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

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

4 Toxicity, animal data

4.1 Acute and short term toxicity
4.2 Long term toxicity
4.3 Reproductive and developmental effects
4.4 Mutagenic and genotoxic effects
4.5 Carcinogenic effects

Test results from studies performed over 30 years ago have been available. The tests do not fully comply with current internationally accepted guidelines, and they have not been performed in accordance with current requirements to good laboratory practice (GLP). Despite the lacks in study design and performance, the studies, in general, appear to be well performed and are considered valid for a toxicological assessment of BAM.

Because of lack of actually measured data default values have sometimes been used in this assessment. WHO lists some default animal weight values in a table showing approximate relation of parts per million (ppm) in the diet to mg/kg body weight per day (WHO, 1987). The values were originally reported in 1954 (Lehman, 1954).

4.1 Acute and short term toxicity

Toxicological studies have been performed with BAM, since BAM is the major residue in plants treated with dichlobenil, and is consumed in the food supply (U.S.Environmental Protection Agency, 1998). It is not an animal metabolite.

Wistar rats (5 of each sex per group) were administered a single dose of BAM (purity not recorded) in 1% w/v aqueous tragacanth by gavage at the following doses: 1000, 2150, 4640 and 10000 mg/kg body weight (bw) (Kemp and van der Linde, 1967). It is an old study without GLP status, and it does not fully live up to today's standard. The surviving rats were observed for 14 days. Deaths occurred 3 – 72 hours after treatment. The acute oral LD₅₀ value was 1470 (951 – 2270) mg/kg for males and 2330 (1430 – 3780) mg/kg for females. The symptoms began to appear 10 min after dosage and disappeared completely 24 – 28 hours later. Symptoms from the CNS dominated the clinically appearance. At the lowest dose administered (1000 mg/kg), the symptoms included: one death, prostrate, limbs relaxed, righting reflex absent but corneal reflex present, miosis, and rapid but shallow respiration. High doses were followed by progressive narcosis and death. No autopsy was performed.

The acute oral LD₅₀ of BAM in mice was 1538 and 1144 mg/kg in males and females, respectively (U.S.Environmental Protection Agency, 1998).

A group of Wistar rats (5 of each sex per group) received BAM (purity not recorded) in 1% w/v aqueous tragacanth by gavage for eight consecutive days (Kemp and van der Linde, 1967). The study is not a guideline study with GLP status. It was designed as a dose range finding study with the following dose levels: 6.25, 12.5, 25, 50, 100, 200, 400, 800 and 1600 mg/kg bw/day. 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 (410 – 1117) mg/kg for males and 574 (363 – 907) mg/kg for females. A significant decrease in skeletal muscle tone was observed from 25 mg/kg bw/day for males and from 100 mg/kg bw/day for females. 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. Only partial regression of symptoms was observed at dose levels above 200 mg/kg bw/day. An increase in liver weights was observed in males at levels above 50 mg/kg bw/day. The weights of adrenal glands were increased at 200 mg/kg bw/day in males and at 400 mg/kg bw/day in both sexes. Animals of 6.25 and 12.5 mg/kg bw/day group, as well as groups severely affected by treatment, 800 and 1600 mg/kg bw/day, were not included in the autopsy. No histopathological changes related to treatment were observed. The growth and food consumption was affected at 400 mg/kg bw/day (800 and 1600 mg/kg bw/day groups were not monitored).

Critical effect: Reduction in skeletal muscle tone
LOAEL: 25 mg/kg bw/day (males) and 100 mg/kg bw/day (females)
NOAEL: 12.5 mg/kg bw/day (males) and 50 mg/kg bw/day (females)

Wistar rats (10/sex/dose) were exposed to BAM for 13 weeks at dietary levels of 0, 50, 180, 600 or 2300 ppm (U.S.Environmental Protection Agency, 1998). The study is not performed according to current guidelines (e.g. with respect to investigated parameters) and does not have GLP status.

