Effects of Subchronic Exposure of Rats to Dichloramine and Trichloramine in Drinking Water

Regulatory Toxicology and Pharmacology 31, 200 –209 (2000) doi:10.1006/rtph.2000.1376, available online at http://www.idealibrary.com on

Effects of Subchronic Exposure of Rats to Dichloramine and Trichloramine in Drinking Water Jamie S. Nakai,* ,1 Raymond Poon,* Pierre Lecavalier, 2 Ih Chu,* Algis Yagminas,* and Victor E. Valli† *Health Canada, Environmental Health Directorate, Ottawa, Ontario K1A 0L2, Canada; and †College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801 Received November 4, 1999

The subchronic toxicity of 0.2–200 ppm dichloramine and 0.2–90 ppm trichloramine in the drinking water of rats was investigated using biochemical, hematological, and histopathological parameters. Animals in the highest dose groups consumed 5–15% less fluid than controls with no significant decrease in body weight gain. No clinical signs of toxicity were observed in either case. Both males and females dosed with 90 ppm trichloramine had significantly increased relative kidney/body weights and the females had increased hepatic glutathione S-transferase and UPD-glucuronosyltransferase activities. No significant changes were detected in other xenobiotic metabolizing enzymes or in serum biochemistry, urine biochemistry, or hematology. Both dichloramine and trichloramine induced minimal to mild adaptive histopathological changes in thyroids and kidneys of animals of both sexes. Dichloramine, but not trichloramine, was associated with histological changes in the gastric cardia characterized by epithelial hyperplasia at concentrations of 2 ppm and above in the males and 200 ppm in the females. This study indicates that dichloramine produced mild histological effects at drinking water concentrations of >0.2 ppm in males (0.019 mg/kg/day) and >2 ppm in females (0.26 mg/kg/day) while trichloramine produced biochemical and mild histological effects at levels of >2 ppm both in males (0.23 mg/kg/day) and in females (0.29 mg/kg/day). © 2000 Academic Press

INTRODUCTION

Chloramination is one alternative to chlorine use for disinfection of the water supply. Unfortunately, one of the main advantages of chloramination, its high residual activity, also has a negative side in that consumers may be exposed to chloramines in their drinking water. 1

To whom correspondence should be addressed at Environmental Health Centre A.L. 0803B, Tunney’s Pature, Ottawa, ON K1A 0L2, Canada. 2 Current address: Department of National Defence, Defence Research Establishment Suffield, Medicine Hat, AB T1A 8K6, Canada. 0273-2300/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Although monochloramine is the primary species targeted for use in water treatment, variations in the quality of the raw water or accidental changes in the charge of either ammonia or chlorine to the water can produce elevated levels of dichloramine and trichloramine in the finished water. Most accounts of the human health effects of trichloramine have dealt with its inhalational toxicology as a result of volatilization from indoor swimming pools (Hery et al., 1995; Massin et al., 1998) or from water used industrially (Sanderson et al., 1995; Hery et al., 1998). Reported symptoms typically consisted of acute eye and upper respiratory irritations starting at a concentration of approximately 0.5 mg/m 3. Similar effects were also observed in rats and mice exposed to trichloramine by inhalation (Barbee et al., 1983; Gagnaire et al., 1994). In a controlled clinical evaluation, human volunteers were exposed to chloramine, chlorine, chlorine dioxide, sodium chlorite, and sodium chlorate in their drinking water for up to 12 weeks (Lubbers et al., 1981). Definitive physiological impacts were not observed after exposure to any of these chemicals, but only chloramine did not produce any statistically significant changes in an extensive battery of biochemical test parameters. These changes were not believed to have immediate consequences, although it is possible that clinical significance might have been achieved with a longer treatment period. African Green monkeys were exposed to monochloramine, chlorine dioxide, sodium chlorite, and sodium chlorate in drinking water for 30 – 60 days (Bercz et al., 1982). A variety of hematological parameters were measured and no effects were detectable at the highest dose of chloramine (100 mg/L). In contrast, a dosedependent decrease in hemoglobin and red cell count and an increase in methemoglobin content were induced by sodium chlorite. A subchronic study of the effects of monochloramine in drinking water with a paired-water control was recently completed by Health Canada (Poon et al., 1997). Male rats displayed no treatment-related adverse ef-

