(S)-7-[[2-O-6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Effect of Methyl Hesperidin on Prenatal Development of Rats

Author:

Kawashima K, Nakaura S, Usami M, Yamaguchi M, Tanaka S, Takanaka A, Omori Y

Year:

1986

Bibliographic source:

Bulletin of the National Institute of Hygienic Sciences, Tokyo, no. 104, pp. 64-68. 1986.

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-(3,4-dimethoxyphenyl)-5-hydroxy-7-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-({[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}methyl)oxan-2-yl]oxy}-3,4-dihydro-2H-1-benzopyran-4-one

Cas Number:

11013-97-1

Molecular formula:

C29H36O15

IUPAC Name:

(2S)-2-(3,4-dimethoxyphenyl)-5-hydroxy-7-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-({[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}methyl)oxan-2-yl]oxy}-3,4-dihydro-2H-1-benzopyran-4-one

Test material form:

solid

Specific details on test material used for the study:

TEST MATERIAL
As for methyl hesperidin, the standardized product according to Japan’s Specification and Standards for Food Addictives (Hamari Chemicals, Lot. No. TO8PG-177) was used.
- Name of test material (as cited in study report): methyl hesperidin
- Molecular formula (if other than submission substance): C29H36O15
- Molecular weight (if other than submission substance): 624.5871
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(OC)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1S/C29H36O15/c1-11-22(32)24(34)26(36)28(41-11)40-10-20-23(33)25(35)27(37)29(44-20)42-13-7-14(30)21-15(31)9-17(43-19(21)8-13)12-4-5-16(38-2)18(6-12)39-3/h4-8,11,17,20,22-30,32-37H,9-10H2,1-3H3/t11-,17-,20+,22-,23+,24+,25-,26+,27+,28+,29+/m0/s1
- Structural formula attached as image file (if other than submission substance): see Fig. in attached document.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan.
- Age at study initiation: 12-13 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: All the pregnant rats were accommodated one by one in aluminum cages (Made by Natsume Seisakusho Co., Ltd)
- Diet: solid feed (Oriental Yeast, MF) ad libitum.
- Water: tap water ad libitum.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2
- Humidity (%): 55± 5
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Methyl hesperidin was dissolved in distilled water and then given to rats.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: In order to get pregnant rats, males were arranged to live together with nulliparous females all night, the female rats with sperm recognized in vaginal smear next morning were provided for the test.
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

10 days

Frequency of treatment:

once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

8 000 mg/kg bw/day (nominal)

Remarks:

8g/16ml/kg

Doses / concentrations 2

Dose / conc.:

4 000 mg/kg bw/day (nominal)

Remarks:

4g/8ml/kg

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

2g/4ml/kg

No. of animals per sex per dose:

19-20 pregnant rats per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Since the toxicity of methyl hesperidin is extremely low, by considering its solubility and the dosage of its solution against rats, the dosage was set by 3 stages with the highest level as 8 g /16 ml/kg. With a spacing factor of 2, the subsequent doses were 4 g/8ml/kg and 2 g/4ml/kg were respectively set.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- The general conditions of the pregnant rats were observed, and the body weight and food consumption were measured on a daily base.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily.

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20.

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: No data
- Number of late resorptions: No data
- Other: uterus was taken out by cesarean section under anesthesia, and corpora lutea count, implantation count and fetal death were checked.

Fetal examinations:

Regarding the living fetuses, the exterior abnormalities were searched by hand-touch and visual examination, and the body weights were measured. Around 1/3 of the living fetuses were fixed with Bouin's solution and their internal organs were examined (Wilson method). About 2/3 of the remaining fetuses were fixed with 90% ethanol, stained with alizarin red, and their skeletons were examined with a magnifying glass to check for abnormalities.

