Application of the threshold of toxicological concern approach for the safety evaluation of calendula flower (Calendula officinalis) petals and extracts used in cosmetic and personal care products.

Calendula flower (Calendula officinalis) (CF) has been used in herbal medicine because of its anti-inflammatory activity. CF and C. officinalis extracts (CFE) are used as skin conditioning agents in cosmetics. Although data on dermal irritation and sensitization of CF and CFE's are available, the risk of subchronic systemic toxicity following dermal application has not been evaluated. The threshold of toxicological concern (TTC) is a pragmatic, risk assessment based approach that has gained regulatory acceptance for food and has been recently adapted to address cosmetic ingredient safety. The purpose of this paper is to determine if the safe use of CF and CFE can be established based upon the TTC class for each of its known constituents. For each constituent, the concentration in the plant, the molecular weight, and the estimated skin penetration potential were used to calculate a maximal daily systemic exposure which was then compared to its corresponding TTC class value. Since the composition of plant extracts are variable, back calculation was used to determine the maximum acceptable concentration of a given constituent in an extract of CF. This paper demonstrates the utility and practical application of the TTC concept when used as a tool in the safety evaluation of botanical extracts.

Absence of adverse effects of sodium metabisulphite in manufactured biscuits: results of subacute (28-days) and subchronic (85-days) feeding studies in rats.

Sulphites are extensively used in the food and drinks industry. Their toxicity has been previously evaluated by addition to the diet or drinking water of laboratory animals. Because interactions between sulphites and food constituents occur, the present work was conducted to determine the subacute and subchronic toxicity of sulphite-bound compounds in a finished product: manufactured biscuits. The studies were performed on Sprague Dawley, rats for 28 and 85 days of dietary exposure. Diets were prepared from sulphited or untreated (controls) biscuits with the addition of sugar, protein, vitamins and minerals according to the nutritional requirements of the animals. Groups of 10 male and 10 female rats were administered diets containing sulphited biscuits at levels of 0, 10, 35 and 75%, corresponding to 10-15, 35-45, 150-170 and 310-340 mg SO2/kg diet. In both studies, no death or clinical abnormalities were reported. Growth rate, food consumption and food conversion efficiency were not affected by treatment. No dose-related changes were observed for haematology, clinical chemistry, ocular examination, renal-function, urinalysis, organ weights or gross and microscopic examinations. The liver concentrations of vitamins A, B1, C and E were not significantly changed except for an increase in vitamin E in high-dose males after 28 days' exposure. Based on these data, the no-observed-adverse-effect level (NOAEL) of sulphites in baked biscuits was judged to be 310 mg SO2/kg diet or 25 mg/kg body weight/day.

The safety assessment of novel foods. Guidelines prepared by ILSI Europe Novel Food Task Force.

The diversity of novel foods and novel ingredients covered by the scope of the EU regulation is such that a check list approach to safety evaluation is inappropriate. Rather, a case-by-case approach is required taking into account the composition of the novel food, its intake, its role in the diet and the intended target group. The SAFEST approach provides a means of targeting the safety evaluation on those aspects, nutritional or toxicological, of a novel food which are of particular concern. Using this approach, novel foods are assigned to one of three classes on the basis of certain background information. For those novel foods which can be shown to be in SAFEST class 1, namely those which are substantially equivalent to a traditional counterpart, no further information is required to demonstrate their safety. For those novel foods in SAFEST class 2, i.e. those sufficiently similar to a traditional counterpart or differing from it only in particular, well defined, characteristics, the evaluation will focus on those differences. Only in the case of novel foods which are not in class 1 or class 2 is extensive testing of the whole food likely to be required. Even in these cases, the testing should follow a scientifically-based hierarchical approach involving: literature reviews; chemical analysis; appropriate in vitro and in vivo tests; and, if necessary, confirmation of safety and nutritional value in humans. Examination of the causes of any adverse effects reported by consumers after the novel food or ingredient has been approved and is introduced into the market may provide additional reassurance of safety.

Differential induction of cytochrome P-450 isozymes by rifampicin in the Chinese hamster, Cricetus griseus.

