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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept 2016 (Protocol approved) to July 2017 (Final report)
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Identity: TiH2
- Label name: Titanium Hydride Powder VM
- CAS no.: 130-20-1
- Batch no.: 75727
- Expiry date: 21 June 2018
- Storage conditions: room temperature
- RTC number: 15068

Method

Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
Chinese hamster V79 cells were obtained from Dr. J. Thacker, MRC Radiobiology Unit,
Harwell, UK. This cell line, V79 4(H) can be traced back directly to the original V79 isolate
prepared by Ford and Yerganian (1958). The karyotype and plating efficiency have been
checked in this laboratory. The cells are checked at regular intervals for the absence of
mycoplasmal contamination and generation time.
Permanent stocks of the V79 cells are stored in liquid nitrogen, and subcultures are prepared
from the frozen stocks for experimental use.
Metabolic activation:
with and without
Metabolic activation system:
One batch of S9 tissue fraction was provided by Trinova Biochem GmbH.
Test concentrations with justification for top dose:
Based on the preliminary solubility assay, dose levels of 10.0, 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391mMwere used for the first main experiment. Since negative results were obtained and no cytotoxicity was observed at any consentration, a second experiment was performed using a continuous treatment in the absence of S9 metabolism and the same dose levels.
Following treatment, the pH and osmolality of the treatment media were determined for both experiments.
Vehicle:
No vehicle - Solutions/suspensions of the test item in culture medium (EMEM complete medium)
Controls
Negative controls:
yes
Remarks:
Untreated culture acted as negative controls
Solvent controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Colchicine
Remarks:
Positive controls were dissolved in sterile water of injectable grade
Details on test system and conditions:
- In the first experiment, both in the absence and presence of S9 metabolism, the cultures were incubated for 3 hours. At the end of treatment, the medium was removed and the flasks were washed twice with Phosphate Buffered Solution (PBS). Fresh culture medium and Cytochalasin B (3 μg/mL) were added and the cultures were incubated for further 21 hours (Recovery Period) before harvesting. Cells were harvested at a time corresponding to approximately
2 cell cycle lengths (approximately 25 hours).

Summary: +/- S9, Treatment time: 3 hours, Harvest time: 24-25 hours, Dose level: 5.00, 2.50, 1.25 mM

- In the second experiment, in the absence of S9 metabolic activation, the treatment medium was added to the flasks. After 3 hours of incubation at 37°C, Cytochalasin B (3 μg/mL) was added to treatment media and the cultures were incubated at 37°C for further 21 hours (24-hour treatment).

Summary: - S9, Treatment time: 24 hours, Harvest time: 24 hours, Dose level: 2.50, 1.25 and 0.625 mM
Evaluation criteria:
* Acceptance criteria
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– The positive control items induce a statistically significant increases in the incidences of micronucleated cells compared with the concurrent negative control
– Adequate cell proliferation is observed in solvent control cultures.
– The appropriate number of doses and cells is analysed.

* Criterion for outcome
In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range. If the increases fall within the range of values normally observed in the negative control cultures, the test item can not be classified as positive. Any significant increases over the concurrent negative controls are therefore compared with historical control values derived from recent studies.
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the dose level shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with an appropriate trend test.
– All the results are inside the distribution of the historical control data.
Statistics:
For the statistical analysis, a modified x2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
- Solubility test
A preliminary solubility trial was performed using sterile distilled water of injectable grade, where a heavy precipitation of the test item was observed at the concentration of 4.99mg/mL (100mM) after approximately 10 seconds vortex mixing and approximately 10 minutes shaking and heating at 37°C in a water bath. A moderate precipitation was observed also at the lower concentration of 2.50mg/mL.
A further trial was performed using EMEM complete medium, where a homogeneous suspension was obtained at the concentration of 0.554mg/mL after approximately 10 minutes heating at 37°C and shaking in a water bath. This concentration permitted to reach the maximum dose level of 10.0mM(499 μg/mL) by using the volume of test item suspension. An opaque solution, considered adequate to prepare serial dilutions, was obtained at the
lower concentration of 0.277mg/mL after approximately 20 minutes heating and stirring at 37°C.

For the first main experiment, slight precipitation was observed at the beginning of treatment at the dose level of 10.0mMboth in the absence and presence of S9. More marked precipitation was observed at the end of treatment, when slight precipitation was also observed at the lower dose level of 5.00mM.

For the second main experiment, slight precipitation was observed at the dose levels of 10.0 and 5.00mMat the beginning and end of treatment.

- Osmolality and pH results
Following treatment with the test item, no remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism.

- Cytotoxicity results
The CBPI was calculated for each of the treatment series. For the first main experiment, since negligible cytotoxicity was observed, scoring of CBPI was interrupted and only five dose levels were analysed. For the second main experiment, scoring of CBPI was performed for all dose levels tested with the exception of the two highest dose levels, where heavy precipitation of the test item onto slides interfered with scoring. Following treatment with the test item, no remarkable toxicity was observed at any analysed dose level in any experiment, in the absence or presence of S9 metabolism.

- Assay results
Following treatment with the test item, no increase in the incidence of micronucleated cells over the concurrent negative control value was observed in the presence or absence of S9 metabolism, at any concentration, in any experiment. The incidences were within the normal distribution range of historical values for negative controls. Marked increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

Applicant's summary and conclusion

Conclusions:
After having tested the genotoxicity of titanium hydride following the OECD guideline N°487, It is concluded that TiH2 does not induce micronuclei in Chinese hamster V79 cells after in vitro treatment.