TOXICITY STUDIES IN ANIMALS

Toxicity studies evaluating the effect of diosmin/hesperidin formulations in animals have documented an excellent safety profile.

Heusser and Oswald conducted several studies examining the subchronic, chronic and teratogenic toxicities of diosmin. In a first study, oral treatment with diosmin, at 200 mg/kg daily, for 50 days, was assessed in 20 white rats. A second study also examined chronic toxicity in 22 white mice. Oral treatment with diosmin wasadministered at a dose of 620 mg/kg daily for 196 days . The observations from both experiments indicated that there were no toxic effects. These findings were confirmed by the blood count, the macroscopic and histo(ogical assessment of the organs, the weight development and biochemical examination . In another experiment, diosmin oral treatment (doses between 50 and 250 mg/kg) was given to male and female mini-pigs for 180 days . There were no systematic deviations in clinical, biochemical or haematological values, suggesting that there was no toxicological effect of diosmin' .

In a teratogenicity study conducted by Heusser and Oswald, mother white mice were given diosmin at a daily dose of 50 mg/kg from the 4th to the 12th postcoital day. The fetuses were delivered by laparotomy on the 19t" day after conception . After laparotomy, the mean number of animals per litter was 12.3 in the control group and 11 .5 in the diosmin treated group. 126 fetuses in each group were assessed and their skeletons were also examined. No pathological findings were observed2' . In another group of mice, diosmin was administered in the same dose and fetuses were delivered by spontaneous birth. The fetuses were examined macroscopically and the development of the animals was carefully monitored. Postnatal mortality (20 days) was 19% in the control group and 26% in the diosmin group. Weight gain, length development, gross behaviour, hair growth and opening of the eyes were similar in both groups. No significant differences between groups in organ weights, macroscopic and histological findings were observed 21 . In a similar study, mother white rats were administered a daily dose of 100 mg/kg of sodium salt from the 4t" to the 14t" postcoital day. The fetuses were delivered by laparotorny on the 21St day after fertilization. The mean number of animals per litter was 13.9 in both the control and diosmin groups. The average weight was 3.56 and 3.17 g respectively . After examination of their skeletons, no pathological findings were observed in the fetuses" . In another group of rats, fetuses were delivered by spontaneous birth. The average number of animals per litter was 12.9 in the control group and 11 .6 in the diosmin group. Mortality within the 20 postnatal days was respectively 11 % and 20%. There was no significant difference between the two groups in terms of weight gain, length development, gross behaviour, hair growth, opening of eyes, organ weights, macroscopic and histological examinations" . Heusser and Oswald concluded from their toxicological experiments that diosmin is well tolerated, even at very high doses, by the different animal species studied .

Animal toxicity studies of diosrnin have also been carefully reviewed and summarised by Meyer' and Buckshee et a1.4. The major findings are detailed below. Diosmin Complex (90% diosmin and 10% hesperidin) was administered to mice, rats and primates as single oral dosing, as well as repeated oral dosing (13 weeks and 26 weeks) . These represent respectively 180 times and 35 times the recommended daily dose in humans. No toxic or lethal effect could be observed. The 50% Lethal Dose (LDSO) was impossible to determine both in the mouse and in the rat, because of the excessively low toxicity; it was assessed as greater than 3000 mg/kg for the active principle".

The good gastrointestinal acceptability was confirmed in the Wistar rat, at oral doses representing 12, 24 and 48 times the recommended daily dose. No impairment of the reproductive function was found in the rat after administration of an oral dose, representing 37 times the recommended daily dose . Fertility, embryotoxicity, perinatal, and postnatal development of the generation born from treated parents were not affected . The absence of genetic toxicity was shown by the following tests: bacteria gene mutation, analysis of metaphases in human lymphocytes in culture, in vitro eukaryote system gene mutation, in vivo clastogenic lesions, and DNA repair.

Transplacental passage in the rat was assessed for a single dose of 10 mg/kg of diosmin and was minimal (0 .003% per fetus, of the dose administered to the mother) . Passage into breast milk was assessed as 1 % of the dose administered to the mother. Accumulation in the uterus was 0.02% . Autoradiography of pregnant females showed that the compound was distributed essentially in the intestine and secondarily in the kidneys. However, there was no uptake of the compound by the genital organs of the mother .

Meyer concluded that these studies, evaluating the possible toxicity on the digestive tract, the lactation and the reproductive function, demonstrated the excellent safety of Diosmin Complex in animals.

Hitzenberger also described several toxicology studies of Daflon 500 mg. Acute symptoms were studied in both mice and rats (oral administration up to a maximum of 3000 mg/kg). However, LDSO could not be determined. No deaths were observed during the 15-day observation period and no substance-related changes were detected during the autopsy". Macaca monkeys were administered an oral dose of 4500 mg/kg . No relevant toxicity symptoms were observed . In addition, subchronic toxicity was tested on rats for a period of 13 weeks. Maximum dosage of 600 mg/kg per day was given without any toxicological symptoms25. Chronic toxicity was studied on rats for a period of 26 weeks, at a dosage of 600 mg/kg per day. No substance-related changes were observed 25 . A similar administration schedule was also used in cynomolgus monkeys, with the same overall results25 . Mutagenicity was examined with various tests and no effect could be shown . Reproduction toxicological test on rats and rabbits, as well as peri- and post-natal toxicity studies and further teratogenic studies on rabbits were negative. Furthermore, fertility was not impaired

In conclusion, chronic toxicity, teratagenicity, mutagenicity, fertility and embryotoxicity studies have clearly demonstrated that diosmin has an excellent tolerability profile in animals at dose ranges superior to the recommended dosage regimen in humans.

Note:This section is cited in the Exquim’s submission to FDA for its brand Diosvein, the only purpose here is to demonstrate the safety of Diosmin to all readers who visit this webiste.