Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

 

In vitro gene mutation in bacteria

There are some experimental in vitro gene mutation studies in bacteria conducted on the main components of the substance available.

For rutin, there are evidences of some negative results but also positive results have been reported in S.typhimurium strains TA98 and TA100 (Hardigree, 1978; Nagao, 1981; Yu, 1986; Crebelli, 1987). For isoquercetin, one study is available in which positive results have been obtained in TA100, TA98 and TA1537(+/-S9) and TA1535 (+S9) (Hobbs, 2018). For quercetin, positive results have been reported in TA100, TA98, TA97, TA102, TA1538 and TA1537 (Stoewsand, 1984; Czeczot, 1990; Cross, 1996; Hardigree & Epler, 1978; NTP, 1992; Crebelli, 1987).

However, there are some studies available with analogue substances found not mutagenic in an Ames test. Naringin was tested on S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 and was assessed to be non-mutagenic. Neohesperidin dihydrochalcone was found not mutagenic when tested in TA1535, TA97, TA98 and TA100. Methyl hesperidin was also found not mutagenic in S. typhimurium strains TA92, TA94, TA100, TA1535 and TA1537.

Based on this information the read-across could be applied and the substance could be considered to be not mutagenic in the Ames test for the strains tested.

 

In vitro cytogenicity in mammalian cells

There are some experimental in vitro cytogenicity studies in mammalian cells conducted on the main components of the substance available.

Rutin was found negative in an in vitro micronucleus test performed in HTC hepatic rat cells (Marcarini, 2011). Isoquercetin was found negative in an in vitro micronucleus test performed in TK6 cells and positive in an in vitro chromosomal aberration assay performed in CHO cells (Hobbs, 2018). Quercetin induced chromosome breakages in both human and Chinese hamster cells (Yoshida, 1980).

Nonetheless, the analogue substance naringin was reported to be non-mutagenic in a chromosomal aberration test with Chinese Hamster Lung fibroblasts (CHL) performed by a method similar to OECD 473.

Based on this information the read-across could be applied and the substance could be considered to be not mutagenic in a chromosome aberration test.

 

In vitro DNA damage

Rutin was found to be positive in 2 comet assays performed in human lymphocyte cells (Anderson, 1997) and HTC hepatic rat cells (Marcarini, 2011). Quercetin was found to induce sister chromatic exchanges (SCE) in both human and Chinese hamster cells (Yoshida, 1980). However, quercetin was negative in an unscheduled DNA synthesis (UDS) assay in rat hepatocytes (Cross1996). kaempferol-3-O-rutinoside was found to produce positive responses in human lymphocytes in an in vitro comet assay (Anderson, 1997).

 

In vitro gene mutation in mammalian cells

There are some experimental in vitro gene mutation studies in mammalian cells conducted on quercetin available.

Quercetin was positive in a gene mutation study at TK locus using mouse lymphoma L5178Y cells (Van der Hoeven, 1984; Meltz, 1981). Quercetin did not induce a consistent, significant increase in gene mutation at the aprt, hgprt, or ATPase loci, but significantly increased mutation frequencies at the tk locus using CHO cells (Carver, 1983). Quercetin was reported positive in a gene mutation study at hprt locus using CV79 Chinese hamster cells (Maruta, 1979).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Weight of evidence from experimental results with individual components:

 

In vivo micronucleus assay

An in vivo micronucleus assay conducted with Madys stigma (10.5% flavonoids including rutoside and quercetin) did not show any genotoxic activity in bone marrow stem cells (Peng, 2016).

Rutoside was negative in two in vivo micronucleus assays performed on bone marrow cells of mice (Sahu, 1981; DaSilva, 2002).

An in vivo micronucleus assay conducted according to OECD Guideline 474 with isoquercetin in mice did not show any genotoxic activity in bone marrow cells at doses up to 2000 mg/kg bw (Hobbs, 2018).

Quercetin was negative in three in vivo micronucleus assays performed on bone marrow cells of mice (Caria, 1995) and wistar rats (Taj, 1996; Utesch, 2008).

 

In vivo chromosome aberration assay

Quercetin was negative in an in vivo chromosome aberration assay performed on bone marrow cells of wistar rats (Taj, 1996)).

 

In vivo Rodent Dominant Lethal Test

Quercetin was found negative in two in vivo Dominant lethal studies carried out in Swiss male mice and Wistar male rats (Aravindakshan, 1985).

 

In vivo Comet assay

Rutoside was negative in an in vivo comet assay performed on bone marrow cells of mice. Increased damage was observed only in the mid dose only and for males. For the other two doses no significant differences with the solvent control were noticed, so this was considered a chance occurrence (DaSilva, 2002).

Isoquercetin did not show any genotoxic activity in liver, duodenum or stomach at doses up to 2000 mg/kg bw in an in vivo comet assay performed in mice according to OECD Guideline 489 (Hobbs, 2018).

 

In vivo unscheduled DNA synthesis assay

Quercetin did not show genotoxic activity in male rat bone marrow cells at doses up to 2000 mg/kg bw in an UDS assay performed according to OECD Guideline 486 (Utesch, 2008).

 

In vivo transgenic rodent mutation assay

Isoquercetin did not show any genotoxic activity in liver or testis at doses up to 5 % in diet in an in vivo transgenic rodent mutation assay performed in mice according to OECD Guideline 488 (Hobbs, 2018).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

There are some positive results from in vitro gene mutation studies in bacteria on the components rutin, isoquercetin and quercetin. Also, there are some positive results from in vitro gene mutation studies in mammalian cells on quercetin. Moreover, rutin was found positive in 2 in vitro comet assays and kaempferol-3-O-rutinoside was found positive in an in vitro comet assay.

However, quercetin was found negative in two in vivo rodent dominant lethal tests and in one in vivo unscheduled DNA synthesis assay. Rutin and isoquercetin were found negative in respective in vivo comet assays. Furthermore, isoquercetin was also found negative in an in vivo transgenic rodent mutation assay. There are no in vivo studies available for kaempferol-3-O-rutinoside but nevertheless it is expected to act in the same way based on its similarities in the chemical structure with the rest of the components.

Moreover, there are positive results from some in vitro cytogenicity studies in mammalian cells on the components isoquercetin and quercetin. Also, quercetin was found positive in an in vitro sister chromatic exchange assay.

However, isoquercetin was found negative in an in vivo micronucleus assay and quercetin was also found negative in at least 3 in vivo micronucleus assays. Furthermore, quercetin was found negative in an in vivo chromosome aberration assay. Also, rutin was found negative in two in vivo micronucleus assays.

Thus, all in vivo genotoxic studies available confirm that none of the components of the test substance is expected to show mutagenicity.

Based on the available data, the substance is not classified for genotoxicity in accordance with CLP Regulation (EC) no 1272/2008.