Dimolybdenum trizinc nonaoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Dimolybdenum trizinc nonaoxide

EC Number:

245-322-4

EC Name:

Dimolybdenum trizinc nonaoxide

Cas Number:

22914-58-5

Molecular formula:

Mo2O9Zn3

IUPAC Name:

dimolybdenum trizinc nonaoxide

impurity 1

Reference substance name:

Molybdenum zinc tetraoxide

EC Number:

237-377-8

EC Name:

Molybdenum zinc tetraoxide

Cas Number:

13767-32-3

Molecular formula:

MoO4Zn

IUPAC Name:

molybdenum zinc oxide

Test material form:

solid

Method

Target gene:

NA

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
- Whether whole blood or separated lymphocytes were used: Whole blood
- Whether blood from different donors were pooled or not: Single volunteer (male age 35) for preliminary toxicity test. Another single volunteer (male age 20) was used for the main experiment.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Cytokinesis block (if used):

NA

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Batch No 31/08/18 PB/βNF S9 was used in this study. The S9 mix was derived from male HSD:CD Sprague Dawley rats dosed with oral phenobarbital/beta-naphtha flavone.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

4 hour without S9: 0, 5, 10, 20, 25, 30, 35 and 40 µg/mL
4 hour with S9 (2%): 0, 5, 10, 20, 40, 60 and 80 µg/mL
24 hour without S9: 0, 10, 20, 40, 50, 60, 70 and 80 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Standard as per OECD guideline
- Justification for percentage of solvent in the final culture medium: standard as per OECD guideline

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine (DC)

Details on test system and experimental conditions:

Test item preparation: The maximum recommended dose level was initially set at 5000 μg/mL. However, due to the presence of precipitate being observed in the solubility test the maximum dose level used in the Preliminary Toxicity Test was limited to 640 μg/mL. The test item was insoluble in Minimal Essential Medium at 50 mg/mL but was partially soluble/ suspendable in DMSO at 500 mg/mL in solubility checks performed in-house. Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Culture conditions: Duplicate lymphocyte cultures (A and B) and quadruplicate for the vehicle were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM (FBS), 0.1 mL Li-heparin, 0.1 mL phytohaemaagglutinin and 0.75 mL heparanized whole blood.

4 hour exposure with metabolic activation (S9): After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL. After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

4 hour exposure without metabolic activation (S9): After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL. After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

24 hour exposure without metabolic activation (S9): The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours. The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format. The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures for the test item dose levels and duplicate cultures for the vehicle controls, whereas the Main Experiment used duplicate cultures and quadruplicate cultures for the vehicle controls.

Cell harvest: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of microscope slides: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative slide assessment: The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

Coding: The slides were coded before analysis using a computerized random number generator. Cytokinesis Block Proliferation Index (CBPI). A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.

Scoring of micronuclei: The micronucleus frequency in 1000 binucleated cells was analyzed per culture (2000 binucleated cells per concentration, 4000 for the vehicle control). In the 4-hour exposure group an additional 1000 binucleated cells were scored from each culture to add weight to the data and confirm the response observed in this exposure group. Cells with 1, 2 or more micronuclei were recorded and included in the total.
Due to the positive response observed after the initial scoring of the binucleate cells in the 4-hour exposure group in the absence of S9 it was considered appropriate to score the mononucleate cells for the incidence of micronuclei to ascertain whether the response was likely to be due to a clastogenic or aneugenic mechanism. The vehicle control, positive control and three dose levels from the 4-hour exposure group in the absence of S9 and vehicle and positive controls from the 4-hour with S9 and the 24-hour exposure were scored for the incidence of micronuclei in 1000 mononucleate cells per culture.
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Rationale for test conditions:

Standard as per OECD guideline

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) There is no dose-related increase.
3) The results in all evaluated dose groups should be within the range of the laboratory historical control data.

Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) There is an increase which can be considered to be dose-related.
3) The results are substantially outside the range of the laboratory historical negative control data.

When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system. There is no requirement for verification of a clear positive or negative response.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity within acceptable limits

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Results:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test. There were binucleate cells suitable for scoring at the maximum dose level of test item, 40 μg/mL in the 4-hour exposure in the absence of S9 and 80 μg/mL in the presence of S9. In the 24-hour exposure group in the absence of S9 the maximum dose level of the test item with binucleate cells suitable for scoring was 60 μg/mL.

Precipitate of the test item was noted in the parallel blood free cultures at the end of exposure at 40 μg/mL in the 4-hour exposure in the absence of S9, at and above 60 μg/mL in the presence of S9 and at and above 70 μg/mL in the 24-hour exposure group in the absence of S9. Precipitate was also seen in the blood cultures at the end of the exposure period at and above 40 μg/mL in the 4-hour exposure groups and at and above 50 μg/mL in the 24-hour exposure group.

