Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies.

Green tea extract and its principal active ingredient, epigallocatechin gallate (EGCG), are gaining attention and increased usage due to their healthful properties. Despite the increasing demand for these products, few studies have examined their safety. The toxicity of purified green tea extracts containing high concentrations of EGCG have been evaluated in a series of studies in order to define the safety of Teavigo, a high-concentration EGCG extract produced by the same novel method. Topical EGCG preparations caused minor dermal irritation in rats and guinea pigs, but not rabbits, and was a moderate dermal sensitizing agent in the guinea pig maximization test. A rabbit eye irritation test produced a strong enough response to not warrant any further testing in this assay. An oral dose delivering 2000 mg EGCG preparation/kg was lethal to rats; whereas, a dose of 200 mg EGCG/kg induced no toxicity. The dietary administration of EGCG preparation to rats for 13 weeks was not toxic at doses up to 500 mg/kg/day. Similarly, no adverse effects were noted when 500 mg EGCG preparation/kg/day was administered to pre-fed dogs in divided doses. This dose caused morbidity when administered to fasted dogs as a single bolus dose, although this model was considered an unrealistic comparison to the human condition. From these studies a no-observed adverse effect level of 500 mg EGCG preparation/kg/day was established.     

Acute, subchronic and genotoxicity studies conducted with Oligonol, an oligomerized polyphenol formulated from lychee and green tea extracts

Oligonol is a phenolic product derived from lychee fruit extract and green tea extract, containing catechin-type monomers and oligomers of proanthocyanidins, produced by a manufacturing process which converts polyphenol polymers into oligomers. The safety of Oligonol was assessed in acute and subchronic studies and genotoxicity assays. In a single dose acute study of Oligonol, male and female rats were administered 2000mg/kg body weight (bw) Oligonol in water by gavage. Oligonol caused no adverse effects and body weight gain and food consumption were within normal range, thus the LD(50) of Oligonol was determined to be greater than 2000mg/kg. A 90 day subchronic study (100, 300 and 1000mg/kgbw/day, oral gavage) in male and female rats reported no significant adverse effects in food consumption, body weight, mortality, clinical chemistry, haematology, gross pathology and histopathology. Similarly, no adverse effects were observed in mice fed diets providing 2, 20 or 200mg/kgbw Oligonol or 200mg/kgbw lychee polyphenol for 90 days. Oligonol did not show any potential to induce gene mutations in reverse mutation tests using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA strains. Oligonol did not induce chromosomal aberrations in cultured Chinese hamster lung cells, but it showed increased polyploidy. In a micronucleus assay in mice, Oligonol did not induce any micronuclei or suppress bone marrow, indicating it does not cause chromosome aberrations. The results from these safety studies and previous reports support the safety of Oligonol for human consumption.     

MNT and Muta™Mouse studies to define the in vivo dose response relations of the genotoxicity of EMS and ENU

Although there are a multitude of in vitro and in vivo studies on the genotoxic activity of EMS, no lifetime carcinogenicity studies, repeat dose mutation data or exposure analysis are available to serve as a solid basis for risk assessment for human exposure cases. The present studies were undertaken to investigate whether a threshold for mutagenic and clastogenic activity in vivo could be established, using the bone marrow micronucleus (MNT) and Muta™Mouse test systems, in the hope to provide reassurance to the patients that their accidental exposure to EMS at doses up to 0.055 mg/kg did not carry a toxicological risk. Dose levels ranging from 1.25 to 260 mg/kg/day were applied orally for up to 28 days. As a reference we included ENU at doses of 1.1–22 mg/kg/day. Our studies showed that daily doses of up to 25 mg/kg/day (bone marrow, GI tract) and 50 mg/kg/day (liver) did not induce mutations in the lacZ gene in the three organs tested. Doses up to 80 mg/kg/day (7-day dosing regime) did not induce micronuclei in mouse bone marrow. The genotoxic activity of EMS became apparent only at higher dose levels. Dose fractionation of EMS (28 times 12.5 mg/kg versus a single high dose 350 mg/kg) provided further evidence for the thresholded dose response of EMS and showed that no cumulation of gene mutations below a threshold was occurring. In contrast, for ENU no threshold was apparent and dose fractionation indicated full additivity of individual dose effects.     

