Methyl cinnamate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

september 11 to December 28, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test with GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
Number of Animals: Group 1: 12 males and 12 females
Group 2: 12 males and 12 females
Group 3: 12 males and 12 females
Group 4: 12 males and 12 females
Total Number of Animals: 50 males and 50 females
Age (at Delivery): 10 weeks
Body Weight Range (at Start of Treatment):
Males: 328 to 370 g
Females: 189 to 226 g
Identification: Cage card and individual animal number (ear tattoo).
Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Was performed after at least three days of acclimatization, using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration.
Acclimatization: 8 days under test conditions after health examina-
tion. Only animals without any visible signs of illness were used for the study.
Room Number, Itingen: E12
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Group-housed in Makrolon type-4 cages during acclimatization and pre-pairing, and group-housed during pairing period or individually thereafter in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Harlan Teklad 2914C (batch no. 20/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. .
Water: Community tap-water from Itingen was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The dose formulations were prepared weekly as indicated by the results of stability analyses in the dose range-finding study.

METHYL CINNAMATE was melted at approx. 35 °C in the water bath overnight and if necessary at approx. 45 °C (50 °C at the most) prior to use in the morning of dose formulation preparation. Thereafter, METHYL CINNAMATE was weighed into a glass beaker on a tared Mettler balance. The vehicle was added and the mixtures were stirred using a magnetic stirrer for approximately 0.5 to 1 hour (or as long as appropriate). The dose formulation were used and stored at room temperature (15 - 25 °C).

Homogeneity of the test item in the vehicle maintained during the daily administration period using a magnetic stirrer.

Storage of Dose Formulations
Stability of Dose Formulations: For at least 8 days, based upon the results of stability analyses performed during the non-GLP Harlan
Laboratories study D61306.
Storage of Dose Formulations: At room temperature (20 ± 5 °C) in glass beakers away from direct sunlight.

Vehicle
Data as supplied by the manufacturer.
Identification: Corn oil
Source: Carl Roth GmbH
Description: Liquid
Batch Numbers: a) 332190038 b) 362190038
Expiry Date (Retest Date): a) 19-Sep-2013 b) 26-Sep-2013
Storage Conditions: At room temperature (20 ± 5 °C) away from direct sunlight.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or

- a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The METHYL CINNAMATE concentrations in the dose formulations ranged from 85.6% to 102.1% with reference to the nominal and were within the accepted range of ±20%.
The homogeneous distribution of METHYL CINNAMATE in the preparations was approved because single results found did not deviate more than 5.4% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In addition, the test item was found to be stable in application formulations when kept up to eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

Duration of treatment / exposure:

Duration of Acclimatization Period: 8 days
Duration of Treatment Period: Males: Minimum 4 weeks, Females: Approximately 7 weeks

Frequency of treatment:

once daily

Details on study schedule:

The rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.
Dose levels were selected in agreement with the Sponsor, based on the results of a non-GLP dose range-finding study (Harlan Laboratories Study D61306).
If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Remarks:

Doses / Concentrations:
0, 100, 300, 1000/600 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Dose Formulations
The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor.

The dose formulations were prepared weekly as indicated by the results of stability analyses in the dose range-finding study.

METHYL CINNAMATE was melted at approx. 35 °C in the water bath overnight and if necessary at approx. 45 °C (50 °C at the most) prior to use in the morning of dose formulation preparation. Thereafter, METHYL CINNAMATE was weighed into a glass beaker on a tared Mettler balance. The vehicle was added and the mixtures were stirred using a magnetic stirrer for approximately 0.5 to 1 hour (or as long as appropriate). The dose formulation were used and stored at room temperature (15 - 25 °C).

Homogeneity of the test item in the vehicle maintained during the daily administration period using a magnetic stirrer.

Positive control:

not applicable

Parental animals: Observations and examinations:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (at least once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.
No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.
Detailed Clinical Observations (Weekly) :
Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.
For the parent animals, special attention was directed at the organs of the reproductive system.

