Guidelines for Canadian Drinking Water Quality: Guideline Technical Document - Haloacetic Acids

10.0 Health effects in laboratory animals and in vitro test systems

10.1 Acute toxicity

Details on the available acute oral and dermal toxicity studies are provided in Tables 7 and 8.

Table 7: Summary table of acute oral toxicity data of the various HAA compounds

Compound	Oral LD50 (mg/kg bw)a				Clinical observations
	Rats	Mice	Rabbits	Guinea pigs
a References are as follows: 1) ECETOC, 1999; 2) Berardi et al., 1987; 3) Woodard et al., 1941; 4) Morrison, 1946; 5) Yount et al., 1982; 6) Tomlin, 1994; 7) WSSA, 1983; 8) Meier et al., 1997; 9) Linder et al., 1994a. b n/a: not available.
MCA acid	90.4 for a 1% solution1) ~200 for a 6% solution1)	2602)	n/ab	n/a	Apathy, hypoactivity, disorders of balance, lacrimation, dyspnoea, cyanosis1)
MCA salt	763)	165-2553), 4)	n/a	803)	Apathy, weight loss3)
DCA salt	44803)	4845-55003), 5)	n/a	n/a	Semi-narcosis, narcosis3)
TCA acid	4006)	n/a	n/a	n/a	n/a
TCA salt	3320-50003), 7)	3640-48703), 7), 8)	40007)	n/a	Semi-narcosis, narcosis3)
MBA salt	1779)	n/a	n/a	n/a	Excessive drinking, hypomobility, laboured breathing, mild diarrhoea9)
DBA salt	17379)	n/a	n/a	n/a	Excessive drinking, hypomobility, laboured breathing, mild diarrhoea9)

Table 8: Summary table of acute dermal toxicity data of the various HAA compounds

Compound	Dermal LD50 (mg/kg bw)a		Clinical observations
	Rats	Rabbits
a References are as follows: 1) ECETOC, 1999; 2) Saghir and Rozman, 2003; 3) Tomlin, 1994; 4) Hoechst AG, 1974. b n/a: not available.
MCA acid	>400 (5% solution)1) 305-800 (40-50% solution)1) 145 (acid)2)	250 (50% solution)1)	Apathy, hypoactivity, lacrimation, piloerection, panting, prone position1)
MCA salt	>2000 (no deaths)1)	n/ab	Decreased activity, squatting posture, stilted and staggering gait, irregular breathing, moist rales, diarrhoea1)
TCA salt	>20003), 4)	n/a

10.2 Short-term exposure

10.2.1 Monochloroacetic acid

In a 13-week gavage study, B6C3F1 mice (20 per sex per dose) received MCA (as an acid in deionized water) at doses of 0, 25, 50, 100, 150 or 200 mg/kg bw per day, 5 days per week. Interim evaluations were done on five mice per dose after 4 and 8 weeks of treatment. Mortality was increased only at the highest dose and mostly in male mice. The final mean body weight and the mean weight gain were significantly less in the 200 mg/kg bw per day females compared with controls. In female mice only, absolute and relative liver weights were significantly increased at the highest dose. Cholinesterase levels were decreased at the two highest dose groups in female mice. Hepatocellular cytoplasmic vacuolations were found following histopathological examination and were related to metabolic derangement occurring in moribund animals. No evidence of peroxisome proliferation in the liver was reported. The authors set the no-observed-adverse-effect level (NOAEL) at 100 mg/kg bw per day (Bryant et al., 1992; NTP, 1992).