Safety studies on epigallocatechin gallate (EGCG) preparations. Part 3: teratogenicity and reproductive toxicity studies in rats.

Green tea and its principal active ingredient, epigallocatechin gallate (EGCG), have been demonstrated to have anticancer properties through interactions with multiple biochemical processes. Since these processes are often crucial in normal fetal development it is important to evaluate the potential effects of EGCG on the fetus. EGCG preparations of >91% purity were administered to pregnant rats during organogenesis and development in order to define the safety of Teavigo, a high-concentration EGCG extract produced by the same novel method. In an initial preliminary study using subcutaneous and gavage routes, there was no evidence of any direct embryo-fetal toxicity, although some maternal toxicity was seen. In the main teratogenicity study, feeding pregnant rats diets supplemented at 1400, 4200 or 14,000 ppm during organogenesis was non-toxic to dams or fetuses. A two-generation study in rats fed 1200, 3600 or 12,000 ppm EGCG preparation showed no adverse effects on reproduction or fertility. The highest dose reduced the growth rate of offspring, and there was a slight increase in pup loss. A growth effect among pups was also seen at 3600 ppm, but in the second generation only. The lowest dose was considered the overall no-observed adverse effect level (NOAEL). As dams consumed twice the amount of feed during the crucial lactation period, the NOAEL was equivalent to 200 mg/kg/day EGCG preparation.     

Effects of (-)-epigallocatechin-3-gallate on pancreatic beta-cell damage in streptozotocin-induced diabetic rats

Oxygen free radicals, which are produced as a result of chronic hyperglycemia, are implicated in many diabetic complications. (-)-Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, has been shown to have both antioxidant and pro-oxidant activities, but its effect on oxidative stress of pancreatic beta cells in vivo is unclear. Here we evaluated the effects of nanomolar concentrations of EGCG on beta-cell survival and the response to high glucose loading in streptozotocin-induced diabetic rats. A 4-day intraperitoneal treatment with EGCG (5 mg/kg/day) further impaired the beta-cell response to high glucose in the diabetic rats. EGCG exacerbated the loss of islet cell mass and insulin-immunoreactivity in beta cells. These results suggest that, even at nanomolar plasma concentrations, EGCG acts as a pro-oxidant rather than an antioxidant, at least in beta cells in vivo.     

Genotoxicity studies on green tea catechin

The beneficial effects of tea catechins are well documented. We evaluated the genotoxic potential of a green tea catechin preparation using established genotoxicity assays, including a bacterial reverse mutation assay (Ames test), a chromosomal aberration assay in cultured Chinese hamster lung cells (CHL/IU), a mouse lymphoma L5178Y/tk assay, and a bone marrow micronucleus (MN) assay in ICR CD mice and SD rats. No significant increases in the number of revertant colonies were observed in the Ames test, but positive responses were observed in two in vitro assays: the chromosomal aberration assay and mouse lymphoma L5178/tk assay. However, the in vivo study demonstrated no significant increase in micronucleated polychromatic erythrocytes (MNPCE) in the bone marrow of both ICR CD mice and SD rats administered a high dose of the green tea catechin preparation up to 2000 mg/kg. Combined with favorable epidemiological information suggesting a chemopreventive effect of tea catechins on carcinogenesis, we conclude that green tea catechin presents no significant genotoxic concern under the anticipated conditions of use. These results are consistent with other genotoxicity studies of tea catechins, which show minimal, if any, genotoxic potential.     

Dose-dependent levels of epigallocatechin-3-gallate in human colon cancer cells and mouse plasma and tissues.