In the 4-hour exposure group in the absence of S9 dose related toxicity was demonstrated with 37%, 44% and 75% cytostasis at 30, 35 and 40 μg/mL, respectively. The maximum dose level selected for scoring binucleate cells was 35 μg/mL where the toxicity was within acceptable limits. The higher dose level of 40 μg/mL was considered too toxic for scoring. In the presence of S9 no marked toxicity was observed and the maximum dose level selected for scoring was the lowest precipitating dose level, 40 μg/mL. The 24-hour exposure group achieved optimum toxicity with 58% cytostasis at 50 μg/mL, which was also the lowest precipitating dose level.

The maximum dose level selected for analysis of binucleate cells was 35 μg/mL for the 4hour exposure group in the absence of S9, 40 μg/mL in the presence of S9 and 50 μg/mL in the 24-hour exposure group. The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item induced statistically significant increases in the frequency of cells with micronuclei in the 4-hour exposure group in the absence of S9, at dose levels which achieved acceptable toxicity. An additional 1000 binucleate cells were scored from each culture (2000 cells per dose level) to confirm the positive response and add weight to the data. The reported data for the 4-hour exposure group includes the additional scoring. As a result of the positive response in the 4-hour exposure group in the absence of S9, additional scoring was performed on the mononucleate cells of selected dose levels to indicate whether the response was due to an aneugenic or clastogenic mechanism. Since there was no marked increase in the number of mononucleate cells with micronuclei it was considered that the response was likely to be due to clastogenic activity.

The 4-hour exposure group in the presence of S9 did not induce any statistically significant increases in the frequency of cells with micronuclei using a dose range which included a dose level which was the lowest precipitating dose level. The maximum dose level scored in the 24-hour exposure group was the lowest precipitating dose level which also achieved optimum toxicity with 58% cytostasis. There were small but statistically significant increases in the frequency of cells with micronuclei. However, since these increases were within the current laboratory historical range for a vehicle and were relatively small they can be considered to be of no toxicological significance.

Discussion:
The maximum dose level tested in the Preliminary Toxicity Test was limited to 640 μg/mL due to the presence of precipitate being observed in the solubility test.

The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level with toxicity also being taken into account in the dose range selection.

Dose related toxicity was demonstrated in both exposure groups in the absence of S9 in the Main Experiment. A statistically significant increase in the frequency of micronuclei in the binucleate cells was demonstrated in the 4-hour exposure group in the absence of S9 at 30 μg/mL and 35 μg/mL with values which exceeded the current laboratory historical control range. Both these dose levels achieved toxicity which was within acceptable limits. The additional scoring performed in this exposure group confirmed that the response seen in the initial scoring of the binucleate cells was correct.

Scoring the mononucleate cells for micronuclei in the 4-hour exposure group in the absence of S9 was considered to indicate that the positive response was due to a clastogenic response rather than an aneugenic response since there were no increases in the number of micronuclei in the mononucleate cells. The scoring of the mononucleate cells of the vehicle and positive control of the 4-hour exposure in the presence of S9 and the 24-hour exposure group demonstrated that the aneugen, demecolcine, was recognized as increasing the numbers of micronuclei in the mononucleate cells and cyclophosphamide (clastogen) did not.

Although the 24-hour exposure group demonstrated some small but statistically significant increases in micronuclei in the binucleate cells when compared to the concurrent vehicle control value these were considered to be of no toxicological significance since they were within the current laboratory historical control range for a vehicle.

The 4-hour exposure group in the presence of S9 did not demonstrate any marked toxicity and the maximum dose level scored for micronuclei in the binucleate cells was the lowest precipitating dose level. There were no statistically significant increases in the frequency of binucleate cells with micronuclei in this exposure group.

Any other information on results incl. tables

Results Summary:

Exposure condition	Treatment/concentration (µg/mL)	Cytostasis (%)	% binucleated cells containing micronuclei
			Mean	P-Value
4 hour –S9	Vehicle (DMSO)	0	0.26
	25	15	0.68	***
	30	37	2.28	***
	35	44	2.30	***
	MMC 0.2	33	3.83	***
4 hour +S9	Vehicle (DMSO)	0	0.35
	10	0	0.25
	20	7	0.40
	40	4	0.20
	CP 5	62	3.10	***
24 hour –S9	Vehicle (DMSO)	0	0.20
	20	0	0.40
	40	13	0.80	***
	50	58	0.55	*
	DC 0.075	40	2.30	***

Applicant's summary and conclusion

Conclusions:

The test substance was considered to be clastogenic to human lymphocytes in vitro under the conditions of the test.