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

6 Summary

The documentation is mainly based on old studies performed over 30 years ago that do not fully comply with current internationally accepted guidelines and GLP. Some study reports have not been available, and results have been cited from the Reregistration Eligibility Decision (RED) document for dichlobenil (U.S.Environmental Protection Agency, 1998).

Description

BAM is a crystalline solid with a melting point of about 200 °C. It is soluble in water (2.7 g/l), and it has a low n-octanol-water partition coefficient (0.77). The vapour pressure of BAM has been estimated by different methods and ranges from 3.5 x 10^-4 Pa to 4.4 x 10^-3 Pa.

Use

BAM is a soil metabolite of the herbicides chlorthiamid (2,6-dichlorothiobenzamide) and dichlobenil (2,6-dichlorobenzonitrile), which were on the Danish market in the periods 1965 – 1980 and 1970 – 1996, respectively. BAM has no known industrial uses.

Environment

BAM has been found in water supply wells. In the period 1992-2001 about 22 % of all water samples analysed for BAM contained BAM in a mean and median concentration of 0.318 and 0.040 µg/l, respectively. The highest concentration detected was 560 µg/l. In 2002 BAM was encountered in 34 – 38 % of small water supply wells in four Danish counties, and the highest concentration encountered was 14 µg/l. BAM is considered stable in water. It is not expected to biodegrade or hydrolyse rapidly in water, and volatilisation from water is not expected to play a role either due to a low estimated Henry's Law constant (1.22 x 10^-9 (atm x m³)/mole.

Human exposure

Intake is considered the most important route of human exposure to BAM (i.e. primarily drinking water containing BAM). Potential dichlobenil exposure may only contribute to BAM exposure to a very limited extent due to minor metabolic conversion of dichlobenil to BAM.

Toxicokinetics

In rats, BAM is well absorbed after oral administration. Over 60 % was eliminated via the urine and 15 % via faeces 48 hours after a single oral dose of radiolabeled BAM. The entero-hepatic circulation plays an important role. The retention of radioactivity in the carcass was about 5 % after 96 hours. Dermal absorption of BAM has not been investigated.

Autoradiographic studies showed that a marked retention of radioactivity was associated with the nasal region (especially in the Bowman's glands) and the contents of the large intestines of rats and mice dosed with ¹⁴C-BAM.

About 25 % of the oral dose was excreted unchanged via the urine in rats. The major route of metabolism of BAM in rats and mice involved conjugation with glutathione in a process, which also involved hydroxylation of the aromatic ring. None of the urinary metabolites of BAM were common to the metabolites of dichlobenil found in the same species.

Human toxicity

No data available.

Animal toxicity – acute effects

Rats

The acute oral LD50 value for rats was 1470 mg/kg for males and 2330 mg/kg for females. The acute oral LD50 for mice was 1538 and 1144 mg/kg in males and females, respectively. CNS effects have been observed: prostrate, limbs relaxed, righting reflex absent, miosis, rapid and shallow respiration, and at high doses unconsciousness. At the lowest dose administered to rats (1000 mg/kg), the symptoms included: one death, prostrate, limbs relaxed, righting reflex absent but corneal reflex present, miosis, and rapid but shallow respiration.

The acute toxicity of BAM after dermal exposure or inhalation has not been studied.

Mice

The toxic effect of BAM was examined in the nasal passages of C57Bl mice following single ip injections (25, 50 or 100 mg/kg bw). No clinical signs of toxicity were observed in any of the BAM treated mice. After administration of 25 and 50 mg/kg bw there was an indication of disturbed function of the Bowman's glands (decreased PAS-staining). This effect was not observed 20 days after the exposure. At 100 mg/kg bw necrosis of the Bowman's glands and neuroepithelium was observed especially in the dorsomedial aspects of the olfactory region. No lesions were observed in other parts of the nasal cavity or in the liver. It was proposed that the lesions are due to a local cytochrome P450-dependent activation and that the more extensive toxic effects of chlorthiamid and dichlobenil in the olfactory mucosa are mediated by common or closely related metabolites, different from those of BAM.

