National Toxicology Program

The nomination by Drs. Gold, Ames, and Slone, University of California, Berkeley, of chlorogenic acid and caffeic acid is based on their occurrence in high concentrations in food and the apparent lack of carcinogenicity data.

Both chlorogenic and caffeic acids are constituents of numerous plant species from the families Umbelliferae, Cruciferae, Cucurbitaceae, Polygonaceae, Compositae, Labiatae, Solanaceae, Leguminosae, Saxifragaceae, Caprifoliaceae, Thaceae, and Valerianaceae. Thus, they occur in many common fruits, vegetables, spices, medicinal plants, and beverages.

Information on the commercial availability of chlorogenic acid was not found, but caffeic acid is available in small quantities from a number of U. S. producers.

No information was found on uses of chlorogenic acid. The use of caffeic acid for treating asthma and allergies has been investigated in drug development studies. Plants containing chlorogenic acid and/or caffeic acid have been used as herbal remedies and possess some of the following pharmacological properties: antiarthritic, antidiarrheal, antiinflammatory, antirheumatic, antitumor, antiviral, astringent, cardiotonic, carminative, chloretic, coronary vasodilatory, diaphoretic, diuretic, gastric sedative, hypotensive, intestinal antiseptic, purgative, and spasmolytic effects. Medicinal plants containing chlorogenic and/or caffeic acid have also been used as remedies for the common cold, hematemesis, hematuria, hemorrhoids, lumbago, neuralgia, tinnitus, and toothache.

Exposure to chlorogenic and/or caffeic acid occurs primarily via the oral route from the ingestion of foods, beverages, and herbal remedies. Inhalation exposure occurs from tobacco smoking.

Chlorogenic and caffeic acids may be expected to be found in the wastes generated by industries making coffee and processed potatoes. No information on the regulatory status of chlorogenic and caffeic acids was found.

In humans, the carcinogenic potency of caffeic acid has been estimated based on an average human intake of 10⁶ ng/kg body wt/day (1 mg/kg body wt/day). The estimated number of cancer cases was less than 1000 per 1 million individuals.

In human immunological studies, both positive and negative results were found when individuals allergic to green coffee were tested for allergic reactions to chlorogenic acid by subcutaneous (s.c.) injection or skin scratch tests. Caffeic acid did not produce allergic reactions when administered s.c. to individuals allergic to green coffee, nor did it induce sensitization when applied by dermal patch to a woman allergic to beeswax.

No information on the chemical disposition, metabolism, or toxicokinetics of chlorogenic acid in humans was found. Ingestion of caffeic acid, however, produces a number of metabolites, including glucuronides of m-coumaric acid and m-hydroxyhippuric acid. Oral administration of caffeic acid to volunteers resulted in rapid (no precise time specified) urinary excretion of O-methylated derivatives (ferulic, dihydroferulic, and vanillic acids), while m-hydroxyphenyl derivatives were excreted later (time not provided).

In one study using rats, chlorogenic acid was hydrolyzed in the stomach and intestine to caffeic and quinic acids. In isolated rat livers that were perfused with caffeic acid, 93% of the caffeic acid appeared unchanged after one liver passage; oxidation, methylation, and cyclization products were found in the perfusion medium, and glucuronides and sulfates of caffeic acid were identified in the bile. Following intravenous (i.v.) administration to rabbits, most of the dose was excreted unchanged in the urine within 2 hours. For caffeic acid, the elimination kinetics fit a two-compartment model when administered orally to rats or i.v. to rabbits.

For both chlorogenic and caffeic acids, the oral LD₅₀ for redwing blackbirds was greater than 100 mg/kg (0.282 and 0.555 mmol/kg, respectively).

Few toxic effects resulting from acute exposure to chlorogenic or caffeic acid were noted in the reviewed studies. In rats dosed intraperitoneally (i.p.), chlorogenic acid at 4000 mg/kg (11.29 mmol/kg) induced death in 4 of 6 animals, and caffeic acid at 1500 mg/kg (8.326 mmol/kg) induced death in 5 of 8 animals, but doses of chlorogenic acid and caffeic acid lower than 2437 mg/kg (6.878 mmol/kg) and 1250 mg/kg (6.938 mmol/kg), respectively, were non-lethal.

