. The compound was not mutagenic in the microbial assays either in the presence or absence of a liver microsomal system, i.e., it did not induce a significant increase over the spontaneous mutation frequency. This study was regarded reliable with restriction since the study does not include full range of recommended strains and there is no confirmation of negative results. However the study method is comparable to OECD 471 guideline.

Cytogenicity Assay (OECD473)

The clastogenic potential of the test item, TYZOR TE 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.

TYZOR OGT was soluble in Ethanol at 200 mg/mL and was found stable in Ethanol at room temperature for 24 hours at the concentrations of 50 and 200,000 µg/mL.

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 2000µg/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 2000µg/mL.

The test item did notprecipitate in the test medium and did not cause any appreciable changes in the pHand 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 2000mg/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,underidentical 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 ETwas not clastogenic at the concentrations tested and under the conditions of testing.

Mutagenicity Assay (OECD476)

The mutagenic potential of the test item TYZOR TE to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

TYZOR TE was soluble in DMSO at 200 mg/mL and was found stable in DMSO at room temperature for 24 hours at the concentrations of 50 and 200,000 µg/mL.

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, Tyzor TE did not show evidence of sgnificant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) at any of the tested concentrations, either in the presence or in the absence of metabolic activation. Based on these observations the highest recommended test concentration of 2000 µg/mL was tested in the gene mutation assay both in the presence and absence of metabolic activation.

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 74, 222, 667 and 2000 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

The results of the forward gene mutation test at the hprt locus with TYZOR TE indicated that the test item was non-mutagenic under the conditions of this study.

Justification for classification or non-classification

Based on the results of in vitro bacterial gene mutation study, in vitro mammalian chromosomal aberration and gene mutation studies no classification is proposed for genotoxicity according to the criteria of CLP regulation 1272/2008.