Acute toxicity and genotoxicity assessment of Ligigeopoong-san

Article information

J Korean Med. 2024;45(3):40-53

Publication date (electronic) : 2024 September 1

doi : https://doi.org/10.13048/jkm.24036

Korean Medicine Non-clinical Study Center, National Development Institute of Korean Medicine

Correspondence to: Jong-Hyun Nho, Korean Medicine Non-clinical Study Center(GLP), National Development Institute of Korean Medicine, Wondogwandeok-gil 27, 59319, Jangheung-eup, Jangheung-gun, Jeollanam-do, Republic of Korea, Tel: +82-61-860-2873, E-mail: nhopaper@gmail.com

Received 2024 June 11; Revised 2024 August 5; Accepted 2024 August 19.

Abstract

Objectives

This study aimed to investigate whether Ligigeopoong-san induces acute toxicity and genotoxicity

Methods

Ligigeopoong-san contains the rhizome of Cnidium officinale MAKINO, an herb with potential teratogenicity. Teratogenicity is closely associated with genotoxicity. We analyzed whether Ligigeopoong-san induces acute toxicity and genotoxicity using various experimental models in accordance with Korean non-clinical test standards for pharmaceuticals and OECD test guidelines.

Results

When Ligigeopoong-san was administered as a single dose to male and female rats, no toxic reactions, including organ damage, were observed at doses up to 2,500 mg/kg. In the bacterial reverse mutation test, no DNA mutations were detected at concentrations up to 5,000 μg/plate. In cell models, Ligigeopoong-san did not induce structural or numerical chromosomal aberrations at concentrations up to 2,000 μg/mL. Additionally, in animal studies, it did not cause bone marrow toxicity or form micronuclei in erythrocytes at doses up to 2,000 mg/kg.

Conclusions

The experiments using various models demonstrated that Ligigeopoong-san did not induce acute toxicity or genotoxicity.

Keywords: Ligigeopoong-san; Acute toxicity; genotoxicity; Korean medicine; safety

Fig. 1

Structural aberration in vitro mammalian chromosomal aberration test

Image captured using E-200(Nikon, Tokyo, Japan) microscope. (A)~(G) 600X magnification, (H) 400X magnification. (A) Distilled water, (B)~(E) Ligigeopoong-san(short-term exposure, −S9), (F) chromosome exchange(Benzo[a]pyrene 20 ug/mL, short-term exposure, +S9), (G) chromatid break(Mitomycin 0.1 ug/mL, short-term exposure, −S9), (H) chromatid exchange(Mitomycin 0.1 ug/mL, continuous exposure, −S9).

Table 1

Information of Ligigeopoon-san

Table 2

Results of the acute toxicity assessment

Table 3

Results of the bacterial reverse mutation test without metabolic activation system

Table 4

Results of the bacterial reverse mutation test with metabolic activation system

Table 5

Results of the in vitro mammalian chromosomal aberration test

Table 6

Results of the mammalian erythrocyte micronucleus test

References

1. Park M. J., Jin S. R., Oh J. H., Song W. S., Lee H. S., Woo J. H., Hwang K. H., Bae G. H., Yun Y. C.. 2022;Peripheral facial palsy due to cerebellar artery infarction is improved by Korean medical treatment: A case report. The Journal of Internal Korean Medicine 43(2):122–129. https://doi.org/10.22246/jikm.2022.43.2.122.

2. Sim S. Y.. 2015;Clinical research of Korean medical treatment for the peripheral facial paralysis. The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology 28(4):62–73. https://doi.org/10.6114/jkood.2015.28.4.062.

3. Lee D. H., Kwon B. I., Yu J. S., Park S. K., Kim J. H.. 2022;Neural mechanisms underlying peripheral facial nerve palsy: A protocol for systematic review and meta-analysis of neuroimaging studies. Medicine 101(48):e32110. https://doi.org/10.1097/MD.0000000000032110.

4. Wang C. C., Li L., Tang L. Y., Leung P. C.. 2012;Safety evaluation of commonly used Chinese herbal medicines during pregnancy in mice. Human Reproduction 27(8):2448–2456. https://doi.org/10.1093/humrep/des180.

