Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Screening for reproductive/developmental toxicity: Data waiving (study scientifically not necessary): According to REACH Annex VIII, column 2, the screening study does not need to be conducted because pre-natal developmental toxicity studies are available.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Weight of evidence approach. Read-across from experimental results in analogue substances


 


There are two available developmental studies on the main components rutoside and quercetin. The results of both studies indicate that these substances do not have teratogenic effects. There is also one study available on rutin hydrate that confirms this result. However, these studies have been assessed to be of low reliability because tests were performed with less than 3 doses and/or less than 16 females with implantations.


Thus, studies on analogue substances neohesperidin dihydrochalcone and methyl hesperidin have been included in the dossier in order to strengthen the weight of evidence approach. The pre-natal developmental studies performed on these analogues did not find maternal or developmental effects and therefore these analogues are considered without teratogenic properties.


Rutoside, isoquercetin and kaempferol-3-O-rutinoside, three of the main components of the substance (91% of total content) are flavonol glycosides. Quercetin, the fourth main component, is a flavonol and it is the aglycone form of the two main components, rutoside and isoquercetin (90% of total content).


Glycosides, when ingested, are hydrolysed by the intestinal microflora, yielding the corresponding aglycones. Thus, absorption of these substances in significant amount will only occur via their hydrolysed derivatives (i.e., quercetin for the two main components).


Likewise, analogue neohesperidin dihydrochalcone is a dihydrochalcone glycoside and methyl hesperidin is a flavanone glycoside. Both glycosides are also hydrolysed to their corresponding aglycone (hesperetin dihydrochalcone or methyl hesperetin) and further absorbed in this form.


These aglycones are absorbed mainly in the bacterially colonized segments of the gastrointestinal tract and are partly conjugated with glucuronic acid and/or sulphate and partly further metabolized by bacterial ring cleavage (of the flavonoids C-ring). Then, the glucuronates, sulphates and bacterial degradation products (phenols and phenolic compounds) are all excreted via the bile and urine (EFSA Journal 2010; 8(9):1065)


As the metabolic pathway is very similar, all these substances are expected to have similar toxicological properties.


The available developmental studies on both analogue substances did not show any developmental effects up to the highest dose tested and are both of good reliability. Thus, as a worst case approach the NOAEL for the test substance was selected based on the lowest NOAEL of the two studies.


In this selected study, the analogue neohesperidin dihydrochalcone was tested in a prenatal developmental study according to OECD 414 (GLP study) and performed in 28 mated female Wistar rats at doses of 1.25, 2.5, and 5% in diet. Results showed that 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. Therefore, the NOAEL of the test item was set at 3300 mg/kg bw/d.


Based on these results, the read-across approach was applied and the substance extract of fava d'anta was determined to not have teratogenic properties and the NOAEL for dams and fetuses was calculated to be 3065.59 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The analogue substance neohesperidin dihydrochalcone which shares the same functional groups with the main components of the target substance Extract of fava d'anta also has comparable values for the relevant molecular properties. See attached the reporting format.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attached the reporting format.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance and main components of the target substance are synthetic or naturally occurring flavonoids found in different plants and foods.
Neohesperidin dihydrochalcone is a dihydrochalcone glycoside mainly used as a sweetener and flavouring agent. It can be prepared by the hydrogenation of neohesperidin, a bitter flavanone found in citrus fruits.
Rutoside, isoquercetin and kaempferol-3-O-rutinoside are naturally occurring flavonol glycosides. Quercetin is a flavonol and it is the aglycone form of rutoside and isoquercetin.
Rutoside is found in many plants including buckwheat, the leaves and petioles of Rheum species, and asparagus. Isoquercetin is a compound naturally found in various plants and foods such as tea, wine and strawberries. Quercetin is widely distributed in many edible plants. Kaempferol-3-O-rutinoside is a bitter-tasting flavonol that can be isolated from the rhizomes of the fern Selliguea feei.

Ingestion of these substances results in the formation of the same range of metabolites. Since the β-glycosidic bond is generally resistant to the action of the mammalian hydrolysing enzymes, glycosides are hydrolysed by the intestinal microflora, yielding the corresponding aglycones. Thus, absorption of these substances in significant amount will only occur via their hydrolysed derivatives (hesperetin dihydrochalcone or quercetin). These aglycones are absorbed mainly in the bacterially colonized segments of the gastrointestinal tract and are partly conjugated with glucuronic acid and/or sulphate and partly further metabolized by bacterial ring cleavage (of the flavonoids C-ring). Then, the glucuronates, sulphates and bacterial degradation products (phenols and phenolic compounds) are all excreted via the bile and urine. As the metabolic pathway is very similar, they are expected to have similar toxicological properties.

