1. Materials

1.1 Subject : FRC001.
1.2 Experimental Animals :

1.2.1 Mouse , Kunming species , healthy, 2~3 months old, male or female

1.2.2 Rat, Wistar species, healthy, 3~4 months old, 180~220g, male or female . Mice and rats were provided by Experimental Animal Center of Tongji University of Medical Science.

1.3 Experimental Strain: Histidine dystrophic of Salmonella typhimurium TA98, TA100 and TA102 were provided by Ames Laboratory of California.

2. Methods:
2.1 Acute toxicity test: According to Horn's method. Fifty mice were randomly divided into five groups :
one control group and four dose groups ( 21.5, 10.0, 4.64 and 2.15g/kg. bw). Each group had ten rats with half male and half female. FRC001 was diluted by distilled water. The rats were fed with drugs endogastricly, one time per day for successive two weeks.

2.2 Increasing dose method : Thirty-two rats were randomly divided into two groups : control and experiment group. Each group had 16 rats with half male and half female. The rats of experiment group were fed with FRC001 endogastricly beginning at 3.0g/kg. bw, then increasing the dosage daily to 5.26 LD50 for successive 30 days. The control group were fed with normal saline the same way as experiment group.

2.3 Micronucleus test : Fifty mice were randomly divided into five groups : one negative control group , one positive group and three dose groups (4.3, 10.8, 21.5g/kg. bw). Each group had ten mice with half male and half female. The negative group were fed with distilled water 5g/kg. bw endogastricly , the positive group were injected with cyclophosphamide 100mg/kg. bw intraperitoneally and they were killed after 30 hrs. The other groups were fed drugs endogastricly one time daily for successive four days, then killed at the fifth day. The sterna were separated and crushed to prepare the smear of bone marrow polychromatic erythrocytes. The polychromatic erythrocyte and micronucleus cell were calculated.

2.4 Sperm aberration test : Forty male mice were randomly divided into five groups: negative group, positive group and three drug groups (4.3 , 10.8, 21.5 g/kg. bw). Each group had eight mice. The mice of negative group were fed with distilled water 21.5g/kg. bw (I.G.), the mice of positive group were fed with cyclophosphamide 21.5g/kg. bw, and the mice of drug groups were fed with FRC001. All of them were fed intragastricly one time daily for successive five days, and killed after 30 days of normal feeding. The epididymides were separated , put into five ml normal saline , cut into pieces, then put into 36.5 °C water bath for 15 min. The total sperms and active sperms in 200 sperms were counted. The sperm aberration rate was calculated by staining smear.

2.5 Ames test: FRC001 was diluted by DMSO into five concentrations: 1:50, 1:200, 1:500, 1:2000 and 1:5000. DMSO was the negative control subject, while Dexon and 2,7-diaminofluorene were the positive control subjects. Dexon was used as direct mutagen and 2,7-diaminofluorene was used as indirect mutagen. To see the detail in reference (4).

1. Acute toxicity test of FRC001:

To do the test according to the method of acute toxicity test. After two week's observation, all animals fed with FRC001 had no abnormal manifestation, were normal food-intake and water-intake , activity. The body weight has no obvious change. No animal died during test period. LD50 of FRC001 by oral administration is more than 21.5g/kg.bw.

2.Cumulative toxicity test of Edfrnn

 

2.2 The influence on main organ coefficients of rats ( Table 1)

Group Number	Organ Coefficients ( X ± SD)
		Heart	Liver	Spleen	Lung	Kidney
Control   Experiment	16	0.45±0.04	3.96±0.60	0.38±0.07	0.96±0.30	0.85±0.08
	6	0.44±0.05	4.14±0.39	0.38±0.07	1.03±0.26	0.88±0.06

Table 1. The influence of FRC001 on Main Organ Coefficients of Wistar Rats

Difference between the two groups was not significant , P>0.05

According to Table 1 and Figure 1,2, FRC001 had no obvious effects on the body weight and the increase value of average body weight with successive 30 days of intragarstric feeding. The food-intake and activity were normal. No toxic reaction was found. Organs were normal by pathological examination. The difference between control group and experimental group was not significant (P>0.05). The cumulative dose was up to 5.26 LD50 in 30 days and no animal died. These results showed that FRC001 had no obvious cumulative toxicity.