Oestrous cyclicity (parental animals):

not applicable

Sperm parameters (parental animals):

not applicable

Litter observations:

Pup Data: The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

Pathology
Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Dams and pups were sacrificed on day 4 post partum.
If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

Necropsy
All animals sacrificed or found dead were weighed and subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

Postmortem examinations (offspring):

Dead pups, except those excessively cannibalized, were examined macroscopically.
All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:

• Means and standard deviations of various data were calculated.

• The Dunnett-test [see References (2)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test [see References (3)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test [see References (4)] was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

gestation index, fertility index and conception rate

Offspring viability indices:

Implantation Rate and Post-Implantation Loss, viability index

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Viability / Mortality:
All animals survived until the scheduled necropsy with the exception of a single male of group 3 at 300 mg/kg bw/day.
One group 3 male (no. 27) was found dead on day 9 of pairing period. Due to single occurrence of this mortality, in absence of a dose-dependent distribution and due to the macroscopic finding of incompletely collapsed lungs at necropsy, this mortality was considered to be not related to the treatment with the test item but considered to be most likely caused by accidental aspiration of the test item during gavage procedure.
General Cageside Observations (Daily) :
• In test item-treated males and females, salivation was slightly increased compared to controls
• In females group 4 at 1000/600 mg/kg bw/day, additionally uncoordinated movements, decreased activity, prostrating and/or ruffled fur occurred during pre-pairing
• In males group 4 at 1000/600 mg/kg bw/day, additionally uncoordinated movements, dragging of limbs or abnormal gait, decreased activity, prostrating and/or ruffled fur occurred pre-pairing
• The remainder of sporadic findings was considered to be within normal background findings which might be seen in animals of this strain, age and study type
Detailed Clinical Observations (Weekly) :
• In males and females group 4 at 1000/600 mg/kg bw/day, clinical signs similar to those of daily observation were noted during detailed weekly clinical observation, such as swaying gait, decreased activity, prostrating and/or ruffled fur
• The remainder of sporadic findings was considered to be within normal background findings which might be seen in animals of this strain, age and study type
Functional Observational Battery :
Grip Strength
None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.
• Sporadic increases in mean values of grip strength (fore- and hind paws) in males gave no indication of test item-related effects.

Locomotor Activity
• Sporadic increases in mean values of locomotor activity in females gave no indication of test item-related effects.

Food Consumption of Males :
Pre-Pairing Period
• In males group 4 at 1000/600 mg/kg bw/day, the mean food consumption (g/animal/day) was slightly reduced during days 1 to 8 of pre-pairing (p<0.05), but recovered thereafter.
Food Consumption of Females:
Pre-Pairing, Gestation and Lactation Periods
• No test item-related changes in food consumption were noted in females throughout study periods.

Body Weights of Females
Pre-Pairing, Pairing, Gestation and Lactation Periods
• In females no test item-related changes were noted in absolute and relative body weights throughout study periods.

Hematology :
Males
• In group 4 at 1000/600 mg/kg bw/day, the white blood cell count (WBC), absolute count of eosinophils, monocytes and large unstained cells (LUC) were statistically significantly decreased (p<0.05 or p<0.01). Large unstained cell count was also decreased in males of group 3 at 300 mg/kg bw/day. Values stayed within ranges of historical control data but were dose-dependently decreased.
The remainder of findings such as decreased hemoglobin distribution width in group 2 males at 100 mg/kg bw/day was considered to stay within ranges of normal biological variance.
Females
• In test item-treated females, the white blood cell count (WBC) and absolute count of lymphocytes was decreased in all groups 2 to 4 (mainly p<0.01). Values of the WBC and lymphocyte count on group 4 females were out of ranges of historical control data.
• Additionally, absolute monocytes and large unstained cells were decreased in females of group 3 and 4 at 300 and 1000/600 mg/kg bw/day (p<0.05 or p<0.01). Values of monocytes were out of and values of large unstained cells were within ranges of historical control data.
• Absolute basophils were decreased and relative neutrophils were increased in females at 1000/600 mg/kg bw/day (p<0.05). Values of basophils and relative neutrophils were out of ranges of historical control data.
• Absolute neutrophils were marginally decreased in test item-treated females with p<0.05 in females of group 2 at 100 mg/kg bw/day with values out of ranges of historical control data.