Critical effects: Decreased body weight gain and food efficiency, increased blood urea nitrogen, and reduced coagulation times.
LOAEL: 600 ppm (49 mg/kg/day).
NOAEL: 180 ppm (14 mg/kg/day)

BAM (purity 97 % w/w) was administered orally to dogs (6 (control) or 4 dogs/sex/group) at dietary concentrations of 0, 100, 300 and 2000 ppm for a period of 13 weeks (Walker, 1967). The study appears to be well performed, though not according to present guidelines (e.g. no ophthalmoscopic examination or tests for clotting potential), and it does not have GLP status. Dogs in the highest dose group lost body weight and bodily condition after 3 – 5 weeks of exposure. Increased liver weights were observed in 300 and 2000 ppm level groups, but the effect was only statistically significant in females. The food consumption was monitored but not stated in the report. The high dose females had a decrease in serum urea and an increase in serum alkaline phosphatase activity and 2-globulin after 13 weeks exposure. Liver function tests carried out in control and high dose groups did not show any difference between the two groups.

Critical effect: Increased liver weights
LOAEL: 300 ppm (approximately 7.5 mg/kg bw/day (WHO, 1987)).
NOAEL: 100 ppm (approximately 2.5 mg/kg bw/day (WHO, 1987)).

The toxic effect of BAM was examined in the nasal passages of C57Bl mice following single intraperitoneal (ip) injections of BAM dissolved in DMSO (0(vehicle), 25, 50 or 100 mg/kg bw) (Brittebo et al., 1991). Two or three animals from almost every dose group were killed after 8 hours, 1 day, 3 days or 20 days. 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) in the dorsal meatus (not lateral) at the 8 hour and 1 day killings, but not after 3 days (50 mg/kg only). Twenty days after the 25 and 50 mg/kg ip injections, the PAS-staining intensity of the Bowman's gland was similar to the controls. At 100 mg/kg necrosis of the Bowman's glands and the neuroepithelium was observed, especially in the dorsomedial aspects of the olfactory region at the 1 day and 3 days killings. There was however signs of restitution of the olfactory epithelium and lamina propria (with Bowman's glands) 3 days after 100 mg/kg, but no animals were observed for a longer period at the 100 mg/kg dose level. Debris was observed in the nasal passages following ip exposure to 100 mg/kg. No lesions were observed in other parts of the nasal cavity including the respiratory or squamous mucosa in the lateral nasal glands, maxillary nasal glands or vomeronasal organ, and there were no morphological lesions observed 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.

Critical effect: Necrosis of the Bowman's glands and the neuroepithelium<
LOAEL: 100 mg/kg bw (ip)
NOAEL: 50 mg/kg bw (ip)
LOEL: 25 mg/kg bw (ip) (decreased PAS-staining)
NOEL: Could not be established because of decreased PAS-staining of Bowman's glands in the dorsal meatus, an apparently reversible effect.

4.2 Long term toxicity

In a combined chronic toxicity/carcinogenicity study BAM (purity unknown) was given to Crl CD rats (35/sex/dose) for 106-107 weeks at dietary levels of 0, 60, 100, 180 or 500 ppm (U.S.Environmental Protection Agency, 1998, Wheldon et al., 1971). The study appears to be well performed, though not according to current guidelines (too small group size, incomplete haematology, serum chemistry, urine analysis and histopathology), and it does not have GLP status. Although the study was reported as a combined chronic toxicity/carcinogenicity study, a group size of 35/sex/dose is too small (min. 50/sex/dose) for an adequate evaluation of carcinogenicity.

The dietary levels were equivalent to an average BAM intake of 0, 2.2, 3.6, 6.5, or 19 mg/kg bw/day in males and 0, 2.8, 4.7, 8.5, or 25 mg/kg bw/day in females calculated from nominal dietary concentrations over 106-107 weeks. When correlated for mean analysed concentrations, the average BAM intake was 0, 2.0, 3.4, 6.0, or 17 mg/kg bw/day in males and 0, 2.6, 4.4, 7.9, or 23 mg/kg bw/day in females. As usual in this kind of studies, the dosages (based on mg/kg bw/day) fell during the first 26 - 30 weeks to about one-third of their initial values, and thereafter remained essentially constant.