200

SUBCHRONIC EFFECTS OF DICHLORAMINE AND TRICHLORAMINE ON RATS

fects following a 13-week exposure to 200 ppm monochloramine in drinking water, based on clinical, biochemical, hematological, immunological, and histopathological endpoints. The reductions in body weight gain observed during monochloramine treatment (Abdel-Rahman et al., 1984; Daniel et al., 1990; Poon et al., 1997) were attributed to decreased food and water consumption and were not believed to be a treatment-related effect. Health Canada has proposed a guideline value of 3.0 ppm for the recommended maximum acceptable concentration (MAC) for chloramines in drinking water (Health Canada, 1995). This MAC was based on a risk evaluation for monochloramine only since it is the predominant form used for water disinfection and because the existing toxicological database was insufficient to establish guidelines for dichloramine and trichloramine. This project was therefore initiated to examine the subchronic toxicity of dichloramine and trichloramine in the drinking water of rats using biochemical, hematological, and histopathological parameters. MATERIALS AND METHODS

Analysis of Free Chlorine, Monochloramine, Dichloramine, and Trichloramine The analyses of free chlorine, monochloramine, dichloramine, and trichloramine were based on the titrimetric method of Palin (1957), utilizing N,N-diethyl1,4-phenylenediamine sulfate as the indicator and standard ferrous ammonium sulfate (FAS) as the titrant, as described in the “Standard Methods for the Examination of Water and Wastewater” (Eaton et al., 1995). Water The water used throughout the study was chlorinefree, organic-free, reagent-grade water (Millipore RO/Q; Millipore Corporation, Ottawa, Ontario, Canada). Chlorine Stock Solution Ultrahigh purity chlorine gas (Matheson Gas, Ottawa, Ontario, Canada) was bubbled into water until saturation. The solution was then titrated with standard FAS to determine the concentration of free chlorine (approximately 8 –10 ⫻ 10 ⫺5 equivalents of chlorine per milliliter). An equimolar quantity of sodium hydroxide was added, followed by enough solid sodium bicarbonate and sodium carbonate to yield an initial concentration of 0.045 M each. This chlorine stock solution was kept at 4°C and titrated with standard FAS before each use to determine the exact chlorine concentration.

201

Dichloramine and Trichloramine Solutions The dichloramine and trichloramine solutions were prepared by stepwise addition of chlorine to ammonium hydroxide and through pH control. First, a monochloramine solution was prepared by combining one equivalent of buffered chlorine solution with one equivalent of ammonium hydroxide solution (approximately 1.6 ⫻ 10 ⫺3 equivalents per milliliter). After reaction, the solution was titrated with standard FAS to determine the concentrations of free chlorine and monochloramine. The dichloramine dosing solutions of each nominal concentration were then prepared in 25-L volumes by combining an appropriate amount of monochloramine solution with an equivalent of chlorine solution and diluting to volume with acetate buffer (0.1 M, pH 4.5). The trichloramine dosing solutions of each nominal concentration were prepared in an analogous manner by combining an appropriate quantity of monochloramine solution with two equivalents of chlorine solution and diluting to 25 L with phosphate buffer (0.1 M, pH 3.0). Both the dichloramine and the trichloramine solutions were stored at 4°C until use. The actual concentrations of the solutions were checked by titration with standard FAS except for the 0.2 ppm solutions which were too dilute, and therefore the nominal concentration of this dosing solution was used for all calculation purposes. The average concentrations for the dichloramine solutions were 1.5, 21, and 195 ppm, and therefore the nominal concentrations of 2, 20, and 200 ppm were used for all calculations. The reactions producing the trichloramine were less efficient and therefore the measured average concentrations of 2, 10, and 90 ppm were used for all calculations. Because these chloramines can be unstable/volatile at room temperature, the contents of the individual water bottles in the dichloramine- and trichloramine-treated groups were discarded and refilled with the refrigerated solutions every 3 or 4 days during the 13-week treatment period. Animal Treatment The treatment of the animals for both the dichloramine and the trichloramine studies was identical, except as noted. Sixty male and 60 female Sprague– Dawley rats were each divided randomly into six groups of 10 animals and housed in individual Health Guard cages (Research Equipment Company, U.S.A.). Lighting was maintained on a 12-h light/dark cycle and the room temperature and humidity were maintained at 21 ⫾ 3°C and 30 –70%, respectively. All animals were fed ad libitum (Lab Chow 5002; PMI Feed, St. Louis, MO). Food consumption was measured weekly and clinical observations were made daily. Control animals for both studies were given the chlorine-free, organic-free, reagent-grade water. An additional control group for the dichloramine study was given 0.1 M

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TABLE 1 Fluid and Feed Consumption and Body Weight Gain Following 13-Week Exposure to Dichloramine or Trichloramine a

Chloramine Dichloramine Male

Female

Trichloramine Male

Female

Concentration in drinking water (ppm)

0 (water) 0 (acetate) e 0.2 2 20 200 0 (water) 0 (acetate) e 0.2 2 20 200 0 (water) 0 (phosphate) e 0.2 2 10 90 0 (water) 0 (phosphate) e 0.2 2 10 90