Clinical Biochemistry :
Males
• In group 4 at 1000/600 mg/kg bw/day, potassium was slightly increased (statistical significance of p<0.05) decreased with values within ranges of historical control data.
Females
• In group 3 and 4 females at 300 and 1000/600 mg/kg bw/day, glucose was statistically significantly increased (p<0.01) with values within ranges of historical control data.
• In group 2 to 4 females at 100, 300 and 1000/600 mg/kg bw/day, calcium levels were statistically significantly decreased (p<0.01) with values within ranges of historical control data.
• In group 4 females at 1000/600 mg/kg bw/day, urea levels were statistically significantly decreased (p<0.05) with values below ranges of historical control data.

Duration of Gestation:
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.6, 21.4, 21.9 and 22.0 days, in order of ascending dose level. The gestation index was 100% in groups 1 to 3 and 83.3% in group 4.

Corpora Lutea Count:
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.6, 14.4, 14.1 and 13.8 in order of ascending dose level) and gave no indication of a test item-related effect.
The fertility index and conception rate were 100% in groups 1, 3 and 4 and 83.3% in group 2 due to two females (nos. 61 and 66) with one mated female and one unmated female which both did not get pregnant and did not show implantation sites at necropsy. In absence of a dose-dependent distribution, the decrease of fertility index to 83.3% and conception rate to 90.) % in group 2 females was not considered to be related to the treatment with the test item but within ranges of normal biological variance.

Mating Performance and Fertility:
The mating performance was 100% in groups 1, 2 and 4 and 91.7% in group 2.
The majority of females in groups 1, 2, 3 and 4 mated within the first pairing period (10 or 11 of 12 females per group). In group 1, 2 and 4, two females of each group were mated during the second pairing period. In group 3, one female was mated during the second pairing period.
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times during the first pairing period were 2.5, 2.7, 4.0 and 3.5 days in groups 1, 2, 3 and 4, respectively. The median precoital time during the first pairing period was 3, 3, 4 and 3 days in order of ascending dose level.
The gestation index was 100% in groups 1 to 3 and 83.3% in group 4.
In group 4, one female (no. 87) was pregnant, came into birth, but did not deliver any pup or cannibalized prior to first litter check. This female showed four implantation sites at necropsy, only. Another female (no. 91) of group 4, did not deliver any pup and showed two implantation sites at necropsy, only. The decrease in the gestation index of group 4 to 83.3% compared to controls was considered to be test item-related due to occurrence in the high dose group and due to test item-related findings of toxicological relevance in hematology, biochemistry and histopathology.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Implantation Rate and Post-Implantation Loss:
The mean number of implantations per dam was similar in all groups.

The total and mean post-implantation loss and the mean incidence of post-implantation loss as a percentage of total implantations were slightly increased in test item-treated groups 3 and 4 at 300 and 1000/600 mg/kg bw/day compared to the control group (no statistical significance).

The mean numbers of implantations per litter were 12.4, 12.5, 11.9 and 12.1 in order of ascending dose level.

