Research#8.txt

Environmental Vol. 46, pp. 57-62, Health 1982 Perspectives and of Controlled Chlorine Chlorate Clinical Dioxide, in Man Evaluations Chlorite by R. Bianchine* Judith R. Lubbers,* Sudha Chauan,* and Joseph To the volunteer trends controlled common healthy administered double-blind sequellae cases, associated consequence. detrimental ingestion with susceptible ingestion glucose-6-phosphate evaluation assess acute a statistically might low adult to of noted the of effects subjects study of with investigational to the physiological chlorine clinical level relative a indeed oxidative One daily male battery disinfectants by treatment; was of any of cannot were significant volunteers. for progressively evaluation dehydrogenase-deficient achieve dioxide safety undertaken. glucose-6-phosphate of of stress; twelve completed the qualitative responses drug rule however, of at participating and proportions therefore, trends chronically out a studies. of Phase consecutive concentration its chlorine increasing The the uneventfully. metabolites, within and in none clinical II possibility Phase certain in quantitative of considered subjects administered dioxide dehydrogenase Phase of the clinical weeks single subjects. I, these of evaluation limits biochemical a chlorite rising III, 5 There that, and or mg/l. doses to trends importance. by the chlorite tests. of its Physiological a does chlorine over the were for the and impact potential small of was may The observing was twelve or tolerance a chlorine study, chlorate, at no longer conducted physiological three be a judged water group on obvious However, concentration consecutive metabolites expected the normal impact phases medical disinfectants treatment investigation, disinfectants was of relative to undesirable in potentially have demonstrated. by subjects of the parameters to was team. weeks. in the this safety of be physiological period, three human assessed 5 absence to controlled in especially In examined mg/l. of Persons clinical normal man, several of phases at-risk these daily male were oral was by of a Introduction sideration chlorine may safety and purpose, dioxide, the was Chlorine The auspices its be designed study of used chlorite by-products water a oral in controlled dioxide the of was routinely human to treatment. USEPA and United conducted evaluate is chlorate must clinical currently ingestion as States HERL a Before the water be in was evaluation three as assessed. acute of under #CR805643. undertaken an disinfectant, chlorine chlorine parts. alternative physiological serious of For chlorine Phase dioxide dioxide under con- this the to I 
ty, OH * The College 43210 
Department of Medicine, of Pharmacology, 333 W. 10th The Avenue, Ohio State Columbus, Universi- tants effects Chronic studied logical susceptible phosphate chlorite. 
administered ofprogressively response in ingestion Phase dehydrogenase, individuals, II. of by to Phase a normal normal small increasing those III to deficient group male healthy assessed chronic doses volunteers of adult in ingestion the of potentially glucose-6- disinfec- physio- males. was of Methods Subject Selection tive adult abnormality For study male Phase participant volunteers in I the and routine for were who Phase clinical exhibited selected. II, serum normal No a significant analysis, prospec- healthy 

58blood count, urinalysis, or electrocardiogram was selected. Subjects manifested no physical abnor- malities at the pretreatment examination, were 21 to 35 years of age, and weighed within ± 10% of normal body weight for their frame and stature. A history of disease or any medical or surgical condi- tion which might interfere with the absorption, excretion, or metabolism of substances by the body precluded inclusion. Regular drug intake prior to the start of the investigation, either therapeutic or recreational, resulted in exclusion from the study. Normal methemoglobin levels, thyroid function, and glutathione levels were mandatory. Written informed consent was obtained from each subject prior to initiation of treatment. For Phase III, volunteers were defined as glucose- 6-phosphate dehydrogenase (G-6-PD)-deficient on the basis ofa hemoglobin G-6-PD level of less than 5.0 IU/GM hemoglobin in the pre-study screening. Phase III subjectswerenormal in all other respects. 
Water Disinfectant Preparation A detailed description of the water disinfectant preparation techniques has been presented by Lub- bers and Bianchine (1). In general, freshly pre- pared stock solutions of chlorine dioxide, sodium chlorite, sodium chlorate, chlorine and chloramine were assayed by the colorimetric techniques of Palin (2) then diluted with organic-free demineral- ized deionized water to appropriate concentrations. Individual bottles were capped and stored in the dark under refrigeration until use. All bottles were coded by an independent observer and the identity of each bottle remained "double-blind" to both the investigative staff and the volunteer subjects. 
