Cell types: L5178Y/Tk ^+/− −3.7.2°C subline, derived from a mouse lymphoma (Clive and Spector, 1975), and TK6, a lymphoblastoid cell line derived from a human genetic disease source (Skopek et al., 1978). Both cell types are grown in suspension culture and maintained in exponential growth for the assay. See (Lorge et al., 2016) for additional information on methods for handling cells.

• The basic medium (F_0P) consists of RPMI 1640 medium supplemented with 100 unit/mL penicillin, 100 mg/ml streptomycin, and 200 mg/ml sodium pyruvate.

Media are filter-sterilized and stored at 4°C wrapped in foil to protect from light. THMG, THG, CHAT and HCT media can be stored at −20°C. The media are warmed to room temperature before use.

The cultures are grown in polycarbonate tissue culture flasks and placed in a 95% humidified incubator with 5% CO₂-in- air at 37°C. For a valid assay the cells need to be maintained in log phase; doubling times are 9–10 h for L5178Y and 11–12 h for TK6. The cultures are routinely diluted with fresh F_10P medium each day to 2 × 10⁵ cells/mL. For longer periods, the cells can be diluted to 7 × 10³ cells/mL Doubling times must be carefully monitored because cultures showing prolonged doubling times should not be used for experiments. Cells can be cryopreserved in liquid N₂ using F_20P containing 5% dimethyl sulfoxide (DMSO).

An essential requirement is elimination of pre-existing TK ^−/− cells that would impact the mutation levels in the negative controls in the test. Cleansing is carried out during the week preceding an assay or prior to freezing many vials for storage in liquid N₂. Note that the cells grow at longer doubling times during cleansing. The cells should not be exposed to test chemicals until they have completely recovered from cleansing, usually after 24 h. Cleansed cells can be cryopreserved at a density of 5 × 10⁶ cells/mL/tube in freezing medium (e.g., 10% DMSO in cell culture medium with serum). New cultures for assays may be started directly from the cryopreserved cleansed stocks, after centrifuging and re-suspended in fresh medium.

To cleanse L5178Y: Cells are treated sequentially with THMG and then THG, as follows: 0.5 ml of THMG (100X stock) is added to 50 ml of cell culture at 2 × 10⁵ cells/mL in F_10P. The cells are incubated at 37°C for 24 h in this medium. After counting (the cell density should be ∼1.0 × 10⁶ cells/mL), cells are centrifuged at 200 x g for 10 min and the pellet is resuspended at a concentration of 2 × 10⁵ cells/mL in 1% THG medium (F_10P medium containing 1% THG stock) and incubated for another 24 h.

To cleanse TK6: Cells are incubated in CHAT medium for 48 h and then transferred into HCT medium for 48 h to kill TK ^−/− cells before starting the TK assay.

To ensure that an assay is valid, positive controls and negative controls are included in each experiment. Chemicals commonly used as positive controls for test of any agent in TK mutagenicity assays include methylmethanesulphonate (MMS) at 10–20 mg/ml and 4-nitroquinoline 1-oxide (NQO) at 0.05–0.1 mg/ml. NQO can be prepared in DMSO as a 100-fold concentrated stock solution and stored as frozen aliquots at −80°C . However, MMS 100X stocks should be freshly prepared with physiological saline. Reagent solutions should be protected from light. If a solvent other than saline or F_0P (culture medium minus serum) is used, the negative control should receive a volume of the solvent equivalent to the highest amount used for a treated culture without exceeding a final volume of 1%. A NM-type positive control, such as Tungsten carbide-cobalt (WC-Co), would be useful; however, validation studies have not been carried out, and widespread use of this or other potential NM positive controls have not occurred. Consensus has not been found yet for a positive NM control for this (or any other genotoxicity) assay. The general requirement that a positive control generate a strong response may prevent the use of WC-Co and other NM as positive controls, since their responses generally are relatively weak. Thus, genotoxicity assessment of NM is carried out using common positive controls for each assay.

If the cytotoxicity of the test NM is unknown in this test system, a preliminary experiment should be performed to define the cytotoxic concentration range. The test NM is suspended in a suitable solvent such as saline, DMSO, or F_0P (cell culture medium with no serum) at appropriate concentrations of stock solutions. Stock solutions are created that can be diluted into the test system to generate the desired concentration. Sets of stock solutions are prepared so that the same dilution into the test system (e.g., 1:10 or 1:100) is made for each concentration. If possible, aqueous vehicles compatible with the cell systems should be chosen. Appropriate amounts of stock solutions are added to the cells in suspension in treatment medium to create the desired final concentrations for each data point in the experiment. The volume added should not exceed 1% (i.e., at least 1:100 dilution) when DMSO or other non-aqueous solvents are used. Such dilutions are necessary to avoid toxic effects of non-aqueous solvents on the test cells. The final volume of solvent or vehicle should be the same in all cultures.