The mean incidence of post-implantation loss as a percentage of total implantations was 8.1, 4.8, 12.6 and 12.8%, in order of ascending dose level. The slight increase in group 3 was caused by a single animal (no. 83) and in group 4 by two animals (nos. 87 and 92), only. Due to occurrence in the mid and high dose group, the increase in post implantation loss was considered to be test item-related, even though the percentage of post-implantation loss was still in the range of findings which might be seen in animals of this strain and age, was not statistically significant, and no clear effect on mean litter size was noted.
Litter Size at First Litter Check :
In group 3 and 4, due to increased incidence of post implantation loss, the birth index (number of pups born alive/ number of implantations) resulted to be slightly reduced (87.4% and 87.2% compared to 91.9% in controls).
The mean litter size at first litter check was similar in all groups (11.4, 11.9, 10.4 and 10.5).
Postnatal Loss Days 0 - 4 Post Partum:
The mean postnatal loss was unaffected by treatment with the test item (mean of 0.2, 0.2, 0.1 and 0.0 in group 1, 2, 3 and 4, respectively).
Correspondingly, the viability index was not altered (98.5, 98.3, 99.2 and 100.0% in group 1, 2, 3 and 4, respectively).

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was higher than 1000/600 mg/kg bw/day.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Conclusions:

It was concluded that the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 100 mg/kg/day, based on a slightly higher post-implantation loss in the mid and high dose group, which did not increase dose-dependently from mid to high dose, was not statistically significantly different from the control group, and was within ranges of historical control data. The NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was higher than 1000/600 mg/kg bw/day, and the NOAEL for systemic toxicity is 300 mg/kg/day.

Executive summary:

This GLP study following OECD guideline 422 was performed to investigante the toxicity of test item by combining the repeated oral-gavage study to rats with reproductive screening test. Test article was administered in corn oil as vehicle at dosages of 100 and 300 mg/kg body weight/day in group 2 and 3, respectively and 1000 mg/kg body weight/day (week 1) and 600 mg/kg body weight/day (from week 2 onwards) in group 4. Controls received the vehicle only. Test item was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

At 1000/600 mg/kg, treatment with the test item resulted in a reduction of absolute body weights and food consumption in males and reversible clinical signs in both sexes. Since absolute body weights did not fully recover - even though showing a tendency of reversibility due to compensatory increases in body weight gains – this finding was considered to be adverse. In hematology, changes were noted dose-dependently and mainly in both sexes, often showing statistical significance of p<0.01. Additionally, decreases in white blood cell populations in males at 300 and 1000/600 mg/kg bw/day and in females at 100, 300 and 1000/600 mg/kg bw/day were not accompanied by obvious microscopic changes in all males or low and mid dose females and only accompanied by microscopic changes of low severity grades in high dose females and therefore considered to be not adverse. Those changes most likely represented a response to stress condition mainly in females. In biochemistry, those changes were considered to be not adverse.

At 1000/600 mg/kg bw/day, liver to body weight ratios were slightly increased with statistical significance in males, only. In absence of microscopic findings, these changes were considered to be adaptive. Microscopically, increased incidence of atrophy of lymphatic tissue (spleen, thymus, lymphnodes) in females, mainly and tubular basophilia of the kidney in males was observed at 1000/600 mg/kg bw/day. Due to low severity grades, these changes were considered to be not adverse. In regard to reproduction data, the post-implantation loss was slightly increased, the birth index slightly reduced in females at 300 and 1000/600 mg/kg bw/day and the gestation index slightly reduced in females at 1000/600 mg/kg bw/day. Litter data or microscopic evaluation of reproductive organs in parent animals did not reveal a test item-related effect.

Based on these results the NOAEL (No Observed Adverse Effect Level) for general toxicity was set at 300 mg/kg bw/day.The NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was higher than 1000/600 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

According to the toxicokinetic assessment, the potential exposure route of target substance is via oral or dermal while the inhalation is an irrelevant route due to the high boiling point and low vapor pressure. The available data by oral route is sufficient for assessment. Thus, there is no safety assessment concern of dermal and inhalation route.

