Hesperidin

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

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

Additional information

No key-studies regarding fertility of hesperidin are available but in sub-acute and subchronic studies no effects on reproductive organs have been observed, although doses applied in these studies were very high (1% in diet food). Hence, there is currently no suspicion of hesperidin affecting impairment of fertility.

In a supporting study reported, the aim was to investigate the effect of hesperidin on male reproductive system in rats to which methotrexate (MTX) was administered. In the study, 28 male Wistar albino rats at the age of 8 weeks and had 250-300 g of live weight were used. Four experimental groups were formed; Group 1 (n=7): The control group, only feed and water were given. Group 2 (n=7): MTX group, a single dose of 20 mg/kg of i.p. MTX was administered. Group 3 (n=7): Hesperidin group, 200 mg/kg of hesperidin was administered by gavage for 7 days. Group 4: MTX + hesperidin group (n=7): Following administration of a single dose of 20 mg/kg i.p. MTX , 200 mg/kg of Hesperidin was administered by oral gavage for 7 days. At the end of the experiment, rats were decapitated and biochemical, histopathological and spermatological parameters were examined. It was observed that in the MTX group, sperm motility and density, the enzymes CAT, GPx and SOD and GSH level decreased, TNF-alpha and IL-1 Beta, as well as MDA, levels were increased, regular structure of spermatogenic cells was impaired, and seminiferous tubules became necrotic and degenerative. It was determined that spermatological parameters improved and, necrotic and degenerative changes diminished by the administration of MTX+hesperidin. These outcomes indicated that hesperidin had a protective effect on destructive effects of MTX in rat testicles. No negative effects in any of the investigated parameters were seen in the hesperidin dose group.

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: fulfill the basical principle, detailed information, performed scientifically

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

All the pregnant rats were accommodated one by one in aluminum cages (Made by Natsume Seisakusho Co., Ltd), where solid feed (Oriental Yeast, MF) and tap-water could be freely taken.
Regarding to the animal feeding and breeding room, the temperature was set as 25 ±2 ℃, the relative humidity was set as 55 ±5%, the air exchange rate was set as 15/hr, and the dark-and-light switch rate was set as 12 hours (light period: 6:00-18:00).

Route of administration:

other: oral administration by stomach tube

Vehicle:

water

Details on exposure:

Methyl hesperidin was dissolved in distilled water and then given to rats. 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, following which, divided by common ratio 2, another 2 stages as 4 g/8 ml/kg and 2 g/4 ml/kg were respectively set. During the period from the 7th day to the 17th day of pregnancy including the period of organogenesis of fetuses, oral administration was performed to 19-20 pregnant rats of each group by stomach tube once a day. In addition, 16 ml/kg distilled water was orally given to the rats of the control group as the same.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

In order to get pregnant rats, males were arranged to live together with the nulliparous females all night, the female rats with sperm recognized in vaginal smear next morning were provided for test and this day was taken as the day 0 of pregnancy.

Duration of treatment / exposure:

10 days

Frequency of treatment:

once a day

Duration of test:

no data

Dose / conc.:

0 mg/kg bw/day

Remarks:

control

Dose / conc.:

2 000 mg/kg bw/day

Dose / conc.:

4 000 mg/kg bw/day

Dose / conc.:

8 000 mg/kg bw/day

No. of animals per sex per dose:

19-20 pregnant rats of each group

Control animals:

yes, concurrent vehicle

Maternal examinations:

The general conditions of the pregnant rats were observed, and the body weight and food consumption were measured on a daily base.

Ovaries and uterine content:

On the 20th day of pregnancy, uterus was taken out by cesarean section under ether anesthesia, and corpora lutea count, implantation count and fetal death were checked.

Fetal examinations:

Regarding to the living fetuses, the exterior abnormalities were searched by hand-touch and visual contact, and the body weights were measured. Regarding to each matrix, around 1/3 of the living fetuses were fixed for about 2 weeks by Bouin solution, during which, internal organs were checked by Wilson method. After about 2/3 of the rest fetuses were fixed by 90% ethanol, alizarin red stain skeleton specimen were made to check the abnormalities of skeletal system under magnifying glass.

Statistics:

The result of test was compared with that of the control group by the levels of P<0.05 and P<0.01 by X2 test (the 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). In addition, as for t test, in case of equal variances, the student’s method was used, in case of unequal variances, the method of Aspin-Welch was used.

Indices:

the 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

Historical control data:

no data

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.