At the highest dose level a statistically significant decrease in mean body weight gains was observed in both males and females (11 % and 20 % less than controls, respectively, at week 52). Food consumption and food conversion efficiency of females was marginally reduced at the highest dose level.

No clinical findings or macroscopic changes were considered related to treatment. The relative liver weights were increased in a dose-related manner in females and at the highest dose level in males. The effect was only statistically significant for the females at the highest dose level, and this was consistent with greater degree of histological changes (slightly increased severity of fat deposition) in the livers of females. There were no treatment-related changes in liver enzyme activities. A non-progressive depression of packed cell volume, erythrocyte counts and haemoglobin was observed in high dose males and female (not examined for low or intermediate dose groups). BAM produced an increased incidence of hepatoma in females at 500 ppm. The frequency of rats bearing hepatoma of the rats subjected to liver histology was 7.4, 0, 6.3, 7.7 and 2.9 % for males and 0, 0, 0, 3.1 and 14.3 % for females in treatment groups 0, 60, 100, 180 and 500 ppm, respectively (Table 4, section 4.5). No hepatocarcinoma was found. The U.S. EPA determined that the high dose female rats demonstrated an increased incidence of hepatoma (14 %), which was of borderline significance (p< 0.049). However, a histopathological examination of livers from the rats needs to be reevaluated before a formal determination can be made on the carcinogenic potential of BAM. In addition the group size appears to be too small for an adequate evaluation of carcinogenicity.

Critical effect: Decreased body weight gain in males and in females.
LOAEL was set at 19 (17 based on analysed feed data) mg/kg/day.
NOAEL was 6.5 (6.0 based on analysed feed data) mg/kg/day.

In a chronic toxicity study in dogs, BAM (purity 97 % w/w) was fed to beagle dogs 4/sex/dose for two years at dietary levels of 0 (control), 60, 100, 180, or 500 ppm (approximately 0, 1.5, 2.5, 4.5, or 12.5 mg/kg/day) (U.S.Environmental Protection Agency, 1998, Wilson and Thorpe, 1971). The study report is very brief. It is not performed according to present guidelines (no food consumption data, incomplete serum chemistry study, and no ophthalomological examination), and it does not have GLP status. Decreased body weight gain in males (58 % of controls at 2 years) and in females (29% of controls at 2 years) was observed at 500 ppm. At the 500 ppm level, the males had an increased relative liver weight. The females had reduced relative kidney and liver weights at and above 60 ppm, but this was not considered related to treatment.

Critical effect: Decreased body weight in both sexes and increased relative liver weight in males.
LOAEL: 12.5 mg/kg/day
NOAEL: 4.5 mg/kg/day

4.3 Reproductive and developmental effects

New Zealand white rabbits (16/dose group) were given BAM at dosing levels of 0, 10, 30, or 90 mg/kg/day by oral gavage on gestational days (GDs) 7 – 19 (U.S.Environmental Protection Agency, 1998). Maternal toxicity was observed at 30 and 90 mg/kg/day. Increased moribundity was observed (2 animals in both the middle and high dose groups compared to 0 in the control). At 90 mg/kg/day, the body weight decreased non-significantly (93%-95% of control) on GDs 13 - 19; weight gain decreased significantly during the dosing period; food consumption decreased significantly (51% of controls) during the dosing period; and the number of abortions increased (3 animals compared to 0 in the control). Post-dosing, compensatory body weight and food consumption increases above controls were observed.

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.

