National Toxicology Program

V. TOXICOLOGICAL EFFECTS

A. Acute

1. Animal Data

Exposure to Cinnamaldehyde had been found to affect the central nervous, cardiovascular, and digestive systems. This compound has also reportedly caused contact urticaria, diarrhea, depression and coma in animals following acute exposure.

Cinnamaldehyde has been found to have both inhibitory and excitatory effects on the central nervous system of mice. Intraperitoneal administration of this compound at doses higher than 100 mg/kg was observed to cause a transient excitation (running fit) followed by a depression in activity [72].

Cinnamaldehyde has been observed to affect the cardiovascular system of dogs and guinea pigs. Intravenous administration of 5-10 mg/kg to male and female Mongrel dogs was found to reduce blood pressure and increase respiratory rate and femoral blood flow. Heart rate was observed to increase simultaneously with the fall in blood pressure, and thereafter to return to baseline.

A fall in blood pressure was also observed in male guinea pigs following intravenous administration of Cinnamaldehyde at a dose of 1 mg/kg. Heart rate was lowered by 15 percent following administration of this compound at a dose of 5 mg/kg, while femoral blood flow was observed to increase. In experiments using isolated guinea pig hearts, Cinnamaldehyde administered at doses ranging from 50 to 500 mg was found to increase heart beat rate and to induce arrhythmias at does greater than 250 mg.

Cinnamaldehyde has also been observed to affect the digestive systems of rats and mice. In male, dd mice, Cinnamaldehyde was found to have an inhibitory effect on intestinal propulsion following intraperitoneal administration at a dose of 250 mg/kg. In addition, Cinnamaldehyde was observed to decrease stress-induced gastric erosion at an intraperitoneal dose of 250 mg/kg. In male, Wistar rats, this compound was found to inhibit spontaneous gastric contraction at an intravenous dose of 5 mg/kg. Oral administration of Cinnamaldehyde at a dose of 500 mg/kg reportedly increased biliary excretion. Cinnamaldehyde did not change the pH value of gastric perfusate at intravenous doses up to 10 mg/kg [18].

Cinnamaldehyde has been found to induce nonimmunologic contact urticaria in guinea pigs, rats and mice, with symptoms ranging from slight erythema to extensive local erythema and edema accompanied by tingling, burning and itching, following application of a 20% solution to the earlobes. The thickness of the earlobes was measured before, during and after the application. Maximal ear swelling was observed 20 to 50 minutes after the application of Cinnamaldehyde and reportedly decreased during the three-hour observation period [32].

Acute expose to Cinnamaldehyde has been found to cause diarrhea and depression in rats. High, acute doses of this compound have induced coma in rats [48]. Acute systemic toxicity values for Cinnamaldehyde are presented in Table 1.

2. Human Data

Acute exposure to Cinnamaldehyde may result in skin, eye [58], respiratory [47] and gastrointestinal irritation. Systemic effects from acute exposure are believed to be limited [16]. Acute toxicity data available for Cinnamaldehyde is restricted primarily to this compound's effect on the skin.

Cinnamaldehyde has been found to cause severe skin irritation followingapplication of 40 mg for 48 hours [48]. A 3 percent solution of Cinnamaldehyde in petrolatum was not found to cause skin irritation after a 48 hour closed-patch test on humans. However, an 8 percent solution was found to be severely irritating to the skin, and the concentration had to be reduced to 2 percent for the test to be completed [56].

The acute toxicity of Cinnamaldehyde has been assessed in vitro using cultured human KB cells. A dose response curve was obtained following a 72-hour, KB cell exposure to various concentrations of Cinnamaldehyde. The 72-hour ID₅₀¹ was determined to be 19.50 mg/ml. This was compared to a 72-hour ID₅₀ value of 70.0 mg/l for Saccharo- myces cerevisie tested under identical conditions[43].

