Bacterial Reverse Mutation Test of CP pharmacopunture

Ji Hye Hwang; Jaseung Ku; Chul Jung; Hwang, Ji Hye; Ku, Jaseung; Jung, Chul

doi:10.13048/jkm.20025

JKM > Volume 41(3); 2020 > Article

Hwang, Ku, and Jung: Bacterial Reverse Mutation Test of CP pharmacopunture

Original Article

The Journal of Korean Medicine 2020; 41(3): 55-68.

Published online: September 01, 2020

DOI: https://doi.org/10.13048/jkm.20025

Bacterial Reverse Mutation Test of CP pharmacopunture

Ji Hye Hwang¹

, Jaseung Ku²

, Chul Jung³^{, *}

¹Department of Acupuncture & Moxibustion Medicine, College of Korean Medicine, Gachon University

²Bogwang korean medical clinic

³Namsangcheon Korean Medicine Clinic

Correspondence to: Chul Jung, Namsangcheon Korean Medicine Clinic, Seoul 06656, Republic of Korea, Email: jcnu2000@hanmail.net

Received June 11, 2020 Revised July 29, 2020 Accepted August 10, 2020

Abstract

Objectives

This study aimed to evaluate the toxicity of CP pharmacopunture using bacterial reverse mutation test.

Methods

To determine the mutagenic potential of CP pharmacopunture, histidine requiring Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and tryptophan requiring Escherichia coli (WP2uvrA, pKM101) strains were used. The negative (normal saline solution) and positive (Sodium azide, 2-Nitrofluorene, 2-Aminoanthracene, 9-Aminoacridine, and 4-Nitroquinoline N-oxide) control groups were used. To determine the dose levels of the main study, a dose range-finding study was conducted.

Results

As a results of the dose range-finding study, the growth inhibition by CP pharmacopunture was not evident at any dose levels in the absence and presence of metabolic activation. As a results of the main study, the mean number of revertant colonies was less than twice when compared to the negative control values at all dose levels of the CP pharmacopuncture in the presence and absence of metabolic activation, showing no dose-related increase. In the positive control group, the number of revertant colonies was markedly increased by more than twice when compared to the negative control group.

Conclusion

According to the results of this study, CP pharmacopunture did not show any signs of mutagenic potential.

Keywords: CP pharmacopunture, capsaicin, toxicity, bacterial reverse mutation test, acupuncture

Acknowledgment

This work was supported by from the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2017R1C1B5076224).

Notes

Conflicts of interest

The authors declare that they have no conflicts of interest.

Table 1

The Number of Revertant Colonies per Plate in the Absence of Metabolic Activation (1^st and 2^nd Main Studies)

