Methyl cinnamate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test article cinnamaldehyde can be metabolized through cinnamic acid, as well as target substance methyl cinnamate. Thus, the study conducted with test article is acceptable to be used for read-across.

Data source

Reference

Reference Type:

publication

Title:

PRE-NATAL (SEGMENT II) TOXICITY STUDY OF CINNAMIC ALDEHYDE IN THE SPRAGUE—DAWLEY RAT

Author:

A. MANTOVANI, A. V. STAZI, C. MACRI, C. RICCIARDI, A. PicciONif and E. BADELLINO

Year:

1989

Bibliographic source:

Fd Chem. Toxic. Vol. 27. No.12, pp. 781-786

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cinnamic aldehyde(CA)

IUPAC Name:

cinnamic aldehyde(CA)

Constituent 2

Reference substance name:

Cinnamaldehyde

EC Number:

203-213-9

EC Name:

Cinnamaldehyde

Cas Number:

104-55-2

IUPAC Name:

3-phenylacrylaldehyde

Test material form:

not specified

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Eighty female virgin Sprague-Dawley rats (175-200 g) were purchased from Charles River (Calco, CO, Italy). They were kept in solid plastic boxes and given water and Open Formula rat diet (D. ri Piccioni, Brescia, Italy) ad lib. They were kept in an environmentally controlled room at 22 ± 0.5 °C and 50-60% relative humidity, with 12-14 air changes/hr and a 12-hr light/dark cycle.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Remarks:

Sigma Tau, Milan, Italy

Details on exposure:

At 09.30 hr on day 7 of pregnancy, the dams were weighed and then given by gavage (Group 0, control), 5 (Group 1), 25 (Group 2) or 250 (Group 3) mg/kg/day. These doses were repeated at 09.30 hr on days 8-17 of pregnancy.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

N/A

Details on mating procedure:

After 1 week of acclimatization, the rats were housed overnight with adult males of proven fertility and of the same strain (with a male:female ratio of 1:2). Vaginal smears were taken the following morning and if spermatozoa were found, mating was considered successful (day 0 of pregnancy). The pregnant rats were then put into individual boxes and randomly assigned to the experimental groups (15, 14, 16 and 15 rats in groups 0 (control), 1, 2 and 3, respectively).

Duration of treatment / exposure:

day 7-17 of pregnant rats

Frequency of treatment:

once daily

Duration of test:

20 days

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 25 and 250 mg/kg body weight/day.
Basis:
actual ingested

No. of animals per sex per dose:

group 0 (control): 15
group 1 (5mg/kg/day):14
group 1 (25mg/kg/day):16
group 1 (250mg/kg/day):15

Control animals:

yes, concurrent vehicle

Details on study design:

Weight and food consumption were recorded on days 0, 7, 18 and 20. On the morning of day 20 the dams were taken in random order and given an ip injection of sodium pentobarbital and killed by cervical dislocation.

Examinations

Maternal examinations:

The following parameters, that might indicate maternal effect of CA were determined: food consumption; weight increase (minus the uterine weight) between days 7 and 20; relative weights of liver and kidney.

Ovaries and uterine content:

On day 20 the ovaries were excised and the corpora lutea were counted. The uterus was removed and the following parameters were recorded: total uterine weight; the number and position of implants, resorptions and dead foetuses in each uterine horn;

Fetal examinations:

the numbers, positions, sex and weight of live foetuses; the occurrence of any external alterations. The postimplantation lethality’ (POLE) was evaluated for each litter as follows: (No. of resorptions + no. of dead foetuses)/total no. of implants × 100
A foetus was defined as a ‘runt, when it weighed <2 SD compared with the average weight of the live foetuses of the same sex in Group 0. The foetuses from each litter were randomly assigned to two groups of almost equal size. One group was fixed in 95% ethanol and underwent double staining with alcyan blue-Alizarin Red S (Whitaker and Dix, 1979) in order to evaluate alterations of bone and cartilaginous structures, and the degree of skeletal ossification (evaluated by determining endpoints such as the extent of ossification of hyoid, anterior phalanxes, sternebrae, metacarpi and coccigeal vertebrae).
The other group of foetuses was fixed in Bouin's fluid and examined for soft-tissue alterations according to Wilson’s serial section method (Wilson and Warkany, 1965).

Statistics:

Statistical analysis was performed using either parametrical (Lison, 1961) or non-parametrical (Siegel, 1967) tests. One-way analysis of variance using Duncan’s method for multiple comparisons was applied to data on feed consumption, weight gains and relative weight of liver and kidney of the dams, and to the numbers of corpora lutea and implantations, the number and weight of foetuses of either sex, and the M:F ratio among foetuses. The 2x2 chi-square test with Yates’ correction or the one-tailed Fisher’s exact test were applied to data on the incidences of pre-implantation loss, resorptions and dead foetuses, and of abnormalities observed during the study. The parameters tested by the chi-square test were also tested by means of the Armitage-Cochran trend test (Armitage, 1975) to assess whether a dose-response relationship was present.
The non-parametric Kruskal-Wallis test, equivalent to the analysis of variance, and then the Mann-Whitney test for pair-wise comparisons were applied to the average numbers (per foetus and per litter) of ossified metacarpi, coccigeal vertebrae and stemebrae as well as to the average incidences, on a ‘litter' basis, of the main abnormalities observed, because of the non-Gaussian distribution of the values for these parameters.