2. Case Reports

Cinnamaldehyde has been found to cause contact urticaria in children. Children being treated for contact urticaria were patch tested for skin reaction to a variety of fragrances and food additives. Children who developed palpable pruritic erythema 20 minutes after exposure were considered positive for contact urticaria reactions. Twelve out of 125 children reportedly had a positive patch test result for Cinnamaldehyde [60].

B. Subchronic/Chronic

1. Animal Data

The data available in the literature concerning the subchronic and chronic toxicology of Cinnamaldehyde in animals primarily concerns the sensitizing effect of this chemical. The contact sensitization potential of Cinnamaldehyde has been tested in female, Balb/C mice maintained on a diet supplemented with vitamin A acetate². The sensitization protocol included an induction period of two weeks followed by a total of six topical applications of a 30 percent Cinnamaldehyde solution to the shaved abdomen and thorax. This was followed one week later by a topical challenge of 15 percent Cinnamaldehyde to both ears. Ear thickness was measured before the challenge as well as 24 and 48 hours after the challenge. The percent increase in ear thickness was determined, and the statistical significance of increased ear thickness was assessed by the Mann Whitney U test. A compound was classified as a sensitizer if the Mann Whitney test was significant at P less than or equal 0.01 or the Mann Whitney test was significant at P not greater than 0.05, and in addition 2 animals had increases in ear thickness twice that of the highest control increase.

One mouse from the group of ten tested was found to have an increase in ear thickness 24 hours after the challenge that was 100 percent greater than the highest increase in the control group, while six mice had increases in ear thickness after the challenge that were determined to be 50 percent greater than the highest increase in the control group. The Mann Whitney test was found to be significant at P< 0.01, classifying Cinnamaldehyde as a contact sensitizer [36].

Effects observed following dietary administration of Cinnamaldehyde to male and female rats over a sixteen week period at a concentration of 10,000 ppm include slight hyperkeratosis of the squamous portion of the stomach lining and slight swelling of the hepatic cells. When administered at doses of 2 mg on alternate days to two generations of rats for 223 and 210 days respectively, Cinnamaldehyde was found to cause an increase in liver weight by 20 percent in the first generation and 22 percent in the second.

The maximum tolerated dose (MTD) of Cinnamaldehyde defined as the maximum single dose tolerated by a group of five mice following six intraperitoneal injections over a two week period was determined to be 0.25 g/kg [56].

2. Case Reports

Numerous case reports describe the skin sensitization potential of Cinnamaldehyde in humans. Skin sensitization has been found to occur following both occupational and consumer exposure to this compound. In some cases, the skin sensitization caused by Cinnamaldehyde has been found to be permanent [58]. The following cases of chronic contact dermatitis from occupational exposure to Cinnamaldehyde are presented in the literature:

A case of allergic contact dermatitis from exposure to Cinnamaldehyde at an air freshener manufacturing plant has been reported. A 43 year old man who had no history of non-occupational exposure to perfumed products developed an itchy eruption on his fingertips which began one month after he began working at the plant. The eruption was confined to his hands and consisted of erythematous scaling patches with indistinct borders on the fingertips and the dorsal surfaces of both hands. In his job, the employee added various fragrances to a dispensing machine that subsequently applied the fragrances to pads used to make household air freshening devices. In addition, the employee served as a maintenance person and was frequently exposed to full-strength perfume concentrates from malfunctioning equipment.

Patch tests were performed on the employee using the European Standard Patch Test Series. The worker only developed an allergic response to Cinnamaldehyde. The eight fragrance concentrates to which the worker was exposed were subsequently analyzed for their Cinnamaldehyde content; three of the eight fragrances were found to have detectable levels of Cinnamaldehyde. It was concluded that the allergic contact dermatitis most likely resulted from repeated skin contamination with full-strength perfume concentrates [52].

