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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003 - 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Source: Zoster S.A. (Zeneta–Murcia, Spain)
- Purity: 96.9% (by HPLC) on a dry matter basis (water content 9.3%)
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- Structural formula attached as image file (if other than submission substance): see Fig. in attached document.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:(WI)WU BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga (Sulzfeld, Germany)
- Age at study initiation: about 11–13 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: For mating, two females were placed together with one male in suspended stainless steel cages fitted with wire-mesh fronts and floors. During gestation, the dams were housed individually in suspended stainless steel cages with wire-mesh fronts and floors.
- Diet: commercial rodent diet was fed ad libitum (RM3 Diet, SDS Special Diets Services, Witham, UK).
- Water: unfluoridated tap water were provided ad libitum.
- Acclimation period: 2-day quarantine and a 10-day acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Starting from day 0 of gestation, the rats received the experimental diets which were prepared by adding NHDC at levels of 0 (control), 1.25, 2.5, and 5% to the powdered RM3 diet.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): Starting from day 0 of gestation, the rats received the experimental diets which were prepared by adding test item to the powdered RM3 diet (RM3 Diet, SDS Special Diets Services, Witham, UK).
- Storage temperature of food: The experimental diets were stored in a refrigerator until use.
- The different test diets were provided ad libitum from day 0 up to and including day 21 of gestation. The test diets were offered as a meal mash in stainless steel cans which were covered by a perforated stainless steel plate to prevent spillage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC analyses in duplicate of the test diets demonstrated that the actual and nominal NHDC levels were similar (deviations of -6, -5, and -5% were found in the food of the low-, mid-, and high-dose group, respectively). From an earlier study it was known that NHDC can be homogenously distributed in the test diet and is stable (Lina et al., 1990; see 'attached background material').
Details on mating procedure:
Two females were placed together with one male in suspended stainless steel cages fitted with wire-mesh fronts and floors. Females showing evidence of mating (vaginal plug or vaginal smears with sperm cells) were randomly assigned in rotation to each experimental group, up to the day when 28 positive females had been allotted to each group. The day on which there was evidence of mating was recorded as day 0 of gestation.
Duration of treatment / exposure:
21 days.
Frequency of treatment:
No data.
Duration of test:
21 days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: %
Remarks:
control group
Dose / conc.:
1.25 other: %
Dose / conc.:
2.5 other: %
Dose / conc.:
5 other: %
Remarks:
3.3 g/kg bw/day
No. of animals per sex per dose:
28 mated females per dose.
Control animals:
yes
Details on study design:
- Dose selection rationale: The high-dose level applied in this study corresponds to that which was tested in a 13-week subchronic toxicity study in rats and an earlier three-generation reproduction and teratogenicity study in rats (Booth, 1974, unpublished; Gumbmann et al., 1978; Lina et al., 1990; see 'attached background material'): In the study by Lina (1990), neohesperidin dihydrochalcone was administered to groups of 20 male and 20 female Wistar rats at dietary levels of 0, 0.2, 1.0 and 5.0% for 91 days. Treatment-related changes were only observed at the high-dose level, and they were considered adaptive responses rather than manifestations of toxicity. It was concluded that the NOEL was ca. 750 mg/kg bw.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 7, 14, and 21 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Food consumption was determined during three consecutive periods (days 0–7, 7–14, and 14–21 of gestation.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (not a drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Macroscopic abnormalities of major organs of the abdominal and thoracic cavity were recorded.
- Organs examined: The ovaries, uterus, and cecum were removed and weighed.
Ovaries and uterine content:
The number of corpora lutea in each ovary was recorded. The number of implantation sites was recorded in both uterine horns. Finally, the empty uterus was weighed.
Fetal examinations:
The fetuses were removed from the uterus, dried of amniotic fluid, weighed, sexed by observing the anogenital distance, and examined for gross abnormalities. The placentas of the live fetuses were weighed and examined for macroscopic abnormalities. Early and late resorptions and dead fetuses were counted. Resorption was classified ‘‘early’’ when only placental tissue was visible and ‘‘late’’ when placental as well as embryonic tissue was visible.
Half of the fetuses randomly selected from each litter were eviscerated, partly skinned, fixed in 70% ethanol, cleared in potassium hydroxide, and stained with Alizarin Red S for examination of the skeleton (Dawson, 1926; Lorke, 1963, 1965). The remaining fetuses were fixed in Bouins fluid for subsequent visceral examination according to a modified technique of Wilson (Barrow and Taylor, 1969). All examinations for fetal abnormalities were performed microscopically. Skeletal and visceral examinations were conducted on fetuses of the control and high-dose group only.
Statistics:
Data on maternal body weights, food consumption, fetal weights, and placenta weights were subjected to one-way analysis of variance (ANOVA) followed by Dunnetts multiple comparison test. Litter data were analyzed by analysis of variance (Kruskal–Wallis) followed by the Mann–Whitney U test. Parental necropsy findings, the number of mated and pregnant females and females with live fetuses, as well as the data on skeletal and visceral anomalies were evaluated by Fishers exact probability test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs of ill health, abnormal behaviour or intolerance were noted in any treatment group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the females died during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean maternal body weights and body weight changes during gestation were similar in all groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Maternal food consumption, when expressed in g/kg bw/day, was slightly yet significantly increased in the mid and high-dose group during the last week of pregnancy. However, this increase was statistically significant only for the relative food intake (expressed in g/kg bw/day) but not the absolute food intake (g/animal/day).
