Methyl cinnamate

Carcinogenicity

Description of key information

Cinnamaldehyde following the same metabolic pathway as methyl cinnamate via cinnamic acid as primary metabolite was non carcinogenic when investigated in a chronic study with rats and mice.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-conducted study, minor restrictions in design and reporting. Cinnamaldehyde is metabolized through cinnamic acid, as well as methyl cinnamate. Thus, the study conducted with test article is accepted to be used for read-across.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

not specified

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female F344/N rats were obtained from Taconic Laboratory Animals and Services (Germantown, NY) for use in the 2-year studies. Rats were quarantined for 11 (males) or 12 (females) days before the beginning of the studies. Five male and five female rats were randomly selected for parasite evaluation and gross observation of disease. Rats were approximately 6 weeks old at the beginning of the studies. The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program.
Cages
Polycarbonate (Lab Products, Inc., Maywood, NJ) changed twice weekly (rats and female mice) or once weekly (male mice)
Bedding
Sani-Chips® (P.J. Murphy Forest Products Corp., Montville, NJ), changed twice weekly (rats and female mice) or once weekly (male mice)
Cage Filters
Dupont 2024 spun-bonded polyester (Snow Filtration Co., Cincinnati, OH), changed every 2 weeks
Racks
Stainless steel (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 weeks
Animal Room Environment
Temperature: 72° ± 3° F
Relative humidity: 50% ± 15%
Room fluorescent light: 12 hours/day
Room air changes: 10/hour

Route of administration:

oral: feed

Vehicle:

other: empty starch microcapsules

Details on exposure:

The dose formulations were prepared at least every 3 weeks by mixing microencapsulated trans-cinnamal­dehyde with irradiated NTP-2000 feed during the 2-year studies. Placebo and/or loaded microcapsules were combined with feed to a concentration of 1.25% (2-year studies) in the diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations used during the 2-year studies were conducted by the study laboratory using HPLC. During the 2-year studies, the dose formulations were analyzed approximately every 9 to 12 weeks; animal room samples of these dose formulations were also analyzed. Based on the original criteria, all formulations were within 10% of the target concentration.

Duration of treatment / exposure:

Rats: 105 or 106 (females) weeks

Frequency of treatment:

once daily

Post exposure period:

not applicable

Remarks:

Doses / Concentrations:
1000, 2100, and 4100 ppm.
Basis:
nominal in diet

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 50 male and 50 female rats were fed diets containing 1000, 2100, or 4100 ppm microencapsulated trans-cinnamaldehyde for 104 to 105 (males) or 105 to 106 (females) weeks. Additional groups of 50 male and 50 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls).

Positive control:

no data

Observations and examinations performed and frequency:

Observed twice daily; animals were weighed initially, on day 8, day 36 (mice), every 4 weeks thereafter, and at the end of the studies. Clinical findings were recorded on day 36, every 4 weeks thereafter, and at the end of the studies. Feed consumption was recorded by cage for a 1-week period approximately every 4 weeks.

Sacrifice and pathology:

Method of Sacrifice:
Carbon dioxide asphyxiation
Necropsy:
Necropsies were performed on all study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.
Histopathology:
Complete histopathology was performed on all rats and mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and/or mesenteric), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
Hippuric Acid – Biomarker of Exposure:
Urine was collected during a 24-hour period from 10 male and 10 female rats from each group at 2 weeks and 3, 12, and 18 months. Parameters evaluated included creatinine and hippuric acid concentrations and volume.

Other examinations:

not applicable

Statistics:

Survival Analyses:
Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.
Analysis of Neoplasm and Nonneoplastic Lesion Incidences
The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance of lower incidences or decreasing trends in lesions is represented as 1-P with the letter N added (e.g., P=0.99 is presented as P=0.01N).
Calculation of Incidence
This survival­adjusted rate (based on the Poly-3 method described below) accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on study, to animals that do not reach terminal sacrifice.
Analysis of Continuous Variables
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, and urinalysis data, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Survival of 4100 ppm males was greater than that of the vehicle control group

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival of 4100 ppm males was greater than that of the vehicle control group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of 4100 ppm males were less than those of the vehicle controls throughout the study

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption by 2100 and 4100 ppm males and 4100 ppm females was less than that by the vehicle controls at the beginning and end of the study

