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JKM > Volume 41(3); 2020 > Article
Hwang, Ku, and Jung: Bacterial Reverse Mutation Test of CP pharmacopunture

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.

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 (1st and 2nd Main Studies)
Strain Test substance Dose (%) 1st Main study 2nd Main study
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 (1st and 2nd Main Studies)
Strain Test substance Dose (%) 1st Main study 2nd Main study
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
+ 101 96.8 ± 11.6 69.2 124.5

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

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

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

TA1537 100 7.8 ± 1.1 4.7 10.9
+ 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
+ 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
+ 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
+ 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
+ 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
+ 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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