Fluid consumption (g/week) b

Calculated intake (mg/kg/day) c

Feed consumption (g/week) b

Body weight gain (g) d

267 ⫾ 16 242 ⫾ 20* 238 ⫾ 21* 250 ⫾ 13* 250 ⫾ 22* 230 ⫾ 18* 223 ⫾ 9 194 ⫾ 11* 197 ⫾ 13* 216 ⫾ 17 198 ⫾ 29* 189 ⫾ 17*

0 0 0.019 ⫾ 0.007 0.19 ⫾ 0.07 1.9 ⫾ 0.8 18 ⫾ 6 0 0 0.025 ⫾ 0.006 0.26 ⫾ 0.04 2.5 ⫾ 0.6 24 ⫾ 4

194 ⫾ 13 190 ⫾ 14 187 ⫾ 15 198 ⫾ 15 193 ⫾ 13 182 ⫾ 17* 127 ⫾ 6 126 ⫾ 5 142 ⫾ 12* 139 ⫾ 10* 132 ⫾ 7 140 ⫾ 13*

403 ⫾ 46 390 ⫾ 16 374 ⫾ 27 424 ⫾ 48 408 ⫾ 28 381 ⫾ 42 156 ⫾ 27 153 ⫾ 16 158 ⫾ 18 158 ⫾ 30 154 ⫾ 28 145 ⫾ 27

311 ⫾ 13 302 ⫾ 25 259 ⫾ 12* 320 ⫾ 26 293 ⫾ 12* 291 ⫾ 21* 260 ⫾ 19 254 ⫾ 21 235 ⫾ 12* 242 ⫾ 12* 237 ⫾ 14* 249 ⫾ 14

0 0 0.020 ⫾ 0.006 0.23 ⫾ 0.08 1.1 ⫾ 0.3 9.6 ⫾ 3 0 0 0.028 ⫾ 0.004 0.29 ⫾ .06 1.3 ⫾ 0.2 13 ⫾ 13

191 ⫾ 7 181 ⫾ 7* 177 ⫾ 6* 193 ⫾ 5 188 ⫾ 4 188 ⫾ 6 128 ⫾ 4 135 ⫾ 6* 130 ⫾ 6 131 ⫾ 5 137 ⫾ 6* 131 ⫾ 8

325 ⫾ 41 295 ⫾ 51 293 ⫾ 33 318 ⫾ 22 328 ⫾ 58 310 ⫾ 56 123 ⫾ 22 139 ⫾ 27 119 ⫾ 22 130 ⫾ 20 139 ⫾ 20 126 ⫾ 24

Mean ⫾ SD of 10 animals per group. Group average weekly consumption over 13 weeks. Calculated intake ⫽ average water consumption ⫻ concentration of test chemical in water ⫼ average body weight. d Body weight gain over 13 weeks. e Contained 0.1 M acetate buffer, pH 4.5, in the dichloramine study, and 0.1 M phosphate buffer, pH 3.0, with 19 ppm chlorine, in the trichloramine study. * Significantly different (P ⬍ 0.05) from water control. a b c

acetate buffer, pH 4.5. The trichloramine dosing solutions were typically found to contain a small amount of free chlorine (average 20 ppm); therefore, an additional control group was given 0.1 M phosphate buffer, pH 3.0, containing an average of 19 ppm chlorine. At Week 12 of the dosing, the animals were transferred to individual metabolism cages and overnight urine samples were collected into ice-chilled beakers containing 1.0 mL of a preservative (100 mM EDTA, 1 g/dL sodium azide, 100 mM potassium phosphate, pH 6.0). The urine was spun at 1500g for 10 min; one portion of the clarified urine was stored at ⫺80°C for enzyme and protein analysis and another portion was preserved with HCl (final concentration 0.4 N) and stored at ⫺80°C for ascorbic acid and creatinine analysis. At the termination of dosing (Week 13), the animals were anesthetized with isoflurane (Aerrane; Anaquest, Ontario, Canada). A 2-mL sample of blood was immediately transferred into a Vacutainer (Becton-Dickinson, Rutherford, NJ) containing EDTA and was used to

measure the hematological parameters. The remaining blood was emptied into a serum separation tube (Becton-Dickinson) for the preparation of serum. A 2.0-g piece of liver was excised and homogenized in 5.0 mL of ice-cold 0.05 M Tris/1.15% KCl buffer, pH 7.4. The homogenate was centrifuged at 10,000g for 20 min and the supernatant (postmitochondrial fraction) was stored at ⫺80°C. Biochemical and Hematological Analysis A Technicon H1E hematology analyzer (Bayer, Toronto, Ontario, Canada) was used for the determination of erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet count, and total and differential white blood cell counts. A Radiometer Medical A/S (Copenhagen, Denmark) blood hemoximeter, Model OSM3, was used to measure methemoglobin levels for the dichloraminetreated animals. A Technicon RA-XT analyzer was