Epigallocatechin-3-gallate (EGCG; molecular formula: C22H18011)is the most abundant catechin in green tea (Camellia sinensis Theaceae). Both EGCG and green tea have been shown to have cancer-preventive activity in a number of animal models, and numerous mechanisms have been proposed based on studies with human cell lines. EGCG has been shown to undergo extensive biotransformation to yield methylated and glucuronidated metabolites in mice, rats, and humans. In the present study, we determined the concentration-dependent uptake of EGCG by HT-29 human colon cancer cells (20-600 microM) and the dose dependence of EGCG plasma and tissue levels after a single dose of EGCG (50-2000 mg/kg i.g.) to male CF-1 mice. The cytosolic levels of EGCG were linear with respect to extracellular concentration of EGCG after treatment of HT-29 cells for 2 h (915.3-6851.6 microg/g). In vivo, EGCG exhibited a linear dose relationship in the plasma (0.03-4.17 microg/ml), prostate (0.01-0.91 microg/g), and liver (0.09-18.3 microg/g). In the small intestine and colon, however, the levels of EGCG plateaued between 500 and 2000 mg/kg i.g. These results suggest that absorption of EGCG from the small intestine is largely via passive diffusion; however, at high concentrations, the small intestinal and colonic tissues become saturated. The levels of 4'-O-methyl-EGCG and 4',4'-di-O-methyl-EGCG parallel those of EGCG with respect to dose. The present study provides information with respect to what concentrations of EGCG are achievable in mice and may guide dose selection for future cancer chemoprevention studies with EGCG.     

Absorption,tissue distribution and elimination of 4-[(3)h]-epigallocatechin gallate in beagle dogs

Polyphenols found in tea are potent antioxidants and have inhibitory activity against tumorigenicity. The purpose of the described study was to assess the absorption, tissue distribution, and elimination of epigallocatechin gallate (EGCG), the principal catechin found in green tea, in a nonrodent species. 4-[(3)H]-EGCG was administered to beagle dogs by intravenous (IV) and oral routes. Following IV administration of 25 mg/kg, radioactivity in the bloodstream resided predominantly in the plasma. Distribution occurred during the first hour, and the plasma levels of total radioactivity declined with a mean half-life of approximately 7 hours. The apparent volume of distribution (0.65 l/kg) indicated wide distribution, and the total body clearance (1.01 ml/min-kg) was low. A subsequent single oral dose (250 mg/kg) was rapidly absorbed, with peak plasma levels at about 1 hour after administration, followed by elimination with a mean half-life of 8.61 hours. The mean area under the curve (AUC) for total radioactivity was approximately 20% of the value following IV administration (corrected for dose administered). Excretion of radioactivity in the feces predominated over urinary excretion following both IV and oral administration of [(3)H]-EGCG. Tissue distribution was determined 1 hour after an IV dose (25 mg/kg) administered after 27 days of oral treatment with EGCG (250 mg/kg/day) to mimic chronic consumption of tea. Radioactivity was distributed to a variety of epithelial tissues; the highest concentrations were observed in the liver and gastrointestinal tract tissues. Repeat dose oral administration of EGCG resulted in significantly lower blood radioactivity compared to the concentration following a single dose. These results are generally in accord with previous studies in rodents and indicate that, after oral administration, EGCG (as parent compound and metabolites) is widely distributed to tissues where it can exert a chemopreventive effect.     

Hepatotoxicity of high oral dose (−)-epigallocatechin-3-gallate in mice

The tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) has been studied for chronic disease preventive effects, and is marketed as part of many dietary supplements. However, case-reports have associated the use of green tea-based supplements with liver toxicity. We studied the hepatotoxic effects of high dose EGCG in male CF-1 mice. A single dose of EGCG (1500 mg/kg, i.g.) increased plasma alanine aminotransferase (ALT) by 138-fold and reduced survival by 85%. Once-daily dosing with EGCG increased hepatotoxic response. Plasma ALT levels were increased 184-fold following two once-daily doses of 750 mg/kg, i.g. EGCG. Moderate to severe hepatic necrosis was observed following treatment with EGCG. EGCG hepatotoxicity was associated with oxidative stress including increased hepatic lipid peroxidation (5-fold increase), plasma 8-isoprostane (9.5-fold increase) and increased hepatic metallothionein and γ-histone 2AX protein expression. EGCG also increased plasma interleukin-6 and monocyte chemoattractant protein-1. Our results indicate that higher bolus doses of EGCG are hepatotoxic to mice. Further studies on the dose-dependent hepatotoxic effects of EGCG and the underlying mechanisms are important given the increasing use of green tea dietary supplements, which may deliver much higher plasma and tissue concentrations of EGCG than tea beverages.     

