The Toxicity Study Of Cinnabar

1. ObjectiveTo investigate hepatoxicity, nephrotoxicity and reproductive toxicity and the dose-time-toxicity relationship by chronic toxicity test and research of Hg accumulation in blood, liver, kidney and brain, in order to provide the scientific basis for safety use of cinnabar in clinic.2.Methods2.1 Test of HgHgS was tested according to Chinese Pharmacopoeia by using of titration method, and total Hg and soluable Hg were tested by using of graphite furnace atomic absorption spectrophotometry.2.2 Acute toxicity testMaximally tolerated Dose of cinnabar (MTD) was tested by single oral administration。2.3 Chronic toxicity of cinnabar220 SD rats were randomly divided into control group and different dose groups of cinnabar (0.025, 0.05, 0.1, 0.4, 0.8 g·kg^-1·d^-1) corresponding to 1/2, 1, 2, 8, 16 times of clinic doses of cinnabar. The rats were treated with the test substances through oral administration once a day for successively 90 days. Urinary qualitative test, blood routine examination, serum chemistry measurement, and histomorphologic observation were conducted at 30, 60, 90 days.Toxic changes related to the treatment of cinnabar and no-observed adverse effect level (NOAEL) were evaluated.2.4 Hg accumulaion in blood, liver, kidney and brain30 SD rats were employed for organ Hg distribution experiment. The rat were fasted for 16 h before administration. Except that 4 rats were served as control, other rats were orally given cinnabar 0.8 g·kg^-1·d^-1, and blood, liver, kidney, brain of each rat were removed at 0.5,1,2,4,8,16,32 h respectively for Hg measurement.For organ Hg accumulaion experiment, 40 SD rats were randomly divided into control group and cinnabar 0.1, 0.4, 0.8 g·kg^-1·d^-1 groups. Cinnabar was orally administered once a day for successively 90 days, and blood, liver, kidney, brain of each rat were accumulated for Hg measurement.2.4 Effect of cinnabar on CCl₄-induced liver indury50 mice were randomly divided into control group, model CCl₄ group and cinnabar 0.04, 0.08, 0.16 g·kg^-1·d^-1 groups. Each cinnabar group was treated with oral administration of cinnabar once a day for successively 7 days. Except for control group, mice in other groups were treated with 1% CCl₄ 10ml·kg^-1 at day 4 and day 6 ip respectively, and biochemistry parameters ALT, AST, BUN and Cr were measured and histomorphological changes of liver and kidney were evaluated at 18 h after last administration.2.5 Reproductive toxicity studyExposure of cinnabar to mice in the last 2/3 period of pregancy: Female mice were placed with male mice (2 female and 1 male in one cage) over night for mating, and semen embolus found in the next morning was considered as successive mating. The successive mating mice were divided into control group, and cinnabar 0.08, 0.4, 4 g·kg^-1·d^-1 groups, 20 pregnant mice for each group, and the mice in cinnabar groups were received oral administration of different doses of cinnabar at 6-19 days post- gestation (G6-19d)。Exposure of cinnabar to mice from 14 days before pregnancy until full lenth of gestation (P14d-G19d): 80 femal mice were divided into control group, and cinnabar 0.08, 0.4, 4 g·kg^-1·d^-1 groups. The mice in cinnabar groups were received oral administration of different doses of cinnabar for 14 days before mating. After 14 day's administration, female mice of all groups were placed with male mice (2 female and 1 male in one cage) over night for mating, and the treatment of cinnabar was continued until the end of gestation.The same examinations were conducted in two experiments.Toxicity reaction and abortion of mother mice were recorded. The number of alive fetals, absorbed embryos and dead fetals was counted at G20d at the moment of caesarean birth. 