Strain	Test substance	Dose (%)	1^st Main study							2^nd Main study
Strain	Test substance	Dose (%)	Individual revertant colony counts					Mean	S.D.	Individual revertant colony counts					Mean	S.D.
TA98	Normal saline injection	0	20	,	20	,	21	20	1	21	,	24	,	24	23	2
	CP	6.25	20	,	17	,	18	18	2	22	,	23	,	23	23	1
		12.5	19	,	17	,	22	19	3	21	,	25	,	22	23	2
		25.0	17	,	18	,	18	18	1	23	,	19	,	22	21	2
		50.0	20	,	19	,	17	19	2	19	,	22	,	24	22	3
		100	18	,	16	,	18	17	1	21	,	21	,	21	21	0
	2-Nitrofluorene (2-NF)	5.0	731	,	737	,	732	733	3	720	,	726	,	716	721	5
TA100	Normal saline injection	0	102	,	101	,	96	100	3	101	,	97	,	98	99	2
	CP	6.25	93	,	99	,	96	96	3	100	,	103	,	109	104	5
		12.5	91	,	100	,	99	97	5	108	,	98	,	100	102	5
		25.0	92	,	98	,	90	93	4	100	,	109	,	112	107	6
		50.0	102	,	96	,	92	97	5	101	,	118	,	115	111	9
		100	99	,	95	,	95	96	2	100	,	102	,	100	101	1
	Sodium azide (SA)	1.5	723	,	738	,	745	735	11	708	,	720	,	723	717	8
TA1535	Normal saline injection	0	16	,	15	,	15	15	1	13	,	16	,	16	15	2
	CP	6.25	16	,	14	,	17	16	2	18	,	14	,	16	16	2
		12.5	16	,	14	,	15	15	1	16	,	19	,	16	17	2
		25.0	16	,	14	,	13	14	2	13	,	14	,	17	15	2
		50.0	15	,	17	,	14	15	2	16	,	14	,	12	14	2
		100	16	,	16	,	14	15	1	17	,	14	,	15	15	2
	Sodium azide (SA)	1.5	579	,	600	,	572	584	15	585	,	585	,	603	591	10
TA1537	Normal saline injection	0	10	,	10	,	9	10	1	10	,	10	,	9	10	1
	CP	6.25	10	,	10	,	13	11	2	11	,	10	,	10	10	1
		12.5	13	,	13	,	10	12	2	10	,	12	,	13	12	2
		25.0	14	,	12	,	11	12	2	14	,	11	,	11	12	2
		50.0	9	,	12	,	9	10	2	10	,	8	,	10	9	1
		100	12	,	11	,	12	12	1	9	,	9	,	11	10	1
	9-Aminoacridine (9-AA)	80.0	610	,	583	,	591	595	14	579	,	587	,	588	585	5
WP2uvrA (pKM101)	Normal saline injection	0	94	,	99	,	101	98	4	106	,	96	,	99	100	5
	CP	6.25	94	,	96	,	95	95	1	114	,	106	,	104	108	5
		12.5	94	,	96	,	89	93	4	103	,	98	,	105	102	4
		25.0	104	,	100	,	103	102	2	101	,	115	,	99	105	9
		50.0	89	,	88	,	95	91	4	109	,	96	,	114	106	9
		100	89	,	94	,	92	92	3	109	,	119	,	108	112	6
	4-Nitroquinoline N-oxide (4-NQO)	0.1	417	,	410	,	414	414	4	543	,	552	,	540	545	6

S.D.: Standard Deviation

Table 2

The Number of Revertant Colonies per Plate in the Presence of Metabolic Activation (1^st and 2^nd Main Studies)

Strain	Test substance	Dose (%)	1^st Main study							2^nd Main study
Strain	Test substance	Dose (%)	Individual revertant colony counts					Mean	S.D.	Individual revertant colony counts					Mean	S.D.
TA98	Normal saline injection	0	36	,	37	,	37	37	1	35	,	36	,	36	36	1
	CP	6.25	33	,	36	,	32	34	2	35	,	35	,	37	36	1
		12.5	36	,	34	,	35	35	1	28	,	29	,	32	30	2
		25.0	33	,	33	,	31	32	1	32	,	30	,	28	30	2
		50.0	34	,	37	,	35	35	2	33	,	30	,	33	32	2
		100	37	,	34	,	36	36	2	33	,	30	,	33	32	2
	2-Aminoanthracene (2-AA)	1.0	381	,	370	,	387	379	9	386	,	394	,	398	393	6
TA100	Normal saline injection	0	106	,	110	,	100	105	5	110	,	115	,	107	111	4
	CP	6.25	113	,	111	,	110	111	2	126	,	124	,	116	122	5
		12.5	109	,	101	,	107	106	4	124	,	135	,	129	129	6
		25.0	111	,	110	,	118	113	4	123	,	128	,	119	123	5
		50.0	102	,	105	,	101	103	2	129	,	125	,	123	126	3
		100	99	,	94	,	100	98	3	127	,	127	,	122	125	3
TA1535	2-Aminoanthracene (2-AA)	2.0	919	,	945	,	960	941	21	942	,	951	,	963	952	11
	Normal saline injection	0	13	,	11	,	13	12	1	14	,	14	,	12	13	1
	CP	6.25	13	,	13	,	13	13	0	13	,	15	,	14	14	1
		12.5	11	,	12	,	12	12	1	13	,	14	,	12	13	1
		25.0	15	,	13	,	12	13	2	13	,	17	,	14	15	2
		50.0	16	,	13	,	16	15	2	14	,	13	,	12	13	1
		100	16	,	11	,	12	13	3	12	,	14	,	15	14	2
TA1537	2-Aminoanthracene (2-AA)	3.0	175	,	185	,	184	181	6	174	,	183	,	176	178	5
	Normal saline injection	0	18	,	20	,	19	19	1	22	,	22	,	21	22	1
	CP	6.25	21	,	18	,	22	20	2	25	,	23	,	22	23	2
		12.5	17	,	20	,	16	18	2	24	,	22	,	20	22	2
		25.0	16	,	16	,	18	17	1	20	,	21	,	25	22	3
		50.0	20	,	18	,	18	19	1	20	,	17	,	19	19	2
		100	19	,	20	,	22	20	2	22	,	19	,	18	20	2
	2-Aminoanthracene (2-AA)	3.0	229	,	231	,	231	230	1	249	,	241	,	242	244	4
WP2uvrA (pKM101)	Normal saline injection	0	131	,	123	,	128	127	4	140	,	132	,	138	137	4
	CP	6.25	122	,	140	,	135	132	9	136	,	138	,	142	139	3
		12.5	131	,	136	,	144	137	7	139	,	133	,	142	138	5