Validation of green tea polyphenol biomarkers in a phase II human intervention trial.

Health benefits of green tea polyphenols (GTPs) have been reported in many animal models, but human studies are inconclusive. This is partly due to a lack of biomarkers representing green tea consumption. In this study, GTP components and metabolites were analyzed in plasma and urine samples collected from a phase II intervention trial carried out in 124 healthy adults who received 500- or 1000-mg GTPs or placebo for 3 months. A significant dose-dependent elevation was found for (-)-epicatechin-3-gallate (ECG) (p<0.001, trend test) and (-)-epigallocatechin-3-gallate (EGCG) (p<0.05, trend test) concentrations in plasma at both 1-month and 3-months after intervention with GTPs. No significant increase of (-)-epicatechin (EC) or (-)-epigallocatechin (EGC) was observed in plasma after GTP intervention. A mixed-effects model indicated significant effects of dose (EGCG) and dose by time interaction (ECG), but not for EC and EGC. Analysis of phase 2 metabolic conjugates revealed a predominance of free GTPs in plasma, up to 85% for EGCG, while a majority of GTPs in urine were sulfated and glucuronidated conjugates (up to 100% for EC and 89% for EGC). These results suggest that plasma ECG and EGCG concentrations are reliable biomarkers for green tea consumption at the population level.     

Assessment of in vivo mutagenic potency of ethylenediaminetetraacetic acid in albino mice.

Ethylenediaminetetraacetic acid (EDTA) disodium salt, a widely used metal chelator, was studied for its potency to induce bone marrow micronuclei, dominant lethal mutations and sperm-head abnormalities in albino mice. The acute oral LD₅₀ dose computed by probit regression was 30 mg/kg body weight in the strain used. Preliminary studies showed that oral administration of EDTA disodium salt at doses of 5, 10 and 15 mg/kg body weight/day on 5 consecutive days did not induce any obvious signs of toxicity. In the bone marrow micronucleus assay acute doses of EDTA disodium salt (5–20 mg/kg body weight) induced a dose-dependent increase in the incidence of micronucleated polychromatic erythrocytes at a 24-hr sampling. However, administration at doses of 5, 10 and 15 mg/kg for 5 consecutive days did not produce any observable effect on either the testicular or epididymal weights and histology. No appreciable alterations were observed in the caudal sperm counts at any of the sampling intervals and there was no treatment-related increase in the incidence of sperm-head abnormalities. Furthermore, treatment of male mice with EDTA disodium salt (10 mg/kg body weight/day for 5 consecutive days) induced no increase in the incidence of post-implantation embryonic deaths, except for a marginal but statistically insignificant increase during wk 2 and 3 of mating.     

Lack of in vivo clastogenic activity of grape seed and grape skin extracts in a mouse micronucleus assay.

Meganatural brand grape seed extract (GSE) and grape skin extract (GSKE), containing proanthocyanidin polyphenolic compounds, are intended for use in food as functional ingredients exhibiting antioxidant activity. Proanthocyanidins, as well as the minor constituent phenolic compounds in GSE and GSKE, are present naturally in many foods such as fruits, vegetables, chocolate, tea, etc., and on average people consume 460-1000 mg/day of these combined substances. While some polyphenolic compounds, tested individually, have demonstrated antitumorigenic or antipromotional activity, at least one minor component of GSE and GSKE, quercitin, has exhibited positive activity in Salmonella and other in vitro mutagenicity assays. As part of a program to investigate the safety of GSE and GSKE, these products were tested for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1(ICR) BR mouse bone marrow. The appropriate test article was dissolved in 0.5% carboxymethylcellulose and dosed by oral gavage to five males/test article/dose level/harvest time point. Animals were dosed at 500, 1000 and 2000 mg/kg. Five animals dosed with either test article at 500, 1000 and 2000 mg/kg dose levels and five animals dosed with the cyclophosphamide (80 mg/kg) positive control were euthanized approximately 24 h after dosing for extraction of bone marrow. Five animals dosed with either test article at the 2000 mg/kg dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 h after dosing for extraction of bone marrow. At least 2000 PCEs per animal were analyzed for frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal. For both GSE and GSKE, no statistically significant increase in micronucleated PCEs was observed at any dose level or harvest time point. GSE produced indication of cytotoxicity (decreased PCE:NCE ratio) at the 2000 mg/kg dose level for the 48-h harvest time point, confirming that the test article reached the target bone marrow in significant amount. Meganatural GSE and Meganatural GSKE were evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.     

