Cinnamyl anthranilate

Three of the compounds evaluated in this volume (di(2-ethylhexyl) phthalate, di(2-ethylhexyl) adipate and cinnamyl anthranilate) are carcinogenic to the liver in mice and/or rats, and have been proposed to act by a mechanism involving peroxisomal proliferation in hepatocytes in those species. The role of peroxisome proliferation in evaluating carcinogenicity in humans has been discussed (lARC, 1995b). When, for any chemical, the relationship between peroxisome proliferation and liver tumours in rats or mice has been established, this should be considered relevant information in the evaluation of the possible risks for cancer in humans, taking into account the following ... 

Cinnamyl anthranilate is not known to be currently commercially available. 

Cinnamyl anthranilate can be assayed by a method based on ester hydrolysis. Bulk samples of food-grade cinnamyl anthranilate have been analysed for purity by thin-layer chromatography and high-performance liquid chromatography. A method has been described for determining the content of this compound in food products by steam distillation followed by paper chromatography and examination under ultraviolet light it has a limit of detection of 1 pg (lARC, 1983). 

Cinnamyl anthranilate can be synthesized by esterification of anthranilic acid with cinnamyl alcohol (Burdock, 1995). Annual production in the United States in the 1970 was in the range of a few hundred kg (lARC, 1983). It has not been commercially available, except for research purposes, since 1985 (Lucas et al, 1999 Food and Drug Administration, 1999). 

In the second mouse lung adenoma assay, cinnamyl anthranilate was administered to 15 females and 15 males under similar experimental conditions, except that redistilled tricaprylin was used as the vehicle. The control group consisted of 80 females and 80 males. The numbers of tumours per mouse were increased at the high dose, being 0.85 0.23 (p < 0.01) in high-dose females, 1.40 0.36 p < 0.001) in high-dose males, 0.54 0.15 (p < 0.05) in low-dose females and 0.47 0.12 in low-dose males compared with 0.20 0.02 in control females and 0.24 0.03 in control males (Stoner etal., 1973). 

Five human volunteers took a single oral dose of 250 mg cinnamyl anthranilate in water and no unchanged compound was detected in the 0-24-h urine, using analytical methods able to detect 0.04% of the dose (Keyhanfar Caldwell, 1996). 

The metabolism of cinnamyl anthranilate in rats and mice has been studied (Keyhanfar Caldwell, 1996). Male Fischer 344 rats and male CD-I mice were given 250 mg/kg bw [3- C] cinnamyl anthranilate by intraperitoneal injection. The majority of the administered C was excreted in the 0-24-h urine (70% of the dose in rats and 78% in mice). A further 10% (rat) and 6% (mouse) was recovered in the 24-72-h urine with 10% (rat) and 7% (mouse) in the 0-72-h faeces. In the rat, the major urinary metabolite was hippuric acid (95% of urinary C), together with much smaller amounts of benzoic acid. However, in mice, the urine contained relatively less hippuric acid ( 80%) and more benzoic acid (16% of urinary i C), together with 2.2% of the dose as unchanged ciimamyl anthranilate. 

In further studies, the effect of intraperitoneal dose size upon the fate of cinnamyl anthranilate in mice was examined over a range of 5-250 mg/kg bw. No intact ester was found in the urine after 5 mg/kg bw, but at 50 mg/kg bw, 3.1 % of the dose was excreted as cinnamyl anthranilate and, at 250 mg/kg, the percentage was 2.2% (Keyhanfar Caldwell, 1996). 

In an earlier study, Caldwell etal. (1985) gave a single oral dose of 500 mg/kg bw to B6C3Fi mice and found 0.3-0.4% of the dose as unchanged cinnamyl anthranilate in the 0-24-h urine, accompanied by 17% as anthranilic acid and 35% as hippuric acid. Cinnamyl anthranilate was detected in the plasma, rapidly declining from peak levels seen at 0.5 h after dosing. The peak levels were some 3.5 times higher in males than in females. 