An increased incidence of fragrance-related occupational dermatitis among a group of coal miners being treated for eczematous skin problems has been documented. Thirty five miners, 55 male non-miners and 30 female non-miners were patch tested over a period of eighteen months using the International Contact Dermatitis Research Group (ICDRG) Standard Series. Forty-five percent of the coal miners were found to be fragrance sensitive while 20 percent of the male, and 13 percent of the female non-miners had positive patch test results. Cinnamaldehyde reportedly caused the highest number of positive responses among the male miners and the male non-miners tested; 14 of the miners and 7 of the non-miners developing positive patch test results after 96 hours. The increased incidence of allergic contact dermatitis among the coal workers is believed to be related to a highly perfumed body lotion used at the coal mine [15].

A high incidence of occupationally-related allergic skin reactions was also reported among factory workers in a Danish spice manufacturing plant. Almost all of the workers exposed to high concentrations of Cinnamaldehyde during the manufacture of cinnamon spice substitutes developed sensitivity to Cinnamaldehyde [56].

During an eight-year study, 66 hairdressers who were being treated by dermatologists for contact dermatitis were patch tested to the North American Contact Dermatitis Group Standard Screening Trays and to a hairdressers' screening tray. Cinnamaldehyde was found to produce allergic skin reactions in 1.5% of the hairdressers tested [34].

The following cases of chronic contact dermatitis from consumer exposure (toothpaste, cosmetics, fragrances) to Cinnamaldehyde are reported in the literature:

Over a six-month period, a 25 year-old woman reportedly developed perioral leukoderma caused by a Cinnamaldehyde-containing toothpaste. The leukoderma around the woman's mouth began at the oral commissures and had spread above and below the lips. Porcelain-white depigmentation of the skin lateral to the oral commissures was observed. In addition, leukoderma of the perioral skin adjacent to the borders of her lips was marginated by a thin border of hyperpigmentation. Patch testing was performed using the routine screening series of the North American Contact Dermatitis Group (NACDG) which included a 2 percent solution of Cinnamaldehyde in petrolatum. A positive (2+) papular reaction to Cinnamaldehyde was observed 48 and 96 hours after exposure.

It was subsequently determined that two years before the onset of the leukoderma, the woman had begun using a Cinnamaldehyde-containing toothpaste. Six months after she switched to a non-Cinnamaldehyde-containing toothpaste, the perioral leukoderma almost completely disappeared [41].

Consumer exposure to Cinnamaldehyde has reportedly caused chronic cheilitis in an 82 year-old woman who had been using both a Cinnamaldehyde-containing toothpaste and a sunscreen lipstick. The woman's symptoms consisted of cracking, swelling and peeling lips, but no cutaneous lesions were observed. Patch testing with the standard fragrances and preservative series utilizing ICDRG standard techniques resulted in a positive reaction only to Cinnamaldehyde. When the woman stopped using the Cinnamaldehyde-containing toothpaste and lipstick her symptoms cleared [35].

Two case reports of cosmetic intolerance among persons being treated for chronic contact dermatitis are described in the literature. In one study, 5202 patients were patch tested using the Belgian Tri-Contact Patch Test Series. Eight percent of the total test population reacted positively to cosmetic patch tests. Perfumes were the principal allergens observed in the group of patients who suffered from pure allergies to cosmetics (156 patients). Of these cases, 5.1 percent were attributed to Cinnamaldehyde.

In the second study, 182 patients suspected of suffering from contact sensitization to cosmetics were patch tested using the standard tray of the ICDRG as well as 22 fragrance raw materials. Cinnamaldehyde was found to produce positive results in 3.7 percent of the patients tested [38].

Over a period of more than three years, 2826 patients at the Göttingen University Hospital for Skin Diseases were tested for skin sensitivity to Cinnamaldehyde. Only 0.74 percent of the patients (21) reacted positively to Cinnamaldehyde. It was noted by the authors that in countries other than Germany, especially England and the United States, allergy to Cinnamaldehyde occurs more frequently. The discrepancy is presumably a result of the variation in consumer exposure to Cinnamaldehyde between different countries [67].