Given that the decreased PAS-staining of Bowman's glands only implies a slight physiological response and does not affect the function of the nasal cavity, an acute NOAEL of 50 mg/kg may be assigned. The LOAEL may therefore be 100 mg/kg.

For an overview of NOAEL's, LOAEL's and critical effects see Table 5.

Rats

A group of Wistar rats (5 of each sex per group) received BAM orally at the following dose levels: 6.25, 12.5, 25, 50, 100, 200, 400, 800 and 1600 mg/kg bw/day for 8 days. Deaths occurred between day 2 and 9 and were associated with loss of righting reflex, corneal and pain reflexes, mydriasis, shallow respiration, bradycardia and hypothermia. The subacute oral LD50 value was estimated to be 677 mg/kg for males and 574 mg/kg for females. The reduction in skeletal muscle tone was the most sensitive parameter in this study. The hypotonus was accompanied by impaired righting reflex, miosis, hypothermia, moderate analgesia and rapid but shallow respiration. These symptoms appeared about 15 min after the treatment and peaked 2 hours later. The NOAEL was 12.5 mg/kg bw/day for males and 50 mg/kg bw/day for females.

Wistar rats (10/sex/dose) were exposed to BAM for 13 weeks at dietary levels of 0, 50, 180, 600 or 2300 ppm. The NOAEL for systemic effects was set at 180 ppm (14 mg/kg/day) and the LOAEL was set at 600 ppm (49 mg/kg/day) based on decreased body weight gain and food efficiency, increased blood urea nitrogen, and reduced coagulation times.

Dogs

Dogs (6 (control) or 4 dogs/sex/group) were fed a diet containing 0, 100, 300 and 2000 ppm BAM for a period of 13 weeks. Dogs in the highest dose group showed reduced body weight, increased liver weights, and in females decreased serum urea and increased serum alkaline phosphatase activity and 2-globulin. At 300 ppm, increased liver weights were observed. The NOAEL in this study was 100 ppm (approximately 2.5 mg/kg bw/day).

Animal toxicity – Chronic effects / carcinogenicity

Rats

In a combined chronic toxicity/carcinogenicity study BAM was given to Crl CD rats (35/sex/dose) for 106-107 weeks at dietary levels of 0, 60, 100, 180 or 500 ppm. At 500 ppm the following was observed: a decrease in mean body weight gain, increased relative liver weight (females) and slightly increased severity of fat deposition in the livers of females. The NOAEL was or 180 ppm or 6.5 (6.0 based on analysed feed data) mg/kg/day. The LOAEL was set at 500 ppm or 19 mg/kg/day (17 mg/kg/day based on analysed feed data).

BAM produced an increased incidence of hepatoma in females at 500 ppm (≈ 25(23 based on analysed female feed data) mg/kg bw/day), which was of borderline significance (p < 0.049).

Dogs

Beagle dogs (4/sex/dose) were studied in a chronic toxicity study. BAM was fed to the dogs for two years at dietary levels of 0 (control), 60, 100, 180, or 500 ppm. The NOAEL was 180 ppm or 4.5 mg/kg/day. The LOAEL was set at 500 ppm or 12.5 mg/kg/day based on decreased body weight gain in males (58 % of controls at 2 years) and in females (29% of controls at 2 years) and increased relative liver weight in males.

Reproductive and developmental effects

Rabbits

In a teratology study, New Zealand white rabbits (16/dose group) were administered BAM at dosing levels of 0, 10, 30, or 90 mg/kg/day by oral gavage on gestational days (GDs) 7 – 19. Developmental toxicity was observed at 90 mg/kg/day as a non-significant decrease (94% of controls) in foetal body weight, which was outside the historical control range. BAM was not teratogenic in this study. The maternal NOAEL was set at 10 mg/kg/day, and the LOAEL was set at 30 mg/kg/day. The developmental toxicity NOAEL was set at 30 mg/kg/day, and the developmental toxicity LOAEL was set at 90 mg/kg/day.