SUBCHRONIC EFFECTS OF DICHLORAMINE AND TRICHLORAMINE ON RATS

used to measure the following serum chemistries: inorganic phosphate, total protein, alkaline phosphatase, aspartate aminotransferase, bilirubin, calcium, cholesterol, glucose, uric acid, creatinine, and blood urea nitrogen. In addition, serum was analyzed for thiobarbituric acid-reactive substances (TBARS) as a measure of lipid peroxidation by the fluorescent method of Yagi (1982). The liver homogenate was used for the measurement of TBARS (Yagi, 1982) and UDP-glucuronosyltransferase (Burchell and Weatherill, 1981) activity. The liver postmitochondrial fraction was assayed for ethoxyresorufin O-deethylase (Lubet et al., 1985), benzyloxyresorufin O-dealkylase, pentoxyresorufin Odealkylase, methoxyresorufin O-dealkylase (Burke et al., 1985), and glutathione S-transferase (Habig et al., 1974) activities. Urine Biochemistry N-Acetylglucosaminidase activity and protein concentration in urine were determined by the method of Poon et al. (1995) and a modified Lowry assay (Sigma Chemical Company, St. Louis, MO), respectively. Urinary ascorbic acid was determined by HPLC (Poon et al., 1994) and creatinine by an enzymic procedure (Boehringer-Mannheim, Montre´al, Quebec, Canada).

TABLE 2 Organ Weight Changes Following 13-Week Exposure to Dichloramine or Trichloramine a

Chloramine Dichloramine Male

Female

Trichloramine Male

Female

Histopathology and Cytology Blood smears were prepared at necropsy and stained with Wright stain. The following tissues and organs were fixed in 10% buffered formalin (pH 7.4): brain, pituitary, thyroid and trachea, salivary glands, thymus, lung, heart, liver, kidneys, adrenals, spleen, pancreas, esophagus, gastric cardia, fundus and pylorus, duodenum, jejunum, ileum, ceacum, colon, urinary bladder, skin, bone marrow, and gonadal tissues. The testes were fixed in Bouin’s solution. The fixed tissues were dehydrated with graded alcohol, cleared, and then impregnated with paraffin. The paraffin blocks were sectioned at 5 ␮m thickness and stained with hematoxylin and eosin for light microscopic examination. Histological changes were assigned average severity scores where 1 was minimal, 2 was mild, 3 was moderate, and 4 was severe. The average scores were obtained by dividing the sum of total scores by the number of tissues examined. For tissue changes that are focal, locally extensive, and multifocal, a score of less than an integer was assigned as follows: minimal focal, 0.25; locally extensive, 0.50; minimal multifocal, 0.75; mild focal, 1.25; mild, locally extensive, 1.50; mild multifocal, 1.75; etc. Statistical Analysis Differences between the control, vehicle control and treated groups were analyzed using one-way analysis of variance and Dunnett’s test (P ⱕ 0.05).

203

Concentration in drinking water (ppm)

0 (water) 0 (acetate) b 0.2 2 20 200 0 (water) 0 (acetate) b 0.2 2 20 200 0 (water) 0 (phosphate) b 0.2 2 10 90 0 (water) 0 (phosphate) b 0.2 2 10 90

Kidney/body weight

Spleen/body weight

0.623 ⫾ 0.068 0.659 ⫾ 0.060 0.673 ⫾ 0.074 0.667 ⫾ 0.075 0.679 ⫾ 0.064 0.693 ⫾ 0.066 0.725 ⫾ 0.094 0.730 ⫾ 0.060 0.752 ⫾ 0.039 0.711 ⫾ 0.048 0.716 ⫾ 0.035 0.755 ⫾ 0.102

0.160 ⫾ 0.015 0.152 ⫾ 0.017 0.154 ⫾ 0.015 0.161 ⫾ 0.016 0.160 ⫾ 0.028 0.155 ⫾ 0.020 0.189 ⫾ 0.025 0.194 ⫾ 0.026 0.180 ⫾ 0.023 0.182 ⫾ 0.023 0.180 ⫾ 0.018 0.183 ⫾ 0.023

0.654 ⫾ 0.040 0.689 ⫾ 0.035 0.667 ⫾ 0.034 0.697 ⫾ 0.049 0.681 ⫾ 0.074 0.750 ⫾ 0.054* 0.684 ⫾ 0.067 0.723 ⫾ 0.074 0.762 ⫾ 0.065* 0.730 ⫾ 0.072 0.753 ⫾ 0.044 0.771 ⫾ 0.062*