C. Carcinogenicity

1. Animal Data

There are limited data available concerning the carcinogenicity of Cinnamaldehyde in animals. Cinnamaldehyde has been tested for its hepatocarcinogenicity in male, B6C3F₁ mice following injection on days 1, 8, 15 and 22 prior to weaning. The concentration of Cinnamaldehyde injected per dose was in the ratio of 1:2:4:12 respectively, for a total dose of 4.8 mmol per mouse. Cinnamaldehyde showed no hepatocarcinogenic activity at the dose levels tested [77].

The remaining information on the carcinogenic effects of this compound concerns its transforming capacity. The transforming potency of Cinnamaldehyde has been demonstrated by in vitro studies using Chinese hamster epithelial cells (CH-B241). The CH-B241 cells were treated with sublethal doses of Cinnamaldehyde (10nM), and the surviving cells were cultivated until they acquired characteristics typically associated with transformed cells; namely 1.) an increase in saturation density in the monolayer culture, 2.) an increase in plating efficiency at a low serum level, or 3.) an increase in colony forming efficiency in soft agar medium. The treated CH-B241 cells that met these in vitro criteria were subsequently analyzed for their ability to induce neoplastic transformation. This was achieved by subcutaneous injection of 1 x 10⁶ cells into a suprascapular region of male, nude mice (BALB/C, JCL, NuNu).

Formation of nodules at the injection site was observed in six out of seven mice treated with Cinnamaldehyde-transformed cells. One mouse produced nodules in the liver and spleen, indicating metastasis. The nodules were first palpable between days 91 and 237 after injection, after which they grew slowly to 2 cm in diameter until day 311. When the tumors at the injection site reached 2 cm in diameter, the animals were sacrificed and the tumors were removed for histological examination. Microscopic examination revealed that the tumors were malignant and consisted of cells with random shaped nuclei and a high frequency of mitosis. Karyotype analysis demonstrated that approximately 45 percent of the tumor cells were polyploid.

In addition, tumors were aseptically removed from the mice, and cells from the tumors were re-injected into mice in order to assess serial transplantability. Tumor formation was observed at the injection site in all animals tested within a considerably shorter latent period (17 to 114 days) than that observed following the primary inoculation. Metastasis of the spleen was observed in three out of four animals injected with tumor cells from the Cinnamaldehyde-treated mice.

Although the in vitro transforming potency of Cinnamaldehyde was demonstrated, the induction mechanism is unclear. Direct or indirect interaction with genetic material is presumably involved because considerable structural chromosomal aberrations, including chromosome and/or chromatid breaks, were observed [27, 29].

Cinnamaldehyde has been tested for its capacity to enhance the transformation of Syrian hamster embryo cells by Simian adenovirus, SA7. Various sub-lethal doses (0.01 mm, 0.02 mm, 0.05 mm, 0.09 mm, 0.19 mm) were diluted in cell culture medium and added to replicate dishes of Syrian hamster embryo cells for 20 hours. After 20 hours, the cells were rinsed and SA7 virus was absorbed for 3 hours. The number of colonies from Cinnamaldehyde and virus treated cells were determined. This number was divided by the number of colonies from virus inoculated control cells in order to determine the surviving fraction. The number of SA7 foci from 2 x 10⁶ plated cells was determined and the enhancement ratio was calculated by dividing the transformation frequency of treated cells by the transformation frequency of the control cells. The Cinnamaldehyde-induced enhancement was found to be statistically significant (P less than or equal 0.05 or P less than or equal 0.01 ) at only one dose level 0.05mM (see Table 2). Therefore, based on standard classification criteria, it was concluded that there is "some evidence" that Cinnamaldehyde enhances viral transformation [21].