3.The influence of FRC001 on bone marrow cell micronucleusrate of mice (Figure 3, 4)

Generally in the micronucleus test, the micronucleus rate of cyclophosphamide (positive control group) is 0~3%, the micronucleus rate of negative control group is lower than 3%. According to Figure 3,4, the micronucleus rate of positive control group and negative control group were 30.3% and 1.4%, respectively. The results showed that this test was dependable. The micronucleus rate of low, middle and high dose of FRC001 were 4.5%, 6.6%, 9.0%, respectively, The difference between Edfrnn groups and negative control group were significant. These findings showed that FRC001 had mutagenic action.

4. The influence of FRC001 on sperm aberration of mice (Figure 5 and Table 2) Table 2 The influence of FRC001 on Sperm Aberration Rate of Mice

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Groups	Number	The Number of Sperm	No. of Active Sperm	Sperm Aberration Rate
Negative	8	237.1	158.9	9.3
4.3g/kg.bw	8	156.3	109.7	11.3
10.8g/kg.bw	8	253.0	177.5	19.4
21.5g/kg.bw	8	212.0	139.2	27.9
Positive	8	378.9	263.1	55.1

Figure 5 The Influence of FRC001 on Sperm Aberration Rate of Mice

According to Table 2, the sperm aberratin rate of three doses of FRC001 and positive control group were higher than that of negative control group. Generally, the sperm aberration rate doubles that of negative control group shows that the sperm aberration rate is positive. In this test, the sperm aberration rate of positive control group was 6 times of negative control group, while that of low middle and high dose of FRC001 were 1.2, 2.1 and 3 times of negative control group. The difference were significant (P<0.05, P<0.01). These findings showed that FRC001 had some harmful and toxic action on the heredity of germ cells.

5. Ames test of FRC001 (Table 3)

	MR (Rt/Rc)
Concentration of    FRC001(ug)	TA97		TA98		TA100		TA102
	+S-9	-S-9	+S-9	-S-9	+S-9	-S-9	+S-9	-S-9
1/50  >	1.02	0.80	0.78	1.26	0.96	1.04	0.94	0.90
1/200	1.18	0.85	1.00	1.24	0.91	1.14	1.16	1.00
1/500	1.02	0.86	1.12	1.04	1.00	1.09	1.12	0.86
1/2000	1.08	1.02	1.25	1.13	1.02	1.13	1.29	1.26
1/5000	1.11	1.03	1.06	1.10	1.07	1.08	1.18	1.13
Dexon(50ug/)	-	11.30	-	15.70	-	4.33	-	3.55
2-AF	3.35	-	5.32	-	5.07	-	2.10	-
DMSO	-	1.14	-	0.74	-	0.96	-	0.84

Table 3 The Results of Ames Test of FRC001

According to Table 3, using incorporatin method to do Ames test of FRC001, the ratio (MR) of mutagenic colony (Rt) and spontaneous reverse mutation colony (Rc) were lower than two. The results showed that FRC001 had no obvious mutagenicaction. The MR of Dexon and 2,7-diaminofluorene were higher than two showed that Ames test was positive and they had mutagenic action. The MR of DMSO was lower than two showed that it had no mutagenic action. These results also showed the test was dependable. In a conclusion, in the toxicology test of FRC001, the acute toxicity test, cumulative toxicity test and Ames test were normal while the micronucleus test and sperm aberration test were abnormal. It is generally thought that the occurance of micronucleus in interphase caused by mutagen, i.e, the micronucleus consists of the pieces of chromosome after the acting of mutagen. The micronucleus rate is closely related to chromosomal aberration. FRC001 induced the increase of sperm aberration rate of mice showed that it had potential damage action on the heredity of male germ cells. According to the estimation procedure and method of toxicology of food and drug issued by government; the subject can not be used as food and drug if there are two positive in heredity toxicity test and it has apparent toxicity in short term feeding test; if the short term feeding test is suspicious positive, the application is determined by the importance and the potential intake of the subject. The positive of micronucleus test and sperm aberration test of FRC001 may be related to its ingredients, the interaction of ingredients and /or the formation of new products during the preparation of FRC001. We suggest that the application departments should think over the mutagensis of FRC001 while they can try to use it in clinic. Whether the toxicity of FRC001 can be decreased by controlling the dose or small amount and repeated administration is awaiting further study.