The exposure concentrations of a test NM are selected according to target toxicity ranges, with a maximum allowable cytotoxicity of 10% growth relative to the negative control [see (OECD 490, 2016)]. The exposure concentration selected is also dependent on agglomeration status of the test article, which may signal an upper limit of exposure (see the Common Considerations paper). Generally, one or more preliminary experiments using half-log dilutions may be useful to aid in determining the concentrations used in the definitive assay. Duplicate (or triplicate) cultures are used for the negative/solvent control. See the Common Considerations paper for additional information on selection of concentrations for study (Front. Toxicol. doi: 10.3389/ftox.2022.859122).

Selection of concentrations for test may have been informed by a preliminary experiment determining reasonable limits of high and low concentrations selected for assessment. In that case, 4-5 useful concentrations with a relatively narrow concentration range (2 or 3-fold) between exposures may be selected. If a preliminary concentration-response assessment has not been made, then additional test concentrations should be used as a better likelihood of finding an informative dose range. Test concentrations ideally range between non-toxic and moderately toxic (90% survival loss). With many NM it may not be feasible to test into a moderately toxic zone, due to agglomeration or other effects.

The cells should always be maintained in logarithmic growth before cell treatment. For treatment, the test NM solution or suspension, positive control chemicals, or control vehicle are added into 50-ml sterile disposable centrifuge tubes containing 6 × 10⁶ cells for L5178Y or 10⁶ cells for TK6 in 10 ml of F_5P (cell culture medium with 5% serum). After gentle mixing, cultures are placed in a CO₂ incubator at 37°C for 4 h. A 24 h treatment should be conducted in parallel, or subsequently if the 4 h treatment is negative. For the 24 h treatment, the cell density should be adjusted to 2 × 10⁵ cells/mL to allow for additional growth over the longer incubation period. Cultures may be placed on a rocker platform during treatment to prevent the NM from settling out. After incubation, the cells are centrifuged at 200 x g for 10 min and the supernatant is discarded. Each culture is then washed twice with F_0P, to remove the NM test article, by centrifuging and resuspending the cells in fresh medium. Notation should be made of visible remaining NM, and a third wash instituted if necessary. After the final centrifugation, the cell pellet is resuspended in 20 ml of fresh F_10P (cell culture medium with 10% serum) at a concentration of 3 × 10⁵ cells/mL.

After treatment, the cells are cultured for an expression period of 48 h for L5178Y or 72 h for TK6 for DNA damage processing and mutation fixation. Cell densities are measured approximately 24 h following treatment and adjusted to 2 × 10⁵ cells/mL with fresh F_10P. On completion of the 48- or 72-h expression period, cell densities are measured again. The cell densities from each expression day are used in calculating the relative suspension growth (RSG) and the relative total growth (RTG) for L5178Y or the RS for TK6 [see (OECD 490, 2016)]. Cultures with cell densities less than 2 × 10⁵/ml are not used for cloning and mutagenicity measurements.

Trifluorothymidine (TFT) stock solution is made with 10 mg TFT in 100 ml physiological saline and stored in a foil-wrapped bottle. The stock solution is filter sterilized and can be dispensed in 15-ml aliquots in sterile tubes and stored at −20°C for up to 3 months.

For cloning, each culture is centrifuged, and the cell pellet resuspended in F_20P (cell culture medium with 20% serum) at a density of 2 × 10⁵ cells/mL. The cells should be single cell suspensions, so that individual cells are plated and the colonies that form are from single cells. The cultures are incubated for 30 min to minimize trauma and allow them to adapt to the medium. The cells are then diluted to the appropriate densities to plate for TFT resistance and cell viability.

For the TFT resistant plating (mutant colonies), the cell concentrations are adjusted to 1 × 10⁴/ml in F_20P for L5178Y and 2 × 10⁵/ml for TK6 cells. Then TFT (3 mg/ml) is added to the selection flask. Using a multichannel pipette, 200 ml of each TFT containing suspension is placed into each well of 4 flat-bottomed 96-well plates. For L5178 the final density is 2000 cells/well; for TK6 it is 40,000 cells/well. Colonies are identified by low power microscope or by visual observation. Small colonies are defined as less than a quarter of the diameter of the well while large colonies are more than a quarter of the diameter of the well. The morphology is generally compact for small colonies and may be diffuse for large colonies.

For the determination of plating efficiency, the cultures are adjusted to 8 cells/mL in media without TFT and 200 µL per well are aliquoted into two 96-well flat-bottom microtiter plates (∼1 or 2 cells/well) for the counting of survivors. The microtiter plates are incubated at 37°C in a humidified incubator with 5% CO2-in-air for 11–14 days for the L5178Y Tk mutants. An additional period of 10–14 days is required for the TK6 slower growing colonies to appear.

The mutant frequency (MF) is determined by the plating efficiencies of mutant colonies (PE_M) and adjusted with plating efficiencies of viable cells (PE_V) from the same culture. See OECD 490 for details (OECD 490, 2016).

• The positive control must demonstrate the assay is properly conducted and that small or late growing colony mutants were detected. This is demonstrated by a significantly induced small colony mutant (MLA) or late growing colony (TK6) mutation frequency in the positive controls.

These criteria are found in OECD 490 and represent the work of several IWGT MLA Workgroups.