Genotoxicity and acute toxicity of Hyunburikyung-tang: assessing the safety of prescribing traditional Korean medicine for dysmenorrhea.

We assessed standard acute toxicity indicators to determine the acute toxicity of orally administered Hyunburikyung-tang in male and female rats, including body weight, general symptoms, and autopsy findings. No statistically significant difference in body weight was observed after a single administration of Hyunburikyung-tang. Detailed individual weights are presented in Tables  2 and 3 . Moreover, no toxicological responses were observed regarding general symptoms 14 days post-administration, including appearance, body position, consciousness, behavior, and nervous system symptoms. Autopsy examination, which included assessment of systemic organs such as the liver, heart, kidneys, and testis (Table  4 ), showed no signs of abnormal reactions (Tables  2 and 3 ). Table 2 Individual body weights in acute toxicity assessment (male) Sex: Male (g) Group/ Dose (mg/kg) Animal ID Day 0 1 3 7 14 B.W G.S P-value B.W G.S P-value B.W G.S P-value B.W G.S P-value B.W G.S P-value G1/0 1101 208.1 Normal - 233.6 Normal - 254.0 Normal - 280.5 Normal - 322.6 Normal - 1102 220.9 Normal 253.7 Normal 280.5 Normal 341.7 Normal 425.8 Normal 1103 216.2 Normal 251.8 Normal 275.2 Normal 320.0 Normal 382.3 Normal 1104 217.7 Normal 249.1 Normal 269.2 Normal 304.2 Normal 367.1 Normal 1105 213.3 Normal 246.9 Normal 266.8 Normal 310.2 Normal 372.8 Normal Mean 215.2 247.0 269.1 311.3 374.1 S.D 4.8 7.9 10.0 22.4 36.9 S.E 2.2 3.5 4.5 10.0 16.5 N 5 5 5 5 5 G2/625 1201 203.1 Normal 0.777 231.2 Normal 0.815 257.0 Normal 0.992 295.5 Normal 0.994 354.9 Normal 0.992 1202 221.2 Normal 250.7 Normal 270.7 Normal 310.6 Normal 367.8 Normal 1203 210.8 Normal 244.6 Normal 271.2 Normal 309.9 Normal 366.2 Normal 1204 209.3 Normal 246.6 Normal 271.4 Normal 312.3 Normal 382.1 Normal 1205 214.9 Normal 243.7 Normal 268.4 Normal 317.8 Normal 380.6 Normal Mean 211.9 243.4 267.7 309.2 370.3 S.D 6.7 7.3 6.1 8.3 11.2 S.E 3.0 3.3 2.7 3.7 5.0 N 5 5 5 5 5 G3/1,250 1301 220.4 Normal 0.986 257.4 Normal 1.000 287.6 Normal 0.881 343.1 Normal 0.766 432.7 Normal 0.696 1302 208.5 Normal 239.0 Normal 260.7 Normal 304.9 Normal 371.8 Normal 1303 219.0 Normal 248.9 Normal 275.6 Normal 321.4 Normal 383.4 Normal 1304 207.3 Normal 242.0 Normal 266.7 Normal 310.0 Normal 375.4 Normal 1305 215.2 Normal 249.6 Normal 274.0 Normal 319.3 Normal 384.8 Normal Mean 214.1 247.4 272.9 319.7 389.6 S.D 6.0 7.2 10.1 14.7 24.7 S.E 2.7 3.2 4.5 6.6 11.0 N 5 5 5 5 5 G4/2,500 1401 197.0 Normal 0.926 228.7 Normal 0.831 250.8 Normal 0.949 295.3 Normal 0.879 359.7 Normal 0.801 1402 218.9 Normal 248.4 Normal 282.1 Normal 332.0 Normal 401.5 Normal 1403 217.6 Normal 248.0 Normal 281.7 Normal 339.9 Normal 427.8 Normal 1404 215.4 Normal 252.9 Normal 273.0 Normal 309.9 Normal 383.1 Normal 1405 216.6 Normal 239.5 Normal 271.7 Normal 311.1 Normal 361.8 Normal Mean 213.1 243.5 271.9 317.6 386.8 S.D 9.1 9.6 12.7 18.1 28.6 S.E 4.1 4.3 5.7 8.1 12.8 N 5 5 5 5 5 N Number of animals, S.D standard deviation, S.E Standard error, B.W Body weight, G.S General symptoms Significance of body weight between groups by analysis of variance with Dunnett’s test ( p  < 0.05) Individual body weights in acute toxicity assessment (male) N Number of animals, S.D standard deviation, S.E Standard error, B.W Body weight, G.S General symptoms Significance of body weight between groups by analysis of variance with Dunnett’s test ( p  < 0.05) Table 3 Individual body weights in acute toxicity assessment (female) Sex: Female (g) Group/ Dose (mg/kg) Animal ID Day 0 1 3 7 14 B.W G.S P-value B.W G.S P-value B.W G.S P-value B.W G.S P-value B.W G.S P-value G1/0 2101 148.8 Normal - 173.8 Normal - 184.8 Normal - 201.1 Normal - 230.3 Normal - 2102 156.9 Normal 171.3 Normal 187.9 Normal 206.4 Normal 237.2 Normal 2103 151.5 Normal 170.2 Normal 185.1 Normal 203.5 Normal 220.3 Normal 2104 153.5 Normal 179.2 Normal 188.9 Normal 205.7 Normal 230.8 Normal 2105 154.0 Normal 173.1 Normal 185.1 Normal 197.1 Normal 225.3 Normal Mean 152.9 173.5 186.4 202.8 228.8 S.D 3.0 3.5 1.9 3.8 6.4 S.E 1.3 1.6 0.8 1.7 2.8 N 5 5 5 5 5 G2/625 2201 158.4 Normal 0.981 181.9 Normal 0.225 193.5 Normal 0.480 212.0 Normal 0.422 231.8 Normal 0.996 2202 155.7 Normal 180.5 Normal 192.3 Normal 216.2 Normal 243.4 Normal 2203 149.8 Normal 174.4 Normal 183.8 Normal 197.9 Normal 219.3 Normal 2204 152.7 Normal 174.2 Normal 184.8 Normal 201.1 Normal 218.3 Normal 2205 151.3 Normal 178.0 Normal 193.6 Normal 212.5 Normal 236.4 Normal Mean 153.6 177.8 189.6 207.9 229.8 S.D 3.5 3.5 4.9 8.0 10.9 S.E 1.5 1.6 2.2 3.6 4.9 N 5 5 5 5 5 G3/1,250 2301 152.6 Normal 0.954 172.6 Normal 0.950 182.7 Normal 1.000 205.5 Normal 0.809 226.0 Normal 1.000 2302 154.2 Normal 181.2 Normal 192.8 Normal 208.4 Normal 233.6 Normal 2303 148.5 Normal 170.4 Normal 186.7 Normal 204.6 Normal 235.5 Normal 2304 149.4 Normal 173.7 Normal 186.9 Normal 211.0 Normal 229.9 Normal 2305 155.6 Normal 174.9 Normal 183.1 Normal 198.4 Normal 218.8 Normal Mean 152.1 174.6 186.4 205.6 228.8 S.D 3.0 4.1 4.1 4.7 6.7 S.E 1.4 1.8 1.8 2.1 3.0 N 5 5 5 5 5 G4/2,500 2401 160.8 Normal 0.615 183.4 Normal 0.421 192.8 Normal 0.954 212.2 Normal 0.974 239.1 Normal 