0.158 ⫾ 0.014 0.176 ⫾ 0.019 0.172 ⫾ 0.023 0.171 ⫾ 0.017 0.164 ⫾ 0.021 0.179 ⫾ 0.018 0.191 ⫾ 0.014 0.190 ⫾ 0.024 0.208 ⫾ 0.022 0.204 ⫾ 0.016 0.186 ⫾ 0.018 0.210 ⫾ 0.014

Mean ⫾ SD of 10 animals per group. Contained 0.1 M acetate buffer, pH 4.5, in the dichloramine study, and 0.1 M phosphate buffer, pH 3.0, with 19 ppm chlorine, in the trichloramine study. * Significantly different (P ⬍ 0.05) from water control. a b

RESULTS

Over the course of the 13-week treatment period, the animals of both sexes in the highest dose dichloramine group (200 ppm) consumed approximately 15% less fluid than the water controls but with no significant decrease in the final body weight gain (Table 1). Animals in the trichloramine-treated groups consumed approximately 5 to 8% less fluid but again, with no significant decrease in body weight gain. No clinical signs of toxicity were observed in either case. At termination, significantly increased kidney weights relative to total body weights were observed in both the male (15%) and the female (13%) animals dosed at 90 ppm trichloramine (Table 2). There was also a trend toward increased spleen weight relative to total body weight but this did not reach statistical significance. Measurement of serum biochemical parameters at termination did not reveal any statistically significant changes for either study. Similarly, there were no sta-

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TABLE 3 Changes in Hepatic Enzyme Activities Following 13-Week Exposure to Dichloramine or Trichloramine a

Chloramine Dichloramine Male

Female

Trichloramine Male

Female

Concentration in drinking water (ppm)

Glutathione S-transferase activity (␮mol/min/mg protein)

UPD-glucuronosyltransferase activity (nmol/min/mg protein)

1.19 ⫾ 0.36 1.67 ⫾ 0.49 1.44 ⫾ 0.50 1.66 ⫾ 0.63 1.58 ⫾ 0.56 1.60 ⫾ 0.45 0.93 ⫾ 0.31 1.01 ⫾ 0.38 1.04 ⫾ 0.29 1.22 ⫾ 0.49 1.04 ⫾ 0.38 1.06 ⫾ 0.48

1.84 ⫾ 0.79 2.21 ⫾ 0.81 2.14 ⫾ 0.81 1.99 ⫾ 0.66 2.01 ⫾ 0.72 1.97 ⫾ 0.84 1.38 ⫾ 0.53 1.29 ⫾ 0.34 1.09 ⫾ 0.33 1.39 ⫾ 0.44 1.44 ⫾ 0.40 1.43 ⫾ 0.47

1.20 ⫾ 0.30 0.93 ⫾ 0.32 1.04 ⫾ 0.13 0.99 ⫾ 0.20 1.02 ⫾ 0.25 1.29 ⫾ 0.29 0.77 ⫾ 0.29 0.91 ⫾ 0.25 0.91 ⫾ 0.18 0.89 ⫾ 0.14 0.89 ⫾ 0.21 1.13 ⫾ 0.27*

2.08 ⫾ 0.41 2.11 ⫾ 0.49 2.19 ⫾ 0.39 2.28 ⫾ 0.35 2.03 ⫾ 0.37 2.20 ⫾ 0.29 1.31 ⫾ 0.30 1.52 ⫾ 0.27 1.59 ⫾ 0.37 1.39 ⫾ 0.26 1.55 ⫾ 0.37 1.76 ⫾ 0.32*

0 (water) 0 (acetate) b 0.2 2 20 200 0 (water) 0 (acetate) b 0.2 2 20 200 0 (water) 0 (phosphate) b 0.2 2 10 90 0 (water) 0 (phosphate) b 0.2 2 10 90

Mean ⫾ SD of 10 animals per group. Contained 0.1 M acetate buffer, pH 4.5, in the dichloramine study, and 0.1 M phosphate buffer, pH 3.0, with 19 ppm chlorine, in the trichloramine study. * Significantly different (P ⬍ 0.05) from water control. a b

tistically significant changes in methemoglobin or any other hematological parameters. Increased hepatic glutathione S-transferase and UDP-glucuronosyltransferase activities were detected in females receiving 90 ppm trichloramine (Table 3). No other statistically significant changes were observed in the activities of the other xenobiotic metabolizing enzymes. No treatment-related changes were observed in the urinary ascorbic acid, total protein, or N-acetylglucosaminidase activity. Both dichloramine and trichloramine induced minimal to mild adaptive histopathological changes in thyroid and kidneys of animals of both sexes (Table 4). Males in the 90 ppm trichloramine group had minimal changes in the kidneys consisting of glomerular adhesions and protein casts in the tubules of the inner cortex (Fig. 1). Females in the 0.2 to 90 ppm trichloramine groups displayed minimal to mild tubular mineralization in the outer cortex of the kidneys but with no clear dose–response over the range examined. Dichloramine, but not trichloramine, was associated with minimal histological changes in the gastric cardia characterized by epithelial hyperplasia at concentra-

tions of 2 ppm and above in the males (Fig. 2) and at 200 ppm in the females. These changes were often accompanied by submucosal eosinophilic reaction. DISCUSSION

