Hesperidin

Carcinogenicity

Description of key information

Bio flavonoids such as hesperidin are not considered carcinogenic. Methyl hesperidin, as structural analogon to hesperidin, sharing common metabolites with hesperidin, has been investigated for carcinogenicity as was found not to show carcinogenic effects. It is therefore conclude that hesperidin is not considered carcinogenic, supported by the fact that hesperidin does also not show any mutagenic effects.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

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

Adequacy of study:

supporting study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: publication, well documented and detailed information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

not specified

GLP compliance:

not specified

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and environment. A total of 300 B6C3F1 mice (Charles River Japan Inc., Kanagawa, Japan), aged 5 wk old at the start of the study, were used. The mice were housed five per cage, under constant conditions, in plastic cages with stainless-steel grid tops and solid bases on white woodchip bedding (Beta Chip Bedding, Northeastern Products Co., NY, USA). Room temperature was maintained at 23 ± 1 °C with a relative humidity of 55 ± 2%. The mice were subjected to a normal 12-hr light/dark cycle and acclimatized to laboratory conditions for 1 wk prior to the start of the study.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

To prevent loss of the compound as dust during diet preparation or feeding, 20 corn oil was added prior to its incorporation. Control diets also contained 2% corn oil.
The mice were assigned to control or treatment groups according to their initial body weights using a computer-assisted classifying procedure designed to provide homogeneity of variance and equality of initial group body weight.
Administration of test substance in the diet of mice for 96 wk at concentrations of 0, 1.25 or 5% was followed by an 8-wk observation period
without chemical supplement. The two dose levels used were chosen based on criteria used in previous studies.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

96 wk

Frequency of treatment:

once a day

Post exposure period:

8 wk

Remarks:

Doses / Concentrations:
dietary concentrations of 0, 1.25 or 5%.
Basis:
nominal in diet

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

To eliminate the possibility of interference by reversible changes due to the compound treatment, a recovery period of 8 wk was set. Sub-
chronic toxicity studies were carried out with test substance prior to the carcinogenicity experiment and the 5% level was chosen as the maximum tolerable dose for life-long treatment.

Positive control:

no data

Observations and examinations performed and frequency:

All mice were observed at least once a day for clinical signs, and body weights were recorded weekly during the first 13 wk and biweekly
thereafter. Food and water consumption were measured over 2-day periods before each weighing and average daily and total intake of test substance were also calculated.
Clinical investigation: During wk 104 of the experiment fresh urine samples were obtained from 10 mice/sex/group. Urinalysis included measurements of pH, protein, glucose, ketone, bilirubin, occult blood, urobilinogen and specific gravity. Specific gravity was determined with a clinical refractometer. The other measurements were made with Multistix III. At the end of the study the mice were fasted overnight. Blood samples were taken from 10 males and 10 females of each group. Haematology parameters evaluated were haemoglobin concentration and haematocrit, and erythrocyte, leucocyte and platelet counts. The blood cell indices of mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were calculated. Differential leucocyte counts and estimation of the percentage of nucleated red blood cells, anisocytosis and polychromasia were performed for each smear. Clinical chemistry parameters determined included glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, alkaline phosphatase, total cholesterol, total protein, albu- min/globulin ratio, and urea nitrogen. These parameters were measured by autoanalyser.

Sacrifice and pathology:

Pathology: Gross and complete histology examinations were performed on all mice that died, were killed on becoming moribund, or survived until the terminal killing. During autopsy, weights were recorded for the following organs: liver, kidneys, brain, heart and testes or ovaries. Relative organ weights (organ-to-body-weight ratios) were calculated. Microscopic examinations were performed after routine processing and staining with haematoxylin and eosin. The above tissue plus the following were collected for histopathological examination: salivary glands, trachea, lungs, thymus, lymph nodes, spleen, stomach, gall bladder, small intestine (duodenum, jejunum, ileum), large intestine (caecum, colon, rectum), pancreas, urinary bladder, pituitary, thyroids, adrenals, prostate, seminal vesicles, uterus, mammary gland, skeletal muscle, eyes, Harderian glands, spinal cord, sciatic nerve and gross lesions.

Statistics:

All measurements were expressed as mean + SD and were analysed where appropriate by the F- and t-tests or the Welch method. The significance of differences in the incidences of non-neoplastic lesions between the different groups was evaluated by the chi-square test or by Fisher's exact probability test. All references to statistical significance in this experiment represent two-tailed P values. The analyses of differences in the survival periods between the treated and control groups were analysed by the Generalized Wilcoxon and Cox-Mantel tests.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details on results

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Details on results:

No treatment-related clinical signs were observed in any of the mice throughout the study. The growth of male mice given 5% test substance showed a slight decrease from the 16th to the 80th week, but this difference was not observed thereafter or at the end of the study. A significant decrease, with a slight dose dependency, in the body-weight gains of female mice given test substance at levels of 1.25 and 5%, was observed from wk 24 to 96. No obvious recovery was evident during the observation period over the last 8 wk after cessation of treatment. However, since survival periods of compound-treated male and female groups were not significantly different from those of controls, body-weight changes were not associated with any effects on the survival rate.

The average daily food consumption of the females was slightly greater than that of the males. This sex difference was reflected in both values of average daily and calculated total test article intake.