0.999 2402 151.8 Normal 175.6 Normal 185.7 Normal 204.4 Normal 221.1 Normal 2403 157.1 Normal 178.3 Normal 186.2 Normal 199.0 Normal 236.7 Normal 2404 152.1 Normal 174.2 Normal 181.4 Normal 197.5 Normal 234.0 Normal 2405 153.8 Normal 172.4 Normal 180.3 Normal 194.2 Normal 209.4 Normal Mean 155.1 176.8 185.3 201.5 228.1 S.D 3.8 4.3 4.9 7.0 12.5 S.E 1.7 1.9 2.2 3.1 5.6 N 5 5 5 5 5 N Number of animals, S.D Standard deviation, S.E Standard error, B.W Body weight, G.S General symptoms Significance of body weight between groups by analysis of variance with Dunnett’s test (p < 0.05) Individual body weights in acute toxicity assessment (female) N Number of animals, S.D Standard deviation, S.E Standard error, B.W Body weight, G.S General symptoms Significance of body weight between groups by analysis of variance with Dunnett’s test (p < 0.05) Table 4 Observation results of major organs Group/Dose (mg/kg) Animal ID Liver Heart Spleen Lung Kidney Adrenal gland Gastrointestinal tract Brain Urinary system Reproductive system Sex: Male  G1/0 1101 N N N N N N N N N N 1102 N N N N N N N N N N 1103 N N N N N N N N N N 1104 N N N N N N N N N N 1105 N N N N N N N N N N  G2/625 1201 N N N N N N N N N N 1202 N N N N N N N N N N 1203 N N N N N N N N N N 1204 N N N N N N N N N N 1205 N N N N N N N N N N  G3/1,250 1301 N N N N N N N N N N 1302 N N N N N N N N N N 1303 N N N N N N N N N N 1304 N N N N N N N N N N 1305 N N N N N N N N N N  G4/2,500 1401 N N N N N N N N N N 1402 N N N N N N N N N N 1403 N N N N N N N N N N 1404 N N N N N N N N N N 1405 N N N N N N N N N N Sex: Female  G1/0 2101 N N N N N N N N N N 2102 N N N N N N N N N N 2103 N N N N N N N N N N 2104 N N N N N N N N N N 2105 N N N N N N N N N N  G2/625 2201 N N N N N N N N N N 2202 N N N N N N N N N N 2203 N N N N N N N N N N 2204 N N N N N N N N N N 2205 N N N N N N N N N N  G3/1,250 2301 N N N N N N N N N N 2302 N N N N N N N N N N 2303 N N N N N N N N N N 2304 N N N N N N N N N N 2305 N N N N N N N N N N  G4/2,500 2401 N N N N N N N N N N 2402 N N N N N N N N N N 2403 N N N N N N N N N N 2404 N N N N N N N N N N 2405 N N N N N N N N N N Urinary system: urinary bladder, preputial gland, coagulating gland, clitoral glandReproductive system: Testis, Epididy, is, prostate, seminal vesicle, ovary, oviduct, uterus, vagnina N Normal Observation results of major organs Urinary system: urinary bladder, preputial gland, coagulating gland, clitoral glandReproductive system: Testis, Epididy, is, prostate, seminal vesicle, ovary, oviduct, uterus, vagnina N Normal A bacterial reverse mutation test was conducted to assess the mutagenic potential of Hyunburikyung-tang on bacterial strains (TA98, TA100, TA1535, TA1537, and WP2 uvrA). Test acceptability was verified using negative and positive controls. The highest concentration tested was 5,000 µg/plate, in line with the OECD guidelines. The results findings were confirmed by performing two individual experiments encompassing a total of five concentrations at approximately half-log (√10) intervals. Hyunburikyung-tang was added to both frameshift-type strains (TA98 and TA1537) and basepair-substitution-type strains (TA100, TA1535, and WP2 uvrA), with and without metabolic activation. The findings are summarized in Tables  5 and 6 . Table 5 Results of the bacterial reverse mutation test without metabolic activation Strain Dose (µg/plate) Primary Secondary Colony Mean S.D Ratio Colony Mean S.D Ratio TA 98 0 10 16 18 15 4.2 [1.0] 18 13 12 14 3.2 [1.0] 61.7 18 13 11 14 3.6 [1.0] 13 13 13 13 0.0 [0.9] 185.2 16 11 15 14 2.6 [1.0] 13 11 17 14 3.1 [1.0] 555.6 10 14 19 14 4.5 [1.0] 17 12 14 14 2.5 [1.0] 1,666.7 15 20 14 16 3.2 [1.1] 20 11 14 15 4.6 [1.0] 5,000.0 14 18 17 16 2.1 [1.1] 16 15 15 15 0.6 [1.1] 2-NF (0.5) 154 145 156 152 5.9 [10.1] 143 118 156 139 19.3 [9.9] TA 100 0 81 87 94 87 6.5 [1.0] 73 89 77 80 8.3 [1.0] 61.7 88 72 85 82 8.5 [0.9] 81 73 77 77 4.0 [1.0] 185.2 72 95 77 81 12.1 [0.9] 73 73 78 75 2.9 [0.9] 555.6 76 94 78 83 9.9 [0.9] 74 86 77 79 6.2 [1.0] 1,666.7 73 88 93 85 10.4 [1.0] 78 87 79 81 4.9 [1.0] 5,000.0 100 76 86 87 12.1 [1.0] 87 83 76 82 5.6 [1.0] SA (1.0) 736 643 608 662 66.2 [7.6] 601 576 632 603 28.1 [7.5] TA 1535 0 15 20 18 18 2.5 [1.0] 14 19 16 16 2.5 [1.0] 61.7 19 18 12 16 3.8 [0.9] 17 14 16 16 1.5 [1.0] 185.2 19 20 13 17 3.8 [0.9] 17 19 14 17 2.5 [1.0] 555.6 15 20 13 16 3.6 [0.9] 20 18 14 17 3.1 [1.1] 1,666.7 12 18 19 16 3.8 [1.0] 14 15 18 16 2.1 [1.0] 5,000.0 20 19 15 18 2.6 [1.0] 19 19 16 18 1.7 [1.1] SA (1.0) 696 719 651 689 34.6 [38.3] 586 612 702 633 60.9 [39.6] TA 1537 0 9 7 5 7 2.0 [1.0] 6 9 9 8 1.7 [1.0] 61.7 6 8 7 7 1.0 [1.0] 9 8 5 7 2.1 [0.9] 185.2 8 6 6 7 1.2 [1.0] 8 6 8 7 1.2 [0.9] 555.6 8 8 6 7 1.2 [1.0] 7 8 6 7 1.0 [0.9] 1,666.7 7 9 9 8 1.2 [1.2] 6 7 6 6 0.6 [0.8] 5,000.0 7 7 8 7 0.6 [1.0] 7 9 8 8 1.0 [1.0] 9-AA (40.0) 218 195 159 191 29.7 [27.3] 128 129 101 119 15.9 [14.9] WP2 uvrA 0 43 40 44 42 2.1 [1.0] 43 39 43 42 2.3 [1.0] 61.7 44 42 43 43 1.0 [1.0] 47 45 49 47 2.0 [1.1] 185.2 52 48 52 51 2.3 [1.2] 46 46 42 45 2.3 [1.1] 555.6 47 45 52 48 3.6 [1.1] 43 41 47 44 3.1 [1.0] 1,666.7 46 53 54 51 4.4 [1.2] 49 49 50 49 0.6 [1.2] 5,000.0 46 53 53 51 4.0 [1.2] 47 52 52 50 2.9 [1.2] 4-NQO (0.5) 820 833 844 832 12.0 [19.8] 778 815 800 798 