Subchronic exposure of mice to chlorogenic or caffeic acid in the diet reduced aryl hydrocarbon hydroxylase (AHH) and glutathione-S-transferase (GST) levels in the intestine, but did not induce clinical symptoms of toxicity. In rats, the effects of feeding chlorogenic or caffeic acid in the diet include reduced kidney and adrenal weights (chlorogenic acid), hyperplasia of the forestomach (chlorogenic and caffeic acids), and increased antioxidant capacity (caffeic acid).

No information on chronic exposure to chlorogenic acid was found, but chronic exposure to caffeic acid in the diet induced hyperplasia of the forestomach (mice, rats, and hamsters), hyperplasia of the kidney (mice and rats), and increased liver and kidney weights (rats).

Chlorogenic acid in the diet inhibited benzo[a]pyrene (BaP)-induced increases in liver aryl hydrocarbon hydroxylase and liver glucuronosyl transferase in mice. In rats chlorogenic acid inhibited paraquat-induced increases in liver catalase, liver glutathione peroxidase, and liver glutathione reductase and peroxidized corn oil-induced increases in serum total cholesterol, serum triglycerides, serum alanine aminotransferase, serum aspartate aminotransferase, and serum and liver lipid peroxides.

Based on one rat study, the adverse reproductive effects of i.p. treatment with chlorogenic or caffeic acid consisted only of fetal rib defects.

In mice, 2% (20,000 ppm) chlorogenic acid in the diet for 96 weeks induced papillomas and carcinomas of the forestomach, alveolar type II-cell tumors of the lung, and renal cell adenomas, while in rats 1 or 2% (10,000 or 20,000 ppm) caffeic acid in the diet for 51 weeks to 2 years induced papillomas of the forestomach and renal adenomas. One study in which rats were exposed to 2% (20,000 ppm) caffeic acid in the diet for two years showed treatment-induced carcinomas of the forestomach, whereas two studies with shorter exposure durations showed no such effect.

Initiation/promotion carcinogenicity studies for chlorogenic acid were not found, but in studies using rats, caffeic acid was shown to exert strong promotion activity for forestomach carcinogenesis when administered in the diet for 51 weeks after a single dose of a carcinogen (e.g., 7,12-dimethylbenz[a]anthracene [DMBA], N-methyl-N'-nitro-N-nitrosoguanidine [MNNG]); caffeic acid treatment for shorter durations (i.e., 35 weeks) did not promote the induction of tumors.

Chlorogenic acid inhibited the number of DMBA-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumors when administered topically to mice concomitantly with the DMBA and TPA, but not when administered prior to the initiator and promoter. Caffeic acid also inhibited formation of DMBA-initiated/TPA-promoted skin tumors, but to a lesser extent than chlorogenic acid. Chlorogenic acid, when administered i.p. to mice, reduced the number of BaP-induced lung tumors, and, when administered in the diet of hamsters, reduced the number of methylazoxymethanol acetate (MAM acetate)-induced colon tumors, colon adenocarcinomas, and hepatocellular foci. In mice, caffeic acid in the diet reduced the number of BaP-induced tumors of the forestomach, while i.p. administration of caffeic acid for ten days after a single s.c. injection of sarcoma-180 cells inhibited sarcoma-180 tumor growth. In rats, caffeic acid in the diet reduced the incidence of 4-nitroquinoline-1-oxide (4-NQO)-induced tongue neoplasms. Dietary treatment of rats with caffeic acid inhibited the formation of neoplasms and preneoplasms of the forestomach induced by treatment with diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), 1,2-dimethylhydrazine (DMH), and 2,2-dihydroxydi-n-propylnitrosamine (DHPN).

