Methyl cinnamate

Endpoint summary

Description of key information

NOAEL (subacute, oral, rat): 300 mg/kg bw/day  resulted from the 28-day OECD 422 test with methyl cinnamate;
NOAEL (subchronic, oral, rat): 4100 ppm (i.e. 275 mg/kg bw/d (male) and 300 mg/kg bw/day (female)) based on the read-across to cinnamaldehyde administered as supporting substance, supported by same study on mice with NOAEL of 4100 ppm (i.e. 650 mg/kg bw/d (male) and 625 mg/kg bw/d (female)).
NOAEL (chronic, oral, rat): 200 mg/kg bw/d (male and female) based on the read-across to cinnamaldehyde administered as supporting substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Expert panel report, published by a reliable scientific institution, contributing to the assessment. Both, Cinnamaldehyde as well as methylcinnamate are metabolised through cinnamic acid as primary metabolite, as shown in the metabolism diagram (see attached background material), and thus fulfill the criterion for read-across as indicated in Annex XI, 1.5(2). An equivalent category approach was used by WHO when assessing dietary uptake on cinnamyl derivates via food and by the European Food Safety Authority in their "Scientific Opinion on Flavouring Group Evaluation 15, Revision 2 (FGE.15Rev2) on Aryl-substituted saturated and unsaturated primary alcohol/aldehyde/ester derivatives from chemical group 22". Thus, the study conducted with cinnamaldheyde is acceptable to be used for read-across to methyl cinnamate as both do metabolise via cinnamic acid.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

all animals were exposed through entire life span (2 years)

GLP compliance:

yes

Limit test:

no

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). On receipt, the rats were 5 to 6 weeks old. Rats were quarantined for 11 (males) or 12 (females) days; rats were approximately 6 weeks old on the first day of the studies. Before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease. Serologic analyses were performed on five male and five female sentinel rats 4 weeks after the study began and on five male and five female untreated control rats at study termination using the protocols of the NTP Sentinel Animal Program.
Animal identification by tail tattoo. Diet and water was available ad libitum.
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 oral exposure:

The dose formulations were prepared at least every 3 weeks by mixing microencapsulated trans-cinnamaldehyde with nonirradiated NTP-2000 feed during the 3-month studies. Placebo and/or loaded microcapsules were combined with feed to a concentration of 10% (3-month 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.
During the 3-month and 2-year studies, problems with animal room samples were encountered due to the animals’ ability to eat around the microcapsules (causing high animal room sample analyses results) and due to contamination of the feed with urine and feces which softened the microcapsules (causing low results). Both problems were more prevalent in the 3-month studies because the animals were younger and smaller and because of the higher concentrations of cinnamaldehyde in the feed.

Duration of treatment / exposure:

104 to 105 (males) weeks or 105 to 106 (females) weeks

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 1000, 2100, 4100 ppm microencapsulated trans-cinnamaldehyde (equivalent to average daily doses of approximately 0, 50, 100 and 200 mg trans-cinnamaldehyde/kg body weight to males and females).
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 core study rats were fed diets containing 1000, 2100 and 4100 ppm microencapsulated trans-cinnamaldehyde for2 years. Additional groups of 50 male and 50 female core study rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls).
Feed and water were available ad libitum. Rats were housed five per cage. Observations were made twice daily; animals were weighed initially, on day 8, every 4 weeks thereafter, and at the end of the study.
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.

Observations and examinations performed and frequency:

Observed twice daily; animals were weighed initially, weekly, and at the end of the studies; clinical findings were recorded on day 8 and every 4 weeks thereafter. 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 core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.
Histopathology
Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and/or mesenteric), mammary gland, 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.

Statistics:

Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. 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.
Calculation of Incidence
The incidences of neoplasms or nonneoplastic lesions were presented as numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. For calculation of statistical significance, the incidences of most neoplasms and all non neoplastic
lesions are given as the numbers of animals affected at each site examined microscopically.
Analysis of Neoplasm
Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. 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).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slightly reduced body weights at 2100 and 4100 ppm in males and at 4100 ppm in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slight effects at 2100 and 4100 ppm n males and 4100 ppm in females