18.6 [19.0] 4-nitroquinoline 1-oxide (4-NQO) Ratio: Indicates the percentage of the mean value compared to the negative control value S.D Standard deviation, 0 Distilled water, 2-NF 2-nitrofluorene, SA Sodium azide, 9-AA 9-amincoacridine Results of the bacterial reverse mutation test without metabolic activation 4-nitroquinoline 1-oxide (4-NQO) Ratio: Indicates the percentage of the mean value compared to the negative control value S.D Standard deviation, 0 Distilled water, 2-NF 2-nitrofluorene, SA Sodium azide, 9-AA 9-amincoacridine Table 6 Results of the bacterial reverse mutation test with metabolic activation Strain Dose (µg/plate) Primary Secondary Colony Mean S.D Ratio Colony Mean S.D Ratio TA 98 0 16 25 20 20 4.5 [1.0] 24 15 18 19 4.6 [1.0] 61.7 23 20 20 21 1.7 [1.0] 12 22 16 17 5.0 [0.9] 185.2 17 24 19 20 3.6 [1.0] 23 21 17 20 3.1 [1.1] 555.6 18 17 24 20 3.8 [1.0] 19 24 20 21 2.6 [1.1] 1,666.7 20 16 21 19 2.6 [0.9] 24 18 25 22 3.8 [1.2] 5,000.0 17 20 19 19 1.5 [0.9] 21 20 21 21 0.6 [1.1] B[a]P (1.0) 103 108 111 107 4.0 [5.4] 131 107 91 110 20.1 [5.8] TA 100 0 77 77 90 81 7.5 [1.0] 91 99 76 89 11.7 [1.0] 61.7 78 72 83 78 5.5 [1.0] 102 88 96 95 7.0 [1.1] 185.2 83 89 72 81 8.6 [1.0] 98 82 96 92 8.7 [1.0] 555.6 74 88 73 78 8.4 [1.0] 87 79 102 89 11.7 [1.0] 1,666.7 85 78 88 84 5.1 [1.0] 81 98 83 87 9.3 [1.0] 5,000.0 80 93 79 84 7.8 [1.0] 88 81 86 85 3.6 [1.0] 2-AA (0.5) 532 591 424 516 84.7 [6.4] 558 584 549 564 18.2 [6.3] TA 1535 0 20 16 14 17 3.1 [1.0] 15 16 13 15 1.5 [1.0] 61.7 14 20 20 18 3.5 [1.1] 14 13 16 14 1.5 [1.0] 185.2 15 22 20 19 3.6 [1.1] 18 16 16 17 1.2 [1.1] 555.6 19 18 21 19 1.5 [1.2] 18 14 18 17 2.3 [1.1] 1,666.7 15 18 15 16 1.7 [1.0] 14 16 17 16 1.5 [1.1] 5,000.0 14 18 17 16 2.1 [1.0] 13 12 19 15 3.8 [1.0] 2-AA (2.0) 353 345 323 340 15.5 [20.0] 279 311 239 276 36.1 [18.4] TA 1537 0 9 9 10 9 0.6 [1.0] 19 19 23 20 2.3 [1.0] 61.7 10 8 9 9 1.0 [1.0] 21 22 18 20 2.1 [1.0] 185.2 8 8 9 8 0.6 [0.9] 26 15 16 19 6.1 [0.9] 555.6 10 10 8 9 1.2 [1.0] 21 19 17 19 2.0 [0.9] 1,666.7 7 10 10 9 1.7 [1.0] 21 18 24 21 3.0 [1.0] 5,000.0 7 10 11 9 2.1 [1.0] 19 17 26 21 4.7 [1.0] 2-AA (2.0) 282 272 299 284 13.7 [31.6] 300 320 328 316 14.4 [15.8] WP2 uvrA 0 46 48 47 47 1.0 [1.0] 51 50 47 49 2.1 [1.0] 61.7 48 40 46 45 4.2 [1.0] 49 46 45 47 2.1 [0.9] 185.2 48 46 48 47 1.2 [1.0] 53 48 47 49 3.2 [1.0] 555.6 47 42 44 44 2.5 [0.9] 44 46 47 46 1.5 [0.9] 1,666.7 49 43 49 47 3.5 [1.0] 45 48 52 48 3.5 [1.0] 5,000.0 43 49 51 48 4.2 [1.0] 47 50 54 50 3.5 [1.0] 2-AA (4.0) 193 172 177 181 11.0 [3.9] 229 226 198 218 17.1 [4.4] Ratio: Indicates the percentage of the mean value compared to the negative control value SD Standard deviation, B[a]P benzo[a]pyrene, 2-AA 2-aminoanthracene) Results of the bacterial reverse mutation test with metabolic activation Ratio: Indicates the percentage of the mean value compared to the negative control value SD Standard deviation, B[a]P benzo[a]pyrene, 2-AA 2-aminoanthracene) Conventionally, the bacterial reverse mutation test yields a mutagenic response when colony numbers show a two-fold increase compared to the negative control or in cases where a dose-response relationship is evident. However, we observed that exposure of the bacterial strains to Hyunburikyung-tang, with/without metabolic activation, did not result in a colony count increase of more than two-fold. These findings suggest no mutagenic potential of Hyunburikyung-tang up to a dose of 5,000 µg/plate. The chromosomal aberration test aimed to assess whether exposure of CHL/IU cells to Hyunburikyung-tang induced chromosomal abnormalities. The highest concentration of the short-time exposure (6 h) and long-time exposure (24 h) without metabolic activation was set to 2,000 µg/mL, and a total of three concentrations were set as twofold intervals. The highest concentration of 2,000 µg/mL was excluded in short-term exposure with metabolic activation to prevent false positive results due to cytotoxicity. Following metabolic activation, Hyunburikyung-tang reduced the relative increase in cell count (RICC) to < 50% after short-term exposure. Therefore, the highest concentration for short-term exposure with metabolic activation was set at 1,000 µg/mL, with four concentrations set at two-fold intervals. We found that Hyunburikyung-tang did not cause medium precipitation or affect pH or osmotic pressure at all the concentrations listed in Table  7 . Statistical analysis revealed significant differences between positive and negative controls for all treatment groups ( p  < 0.001). Hyunburikyung-tang exposure did not lead to structural chromosomal abnormalities during short-term or long-term exposure, with or without metabolic activation. Additionally, the Cochran–Armitage test indicated no significant concentration-response relationship ( p  > 0.05). Therefore, Hyunburikyung-tang did not induce chromosomal aberrations under the present experimental conditions (Fig.  1 ). Table 7 Results of the chromosomal aberration test Substance Dose(µg/mL) Metabolic activation Treatment-Recovery time pH Osmotic pressure RICC(%) Chromosome Chromatid Others Aberrant cell/total cell Total Aberration(%) P-value Trend test p-value Del Exc Del Exc Distilled water 0 + 6–18 8.38 290 100.0 0 0 2 0 0 2/300 