Regul Toxicol Pharmacol. 2017 Mar;84:26-34.

In Vitro Metabolism of [14C]-Benalaxyl in Hepatocytes of Rats, Dogs and Humans

Authors and Affiliation

Gopinath C Nallani1*, Shaaban F ElNaggar1, Li Shen2 and Appavu Chandrasekaran1

1Global Regulatory Science, FMC Agricultural Solutions, 701 PrincetonSouth Corporate Center, Ewing, NJ, 08628, USA; Phone: (609) 963-6200, Fax: (609) 538-6823

2Frontage Laboratories, 700 Pennsylvania Drive, Exton, PA 19341

* Corresponding Author; Email:



The in vitro comparative animal metabolism study is now a data requirement under EU Directive 1107/2009 for registration of plant protection products. This type of study helps determine the extent of metabolism of a chemical in each surrogate species and whether any unique human metabolite(s) are formed. In the present study, metabolism of racemic [14C]-benalaxyl, a fungicide was investigated in cryopreserved rat, dog and human hepatocytes. The metabolites generated were identified/ characterized by LC/MS/MS with radiometric detection and comparison with reference standards. [14C]-glucuronide conjugates of benalaxyl metabolites in rat, dog and human hepatocytes were confirmed via additional experiments in which known reference standards were incubated with dog liver microsomes in the presence of UDPGA. After 4 hours of incubation, benalaxyl was extensively metabolized in all the species with the following trend: dog (100%) > human (86%) > rat (75%). In all species, the major metabolic pathways consisted of hydroxylation of the methyl group in the xylene moiety to 2-hydroxymethyl-benalaxyl, further oxidation to its carboxylic acid analogue (benalaxyl-2-benzoic acid), and hydrolysis of the methyl ester to yield benalaxyl acid or 2-hydroxymethyl benalaxyl acid. In addition, glucuronidation of phase I metabolites occurred in all species, to a higher extent in dog hepatocytes in which 2-hydroxymethyl-benalaxyl-glucuronide conjugate constituted the most significant metabolite. No major unique metabolite was observed in human hepatocytes. Also, benalaxyl did not undergo stereo-selective metabolism in rat or human hepatocytes.

Keywords: benalaxyl, comparative in vitro metabolism, cryopreserved, hepatocytes



Under the current EU regulatory framework EC 1107/2009, in vitro comparative animal metabolism study is required to be submitted as part of submission of new or renewal of plant protection products, primarily to obtain information on the presence, if any, of unique human metabolites [1]. Benalaxyl (methyl-N-phenylacetyl-N-2,6,-xylyl alaninate) is a broad-spectrum fungicide of phenylamide class that inhibits growth of fungi and germination of spores. It is a foliar applied product and is very efficient in controlling fungal diseases in potatoes and grapes. The present study examined in vitro metabolism of Benalaxyl (racemic) in cryopreserved hepatocytes of rat, dog and human.

This study used cryopreserved hepatocytes of animals (rat, dog and human) as the test system. Cryopreservation of primary hepatocytes reduces the need for fresh liver tissues. In addition, these test system have previously been proven to exhibit good metabolic activity close to those of fresh liver hepatocytes [2].

Metabolism of Benalaxyl was qualitatively similar across the three species tested and there were no unique human metabolites. After 4 hours of incubation the extent of metabolism was highest in dog (100%) followed by human (86%) and rat (75%). Benalaxyl metabolized primarily via oxidation and to a lesser extent through hydrolysis. Hydroxylated metabolites were further conjugated. Benalaxyl did not undergo stereo-selective metabolism in rat or human hepatocytes as shown in Figure 1.



Figure 1. Chiral separation of enatiomers of Benalaxyl before and after 4 h incubation with rat and human hepatocytes



  1. EU, 2013. Commission Regulation (EU) No 283/2013 of March 2013 setting out data requirements for active substances, in accordance with Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on . Official Journal of the European Union L 93, 3 April 2013, pp. 1-84.
  2. Plant, N., 2004. Strategies for using in vitro screens in drug metabolism. Drug Discov. Today. doi:10.1016/S1359-6446(03)03019-8.