The toxicity of behenyl alcohol. I. Genotoxicity and subchronic toxicity in rats and dogs

The genotoxic potential of behenyl alcohol, a saturated long-chain (C22:0) fatty alcohol, was examined in the Ames Salmonella typhimurium reverse mutation assay, the gene mutation, and chromosome aberrations assays in Chinese hamster V79 cells, and the micronucleus assay in NMRI mice. Behenyl alcohol did not increase the number of revertants per plate compared to controls in the S. typhimurium assay, with or without metabolic activation. No significant increases in the number of mutant colonies or in structural chromosome aberrations were observed in Chinese hamster V79 cells. In addition, behenyl alcohol did not increase the frequency of bone marrow polychromatic erythrocyte (PCE) micronuclei in mice in vivo. In two subchronic toxicity studies, CD rats and beagle dogs were administered behenyl alcohol by oral gavage for at least 26 weeks at doses of 0, 10, 100, or 1000 mg behenyl alcohol/kg body weight/day for rats and 0, 20, 200, or 2000 mg behenyl alcohol/kg body weight/day for dogs. Adverse effects were not observed following gross and histopathological evaluations of dosed rats. Compound-related effects in dogs were limited to observations of pale feces, indicative of unabsorbed behenyl alcohol, at doses of 2000 mg/kg body weight/day. There were no histopathological changes observed in dogs dosed with behenyl alcohol. The no-observed-adverse-effect-level (NOAEL) for behenyl alcohol was 1000 mg/kg body weight/day for rats, and 2000 mg/kg body weight/day for dogs, the highest doses used in these studies.     

Hydroquinone: genotoxicity and prevention of genotoxicity following ingestion.

Plant-derived polyphenolics and other chemicals with antioxidant properties have been reported to inhibit the expression of genotoxic activity by pro-oxidant chemicals (Sai et al., 1992, 1994; Teel and Castonguay, 1992). In vitro and in vivo studies with ionizing radiation suggest that hydroquinone (HQ) may have similar protective effects (Babaev et al., 1994). The present study was conducted to determine whether HQ is capable of inhibiting the induction of micronuclei in the bone marrow of mice following exposure to an oxidant, potassium bromate or KBrO3 (Nakajima et al., 1989; Sai et al., 1992, 1994). To be able to interpret the results of this work, it was also necessary to determine whether HQ is itself genotoxic when fed in the diet. HQ diets (0.8%) fed to mice for 6 days reduced the background incidence of micronuclei compared with the basal diet. KBrO3 dosed ip (12.5-100 mg/kg) produced a dose-dependent increase in micronuclei as reported by others. Mice fed 0.8% HQ diets 6 days, and then dosed intraperitoneally with KBrO3, showed a 36% reduction in micronuclei across the range of KBrO3 dose levels. This effect was associated with a reduction in the background micronucleus response as well as a reduction in response to KBrO3. Statistical significance (P < or = 0.05), observed at a dose of 25 mg/kg KBrO3 in the mice fed the control diet, was abolished in the group fed 0.8% HQ. When mice were given 50 mg HQ/kg by oral gavage and then given 50 mg KBrO3/kg ip 20 min later, the micronucleus response induced by KBrO3, was lower in animals given HQ. The results of this study demonstrate that large doses of HQ may be given orally without induction of micronuclei or bone marrow depression, that HQ reduces the background micronucleus response in animals fed a basal diet, and that the HQ reduces the micronucleus response to KBrO3 as well as background incidence of micronuclei in KBrO3-dosed animals. The protective effect of HQ may be due to enzyme induction or a direct antioxidant effect of HQ against oxidants commonly present in the diet.     