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Survival of 4,100 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 4,100 ppm males were less than those of the vehicle controls throughout the study, mean body weights of 2,100 ppm males were less after week 94, and mean body weights of 4,100 ppm females were less after week 18 (Figure 3; Tables 6 and 7). Feed consumption by 2,100 and 4,100 ppm males and 4,100 ppm females was less than that by the vehicle controls at the beginning and end of the study. Dietary concentrations of 1,000, 2,100, or 4,100 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 trans-cinnamaldehyde exposure.
Hippuric acid excretion in urine expressed as the hippuric acid to creatinine ratio was proportional to dose, indicating that neither absorption, metabolism, nor excretion was saturated in either male or female rats exposed to dosed feed containing 1,000 to 4,100 ppm trans-cinnamaldehyde.
Preputial and Prostate Glands:
The incidences of adenoma of the preputial gland (vehicle control, 5/50; 1,000 ppm, 1/49; 2,100 ppm, 2/50; 4,100 ppm, 0/50) and prostate gland (4/50, 0/49, 0/49, 0/50) in 4,100 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). 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 4,100 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.

Dose descriptor:

NOAEL

Effect level:

4 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no clinical findings related to trans-cinnamaldehyde exposure at all doses up to 4100 ppm (200 mg/kg bw/d males and females) and thus the high dose may be seen as NOAEL in this study with rats.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no clinical findings related to trans-cinnamaldehyde exposure at all doses up to 4100 ppm (200 mg/kg bw/d males and females) and thus the high dose may be seen as NOAEL in this study with rats.

Critical effects observed:

not specified

Conclusions:

Based on the available data it was concluded that trans-cinnamaldheyde did not cause adverse effects to rats at concentrations of 50, 100 and 200 mg/kg bw/d compared with untreated control group. The NOAEL may be set to 4100 ppm (200 mg/kg bw/d males and females).

Executive summary:

Groups of 50 male and 50 female F344/N rats were fed diets containing 1,000, 2,100, or 4,100 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 1,000, 2,100, or 4,100 ppm delivered average daily doses of approximately 50, 100, or 200 mg/kg to males and females. Survival of 4,100 ppm males was greater than that of the vehicle controls. Mean body weights of 4,100 ppm males and females were generally less than those of the vehicle controls throughout the study (91% males and 92% females at end of study, compared to control). Feed consumption by 2,100 and 4,100 ppm males and 4,100 ppm females was less than that by the vehicle controls at the beginning and end of the study, whereas survival in the highest dose group was s higher, particularly for male rats. There were no neoplasms or nonneoplastic lesions that were attributed to exposure to trans-cinnamaldehyde at all dose groups. The incidence of mononuclear cell leukemia in 4,100 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. Thus, the NOAEL for repeated dose toxicity is set to 4100 ppm (i.e. 200 mg/kg bw/d) in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Although this key study was not performed on the substance itself but on cinnamaldheyde, the study was performed chronic over 2 years on cinnamaldehyde, having the same primary metabolite (cinnamic acid) as methyl cinnamate. The findings are supported by a subacute study on rats (as part of an OECD 422 study) with methyl cinnamate, resulting in a NOAEL of 300 mg/kg bw/d and by a subchronic study by the same authors using rats and mice as species with identical NOAEL of 4100 ppm (275 mg/kg bw/d male rats respectively 300 mg/kg bw/d female rats and 650 mg/kg bw/d male mice and 625 mg/kg bw/d female mice).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The primary metabolite of methyl cinnamate is cinnamic acid and this holds true also for cinnamaldehyde. Therefore, the read-across to the subchronic feeding study with cinnamaldehyde is justifiable (see further details below). Although the key study was not performed on the substance itself but on cinnamaldehyde, the study was performed subchronic and chronic on cinnamaldehyde. The findings are supported by a subacute study on rats (as part of an OECD 422 study) with methyl cinnamate, resulting in a NOAEL value of 300 mg/kg bw/d and by a subchronic study by the same authors on cinnamaldehyde using rats (NOAEL 4100 ppm equivalent to 275 (males) and 300 mg/kg bw/d (females)) and using mice as species with identical NOAEL (4100 ppm) but due to different species twice as high NOAEL when based on amount per kg body weight (650 mg/kg bw/d males and 625 mg/kg bw/d females). In addition a chronic study is available using cinnamaldehyde which resulted in a NOAEL (male and female) of 200 mg/kg bw/d (4100 ppm) which was the highest dose group in this study. Thus, it can be reasonably concluded that findings in chronic studies with rats are representative for methyl cinnamate too and the NOAEL based on the chronic study with rats is set to 200 mg/kg bw/d (males/females). The acute effects of the second metabolite methanol are sufficiently covered by the OECD 422 subacute study on methyl cinnamate.