4. DATA MATRIX
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Species:
rat
Key result
Dose descriptor:
NOAEL
Effect level:
4.64 other: % in diet
Based on:
other: Read-across from an analogue
Basis for effect level:
other: no significant effects observed
Remarks on result:
other: read-across from an analogue for which NOAEL = 5% in diet (equivalent to 3300 mg/kg bw)
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
4.64 other: % in diet
Based on:
other: Read-across from an analogue
Sex:
male/female
Basis for effect level:
other: no significant effects observed
Remarks on result:
other: read-across from an analogue for which NOAEL = 5% in diet (equivalent to 3300 mg/kg bw)
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on read-across approach from the analogue neohesperidin dihydrochalcone, the substance extract of fava d'anta was determined to not have teratogenic properties and the NOAEL for maternal and fetus toxicity was calculated to be 3065.59 mg/kg bw/d.
Executive summary:

A study was conducted to assess the embryotoxic, fetotoxic, and teratogenic potential of the oral administration of the test item 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 test item in rats was set at 3300 mg/kg bw/d.


 


Based on these results, the read-across approach was applied and the substance extract of fava d'anta was determined to not have teratogenic properties and the NOAEL for dams and fetuses was calculated to be 3065.59 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
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%)
Species:
rat
Strain:
Wistar
Remarks:
Crl:(WI)WU BR
Details on test animals or test system 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
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
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.
Dose / conc.:
0 other: %
Remarks:
control group
Dose / conc.:
1.25 other: %
Dose / conc.:
2.5 other: %
Dose / conc.:
5 other: %
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 bacground 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.
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

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 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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter (control and high-dose group only)
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes (only to identify sex)
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.
Indices:
Fertility index (%) = (no. of pregnant females/no. of mated females) x 100.
Gestation index (%) = (no. of females with live fetuses/no. of females pregnant at C-section) x 100.
Clinical signs:
no effects observed
Description (incidence and severity):
No signs of ill health, abnormal behavior 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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine 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 examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
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 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
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean number of early and late resorptions id not differ between any of the treated groups and the controls.
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.
Key result
Dose descriptor:
NOAEL
Effect level:
5 other: % in diet
Based on:
test mat.
Basis for effect level:
other: no significant effects observed
Remarks on result:
other: equivalent to 3300 mg/kg bw
Key result
Abnormalities:
no effects observed
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.
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
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. 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):
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.
Key result
Dose descriptor:
NOAEL
Effect level:
5 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects observed
Remarks on result:
other: equivalent to 3300 mg/kg bw
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
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 test item 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 test item in rats was set at 3300 mg/kg bw/d.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
3 065.59 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A weight of evidence approach has been applied. A couple of experimental studies are available with a Klimisch score of 2.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Weight of evidence from experimental results with individual components and analogue substances:


 


Rutoside was tested at 1% diet in a developmental toxicity study with albino rats. No adverse effects were seen in the litters, thus a NOAEL = 1% in diet (equivalent to 500 mg/kg bw day) can be stated.


 


A teratogenic test similar to OECD 414 was conducted for quercetin in Sprague-Dawley rats at doses of 2, 20, 200, 2000 mg/kg bw/d. Doses were given by oral gavage at a single dose in day 9 of gestation and during gestation days 6 to15. The results indicated that neither a single oral exposure during a critical stage of embryonic development nor multiple oral doses of test item given throughout organogenesis represented a teratogenic threat in the rat. Therefore, it is concluded that the test item is not teratogenic in rats and the NOAEL is 2000 mg/kg bw per day.


 


Rutin hydrate was tested in a reproduction and developmental study conducted in C57BL/6J mice at doses of 1 and 2% of test item in diet. Results proved that the consumption of maternal diets throughout gestation and lactation did not affect litter size, fetal development, postnatal survival, skeletal development or postnatal body weight of the offspring. Therefore, the NOAEL of the test item in C57BL/6J mice for fetal growth and development was set at 2% in diet (equivalent to 1000 mg/kg bw day).


 


A prenatal developmental study according to OECD 414 (GLP study) was conducted on neohesperidin dihydrochalcone in 28 mated female Wistar rats at doses of 1.25, 2.5, and 5% in diet. Results showed that 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. Therefore, the NOAEL of the test item in rats was set at 3300 mg/kg bw/d.


 


A prenatal developmental toxicity test was conducted on methyl hesperidin by a method similar to OECD 414 through oral administration of the test item to 19 -20 Wistar rats per group at doses of 2, 4 and 8 g/kg, during gestation days 7 to17. No effects were observed in any of the maternal or developmental parameters examined. Thus, a NOAEL for maternal and developmental toxicity in rats was set at 8000 mg/kg bw. The test item was not teratogenic under test conditions.

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.

Additional information