NOTE: The entire experiment must be performed in a clean isolated laboratory maintained at 20 °C with low dust and with minimization of contamination during worm and bacterial handling. For this purpose, experiments must be performed under the flame of an alcohol lamp or using a clean bench.

1. Maintenance of C. elegans and Egg Preparation for the Chemical Test

1. Maintain C. elegans N2 (var. Bristol) on an NGM agar plate fed with live E. coli OP50 at 20 °C^12,13.
2. Prepare age-synchronized eggs using either bleach solution treatment⁵ or the time egg laying method^6,14,15.
   NOTE: Before egg preparation, coat the etoposide-containing NGM plate with heat-inactivated E. coli OP50 as described below. Heat-inactivated E coli OP50 is used to minimize the metabolic activity of E. coli^6,16.

2. Preparation of Heat-inactivated E. coli OP50 to Feed C. elegans During the Toxicity Test

1. Prepare 500 mL of double yeast tryptone (DYT) medium as described in Table 1, and inoculate a single colony of E. coli OP50 cultured on a Luria-Bertani (LB) agar plate⁵.
2. Incubate the E. coli OP50 in a shaking incubator for 14 h at 37 °C and 150 rpm.
3. After centrifugation for 30 min at 3,220 x g and 20 °C, remove all supernatant DYT medium, and add a pre-mixture of 25 mL of S-buffer, 250 µL of 1 M MgSO₄, and 25 µL of 5 mg/mL cholesterol (dissolved in ethanol). All these solutions are sterile.
4. Resuspend the bacteria and transfer them to a 50-mL disposable plastic tube.
5. For the heat-inactivation, incubate the resuspended E. coli OP50 in a water bath for 30 min at 65 °C.
6. Cool down to room temperature and store at 4 °C until use. This can be stored for up to one month.

3. Preparation of NGM Plates Containing Either 1% DMSO or 750 µM Etoposide

1. Prepare two clean 200 mL glass bottles. Add 0.6 g of NaCl, 0.5 g of peptone, 3.4 g of agar, and 195 mL of distilled water, and a magnetic stirrer to one bottle. Add a magnetic stirrer to the other empty bottle.
2. Autoclave these two bottles for 15 min at 121 °C, and then cool down the bottles in a water bath for 30 min at 55 °C.
3. Add 0.2 mL of 1 M CaCl₂, 0.2 mL of 5 mg/mL cholesterol, 0.2 mL of 1 M MgSO₄, and 5 mL of 1 M KPO₄ (all these solutions are sterile) and then mix them by magnetic stirring on a hot plate at 55 °C.
4. Divide the mixed NGM medium into two bottles with the same volume (100 mL each). Next, add 1 mL of DMSO to one bottle, mix it again using the magnetic stirring. Store the other bottle in a water bath at 55 °C until the next step. Aliquot 3 mL of the DMSO-containing medium to each 35 x 10 mm² Petri dish. Approximately 33 DMSO-containing NGM plates can be made from this step.
5. Mix the other bottle using the magnetic stirring, add 1 mL of 75 mM etoposide (stock dissolved in DMSO), mix again, and repeat the aliquot procedure (step 3.4). Approximately 33 etoposide (750 µM)-containing NGM plates can be made from this step.
   NOTE: In the correlated previous paper⁶, we tested 0, 250, 500, and 1,000 µM of etoposide. Based on that data, we chose the 750 µM of etoposide dose in this paper.
6. Cool down the chemical-containing NGM plates to room temperature by leaving them at room temperature for approximately 3 h, and store them at 4 °C until use.
   NOTE: In the case of light-sensitive chemicals, block out light to minimize the light-induced decomposition.
   NOTE: Optionally, make a normal NGM plate without chemicals for the egg laying assay, which can be performed using two different chemical treatment conditions as described below.
7. Add 100 µL of heat-inactivated E. coli OP50 (as described in section 2) and spread on each NGM plate. Allow to dry overnight in a C. elegans incubator at 20 °C for the chemical test.

4. Measurement of C. elegans Growth

1. Transfer the age-synchronized C. elegans eggs to the NGM plate supplemented with 1% DMSO or 750 µM etoposide.
2. Incubate C. elegans in an incubator at 20 °C for 4 days. Observe the worms using a stereo microscope, and take microscopic images every day. Usually 20X magnification (1X objective lens, 2X zoom magnification, and 10X eyepiece lens) is appropriate for the growth observation of C. elegans. Also take a microscopic image of the microscope stage micrometer for the worm size calibration.
   NOTE: Starvation affects the toxicity test. Therefore, feed with sufficient bacteria or adjust the transferred C. elegans egg numbers at the beginning. Observe until 3 days to exclude the possibility of starvation.
3. Measure the body length of C. elegans treated with DMSO or etoposide at each time point using ImageJ software.
   NOTE: For each sample measurement, 50 worms are sufficient for the statistical analysis.
   1. In ImageJ, open the microscope stage micrometer image ('File' → 'Open').
      NOTE: The micrometer image and worm images (step 4.2) must be the same magnification.
   2. Drag a line of 1,000 µm on the micrometer calibration image by using 'Straight Line' tool.
   3. Click 'Analyze' → 'Set Scale' and input 1,000 and µm into the 'Known distance' box and 'Unit of length' box, respectively. Check 'Global' and click 'OK'.
   4. Open the worm images ('File' → 'Open'). Draw a line along the feature of each worm by using 'Freehand Line' tool. Obtain the body length data by clicking 'Analyze' → 'Measure'.
   5. Repeat these steps for 50 worms.

5. C. elegans Egg Laying Assay

1. Incubate the age-synchronized eggs on the NGM plate containing DMSO or etoposide for 64 h (before first birth) until the young adult stage at 20 °C.
   NOTE: It is optional to use the worms from the growth measurement experiments. It is convenient to do the growth measurement and egg laying assay together.
2. Transfer 5 adult worms to the new NGM plates without chemicals (condition 1, chemical treatment within a certain period of time, from eggs to young adult stage) or new NGM plate containing the same chemical pretreatment (condition 2, continuous exposure of chemicals through the overall experimental period), and then incubate them for 24 h.
   NOTE: It is recommended to make replicates using 3-5 NGM plates for each treatment; these replicates can be used for statistical analysis.
3. Transfer to the new NGM plate as described above, and count the number of eggs every day. Count the worms that crawled off, died, and were internally hatched.
4. Repeat these steps until the worms lay no more eggs, usually in 5 days.
5. Calculate the number of eggs laid per worm from each plate, and sum these values every day for 5 days to determine the total number of eggs laid.
   NOTE: Exclude the number of worms that crawled off and internally hatched from the calculation.