- External examinations: Yes:all per litter
- Soft tissue examinations: Yes
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: Yes

Statistics:

The results were compared with those of the control group by the X2 test (death rate of maternal rats), t test (body weight, food consumption, corpora lutea count, implantation count, number of fetuses and weight of fetuses) and rank sum test (death rate of fetuses, occurrence rate of morphological abnormality, number of ossification). Statistically significant values had levels of P<0.05 and P<0.01. For the t test, Student's method was used for equal variance, and Aspin-Welch's method was used for unequal variance.

Indices:

Death rate of maternal rats, death rate of fetuses, occurrence rate of morphological abnormality, body weight, food consumption, corpora lutea count, implantation count, number of fetuses and weight of fetuses, number of ossification

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

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:

no effects observed

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

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

A maternal rat in which all of the litter had died was found in 4 g / kg group, but there was no significant difference in incidence.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

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

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: One death animal in the mid-dose group, not considered treatment related

Details on maternal toxic effects:
No change in particular was found in food consumption in all the groups with the dose of methyl hesperidin through the entire dosing period, and the body weight gain was also smooth. No abnormality was recognized in general condition either.
As for the maternal rat with all the litter dead, 1 case was recognized in the group of 4 g/kg, however, no significant difference was recognized in occurrence rate. Regarding to the average corpora lutea count, average implantation count and implantation rate, no significant change was recognized in between the control group and the groups with the dose of methyl hesperidin.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

8 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal and developmental toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

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

Skeletal malformations:

no effects observed

Description (incidence and severity):

A skeleton deformity, 1 case (0.7%) of the front vertebra of sacral vertebra was observed out of 27 fetuses in the group of 4g/kg, however, no significant difference was recognized in occurrence rate.
Regarding to the fetuses which have skeletal mutation, 113 cases (79.0%) were observed in the control group, 112 cases (76.0%) were observed in the group of 2g/kg, 132 cases (78.8%) were observed in the group of 4g/kg and 139 cases (79.8%) were observed in the group of 8g/kg. Comparing with the control group, no significant difference was recognized in the occurrence rate of each group with the dose of methyl hesperidin. The types of skeletal mutation observed and their occurrence rates were shown as follows. Cervical rib was observed in each group including the control group by 1.7-3.4%.The deformity of cervical lordosis was observed in the group of 8g/kg by 0.6%, the abnormality of thoracic body (deformity, separation) was observed in each group including the control group by 14.2%-18.6%, however, no significant difference was observed in their occurrence rate. In addition, the abnormality of sternebra (deformity, separation and damage) was observed by 43.2%-61.2% in each group including the control group. Lumbar rib (including rudimentary rib) was observed in each group including the control group by 31.0%-42.0%. However, no significant difference was recognized in its occurrence rate.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The cases of dilatation of renal pelvis were observed in each group including the control group by 1.7-4.9%, however, no significant difference was recognized in occurrence rate.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

No significant change was recognized in between the control group and the groups with the dose of methyl hesperidin in average number of fetuses, sex ratio, and average body weight of fetuses. No abnormal case was observed including the control group in results of visual test. Ossification status was judged by checking the number of metatarsal bones, sacral vertebrae and caudal vertebra, however, no significant difference was recognized from the control group regarding to all of these.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

8 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant effects observed at any dose level.

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 was found to be non-teratogenic under test conditions, with a NOAEL of 8000 mg/kg bw/d for both maternal and developmental toxicity.

Executive summary:

A prenatal developmental toxicity test was conducted by a method similar to OECD 414. Daily oral administration of the test item to 19 -20 Wistar rats per group at doses of 2, 4 and 8 g/kg, from the 7th day of pregnancy to the 17th of pregnancy was performed. A control group receiving 16 ml/kg distilled water was run in parallel. No changes in death rate, food consumption or body weight gain were observed for any of the treated groups. There was no significant change in mean fetal number, sex ratio and fetal mortality in any of the treated fetuses, no abnormalities related to the test item were observed. Based on the results, the NOAEL for maternal and developmental toxicity in rats was set at 8000 mg/kg bw. The test item was not teratogenic under test conditions.