Both chlorogenic acid and caffeic acid induced strand breaks in DNA in acellular test systems that favored formation of oxygen radicals, particularly in the presence of transition metals. These chemicals were not mutagenic in standard bacterial mutagenicity assays. However, caffeic acid, in the presence of Mn²⁺ and in the absence of S9 activation induced mutations in Salmonella typhimurium strains TA98 and TA100; removal of the transition metal ions or addition of liver S9 eliminated the mutagenic response. Both chlorogenic acid and caffeic acid induced mitotic gene conversion in Saccharomyces cerevisiae strain D7 under conditions of alkaline pH and in the absence of S9. Caffeic acid was also able to induce gene conversion at normal pH, without S9, although the response was weaker. As with the other genotoxicity assays, addition of transition metal ions enhanced the recombinogenic response in S. cerevisiae, but S9 eliminated all activity, even in the presence of metal ions. Neither chlorogenic acid nor caffeic acid induced 8-azaguanine resistance in Chinese hamster V79 cells, but both compounds were clastogenic in mammalian cells in vitro. Induction of chromosomal aberrations was seen in Chinese hamster ovary (CHO) cells treated with chlorogenic acid or caffeic acid in the absence of S9; addition of S9 eliminated the clastogenicity. Addition of Mn²⁺ enhanced the response seen with caffeic acid. Both chemicals induced forward mutations at the tk locus in mouse lymphoma L5178Y cells, but chlorogenic acid required S9 for a positive response and caffeic acid was only positive in the absence of S9. Chlorogenic acid or caffeic acid did not induce chromosomal damage in mice or rats in vivo.

Co-mutagenicity data was limited to a single study of the clastogenicity of chlorogenic acid with and without arecoline, in CHO cells; there was a significant enhancement of clastogenic activity when both chemicals were administered in combination, compared to the responses elicited by the individual chemicals. The addition of Mn²⁺ further enhanced the clastogenic response.

Caffeic acid inhibited the induction of DNA single strand breaks in phage ØX174 DNA by H₂O₂ and cytochrome c. Both positive and negative results were observed in tests of the antigenotoxicity of chlorogenic acid and caffeic acid in S. typhimurium strains TA98 and TA100. In S. typhimurium strain TA1535, however, both compounds were reported to inhibit the mutagenicity of MNNG, and chlorogenic acid also inhibited the mutagenicity of nitrosation products of nitrosoproline in the absence of S9. Neither chlorogenic acid nor caffeic acid inhibited the mutagenicity of ultraviolet (UV) radiation in E. coli. In Chinese hamster V79 cells, both chlorogenic acid and caffeic acid inhibited the mutagenicity of B[a]P 7,8-diol-9,10-epoxide-2 in the absence of S9. In in vivo studies, oral administration of chlorogenic acid to gamma-irradiated mice significantly reduced the incidence of micronuclei in bone marrow erythrocytes.

In in vitro immunotoxicity tests using rat mast cells, both chlorogenic and caffeic acid inhibited histamine release induced by compound 48/80 or by concanavalin A plus phosphatidylserine, although caffeic acid appeared to be more effective than chlorogenic acid. Chlorogenic acid reduced serum complement activity in normal human serum and caffeic acid reduced guinea pig serum complement activity. Caffeic acid also inhibited leukotriene production in mouse peritoneal macrophages.

Chlorogenic acid, administered by i.v. injection, did not induce allergic reactions in monkeys that were first sensitized by topical applications of sera from humans who were allergic to green coffee. In mice, topical application of chlorogenic acid, but not caffeic acid, inhibited TPA-induced edema of the ear. Similarly, i.p. injection of caffeic acid to rats inhibited edema induced by carrageenan or formalin.

Other data reviewed on chlorogenic and caffeic acid included antibacterial activity, cytotoxicity, effect on cell proliferation, effect on enzymes in vitro, hepatoprotective activity in vitro, inhibition of the nitrosation reaction in vitro, inhibition of oxidation in vitro, and miscellaneous effects identified in human studies. Caffeic acid was more potent than chlorogenic acid in its ability to inhibit nitrosamine formation and reduce nitrite levels in vitro. In contrast to caffeic acid, treatment with the O-methylated metabolite ferulic acid in the diet did not induce rat forestomach carcinogenesis, but ferulic acid, like caffeic acid, was found to be a potent antioxidant in vitro. Caffeic acid phenethyl ester was a more potent inhibitor of leukotriene production than caffeic acid in calcium ionophore A23187-stimulated murine peritoneal macrophages.