0.67 - 0.419 Hyunburikyung-tang 125 + 6–18 8.20 286 78.4 0 0 1 0 1 2/300 0.67 1.000 250 8.18 291 76.3 0 0 3 0 0 3/300 1.00 0.686 500 8.10 288 69.8 1 0 2 0 0 2/300 0.67 1.000 1,000 8.26 283 63.3 0 0 0 1 0 1/300 0.33 0.624 B[a]P 20 + 6–18 8.28 455 69.8 4 0 11 126 1 42/300 14.00 * 0.000 Distilled water 0 - 6–18 8.39 268 100.0 0 0 0 0 0 0/300 0.00 - 0.559 Hyunburikyung-tang 500 - 6–18 8.39 267 100.0 1 0 2 0 0 3/300 1.00 0.249 1,000 8.33 284 95.5 0 0 1 1 0 2/300 0.67 0.249 2,000 8.33 269 84.2 0 0 1 1 0 2/300 0.67 0.499 MMC 0.1 - 6–18 8.32 282 61.7 2 0 7 26 5 39/300 13.00 * 0.000 Distilled water 0 - 24 − 0 8.37 260 100.0 1 0 0 1 0 2/300 0.67 - 0.434 Hyunburikyung-tang 500 - 24 − 0 8.51 304 100.0 0 0 1 1 0 2/300 0.67 1.000 1,000 8.28 265 95.5 1 0 1 0 0 2/300 0.67 1.000 2,000 8.25 276 91.0 0 0 0 1 0 1/300 0.33 0.624 MMC 0.1 - 24 − 0 8.33 294 63.9 4 0 16 43 0 56/300 18.67 * 0.000 B[a]P Benzo[a]pyrene), MMC Mitomycin C, RICC Relative increase in cell count, Del Deletion, Exc Exchange, Others Fragmentation and multiple aberrations Significance of total aberration between groups by Fisher’s exact test ( * p  < 0.05) The trend test p-value represents the significance level of the Cochran-Armitage trend test Results of the chromosomal aberration test B[a]P Benzo[a]pyrene), MMC Mitomycin C, RICC Relative increase in cell count, Del Deletion, Exc Exchange, Others Fragmentation and multiple aberrations Significance of total aberration between groups by Fisher’s exact test ( * p  < 0.05) The trend test p-value represents the significance level of the Cochran-Armitage trend test Fig. 1 Observation results of metaphase cells by treatment group. A  Short-term exposure (6 h) with metabolic activation. B  Short-time exposure (6 h) without metabolic activation. C  Long-time exposure (24 h) without metabolic activation. The representative image was captured using a microscope (600X magnification). The black arrow indicates chromosomal aberration. Abbreviation: CSB (chromosome break), CTE (chromatid exchange), CTB (chromatid break) Observation results of metaphase cells by treatment group. A  Short-term exposure (6 h) with metabolic activation. B  Short-time exposure (6 h) without metabolic activation. C  Long-time exposure (24 h) without metabolic activation. The representative image was captured using a microscope (600X magnification). The black arrow indicates chromosomal aberration. Abbreviation: CSB (chromosome break), CTE (chromatid exchange), CTB (chromatid break) The micronucleus test aimed to assess micronucleus formation in bone marrow following oral administration of Hyunburikyung-tang. Administration was performed for 2 days at 24 h intervals, and general symptoms due to Hyunburikyung-tang administration were observed immediately post-administration and before sacrifice. No weight changes or toxic signs were observed among the groups after Hyunburikyung-tang administration (Table  8 ). The PEC/NCE ratio was assessed to evaluate bone marrow toxicity. No differences in the PEC/NCE ratio were observed in the bone marrow of experimental animals, and the micronucleus frequency did not reach statistical significance. Therefore, twice-daily administration of Hyunburikyung-tang up to 2,000 mg/kg did not induce micronuclei formation (Fig.  2 ). Table 8 Results of the micronucleus test Group/Dose (mg/kg) 1 st dosing 2nd dosing Sacrifice mnPCE Frequency (%) P -value PCE/(PCE + NCE) B.W G.S B.W G.S B.W G.S PCE NCE Ratio (%) G1/0 36.32 N 35.96 N 35.98 N 3 0.08 - 237 263 47.4 34.67 N 34.46 N 33.35 N 3 0.08 248 252 49.6 34.36 N 33.79 N 32.96 N 3 0.08 237 263 47.4 33.83 N 34.59 N 32.98 N 5 0.13 242 258 48.4 33.51 N 33.68 N 33.53 N 2 0.05 239 261 47.8 Mean 34.54 - 34.50 - 33.76 - 3.2 0.08 240.6 259.4 48.1 S.D 0.98 - 0.81 - 1.13 - 1.1 0.03 4.6 4.6 0.9 G2/500 36.28 N 34.95 N 35.39 N 2 0.05 0.999 242 258 48.4 34.68 N 35.47 N 35.38 N 1 0.03 233 267 46.6 34.20 N 34.56 N 35.38 N 1 0.03 233 267 46.6 33.88 N 34.27 N 34.11 N 2 0.05 246 254 49.2 33.40 N 32.67 N 33.24 N 3 0.08 242 258 48.4 Mean 34.49 - 34.38 - 34.70 - 1.8 0.05 239.2 260.8 47.8 S.D 0.99 - 0.95 - 0.88 - 0.8 0.02 5.9 5.9 1.2 G3/1,000 35.58 N 35.08 N 34.50 N 1 0.03 1.000 233 267 46.6 35.15 N 35.24 N 35.54 N 2 0.05 246 254 49.2 34.13 N 33.42 N 32.68 N 5 0.13 239 261 47.8 33.92 N 33.33 N 32.62 N 3 0.08 237 263 47.4 33.26 N 32.40 N 31.60 N 3 0.08 241 259 48.2 Mean 34.41 - 33.89 - 33.39 - 2.8 0.07 239.2 260.8 47.8 S.D 0.84 - 1.09 - 1.43 - 1.5 0.04 4.8 4.8 1.0 G4/2,000 35.57 N 35.22 N 34.13 N 3 0.08 1.000 242 258 48.4 35.17 N 35.02 N 33.56 N 4 0.10 239 261 47.8 34.08 N 34.35 N 33.67 N 2 0.05 242 258 48.4 33.93 N 34.01 N 33.12 N 1 0.03 232 268 46.4 33.00 N 33.45 N 32.13 N 6 0.15 241 259 48.2 Mean 34.35 - 34.41 - 33.32 - 3.2 0.08 239.2 260.8 47.8 S.D 0.92 - 0.65 - 0.68 - 1.9 0.05 4.2 4.2 0.8 G5/MMC 2 35.38 N 34.58 N 34.30 N 458 11.5 0.000 147 353 29.4 35.30 N 35.94 N 34.01 N 430 10.8 152 348 30.4 34.07 N 34.54 N 32.63 N 403 10.1 160 340 32.0 34.01 N 34.45 N 34.02 N 479 12.0 162 338 32.4 32.87 N 33.08 N 32.11 N 433 10.8 144 356 28.8 Mean 34.33 - 34.52 - 33.41 - 440.6 11.02 * 153.0 347.0 30.6 S.D 0.93 - 0.91 - 0.87 - 29.0 0.72 7.9 7.9 1.6 B.W Body weights, G.S General symptoms, mnPCE Micronucleated polychromatic erythrocytes, PCE