Dosing rats with dichloramine or trichloramine in their drinking water did not cause a reduction in their weight gain relative to controls. Poon et al. (1997) deduced that the reduction in weight gain usually associated with monochloramine treatment (Abdel-Rahman et al., 1984; Daniel et al., 1990; NTP, 1992) was likely the result of a significant decrease in fluid consumption (42% relative to controls) and not a treatment-related effect. In the present work, the decrease in fluid consumption relative to controls observed for the highest dose groups (5–15%) may be attributable in part to the buffers used to stabilize the chloramines since some reductions were also observed in consumption of the buffer controls. An increase in kidney weight relative to body weight was observed for both males (15%) and females (13%) dosed with 90 ppm trichloramine but not 200 ppm dichloramine. Previously, similar in-

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TABLE 4 Histopathology of Rats Following 13-Week Exposure to Dichloramine or Trichloramine Tissue

Condition

Dichloramine 0 ppm 0 ppm (water) (acetate) a

0.2 ppm

2 ppm

Trichloramine 20 ppm

200 ppm

0 ppm 0 ppm (water) (phosphate) b

0.2 ppm

2 ppm

10 ppm

90 ppm

10 (1.6) 5 (0.3) 6 (0.4)

7 (1.3) 6 (0.6) 8 (0.6)

8 (1.4) 10 (2.2) 2 (0.2) 7 (0.7) 9 (1.0) 9 (1.3)

Male Thyroid Reduced follicle size Reduced colloid density Vesiculation of nuclei Liver Anisokaryosis Hyperchromicity of nuclei Increased perivenous homogeneity Kidneys Glomerular adhesion Protein cast, inner cortex Tubular mineralization, outer cortex Spleen Sinus hyperplasia Arterial cuff atrophy Gastric cardia Epithelial hyperplasia Submucosal eosinophilic reaction

8 c (1.2) b 10 (1.8) 1 (0.1) 4 (0.3) 5 (0.3) 7 (0.5)

10 (1.9) 4 (0.4) 10 (0.9)

8 (1.1) 10 (1.4) 10 (1.6) 5 (0.5) 5 (0.6) 8 (1.0) 7 (0.8) 10 (1.7) 10 (2.5)

8 (1.0) 1 (0.1) 2 (0.2)

10 (1.3) 2 (0.1) 7 (0.5)

2 (0.2) 0

2 (0.1) 0

1 (0.2) 0

4 (0.4) 5 (0.6)

7 (0.5) 9 (1.3)

9 (1.6) 9 (1.7)

3 (0.2) 2 (0.2)

8 (0.6) 8 (1.0)

10 (1.7) 10 (2.0) 10 (2.3) 10 (2.5) 3 (0.5) 5 (0.7) 1 (0.1) 7 (1.4)

5 (0.3)

3 (0.2)

8 (0.4)

8 (0.6)

6 (0.4) 10 (0.7)

7 (0.6)

5 (0.5)

10 (1.1) 10 (1.0) 10 (1.3) 10 (2.0)

5 (0.6) 0

6 (0.5) 0

4 (0.4) 0

3 (0.3) 0

2 (0.1) 0

5 (0.5) 0

3 (0.2) 0

5 (0.6) 1 (0.2)

5 (0.7) 1 (0.4)

7 (0.9) 1 (0.2)

8 (0.9) 0

9 (1.2) 5 (0.7)

0

0

0

0

0

0

0

0

0

0

1 (0.2)

0

0 0

0 1 (0.1)

0 0

0 0

0 0

0 1 (0.1)

0 0

0 0

1 (0.2) 2 (0.4)

1 (0.1) 1 (0.2)

6 (0.8) 6 (0.7)

8 (1.1) 6 (1.0)

0 0

0 0

0 0

7 (1.3) 2 (0.4)

9 (1.4) 9 (1.0)

9 (1.7) 7 (1.0)

0 0

0 0

0 0

0 0

0 0

0 0

Female Thyroid Reduced follicle size Reduced colloid density Vesiculation of nuclei Liver Anisokaryosis Hyperchromicity of nuclei Increased perivenous homogeneity Kidneys Glomerular adhesion Protein cast, inner cortex Tubular mineralization, outer cortex Spleen Sinus hyperplasia Arterial cuff atrophy Gastric cardia Epithelial hyperplasia Submucosal eosinophilic reaction

