Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2016 (Protocol signed) - April 2017 (Final report signed)
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
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
28 july 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian cell gene mutation test

Test material

1
Chemical structure
Reference substance name:
Zirconium dihydride
EC Number:
231-727-3
EC Name:
Zirconium dihydride
Cas Number:
7704-99-6
Molecular formula:
H2-Zr
IUPAC Name:
zirconium dihydride
Specific details on test material used for the study:
Identity ZrH2
Alternative name Zirconium Hydride S
Batch no. 74031
Expiry date 25 February 2018
Storage conditions room temperature

Method

Target gene:
hypoxanthine-guaninphosphoribosyl-transferase (HPRT) gene
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (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. The karyotype, generation time, plating efficiency and
mutation rates (spontaneous and induced) have been checked in this laboratory. The cells
are checked at regular intervals for the absence of mycoplasmal contamination. Permanent
stocks of the V79 cells are stored in liquid nitrogen. Subcultures are prepared from the frozen stocks for experimental use.
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone
Test concentrations with justification for top dose:
466, 186, 74.6, 29.8, 11.9 and 4.77 µg/mL the doses are based on the results obtained in the preliminary cytotoxicity assay.
Vehicle / solvent:
No vehicle
Note: suspensions/solutions were prepared using EMEM Minimal medium
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Mutation assay

- Treatment of cell cultures
A Main Assay was performed including negative and positive controls, in the absence and presence of S9 metabolising system. The two treatment series were assayed in separate runs. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. For each run, two additional control cultures were included in the experimental scheme, in order to evaluate baseline count. Two days before the experiment, sufficient numbers of 175cm2 flasks were inoculated with 5 million freshly trypsinised V79 cells from a common pool, in order to have at least 20 million of cells for treatment. At the treatment time, the growth medium was removed from the flasks and replaced with treatment medium; cultures were incubated at 37°C for three hours.

- Determination of survival (Day 0)
At the end of treatment, the medium was removed and the cell monolayers were washed with PBS. The cultures were trypsinised, counted and an aliquot from each culture was diluted and plated to estimate the viability of the cells. Each cell suspension was re-plated (2×106 cells/F175) in order to maintain the treated cell populations. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere
(100% nominal relative humidity) to allow for expression of the mutant phenotype.

- Subculturing
For the treatment series in the absence of S9 metabolic activation, on Days 2, 5 and 7, the cell populations were subcultured in order to maintain them in exponential growth. The cultures were trypsinised, counted and the number of cells taken forward was adjusted to give two million viable cells seeded in 225cm2 flasks (Day 2) or in 175cm2 flasks (Day 5 and Day 7). For the treatment series in the presence of S9 metabolic activation, the cell populations were subcultured on Day 2 and Day 5. The cultures were trypsinised, counted and the number of cells taken forward was adjusted to give two million viable cells seeded in 225cm2 flasks.

- Determination of mutant frequency (Day 9 or Day 8)
At the expression time (Day 9 in the absence of S9 metabolism, Day 8 in its presence), each culture was trypsinised, resuspended in complete medium and counted by microscope. After dilution, an estimated 1×105 cells were plated in each of twenty 100mmtissue culture petridishes containing medium supplemented with 6-thioguanine (at 7.5 µg/mL). These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants.
After dilution, an estimated 200 cells were plated in each of three 60mmtissue culture petri dishes. These plates were used to estimate Cloning Efficiency (CE).
Rationale for test conditions:
preliminary cytotoxicity assay was performed. In this assay, the test item was assayed at a maximum dose level of 932 µg/mL and at a wide range of lower dose levels: 466, 233, 117, 58.3, 29.1, 14.6, 7.28 and 3.64 µg/mL. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. Upon addition to the test item, precipitation was observed at the highest concentration. At the end of treatment, precipitation of the test item was noted starting from 29.1 and 58.3 µg/mL in the absence and presence of S9 metabolism, respectively.
Evaluation criteria:
* Acceptance criteria
The assay was considered valid if the following criteria were met:
– The mutant frequency of the solvent/vehicle control is within the 95% control limits of the distribution of the laboratory’s historical control database.
– The positive controls induce responses that are compatible with those generated in the historical control database and produce a statistically significant increase in mutant frequency, compared with the concurrent solvent/vehicle control.
– Two experimental conditions are tested (i.e. with and without metabolic activation), unless one gives positive results.
– Adequate number of cells (i.e. at least 20×106 at treatment time and at least 2×106during the expression period) and concentrations (at least 4 with appropriate cytotoxicity) are analysable.
– The selection of dose levels is consistent with those indicated in cytotoxicity assay.