5. Wang H., Bao Q., Yi H., Xia Q.. The evaluation of embryotoxicity of Ligusticum chuanxiong on mice and embryonic stem cells. Journal of Ethnopharmacology 15(239):111895. https://doi.org/10.1016/j.jep.2019.111895.

6. Choi J. S., Han J. Y., Koren G., Cho Y. K.. 2021;Evaluation of fetal and neonatal outcomes after ingestion of Cnidium root (Cnidium officinale Makino) during pregnancy. Early Human Development 161:105456. https://doi.org/10.1016/j.earlhumdev.2021.105456.

7. Mohamed H. R. H., El-Atawy R. H., Ghoneim A. M., El-Ghor A. A.. 2020;Induction of fetal abnormalities and genotoxicity by molybdenum nanoparticles in pregnant female mice and fetuses. Environmental Science and Pollution Research 27(19):23950–23962. https://doi.org/10.1007/s11356-020-08137-0.

8. Argüelles-Velázquez N., Alvarez-González I., Madrigal-Bujaidar E., Chamorro-Cevallos G.. 2013;Amelioration of cadmium-produced teratogenicity and genotoxicity in mice given arthrospira maxima (Spirulina) treatment. Evidence-Based Complementary and Alternative Medicine 2013:604535. https://doi.org/10.1155/2013/604535.

9. Phillips D. H., Arlt V. M.. 2009;Genotoxicity: damage to DNA and its consequences. EXS 99:87–110. https://doi.org/10.1007/978-3-7643-8336-7_4.

10. De Assis K. R., Ladeira M. S., Bueno R. C., Dos Santos B. F., Dalben I., Salvadori D. M.. 2009;Genotoxicity of cigarette smoking in maternal and newborn lymphocytes. Mutation Research 679(1–2):72–78. https://doi.org/0.1016/j.mrgentox.2009.02.006.

11. Erhirhie E. O., Ihekwereme C. P., Ilodigwe E. E.. 2018;Advances in acute toxicity testing: strengths, weaknesses and regulatory acceptance. Interdisciplinary Toxicology 11(1):5–12. https://doi.org/10.2478/intox-2018-0001.

12. Nair A. B., Jacob S.. 2016;A simple practice guide for dose conversion between animals and human. Journal of Basic and Clinical Pharmacy 7(2):27–31. https://doi.org/10.4103/0976-0105.177703.

13. Hayashi M.. 2022;Opinion: regulatory genotoxicity: past, present and future. Genes and Environment 44(1):13. https://doi.org/10.1186/s41021-022-00242-5.

14. Levy D. D., Zeiger E., Escobar P. A., Hakura A., van der Leede B. M., Kato M., Moore M. M., Sugiyama K. I.. 2019;Recommended criteria for the evaluation of bacterial mutagenicity data (Ames test). Genetic Toxicology and Environmental Mutagenesis 848:403074. https://doi.org/10.1016/j.mrgentox.2019.07.004.

15. Sims P., Grover P. L., Swaisland A., Pal K., Hewer A.. 1974;Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature 252(5481):326–328. https://doi.org/10.1038/252326a0.

16. Theisen A., Shaffer L. G.. 2010;Disorders caused by chromosome abnormalities. The Application of Clinical Genetic 3:159–174. https://doi.org/10.2147/TACG.S8884.

17. Kang S. H., Kwon J. Y., Lee J. K., Seo Y. R.. Recent advances in in vivo genotoxicity testing: prediction of carcinogenic potential using comet and micronucleus assay in animal models. Journal of Cancer Prevention 18(4):277–288. https://doi.org/10.15430/jcp.2013.18.4.277.