Treatment of male and female Chinese hamsters with rifampicin at intraperitoneal doses of 25 and 50 mg/kg did not increase the cytochrome P-450 content of the liver except for a 1.3-fold increase in male hamsters at a dose of 50 mg/kg. Enhancement of the activities of erythromycin N-demethylase and testosterone hydroxylases, except for 15 alpha-hydroxylation, was observed in the livers of both male and female hamsters treated with rifampicin at both doses. Western blot analysis revealed that rifampicin caused no change in the content of CYP3A subfamily proteins in the liver, whereas changes in that of CYP2A subfamily proteins were evident.

Characterization of cytochrome P450-monooxygenases of Chinese hamsters with respect to aflatoxin B1 activation.

Monooxygenases of Chinese hamster livers were characterized with respect to aflatoxin B1 activation and cytochrome P450 isozymes. Polychlorinated biphenyl (PCB)-treatment induced higher activity for aflatoxin B1 activation in Chinese hamsters than in rats and this activity was more elevated by the treatment with phenobarbital than with 3-methylcholanthrene, which is contrary to the observation in Syrian hamsters and rather similar to that in rats. Western blot analysis demonstrated that the treatment of Chinese hamsters with phenobarbital markedly elevated the level of cytochrome P450 isozyme belonging to the CYP2A family and only slightly that immuno-related to CYP2B1. In parallel with this, the activities of testosterone 15 alpha-hydroxylase and 7-ethoxycoumarin O-deethylase were significantly induced by phenobarbital treatment. These suggest that phenobarbital mainly induces an isozyme related to CYP2A2 which is responsible for aflatoxin B1 activation in Chinese hamsters.

Effects of dexamethasone on the hepatic cytochrome P450IIIA subfamily in two hamster strains Mesocricetus auratus and Cricetus griseus.

1. Administration of dexamethasone significantly reduced the amount of hepatic cytochrome P450 in Syrian golden and Chinese hamsters, while it increased the amount in rats. 2. In contrast to rats, the induction rate of the activities of erythromycin and troleandomycin N-demethylases by dexamethasone was relatively low, while that of the testosterone 6 beta-hydroxylase was high in the two hamster strains. 3. Western blot analysis revealed that dexamethasone did not modify markedly the pattern of the P450IIIA subfamily proteins in the two hamster strains.

Clotrimazole-induced modulation of hepatic cytochrome P450 enzymes in Syrian and Chinese hamsters.

Clotrimazole, an imidazole antifungal drug, is known to induce cytochrome P450 isozymes of the P450IIIA and P450IIB subfamilies in rats. This agent modulated hepatic cytochrome P450 enzymes differently in golden Syrian and Chinese hamsters and also in hamsters and rats. Clotrimazole at a daily intraperitoneal dose of 100 mg/kg for three days increased the amount of cytochrome P450 in the livers of the two hamster strains. In Syrian hamsters, clotrimazole significantly induced the activities of 7-pentoxyresorufin O-dealkylase, coumarin 7-hydroxylase, benzphetamine N-demethylase and testosterone 15 alpha- and 16 alpha-hydroxylases, but reduced those of testosterone 15 beta-, 7 alpha-, 6 beta-, 2 alpha- and 2 beta-hydroxylases. In Chinese hamsters, clotrimazole markedly stimulated the activities of coumarin 7-hydroxylase and testosterone 15 alpha, 16 alpha- and 2 alpha-hydroxylases as well as the formation of androstenedione. Western blot analysis revealed that clotrimazole treatment induced mainly cytochrome P450 isozymes immunorelated to the P450IIB and P450IIA subfamilies in Syrian hamsters and isozymes immunorelated to the P450IIA subfamily in Chinese hamsters. In contrast, in both hamster strains, clotrimazole did not induce the isozymes corresponding to the P450IIIA subfamily.

Identification of a constitutive form of cytochrome P-450 with high activity towards macrolide antibiotics in Syrian golden hamster liver.

