Registration Dossier

Administrative data

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

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March 2018 - 29 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
(a) 50, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 3200 and (h) 5000 ug/plate The test item did not show toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates, both in the presence and absence of metabolic activation.

Based on these observations, a top dose of 5000 ug/plate was tested in the mutation assay, as recommended by the OECD 471 guideline.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoantharacene
Details on test system and experimental conditions:
In the initial mutation assay, which was a plate incorporation mode of exposure, the bacterial suspensions were exposed to the test item, vehicle and the positive controls in the presence and absence of an exogenous metabolic activation system. These bacterial suspensions were then mixed with overlay agar and plated immediately onto minimal medium viz., his- for Salmonella typhimurium and trp- for Escherichia coli, respectively.

In the confirmatory assay, which was a pre-incubation mode of exposure, the test constituents were mixed with the bacteria inside a tube, incubated in an incubator shaker, mixed with overlay agar and plated immediately onto minimal medium his- for Salmonella typhimurium and trp- for Escherichia coli, respectively.

After 67 hours of incubation, the revertant colonies were counted and compared with the number of spontaneous revertants in the vehicle control plates.


DURATION
- Preincubation period: 20 min
- Exposure duration: 67h
Evaluation criteria:
To determine a positive result, there should be a dose related increrase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the testi item either in the presence or absence of metabolic activation system. The test will be judged postive if the increase in mean revertants at the peak of the dose respomse is equal to or greater than two times the mean vehicle control values for strains TA98, TA100 or WP2uvrA(pKM101) or equal to or greater than three times the mean vehicle control values for strains TA1535 and TA1537. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respecitive threshold cited above or a non-dose responsive increase that is equal to or greated than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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:
The test item tetrakis(2-ethylhexane-1,3-diolato)titanium was not mutagenic in the bacterial reverse mutation test up to the OECD 471-recommended top dose of 5000 µg/plate under the conditions of testing employed.
Executive summary:

Both the Salmonella typhimurium and Escherichia coli tester strains were found to be reliable and responsive to the different genotypic characterization tests like the amino acid requirement, rfa mutation, uvr mutation and the R-factor plasmids. Similarly, the spontaneous revertant counts of the vehicle control groups of these tester strains were in the ranges of the test facility’s historical control data.  

The positive controls produced a more than 3-fold increase in the mean numbers of revertant colonies when compared to the respective vehicle controls, demonstrating the sensitivity of the assay procedure.  

The test item at doses up to 5000 µg/plate did not cause a two fold increase in the mean numbers of revertant colonies in the strains TA98, TA100 and WP2uvrA (pKM101) or three fold increase in the mean numbers of revertant colonies in the strains TA1535 and TA1537 either in the presence or absence of the metabolic activation system when compared to the respective vehicle control plates.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3rd April 2018 - 23rd May 2018
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Cyclophosphamide (CPA)
Test concentrations with justification for top dose:
Following concentrations of the test item were used in the preliminary cytotoxicity test:
Experiment 1 and 2: 5.7, 40, 280 ug/ml
Experiment 3: 5, 30 and 180 ug/ml

Justification for the top dose; percipitation and cytotoxicity


Vehicle / solvent:
Ethanol
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 106 cells in 75 cm2 flask in duplicate with 15 mL F-12 FBS10 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, exhibited the required level of cytotoxicity as RICC between 160 and 320 µg/mL in the presence and absence of metabolic activation with 3-hour exposure, as well as in the absence of metabolic activation with 21-hour exposure. The test item precipitated in the test medium at and above 320 µg/mL but did not cause any appreciable changes in the pH and osmolality of the test medium. Based on these observations, a maximum of 280 µg/mL in Experiments 1 & 2 and a maximum of 180 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

 

Table 1. Results of cytotoxicity test.

