Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 16, 1998 to August 18, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Study design is considered to follow OECD guideline Test Guideline No 414 (2001).
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Storage condition of test material: 5°C protected from light

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: Not reported
- Weight at study initiation: 232-275 g on gestational day 0
- Fasting period before study: Not reported
- Housing: Sani-Chip hardwood cage litter
- Diet (e.g. ad libitum): Purina Certified Roden Chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-23.3
- Humidity (%): 39.8-59.9
- Air changes (per hr): Not reported environmental conditions monitored and controlled by computer
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 ml/kg (administered dose volume)
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not reported
- Further matings after two unsuccessful attempts: not reported
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy:sperm found in vaginal lavage referred to as day 0 of pregnancy
- Any other deviations from standard protocol: not reported
Duration of treatment / exposure:
Day 6 through day 19 of gestation
Frequency of treatment:
Daily
Duration of test:
13 days
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 pregnant females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on screening study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily at dosing, and at 0.5 and 2 hours after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Gestational Day 0, 6 through 19, 20 and immediately following termination at day 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Gestational Day 0, 6, 9, 12, 15, 18, 19, and 20

WATER CONSUMPTION: Yes
- Time schedule for examinations: Gestational Day 0, 6, 9, 12, 15, 18, 19, and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: liver, gravid uterus, thoracic and abdominal cavities
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of dead and live fetuses
Fetal examinations:
- External examinations: Yes: all live fetuses
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
SAS software
Indices:
% Resorptions or fetal deaths/litter
% Litters with resorptions or deaths
% Litters with 100% prenatal mortality
No. live fetuses/litter
Average female body weight/litter
Average male body weight/litter
% Malformed fetusesllitter (all, extemal, visceral, or skeletal)
% Litters with malformations (all, extemal, visceral, or skeletal)
% Fetuses with any variations/litter
% Litters with any variations
% Fetuses with skeletal variations
Historical control data:
None

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Notable changes included clinical signs of piloerection and sedation at 500 and 1,000 mg/kg body weight/day.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compared to control, findings included lower maternal body weight (4.6%, 11.6%, and 9.9% lower at dose levels of 250, 500, and 1,000 mg/kg body weight/day, respectively) when measured on GD 20 and statistically significant and dose-dependent lower gestation body weight gain (13.6%, 22.6%, and 30.5% lower at dose levels of 250, 500, and 1,000 mg/kg body weight/day, respectively) when measured over GD 0 to 20 .
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Changes included lower maternal relative food intake (statistically significant values of 15.3% at 1,000 mg/kg body weight/day).
Food efficiency:
not examined
Description (incidence and severity):
Gavage
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Maternal relative water intake in the high dose group was reduced on gd 6-9, but was significantly elevated in the high dose group throughout the treatment and gestation periods. In the mid dose group, relative maternal water consumption was elevated during every time period from gd 9-20 with the exception of gd 18-19.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lower gravid uterine weight (statistically significant values of 8.54% and 10.5% at 500 and 1,000 mg/kg body weight/day, respectively). Maternal absolute liver weight showed a decreasing trend, but relative liver weight was significantly increased at all dose levels.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment-related group mean differences
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No treatment-related group mean differences
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Confinned pregnancy rates were 92-100% per group.
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related group mean differences for the following reproductive parameters: number of corpora lutea per dam, number of implantation sites per dam, average litter size, and percentage of male foetuses per litter

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The only developmental effect noted was a 7 to 9% decrease in body weight in high-dose foetuses when compared with control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The only developmental effect noted was a 7 to 9% decrease in body weight in high-dose foetuses when compared with control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live litter size in treated groups was 94-98% of the control mean.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No statistically significant differences
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant differences among groups for the incidences of foetal malformations (i.e., total, external, or skeletal); however, the incidence of skeletal variations (i.e., unossified sternebrae) was significantly increased in high-dose foetuses with 1 (1/184), 4 (1/180), 3 (3/184), and 14 (14/179) fetuses in the 0, 250, 500, and 1000 mglkg/day dose groups exhibiting unossified sternebrae. I
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
ncidental occurrences of visceral malformations were noted in one high-dose foetus (1/179); however, these did not occur in a dose-related manner and were therefore considered not to be relate to the test article; these variations included enlarged lateral ventricle, enlarged nasal sinus, agenesis of the innominate artery, and distended ureter.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
Description (incidence and severity):
1 (1/184), 4 (1/180), 3 (3/184), and 14 (14/179) fetuses in the 0, 250, 500, and 1000 mglkg/day dose groups exhibiting unossified sternebrae