Study Design: Phase I The 60 volunteers in Phase I were divided at random into six treatment groups (1). Ten persons were assigned to receive each of the disinfectants; the ten members of the control group received untreated water. The study involved a series of six sequences of three days each. Treatment concen- trations were increased for each treatment. The specific concentrations or disinfectant administered to the study participants are listed in Table 1. A clinical evaluation of the collection of blood and urine samples for determination of pretreatment baseline laboratory values preceded the first treat- ment. On the first day of each three day treatment sequence, each volunteer ingested 1000 ml of the water in two portions. The second 500 ml portion aliquot was administered 4 hr after the first. Each 500 ml portion was consumed within 15 min. Only two doses of disinfectant were administered on the 
LUBBERS, CHAUAN AND BIANCHINE first day of each treatment sequence. No disinfec- tant was administered on the second and third day of each sequence, since these two days were to serve as followup observation days. The second day of the treatment sequence consisted of a physical examination and collection of blood and urine sam- ples for determination of posttreatment laboratory values. On the third day, each volunteer was given aphysical examination to determine residual effects of treatment with the water disinfectants and byproducts. Taste evaluations were obtained at each dose level. Study participants were asked to rate the treated water as very unpleasant, slightly unpleas- ant, not pleasant, pleasant, or tasteless. 
Study Design: Phase II The sixty volunteers of Phase II were divided at random into six treatment groups of ten subjects each (3). In order to assure efficient management of the 60 subjects, they were randomly assigned to three subsets. These subsets were sequentially entered into the study on three successive days and exited from this study in a similar fashion. For all of the treatment groups, the concentration of disin- fectants ingested was 5 mg/l. The control group received untreated water. Each subject received 500 ml daily for 12 weeks. Physicals, collection of blood and urine samples for laboratory assays, and taste evaluations were conducted on a weekly basis during the treatment period and for8 weeks follow- ing cessation of treatment. 
Study Design: Phase III The three glucose-6-phosphate dehydrogenase- deficient subjects of Phase III were given sodium chlorite at a concentration of 5 mg/I. chlorite (4). The treatment protocol was identical to that of Phase II, with daily administration of 500 ml of solution to each volunteer. 
Evaluation Procedures An extensive battery of parameters was moni- tored to assess the biochemical and physiological response to the oral ingestion of the water disinfec- tants and water treatment by-products (Table 2). All laboratory determninations of biochemical param- eters were conducted by a licensed medical labora- tory, Consolidated Biomedical Laboratories, Inc. (CBL), Columbus, Ohio, HEW license number 34-1030. For each volunteer, pretreatment baseline values and six sets of posttreatment values were compiled. Laboratory tests were carefully chosen. 

ORAL INTAKE OF CHLORINE DISINFECTANTS IN MAN 
Table 1. Concentration of disinfectants in phase I: acute rising dose tolerance.a 
Disinfectant concentration, mg/l. Water disinfectant Day 1 Day 4 Day 7 Day 10 Day 13 Day 16 Water Chlorate Chlorine Chlorite Chlorine Chloramine control dioxide 0.01 0 0.1 0.01 0.1 0.01 0.1 0 0.1 1.0 1.0 1.0 0.5 0 5.0 0.5 5.0 8.0 10.0 10.0 18.0 0 1.0 1.0 24.0 18.0 18.0 0 1.8 1.8 24.0 24.0 2.4 0 2.4 aFor each dose, two portions of 500 ml each were administered at 4-hr intervals. 
Table 2. Biochemical parameters assayed in the controlled clinical evaluation of chlorine dioxide, chlorite and chlorate in man. 