Indices:

'postimplantation lethality’ (POLE): (No. of resorptions + no. of dead foetuses)/total no. of implants × 100

Historical control data:

NA

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: there was a significant decrease in body-weight gain between days 7 and 20 in Groups 2 (p < 0.01) and 3 (p < 0.05).

Details on maternal toxic effects:
Effects on the dams
No clinical signs attributable to the treatment were observed, but there was a significant decrease in body-weight gain between days 7 and 20 in Groups 2 (p < 0.01) and 3 (p < 0.05). CA treatment had no effect on relative liver or kidney weight except for a marginally significant (P = 0.05) decrease in the relative weight of the liver in rats in Group 1. This finding was considered to be incidental.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: a significant (P < 0.001) increase in pre-implantation loss in the controls in comparison with the treated groups was not considered treatment related.

Details on embryotoxic / teratogenic effects:
General parameters of embryotoxicity
No statistically significant differences were detected except for a significant (P < 0.001) increase in pre-implantation loss in the controls in comparison with the treated groups.
A few dead foetuses, mostly oedematous, and a fairly high incidence of external haemorrhages and haematomata were observed, but there were no significant differences between the groups in the incidences of these phenomena. The slight increase in the incidence of runts that was observed in Groups 2 and 3 was not high enough to attain statistical significance.
Very few gross malformations of foetuses were observed. They included two tail-less foetuses (one each in Group 0 and Group 3) and one case of facial hemiaplasia in Group 3.
Skeletal examination:
Several common anomalies and variants of sternebrae (e.g. asymmetric, malaligned) and, with a lower incidence, vertebrae (e.g. dumb-bell) were observed, with no significant differences between treated groups and controls. However, in Group 2 there was a highly significant (p<0.001) increase in the incidence of foetuses with >2 abnormal sternebrae. The Kruskal-Wallis test showed a significant (P < 0.01) difference between the groups in the incidence, on a ‘litter’ basis, of >=2 abnormal sternebrae. The average rate/litter of foetuses affected were (mean ± SEM): i).064± 0.113 (controls); 0.072 ±0.104; 0.234 ±0.183;0.113 ±0.109. According to the Mann-Whitney test, Group 2 showed a significant increase in comparison with the controls (P <0.01). The rate of litters affected was also increased (P = 0.05, Fisher’s exact test).
Some foetuses had rudimentary (not supernumerary) ribs, but the incidence was not statistically significant.
There were a few cases of unusual bone anomalies in the treated groups: ‘narrowed’ sternebrae~horizontally bipartite (one case in each group); bipartite-asymmetric coccigeal body (one case, Group 1); bipartite ossification centre in the hyoid (one case, Group 2); split ossification centre in the ilium (one case, Group 3).
The ossification parameters did not show any significant differences, with the notable exception of cranial ossification defects: reduced and/or asymmetric ossification of interparietal and/or supraoccipital bones was observed to be significantly increased in all treated groups (P < 0.01, P < 0.05 and P < 0.025 for Groups 1, 2 and 3 respectively), without any dose-related trend. In one Group-2 foetus with a poorly ossified cranium, the atlas and epistropheum were also unossified. There was a significantly higher rate of poor ossification of the tympanic bulla, either mono- or bilateral, in Groups 2 and 3 (P < 0.05, Fisher’s exact test) than in Groups 1 and 0 (controls) in which no cases were detected. A small number of cases of unossificd pelvis were observed in treated groups, but not in controls.
Soft-tissue examination
Cases of internal haemorrhages and generalized oedema were observed, but there were no significant differences between groups in the incidences of these phenomena. Besides these, the only soft-tissue alterations that were observed were those of the excretory system. The incidence of renal variants (reduced papilla, dilated pelvis) and of dilated ureters were significantly higher in Groups 1 and 2 (renal variants, P < 0.025 for both groups; dilated ureters, P < 0.025 for Group 1 and P < 0.05 for Group 2). The increases observed in Group 3 did not attain statistical significance.
In Group 3 a few cases of hypoplastic-dysplastic kidneys (with absent or abnormal pelvis) were observed in two litters (out of 15). One of these litters included the grossly abnormal foetus with facial hemiaplasia: a close examination of the affected side revealed a hypoplastic, skin-covered eye, distorted nare without opening, and monolateral absence of mouth fissure. Finally, the incidence of foetuses with >=2 variants of the urinary system (e.g. bilateral reduced papilla) was also assessed and showed a marginal, non-significant increase in the treated groups.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It is estimated that the no-observed-adverse-effect-level (NOAEL) of maternal and developmental toxicity of test article is 250 mg/kg/day based on the available data.

Executive summary:

In this publication, study was performed to investigate the effects of test article on organogenesis using the prenatal development toxicity test. Test article was administered by gavage to Sprague-Dawley rats on days 7-17 of pregnancy at doses of 5, 25 and 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.

Therefore, the dose of 250 mg/kg/day was selected to be the NOAEL of maternal and developmental toxicity based on no overt sign of maternal toxicity when administered by gavage at doses of up to 250 mg/kg body weight/day to rats on days 7 to 17 of pregnancy.Though significantly higher rate of poor ossification of the tympanic bulla, either mono- or bilateral, in Groups 2 and 3 (P < 0.05, Fisher’s exact test) than in Groups 1 and 0 (controls), no dose dependent trend was revealed.