Mean daily test item intake, calculated on basis of the nominal dietary levels, ranged during the three measurement periods from 0.8 to 0.9, 1.6 to 1.7, and 3.1 to 3.4 g/kg bw/day for the low-, mid-, and high-dose group, respectively.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy the absolute and relative weight of the full cecum was significantly increased in all treatment groups in a dose-dependent manner. The weight of the empty cecum was significantly increased in the high-dose group. Cecal enlargement in rats and mice is a fully reversible, adaptive phenomenon which lacks relevance for human safety. Its occurrence demonstrated that the test item is not readily digested and absorbed.
No statistically significant differences were observed in gravid and empty uterus weights, ovary weight and placenta weight. No other gross changes were observed that could be attributed to the treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females aborted during the study. Two rats (one of the low- and one of the high-dose group) delivered just before Cesarean section.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean numbers of corpora lutea and implantation sites, the pre-implantation loss, the number of live fetuses, the mean number of early and late resorptions, and the post-implantation loss did not differ between any of the treated groups and the controls.
Total litter losses by resorption:
no effects observed
Effects on pregnancy duration:
effects observed, non-treatment-related
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus of the mid-dose group was dead.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
The fertility and reproductive performance did not show any treatment-related effects.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 3 300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no significant effects observed.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal body weights of the viable fetuses were similar in all groups. Large fetuses were found in the control group (2 fetuses from 2 litters), in the low-dose group (9/2) and high-dose group (2/2). Small fetuses were found in the low-dose group (2/2).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio was, within normal limits, close to 1 in all groups.
Fetal/pup body weight changes:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations and anomalies were not observed. The few skeletal variations included mainly irregularly shaped sternebrae. However, the incidence was low and not different between the high-dose group and the controls.
Variations in ossification (incomplete or absent ossification) occurred at a high incidence. However, the incidence of incomplete ossification was significantly increased in the high-dose group only for the front and hind proximal phalanges but not any other part of the skeleton (data not shown). For the high-dose group a significantly increased number of fetuses had 1–4 incompletely ossified digits and 3–6 unossified digits of the proximal hind phalanges. However, these differences are compensated when taking the categories 5–8 incompletely ossified digits and 7–10 unossified digits into account. Moreover, the differences were only observed at fetal incidence but not litter incidence. Therefore, the observed differences are considered to be fortuitous findings.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral malformations were not found in any group. None of the observed visceral anomalies and variations was considered to be treatment related.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus of the mid-dose group showed a subcutaneous hemorrhagic area.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects: Mean fetal body weights of the viable fetuses were similar in all groups. The sex ratio was, within normal limits, close to 1 in all groups. Large fetuses were found in the control group, in the low-dose group and high-dose group. Small fetuses were found in the low-dose group. One fetus of themid-dose group showed a subcutaneous hemorrhagic area. Skeletal malformations and anomalies were not observed. The few skeletal variations included mainly irregularly shaped sternebrae. Variations in ossification (incomplete or absent ossification) occurred at a high incidence. However, the incidence of incomplete ossification was significantly increased in the high-dose group only for the front and hind proximal phalanges but not any other part of the skeleton).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 3 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects observed.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item, at a dietary level of up to 5% (ca. 3.3 g/kg bw/day) did not exhibit any maternal toxicity, fetotoxicity, embryotoxicity, or teratogenicity in female Wistar Crl:(WI)WU BR rats. Therefore, the NOAEL in rats was set at 3300 mg/kg bw/d.
Executive summary:

A study was conducted to assess the embryotoxic, fetotoxic, and teratogenic potential of the oral administration of the analogue substance neohesperidin dihydrochalcone in rats by a method according to OECD 414 (GLP study). Starting from day 0 of gestation, 28 mated female Wistar Crl:(WI)WU BR rats per group received experimental diets containing the analogue substance at levels of 0 (control), 1.25, 2.5, and 5%. The top dose was selected on the basis of previous studies conducted. Maternal examinations performed included detailed clinical observations, body weights, food consumption and compound intake, gross necropsy, and organ weights. The fetuses were removed from the uterus, dried of amniotic fluid, weighed, sexed by observing the anogenital distance, and examined for gross abnormalities. The placentas of the live fetuses were weighed and examined for macroscopic abnormalities. Early and late resorptions and dead fetuses were counted. Half of the fetuses randomly selected from each litter were eviscerated, partly skinned, fixed in 70% ethanol, cleared in potassium hydroxide, and stained with Alizarin Red S for examination of the skeleton. The remaining fetuses were fixed in Bouins fluid for subsequent visceral examination. All examinations for fetal abnormalities were performed microscopically. Skeletal and visceral examinations were conducted on fetuses of the control and high-dose group only. Based on the test results, the test item, at a dietary level of up to 5% (corresponding to an intake of about 3.3 g/kg bw/day) did not exhibit any maternal toxicity, fetotoxicity, embryotoxicity, or teratogenicity in Wistar Crl:(WI)WU BR rats. Therefore, the NOAEL of the analogue substance neohesperidin dihydrochalcone in rats was set at 3300 mg/kg bw/d.