In Vivo Micronucleus Test

The oral administration of LK1 at a dose of 2,500 mg/kg did not produce overt toxicity in rats. Thus, 2,500 mg/kg was selected as the maximum dose. Cyclophosphamide monohydrate (CPA) (Sigma-Aldrich Co.) was used as the positive control, which is recommended in OECD guideline TG474. Hsd:ICR (CD-1) SPF mice (Koatech, Korea) were visually inspected upon arrival and acclimatized for 7 days. The animal laboratory conditions, diet, and water were the same as those described above in the single-dose oral toxicity study using rats. To determine optimal doses in a preliminary study, LK1 was administered (20 ml/(kg·day)) orally to three male and three female mice once daily for 2 days at 625, 1,250, or 2,500 mg/(kg·day). Observations were conducted for 4 days including the treatment day. All animals showed compound-colored stool on Days 2 and 3, but no abnormality was observed by Day 4.

Accordingly, there were three dose groups that were administered LK1 (0 mg/(kg·day), 1,250 mg/(kg·day), and 2,500 mg/(kg·day)) once daily for 2 days, with six males per group. The positive control CPA (70 mg/(kg·day)) was intraperitoneally injected once on Day 2. The mice were euthanized by CO₂ gas inhalation 24 h after the last administration. Bone marrow specimens were collected from each euthanized mouse using a 23G syringe and suspended in fetal bovine serum (HyClone; GE Healthcare Life Sciences, USA) [20]. The cell suspensions were centrifuged, smeared on slides, dried, and fixed in methanol for 5 min. Two slides were prepared per mouse. The mounted specimens were stained with acridine orange solution (0.05%) diluted in Sorensen's buffer (1:4 v/v, pH 6.8) and observed under a fluorescence microscope at 400× magnification. The numbers of micronucleated polychromatic erythrocytes (MNPCEs) were counted among 4000 polychromatic erythrocytes (PCEs) per animal, and the MNPCE frequency was calculated as the average number of MNPCEs per 4000 PCEs. Morphological examinations were conducted according to the method described by Hayashi et al. [21] and were regarded as valid when the following conditions were satisfied. 1) More than five animals were alive at autopsy in every dose group. 2) The average of the PCE/red blood cell (RBC) ratios in the control and dose groups was more than 20% of the value in the negative control group. 3) Among 4000 PCEs, the average MNPCE frequencies in the positive and negative control groups were similar within the range of historical control data, and the positive control result was significantly higher than the negative control result.