[1] Qian Jiaqing, Song Ruikun, The Materia Medica and Toxicology Basis. 1st Edition , The People's Medical Publishing House, Beijing, 1986,258

[2] Ding Kexiang, Song Ruikun, The Acute Toxicity Test of SOD Atomic Energy Press , Beijing, 1991:84

[3] Ding Kexiang, Yao Shuren, Song Ruikun, The Cumulative Toxicity Test of SOD Complex Enzyme and Its Preparation, Application Study on SOD, Atomic Energy Press, Beijing, 1991,94

[4] Qian Jiaqing, Song Ruikun, Pharmacology and Toxicology Basis, The People's Medical Publishing House, Beijing, 1986,225

[5] Yu Shouyang, Liu Yugu, Animal Experiment in the Food Toxicologic study, The People's Medical Publishing House, Beijing,1981:379~383

[6] Cai Hong Dao, Wan Jialing, Enviromental Microorganism, Tongji University of Medical Science Press, Wuhan, 1987:269~275

[7] Ding Kexiang, Ames test of SOD Complex Enzyme and its Preparation, Application Study on SOD, Atomic Energy Press, Beijing, 109~113

[8] Zhu Honghong, Huang Xingshu, PCD Method and Its Application in the study of the Influence of Exogenous Hormone on PCD of Testicular Cells. Canceration, Aberration Mutation 1997,9(1):23

[9] Yang Enpu, Yang Cangzhen, The Aberration Effects of Lead and X-ray on rats. Canceration Aberration Mutation 1996,8(6): 18

[10] Zhang Chen, Yao Hua, Ling Bing, et al. The Effects of Arsenic on Reproduction and Filial Generation Development of Rats. Canceration. Aberratin Mutation, 1997,9(1):32

[11] He Qingyu, Yin Muquan, Lu Dun, et al. Study of the Aberration of on Rats Canceration Aberration Mutation, 1997,9(1)38

[12] Liu Yugu, Health Toxicology Basis, The People's Medical Publishing House, Beijing, 1996; 56

[13] Maron D. M. et al. Revised methods for the Salmonella Mutgenicity Test. Mutation Res, 1983;113:173-190

[14] Blakey BR. Et al. The Effect of Methylmerury Tetrathyl lead and sodium arsente on the humoral immune respone in mice. Toxicol Appl Pharmacol. 1980,52(2):245

[15] Koller LD. Chemical induced immunomodulation. J Am Vet Med Assoc. 1982, 181(10): 1102

[16] The Health Ministry of China, The Estimation Procedure and Method of Toxicology and Safety of Food, 1994,8:10

[17] Eudak E. Jacobs PA, Yangangimachi R, et al. Direct analysis of the chromosome constitution of human spermatozoa. Nature, 1978; 274:911

[18] Martin RH, Hildebrand K, Yamamoto J, et al An increased frequency of human sperm chromosomal abnormalities after radiotheraphy. Mutat res,1986; 14:219

[19] Brandriff B, Gordon LA, Sharlip I, et al. Sperm chromosomal analysis in a survivor of seminoma and associated radiotheraphy. Environ Mol Mutagen, 1987;9(Suppl 8):19

[20] Jenderny J, Rohrborn G. Chromosome analysis of human sperm. 1. First results with a modified method, Hum Genet, 1987;76:385

[21] Genesca A, Miro R, Caballin MR, et al. Sperm chromosome studies in individuals treated for testicular cancer. Ilum Reprod, 1990;5(3):286