In the same study as above, six groups of F344 rats (20 per sex per dose) received MCA (as an acid in deionized water) by gavage at 0, 30, 60, 90, 120 or 150 mg/kg bw per day, 5 days per week, for 13 weeks. Interim evaluations were done on five rats per dose group after 4 and 8 weeks of treatment. Toxic effects were observed at every dose level. Almost all animals died at 90 mg/kg bw per day and above. Relative heart weights were significantly decreased in both sexes at 60 mg/kg bw per day and only in females at 30 mg/kg bw per day. A dose-related increase in the incidence and severity of cardiomyopathy was observed in both sexes at 60 mg/kg bw per day and above. In male rats, relative liver and kidney weights were increased at 30 and 60 mg/kg bw per day; in female rats, relative liver weight was increased at 60 mg/kg bw per day only. During the treatment, blood urea nitrogen levels were increased in male rats at 90 mg/kg bw per day and up and in female rats at 60 mg/kg bw per day and up; however, there was no microscopic evidence of kidney or liver damage. Significant dose-related increases in serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were seen in both sexes at 60 mg/kg bw per day and above. Lymphocyte counts were decreased at 30 mg/kg bw per day and above, but were related to stress. The authors set the NOAEL at 30 mg/kg bw per day based on cardiac effects in both sexes (Bryant et al., 1992; NTP, 1992).

10.2.2 Dichloroacetic acid

In a 7-week drinking water study, male Sprague-Dawley rats received DCA (sodium salt) at dose levels of 50 or 1100 mg/kg bw per day (Stacpoole et al., 1990). The high-dose rats had severe hind limb weakness; however, light microscopic examination of the peripheral nerves did not detect any changes. Thiamine deficiency was also detected at the high dose, as measured by transketolase activity in red cells. No clinical signs or effects on transketolase activity were seen at the low dose. The authors stated that the toxicity seen in rats in this study is associated with signs typical of thiamine deficiency and that hind limb weakness and other neuropathic manifestations of chronic thiamine deficiency in animals are considered to be due to changes in central, rather than peripheral, nervous system structure and function.

In a 12-week dietary study, hind limb weakness and abnormal gait also appeared in male Wistar rats (n = 6) exposed to DCA (neutralized). Approximate doses varied from 4 mmol/kg bw per day (516 mg/kg bw per day) at the beginning of the study to about 2.5 mmol/kg bw per day (323 mg/kg bw per day) at the end of the study (Yount et al., 1982). Decreased nerve conduction velocity was also detected in several nerves (sural and motor), as well as a decrease in the diameter of the tibial nerves. Decreased weight gain as a result of decreased food consumption and the presence of hepatomegaly were among the other toxic effects observed.

In a 3-month oral gavage study, DCA (sodium salt) was administered to Sprague-Dawley rats (10 per sex per dose) at dose levels of 0, 125, 500 or 2000 mg/kg bw per day, with an additional five rats per sex dosed with 0 or 2000 mg/kg bw per day allowed a 4-week recovery period (Katz et al., 1981). Death was seen in two rats per sex at the high dose. Hind limb paralysis occurred in 27% of rats of both sexes at the high dose. In the 4-week recovery group, one rat per sex afflicted by the paralysis seemed to have recovered completely. Histopathological examination showed the brain and testes as the main target organs. Oedematous brain lesions, characterized by vacuolation of the myelinated white tracts, were seen in the cerebrum and, to a lesser extent, the cerebellum. Combined incidence rates for these were 60% at the low dose and 100% at the middle and high doses. In the high-dose recovery group, brain lesions persisted in three of eight rats. Body weight was decreased in all treated rats and was associated with reduced food consumption. A significant increase was observed in relative liver weight (both sexes, all doses), relative kidney weight (females, all doses) and relative adrenal weight (males, 500 mg/kg bw per day and above; females, 2000 mg/kg bw per day). Clinical chemistry showed a mild depression of the erythroid parameters at the middle and high doses and a decrease in blood glucose and lactate levels at all levels. Testicular effects were also seen and are discussed in Section 10.5.2.