One GLP study (A. Brunke, 2012) following OECD guideline 422 was conducted to assess the reproductive toxicity of target article. In this study, test article was administered in corn oil as vehicle at dosages of 100 and 300 mg/kg body weight/day in group 2 and 3, respectively and 1000 mg/kg body weight/day (week 1) and 600 mg/kg body weight/day (from week 2 onwards) in group 4. Controls received the vehicle only. Test item was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. At 1000/600 mg/kg, treatment with the test item resulted in a reduction of absolute body weights and food consumption in males and reversible clinical signs in both sexes. This finding was considered to be adverse. In hematology, changes were noted dose-dependently and mainly in both sexes, often showing statistical significance of p<0.01. At 1000/600 mg/kg bw/day, liver to body weight ratios were slightly increased with statistical significance in males, only. Due to low severity grades, these changes were considered to be not adverse. Litter data or microscopic evaluation of reproductive organs in parent animals did not reveal a test item-related effect.

Based on these results the NOAEL for reproduction/developmental toxicity was higher than 600 mg/kg bw/day and thus higher than the NOAEL for general toxicity (300 mg/kg bw/d).

Short description of key information:
NOAEL of the reproductive toxicity is considered to be 600 mg/kg/day based on the combined repeated oral-gavage study with reproductive screening test.

Justification for selection of Effect on fertility via inhalation route:
According to the toxicokinetic assessment, the potential exposure route of methyl cinnamate is via oral or dermal while the inhalation is an irrelevant route due to the high boiling point and low vapor pressure of methyl cinnamate. And the available data is sufficient for assessment. Thus, there is no safety assessment concern of this route.

Justification for selection of Effect on fertility via dermal route:
According to the toxicokinetic assessment, the potential exposure route of methyl cinnamate is via oral or dermal. And the available data is sufficient for assessment. Thus, there is no safety assessment concern of this route.

Effects on developmental toxicity

Description of key information

NOAEL of developmental toxicity is estimated to be 250 mg/kg/day based on the prenatal toxicity test of supporting substance.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Greater 250 mg/kg bw/d was set as NOAEL in one study investigating cinnamaldehyde as this was the high dose group showing no developmental effects.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are two publications studied with supporting substance cinnamic aldehyde, which is metabolized via cinnamic acid and therefore a basis for a weight of evidence approach was given.

In one of these publications (A. Mantovani, 1989), a study was performed to investigate the effects of cinnamic aldehyde on organogenesis using the prenatal development toxicity test. Cinnamaldehyde was administered by gavage to Sprague-Dawley rats on days 7-17 of pregnancy at doses of 5, 25or 250 mg/kg body weight/day. Significantly lower weight gain of the dams was observed at the two higher dose levels. No significant dose-related increase of abnormalities was observed: the incidence of poor cranial ossification was significantly increased in all treated groups, while reduced ossification of the tympanic bulla was increased at 25 or 250 mg/kg/day. Significant increases of the incidences of dilated pelvis/reduced papilla in the kidney, dilated ureters and>=2 abnormal sternebrae per foetus were detected in the 25mg/kg group, which had the highest overall prevalence of minor abnormalities. However, the results were considered to be unexpected since there was no evidence of dose depended trend. Thus, the NOAEL of maternal and developmental toxicity of test article was concluded to be 250 mg/kg/day based on the results in this study.

Another supportive study (Bryan D. Hardin, 1987) supported this conclusion as discussed above, where cinnamaldehyde caused negative developmental response at a single dose of 1200 mg/kg/day during the test period. That report was conducted to investigate the primary developmental toxicity of 60 chemicals including test article. There is no significant difference between cinnamaldehyde-group and control group in the maternal and neonatal response.

Therefore, the dose of 250 mg/kg/day was considered to be the NOAEL of maternal and developmental toxicity of target article based on the available data under the present conditions.

Thus, in line with the criteria in REACH, Annex IX 8.7.3, column 1 there is no need at this tonnage level to further investigate developmental toxicity by a multi-generation study and such a study is waived.

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

 

Hypothesis for the analogue approach from cinnam aldehyde 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	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 developmental toxicity of methanol. The overview given here is based on the information summarized by NIH-publication No. 03-4478, September 2003, provided by the National Toxicology Program, US Department of Health and Human Services, focusing on the oral exposure study by Rogers et al. that was given highest confidence by the assessment panel as provided in the report.