The influence of dose size was also examined in male and female B6C3Fi mice given 0, 10, 100, 1000, 5000, 15 000 or 30 000 ppm (mg/kg diet) ciimamyl anthranilate in the diet for four days (Caldwell et al., 1985). The urinary excretion of cinnamyl anthranilate, hippuric acid and anthranilic acid within 24-h after removal of the test diet rose with increasing cinnamyl anthranilate dose. Cinnamyl anthranilate was detected in increasing quantities in the urine of male mice at 1000 ppm and above. In females, it was only seen at 5000 ppm and above and the levels were two- to nine-fold lower. 

The hepatic effects of cinnamyl anthranilate were evaluated in male CD 1 mice and male Fischer 344 rats treated by intraperitoneal injection for three consecutive days (Viswalingam Caldwell, 1997). At doses of 100 and 1000 mg/kg bw per day, relative liver weights of mice increased by 22% and 50%, respectively, 24 h after the final dose and peroxisomal (cyanide-insensitive) palmitoyl-coenzyme A (CoA) oxidation activity increased fivefold at both levels. Microsomal lauric acid 11- and 12-hydroxylase activity (CYP4A) was increased 15-fold at 100 mg/kg bw per day and 17-fold at 1000 mg/kg bw per day. Limited evaluation indicated that cirmamyl anthmilate increased the size and number of peroxisomes in electron micrographs of hepatocytes of treated mice. In rats, relative liver weights and peroxisomal palmitoyl-CoA oxidation activity were significantly increased only at 1000 mg/kg bw per day (22% and twofold, respectively). 

In a separate experiment, groups of male CDl mice were given intraperitoneal injections of 0-200 mg/kg bw ciimamyl anthranilate daily for three days. At doses of 20 mg/kg bw and above, there were dose-dependent increases in relative liver weight, total cytochrome P450, and cyanide-insensitive palmitoyl-CoA oxidation. The hepatic effects of cinnamyl anthranilate are apparently due to the intact ester, since neither its expected metabolites alone nor an equimolar mixture of the hydrolysis products, cinnamyl alcohol and anthranilic acid, had a significant effect on the weight or marker enzyme content of mouse liver (Viswalingam Caldwell, 1997). 

Table 1. Comparison of responses in liver of female mice and rats following four weeks of cinnamyl anthranilate treatment...

Table 2. Genetic and related effects of cinnamyl anthranilate...

The weight of evidence for cinnamyl anthranilate and for other rodent peroxisome proliferators in general, demonstrates that they do not act as direct DNA-damaging... 

Chronic administration of peroxisome proliferators to rodents results in sustained oxidative stress due to overproduction of peroxisomal hydrogen peroxide. The induction of peroxisomal fatty acid P-oxidation by cinnamyl anthranilate in vivo under bioassay conditions (Lake et al, 1997) supports this h othesis. Other data on the induction of oxidative stress are not available for cinnamyl anthranilate. 

Similarly, the modulation of hepatocellular proliferation by peroxisome proliferators has been implicated in the mechanism of carcinogenesis. This can theoretically result in increased levels of mutation by increasing the frequency of replicative DNA synthesis as well as increasing the number of hepatocytes at risk. Furthermore, hepatocellular proliferation is likely to be involved in the promotion of growth of preneoplastic hepatocytes. There is clear evidence that cinnamyl anthranilate causes acute and sustained levels of hepatocellular proliferation under bioassay conditions which resulted in liver tumours in mice. Interestingly, the magnitude and duration of hepatocellular proliferation were limited in rats, which did not respond with liver tumours in the bioassay (Lake et al., 1997). 

Marked species differences in hepatic peroxisome proliferation have been reported (Ashby et al, 1994 lARC, 1995 Lake, 1995a,b Cattley et al, 1998). No study has yet compared the responsiveness of human versus rodent livers in vivo or hepatocytes in vitro to cinnamyl anthranilate however, a growing body of evidence concerning the molecular basis of peroxisome proliferation indicates that human livers and hepatocytes would be refractory to induction of peroxisome proliferation by cinnamyl anthranilate (Doull et al., 1999). 

