Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March to September 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study weas performed according OECD guideline no. 414 and under GLP conditions.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Specification: The weight of the virgin females at the time of mating was between 185 g and 230 g. The weight of male animals was more than 200 g. All rats were acclimated to housing conditions at least 6 days prior to mating.

Housing of the animals: The rats were kept at an average room temperature of 22°C (21°C - 24°C) and a relative humidity of 40- 60%; they were exposed to the natural light cycle. Both prior to and after mating, the female rats were housed in "Ebeco BKFL II" individual cages, company E. Becker & Co GmbH, D-4620 Castrop-Rauxel, the male animals in "Ebeco Makrolon MIII" individual cages, company E.Becker & Co GmbH, D-4620 Castrop-Rauxel. Prior to study initiation, the cages had been autoclaved; they were cleaned twice a week during the experiment.

Feed and drinking water: During the entire study period, male and female animals received drinking water and feed ad libitum. The feed administered was "Altromin 1324"-Haltungsdiät (maintenance diet), company Altromin GmH & Co KG, Lange Straße 42, D-4937 Lage. It was used in the form of pellets (Batch 110/88/1326).

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
olive oil
Details on exposure:
The substance was administered once daily by probang.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no details given
Details on mating procedure:
Prior to mating, vaginal smears from the rats were prepared and evaluated. Mating was carried out exclusively during the fertile phases of the cycle (oestrus and/or pro-oestrus). For this purpose, one female in each case was placed in the cage of the male for 12 h (overnight). On the next day the female was placed back into its own individual cage and the vaginal smear was examined for the presence of sperms. The day of the positive sperm proof was defined as day 0 of gestation for this animal. All further chronological information was related to this day, days prior to gestation were marked by a negative sign.
Duration of treatment / exposure:
Treatment of sperm-positive females was performed from day 6 post-coitum up to and including day 15 post-coitum.
Frequency of treatment:
The substances were administered once daily by probang.
Duration of test:
The study duration was 20 d after day 0. The rats were sacrificed on day 20 post-coitum.
No. of animals per sex per dose:
Control: 21 females
0.25 mg/kg/d: 22 females
0.75 ml/kg/d: 21 females
1.25 ml/kg/d: 21 females
Positive control: 19 females
Control animals:
yes, concurrent vehicle
other: Positive control: 3 mg/kg/d tretinoin
Details on study design:
On day 20 p.c., the animals, in compliance with the regulations for the protection of animals, were sacrified by ether anesthesia and examined.

Examinations

Maternal examinations:
- mortality
- body weight
- behaviour and signs of intoxication
- liver weight
Ovaries and uterine content:
- number of corpora lutea of pregnancy per ovary
- location of foetuses in each horn of uterus
- placenta weight
Fetal examinations:
- number of living offspring
- number of early inta-uterine deaths
- number of late intra-uterine deaths
- sex of living offspring
- weight of offspring
The offspring were examined for external deformations. Irrespective of the stage of development and the sex of the gestational products the positions were consecutively counted from the proximal and to the right horn of the uterus. The odd-numbered positions of the litter of fully developed foetuses were eviscerated and fixed in 70 % alcohol (alizarin staining method according to Staple). The offspring of the even numbered positions of the litter were fixed in Bouin's fluid for the purpose of a later examination for visceral defects (method according to Wilson). In case no implantations could be found in the uterus of a sperm-positive female, the uterus was transferred in its native state into 10 % ammonium sulfide solution in order to stain existing implantation sites (method according to Salewski). Animals that died during the study were necropsied.
Statistics:
Calculations of relevant data basically included the mean value, standard deviation, mean deviation of the mean values, group means and/ or median. Common procedures were used.
The significance level was estimated using the U test according to Wolcoxon, Man and Whitney (two-sided).
Indices:
- Proportion of early intra-uterine deaths
- Proportion of late intra-uterine deaths
- Proportion of living offspring
- Intra-uterine mortality rate