Polychromatic erythrocytes, NCE Normochromatic erythrocytes, N Normal The mnPCE frequency in 4,000 PCE per animal significance of frequency between groups by Dunnett’s test ( * p  < 0.05) Results of the micronucleus test B.W Body weights, G.S General symptoms, mnPCE Micronucleated polychromatic erythrocytes, PCE Polychromatic erythrocytes, NCE Normochromatic erythrocytes, N Normal The mnPCE frequency in 4,000 PCE per animal significance of frequency between groups by Dunnett’s test ( * p  < 0.05) Fig. 2 Observation results of polychromatic erythrocytes in bone marrow. The red arrow indicates the micronucleated polychromatic erythrocyte. The representative image was captured using a microscope (600X magnification) Observation results of polychromatic erythrocytes in bone marrow. The red arrow indicates the micronucleated polychromatic erythrocyte. The representative image was captured using a microscope (600X magnification)

Hyunburikyung-tang was purchased from the Hocburi (The facility standard of herbal dispensaries, Gyeonggi Province, Korea), composed of 15 medicinal plants. Medicinal plants used in the preparation of Hyunburikyung-tang were analyzed by Good Manufacturing Practice (GMP)-certified pharmaceutical companies to determine whether they met the acceptance criteria of the Korean pharmacopoeia (KP) and Korean herbal pharmacopoeia (KHP) of the Ministry of Food and Drug Safety (MFDS) of the Republic of Korea. Medicinal plants that were confirmed to meet the legal acceptance criteria were used for extraction (see Table  1 for composition and analysis data). Table 1 Composition and analysis data of Hyunburikyung-tang Number Scientific name Part Weight (g) Ratio (%) Origin Description Ash (%) SO 2 (ppm) Aflatoxin Quantification Heavy metal (ppm) Pesticide residues Company Name Ratio (%) 1 Angelica gigas Nakai Radix 3.8 6.1 Korea Suitable 5.8 7.0 - Decursin+Decursinol angelate 8.000 < 0.0 N.D A 2 Prunus persica Batsch Semen 3.8 6.1 South Africa Suitable 4.7 2.6 N.D Amygdalin 2.400 < 0.10 N.D B 3 Aucklandia lappa Decne. radix 2.6 4.2 China Suitable 3.4 8.4 - Costunolide+Dehydrocostus lactone 7.130 < 0.13 N.D B 4 Zingiber officinale Roscoe. Rhizoma Recens 1.5 2.4 Korea Suitable 1.0 1.0 - - - < 0.0 - A 5 Curcuma zedoaria (Berg.) Rosc Rhizoma 3.8 6.1 China Suitable 4.8 2.6 - - - < 4.92 N.D B 6 Lindera strichnifolia Fernandez-Villar Radix 5.6 9.0 China Suitable 0.6 11.4 - - < 1.16 N.D B 7 Cinnamomum cassia Blume Cortex Interior 2.6 4.2 Vietnam Suitable 2.0 4.0 - Cinnamic acid 0.100 < 1.00 N.D B 8 Paeonia lactiflora Pallas Radix Alba 3.8 6.1 Korea Suitable 2.8 3.0 - Albiflorin+Paeoniflorin 3.800 < 0.0 N.D A 9 Citrus aurantium Linné Fructus Immaturus 3.9 6.3 China Suitable 2.6 5.4 - - - < 0.0 N.D A 10 Citrus unshiu Markovich Pericarpium 3.8 6.1 Korea Suitable 2.2 7.8 - Hesperidin 14.300 < 0.0 N.D B 11 Atractylodes lancea De Candlle Rhizoma 5.6 9.0 China Suitable 5.2 6.4 - - - < 0.00 N.D A 12 Cnidium officinale Makino Rhizoma 3.8 6.1 Korea Suitable 4.4 2.0 - - - < 0.2 N.D A 13 Cyperus rotundus Linné Rhizoma 11.3 18.1 Korea Suitable 2.5 3.0 - - - < 0.0 N.D A 14 Corydalis ternata Nakai Tuber 3.8 6.1 China Suitable 2.2 6.0 N.D Coptisine+Tetrahydropalmatine 0.410 < 0.01 N.D A 15 Carthamus tinctorius Linn Flos 2.6 4.2 China Suitable 7.1 15.2 N.D Kaempferol 0.094 < 0.30 N.D A - 62.3 100.0 - N Number, N.D Non-detection, Suitable Suitability with Korean herbal standards Heavy metals: Sum of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) Pesticide residues: Total DDT, dieldrin, methoxychlor, total BHC, azocyclotin, azoxystrobin, aldrin, endosulfan, endrin, tebuconazole, pendimethalin, fenpropathrin, sethoxydim, fluazifop-butyl, etc Company: Hando pharmaceuticals (A, Gyeonggi Province, Korea), Hyeongyoul pharmaceuticals (B, Gyeonggi Province, Korea) Composition and analysis data of Hyunburikyung-tang N Number, N.D Non-detection, Suitable Suitability with Korean herbal standards Heavy metals: Sum of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) Pesticide residues: Total DDT, dieldrin, methoxychlor, total BHC, azocyclotin, azoxystrobin, aldrin, endosulfan, endrin, tebuconazole, pendimethalin, fenpropathrin, sethoxydim, fluazifop-butyl, etc Company: Hando pharmaceuticals (A, Gyeonggi Province, Korea), Hyeongyoul pharmaceuticals (B, Gyeonggi Province, Korea) The preparation involved the addition of distilled water at 10 times the weight of the herbal materials, extraction at 115 °C for 4 h, and filtration through a nonwoven material. The extract was subsequently concentrated to 10°Bx at 120 °C and sterilized at 100 °C for 30 min. The sterile extract was spray-dried into powder form, dissolved at appropriate concentrations, and utilized in the experiments. The specific pathogen-free animal facility used in this study was maintained at a temperature of 22 ± 3 °C, humidity of 30–70%, noise < 60 dB, ammonia concentration < 20 ppm, and a 12-hour light-dark cycle with 150–300 lx. The room received over 10 air changes per h of HEPA-filtered air and was cleaned with a disinfectant daily. Rats were housed individually in stainless steel cages for acute toxicity assessments, whereas mice for the in vivo micronucleus test were housed five per group in polycarbonate