In a study by Trubek et al. a mix of 5 cinnamates used as flavors in human food was investigated in a subchronic oral study on rats, amongst them methyl cinnamate (2.5%) but mainly consisting of cinnamaldehyde (90%). Besides reduced food consumption, no significant effects were noted at 100 ppm. These findings were later supported by studies performed by Zaitsev et al. investigating ethyl cinnamate at a limit dose of 80 mg/kg bw/d over 16 weeks, which also revealed no adverse effects and by Hagan et al. investigating cinnamaldehyde over 16 weeks to rats (NOAEL 166.7 mg/kg bw/d, LOAEL 666.7 mg/kg bw/d).

Thus it can be concluded that repeat dose toxicity information is based on a wider basis of substance and surrogate data and no further tests are required.

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	Methyl cinnamate
CAS No	104 -55 -2; 14371-10-9 (trans)	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.

There is publicly available data about the toxicity of methanol. The overview given here is based on the information disseminated on ECHA’s website. The majority of available oral repeated dose toxicity studies were conducted for very short exposure durations of 3 to 6 days. The few longer-term repeated dose studies were conducted via the inhalation route. Effects seen in repeated dose toxicity studies included histological changes in the liver and kidneys of experimental animals, but also in the brain and reproductive organs. It appears that the reproductive organs are affected at lower doses than other organs. Rats were slightly more sensitive to repeated inhalation exposure than monkeys. The DNELs for acute and long-term exposure of workers and the general public to methanol

are given on ECHA’s dissemination website (Table 1).

Table 1: DNELs for methanol formed by hydrolysis of methyl cinnamate

DNEL	Worker	General public
Acute – systemic, dermal	40 mg/kg/day	8 mg/kg/day
Acute – systemic, inhalation	260 mg/m³	50 mg/m³
Acute – systemic, oral	Not applicable	8 mg/kg/day
Long-term – systemic, dermal	40 mg/kg/day	8 mg/kg/day
Long-term – systemic, inhalation	260 mg/m³	50 mg/m³
Long-term – systemic, oral	Not applicable	8 mg/kg/day

The corresponding DNELs for exposure of workers or the general population to methyl cinnamate are given in Table 2.

Table 2. DNELs for methyl cinnamate

DNEL	Worker	General public
Acute – systemic, dermal	no threshold derived	no threshold derived
Acute – systemic, inhalation	no threshold derived	no threshold derived
Acute – systemic, oral	Not applicable	no threshold derived
Long-term – systemic, dermal	3 mg/kg/day	1.5 mg/kg/day
Long-term – systemic, inhalation	21.2 mg/m³	5.22 mg/m³
Long-term – systemic, oral	Not applicable	1.5 mg/kg/day

The DNELs for methyl cinnamate are lower than those for methanol for all endpoints, except for acute exposure, where no threshold was derived due to lack of hazard (see data on acute toxicity above for example). Acute worker DNELs for methanol are more than a factor of 10 lower than long-term DNELs of methyl cinnamate and thus a significant margin of safety is provided. For consumer DNELs, where the margin of safety provided by the long-term DNEL is approx. 5 for dermal and 10 for inhalative route however, it should be considered that end-products used by the general public contain only low amounts of methyl cinnamate, generally below 1% (w/w). Elevated acute inhalation exposure of consumers to methyl cinnamate therefore is highly unlikely. Safe conditions of use of methyl cinnamate or products containing methyl cinnamate therefore ensure that potential exposure of workers or the general population to methanol will remain below the respective DNELs for methanol.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Although this key study was not performed on the substance itself but on cinnamaldehyde, the study was performed chronic on cinnamaldehyde, having the same primary metabolite (cinnamic acid) as methyl cinnamate.

Justification for classification or non-classification

Based on the subacute NOAEL from the OECD 422 study with methyl cinnamate as well as based on the chronic and subchronic studies with rats and mice on cinnamaldehyde, it can be concluded that classification for Specific Target Organ Toxicity (Repeat Exposure) STOT SE according to CLP (Regulation EC No 1272/2008) is not required, nor is a classification for repeated dose toxicity according to DSD (Directive 67/548/EEC) required.