		25.0	133	,	135	,	134	134	1	142	,	140	,	138	140	2

		50.0	131	,	133	,	133	132	1	131	,	139	,	134	135	4
		100	132	,	134	,	129	132	3	145	,	150	,	147	147	3
	2-Aminoanthracene (2-AA)	2.0	360	,	361	,	359	360	1	447	,	449	,	454	450	4

S.D.: Standard Deviation

Table 3

Historical Control Data

Historical negative control values of revertant colonies

Strain	S9 mix	N	Mean ± S.D.	Range

				Lower	Upper
TA100	−	101	86.2 ± 10.5	59.0	113.5
TA100	+	101	96.8 ± 11.6	69.2	124.5

TA1535	−	100	10.9 ± 1.8	5.6	16.2
TA1535	+	100	10.2 ± 1.6	5.8	14.6

WP2uvrA (pKM101)	−	100	123.5 ± 16.0	81.5	165.5
WP2uvrA (pKM101)	+	100	156.3 ± 17.3	113.6	199.0

TA98	−	101	18.5 ± 2.9	10.3	26.6
TA98	+	101	27.4 ± 3.6	16.9	37.9

TA1537	−	100	7.8 ± 1.1	4.7	10.9
TA1537	+	100	14.7 ± 2.4	7.2	22.1

Historical positive control values of revertant colonies

Strain	S9 mix	Positive control	Dose (μg/plate)	N	Mean ± S.D.	Range

						Lower	Upper
TA100	−	SA	1.5	101	621.1 ± 49.5	487.2	755.0
TA100	+	2-AA	2.0	93	744.7 ± 155.2	462.0	1,027.5

TA1535	−	SA	1.5	101	496.1 ± 39.1	387.2	605.1
TA1535	+	2-AA	3.0	100	137.6 ± 21.0	88.7	186.6

WP2uvrA (pKM101)	−	4-NQO	0.1	86	679.2 ± 124.9	327.2	1,031.2
WP2uvrA (pKM101)	+	2-AA	2.0	100	499.4 ± 52.7	348.1	650.6

TA98	−	2-NF	5.0	101	554.6 ± 78.6	359.1	750.1
TA98	+	2-AA	1.0	93	392.3 ± 52.3	275.3	509.4

TA1537	−	9-AA	80.0	101	408.1 ± 127.6	201.0	615.1
TA1537	+	2-AA	3.0	92	186.7 ± 28.1	121.4	252.0

Negative control: Water for injection, Dimethyl sulfoxide, Acetone, Tetrahydrofuran, Normal saline injection, etc.

SA: Sodium azide

2-AA: 2-Aminoanthracene

4-NQO: 4-Nitroquinoline N-oxide

2-NF: 2-Nitrofluorene

9-AA: 9-Aminoacridine

N: The total number of bacterial reverse mutation test

S.D.: Standard Deviation

The above historical control values were obtained from the data pooled from Dec. 10, 2015 to May 18, 2017 (4-NQO).

The above historical control values were obtained from the data pooled from Nov. 19, 2015 to May 18, 2017.

The range was calculated by the control limit of X derived from X̄-R̄-R̄s value.

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