 Treatmeant (ug/ml)     Presence of metabolic activation (3 h exposure)     Absence of metabolic activation (3 h exposure) Absence of metabolic activation     (21 h exposure)
   Cell growth index RICC (%) Cell growth inhibition (%)  Cell growth index RICC (%)  Cell growth inhibition (%)     Cell growth index RICC (%)   Cell growth inhibition (%)
 Ethanol  100  -  100  -  100  -
 5  90  10  91  9  89  11
 10  77  23  75  25  82  18
 20  68  32  69  31 70   30
 40  63 37  60  40  64  36
 80  52 48  54  46  59  41
 160  46 54  48  52  46  54
 320  42 58  42  58  23  77
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, tetrakis(2-ethylhexane-1,3-diolato)titanium, TYZOR OGT is not clastogenic at the concentrations tested and under the conditions of testing.
Executive summary:

The clastogenic potential of the test item, tetrakis(2-ethylhexane-1,3-diolato)titanium, TYZOR OGT 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, exhibited the required level of cytotoxicity as RICC between 160 and 320 µg/mL in the presence and absence of metabolic activation with 3-hour exposure, as well as in the absence of metabolic activation with
21-hour exposure. The test item
precipitated in the test medium at and above 320µg/mL but did not cause any appreciable changes in the pH and osmolality of the test medium. Based on these observations, a maximum of 280 µg/mL in Experiments 1 & 2 and a maximum of 180 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 5.7, 40 and 280 mg/mL in Experiments 1 and 2 and at 5, 30 and 180 µg/mL in Experiment 3 of the chromosomal aberration assay. Concurrent vehicle (ethanol) 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 56, 55 and 54 % 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, tetrakis(2-ethylhexane-1,3-diolato)titanium TYZOR OGTwas not clastogenic at the concentrations tested and under the conditions of testing.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3rd April -17th May 2018
Reliability:
1 (reliable without restriction)
Qualifier:
according to
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:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Dorf Ketal Speciality Catalyst Private Limited
Plot No. 2, Block-F, Sector-12N
Adani Ports & SEZ, Taluka-Mundra
District-Kutch, Gujarat-370421
lot 334-300153/000012
- Expiration date of the lot/batch: Sept 2019


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was tested for its solubility in Ethanol and found soluble at 200 mg/mL. Stability of the test item was established in Ethanol at 50 and 200,000 µg/mL for 24 hours at room temperature.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The treatment mixture was prepared by mixing the required quantity of the test item or the vehicle (Ethanol) with treatment medium.
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, there was no colony growth at 800 and 2000 µg/mL both in the presence and absence of metabolic activation. The test item showed evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) between 128 and 320 µg/mL both in the presence and absence of metabolic activation. The test item precipitated in the test medium at and above 320 g/mL but did not cause any appreciable change in the pH and osmolality of the test medium. Based on these observations a maximum of 200 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: ethanol
Ethanol is one of the organic vehicles compatible with this test system. Hence, based on the results of the solubility test, Ethanol was selected as the vehicle of choice for the gene mutation test.
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): 10 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
- Any supplementary information relevant to cytotoxicity: The test item precipitated in the test medium at and above 320 ug/mL but did not cause any appreciable change in the pH and osmolality of the test medium.

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:

No. of cells at the end of treatment
Adjusted CE (ACE) = CE x ---------------------------------------------------- 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 7.01 and 7.15 with 7.10 in the Ethanol control. Similarly, in the absence of metabolic activation, the pH of the test medium ranged between 7.10 and 7.18 with 7.16 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 0.474 OSMOL/kg; at the lowest precipitating test item level (320 ug/mL) was 0.473 OSMOL/kg and the highest soluble test item level (128 g/mL) was 0.469 OSMOL/kg.

Similarly, at the end of 3-hour exposure, in the absence of metabolic activation, osmolality of the test medium at the highest test item treatment level (2000 ug/mL) was 0.531 OSMOL/kg; at the lowest precipitating test item level (320 ug/mL) was 0.441 OSMOL/kg and the highest soluble test item level (128 ug/mL) was 0.458 OSMOL/kg.

The corresponding osmolality in the Ethanol control was 0.471 and 0.453 OSMOL/kg in the presence and absence of metabolic activation, respectively.