Article information Continued

Fig. 1

Structural aberration in vitro mammalian chromosomal aberration test

Table 1

Information of Ligigeopoon-san

Number	Name	Weights (g)	Ratio (%)	Origin	Ash (%)	SO₂ (ppm)	Aflatoxin	Quantification analysis		Heavy metal (ppm)	Pesticide residues	Analyzation Company^*
Number	Name	Weights (g)	Ratio (%)	Origin	Ash (%)	SO₂ (ppm)	Aflatoxin	Substance	Ratio (%)	Heavy metal (ppm)	Pesticide residues	Analyzation Company^*
1	Root of Glycyrrhiza uralensis Fisch. ex Dc.	3	6.25	China	5.5	7.5	Suitable	Glycyrrhizic acid Liquiritigenin	3.37 0.83	Suitable	Suitable	1
2	Root of Ostericum koreanum Maximowicz	3	6.25	Korea	7.0	9.9	-	-	-	Suitable	Suitable	1
3	Immature fruit of Citrus aurantium Linné	3	6.25	China	3.3	16.2	-	-	-	Suitable	Suitable	1
4	Root of Platycodon grandiflorum A. De Candolle	3	6.25	Korea	5.5	8.0	-	-	-	Suitable	Suitable	2
5	Rhizome of Arisaema amurense Maximowicz	3	6.25	China	4.4	-	-	-	-	Suitable	Suitable	3
6	Root of Aralia continentalis Kitagawa	3	6.25	Korea	4.5	6.0	-	Kaurenoic acid + Continentalic acid	2.00	Suitable	Suitable	1
7	Tuber of Pinellia ternata Breitenbach	3	6.25	China	2.5	14.0	Suitable	-	-	Suitable	Suitable	4
8	Root of Saposhnikovia divaricata (Turcz.) Schischkin	3	6.25	China	2.5	9.0	-	-	-	Suitable	Suitable	1
9	Root of Paeonia lactiflora Pallas	3	6.25	Korea	2.8	3.0	-	Albiflorin + Paeoniflorin	3.80	Suitable	Suitable	1
10	Root of Angelica dahurica Bentham et Hooker	3	6.25	Korea	6.9	3.9	-	Oxypeucedanin + Imperatorin + Isoimperatorin	-	Suitable	Suitable	1
11	Root of Lindera strichnifolia Fernandez-Villar	3	6.25	China	0.6	11.4	-	-	-	Suitable	Suitable	1
12	Pericarp of Citrus unshiu Markovich	3	6.25	China	2.2	7.8	-	Hesperidin	14.3	Suitable	Suitable	1
13	Rhizome of Cnidium officinale MAKINO	3	6.25	Korea	4.4	2.0	-	-	-	Suitable	Suitable	2
14	Rhizome of Gastrodia elata Blume	3	6.25	China	2.9	7.7	-	Gastrodin + Gastrodigenin	-	Suitable	Suitable	1
15	Immature pericarp of Citrus unshiu Markovich	3	6.25	China	3.3	16.2	-	-	-	Suitable	Suitable	1
16	Floral axis of Schizonepeta tenuifolia Briquet	3	6.25	Korea	6.0	19.2	-	-	-	Suitable	Suitable	1
		48.0	100

1: Hyeongyoul Pharmaceuticals (Gyeonggi Province, Republic of Korea) 2: Hando Pharmaceuticals (Gyeonggi Province, Republic of Korea) 3: Puleunmuyak Pharmaceuticals (Seoul, Republic of Korea) 4: Kwangduk Pharmaceuticals (Chungnam Province, Republic of Korea).

Table 2

Results of the acute toxicity assessment

Sex: Male							(g)

Group	Animal	Day

		0	1	3	7	14
Negative control	1	215.9	247.8	272.4	305.4	363.5
	2	235.8	266.3	295.4	337.9	421.0
	3	218.9	252.2	279.4	317.7	384.6
	4	222.1	254.4	279.0	313.2	376.4
	5	223.1	258.0	285.6	324.7	405.5

	Mean	223.2	255.7	282.4	319.8	390.2
	S.D	7.6	7.0	8.7	12.3	23.0

Ligigeopoong-san (625 mg/kg)	1	232.7	263.1	288.3	318.3	385.4
	2	231.0	269.5	300.8	339.7	415.6
	3	223.3	255.2	285.1	318.7	373.6
	4	233.0	267.5	294.7	334.2	411.8
	5	225.3	259.0	281.8	314.5	378.4

	Mean	229.1	262.9	290.1	325.1	393.0
	S.D	4.5	5.9	7.6	11.1	19.4

Ligigeopoong-san (1,250 mg/kg)	1	230.5	268.6	300.1	345.4	429.9
	2	220.7	255.0	275.6	316.1	386.2
	3	221.2	258.1	247.4	326.7	391.3
	4	223.7	259.5	280.2	313.7	389.6
	5	226.7	261.3	283.2	331.4	402.1