Roger et al. exposed pregnant 20 mice per dose group by oral route to doses up to 4000 mg/kg bw/d (twice daily 2000 mg/kg bw/d) during gestation day 6-15 and 8 mice as negative control (gavaged with water). The dose was selected to produce blood methanol levels observed in the inhalation study at the higher doses. Twice daily gavage doses of 2,000 mg/kg methanol (8 mice) on gd 6 –15 gave a pattern of response similar to that seen in the mouse group exposed to 10,000 ppm by inhalation. Mean maternal blood methanol levels 1 hour following the second daily exposure (3,856 mg/L) were slightly lower than blood levels in dams inhaling 10,000 ppm methanol in a previous experiment (4,204 mg/L). Fetal effects in the treated group included decreased fetal weight, increased resorptions, decreased live fetuses, and an increased incidence of fetuses/litter with cleft palate or exencephaly. Statistical significance of effects is indicated in

Rogers et al. identified a developmental LOAEL of 2,000 ppm and a NOAEL of 1,000 ppm based on the inhalative exposure study which dosed 0, 1000, 2000, 5000, 10000 and 15000 ppm by inhalative route to derive to NOAEL and LOAEL. These data that can be correlated by measured blood levels of methanol to an oral dose of 872 mg/kg bw/d as LOAEL and 436 mg/kg bw/d as NOAEL by oral route.

Taking into consideration that the NOAEL of 436 mg/kg bw/d corresponds to 13.6 mol/kg bw/d of methanol, an equivalent of 2210 mg/kg bw/d of methyl cinnamate would be considered as NOAEL when looking at the methanol moiety and its effects on developmental toxicity. This value provides a comfortable margin of safety to the NOAEL of 300 mg/kg bw/d observed in the OECD 422 study performed with methyl cinnamate.

Considering in addition that the NOAEL found in rats in one study using cinnamaldehyde was estimated being 1200 mg/kg bw / d and in another study being 250 mg/kg bw/d (highest tested dose in both studies), equivalent of 9.1 mol/kg bw/d respectively 1.9 mol/kg bw/d and in comparison to 13.6 mol methanol/kg bw/d as NOAEL, it can be concluded that methanol release resulting from methyl cinnamate cleavage in the gastrointestinal tract would be underestimated by read-across to the cinnamaldehyde studies.

In addition it should be noted that the DNELs for methanol, as described in the section on repeated dose toxicity, were derived based on the acute toxicity data of methanol which were considered most relevant even for long term DNEL derivation and thus a significant margin of safety is provided as described in the endpoint summary on repeated dose toxicity.

Thus, it is concluded that read-across to cinnamaldehyde to assess developmental toxicity of methyl cinnamate is a valid approach to follow.

Justification for selection of Effect on developmental toxicity: via oral route:
see discussion section for weight of evidence based on cinnamaldehyde as read-across substance

Justification for selection of Effect on developmental toxicity: via inhalation route:
According to the toxicokinetic assessment, the potential exposure route of methyl cinnamate is via oral or dermal while the inhalation is an irrelevant route due to the high boiling point and low vapor pressure of methyl cinnamate. And the available data is sufficient for assessment. Thus, there is no safety assessment concern of this route.

Justification for selection of Effect on developmental toxicity: via dermal route:
According to the toxicokinetic assessment, the potential exposure route of methyl cinnamate is via oral or dermal. And the available data is sufficient for assessment. Thus, there is no safety assessment concern of this route.

Justification for classification or non-classification

Based on the available data discussed above, methyl cinnamate caused no effects of reproductive/developmental toxicity and thus does not require classification for reproductive toxicity according to CLP (Regulation EC No.1272/2008) or DSD (Directive 67/548/EEC). Data on effects via lactation are not available..