| Front page | | Contents | | Previous | | Next |

Evaluation of health hazards by exposure to BAM (2,6-Dichlorobenzamide) and risk characterisation of drinking water exposure

2 Toxicokinetics

2.1 Absorption, distribution
2.2 Metabolism and excretion
2.3 Toxicological mechanisms

No human data are available and the following is based on results from animal studies.

2.1 Absorption, distribution

The metabolism and tissue distribution of ¹⁴C-labelled BAM (2,6-dichlorobenzamide-carbonyl-¹⁴C) was studied in Sprague-Dawley rats (Bakke et al., 1988a). C57B1 mice were used in autoradiography studies only. Rats and mice received single doses of ¹⁴C-labelled BAM according to Table 2. Rats were given a single oral dose (5 mg) of labelled BAM followed by the collection of urine, bile, and faeces for metabolite analysis. Mice were given an intravenous injection of 7 mg/kg labelled BAM and sacrificed at 10 minutes, 1, 4 and 24 hours, and 4 and 12 days for autoradiographic analysis. Juvenile rats were given an intravenous injection of 2 mg/kg labelled BAM and sacrificed at 10 minutes, 4 and 24 hours. In addition one mouse and one juvenile rat were dosed intravenously with 7 or 2 mg/kg, respectively, of ¹⁴C-labeled BAM and killed 4 hours later, for special examination of the nasal region (microautoradiography).

Table 2. BAM metabolism and distribution studies in rats and mice (Bakke et al., 1988a)

BAM is extensively absorbed from the gastrointestinal tract, because a total of 85 percent of the radioactivity was found in the bile, urine, and tissues 48 hours following the oral administration of BAM to bile duct cannulated rats (total recovery about 90 %). Non-cannulated control rats excreted much more ¹⁴C from BAM in the urine as did the cannulated rats indicating that entero-hepatic circulation was occurring. About 13 – 23 % was excreted with the faeces. Over 60 % of the radioactivity was eliminated via the urine of non-cannulated rats after 48 hours and over 70 % after 96 hours. The excretion of about 7 times more radioactivity in the faeces of non-cannulated compared to cannulated rats indicate that biliary metabolites were precursors to the faecal radioactivity.

Whole body autoradiography and nasal microautoradiography of rats and mice showed the chemical to be homogeneously distributed throughout the body at times between 10 minutes and 4 hours post injection. Radioactivity appeared in the liver, tracheobronchial mucosa, esophageal mucosa, and adrenal and kidney cortices between 24 hours and 4 days post injection. A marked retention of radioactivity was associated with the lateral nasal gland (Steno's gland) that persisted for 4 days. The radioactivity in the olfactory mucosa persisted 12 days after injection, and a marked retention of radioactivity was observed in skeletal muscles for up to 12 days after injection.

Contrary to animals killed 10 minutes after injection much of the radioactivity of the olfactory mucosa and in the contents of the large intestine could not be extracted. The nasal microautoradiography revealed high level of non-extractable radioactivity in Bowman's glands situated beneath the olfactory epithelium, especially in the mouse but in the rat the level also exceeded the background level.

2.2 Metabolism and excretion

Plant metabolism studies on apples and grapes indicate that BAM is the major residue after treatment with dichlobenil (U.S.Environmental Protection Agency, 1998). In addition, a small residue fraction was identified as 4-hydroxy-BAM. US EPA has established tolerances for the combined residues of the herbicide dichlobenil and its metabolite BAM in or on (U.S.Environmental Protection Agency, 2001):

Apples and pears:	0.5 ppm
Blueberries, grapes, and stone fruits:	0.15 ppm
Blackberries, cranberries, and raspberries:	0.10 ppm

Today, no corresponding tolerances are available in Denmark or as harmonised maximum residue levels (MRLs) in the European Union.

BAM was not found as a metabolite or transitory intermediate of dichlobenil in ruminant or poultry studies (U.S.Environmental Protection Agency, 1998). In general, arylnitriles are metabolised to only a small extent by reactions involving the cyano group (Griffiths et al., 1966):

Ar•CN → Ar•CO•NH2 → Ar•COOH

The extent to which this reaction occurs depends on the nature of the substituents in the benzene nucleus. Early studies with radiolabeled dichlobenil indicate that less than 2 % is metabolised in vivo to BAM plus 2,6-dichlorobenzoic acid after oral administration to rats or goats (Bakke et al., 1988b, Griffiths et al., 1966).