2. Human Data

There are no data available on the carcinogenicity of Cinnamaldehyde in humans. However, the in vitro transforming potency of this chemical has been studied. Cinnamaldehyde was not found to induce transformation of the human fibroblast cell line HAIN-55 following treatment with various concentrations ranging from 5-80 nM [29].

D. Mutagenicity/Genetic Toxicology

1. Animal Data

There are conflicting reports concerning the mutagenicity of Cinnamaldehyde. This compound has been found to be mutagenic to Bacillus subtilis, Drosophila melanogaster, Chinese hamster ovary cells, mouse leukocytes, hamster fibroblasts, and Salmonella typhimurium (strain TA100). However, other sources report that Cinnamaldehyde was non-mutagenic to rat hepatocytes, Escherichia coli and several strains of Salmonella typhimrium, including TA100.

Standard Ames reverse mutation assays were carried out using Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of liver microsome fraction Cinnamaldehyde was added at six different concentrations (10, 20, 50, 100, 200 and 500 mg/ml) per plate, and the number of revertant colonies was scored after incubation at 37°C for two days. Cinnamaldehyde induced 222 revertants at 0.5 mg/plate as compared to 146 in the control plates and 318 revertants (139 in the control) at 0.1 mg/plate in strain TA100 with and without metabolic activation, respectively (see Figure 1). Cinnamaldehyde was non-mutagenic in the other Salmonella strains tested [25].

Cinnamaldehyde has been found by other authors to be non-mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 as well as TA100 in the presence and absence of metabolic activation [45, 59].

FIGURE 1: DOSE RESPONSE CURVE FOR CINNAMALDEHYDE EVALUATED IN THE AMES TEST WITH SALMONELLA STRAIN TA100

•Assay performed without S-9
o Assay performed with S-9

Cinnamaldehyde was also reportedly nonmutagenic to S. typhimurium strain TA104 in the absence of metabolic activation [40].

In order to detect its DNA-damaging potential, Cinnamaldehyde was tested in the spore rec- assay with Bacillus subtilis strains M45 (rec-) and H17 (rec+). The DNA damaging activity was assessed by growth inhibition zone measurements. Cinnamaldehyde was found to be mutagenic at a maximal dose of 10 ml per disk [80].

In addition, Cinnamaldehyde has been tested for its mutagenic activity in germ cells of Drosophila melanogaster using the sex linked recessive lethal mutation and the reciprocal translocation tests. Cinnamaldehyde was negative in the recessive lethal mutation test when tested by adult feeding methods. However, when tested by adult injection at 20,000 ppm, Cinnamaldehyde was found to induce sex-linked recessive lethal mutations in meiotic and post-meiotic germ cell stages. Cinnamaldehyde was negative in the reciprocal translocation test [78].

Cinnamaldehyde has been found to induce chromosomal aberrations in Chinese hamster fibroblast cells at concentrations of 0.01 mg/ml after a 48 hour exposure and 0.015 mg/ml following a 24 or 48 hour exposure in the absence of metabolic activation. In order to obtain a quantitative evaluation of the clastogenic potential of Cinnamaldehyde, the D₂₀⁴ and TR⁵ values were calculated. Cinnamaldehyde was determined to be mutagenic at relatively low dose levels (D₂₀=0.01) and was found to have the highest TR value (TR=2133) among a total of 190 food additives tested. TR values are generally reported to be high for chemicals having carcinogenic potential in animals [25].

Cinnamaldehyde has been tested for its ability to induce sister chromatid exchange in Chinese hamster ovary cells in the presence and absence of metabolic activation. Cinnamaldehyde was found to be weakly positive in the sister chromatid exchange (SCE) test with and without metabolic activation, at a least effective concentration (LEC)⁶ of 0.34 mg/ml (See Table 3). In the test system without metabolic activation, a low dose of mitomycin C was used as a "weak positive" control. In test system with metabolic activation, a low dose of cyclophosphamide was utilized. These "weak positive" controls were designed to give a small (20-40%) increase in SCEs and were included to assess the ability of the system to detect small increases in sister chromatid exchange. There was no evidence that Cinnamaldehyde induced chromosomal aberrations in Chinese hamster ovary cells [13]. Cinnamaldehyde has been found to cause DNA inhibition in mouse leukocytes in vitro when tested in the L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay assay. In the presence and absence of activation, Cinnamaldehyde reportedly induced a "questionable" mutagenic response [57]. No additional information was provided.