In a 90-day drinking water study, male Sprague-Dawley rats (n = 10) were treated with DCA (neutralized) at doses of 0, 50, 500 or 5000 mg/L (0, 4, 35 or 350 mg/kg bw per day) (Mather et al., 1990). Decreased body weight and water consumption were observed at 35 mg/kg bw per day and above. Relative liver and kidney weights were decreased at doses of 35 mg/kg bw per day and above. However, histological and biochemical signs of liver and kidney damage, as well as an increase in the hepatic peroxisomal beta-oxidation activity, were seen only in the highest dose group. Increases in relative spleen weight were seen at the top dose in the absence of histopathological effects. No effects on immunological functions were seen.

In a 13-week drinking water study (NTP, 2000), B6C3F1 mice (10 per sex per dose) were exposed to DCA (neutralized to pH 5) doses of 0, 67, 125, 250, 500 or 1000 mg/L (0, 9, 16, 32, 61 and 124 mg/kg bw per day for males and 0, 10, 18, 38, 72 and 132 mg/kg bw per day for females). Dose-related increases were seen in liver weight and incidence of cytoplasmic vacuolation change in hepatocytes (females at 10 mg/kg bw per day and above; males at 32 mg/kg bw per day and above). No clinical signs or deaths were observed during the study. In the high-dose group, mild leukopenia, neutropenia and monocytopenia were observed in some male mice, which were potentially chemical related. The authors set NOAELs of <32 mg/kg bw for males and <10 mg/kg bw for females, both based on microscopic liver lesions.

NTP (2000) also dosed Fischer-344 rats (10 per sex per dose) with the same protocol and doses as above (calculated doses: males: 0, 5, 9.3, 18.8, 39.2 and 81.4 mg/kg bw per day; females: 0, 5.9, 10.0, 20.9, 43.8 and 94.7 mg/kg bw per day). A significant decrease in body weight gain was noted in high-dose males during weeks 4-13. No other significant effects were observed.

In a 13-week subchronic study (Katz et al., 1981), 10- to 12-month-old beagle dogs (3-4 per sex per dose) received DCA (sodium salt) in gelatin capsules at doses of 0, 50, 75 or 100 mg/kg bw per day, with one additional dog per sex dosed with 0 or 100 mg/kg bw per day and allowed a 4-week recovery period. In female dogs only, food consumption was reduced at all doses; however, dose-dependent weight losses at all dose levels were observed in both sexes during the treatment but reversed upon cessation. One female and one male died at the 75 and 100 mg/kg bw per day dose, respectively, and signs of adverse effects observed prior to their deaths included anorexia, ataxia, hind limb weakness and reduced activity. Other adverse effects related to the treatment include emesis (75 and 100 mg/kg bw per day), bloody stools (100 mg/kg bw per day) and paralysis (100 mg/kg bw per day). Also reported in all dose groups (both sexes) were a high incidence of ocular anomalies: bilateral lenticular opacities, injected bulbar conjunctivae and superficial corneal vascularization with a tendency for keratoconjunctivitis sicca. Haematological parameters were depressed at all dose levels in both sexes. Liver and kidney parameters were not affected by the DCA treatment in dogs. An increase in the incidence of lung consolidation was observed in all doses (both sexes). Histopathological examination of the brain revealed that dogs (in all treated groups) suffered slight to moderate vacuolization of white myelinated tracts in the cerebrum and, to a lesser extent, in the cerebellum. Increases in the incidence of haemosiderin-laden Kupffer cells in the liver and cystic mucosal hyperplasia in the gall bladder were observed at all dose levels, even 5 weeks after cessation of the treatment. Prostate and testicular changes were also seen at 50 mg/kg bw per day and above and are discussed in Section 10.5.2. The authors noted that beagle dogs are more susceptible to cataract formation than any other species (Katz et al., 1981).