The presence of cytochromes P450 belonging to the P450IIIA family was investigated in the liver of untreated Syrian golden hamsters and compared to those of untreated, phenobarbital- and dexamethasone-treated rats. The microsomal preparations from untreated Syrian golden hamster livers exhibited higher activities of N-demethylation towards the macrolide antibiotics, erythromycin and troleandomycin, than those from untreated and phenobarbital-treated rats. Western blots analysis revealed larger amounts of proteins immunorelated to the rat cytochrome P450IIIA1 isozyme in Syrian golden hamster liver than in untreated and phenobarbital-treated rats. The N-demethylation of erythromycin and troleandomycin were strongly inhibited after preincubation of the microsomes from Syrian golden hamsters with anti-P450IIIA1. We conclude that a cytochrome P450 belonging to the P450IIIA family is expressed constitutively in the liver from Syrian golden hamsters, whereas it is not in rat liver.

In vitro effects of an imidazole antifungal, prochloraz, on spectral and catalytic properties of microsomal cytochromes P-450.

1. The imidazole antifungal agent, prochloraz, elicited type II spectral interactions with microsomal cytochromes P-450 from rats pretreated with phenobarbital (PB), 3-methyl-cholanthrene (MC) and dexamethasone (DEX). 2. Prochloraz interacts strongly with type I binding sites of both PB- and DEX-induced cytochromes P-450 and to a lesser extent with cytochromes P-450 from MC-induced rats. 3. The antifungal derivative was a more potent inhibitor of the troleandomycin-nitrosoalkyl-cytochrome P-450 complex formation in DEX-induced microsomes than of the isosafrole-carbene-cytochrome P-450 complex formation in MC-pretreated rats. 4. Prochloraz is a strong inhibitor of the cytochrome P-450-dependent mixed-function oxidases in rat liver microsomal preparations.

Prochloraz as potent inhibitor of benzo[a]pyrene metabolism and mutagenic activity in rat liver fractions.

We have determined how prochloraz, an imidazole antifungal agent, affects the metabolism of benzo[a]-pyrene by hepatic microsomes from 3-methylcholanthrene treated male rats. The prochloraz-like 7,8-benzoflavone was a potent inhibitor of total benzo[a]pyrene metabolism while miconazole was a weak inhibitor. The proportion of benzo[a]pyrene dihydrodiols formed was decreased whereas phenols were increased by the in vitro addition of prochloraz. Furthermore, a good correlation was obtained between the effects of prochloraz on the microsomal formation of benzo[a]pyrene metabolites and on the mutagenic activity of benzo[a]pyrene in the Salmonella typhimurium test.

Effects of prototypic PCBs on benzo[a]pyrene mutagenic activity related to vitamin A intake.

The effects of vitamin A dietary intake (2 and 20 IU */g of food) on the mutagenic activity of benzo[a]pyrene (B(a)P) toward Salmonella typhimurium (TA98) were studied either in control rats or in animals treated by the PCB congeners 2,4,5,2',4',5'-hexachlorobiphenyl [2,4,5)2Cl) and 3,4,3',4'-tetrachlorobiphenyl [3,4)2Cl). (3,4)2Cl (a planar compound) strongly increased B(a)P monooxygenase (B(a)PMO) activity and glutathione transferase, (2,4,5)2Cl (a non-planar PCB) was a strong inducer of epoxide hydrolase and a weak inducer of B(a)PMO. Enzyme induction was not modified by changes in vitamin A dietary intake. A higher mutagenic effect was observed in the (3,4)2Cl group than in the (2,4,5)2Cl one. This could be related to the specific form of cytochrome P-450 induced by (3,4)2Cl. In the untreated animals, the activation of B(a)P was higher in the 2-IU group than in the 20-IU one. Conversely, in PCB-treated rats the mutagenic activity of B(a)P was higher in the 20-IU group than in the 2-IU one. PCB induction increased the liver content of vitamin C in both the 2-IU and the 20-IU groups but only increased the glutathione levels in the 2-IU groups. This suggests that glutathione content in cellular fractions may be one of the determining parameters for the mutagenic activity of B(a)P.