Rats

In a three generation reproduction study with two litters per generation, BAM was given to 10 male and 20 female Long-Evans rats per dose group at dietary levels of 0, 60, 100, or 180 ppm (equivalent to about 0, 3-6, 5-10, or 9-18 mg/kg bw/day). The NOAEL was 60 ppm (3-6 mg/kg bw/day), and a LOAEL was set at 100 ppm (5-10 mg/kg/day).

Mutagenic and genotoxic effects

BAM was negative in a bacterial reverse mutation assay, in a test for unscheduled DNA synthesis in primary rat hepatocytes, and in an in vivo mouse micronucleus assay using a single dose of BAM (250 mg/kg).

Table 5. Summary of NOAEL's and LOAEL's

BAM versus dichlobenil

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 may be comparable to that of BAM, but neither the -CONH2 group of BAM nor the –CN group of dichlobenil is changed. The reaction:

Ar•CN → Ar•CO•NH2 → ( Ar•COOH)

that could result in common metabolites does apparently not occur to a measurable degree. In addition, a high fraction of absorbed BAM is excreted unchanged in the urine (about 25%) whereas dichlobenil is apparently not eliminated unchanged via the urine.

Like BAM, dichlobenil generally is of low acute toxicity. It is slightly toxic by the oral, dermal, and inhalation routes (U.S.Environmental Protection Agency, 1998).

The effect on the olfactory mucosa after a single ip injection to mice has been investigated. Necrosis of the Bowman’s glands and the neuroepithelium was observed from 12 mg dichlobenil/kg (lowest dose), from 12 mg dichlorothiobenzamide/kg (but not 6 mg/kg), and at 100 mg BAM/kg (but not 50 mg/kg). Dichlobenil was considerably more toxic to the olfactory mucosa when compared to BAM (Brandt et al., 1990, Brittebo et al., 1991).

The critical effects identified for chronic exposure to dichlobenil were derived from a two-year dog feeding study where systemic toxicity was observed (U.S.Environmental Protection Agency, 1998). The NOAEL was 1.25 mg/kg/day. The LOAEL for systemic toxicity was 8.75 mg/kg/day based on:

1. an increase in absolute and relative liver and thyroid weights in both sexes;
2. an increase in serum alanine aminotransferase in females, and serum alkaline phosphatase in both sexes;
3. an increase in liver enzyme glucose-6-phosphatase and glucose-6-phosphatase dehydrogenase activity in both sexes; and
4. leucocytic infiltration and fibrinoid degeneration around the central hepatic veins of both sexes.

The critical effects identified for chronic exposure to BAM was determined from a two-year dog feeding study based on decreased body weight gain in both males and females. The NOAEL of 4.5 mg/kg/day has been determined, and the LOAEL was 12.5 mg/kg/day. The liver appears to be the target organ for both BAM and dichlobenil.

Neither dichlobenil nor BAM have demonstrated mutagenic potential in a variety of mutagenicity tests (U.S.Environmental Protection Agency, 1998).

There is only limited evidence for carcinogenicity of dichlobenil that is classified like BAM by US EPA as a Group C, “possible human carcinogen” (U.S.Environmental Protection Agency, 1998). Liver tumours were observed in one out of two hamster studies and in one study with Fisher rats. Available data for BAM suggests that its potential carcinogenicity (also liver tumours) does not exceed that of dichlobenil (and may be lower).

Dichlobenil is not considered to be specific toxic to reproduction based on two teratogenicity studies (rabbits and rats) and one two-generation study with Sprague-Dawley rats. Although not fully investigated, this also appears to be the case for BAM.

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Version 1.0 November 2004, © Danish Environmental Protection Agency