[22] Kamiguchi Y, Tateno H, Shimada M, et al. X-ray induced chromosome aberrations in human spermatozoa. In: Mohri H, ed. New Horizons in Sperm Cell Research. New York: Japan Sci Soc Press, Tokyo/Gordon and Breach Sci Publ, 1987:117-123

[23] Kamiguchi Y, Tatcno H, Mikamo K. Dose-response relationship for the induction of structural chromosome aberrations in human spermatozoa after in vitro exposure to tritium ß-rays. Mutat Res. 1990;228:125

[24] Brandriff BF, Gordon LA, Ashworth LK, et al. Chromosomal aberrations induced by vitro irradiations: Comparison between human sperm and lymphocytes. Emviron Mol Mutagen, 1988;12(2)167

[25] Kamiguchi Y, Tateno H, Mikamo K. Types of structural chromosome aberrations and their incidences in human spermatozoa X-irradated in vitro. Mutat Res, 1990;228:133

[26] Yanagimachi R. Katayose H, Matauda J, et al. Stability of mammalian sperm nuclei. In:Spera G>et al, eds. Molecular and cellular biology of reproduction New York: Raven Press, 1992:157-168

[27] Lauria A, Gandolfi F. Recent advance in sperm cell mediated gene transfer. Molecular Reproduction and Development, 1993;36:255

[28] Kaufman MH, Analysis of the first cleavage division to determine the sex-ratio and incidence of chromosome anomalics at conception in the mouse, J Reprod Fertil, 1973;35:67-72

[29] Martin-Deleon PA, et al. Spontaneous heteroploidy in one-cell mouse embryes. Cytogenet Cell Genet, 1983;35:57-63

[30] Akira Nishio. Sister - chromatd exchange and chromosomal aberrations by DHAQ and related anthraquinone derivatives in Chinese hamster ovary cells. Mutat Res,1982;101:77

[31] Kaufman MH. Analysis of the first cleavage division to determine the
sex-ratio and incidence of chromosome anomalies at conception in the mouse.
J Eprod Fertil, 1973;35:67

[32] Martin-Deleon PA, et al. Spontaneous heteroploidy in one-cell mouse embryes. Cytogenet cell Genet, 1983;35:57

[33] Dunkel VC, et al. Comparative neoplastic transformation responses of Balb/3T3 cell, syrian hamster embryo cells and Rauscher Murine leukemin Virus-infected Fisher 344 rat embryo cells to chemical compounds. INCI, 1981;67:1303

[34] Tates AD, Dietrich AJJ, de Voger N, et al. Micronucleus method for detertion of meiosis micronuclei in male germ cells of mammals. Mutat Res, 1983;221:131-138

[35] Lahdetie J, Parvinen M. Meiotic micronuclei induced by X-rays in early spermatids of the rat. Mutat Res, 1981;81:103-115

[36] Oakberg EF. Duration of spermatogenesis in the mouse. Nature (London), 1957;
180:1137-1138

[37] Maron BM, et al. Revised methods for the Salmonella mutageniecity test. Mutat Res, 1983;113(3):173

[38] Matsuoka A, et al. Chromosomal aberrations tests on 29 chemicals combined with S9 mix in vitro. Mutat test, 1979:66(3):277

[39] Jones PD, et al. Efficiency of fuconazole in cryptococcal meningitis. Diagn Microbiol Infect Dis, 1989:12(4Suppl)235s

[40] Lee JW, et al. Safety and pharmmacokinetics of fluconazote in children with neoplastic diseases, Pediatr.1992:120(6):987

[41] Vicki L, et al. Review of the mutagenicity of ethylene oxide. Envirommental and molecular mutagenesis, 1990:16:85-103

[42] Ehrenberg L and Bowman Ko. Tables for determining the satistical significance of mutation. Mutat Res,1970:9:527-549

[43] Kastenbaum MA and Bowman KO. Tables for determining the statistical significance of mutation. Mutat Res. 1970:9:527-549