As mentioned earlier the metabolism of ¹⁴C-labeled BAM (2,6-dichlorobenzamide-carbonyl-¹⁴C) was studied in Sprague-Dawley rats (Bakke et al., 1988a). The metabolism was not studied in mice. The major metabolites formed in rats were identified by mass spectrometry and proton magnetic resonance spectrometry.

Oral doses of ¹⁴C-labeled BAM were excreted by rats as (see also Table 3):

• unchanged BAM (about 25 % in urine)
• two monohydroxy BAM's: 3-hydroxy-2,6-dichlorobenzamide (CAS No. 22818-74-2) and 4-hydroxy-2,6-dichlorobenzamide (CAS No. 7446-00-6) (about 5 % combined in urine).
• 2-chloro-5-hydroxy-6-(methylthio)benzamide (or 6-chloro-3-hydroxy-2-(methylthio)benzamide, CAS No. 117415-43-7) (about 6 % in urine)
• 2-chloro-5-hydroxy-6-[S-(N-acetyl)cysteinyl]benzamide (mercapturic acid) (or N-acetyl-S-[2-(aminocarbonyl)-3-chloro-6-hydroxyphenyl]-L-cysteine, CAS No. 117384-43-7) (about 23 % in urine)
• The metabolites isolated from urine and bile from rats dosed with BAM are also listed in Table 3. The theoretical metabolite, 2,6-dichlorobenzoic acid was not identified. Faecal metabolites were not characterised.

Table 3. Metabolites isolated from urine and bile from rats dosed with BAM showing the percentages of the oral doses excreted as each metabolite (Bakke et al., 1988a)

1 Present as mercapturic acid pathway metabolites
2 Cysteine conjugate

Enterohepatic circulation and metabolism by intestinal microflora were involved because germ free and cannulated rats excreted neither 2-chloro-5-hydroxy-6-thiolobenzamide nor 2-chloro-5-hydroxy-6-(methylthio)-benzamide in the urine, whereas 2-chloro-5-hydroxy-6-thiolobenzamide, a presumable precursor for 2-chloro-5-hydroxy-6-(methylthio)benzamide, was excreted in the bile. Intestinal microfloral metabolism was involved in the formation of 2-chloro-5-hydroxy-6-(methylthio)benzamide, and the mercapturic acid served as a precursor.

The urinary metabolite profile of BAM in rats did not resemble that of dichlobenil (2,6-dichlorobenzonitrile) (Bakke et al., 1988b). The metabolic pathway(s) of dichlobenil is comparable to that of BAM, but as for the -CONH2 group of BAM the -CN group of dichlobenil is not changed. The reaction:

Ar•CN → Ar•CO•NH2 (→ Ar•COOH)

resulting in common metabolites does apparently not occur to a measurable degree. A high fraction of absorbed BAM is excreted unchanged in the urine (about 25%) whereas dichlobenil is apparently not eliminated unchanged via the urine. Based on the metabolites identified, two general metabolic pathways may be proposed: (1) hydroxylation at the 3 or 4 position of the phenyl moiety followed by sulphation or glucoronidation and (2) conjugation with glutathione through displacement of the chlorine atom followed by the mercapturic acid pathway.

Lactating goats were dosed with [U-phenyl]¹⁴C-BAM at a dose level of 10 ppm for five days (U.S.Environmental Protection Agency, 1998). The primary residue found in milk, kidney, fat, and muscle was unchanged BAM. The major residue found in liver was the glutathione conjugate 6-chloro-3-hydroxy-2-mercaptobenzamide.

Laying hens were also dosed with [U-phenyl]¹⁴C-BAM at a dose level of 10 ppm for five days (U.S.Environmental Protection Agency, 1998). The primary residue found in all matrices collected was unchanged BAM.

2.3 Toxicological mechanisms

The toxicological mechanism is unknown.

| Front page | | Contents | | Previous | | Next | | Top |

Version 1.0 November 2004, © Danish Environmental Protection Agency