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The incidence of mononuclear cell leukemia in 4100 ppm males was significantly decreased.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Survival of 4100 ppm males was greater than that of the vehicle control group; survival of other exposed groups of males and of exposed females was similar to that of the vehicle control groups.
Mean body weights of 4100 ppm males were less than those of the vehicle controls throughout the study, mean body weights of 2100 ppm males were less after week 94, and mean body weights of 4100 ppm females were less after week 18. Feed consumption by 2100 and 4100 ppm males and 4100 ppm females was less than that by the vehicle controls at the beginning and end of the study. Dietary concentrations of 1000, 2100, or 4100 ppm delivered average daily doses of approximately 50, 100, or 200 mg/kg body weight to males and females. There were no clinical findings related to test article exposure.
Preputial and Prostate Glands: The incidences of adenoma of the preputial gland (vehicle control, 5/50; 1000 ppm, 1/49; 2100 ppm, 2/50; 4100 ppm, 0/50) and prostate gland (4/50, 0/49, 0/49, 0/50) in 4100 ppm males were significantly decreased compared to those in the vehicle controls. The incidences of preputial gland adenoma in the exposed and vehicle control groups were within the historical range in controls (all routes) given NTP-2000 diet [45/907 (4.2% ± 3.5%), range 0%-13%] . Similarly, the incidences of carcinoma of the preputial gland (1/50, 2/49, 3/50, 1/50) were within the historical range in controls given NTP-2000 diet [27/907 (3.3% ± 3.0%), range 0%-10%]. The incidence of prostate gland adenoma in the vehicle controls (4/50) exceeded the historical control range [13/906 (1.4% ± 1.7%), range 0%-4%] (Suwa et al., 2001).
Mononuclear Cell Leukemia: The incidences of preputial and prostate gland adenomas likely represent biologic variation unrelated to exposure to trans-cinnamaldehyde. Mononuclear Cell Leukemia: The incidence of mononuclear cell leukemia in 4100 ppm males was significantly decreased (18/50, 15/50, 21/50, 9/50; ), was considered unrelated to trans-cinnamaldehyde exposure, and may have contributed to the increased survival in this group. The historical control incidence for vehicle controls given NTP-2000 diet is 401/909 (44.1% ± 11.8%) with a range of 22% to 68%. Mononuclear cell leukemia is one of the most common neoplasms of F344/N rats in 2-year studies.

Relevance of carcinogenic effects / potential:

Under the conditions of this 2-year feed study, there was no evidence of carcinogenic activity of test article in male or female F344/N rats exposed
to 1000, 2100, or 4100 ppm.

Dose descriptor:

other:

Sex:

male/female

Basis for effect level:

other: Under the conditions of this 2-year feed study, there was no evidence of carcinogenic activity of test article in male or female F344/N rats exposed to 1000, 2100, or 4100 ppm.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Conclusions:

It is concluded that test item had no carcinogenicity potential for mammalian animals under the available conditions.

Executive summary:

This study was performed to investigate the carcinogenicity potential of test item.Groups of 50 male and 50 female F344/N rats were fed diets containing 1000, 2100, or 4100 ppm microencapsulated trans-cinnamaldehyde for 2 years. Additional groups of 50 male and 50 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 1000, 2100, or 4100 ppm delivered average daily doses of approximately 50, 100, or 200 mg/kg to males and females. Survival of 4100 ppm males was greater than that of the vehicle controls. Mean body weights of 4100 ppm males and females were generally less than those of the vehicle controls throughout the study. Feed consumption by 2100 and 4100 ppm males and 4100 ppm females was less than that by the vehicle controls at the beginning and end of the study. There were no neoplasms or non-neoplastic lesions that were attributed to exposure to trans-cinnamaldehyde.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Quality of whole database:

The non-adverse findings in this study were supported by the equivalent study using mice instead of rats with virtually the same outcome.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on data from a read-across substance, classification of methyl cinnamate for carcinogenicity is not required according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC).

Additional information

The carcinogenicity study was performed by the US National Toxicology Program NTP to investigate the carcinogenicity potential of cinnamic aldehyde, as this substance is widely used in food, drinks and cosmetics. Groups of 50 male and 50 female F344/N rats and mice were fed diets containing 1000, 2100, or 4100 ppm microencapsulated trans-cinnamaldehyde for 2 years. Additional groups of 50 male and 50 female rats and mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls). Dietary concentrations of 1000, 2100, or 4100 ppm delivered average daily doses of approximately 50, 100, or 200 mg/kg to males and female rats respectively 125, 270, or 550 mg/kg to males and female mice.

Survival of 4100 ppm male rats was greater than that of the vehicle controls, whereas survival of male rats in the 2100 ppm group was less than that of the respective vehicle control. There were no neoplasms or non-neoplastic lesions that were attributed to exposure to trans-cinnamaldehyde, neither in rats nor in mice.