	Mean	224.6	260.5	277.3	326.7	399.8
	S.D	4.1	5.1	19.1	12.8	17.8

Ligigeopoong-san (2,500 mg/kg)	1	229.4	262.3	290.5	322.5	382.8
	2	233.0	268.8	293.3	326.7	398.9
	3	221.6	258.8	279.0	309.2	363.5
	4	217.9	255.8	279.8	317.0	396.3
	5	221.7	258.0	285.0	277.8	362.5

	Mean	224.7	260.7	285.5	310.6	380.8
	S.D	6.2	5.1	6.3	19.5	17.4

Sex: Female							(g)

Group	Animal	Day

		0	1	3	7	14

Negative control	1	168.6	193.1	197.2	220.8	250.4
	2	152.3	174.6	187.7	202.4	230.2
	3	153.2	176.0	186.5	196.8	219.2
	4	157.1	182.1	196.3	200.8	225.7
	5	157.9	183.6	193.4	206.5	231.3

	Mean	157.8	181.9	192.2	205.5	231.4
	S.D	6.5	7.4	4.9	9.3	11.7

Ligigeopoong-san (625 mg/kg)	1	172.5	196.3	215.2	236.6	274.7
	2	154.8	175.6	187.6	201.2	232.7
	3	154.7	177.8	193.5	206.5	235.5
	4	152.4	174.6	188.8	201.7	232.7
	5	163.4	187.3	193.7	196.2	221.8

	Mean	159.6	182.3	195.8	208.4	239.5
	S.D	8.4	9.3	11.2	16.2	20.4

Ligigeopoong-san (1,250 mg/kg)	1	164.9	190.8	203.6	213.0	245.0
	2	151.7	171.7	184.5	189.9	222.3
	3	161.5	181.6	195.8	215.2	249.8
	4	158.3	180.6	189.6	201.3	219.4
	5	160.4	177.9	196.5	207.1	238.6

	Mean	159.4	180.5	194.0	205.3	235.0
	S.D	4.9	6.9	7.3	10.2	13.6

Ligigeopoong-san (2,500 mg/kg)	1	152.9	176.7	181.8	188.5	212.7
	2	165.3	189.6	203.4	225.3	266.3
	3	152.8	178.9	190.6	197.6	225.7
	4	157.3	179.9	196.0	208.5	238.2
	5	155.0	180.3	195.3	212.2	243.4

	Mean	156.7	181.1	193.4	206.4	237.3
	S.D	5.2	5.0	7.9	14.1	20.1

S.D (Standard deviation)

Table 3

Results of the bacterial reverse mutation test without metabolic activation system

Strain	Dose (μg/plate)	Primary experiment								Secondary experiment

		PPT	TOX	Revertant colony			Mean	S.D	Ratio	PPT	TOX	Revertant colony			Mean	S.D	Ratio

				1	2	3						1	2	3
TA98	0	-	-	19	25	18	21	3.8	[1.0]	-	-	17	17	13	16	2.3	[1.0]
	6.86	-	-	18	25	25	23	4.0	[1.1]	-	-	-	-	-	-	-	-
	20.6	-	-	17	24	23	21	3.8	[1.0]	-	-	-	-	-	-	-	-
	61.7	-	-	25	27	24	25	1.5	[1.2]	-	-	11	8	14	11	3.0	[0.7]
	185.2	-	-	25	26	18	23	4.4	[1.1]	-	-	13	19	14	15	3.2	[1.0]
	555.6	-	-	21	18	27	22	4.6	[1.1]	-	-	11	12	20	14	4.9	[0.9]
	1,666.7	-	-	17	18	24	20	3.8	[1.0]	-	-	18	14	22	18	4.0	[1.1]
	5,000.0	-	-	18	25	21	21	3.5	[1.0]	-	-	25	22	19	22	3.0	[1.4]
	2-NF (0.5)	-	-	160	129	145	145	15.5	[7.0]	-	-	160	287	116	188	88.8	[12.0]