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: At highest dose

Details on maternal toxic effects:
Mortality of dams: During the study, no animal died in the control, the low, and the intermediate dose group (0.25 and 0.75 ml/kg/d) and the positive control group. In the group with the highest dose of Neo Heliopan E 1000, 2 of 21 sperm-positive and treated animals died (10%). These animals died on day 11 p.c. and day 13 p.c., respectively. Necropsy findings revealed that in both cases, misdosing can be excluded as the cause of death. The probable cause of death is substance-related erosion of the epithelium of the gastro-intestinal tract wich led to gastro-intestinal bleedings.
Body weight: The group with the highest dose of Neo Heliopan E 1000 showed a decrease in body weight during treatment. During the course of treatment, significant differences with respect to the control group and the other treatment groups were observed from the 2nd day of treatment on. After termination of treatment, the body weight increased. These effects can be regarded as a sign of a general toxic effect at high dosages (2.25 ml/kg/day).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
0.75 other: ml/kg/d
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
0.25 other: ml/kg/d
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
In all groups, the number of total implantations was in the same order of magnitude that is usually met with this strain of rats. In the highest dose group, a marked and significant increase in intra-uterine mortality was conspicuous (77.2 and 67.1 % respectively). Only 4 of 16 pregnant dams had still living offspring on day 20 p.c. These findings together with the other observations can be explained by a general toxic effect on the dam at a high test substance dosage (2.25 mL/kg/day). In the high dose group, a significant reduction in foetal weights was observed. The offspring of the positive control also showed a significant decrease in body weight. A significant reduction in placental weights could be observed in the high dose group. A significant increase was recorded for the positive control.
No teratogenic effects were observed. Skeletal variations observed were consistent with delayed development in the highest dose group, which is consistent with the maternally toxic effects observed at this dose.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
0.75 other: ml/kg/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
2.25 other: ml/kg/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Mortality of dams = During the study, no animal died in the control, the low, and the intermediate dose group (0.25 and 0.75 ml/kg/d) and the positive control group. In the group with the highest dose of isopentyl p-methoxycinnamate, 2 of 21 sperm-positive and treated animals died (10%). These animals died on day 11 p.c. and day 13 p.c., respectively. Necropsy findings revealed that in both cases, misdosing can be excluded as the cause of death. The probable cause of death is substance-related erosion of the epithelium of the gastro-intestinal tract wich led to gastro-intestinal bleedings.

Body weight = The group with the highest dose of isopentyl p-methoxycinnamate showed a decrease in body weight during treatment. During the course of treatment, significant differences with respect to the control group and the other treatment groups were observed from the 2nd day of treatment on. After termination of treatment, the body weight increased. These effects can be regarded as a sign of a general toxic effect at high dosages (2.25 ml/kg/day).

Drinking water consumption and feed consumption = From the start of treatment on, the animals of the highest dose group (2.25 ml/kg/d) showed a significant increase in drinking water consumption when compared to the other groups. This increase remained at a significant level until the end of gestation. In the second half of the treatment period, significantly increased water consumption could also be observed in the low (0.25 ml/kg/d) and intermediate dose group (0.75 ml/kd/d) in comparison to the control. After termination of the treatment the consumed water amount was only slightly increased in these groups as against the control and did not reach a significantly deviating level. At no time did the positive control show a significant deviation in water consumption from that of the control.

During the first half of the treatment period, feed consumption in the intermediate (0.75 ml/kg/d) and high dose group (2.25 ml/kg/d) was significantly lower than that of the control group. During the second half of the treatment period, only the animals of the high dose group (2.25 ml/kg/d) showed significantly reduced feed consumption. After termination of treatment, no significant differences could be observed. The observed effects on water and feed consumption are most probably due to substance-related local irritations of the gastro-intestinal tract.

Clinical observations = Changes in the coat including a localized loss of hair are almost regularly observed and happen also spontaneously in the Wistar strain employed. The increasing frequency in the intermediate and high dose group could be considered to be a first sign of a test substance effect that, before anything else, becomes evident in a site of predilection.

Necropsy findings = Necropsy findings revealed no macroscopic abnormalities.

Applicant's summary and conclusion

Conclusions:
A dosage of 0.25 mL/kg/d after oral administration under the experimental conditions in the rat can be regarded as a safe "no observed effect level", if strict standards concerning general toxicity are applied. The intermediate test substance dosage (0.75 mL/kg/d) can be regarded as NOAEL
Executive summary:

The Institute of Pharmacology and Toxicology of the University of Münster designed and conducted a study in Wistar rats in compliance with the regulations of OECD/EC. This study was geared to determining teratogenicity and embryotoxicity of isopentyl p-methoxycinnamate after oral administration of the doses 0.25 mL/kg/d, 0.75 mL/kg/d, and 2.25 mL/kg/d; Tretinoin was used as a positive control. The study showed that, under the conditions described, the test substance has no teratogenic properties at any of the dosages tested. Embryotoxicity observed at the highest dose (2.25 mg/kg/d) is due to a general toxic effect on the dam (maternal toxicity).

On the basis of the above-mentioned results, a dosage of 0.25 mL/kg/d after oral administration under the experimental conditions in the rat can be regarded as a safe "no observed effect level", if strict standards concerning general toxicity are applied. Even at an (oral) dose of 2.25 mL/kg/day, no teratogenic effects could be observed; the ascertained embryotoxic effect can be explained by intoxication of the maternal organism (maternal toxicity after oral administration). In accordance with the conventional standards of assessment acknowledged by FDA and other national authorities - among others the Federal Health Office in Berlin - as well as other international administrative bodies, the intermediate test substance dosage (0.75 mL/kg/d) can be regarded as NOAEL . The increased loss of hair observed in this treatment group as deviating from the control group is no serious finding in the evaluation and assessment of the total result.