Critical maternal effect: Increased mortality
Critical foetal effect: Decrease in foetal body weight and body weight gain
LOAEL, maternal: 30 mg/kg/day
LOAEL, foetal: 90 mg/kg/day
NOAEL, maternal: 10 mg/kg/day
NOAEL, foetal: 30 mg/kg/day

In a three generation reproduction study with two litters per generation, BAM (purity 99.5 %, phenol 0.1%) 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 (WHO, 1987)) (Hine, 1970, U.S.Environmental Protection Agency, 1998). The study is not performed according to current guidelines (e.g. no diet analysis or food consumption data) and does not have GLP status. The study appears to be well performed, but individual animal data could not be provided. A significant decrease in weights of weanlings (85 – 88 % of the control animals at day 21 only for F2a, F3a and F3b generations) was observed at the highest dose level. The terminal weight of the last parent generation (F2b) females was significant less at the highest dose level compared to the controls (94% of the control animals). The F3b weanlings were autopsied, and at 180 ppm level the relative kidney weight of females and relative liver weights of both sexes were increased. The 100 ppm females also had a marginal, but statistically significantly increased relative liver weight.

Critical effect: Increased relative liver weight
LOAEL: 100 ppm (5-10 mg/kg/day)
NOAEL: 60 ppm (3-6 mg/kg bw/day)

4.4 Mutagenic and genotoxic effects

Three mutagenicity studies have been assessed by the US EPA and reported in the Reregistration Eligibility Decision (RED) document for dichlobenil. No details are available. No other mutagenicity studies have been identified for BAM.

“BAM was negative for inducing reverse gene mutation (his- to his+) in TA strains of Salmonella typhimurium exposed, in the absence and presence of mammalian metabolic activation (rat S9 mix), up to 5000 µg/plate” (U.S.Environmental Protection Agency, 1998).

“BAM was negative for inducing repair of DNA damage as measured by unscheduled DNA synthesis (UDS), as determined by net nuclear silver grain count in primary rat hepatocytes, exposed up to cytotoxic doses (1000 µg/ml)” (U.S.Environmental Protection Agency, 1998).

“A mouse micronucleus assay using a single dose of BAM (250 mg/kg) was negative. This dose was selected based on a single dose study in which the group of mice treated at 250 mg/kg displayed mild neurotoxic effects (lethargy and ataxia); the group treated at 500 mg/kg were severely affecte (becoming comatose), and the groups treated at 1000 mg/kg and higher died in extremis “ (U.S.Environmental Protection Agency, 1998). The route was not mentioned.

4.5 Carcinogenic effects

As mentioned above, a combined chronic toxicity/carcinogenicity study has been performed with BAM. It was administered to Crl CD rats (35/sex/dose) for 106-107 weeks at dietary levels of 0, 60, 100, 180 or 500 ppm (Wheldon et al., 1971). The frequency of rats bearing hepatoma of the rats subjected to liver histology was 7.4, 0, 6.3, 7.7 and 2.9 % for males and 0, 0, 0, 3.1 and 14.3 % for females in treatment groups 0, 60, 100, 180 and 500 ppm, respectively (Table 4). No hepatocarcinoma was diagnosed, or clear distinction between hepatocellular preneoplastic foci, adenomas and carcinomas was not made. Not all animals were subjected to liver histology. The incidence of this common tumour type among historical controls of this particular strain of rat has not been reported. The study is inadequate (e.g. the group size is too small) for a full evaluation of carcinogenicity.

As part of the review process of US EPA, clarification of the tumour types and data on the stability/homogeneity of the test compound was requested (U.S.Environmental Protection Agency, 1998). This information was submitted to US EPA. With this additional data from reclassified liver slides, the Agency determined that the high dose female rats demonstrated an increased incidence of adenomas (14%), which was of borderline significance (p< 0.049). However, a formal US EPA assessment of the carcinogenic potential of BAM awaits peer review results of the histopathological examination of livers from the rats.

Table 4. Hepatoma

* Rats sacrificed at 107 weeks only

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

Version 1.0 November 2004, © Danish Environmental Protection Agency