TA100	0	-	-	108	93	109	103	9.0	[1.0]	-	-	81	68	81	77	7.5	[1.0]
	6.86	-	-	95	102	91	96	5.6	[0.9]	-	-	-	-	-	-	-	-
	20.6	-	-	92	105	107	101	8.1	[1.0]	-	-	-	-	-	-	-	-
	61.7	-	-	96	93	96	95	1.7	[0.9]	-	-	81	82	86	83	2.6	[1.1]
	185.2	-	-	85	98	95	93	6.8	[0.9]	-	-	87	83	81	84	3.1	[1.1]
	555.6	-	-	94	91	94	93	1.7	[0.9]	-	-	73	89	86	83	8.5	[1.1]
	1,666.7	-	-	100	103	107	103	3.5	[1.0]	-	-	97	89	87	91	5.3	[1.2]
	5,000.0	-	-	100	118	121	113	11.4	[1.1]	-	-	130	115	105	117	12.6	[1.5]
	SA (1.0)	-	-	682	726	700	703	22.1	[6.8]	-	-	602	595	644	614	26.5	[8.0]

TA1535	0	-	-	10	9	9	9	0.6	[1.0]	-	-	11	8	9	9	1.5	[1.0]
	6.86	-	-	9	9	9	9	0.0	[1.0]	-	-	-	-	-	-	-	-
	20.6	-	-	10	10	9	10	0.6	[1.0]	-	-	-	-	-	-	-	-
	61.7	-	-	10	8	8	9	1.2	[0.9]	-	-	11	9	11	10	1.2	[1.1]
	185.2	-	-	9	8	9	9	0.6	[0.9]	-	-	7	9	11	9	2.0	[1.0]
	555.6	-	-	10	8	9	9	1.0	[1.0]	-	-	10	9	9	9	0.6	[1.0]
	1,666.7	-	-	9	11	10	10	1.0	[1.1]	-	-	8	10	7	8	1.5	[0.9]
	5,000.0	-	-	9	9	11	10	1.2	[1.0]	-	-	9	10	11	10	1.0	[1.1]
	SA (1.0)	-	-	490	495	484	490	5.5	[52.5]	-	-	422	459	441	441	18.5	[47.2]

TA1537	0	-	-	6	7	7	7	0.6	[1.0]	-	-	11	12	12	12	0.6	[1.0]
	6.86	-	-	8	6	6	7	1.2	[1.0]	-	-	-	-	-	-	-	-
	20.6	-	-	6	6	6	6	0.0	[0.9]	-	-	-	-	-	-	-	-
	61.7	-	-	6	7	6	6	0.6	[1.0]	-	-	8	9	10	9	1.0	[0.8]
	185.2	-	-	6	7	7	7	0.6	[1.0]	-	-	8	10	12	10	2.0	[0.9]
	555.6	-	-	7	8	9	8	1.0	[1.2]	-	-	12	9	12	11	1.7	[0.9]
	1,666.7	-	-	7	8	7	7	0.6	[1.1]	-	-	12	12	9	11	1.7	[0.9]
	5,000.0	-	-	7	10	9	9	1.5	[1.3]	-	-	12	9	14	12	2.5	[1.0]
	9-AA (40.0)	-	-	201	236	253	230	26.5	[34.5]	-	-	170	192	216	193	23.0	[16.5]

WP2 uvrA	0	-	-	46	49	42	46	3.5	[1.0]	-	-	50	52	49	50	1.5	[1.0]
	6.86	-	-	48	45	46	46	1.5	[1.0]	-	-	-	-	-	-	-	-
	20.6	-	-	45	49	43	46	3.1	[1.0]	-	-	-	-	-	-	-	-
	61.7	-	-	43	40	42	42	1.5	[0.9]	-	-	41	46	42	43	2.6	[0.9]
	185.2	-	-	49	40	46	45	4.6	[1.0]	-	-	36	49	44	43	6.6	[0.9]
	555.6	-	-	45	45	45	45	0.0	[1.0]	-	-	39	50	39	43	6.4	[0.8]
	1,666.7	-	-	44	41	41	42	1.7	[0.9]	-	-	48	49	50	49	1.0	[1.0]
	5,000.0	-	-	45	49	41	45	4.0	[1.0]	-	-	43	52	49	48	4.6	[1.0]
	4-NQO (0.5)	-	-	760	736	680	725	41.1	[15.9]	-	-	762	764	680	735	47.9	[14.6]