* Criteria for outcome of assay
A test item is considered to be clearly positive if:
– At least one of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
– The increase is concentration-related.
– Any of the results are outside the distribution of the historical negative control data (95% confidence limits).
A test item is considered to be clearly negative if:
– None of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
– There is no concentration-related increase.
– All results are inside the distribution of the historical negative control data (95% confidence limits).
Historical control data are used to demonstrate biological relevance of the results obtained.
Statistics:
The individual mutation frequency values at each test point were transformed to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y = (x +a)b where a = 0 and b = 0.275.
The mutant frequency in the solvent control and treated cultures was compared using the Dunnett’s test (one-tailed).
For each experimental point, the corrected sum of squares of transformed mutation frequencies
was calculated as follows:
SSy = Sum y 2 − (Sum y)2/r

where:
r = number of replicate cultures
The error mean square (EMS) was calculated as the sum of SSy values divided by the sum of degrees of freedom.

For each experimental point the t value was calculated using the following formula:
t =(yd-yc)/ Æ ((EMS/rd) + (EMS/rc)
where:
yd = mean of transformed mutation frequencies of treated cultures
yc = mean of transformed mutation frequencies of solvent/vehicle control cultures
rd = number of replicate cultures at the analized concentration
rc = number of replicate cultures of solvent/vehicle control
For each comparison of treatment with control, the calculated t value was compared with tabulated critical values for the one tailed Dunnett’s test.
The results of the experiment were subjected to an Analysis of Variance in which the effect of replicate culture and dose level in explaining the observed variation were examined. For each experiment, a two way analysis of variance was performed (without interaction) fitting to two factors:
– Replicate culture: to identify differences between the replicate cultures treated.
– Dose level: to identify dose-related increases (or decreases) in response.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Upon addition to the test item and by the end of treatment, precipitation was observed at the two highest concentrations tested in the absence of S9 metabolism. In its presence, slight precipitation was seen by the end of treatment only at 466 µg/mL.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Upon addition to the test item, precipitation was observed at the highest concentration. At the end of treatment, precipitation of the test item was noted starting from 29.1 and 58.3 µg/mL in the absence and presence of S9 metabolism, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
* Solubility test
A preliminary solubility trial was performed in sterile water for injection and EMEM Minimal medium. A suspension with slight precipitate, suitable for use but not for serial dilutions, was obtained at 1.86mg/mL. This permitted the maximum concentration of 932 µg/mL in the final treatment mixture, which corresponds to 10mM, the highest concentration indicated in the Study Protocol.

* Osmolality and pH
The pH values and osmolality of the treatment media were determined and are available in the main report. The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

* Survival after treatment
No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism, with the exception of the concentration of 186 µg/mL, in the absence of S9 metabolism, where reduction in Relative Survival to 43% of Negative Control value was observed.

* Mutation results
No relevant increases over the spontaneous mutation frequency were observed at any treatment level in the absence or presence of S9 metabolic activation and all results were inside the distribution of the historical control data. No statistically significant increase over the concurrent vehicle control was observed at any dose level. Analysis of variance indicated that dose level and replicate culture were not significant factors in explaining the observed variation in the data, both in the absence and presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:
It is concluded that ZrH2 does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

The test item ZrH2 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item suspensions/solutions were prepared using EMEM Minimal medium.

A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 932 µg/mL (10mM) and at a wide range of lower dose levels: 466, 233, 117, 58.3, 29.1, 14.6, 7.28 and 3.64 µg/mL. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. Precipitation of the test item was noted by the end of treatment starting from 29.1 and 58.3 µg/mL, in the absence and presence of S9 metabolism, respectively.

A Main Assay for mutation to 6-thioguanine resistance was performed. Cells were treated for 3 hours, both in the absence and presence of S9 metabolism and maintained in growth medium for 9 days to allow phenotypic expression of induced mutation. The following dose levels were used: 466, 186, 74.6, 29.8, 11.9 and 4.77 µg/mL.

No relevant increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system.

It is concluded that ZrH2 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.