Serum chemistry 
Blood count 
Urinalysis 
Special tests 
tory 59 glutamic-oxaloacetic Plasma acid, cholesterol, calcium, glucose, triglycerides, phosphorus, sodium, transaminase, potassium, total alkaline protein phosphatase, serum chloride, albumin, glutamic-pyruvic urea gammaglutamyl globulin, nitrogen, albumin/globulin creatinine, transaminase, transferase, BUN/creatinine ratio, lactic total iron bilirubin, dehydrogenase, ratio, serum uric hemoglobin phils, Platelet corpuscular basophils, count, concentration, volume, white large mean unstained blood high corpuscular cell peroxidase cells count, volume, red activity, blood mean neutrophils, cell corpuscular count, lymphocytes, hemoglobin, hemoglobin, monocytes, hematocrit, mean corpuscular eosino- mean blood Color,a count, appearance,a casts,a crystals,a specific gravity, bacteria,a pH, mucus*, protein, amorphous sugar,a acetone, cells,a epithelial blood,a white cells blood count, red test,a Serum hemoglobin haptoglobin, electrophoresis,a sickle cell,a T-3 methemoglobin, (uptake), T-4 (RIA), glucose-6-phosphate free thyroxine index, dehydrogenase, electrocardiograma Coombs Physical exam Systolic blood pressure, diastolic blood pressure, respiration rate, pulse rate, oral temperature 
aThese parameters yielded qualitative data only; no statistical analysis was performed. 
pected thyroid was ties. to On oxidants was cally and in The assayed individual Red free glucose-6-phosphate Bianchine and for designed back. differed State standard The provide evaluation the glutathione specifically Medical analyzed used blood thyroxine; A specific monitored University Any battery biochemical numerical basis function from were have deviations to programs cell (1). value supplementary value detect serum, of Education by surface of defined; the of devoted and been levels. the peripheral which by utilizing by overall Division for values group response possible response dehydrogenase, literature facilitated was T-3 the blood discussed antibody an a Hemoglobin to fell portion (uptake), and qualitative noted. mean physiological individual monitoring the obtained information parameters of outside hemoglobin and to to facilities Research. Computing ingestion (5-8), formation rapid oxidative by of In urine by the T-4 more addition, electrophoresis were normal methemoglobin subject this Coombs Lubbers areas clinical and test was of parameters (RIA), well-being. abnormali- of response. was than stress The Specially collected Services to chlorine assayed battery labora- 
of which every assist feed- clini- test; Ohio sus- two and and by abnormal. ful basis exhibited sensitivity ated able cal Package analyses multiple lized t-tests Statistical scrutiny; Computer computer ranges for throughout BMDP2V. and each for linear of abnormal simple Chemical to variance for the the analyses Programs the of packages, regression that safety t-tests, Social these the BMDP1R disinfectant values parameters parameter study. with utilized and individuals Sciences (BMDP) SPSS-T-test specifically, were the repeated analyses. was possibility commercially agents for was subjected and (SPSS). used on volunteers was measures designated the For the a were to continuing employed. Statistical Two-way of Biomedi- pairwise perform to hyper- evalu- avail- care- who uti- as Results Qualitative and The tions tative accumulated An continued general alone important laboratory with clinical in medical each aspect subjective tests histories subject's of throughout observation this observations and medical study physical this was of file. all study the and examina- A subjects. careful carefulquali- were 
60inspection of each of these medical files presented a review of the general clinical health of each subject. The careful clinical evaluation of every subject in Phases , II and III failed to reveal any clinically important impact upon the medical well-being of any subject as a result of disinfectant ingestion. Further, there was no apparent grouping of the minor subjective symptoms and objective signs noted throughout the study; the "colds," "lymphadeno- pathy," "sore throats" and "flu" problems noted episodically appear to be randomly dispersed among the treatment groups. All subjects remained negative with respect to the Coombs tests and the sickle cell tests during the investigation. Hemoglobin electro- phoresis results indicated that, in Phase II, a small number of subjects yielded abnormal hemoglobin distributions but these individuals were found to be randomly distributed in both the treatment groups and in the control group. Examination of electro- cardiograms revealed no abnormalities. Vital signs (blood pressure, pulse rate, respira- tion rate and body temperature) were measured on a regular basis to provide immediate feedback to the monitoring physician on the acute physiological response of study participants to treatment. The statistical analysis of the vital signs was limited to the calculation of arithmetic group means and stan- dard deviations from the mean. The compiled vital signs were examined for evidence of consistent response to treatment. No such evidence was found. The subjective evaluations of palatability indi- cated that few subjects found the test substances to have an objectionable taste at levels up to 24 mg/l. 