PPT (Precipitation), TOX (Toxicity to bacteria), S.D (Standard deviation), Ratio (The percentage of the mean value compared to the negative control value)

Table 4

Results of the bacterial reverse mutation test with metabolic activation system

Strain	Dose (μg/plate)	Primary experiment								Secondary experiment

		PPT	TOX	Revertant colony			Mean	S.D	Ratio	PPT	TOX	Revertant colony			Mean	S.D	Ratio

				1	2	3						1	2	3
TA98	0	-	-	38	30	34	34	4.0	[1.0]	-	-	15	13	20	16	3.6	[1.0]

	6.86	-	-	37	33	28	33	4.5	[1.0]	-	-	-	-	-	-	-	-
	20.6	-	-	33	31	30	31	1.5	[0.9]	-	-	-	-	-	-	-	-
	61.7	-	-	36	33	34	34	1.5	[1.0]	-	-	22	22	13	19	5.2	[1.2]
	185.2	-	-	30	31	36	32	3.2	[1.0]	-	-	27	22	13	21	7.1	[1.3]
	555.6	-	-	34	38	31	34	3.5	[1.0]	-	-	22	22	14	19	4.6	[1.2]
	1,666.7	-	-	38	32	29	33	4.6	[1.0]	-	-	20	17	17	18	1.7	[1.1]
	5,000.0	-	-	39	32	38	36	3.8	[1.1]	-	-	27	15	22	21	6.0	[1.3]

	2-NF (0.5)	-	-	152	146	136	145	8.1	[4.3]	-	-	156	135	146	146	10.5	[9.1]

TA100	0	-	-	91	82	88	87	4.6	[1.0]	-	-	79	76	77	77	1.5	[1.0]

	6.86	-	-	87	92	92	90	2.9	[1.0]	-	-	-	-	-	-	-	-
	20.6	-	-	90	97	93	93	3.5	[1.1]	-	-	-	-	-	-	-	-
	61.7	-	-	93	93	92	93	0.6	[1.1]	-	-	91	90	96	92	3.2	[1.2]
	185.2	-	-	102	100	98	100	2.0	[1.1]	-	-	89	93	93	92	2.3	[1.2]
	555.6	-	-	116	105	106	109	6.1	[1.3]	-	-	102	93	92	96	5.5	[1.2]
	1,666.7	-	-	100	99	105	101	3.2	[1.2]	-	-	81	110	85	92	15.7	[1.2]
	5,000.0	-	-	126	119	103	116	11.8	[1.3]	-	-	89	108	95	97	9.7	[1.3]

	SA (1.0)	-	-	670	738	660	689	42.4	[7.9]	-	-	395	479	429	434	42.3	[5.6]

TA1535	0	-	-	9	10	7	9	1.5	[1.0]	-	-	12	10	13	12	1.5	[1.0]

	6.86	-	-	10	9	10	10	0.6	[1.1]	-	-	-	-	-	-	-	-
	20.6	-	-	10	9	8	9	1.0	[1.0]	-	-	-	-	-	-	-	-
	61.7	-	-	10	10	8	9	1.2	[1.1]	-	-	9	10	11	10	1.0	[0.9]
	185.2	-	-	8	8	8	8	0.0	[0.9]	-	-	8	11	9	9	1.5	[0.8]
	555.6	-	-	9	8	9	9	0.6	[1.0]	-	-	10	11	9	10	1.0	[0.9]
	1,666.7	-	-	10	9	11	10	1.0	[1.2]	-	-	12	10	11	11	1.0	[0.9]
	5,000.0	-	-	9	11	9	10	1.2	[1.1]	-	-	11	10	12	11	1.0	[0.9]

	SA (1.0)	-	-	275	256	231	254	22.1	[29.3]	-	-	272	256	289	272	16.5	[23.3]

TA1537	0	-	-	12	13	11	12	1.0	[1.1]	-	-	13	12	12	12	0.6	[1.0]

