Methyl cinnamate

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Methyl cinnamate

EC Number:

203-093-8

EC Name:

Methyl cinnamate

Cas Number:

103-26-4

Molecular formula:

C10H10O2

IUPAC Name:

methyl 3-phenylacrylate

Test material form:

other: solid

Test animals

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.

Administration / exposure

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.

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

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'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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).

Effect levels (F1)

1

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.

2

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'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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.