 

The primary metabolite of methyl cinnamate is cinnamic acid and this holds true also for cinnamaldehyde. Therefore, the read-across to the carcinogenicity toxicity study with cinnamaldehyde is justifiable (see further details below). Regards carcinogenicity, cinnamaldehyde, undergoing the same metabolic pathway via cinnamic acid as methyl cinnamate, was investigated in a published carcinogenicity study using rats and mice (two endpoint study records) and with both species no such effects were observed. Thus, it can be concluded that also methyl cinnamate would not pose carcinogenicity effects.

 

Hypothesis for the analogue approach from Cinnamaldehyde to methyl cinnamate

Both, cinnamaldehyde and cinnamic esters such as methyl cinnamate are readily absorbed in the gastrointestinal tract. Whereas cinnamaldehyde is oxidised to cinnamic acid in the gastrointestinal tract, methyl cinnamate becomes enzymatically cleaved by non-specific esterase to cinnamic acid and methanol. Methanol follows established de-toxification pathways whereas the cinnamic acid mainly follows metabolism to benzoic acid and is rapidly excreted (Nutley et al, 1994).

Source Chemical and Target chemical

	Cinnamaldehyde	Methylcinnamate
CAS No	104 -55 -2	103 -26-4
Mol. weight	132.16	162.19
Smiles code	O=CC=Cc(cccc1)c1	O=C(OC)C=Cc(cccc1)c1
Water solubility	soluble (10 g/L)	moderately soluble (286 mg/L)
logPOW	1.83	2.68
Melting point	-7.5 °C	34.9 °C
Purity	Typically >99 %	Typically >99%

Analogue Approach Justification

Both, cinnamic acid and methyl cinnamate are well absorbed in the gastrointestinal tract and metabolistic studies have shown that >90% labelled material can be found mainly in urine of rats within 72 hours whereas the remainder is found in feces as hippuric acid and benzoyl glucuronide.

In general, ester compounds are hydrolysed in mammals. Ester hydrolysis is catalysed by classes of enzymes recognised as carboxylesterases or acetylesterases (Heymann, 1980; WHO, 1999). These enzymes occur in most mammalian tissues (Heymann, 1980; WHO, 1999) but predominate in hepatocytes (Heymann, 1980). In humans, methyl cinnamate is anticipated to be hydrolysed to cinnamic acid and methanol.

Cinnamaldehyde is oxidized to cinnamic acid too and hence, both cinnamaldehyde and methyl cinnamate do share the same primary metabolite, cinnamic acid. This is supported by comparable metabolism and distribution as outlined in the toxicokinetic assessment, also addressing metabolism of methyl cinnamate and cinnamic acid. Further on, this analogues approach was used by the World Health Organisation (see WHO FOOD ADDITIVES SERIES 46:Cinnamyl Alcohol and Related Substances) and the European Food Safety Authority (see SCIENTIFIC OPINION Flavouring Group Evaluation 15, Revision 2 (FGE.15Rev2): Aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives from chemical group 22).

Also, the comparable acute toxicity of both substances support this approach. Whereas the LD50 (oral rat) of cinnamaldehyde was determined being 2220 mg/kg bw (16.8 mmol), methyl cinnamate was found having an oral LD50 (rat) of 2610 mg/kg bw (16.1 mmol) and thus showing almost identical acute oral toxicity on a molar basis, which is indicative of comparable absorption and metabolism.

Conclusion on bioavailability and metabolism of source and target substance

Both, cinnamaldehyde and methyl cinnamate are well absorbed and metabolized to cinnamic acid in the gastrointestinal tract. Thus, their primary metabolite is identical following established metabolistic pathway for further metabolisation and excretion as outlined by WHO (see attachment).

Considerations on exposure to methanol released by hydrolysis of ester

The hydrolysis of methyl cinnamate in organisms releases cinnamic acid and methanol. Since methanol will be released by an enzyme-mediated step, it will be available only within the body and not instantaneously. Potential acute and local effects of methanol thus do not need to be considered. The available DNELs for methanol derived for systemic effects should be considered. Each mol of hydrolysed methyl cinnamate releases one mol of cinnamic acid and one mol of methanol. Methanol is known to exhibit significant toxicity in humans. Human exposure to the read-across substances cinnamaldehyde does not lead to exposure to methanol. Therefore, potential adverse effects due to exposure to methanol should be taken into account when assessing the potential toxicity of methyl cinnamate. However, as carcinogenicity of methanol is not a concern compared to its acute toxicity, the argumentation presented under the repeated dose toxicity section comparing DNELs of methyl cinnamate and methanol provides a significant margin of safety not requiring additional assessment of carcinogenic properties of methanol.