	6.86	-	-	10	12	10	11	1.2	[0.9]	-	-	-	-	-	-	-	-
	20.6	-	-	14	13	15	14	1.0	[1.2]	-	-	-	-	-	-	-	-
	61.7	-	-	13	14	15	14	1.0	[1.2]	-	-	14	12	13	13	1.0	[1.1]
	185.2	-	-	14	13	15	14	1.0	[1.2]	-	-	9	12	14	12	2.5	[0.9]
	555.6	-	-	15	14	14	14	0.6	[1.2]	-	-	12	9	14	12	2.5	[0.9]
	1,666.7	-	-	15	13	14	14	1.0	[1.2]	-	-	16	11	15	14	2.6	[1.1]
	5,000.0	-	-	15	15	14	15	0.6	[1.2]	-	-	9	11	12	11	1.5	[0.9]

	9-AA (40.0)	-	-	380	369	324	358	29.7	[29.8]	-	-	270	209	281	253	38.8	[20.5]

WP2 uvrA	0	-	-	46	46	45	46	0.6	[1.0]	-	-	48	55	50	51	3.6	[1.0]

	6.86	-	-	45	42	42	43	1.7	[0.9]	-	-	-	-	-	-	-	-
	20.6	-	-	48	39	49	45	5.5	[1.0]	-	-	-	-	-	-	-	-
	61.7	-	-	47	43	53	48	5.0	[1.0]	-	-	44	49	46	46	2.5	[0.9]
	185.2	-	-	49	40	45	45	4.5	[1.0]	-	-	44	50	50	48	3.5	[0.9]
	555.6	-	-	39	39	47	42	4.6	[0.9]	-	-	51	50	53	51	1.5	[1.0]
	1,666.7	-	-	43	41	53	46	6.4	[1.0]	-	-	50	47	50	49	1.7	[1.0]
	5,000.0	-	-	43	40	54	46	7.4	[1.0]	-	-	55	55	54	55	0.6	[1.1]

	4-NQO (0.5)	-	-	230	210	218	219	10.1	[4.8]	-	-	154	161	198	171	23.6	[3.4]

PPT (Precipitation), TOX (Toxicity to bacteria), S.D (Standard deviation), Ratio (The percentage of the mean value compared to the negative control value)

Table 5

Results of the in vitro mammalian chromosomal aberration test

Test substance	μg/mL	Metabolic Activation	Treatment (hour)	pH	Osmotic pressure	RICC (%)	Chromosome Type		Chromatid Type		Multiple aberration	Cells with structural aberration (%)	Numerical aberration

							DEL	EXC	DEL	EXC			PP	ED
Distilled water	0	+	6	8.30	289	100.0	0	0	1	1	0	0.67	0	0

Ligigeopoong-san	250	+	6	7.64	273	78.6	0	0	1	0	0	0.33	1	0
	500			7.41	268	72.7	0	0	0	0	1	0.33	0	0
	1,000			7.59	287	66.9	0	0	0	0	0	0.00	0	0
	2,000			8.10	288	59.1	0	0	1	0	0	0.33	0	1

Benzo[a]pyrene	20	+	6	8.34	430	66.9	4	0	10	27	0	12.00^*	1	0

Distilled water	0	−	6	8.22	253	100.0	0	0	0	0	0	0.00	1	0

Ligigeopoong-san	250	−	6	8.12	251	82.2	0	1	1	0	0	0.67	0	0
	500			8.00	250	78.7	0	0	1	1	0	0.67	0	0
	1,000			8.05	252	75.1	0	0	0	1	0	0.33	0	0
	2,000			8.06	297	59.2	0	0	2	0	0	0.67	0	0

Mitomycin C	0.1	−	6	8.21	279	62.7	0	0	13	23	0	11.00^*	1	0

Distilled water	0	−	24	7.83	251	100.0	0	0	0	1	0	0.33	1	0

Ligigeopoong-san	250	−	24	7.53	256	82.6	0	0	0	0	0	0.00	0	0
	500			7.47	266	75.6	0	0	0	0	0	0.00	1	0
	1,000			7.95	257	70.3	0	0	1	0	0	0.33	0	0
	2,000			7.55	260	58.1	1	0	2	0	0	1.00	0	0

Mitomycin C	0.1	−	24	7.77	281	59.9	2	0	8	28	1	12.00^*	4	0

RICC (Relative increase in cell count), DEL (Deletion), EXC (Exchange), PP (Polyploidy), ED (Endoreduplication)

Significant difference (p < 0.05) from negative control group (Fisher’s exact test).