Dose descriptor:

NOAEL

Effect level:

8 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

8 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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.
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.
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.
As 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.

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

There was no evidence of an increase in fetal death or of malformation attributable to the treatment with methyl hesperidin in any of dose levels examined. It is concluded that test substance has no teratogenic effect in rats under the present experimental conditions.

Executive summary:

The prenatal developmental toxicity test was conducted by orally with Wistar rats. 2, 4 and 8 g/kg of methyl hesperidin were given to the pregnant rats from the 7th day to 17th day of pregnancy including the period of organogenesis of infuses once a day, the effect on the maintenance of pregnancy and the occurrence of fetuses was observed. There was no change recognized in death rate, food consumption and body weight gain for pregnant matrix in all the groups with the dose of test item. Regarding to the fetuses of the 20thday of pregnancy, no significant change was recognized in average number of fetuses, sex ratio and the death rate of fetuses in all the group with the dose of methyl hesperidin. Regarding to the inspection on the outside appearance, skeleton and internal organs, no abnormal case supposed to be derived from the dose of test substance was observed.

Based on the description above, it is concluded that methyl hesperidin is not a teratogen. According to CLP (Regulation EC No.1272/2008), test substance shall not be classified.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

8 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

A prenatal developmental toxicity of methyl hesperidin was conducted by oral application with Wistar rats 2, 4 and 8 g/kg bw. the test substance was given to the pregnant rats from the 7th day to 17th day of pregnancy including the period of organogenesis of infuses once a day and the effect on the maintenance of pregnancy and the occurrence of fetuses was observed. There was no change recognized in death rate, food consumption and body weight gain for pregnant matrix in all the groups with the dose of test item. Regarding to the fetuses of the 20^thday of pregnancy, no significant change was recognized in average number of fetuses, sex ratio and the death rate of fetuses in all the group with the dose of methyl hesperidin. Regarding to the inspection on the outside appearance, skeleton and internal organs, no abnormal case supposed to be derived from the dose of test substance was observed. In another study (D.H. Waalkens-Berendsen, 2004), the test article 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 Crl:(WI)WU BR.

Both substances, methyl hesperidin and neohesperidin dihydrochalcone, are structurally very similar to hesperidin. Whereas methyl hesperidin shares the common metabolite hesperetin with the metabolism of hesperidin and thus can be considered as appropriate surrogate for hesperidin, also neohesperidin dihydrochalcone, sharing the next metabolite with hesperidin, resulting from ring cleavage of the central ring in hesperitin is an appropriate surrogate. Methyl hesperidin as structurally more similar to hesperidin than neohesperidin dihydrochalcone has been selected as closest surrogate.

In another supportive study (Gustavo Tadeu Volpato, et al., 2006), a mixture of vitamin C, hesperidin and piperidol was applied to Wistar pregnant rats by oral gavage with 166.5 mg/kg/day to evaluate the reproductive performance and the development of their offspring during the organogenic period (from day 5 to 14 of pregnancy; positive vaginal smear = day 0). There was no alteration in maternal reproductive performance observed, but an increase of the number of fetuses presenting dilated urether, hydronephrosis, and reduced ossification of skull due to the treatment of female rats with the mixture was noted. These abnormalities were considered transitory and may not interfere on offspring development.

No other types of major malformation were observed, neither the appearance of fetuses presenting atrophy of upper limbs that it could be associated to use of this mixture containing piperidol.

Justification for selection of Effect on developmental toxicity: via oral route:
The study on hethylhesperidin has been selected as key study, as this substance is structurally extremely similar to hesperidin. It shares the common metabolite hesperetin with the metabolism of hesperidin and thus can be considered as appropriate surrogate for hesperidin. This study is supported by an equivalent study on neohesperidin dihydrochalcone, sharing the next metabolite with hesperidin, resulting from ring cleavage of the central ring in hesperetin.

Toxicity to reproduction: other studies

Additional information

In the absence of any findings regards fertility in sub-acute and sub-chronic studies and given that strucutral analogues such as methyl hesperidin and neohesperidin dihydrochalcone, both sharing similar metabolic pathways with hesperidin (see read across justification in section 13), did not show teratogenic effects, further studies on reproductive toxicity are not required.

Justification for classification or non-classification

Based on the description above, it is concluded that methyl hesperidin is not a teratogen. According to CLP (Regulation EC No.1272/2008) as well as DSD (Directive 67/548/EEC), test substance shall not be classified.