6 (0.4) 0 0

4 (0.3) 0 0

2 (0.2) 0 1 (0.1)

5 (0.6) 2 (0.1) 6 (0.4)

9 (1.3) 5 (0.9) 2 (0.1) 1 (0.1) 9 (0.7) 10 (1.0)

5 (0.6) 0 4 (0.2)

8 (1.3) 2 (0.2) 8 (0.5)

8 (1.3) 8 (0.9) 10 (1.2)10 (1.5) 1 (0.1) 0 3 (0.5) 5 (0.3) 9 (0.7) 10 (0.8) 10 (1.2) 10 (1.3)

8 (0.8) 9 (1.0)

9 (1.3) 9 (1.3)

8 (1.2) 10 (1.2) 10 (1.9) 10 (2.2) 10 (2.0) 8 (1.4) 10 (1.8) 10 (2.0) 10 (2.0) 10 (1.3)

10 (2.4) 5 (0.9)

10 (2.1) 10 (2.4) 10 (2.1) 10 (2.4) 5 (0.6) 7 (1.1) 10 (1.1) 7 (0.8)

2 (0.1)

4 (0.2)

3 (0.3)

4 (0.2)

9 (0.7) 10 (1.1)

7 (0.4)

10 (1.1)

10 (0.9)

6 (0.7) 0

5 (0.5) 0

1 (0.2) 0

1 (0.2) 0

2 (0.1) 0

5 (0.5) 10 (0.9) 0 0

7 (0.7) 0

8 (0.6) 0

5 (0.7) 0

7 (0.9) 0

8 (0.6) 0

0

0

0

0

0

0

0

1 (0.3)

8 (1.9)

5 (0.8)

6 (0.9)

8 (1.3)

0 0

1 (0.1) 0

0 0

0 0

0 1 (0.1)

0 0

8 (0.9) 0

5 (0.5) 1 (0.1)

4 (0.5) 0

1 (0.1) 0

4 (0.4) 0

3 (0.4) 1 (0.2)

0 1 (0.2)

0 0

0 0

0 0

2 (0.2) 2 (0.3)

6 (0.7) 3 (0.3)

0 0

0 0

0 0

0 0

0 0

0 0

9 (0.5) 10 (1.1) 10 (1.2)

a Contained 0.1 M acetate buffer, pH 4.5, in the dichloramine study, and 0.1 M phosphate buffer, pH 3.0, with 19 ppm chlorine, in the trichloramine study. b Numbers in parentheses denote average severity index (1 ⫽ minimal, 2 ⫽ mild, 3 ⫽ moderate, 4 ⫽ severe). c Number of animals showing abnormality (10 per group).

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FIG. 1. Renal cortex from male rats receiving (A) untreated water and (B) 90 ppm trichloramine in drinking water. In (A), there are normal glomeruli with a normal thickness of the glomerular capsule and intact urinary space. In (B), the Bowman’s capsule is thickened and there are multiple adhesions between glomerular tuft capsule obliterating the urinary space. Hematoxylin and eosin, ⫻400.

creases were observed for monochloramine treatment of rats and mice of both sexes for 90 days by Daniel et al. (1990, 1991), in both male and female rats after exposure for 14 weeks and in female rats after 66 weeks (NTP, 1992). In another study, a 13-week treatment with monochloramine in the drinking water of rats did not induce a significant change in this ratio (Poon et al., 1997). The increased relative kidney weight observed in this study had no effect on urinary total protein or N-acetylglu-

cosaminidase activity, suggesting a lack of biochemical evidence for damage to the kidney proximal tubules (Price, 1982; Bernard and Lauwerys, 1991). The histopathology of the kidneys indicated that the males developed some glomerular adhesions and protein casts in the tubules of the inner cortex at the highest dose level (Fig. 1) while the females developed tubular mineralization in the outer cortex for all treatment groups but with no clear dose response for either incidence or severity over a wide dose

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FIG. 2. Gastric cardia from male rats receiving (A) untreated water and (B) 2 ppm dichloramine in drinking water. In (A), the basal and keratinized layers of epithelium are of normal thickness and the epithelial layer and deeper tissues are free of inflammatory cells. In (B), there is epithelial hyperplasia with a thickened adherent keratinized layer. Inflammatory cells, primarily eosinophil, are present between the layers of smooth muscle, in the lamina propria and in the deeper layers of the epithelium. Hematoxylin and eosin, ⫻100.