cages, with cages and water bottles replaced at least twice weekly. The rodents had free access to solid feed (PMI LabDiet ® 5053) and UV-disinfected, reverse osmosis-purified water in polycarbonate bottles. The acute toxicity assessment was authorized by the Institutional Animal Care and Use Committee (IACUC) of the Korean Medicine Non-clinical Study Center (Approval number: NIKOM-2022-07) and the steering committee of the Good Laboratory Practice (Approval study number: N22006 ). The acute toxicity assessment was conducted in accordance with the Toxicity Test Guidelines for Drugs, etc. (Notification No. 2022-18) issued by the Ministry of Food and Drug Safety (MFDS) of the Republic of Korea [ 32 ]. The main outcomes specified in the guideline are as follows: approximate lethal dose, observation records of general symptoms, gross necropsy findings after the observation period, and histopathological examination of organs and tissues showing gross abnormalities. Six-week-old Sprague-Dawley (SD) rats from Orient Bio (Gyeonggi Province, Korea) were acclimatized for five days. To determine the approximate lethal dose and observe toxic responses according to the Toxicity Test Guidelines for Drugs, etc. of Republic of Korea [ 32 ], the highest dose was set at 2,500 mg/kg based on various references [ 33 – 35 ]. Five animals per group were used to enable statistical interpretation of the experimental results, and they were randomly assigned to the following groups: negative control (distilled water), low-dose (625 mg/kg), medium-dose (1,250 mg/kg), and high-dose (2,500 mg/kg) groups (5 males and 5 females per group, total 40 animals). Hyunburikyung-tang was administered as a single dose after a 12 h fast, excluding water, using a sonde attached to a syringe. Feed was reintroduced 4 h post-administration. General symptoms were assessed shortly after dosing and daily thereafter until autopsy, The detailed observation items were based on the methods described by Hwang et al. [ 36 ]. Body weights were recorded on the day of administration and days 1, 3, 7, and 14 post-administration. Surviving animals were humanely euthanized with CO 2 , followed by a thorough autopsy to examine organs. The bacterial reverse mutation test was conducted following the OECD TG 471 guidelines using five strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA (MOLTOX, USA). Groups included a negative control, a positive control, and test substance treatment groups (61.7, 185.2, 555.6, 1,666.7, and 5,000 µg/plate). Three plates per group or concentration were used. The materials included 0.1 mL of the test substance or solvent, S9 mix for metabolic activation or phosphate buffer for no metabolic activation, 0.5 mL of strain culture, and 2 mL of top agar. These materials were mixed and incubated at 120 rpm and 37 °C for 20 min, plated on minimal glucose agar (MGA) plates, and incubated for 48 h. Colonies were counted visually after the completion of the incubation period. Chromosomal aberration tests were conducted following OECD TG 473 guidelines using Chinese hamster lung (CHL/IU) cells. Cells were plated at a density of 5 × 10 5 cells/mL, cultured overnight in a 60 mm dish, and treated in duplicates for either 6 h (with or without metabolic activation) or 24 h (without metabolic activation). Following the 6 h treatment, cells were replenished with a normal medium devoid of the test substance and further incubated for 18 h. Two hours prior to harvesting, cells were treated with colcemid for 2 h and collected in a KCl solution. After fixation with an acetic acid/methanol solution (1:3), cells were stained with a 5% Giemsa solution. The number of cells exhibiting chromosomal aberrations per 300 cells was determined using two slide glasses obtained from each cell culture dish. The micronucleus test was conducted following OECD TG 474 guidelines and approved by the IACUC of the Korean Medicine Non-clinical Study Center (Approval number: NIKOM-2022-29) and the steering committee of the Good Laboratory Practice (Approval study number: T22043 ). Six-week-old Institute of Cancer Research (ICR) mice were procured from Orient Bio (Gyeonggi Province, Korea) and acclimatized for five days. Five male mice (5 per group, total 25 animals) were allocated to the concurrent negative control group (distilled water without the test substance), concurrent positive control group (Mitomycin C), low-dose group (500 mg/kg), medium-dose group (1,000 mg/kg), and high-dose group (2,000 mg/kg). Hyunburikyung-tang was orally administered daily for two days. The concurrent positive control group received intraperitoneal injections of mitomycin C (2 mg/kg) 24 h before euthanasia. Animals were euthanized 24 h after the last administration, and bone marrow was collected by perfusion of the femur with fetal bovine serum. Cells were plated onto glass slides and stained with a 5% Giemsa solution following methanol fixation. The number of micronucleated polychromatic erythrocytes (mnPCE) per 4,000 PCE was determined using two slides per mouse. The humane endpoints should be considered when experimental animals experience severe suffering and pain or are imminent at death, and detailed symptoms are as follows. (1) When