Table 6

Results of the mammalian erythrocyte micronucleus test

Group / Dose (mg/kg)	Animal	Dosing (1st)		Dosing (2nd)		Sacrifice		mnPCE	Frequency (%)	PCE/NCE ratio

		BW	GS	BW	GS	BW	GS			PCE	NCE	Ratio (%)
Negative control (Distilled water)	1	36.5	N	37.0	N	37.4	N	3	0.08	233	267	46.6
	2	34.9	N	35.4	N	35.1	N	5	0.13	233	267	46.6
	3	34.9	N	34.3	N	33.0	N	3	0.08	239	261	47.8
	4	33.2	N	33.4	N	33.3	N	3	0.08	246	254	49.2
	5	33.1	N	32.1	N	33.4	N	1	0.03	231	269	46.2

Mean	-	34.5	-	34.4	-	34.4	-	3.0	0.08	236.4	263.6	47.3
S.D	-	1.4	-	1.9	-	1.8	-	1.4	0.04	6.1	6.1	1.2

Ligigeopoong-san (500 mg/kg)	1	36.4	N	36.9	N	36.6	N	4	0.10	231	269	46.2
	2	35.0	N	34.5	N	34.8	N	1	0.03	239	261	47.8
	3	34.4	N	35.7	N	33.9	N	3	0.08	233	267	46.6
	4	33.4	N	33.9	N	34.2	N	3	0.08	230	270	46.0
	5	32.7	N	33.7	N	31.7	N	3	0.08	240	260	48.0

Mean	-	34.4	-	34.9	-	34.2	-	2.8	0.07	234.6	265.4	46.9
S.D	-	1.5	-	1.3	-	1.8	-	1.1	0.03	4.6	4.6	0.9

Ligigeopoong-san (1,000 mg/kg)	1	36.3	N	35.7	N	37.0	N	3	0.08	236	264	47.2
	2	35.1	N	35.1	N	33.8	N	5	0.13	247	253	49.4
	3	34.2	N	34.6	N	33.4	N	2	0.05	232	268	46.4
	4	33.5	N	33.6	N	33.8	N	6	0.15	239	261	47.8
	5	32.6	N	33.1	N	32.1	N	2	0.05	247	253	49.4

Mean	-	34.4	-	34.4	-	34.0	-	3.6	0.09	240.2	259.8	48.0
S.D	-	1.4	-	1.1	-	1.8	-	1.8	0.05	6.7	6.7	1.3

Ligigeopoong-san (2,000 mg/kg)	1	36.0	N	36.0	N	34.8	N	2	0.05	233	267	46.6
	2	35.2	N	35.2	N	34.3	N	4	0.10	232	268	46.4
	3	33.8	N	33.8	N	33.3	N	1	0.03	230	270	46.0
	4	33.5	N	33.5	N	32.8	N	2	0.05	238	262	47.6
	5	31.9	N	31.9	N	32.0	N	2	0.05	231	269	46.2

Mean	-	34.4	-	34.1	-	33.4	-	2.2	0.06	232.8	267.2	46.6
S.D	-	1.5	-	1.6	-	1.2	-	1.1	0.03	3.1	3.1	0.6

Positive control Mitomycin C (2 mg/kg)	1	34.8	N	34.8	N	34.6	N	443	11.1	152	348	30.4
	2	35.0	N	35.0	N	34.5	N	436	10.9	152	348	30.4
	3	34.1	N	34.1	N	33.2	N	438	11.0	135	365	27.0
	4	33.8	N	33.8	N	32.7	N	425	10.6	149	351	29.8
	5	32.3	N	32.2	N	31.6	N	423	10.6	155	345	31.0

Mean	-	34.4	-	34.0	-	33.3	-	433^*	10.8	148.6	351.4	29.7
S.D	-	1.4	-	1.1	-	1.3	-	8.6	0.22	7.9	7.9	1.6

GS (General symptoms), N (Normal), mnPCE (Micronucleated polychromatic erythrocyte), PCE (Polychromatic erythrocyte), NCE (Normochromatic erythrocyte), Ratio (NCE/PCE+NCE)

Significant difference (p < 0.05) from negative control group (Dunnett’s test).