In another subchronic dog study (Cicmanec et al., 1991), 4-month-old male and female beagle dogs (five per sex per dose) received DCA (neutralized to pH 7.4) in gelatin capsules at 0, 12.5, 39.5 or 72 mg/kg bw per day for 90 days. The controls received encapsulated distilled water. Three high-dose animals died as a result of dehydration and pneumonia. Overt clinical signs such as dyspnoea (middle and high doses) and partial paralysis (high dose only) were observed in both sexes. Diarrhoea was present in the mid- and high-dose animals; some dogs that were highly dehydrated required fluid therapy. Inflammation of the ocular membranes was accompanied by swelling; discharge was clear at the low and middle doses and became purulent in the high-dose group. A dose-related decrease in body weight gain was observed in all treated animals. Relative liver weights were increased in all female dose groups; however, kidney and lung weights were increased only in the high-dose group. In male dogs, the effects in these organs were less consistent. Apparent increases in ALT, AST and lactate dehydrogenase were observed in both sexes in the high-dose group. Erythrocytes and haemoglobin concentrations were significantly reduced in both sexes in the high-dose group from day 30 and beyond. Upon microscopic examination, lesions were apparent in the liver, brain, lung, pancreas and testes. Hepatic vacuolization was observed in most of the treated dogs (both sexes) as well as in a few control animals. In the brain, mild vacuolization of the white myelinated tracts of the cerebrum, cerebellum and/or spinal cord was present in all exposed groups. Pneumonia and bronchopneumonia was observed in most treated dogs; more severe effects were seen in the mid- and high-dose groups. Pancreatic acinar associated with chronic inflammation was observed in many mid-and high-dose animals (both sexes). Testicular (all doses) and prostatic effects (middle and high doses) were seen and are discussed in Section 10.5.2.

10.2.3 Trichloroacetic acid

In a 90-day study (Mather et al., 1990), male Sprague-Dawley rats (10 per dose) were treated with TCA (neutralized) in their drinking water at dose levels of 0, 50, 500 or 5000 mg/L (0, 4.1, 36.5 or 355 mg/kg bw per day). Water consumption was decreased (statistically significant) at the middle and high doses compared with controls. A decrease in body weight was observed for all dose groups, but was not statistically significant. The high-dose group also had an increase in relative liver and kidney weights and a significant increase in hepatic peroxisomal activity.

10.2.4 Monobromoacetic acid

No subchronic studies on MBA were located in the literature.

10.2.5 Dibromoacetic acid

To determine the effects on the liver, male B6C3F1 mice (five per dose) were administered DBA (neutralized) at 0, 300, 1000 or 2000 mg/L in drinking water for up to 12 weeks (Kato-Weinstein et al., 2001). All animals were sacrificed at 20 weeks of age. Decreased water consumption and body weight were seen at the highest dose. A dose-related increase in relative liver weights was seen at 1000 mg/L and above at 12 weeks. Increased absolute and relative liver weights were seen at all time points (4, 8 and 12 weeks) at the highest dose. A significant increase in the total liver glycogen content was seen at the highest dose at 12 weeks. Dose-related decreases in serum glucose and serum insulin concentrations were seen at 1000 mg/L and above at 12 weeks (Kato-Weinstein et al., 2001). DBA displayed effects similar to those of DCA; both caused an increase in glycogen content and a depression of serum insulin concentrations. This may suggest that common mechanisms are involved with dihaloacetates.

A 13-week drinking water study in both B6C3F1 mice and F344 rats was reported by Melnick et al. (2007) and in greater detail in NTP (2007). Animals (10 per dose per sex per species) were exposed to DBA (neutralized to pH 5) in drinking water at doses of 0, 125, 250, 500, 1000 or 2000 mg/L (equivalent to 0, 10, 20, 40, 90 and 166 mg/kg bw per day in male rats and 0, 12, 23, 48, 93 and 181 mg/kg bw per day in female rats; 0, 16, 30, 56, 115 and 230 mg/kg bw per day in male mice and 0, 17, 34, 67, 132 and 260 mg/kg bw per day in female mice). No clinical signs or deaths were observed. A significant decrease in final mean body weights were seen in both species and sexes at the highest dose, however only a slight decrease in water consumption was seen at weeks 1 and/or 13. Dose-related increases in liver weights were noted in both sexes of mice at 500 mg/L and above and in rats at 125 mg/L and above. Dose-related increases in the severity of cytoplasmic vacuolation in hepatocytes were seen in mice (both sexes at 1000 mg/L and above), although these lesions were present in both controls and treated mice. It was noted that the increase in severity corresponded to increased liver weights. In rats, an increased incidence of cytoplasmic vacuolation in hepatocytes was noted in males at 500 mg/L and above and in females at 2000 mg/L. Haematopoietic cell proliferation (mild to minimal) was observed in the spleen of high-dose female rats. A significant increase in the incidence of cellular hypertrophy of the pituary gland was observed in high-dose male rats; however, these effects were considered secondary to testicular atrophy. Testicular effects were seen in rats and mice and are outlined in Section 10.5.5.