unable to consume water or food, or recumbency, convulsions, or tremors. (2) When observed continuously pain, vocalization or self-mutilation. Weight measurement results in the acute toxicity assessment were expressed as means and standard deviations. Statistical analysis between groups was conducted using SPSS Statistics 25 (IBM Corp., Armonk, N.Y., USA). The significance of body weight differences among groups was assessed via one-way analysis of variance (ANOVA) with Dunnett’s test ( p  < 0.05). Fisher’s exact test was utilized in the chromosomal aberration test. Dunnett’s test was used in the micronucleus test to compare the frequency of micronucleus formation between the negative control group and the Hyunburikyung-tang treated or positive control groups. The Cochran–Armitage test was used to assess the concentration-response relationship among the groups, excluding the negative and positive control groups.

Dysmenorrhea is a prevalent gynecological issue, affecting approximately 90% of menstruating women [ 1 ]. Studies indicate that 78% of young women experience menstrual pain, which negatively impacts their quality of life [ 2 , 3 ]. The resultant decrease in academic or work efficiency among women due to menstrual pain results in socioeconomic loss. Modern medical research suggests that dysmenorrhea results from reduced intrauterine blood flow caused by uterine contractions. Studies have shown an increased ratio of prostaglandin F2a to prostaglandin E2 (PGE 2 ) and vasopressin in the plasma of women with dysmenorrhea, indicating that these substances may be linked to uterine contractions and menstrual pain. The use of anti-prostaglandins alleviates pain in approximately 80% of affected women, highlighting the role of prostaglandins and vasopressin in dysmenorrhea [ 4 – 6 ]. Several medications have been formulated to mitigate menstrual pain, e.g., prostaglandin synthase inhibitors, which offer analgesic and anti-inflammatory effects. However, these are not effective for all women (20–25%), and their prolonged use can result in side effects, such as liver, kidney, and gastrointestinal complications [ 7 ]. Oral contraceptives are not an option for women seeking pregnancy and may cause side effects including nausea, vomiting, and swelling with long-term use [ 8 ]. Gonadotropin-releasing hormone agonists may induce menopausal symptoms, reduce bone mineral density, and elevate the risk of coronary artery disease [ 9 ]. Among tocolytics, nitroglycerin is weakly effective for suppressing menstrual pain and causes side effects such as headaches. Nifedipine can effectively suppress menstrual pain but may lead to side effects such as facial redness, tachycardia, and pain [ 10 , 11 ]. Hyunburikyung-tang, a traditional prescription in Korea, suppresses PGE 2 , nitric oxide and pro-inflammatory cytokines in cell models, and combined administration with piroxicam, a drug used for menstrual pain, is reported to increase the anti-inflammatory effect [ 12 ]. Moreover, various studies have been conducted on the medicinal plants comprising Hyunburikyung-tang in relation to women’s health. The root of Angelica gigas Nakai has been shown to alleviate pain and inflammatory responses [ 13 , 14 ]. One of its primary constituents, decursin, has been reported to inhibit Nuclear factor kappa B-mediated inflammatory responses [ 15 ], while decursinol enhances endometrial receptivity during embryo implantation, thereby potentially improving infertility [ 16 ]. Paeoniflorin, a major component of Paeonia lactiflora Pallas, not only exhibits analgesic properties through interaction with NMDA receptors but is also known to inhibit COX-2, which is related to inflammation and pain, in various experimental models [ 17 , 18 ]. The rhizome of Cnidium officinale Makino has anti-osteoclastogenesis, downregulates MMP-9 inhibiting NFATc1/c-Fos signaling pathway [ 19 ]. MMP-9 is a major factor in endometriosis and is closely associated with pelvic pain and dysmenorrhea [ 20 ]. In clinical settings, Hyunburikyung-tang is widely recognized in traditional Korean medicine as a treatment for dysmenorrhea and menstrual disorders. It improves visual analog scale (VAS) scores and multidimensional verbal rating scale (MVRS) scores and is commonly used in Korea [ 21 – 24 ]. The mechanisms of Hyunburikyung-tang on dysmenorrhea are not clear, but the medicinal plants it comprises possess anti-inflammatory and analgesic properties, suggesting potential efficacy in treating dysmenorrhea. However, Hyunburikyung-tang is potentially dangerous because it contains peach seeds ( Prunus persica Batsch), which are known to cause deformities, and safflower (Carthamus tinctorius Linn), which is known to be toxic to the testes and embryos [ 25 – 27 ]. Multiple studies have demonstrated that genotoxicity and teratogenicity are interconnected. Various parental factors, such as lifestyle habits, environmental toxin exposure, smoking, and alcohol consumption, can induce genotoxic effects in reproductive cells, potentially leading to congenital abnormalities in offspring [ 28 – 31 ]. Therefore, we aimed to determine whether Hyunburikyung-tang causes genotoxicity or acute toxicity and provide safety information.