Cinnamyl anthranilate produces liver tumours in mice. 

Under conditions of the bioassays, cinnamyl anthranilate induced peroxisome proliferation and cell replication in the liver that are characteristic of a peroxisome proliferator in mice and, to a limited extent, in rats. 

The species difference in peroxisome proliferation in response to cinnamyl anthranilate is associated with a species difference in its metabolism the compound is completely hydrolysed by rats but not mice. 

Hepatic peroxisome proliferation has not been evaluated in studies of human subjects or systems treated with cinnamyl anthranilate. However, interspecies comparisons with other peroxisome proliferators, along with the role of PPARa in this response, indicate that humans can reasonably be predicted to be refractory to induction of peroxisome proliferation and hepatocellular proliferation by cinnamyl anthranilate. This conclusion is further supported by the failure to detect intact cinnamyl anthranilate in the urine of human volunteers given large single doses. 

Overall, these findings suggest that the increased incidence of liver tumours in mice treated with cinnamyl anthranilate results from a mechanism that is not expected to operate in humans, although studies of human systems have not been performed with this compound. 

Cinnamyl anthranilate was tested for carcinogenicity in one experiment in mice and in one experiment in rats by oral administration in the diet. In mice, a dose-related increase in the incidence of hepatocellular tumours was found, but there was no increased incidence of tumours in rats. In a mouse lung tumour bioassay, an increased multiplicity of lung tumours was found. 

Cinnamyl anthranilate is metabolized by hydrolysis to anthranilic acid and cinnamyl alcohol, which is oxidized to benzoic acid. In mice, but not in rats or humans, the hydrolysis is saturated at high doses, leading to excretion of unchanged cinnamyl anthranilate in the urine. 

Cinnamyl anthranilate has the characteristic effects of a peroxisome proliferator on mouse liver, increasing the activity of peroxisomal fatty acid-metabolizing enzymes and microsomal CYP4A and increasing hepatocellular proliferation. These effects are mediated by the intact ester, and were not seen after administration of the hydrolysis products, cinnamyl alcohol and anthranilic acid. The corresponding effects on rat liver were very much weaker. No relevant data from humans were available. 

The only standard genotoxicity assay in which cinnamyl anthranilate was active was the mouse lymphoma mutation assay. 

No epidemiological data relevant to the carcinogenicity of cinnamyl anthranilate were available. 

There is limited evidence in experimental animals for the carcinogenicity of cinnamyl anthranilate. 

Cinnamyl anthranilate is not classifiable as to its carcinogenicity to humans (Group 3). 

Caldwell, J. (1992) Problems and opportunities in toxicity testing arising from species differences in xenobiotic metabolism. Toxicol. Lett., 64/65, 651-659 Caldwell, J., Anthony, A., Cotgreave, I.A. Sangster, S.A., Sutton, J.D., Bernard, B.K. Ford, R.A. (1985) Influence of dose and sex on the disposition and hepatic effects of cinnamyl anthranilate in the B6C3F1 mouse. Food chem. Toxicol., 23, 559-566 Cattley, R.C., DeLuca, J., Elcombe, C., Fenner-Crisp, R, Lake, B.G, Marsman, D.S., Pastoor,... 

Lake, B.G (1995b) Mechanisms of hepatocarcinogenicity of peroxisome-proliferating drags and chemicals. Ann. Rev. Pharmacol. Toxicol., 35, 483-507 Lake, B.G, Price R.J., Cunninghame, M.E. Walters D.G (1997) Comparison of the effects of cinnamyl anthranilate on hepatic peroxisome proliferation and cell replication in the rat and mouse. Fundam. appl. Toxicol., 39, 60-66... 

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