Lithium nickel potassium oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

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:

Lithium nickel potassium oxide

Cas Number:

210352-95-7

Molecular formula:

Li.K.Ni.O.H2O

IUPAC Name:

Lithium nickel potassium oxide

Test material form:

solid: particulate/powder

Details on test material:

Lithium nickel potassium oxide (KDLNO)
CAS Number:
210352-95-7
Appearance/Physical state:
Black powder
Storage: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells
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.

The details of the donors used are:
Preliminary Toxicity Test: female, aged 35 years
Main Experiment: female, aged 35 years
Cell Culture
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):

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. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100 - 100{(CBPIT – 1) / (CBPIC – 1)}
Where: CBPI = (Number of monucleate cells + (2*Number of Binucleate cells) + (3*Number of multinucleate cells) / (Total Number of cells)

Key:
T = test chemical treatment culture
C = vehicle control culture

Scoring of Micronuclei
The micronucleus frequency in 1000 binucleated cells was analyzed per cell culture, i.e. 2000 binucleate cells for the test item dose levels and the positive control and 4000 binucleate cells for the vehicle in each exposure group. Cells with 1, 2 or more micronuclei were recorded and included in the total. In the 4-hour exposure group in the presence of S9 positive control ‘A’ culture did not demonstrate a satisfactory response after the first 1000 cells were scored so to add weight to the data and confirm the response, a further 1000 cells were scored from each of the positive control replicates in this exposure group.

Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

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. Both nuclei could be attached by a fine nucleoplasmic bridge approximately no greater than one quarter of the nuclear diameter.

Metabolic activation:

with and without

Metabolic activation system:

Culture conditions
Duplicate lymphocyte cultures (A and B) for the test item dose levels and quadruplicate cultures for the vehicle control were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.06 mL MEM,
10% (FBS) 0.1 mL Li-heparin;
0.1 mL phytohaemagglutinin;
0.74-0.75 mL heparinized 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 1.0 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 1.0 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 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 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 for the test item and quadruplicate cultures for the vehicle controls.

Test concentrations with justification for top dose:

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 2.5, 5, 10, 20, 40, 80, 160, 320 and 640 μg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the exposure groups of the main experiment.

Vehicle / solvent:

Controls
Vehicle and positive controls were used in parallel with the test item.
The vehicle control used was as follows:
Identity:
Minimal Essential Medium (MEM)
Supplier:
Gibco
Preliminary Toxicity Test
Batch number:
2041445
Expiry Date:
30 November 2019
Main Experiment
Batch Number:
2049804
Expiry Date:
31 December 2019

The positive control items were as follows:

Absence of S9-mix:

Identity:
Mitomycin C (MMC)
CAS No.:
50-07-7
Supplier:
Sigma Aldrich
Batch Number:
SLBN5647V
Purity:
100%
Expiry Date:
01 October 2019
Solvent:
Minimal Essential Medium
Concentration:
0.2 μg/mL for 4-hour exposure

Identity:
Demecolcine (DC)
CAS No.:
477-30-5
Supplier:
Sigma-Aldrich
Batch Number:
BCBV3422
Purity:
99%
Expiry Date:
02 July 2023
Solvent:
Sterile distilled water
Concentration:
0.075 μg/mL for 24-hour continuous exposure

Presence of S9-mix:
Identity:
Cyclophosphamide (CP)
CAS No.:
6055-19-2
Supplier:
Acros Organics
Batch Number:
A0389646
Purity:
99.9%
Expiry Date:
01 October 2022
Solvent:
DMSO
Concentration:
5 μg/mL for 4-hour exposure

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium (MEM)

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Demecolcine (DC)

Details on test system and experimental conditions:

Test Item Preparation
The test item is a UVCB, therefore the maximum proposed dose level in the solubility test was initially set at 5000 μg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. The test item formed a suspension in culture medium at 50 mg/mL, which was considered acceptable for dosing in the solubility checks.
The solubility of the test item was investigated in the Mouse Lymphoma Assay, Study Number MS22GN.
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 .

The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Due to test item precipitate being observed in the solubility test at and above 312.5 μg/mL, the maximum dose level selected for the Preliminary Toxicity Test was limited to 640 μg/mL.

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Batch No PB/βNF S9 28/10/18 was used in this study. A copy of the S9 Certificate of Efficacy is presented in Appendix 2.
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%.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.

The dose range of test item used for all three exposure groups was 5, 10, 20, 40, 80, 160 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

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.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

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.

Data Evaluation
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.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions (i.e. S9 concentration or S9 origin)) could be useful.

Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.

Rationale for test conditions:

Acceptability Criteria
The following criteria were used to determine a valid assay:
• The concurrent negative control was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• Cell proliferation criteria in the solvent control were considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations was analyzed.

Evaluation criteria:

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.

Scoring of Micronuclei
The micronucleus frequency in 1000 binucleated cells was analyzed per cell culture, i.e. 2000 binucleate cells for the test item dose levels and the positive control and 4000 binucleate cells for the vehicle in each exposure group. Cells with 1, 2 or more micronuclei were recorded and included in the total. In the 4-hour exposure group in the presence of S9 positive control ‘A’ culture did not demonstrate a satisfactory response after the first 1000 cells were scored so to add weight to the data and confirm the response, a further 1000 cells were scored from each of the positive control replicates in this exposure group.

Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

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.

Statistics:

Statistical Analysis
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.

Major Computerized Systems
The following computerized system was used in this study:
• An in-house validated statistical package (UK0279)

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Conclusion

The test item, Lithium nickel potassium oxide (KDLNO) was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

SUMMARY

Introduction

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Methods

Duplicate cultures of human lymphocytes, treated with the test item, and quadruplicate cultures for the vehicle were evaluated for micronuclei in binucleate cells at four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

 

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by precipitate.

 

Results

All vehicle (Minimal Essential Medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

 

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 did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level.

 

Conclusion

The test item, Lithium nickel potassium oxide (KDLNO) was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.