No biologically significant differences in urinalysis, haematology or clinical chemistry values were noted between control and treatment groups. A significant decrease in absolute organ weight was observed for the kidneys of females given 5,0% (P <0.01). However, evaluation of the relative organ weight revealed increases in the brain, heart and kidneys of 1.25 and 5.0% females, and decreases were evident in the brain and testes of males given 1.25% and in the kidneys of males given 1.25 and 5%. Although these changes were statistically
significant, the differences in each value between the control and experimental groups were small and no dose dependency was apparent.

Of the observed non-neoplastic findings, thyroid cysts, cyst formation and atrophy in the ovary and cystic endometrial hyperplasia of the uterus were frequent but with no treatment-related effects. Testicular atrophy and prostatitis were also observed equally in all groups. The incidences of spontaneously occurring lymphocytic accumulation in the kidneys and urinary bladder were relatively high in the female groups, but were not clearly dose related. Chronic nephropathy was observed in the male groups, at highest levels in the controls. Thus, none of the above-mentioned findings, which included age-related degenerative alterations, were considered to be due to test substance treatment.

In males, predominant lesions were liver hyperplastic nodules (hepatocellular adenoma or neoplastic nodule) and hepatocellular carcinomas and adenoma and adenocarcinoma in the lung. In females, the most common tumour was malignant lymphoma/leukaemia, which infiltrated into many organs and tissues. Other minor neoplastic lesions included adenomas in the pituitary of female mice, endometrial stromal sarcomas in the uterus and adenomas in the Harderian glands of both sexes. However, these lesions occurred at comparable incidences in both control and treated mice with no apparent dose-response relationship. Thus, the observed tumours should be regarded as being of spontaneous origin and not attributable to test- treatment.

Relevance of carcinogenic effects / potential:

No data

Dose descriptor:

NOAEL

Effect level:

5 other: percent in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The observed tumours (in test and control groups) were regarded as being of spontaneous origin and not attributable to methyl hesperidin treatment

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Conclusions:

Based on the available data, it is concluded that test substance has no carcinogenicity for B6C3F1 mice in the 96-week feeding regimen.

Executive summary:

This long-term carcinogenicity study of test substance was carried out in B6C3F1 mice receiving dietary concentrations of 0, 1.25 or 5%. Administration was continued for 96 wk and then the mice were maintained on basal diet for additional 8 wk. Growth retardation during the experiment with final changes in organ weights were observed in females given the 1.25% dose of methyl hesperidin and in both sexes receiving the 5.0% treatment. However, no biologically significant effects were evident with respect to mortality or clinical signs. Furthermore, treatment with methyl hesperidin did not result in any changes in haematology, clinical chemistry and urinalysis data. On histological examination, no significant alteration of non-neoplastic and neoplastic lesion incidence was observed in treated mice.

Thus, the results demonstrated that test substance lacked any carcinogenicity for B6C3F1 mice in the 96-wk feeding regimen used in this study. As methyl hesperidin is structurally very similar to hesperidin (only diffeence is one hydroxy function of hesperidin being methylated) and methyl hesperidin as well as hesperidin do share direct metabolites such as Hesperetin dihydrochalcone (see section on toxicokinetics), the results can be considered representative for hesperidin too.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

mouse

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Kurata et al. investigated in a long-term carcinogenicity study of methyl hesperidin which was carried out in B6C3F1 mice receiving dietary concentrations of 0, 1.25 or 5% the carcinogenic potential of flavanones. Administration was continued for 96 wk and then the mice were maintained on basal diet for additional 8 wk. Growth retardation during the experiment with final changes in organ weights were observed in females given the 1.25% dose of methyl hesperidin and in both sexes receiving the 5.0% treatment. However, no biologically significant effects were evident with respect to mortality or clinical signs. Furthermore, treatment with methyl hesperidin did not result in any changes in haematology, clinical chemistry and urinalysis data. On histological examination, no significant alteration of non-neoplastic and neoplastic lesion incidence was observed in treated mice.

Thus, the results demonstrated that methyl hesperidin lacked any carcinogenicity for B6C3F1 mice in the 96-wk feeding regimen used in this study. As methyl hesperidin is structurally very similar to hesperidin (only difference is one hydroxy function of hesperidin being methylated) and methyl hesperidin as well as hesperidin do share direct metabolites such as Hesperetin dihydrochalcone (see section on toxicokinetics), the results can be considered representative for hesperidin too. In addition, in various studies the anti-tumorgenic effect of hesperidin has been investigated, showing that hesperidin and other flavonoids show a suppresive resp. preventive effect on cancer development such as bladder cancer (see M. Yang et al., Int.J.Cancer 73, 719 -724,(1997)), oral (tongue) cancer (see T. Tanaka et al., Cancer Res 1994;54:4653-4659 and Cancer Res 1997;57:246-252), skin cancer (see B. Berkarda et al., Res Exp Med (Berl). 1998 Aug;198(2):93-9) just to name a few. In none of these studies a promoting effect of cancer development has been observed but as these studies are not classical carcinogenicity studies, their robust study summaries were not included in this dossier. In summary it can be concluded, that flavonoids such as hesperidin, are not considered carcinogenic and contrary have anti-tumorgenic effects and for this reason have been investigated as chemopreventive and anti-tumorgenics for many years (see also read across justification in section 13).

Justification for selection of carcinogenicity via oral route endpoint:
Study on a structural analogon to hesperidin, investigating carcinogenicity at elevated doses such as 5% in diet, not showing any carcinogenic effects. Methyl hesperidin and hesperidin do show common metabolites as described in the section on toxicokinetics and metabolism.