Transdermal delivery of (-)-epigallocatechin-3-gallate, a green tea polyphenol, in mice

Epigallocatechin-3-gallate (EGCG) is the most studied catechin in green tea (Camellia sinensis). EGCG and green tea are cancer preventive in many animal models, and numerous mechanisms have been proposed in cell lines. EGCG is poorly bioavailable in man and rodents. We hypothesized that transdermal delivery of EGCG could result in improved bioavailability. Following application of EGCG transdermal gel (50 mg kg(-1), t.d.) to SKH-1 mice, EGCG was observed in the epidermis (1365.7-121.0 ng g(-1)) and dermis (411.2-42.6 ng g(-1)). The maximum plasma concentration (Cmax) of EGCG was 44.5 ng mL(-1). The t(1/2) (94.4 h) and AUC(0-->24 h) (881.5 ng mL(-1) h) of EGCG were greater than values previously reported for oral EGCG. The t(1/2) and area under the concentration-time curve up to 24 h (AUC(0-->24 h)) in the liver, small intestine and colon were 21.3-74.6 h and 715-2802 ng g(-1)h, respectively. Stability studies showed that the transdermal formulation was stable at 4 degrees C and had a half-life (t(1/2)) of 47.1 and 20.2 h at 25 degrees C and 37 degrees C, respectively. These data indicate that transdermal EGCG is useful for delivering prolonged levels of EGCG to plasma and tissues, and may provide an alternative to tea consumption as a dosage form of EGCG.     

Genotoxicity studies on licorice flavonoid oil (LFO)

Licorice flavonoid oil (LFO) is a new functional food ingredient. In this study, the genotoxicity of LFO was investigated using a test battery of three different methods. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, LFO did not increase the number of revertant colonies in any tester strain with or without metabolic activation by rat liver S9 mix. In a chromosomal aberration test using Chinese hamster lung (CHL/IU) cells, LFO did not induce any chromosomal aberrations either in the short period test without rat liver S9 mix or in the continuous treatment (24 h or 48 h) test. However, in the short-period test with rat liver S9 mix, LFO induced structural chromosomal aberrations at concentrations higher than 0.6 mg/mL. A bone marrow micronucleus test using male F344 rats was initially conducted. The animals were dosed by oral gavage at doses up to 5000 mg/kg/day. No significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were observed and the high dose suppressed the ratio of polychromatic erythrocytes (PCE) to total erythrocytes. Subsequently, a liver and peripheral blood micronucleus test using male F344 rats was conducted. No micronuclei induction either in hepatocytes or PCE was observed even at the highest dose of 5000 mg/kg/day. From the findings obtained from the genotoxicity assays performed in this study and the published pharmacokinetic studies of LFO, it appears unlikely that dietary consumption of LFO will present any genotoxic hazard to humans.     

An assessment of the genotoxicity and human health risk of topical use of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one].