- Precipitation: At the end of 3-hour exposure, the test item precipitated in the test medium at and above 320 ug/mL.

RANGE-FINDING/SCREENING STUDIES: The test item showed evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) between 128 and 320 µg/mL both in the presence and absence of metabolic activation.

Based on these observations it is decided to test up to a maximum of 200 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 Ethanol control was within the range of the in-house historical control data
Conclusions:
It is concluded that the test item, TYZOR OGT 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 assay conditions fulfilled all the specifications as per the OECD 476 guideline.

All the acceptance criteria for a valid test were met.

The Cloning Efficiency of the vehicle controls was 0.95 and 0.94. The mean mutant frequency of the vehicle controls was 7.37 and 5.32 mutants per 10 6 clonable cells in the presence and in the absence of metabolic activation, respectively.

In experiment in the presence of metabolic activation, the positive control chemical produced a statistically significant increase in the mutant frequencies, demonstrating both the activity of the metabolic activation system and the responsiveness of the test system.

No evidence for the induction of gene mutation was observed in any of the concentrations of the test item either in the presence or in the absence of metabolic activation.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames (OECD471)

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

The test item, TYZOR OGT was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay. The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

TYZOR OGT was soluble in Ethanol at 50 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 toxicity test for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates at any of the tested doses. The test item did not cause toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, a top dose of 5000 g/plate was tested in the mutation assay as recommended by the OECD 471 guideline.

The bacterial tester strains were exposed to TYZOR OGT in triplicate at 50, 158, 500, 1581 and 5000 µg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure whereas the confirmatory mutation assay was carried out using the pre-incubation mode of exposure. The vehicle control (Ethanol) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

The results of the study from both the initial and confirmatory mutation assay showed that, TYZOR OGT did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

The results of the concentration analysis of the dose formulation samples of both the initial and confirmatory mutation assays confirmed that the top dose level of 5000 µg/plate was achieved in both assays and the results support the validity of the study conclusion.

The study indicated that TYZOR OGT was not mutagenic in this Bacterial Reverse Mutation Assay up to the OECD 471-recommended top dose of 5000 g/plate, under the conditions of testing employed.

Cytogenicity Assay (OECD473)

The clastogenic potential of the test item, TYZOR OGT 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, exhibited the required level of cytotoxicity as RICC between 160 and 320 µg/mL in the presence and absence of metabolic activation with 3-hour exposure, as well as in the absence of metabolic activation with

21-hour exposure. The test item precipitated in the test medium at and above 320 µg/mL but did not cause any appreciable changes in the pH and osmolality of the test medium. Based on these observations, a maximum of 280 µg/mL in Experiments 1 & 2 and a maximum of 180 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 5.7, 40 and 280 g/mL in Experiments 1 and 2 and at 5, 30 and 180 µg/mL in Experiment 3 of the chromosomal aberration assay. Concurrent vehicle (Ethanol) 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 56, 55 and 54 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.

A total of 300 metaphases each from the Ethanol control, each treatment level and the positive controls were evaluated for chromosomal aberrations.

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 results of the concentration analysis of the dose formulation samples confirmed that the top dose level equivalent to 28000 µg/mL was achieved and the results support the validity of the study conclusion.

The study indicated that the test item, TYZOR OGT was not clastogenic at the concentrations tested and under the conditions of testing.

Mutagenicity Assay (OECD476)

The mutagenic potential of the test item TYZOR OGT 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 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 gene mutation assay, there was no colony growth at 800 and 2000 µg/mL both in the presence and absence of metabolic activation. The test item showed evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) between 128 and 320 µg/mL both in the presence and absence of metabolic activation. The test item precipitated in the test medium at and above 320 g/mL but did not cause any appreciable change in the pH and osmolality of the test medium. Based on these observations a maximum of 200 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 5, 16, 57 and 200 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 (Ethanol) 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 concentration analysis of the dose formulation samples confirmed that the top dose level equivalent to 200 µg/mL was achieved and the results support the validity of the study conclusion.

The results of the forward gene mutation test at the hprt locus with TYZOR OGT 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.