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
LOAEL maternal toxicity = 250 mg/kg bw/day, based on dose-dependent reduced body weight gain.
NOAEL developmental toxicity = 500 mg/ kg bw/day, based on intra-uterine growth retardations mildly delayed skeletal ossification observed at 1000 mg/kg bw/day. However, this finding is likely secondary to maternal toxicity and not indicative of a teratogenic effect.
Executive summary:

In an embryo-foetal developmental study with E-Isoeugenol, the oral (gavage) administration of 0 (control), 250, 500, or 1,000 mg /kg body weight/day to female CD rats on gestation day (GD) 6 to 19 resulted in a number of test article-related maternal effects at all dose levels. These findings included lower maternal body weight (4.6%, 11.6%, and 9.9% lower at dose levels of 250, 500, and 1,000 mg/kg body weight/day, respectively) when measured on GD 20, and statistically significant and dose-dependent lower gestation body weight gain (13.6%, 22.6%, and 30.5% lower at dose levels of 250, 500, and 1,000 mg/kg body weight/day, respectively) when measured over GD 0 to 20 compared to control group.

Other effects in maternal animals included clinical signs of piloerection and sedation at 500 and 1,000 mg/kg body weight/day, and lower gravid uterine weight (statistically significant values of 8.54% and 10.5% at 500 and 1,000 mg/kg body weight/day) and lower maternal relative food intake (statistically significant values of 15.3% at 1,000 mg/kg body weight/day) compared to control group.

There were no treatment-related group mean differences for the following reproductive parameters: number of corpora lutea, number of implantation sites, percent preimplantation loss, percent preimplantation loss, resorptions, late foetal deaths, average litter size, and percentage of male foetuses per litter.

The only developmental effect noted was 7 to 9% lower body weight in high-dose foetuses when compared with Controls. There were no statistically significant differences among groups for the incidences of foetal malformations (i.e., total, external, or skeletal); however, the incidence of skeletal variations (i.e., unossified sternebrae) was significantly increased in high-dose foetuses with 1 (1/184), 4 (1/180), 3 (3/184), and 14 (14/179) fetuses in the 0, 250, 500, and 1000 mglkg/day dose groups exhibiting unossified sternebrae. Incidental occurrences of visceral malformations were noted in one high-dose foetus (1/179); however, these did not occur in a dose-related manner and were therefore considered not to be related to the test article; these variations included enlarged lateral ventricle, enlarged nasal sinus, agenesis of the innominate artery, and distended ureter.

The findings of unossified sternebra(e) at the high dose of 1,000 mg isoeugenol/kg body weight/day in the embryo-foetal developmental study are likely secondary to maternal toxicity and are not indicative of a teratogenic effect.  It is widely accepted that findings of unossified sternebra(e) are strongly influenced by alterations in maternal factors such as body weight, food consumption, and physiology (Carney and Kimmel, 2007).  In this particular study, the findings of unossified sternebra(e) were only reported at the dose level with the maternal toxicity.  Furthermore, even in the absence of maternal toxicity, delayed ossification is considered to be indicative of a foetotoxic effect, but not a teratogenic effect, as ossification usually will complete in the postnatal period and this finding has a relatively high background incidence.

According to the conclusion drawn by NTP, a maternal NOAEL could not be established; however, the lowest-observed-adverse-effect level (LOAEL) was reported to be the low dose level of 250 mg/kg body weight/day. The NOAEL for developmental toxicity was reported to be 500 mg/kg body weight/day, based on findings of growth delay and mildly delayed skeletal ossification in the high-dose foetuses.