1/3 fetals of each litter were treated in Bouin liquid for viscera examination, and other 2/3 fetals were dyed by using alizarin dying process for skeleton examination.3.Results3.1 HgS, total Hg and absoluble Hg contents of cinnabar were 98.1%, 87% and 21.5μg·g-1 respectively.3.2 Acute toxicity of cinnabar MTD of cinnabar with oral administration was 24 g·kg^-1 and this dose was about 300 times of clinic dose(corresponding to 516μg·kg^-1 absoluble Hg) and no obvious adverse effect was showed at this dose. The results manifested that single dose of cinnabar was safe.3.3 Reapeated-dose toxicity of cinnabar and Hg accumulation in blood, liver,kidney and brainCinnabar can cause kidney pathological changes at 0.4 g·kg^-1·d^-1 dose level when it was used for successive 30 days (accumulative intake of soluable Hg 258μg·kg^-1),but the dose to cause liver toxicity was doubled to 0.8 g·kg^-1·d^-1 (accumulative intake of soluable Hg 516μg·kg-1) for 30 day administration. When administration time was reached or longer than 60 days, hepatoxicity and nephrotoxicity occurred even at the lower dose of cinnabar (0.1 g·kg^-1·d^-1). Except for liver and kidney, other organs including heart, lung, brain, speen etc. did not show significant phathological changes. The results indicated that kidney was more sensitive to cinnabar than liver. From the result in Hg accumulation experiment,Hg accumulated in kidney was much higher than that in liver, which can explain why the same dose of cinnabar caused nephrotoxicity earlier than hepotoxicity. Hg accumulation in brain was also higher than that in liver, but there was no pathological changes found in brain, and it should have a further study in future. The order of Hg accumulation was: kidney>brain>liver>blood.Based on repeated dose toxicity study, NOAELs were 0.1 g·kg^-1·d^-1,0.05g·kg^-1·d^-1)respectively for 30 day and 90 day treatment, and those were approximately accumulative intake of absoluble Hg 64.5μg·kg-1and 96.76μg·kg-1. Thus, for safety use of cinnabar, the acceptable daily intake (ADI) of cinnabar was 0.0009～0.0017 g·kg^-1·d^-1 , namely daily dose 0.05～0.1 g for a 60kg person. Considering the difference of drug sensitivity and lifecircle between human and rats, we suggest that cinnabar which contains absoluble Hg≤21μg·g^-1 should be used for no longer than 2 weeks at daily dose 0.05-0.1 g.3.4 Effect of cinnabar on CCl₄-induced liver injuryOn CCl₄-induced liver injury mouse model, cinnabar did not aggravate the extent CCl₄-induced liver injury, but on the contrary,3.5 Reproductive toxicity of cinnabarWhen pregnant mice orally took cinnabar at doses of 0.08,0.4,4.0 g·kg^-1·d^-1 from gestation 6d to 19d, mother mice and fetals showed no toxic effect even though the highest dose of cinnabar was about 50 times of clinic dose. However, if female mice were given cinnabar at doses of 0.08,0.4,4.0 g·kg^-1·d^-1 from 14 days before mating until full lenth of gestation , all dose groups can cause fetal skeleton abnormality in a dose dependent manner. The results indicated that exposure to cinnabar prior impregnation and at the early phase of gestation can be more sensitive for embryo abnormality, but middle and late phase of gestation was comparatively less sensitive to cinnabar.4.Conclusion- Single dose of cinnabar is safe.- Chronic use of cinnabar can cause Hg accumulation in kidney and liver, and thus resulting in kidney and liver pathological change, so the dose and application time should be limited. The suggestion for cinnabar application was: for cinnabar with absoluble Hg≤21μg·g^-1, the daily dose should be restricted at 0.05-0.1 g, and it should used no more than 2 weeks at above doses.- Cinnabar can cause repeoductive toxicity, especially when it is used prior impregnation and at the early phase of gestation. So it is suggested that cinnabar should be forbidden during gestation and it should be also forbidden for at least 2 month before gestation.- On CCl₄-induced liver injury mouse model, cinnabar did not aggravate the extent CCl₄-induced liver injury, but on the contrary,- There is accumulative effect in kidney, brain and liver, obviously in kidney and brain. The toxicity in nerve system is worth studying in the future.