Cinnamaldehyde was not mutagenic in an in vivo test for the induction of unscheduled DNA synthesis in rat hepatocytes following administration by gavage [42]. In addition, Cinnamaldehyde did not cause micronucleus induction in an in vivo micronucleus test with bone marrow mouse cells [22].

2. Human Data

There are no data available in the literature concerning the mutagenicity of Cinnamaldehyde in humans.

E. Teratology/Reproductive Toxicology

1. Animal Data

The reproductive effects of Cinnamaldehyde have been examined in rats and mice, and in both species Cinnamaldehyde was found to be negative for all parameters tested. However, there are conflicting reports concerning the teratogenic effects of Cinnamaldehyde.

Teratogenic parameters have been evaluated following administration of Cinnamaldehyde to pregnant, CD-1 mice at a dose level of 1,200 mg/kg/day in corn oil. Parameters included the number of females producing viable litters, the number of females with resorbed or nonviable litters, the number of proven pregnant females and the reproductive index⁷. In addition, group litter and viability data were evaluated, including the number of live pups per litter, the number of dead pups per litter, the litter weight and the mean pup weight. No significant differences from the control group were observed in any of the criteria examined [23].

In another study, CD-1 mice were dosed by gavage at 1,200 mg/kg/day of Cinnamaldehyde during mid-pregnancy. Litter size, birth weight, neonatal growth and survival to postnatal day three were recorded as indices of potential developmental toxicity. Both the maternal response variables and the neonatal response variables tested were not found to differ significantly from the control [20].

Cinnamaldehyde was not found to affect body weight gain, reproductive ability, or the development and viability of offspring following administration of 2 mg on alternate days to two generations of rats for 223 and 210 days respectively [56].

Suprablastodermic administration of a single dose of Cinnamaldehyde to 3 day-old chick embryos (white Leghorn x Rhode Island red strain) was reportedly teratogenic. The Optimal Teratogenic Dose (OTD)⁸ was found to be 0.50 mM per embryo. At this concentration, the most common teratogenic effects observed included limb malformations, primarily limb size reduction. Malformations of the axial skeleton including spina bifida, anoura (tail absence) or haemisomia were noted in several cases [1].

2. Human Data

There are no data available in the literature concerning the reproductive or teratogenic effects of Cinnamaldehyde on humans.

F. Immunotoxicity

1. Animal Data

There are no data available in the literature concerning the Immunotoxicity of Cinnamaldehyde in animals.

2. Human Data

There are no data available in the literature concerning the immunotoxicity of Cinnamaldehyde in humans.

¹ The ID₅₀ value represents the dose required to inhibit cell growth by 50%.

² Vitamin A acetate was added to the diet in order to amplify the presentation of immunogenic agents.

⁴ The D₂₀ value represents the dose (mg/ml) at which structural abberations, including gaps, were detected in 20% of the metaphase chromosomes observed.

⁵ The TR value indicated the frequency of cells with exchange type aberrations per unit dose (mg/ml).

⁶ The least effective concentration (LEC) represents the lowest dose to give a statistically significant increase in aberrations or a 20% increase in SCEs.

⁷ The reproductive index is a measurement of the number of females that produced viable litters, divided by the number of proven pregnant females (multiplied by 100).

⁸ The Optimal Teratogenic Dose (OTD) is defined as the concentration that induces a maximum teratogenic effect beyond the limits of the embryonic LD₅₀.