range. All observed effects on the kidneys were minimal to mild and may not have apparent clinical manifestations. Poon et al. (1997) observed a decrease in relative liver weight in males dosed with monochloramine which was not observed in this study when dosing with either dichloramine or trichloramine. Although the liver enzymes tested were not affected by dichloramine treatment, significant increases in glutathione S-transferase and UPD-glucuronosyltransferase activ-

ity were observed for the females dosed with 90 ppm trichloramine. This suggested that these two phase II drug metabolizing enzymes may play an important role in the metabolism of chloramines at higher dose levels. An absence of change in urinary ascorbic acid, a noninvasive biomarker of cytochrome P450 induction (Burns et al., 1960; Poon et al., 1994) further supports the absence of significant hepatic change in regard to xenobiotic metabolism. Histologically, the increased anisokaryosis, hyperchromicity of nuclei, and

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perivenous homogeneity were relatively mild and can be considered adaptive. A trend for increased relative spleen weight was observed during trichloramine treatment but not with dichloramine. This trend did not achieve statistical significance and changes were not observed in the various hematological parameters measured nor were there significant changes in the histopathology of the spleen. Chloramines have been implicated as a factor inducing hemolytic anemia, methemoglobinemia, and Heinz body formation in hemodialysis patients (Eaton et al., 1973; Kjellstrand et al., 1974; Pyo et al., 1993). Similarly, marked increases in methemoglobin have been observed during in vitro incubation of red cells with 0.5 ppm of monochloramine (Grisham et al., 1984). However, human volunteers receiving up to 24 ppm of monochloramine for 15 days or 5 ppm for 12 weeks did not have elevated methemoglobin (Lubbers et al., 1981). Poon et al. (unpublished observations) also did not detect an elevated level of methemoglobin when rats were gavaged with 20 mg/kg body wt/day of monochloramine for 21 days. In this work, an increase in methemoglobin levels was not observed for animals dosed with up to 200 ppm dichloramine. Blood smears from rats dosed with 200 ppm monochloramine for 13 weeks did not contain irregularly shaped cells such as schizocytes and padlock cells, and normal levels of polychromatic red cells (reticulocytes), red cell counts, and hemoglobin levels were observed (Poon et al., 1997). A similar absence of response was observed in this work when rats were dosed with up to 200 ppm of dichloramine or 90 ppm of trichloramine in their drinking water. Lipid peroxidation may be induced when oxidating agents are administered acutely. Although the chloramines are oxidating agents, no significant changes were induced in the level of serum or liver TBARS as a result of chloramine treatment, suggesting that lipid peroxidation as a result of oxidative stress was not increased. Minimal histological changes in the gastric cardia characterized by epithelial hyperplasia were induced by treatment with dichloramine at a level of 2 ppm in the males (Fig. 2) and 200 ppm in the females. These effects were not observed with up to 90 ppm trichloramine or in an earlier study with 200 ppm monochloramine (Poon et al., 1997). Various injuries to the stomach have previously been associated with acute monochloramine treatment. For example, oral administration of ⬎60 mM monochloramine produced severe hemolytic lesions in stomachs of unanesthetized rat (Kato et al., 1997; Nishiwaki et al., 1997). Monochloramine has also been implicated in a possible mechanism of Heliobacter pylori-associated gastric carcinogenesis in rats (Iishi et al., 1997), possibly by inducing doublestrand DNA breaks (Suzuki et al., 1997). Monochlora-

mine also inhibited cell growth and induced apoptosis in a rat gastric mucosal cell line (Naito et al., 1997). Rats treated with 200 ppm dichloramine, 90 ppm trichloramine (this study), or 200 ppm monochloramine (Poon et al., 1997) in their drinking water for 13 weeks did not develop hemolytic lesions or gastric cancers; however, the damage to the gastric cardia resulting from dichloramine treatment suggests that it is possible that such endpoints may have been induced either with a longer treatment period or at higher concentrations of the chloramines. In conclusion, dichloramine and trichloramine were found to induce only minimal changes that did not have apparent clinical manifestations over the dose ranges examined. Dichloramine was found to induce mild histopathological effects on the gastric cardia at drinking water levels of ⬎0.2 ppm in males (0.019 mg/kg/day) and ⬎2 ppm in females (0.26 mg/kg/day). Trichloramine was found to induce hepatic enzyme changes in females and increased relative kidney weight and minimal to mild histopathological alterations in the thyroid, kidneys, and liver of males at drinking water levels of ⬎2 ppm for both males (0.23 mg/kg/day) and females (0.29 mg/kg/day). ACKNOWLEDGMENTS The authors thank Ms. Brita Nadeau and Mr. James Elwin for excellent technical assistance and Dr. Robert Liteplo and Dr. Ranjan Bose for their review of the manuscript.

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