Kojic acid (KA), a natural substance produced by fungi or bacteria, such as Aspergillus, Penicillium or Acetobacter spp, is contained in traditional Japanese fermented foods and is used as a dermatological skin-lightening agent. High concentrations of KA (>or=1000 microg/plate) were mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535, TA102 and E. coli WP2uvrA, but not in TA 1537. An Ames test following the "treat and plate" protocol was negative. A chromosome aberration test in V79 cells following a robust protocol showed only a marginal increase in chromosome aberrations at cytotoxic concentrations after prolonged (>or=18 h) exposure. No genotoxic activity was observed for hprt mutations either in mouse lymphoma or V79 cells, or in in vitro micronucleus tests in human keratinocytes or hepatocytes. All in vivo genotoxicity studies on KA doses were negative, including mouse bone marrow micronucleus tests after single or multiple doses, an in vivo/in vitro unscheduled DNA synthesis (UDS) test, or a study in the liver of the transgenic Muta(TM) Mouse. On the basis of pharmacokinetic studies in rats and in vitro absorption studies in human skin, the systemic exposure of KA in man following its topical application is estimated to be in the range of 0.03-0.06 mg/kg/day. Comparing these values with the NOAEL in oral subchronic animal studies (250 mg/kg/day), the calculated margin of safety would be 4200- to 8900-fold. Comparing human exposure with the doses that were negative for micronuclei, UDS and gene mutations in vivo, the margins of safety are 16000 to 26000-fold. In conclusion, the topical use of KA as a skin lightening agent results in minimal exposure that poses no or negligible risk of genotoxicity or toxicity to the consumer.     

Sister chromatid exchanges and micronuclei formations induced by sorbic acid and sorbic acid-nitrite in vivo in mice

The in vivo induction of sister chromatid exchanges and micronuclei formations by acute treatment with different concentrations of sorbic acid and by nitrite, individually and in combination, was studied in bone marrow cells of mice. A significant increase in the frequency of sister chromatid exchanges was only observed with the three higher concentrations of sorbic acid when compared to a distilled water control. Sodium nitrite produced a significant increase at all doses tested. A combination of half the concentration of sorbic acid and of sodium nitrite gave an additive effect over that of sorbic acid or sodium nitrite alone. In the micronucleus assay, the highest dose of sorbic acid (150 mg/kg body weight) produced a significant increase in micronuclei formations compared to the distilled water control. Sodium nitrite alone induced significant numbers of micronuclei at all concentrations tested when compared to the negative control. However, a combination of half the concentration of sorbic acid and of sodium nitrite gave synergistic effects which could possibly be ascribed to the formation of certain genotoxic compounds in vivo.     

Synthesis and structure identification of thiol conjugates of (-)-epigallocatechin gallate and their urinary levels in mice

(-)-Epigallocatechin gallate (EGCG), the most abundant and most biologically active compound in tea, has been proposed to have many beneficial health effects. The metabolic fate of EGCG, however, is not well understood. In the present study, we found that EGCG can be oxidized by peroxidase and hydrogen peroxide and then reacted with cysteine or glutathione to form conjugates. The structures of the cysteine and glutathione conjugates of EGCG were identified using 2D NMR and MS. Two thiol conjugates of EGCG (2'-cysteinyl EGCG and 2' '-cysteinyl EGCG) were identified by ESI-LC-MS/MS analysis from the urine samples of mice administered 200 or 400 mg/kg EGCG, i.p. These conjugates were not found in urine samples of mice after receiving EGCG at 50 mg/kg i.p., or 2000 mg/kg i.g., or in human urine following consumption of 3 g of decaffeinated green tea solids (containing 333 mg EGCG). At high doses, EGCG is believed to be oxidized to form EGCG quinone, which can react with glutathione to form the thiol conjugates. These results suggest that detectable amounts of thiol conjugates of EGCG are formed only after rather high doses of EGCG are given to the mice.     

Mutagenicity tests of the antithyroid agent thiamazole. Cytogenetic studies on male mice

The mutagenic potential of thiamazole, an antithyroid agent, was investigated by an in vivo cytogenetic test and was compared with those of mitomycin C and vincristine. These drugs were subcutaneously injected into slc-ICR male mice either as a single dose or as multiple doses for 5 successive days. Thiamazole (90 or 180 mg/kg) did not increase the number of micronuclei in bone marrow cells. This drug also did not induce chromosomal aberrations in spermatogonium, spermatocyte, or bone marrow cells. On the other hand, mitomycin C (3.0 mg/kg) increased the appearance of chromosomal aberrations and micronuclei. Vincristine (0.2 mg/kg) induced bone marrow cells with a so called large micronucleus (d greater than or equal to D/4). These results suggest that thiamazole may not have significant effects on the genetic systems of mice.