In this study, the acute toxicity and genotoxicity of Hyunburikyung-tang were evaluated using bacteria, cells, and rodents. Previous toxicity studies have reported side effects associated with medicinal plants or ingredients found in Hyunburikyung-tang. Some medicinal plants are known to induce side effects such as teratogenicity, dyspnea, and arrhythmia in non-clinical studies and acute headaches, weakness, and dry mouth in clinical studies [ 37 – 42 ]. Peach seeds contain components such as tocopherols, cyanogenic glycosides, and carotenoids, which are experimentally reported to have anti-inflammatory, anti-oxidative, and anti-cancer effects. However, peach seeds also contain amygdalin (laetrile), which is known to cause toxicity [ 43 ]. Oral administration of amygdalin to humans causes cyanide poisoning, and combined administration with vitamin C decreases cysteine ​​levels in the body and inhibits the detoxification effect of amygdalin [ 44 ]. Moreover, teratogenic effects have been reported with oral but not intravenous administration in animal studies [ 45 ]. Malformations can result from various mechanisms, including oxidative stress, endocrine disruption, or enzyme-mediated induction. Genotoxicity is interconnected to teratogenicity and can arise from various mechanisms including oxidative stress and enzyme-mediated activation [ 46 – 48 ]. Multiple non-clinical studies have demonstrated the potential effect of genotoxicity induced in the parent generation on offspring; therefore, evaluating the safety, including genotoxicity, of consumed drugs and foods is crucial [ 49 – 53 ]. Amygdalin is hydrolyzed by beta-glucuronidase and converted to cyanide in the small intestine, and a 500 mg oral dose is reported to contain up to 30 mg of cyanide. Cyanide toxicity causes cell death by interfering with mitochondrial oxygen utilization. Cyanide binds to the ferric ion of cytochrome oxidase, inhibiting electron transport and oxidation mechanisms and causing hypoxia and lactic acidosis [ 54 ]. Hypoxia and lactic acidosis are related to genotoxicity. Reactive oxygen species caused by hypoxia can cause genotoxicity, and lactic acidosis, which is a tumor microenvironment factor, causes instability of mitotic chromosomes with concomitant glucose deficiency, resulting in centrosome amplification, micronuclei, or aneuploidy [ 55 , 56 ]. The bacterial reverse mutation test, in vitro chromosome aberration test, and in vivo micronucleus test are standard methods for evaluating genotoxicity. The bacterial reverse mutation test detects point mutations, including DNA base or base pair additions, deletions, or substitutions [ 57 ]. Such mutations are implicated in numerous genetic diseases, and evidence suggests that mutations in somatic cell tumor suppressor genes and oncogenes contribute to tumor formation in animals and humans [ 58 ]. Chromosomal abnormalities and micronuclei are commonly associated with cancer development [ 59 , 60 ]. Benzo[a]pyrene is hydrolyzed by beta-glucuronidase and causes genotoxicity only if metabolic activation occurs [ 61 ]. Amygdalin, which is similar to benzo[a]pyrene, is hydrolyzed by beta-glucuronidase and causes cyanide toxicity. Standard testing methods for genotoxicity evaluation were performed in this study, with no mutagenesis or genotoxicity observed with the administration of metabolic-activated Hyunburikyung-tang. Safflower ( Carthamus tinctorius Linn) affects developmental stages in mice and causes structural abnormalities in the placenta; however, the mechanisms of these effects remain unknown [ 62 ]. No genotoxicity was induced by treatment with Hyunburikyung-tang; however, a chromosomal aberration test confirmed that the cell toxicity increased with metabolic activation. Zebrafish models exposed to safflower extract were reported to have increased levels of antioxidant-related proteins and malonaldehyde (MDA) [ 63 ]. Malondialdehyde is a byproduct of lipid oxidation and an indicator of oxidative stress. Oxidative stress damages the syncytial trophoblast membrane, weakens placental function, and adversely affects uterine and embryonic development [ 63 – 65 ]. Hyunburikyung-tang containing safflower did not induce genotoxicity in this study; nevertheless, further studies are required to determine whether it can induce oxidative stress with metabolic activation. An acute toxicity assessment was conducted to determine the lethal dose and potential side effects following a single dose [ 66 ]. In this study, several endpoints were measured to confirm the genotoxicity and acute toxicity of Hyunburikyung-tang, and no toxic reactions were observed. Based on our findings, the approximate lethal dose of Hyunburikyung-tang was expected to exceed 2,500 mg/kg in both male and female rats. Furthermore, Hyunburikyung-tang did not appear to induce genotoxicity under these test conditions. A limitation of this study is that during the acute toxicity assessment, major organs such as the heart, liver, and kidneys were examined only visually, without histopathological or clinical pathology evaluations. Therefore, toxic responses at the tissue or blood level could not be determined. Although Hyunburikyung-tang did not induce genotoxicity in this study, potential effects on embryonic cells or fetal development remain unknown. Therefore, further studies are needed, including reproductive toxicity assessments and investigations of toxicological mechanisms using embryonic cells.

In summary, Hyunburikyung-tang, a traditional medical prescription commonly used clinically for dysmenorrhea due to its anti-inflammatory effects, is potentially dangerous because it contains safflower and peach seeds, which are known to cause deformities. Hyunburikyung-tang did not cause acute toxicity or genotoxicity under the test conditions; however, increased cytotoxicity was observed with metabolic activation in an in vitro chromosomal aberration test. We anticipate that the findings from this study will serve as fundamental data for subsequent toxicity tests and provide non-clinical safety information for Hyunburikyung-tang.