Moser et al. (2004) and Phillips et al. (2002) examined the neurotoxic potential of DBA. Adolescent male and female F344 rats (12 per sex per group) were administered DBA (acid) at 0, 200, 600 and 1500 mg/L (estimated as 0, 20, 72 and 161 mg/kg bw per day) for 6 months in drinking water. No treatment-related deaths were noted. A decrease in weight gain was noted in the high-dose rats (both sexes). Functional observational battery test results revealed dose-related neuromuscular toxicity at the middle and high doses, such as limb weakness, mild changes in gait and hypotonia. Sensorimotor responses were depressed at all treatment levels. Decreased activity and chest clasping were also noted at the highest dose. Neuropathological evaluation revealed degeneration of spinal cord nerve fibres in the mid- and high-dose groups. Neuronal vacuolization of the spinal cord (mostly in grey matter and occasionally in white matter) increased in severity and incidence at the middle dose and above. The authors set a noobserved-effect level (NOEL) of 20 mg/kg bw per day based on neuropathological effects but were unable to establish one for neurobehavioural effects due to sensorimotor changes at that level.

Clinical signs of neurotoxic effects were also seen in a male reproductive study (Linder et al., 1995). Male rats (n = 10) were dosed with 250 mg DBA/kg bw daily for up to 42 days, after which dosing was discontinued due to severe toxicity; clinical signs consisted of abnormal posturing, light tremor, atypical movement of limbs and difficulty in moving hind limbs. Rats were allowed to recover for a 6-month interval; some of the effects either diminished (abnormal gait) or disappeared (tremors). No other overt signs of toxicity were seen at the lower doses in male rats (Linder et al., 1995).

NTP (1999a) examined the immunotoxicity of DBA in female mice in four separate studies investigating different end-points. Groups of female B6C3F1 mice (eight per dose) were exposed to DBA in drinking water at concentrations of 0, 125, 250, 500, 1000 or 2000 mg/L per day (calculated as 0, 14-20, 33-39, 68-73, 132-150 or 236-285 mg/kg bw per day) for a period of 28 days. No signs of overt toxicity were seen, and water consumption was unaffected. Only high-dose females had a 40% decrease in body weight gain compared with controls. In one experiment, an increase in spleen weight was observed, while none was seen in a second experiment using similar doses. Several indicators of immunological response were affected. A statistically significant dose-related increase in the number of spleen macrophages was seen at 500 mg/L and above, indicating an immunotoxic response in the spleen. Humoral immunity was also affected, as seen by a decrease of spleen immunoglobulin M antibody-forming cell response to sheep erythrocytes at doses of 500 mg DBA/L and above. A lowest-observed-adverse-effect level (LOAEL) of 500 mg/L (68-73 mg/kg bw per day) and a NOAEL of 250 mg/L (33-39 mg/kg bw per day) were established for immunotoxicity by U.S. EPA (2005a).

McCay et al. (2000) reported in an abstract that no effects on the immune system were seen in a 28-day study in which female B6C3F1 mice were dosed daily via drinking water with DBA concentrations of 0, 250, 500 or 1000 mg/L. Changes in relative and/or absolute liver and thymus weights were the only effects seen.