[44] Riberio LR, et al. Cytogenetic effects of inhaled ethylene oxide in somatic and germ cells of mice. Arch. Toxical 1987:59:332-335

[45] Tates AD, et al. Biological and chemical monitoring of occupational exposure to ethylene oxide. Mutat Res,1991,250:493-497

[46] Hogstedt B, et al. Chromosome aberrations and micronuclei in bone marrow cells and peripheral blood lymphocytes in humans exposed to ethylene oxide. Heredites, 1983:98:105-113

[47] Symons JM et al. National organics recomaissance survay for halogenated organics. Water works Assoc., 1975:67(11):634

[48] Puck TT, et al. Clone growth of mammalian cells in vitro growth characteristics of colonies from single hela cells with and without "Feeder" layer. J Exptl Med, 1956:103:273

[49] Dipaolo JA, et al. Transformation of hamster cells in vitro by polycyclic hydrocarbons without cytotoxicity. Proc Nat Acad Sci U.S.A. 1971:68(12):2958

[50] Bruce cc. Et al. Enhancement of adenovirus transformation by pretratment of hamster cells with carcinogenic polycyclic bydrocabons. Cancer Res, 1973:33:819

[51] Dipaolo JA, et al. Quantitative studies of in vitro transformation by chemical carcinogens. J Nat Cancer Inst, 1965:35:867

[52] Geoge EM, et al neoplastic transformation of human epithelial cells in vitro after exposure to chemical carcinogens. Cancer Res. 1981:41:5096

[53] Dipaoli JA, et al. In vitro transformation of syrian hamster embryo cells by diverse chemical carcinogens. Nature, 1972:235:278

[54] Maron DM, BN Ames. Revised methods for the Salmonella Mutagenicity test. Mutat Res, 1983:113:173

[55] Quillardet P. Hofnung. M, The SOS Chromotest, a colorimetric bacterial asay for genotoxins procedures. Mutat Res, 1985:147:65

[56] Memahon RE, Cline JC, Thmopson, CZ. Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens. Cancer Res. 1979:39(3):682

[57] Rudak E, Jacobs PA, Yanagimachi R, et al. Direct analysis of the chromosome constitution of human spermatozoa. Nature, 1978:274:911

[58] Martin RH. Hildebrand K, Yamamoto J, et al. An increased frequency of human sperm chromosomal abnormalities after radiotherapy. Mutat Res. 1986:174:219

[59] Brandriff B, Gordon LA, Sharlip I, et al. Sperm chromosomal analysis in a survivor of seminoma and associated radiotherapy. Environ Mol Mutagen , 1987:9(Suppl 8):19

[60] Jenderny J, Rohrborn G. chromosome analysis of human sperm. I. First results with a modified method. Hm Genet, 1987:76:385

[61] Genesca A, Miro R. Caballin MR, et al. Sperm chromosome studies in individuals treated for testicular cancer. Ilum Reprod, 1990:5(3):286

[62] Kamiguchi Y, Tateno H, Shimada M, et al. X-ray induced chromosome aberrations in human spermatozoa. In:Mohri H, ed. New Horizons in Sperm Cell Research New York :Japan Sci Soc Press. Tokyo/Gordon and Breach Sci Publ. 1987:117-123

[63] Brandriff BF. Gordon LA, Ashworth LK, et al. Chromosomal aberrations induced by vitro irradiation:L comparison between human sperm and lymphocytes. Environ Mol
Mutagen, 1988:12(2):167

[64] Kamiguchi Y. Tateno H. Mikamo K. Types of structural chromosome aberrations and their indicences inhuman sermatozoa X-irradiated in vitro. Mutat Res, 1990:228:133

[65] Kamiguchi Y, Tateno H. Mikamo K. Micronucleus test in 2-cell embryos as a simple assay system for human sperm chromosome aberrations. Mutat Res, 1991:252:297

[66] Yanagimachi R, Katayose H, Matsuda J. et al. Stability of mammalian sperm nuclei. In: Spera G et al. eds. Molecular and cellular biology of reproduction. New York; Raven Press, 1992:157-168