Quantitative Forthe Phase I acute rising-dose tolerance study, a two-way analysis of variance with repeated mea- sures was used to compare the treatment group values of each biochemical parameter to the corres- ponding values of the control group. The analysis of variance allowed distinctions to be made among the possible sources of variation. Differences between two groups that existed prior to treatment, parallel variations in quantitative chemical values due to laboratory drift and authentic treatment-related changes in physiological parameters could be dis- tinguished. Three probabilities were calculated for each case: the group main effect (G), the time main effect (R), and group-time interaction (RG). The treatment groups and the corresponding biochemi- cal parameters for which a strong probability of treatment-related change was computed (that is, RG - 0.05) are listed in the first column of Table 3. To assist in determining the clinical importance of the statistically significant group time interac- tions, the group, mean and standard deviations 
LUBBERS, CHAUAN AND BIANCHINE from the mean were examined for the pretreatment baseline assay and each posttreatment assay for each of the treatment groups. In all instances, the group mean values remain well within the estab- lished normal ranges. On the basis of the small magnitude of change within the normal range and the duration of the study, it was concluded that the trends identified by the analysis of variance are unlikely to be of clinical importance. The possibility that the trends might become clinically important with increased exposure cannot be excluded. Alternative statistical techniques were employed for Phase II. An omnibus testing technique was used initially. Totestthe hypothesis that the response ofone or more of the groups was different to that of the rest of the groups, an analysis of variance with repeated measures was performed in which values for all six treatment groups were included. For the parameters urea nitrogen and mean corpuscular hemoglobin, RG-values < 0.05 were obtained. Sup- plementary tests were performed. Analyses of vari- ance with repeated measures in which the values of each treatment group were compared to the cor- responding values of the control group were cho- sen. The use of the analysis of variance in this manner is flawed by the common control group. However, the results of the analyses may be used with caution. The analysis of variance yielded sta- tistically significant RG-values in the comparison of the group mean corpuscul ar hemoglobin values for the chlorite and the chlorate groups and of the group mean urea nitrogen values of chlorate and chlorine dioxide treatment groups to the corres- ponding control group values, as shown in Table 3. No linear trends were detected by linear regres- sion analysis of the chlorite group's mean corpus- cular hemoglobin values, the chlorate group's urea nitrogen levels or the chlorite group's ureanitrogen values. Mean corpuscular hemoglobin levels in the chlo- rate group yielded a probability of 0.01 upon linear regression analysis. The relative slope associated with thechange duringthe 12-weektreatmentperiod was approximately 1% of the normal physiological range per week. We believe that no physiological importance may be attributed with confidence to the variation. However, it is impossible on the basis of this study to rule out the potential physiological significance of the trend. Further study is warrant- ed.The small number ofsubjects (three) in Phase III negated the value of many statistical procedures. Linear regression analyses were chosen. The third column of Table 3 lists the biochemical parameters for which a high probability of change with respect to time was calculated. The p-values computed by 

ORAL INTAKE OF CHLORINE DISINFECTANTS IN MAN 
Table 3. Biochemical parameters and which treatment could be groups attributed in which to ingestion statistical of analyses disinfectant. indicated a high probability of change Test Phase Ia Phase l1b Phase ITIC Urea nitrogen (BUN) Chlorite Creatinine Gamma Total Albumin/globulin T-4 BUN/creatinine Ratio Uric Calcium Methemoglobin Free Mean Iron (RIA) acid thyroxine bilirubin corpuscular glutamyl index transferase ratio hemoglobin Chlorite Chlorine Chlorite Chlorine Chlorine Chlorine Chlorate dioxide Chlorate Chlorate Mean Lymphocytes corpuscular hemoglobin concentration Chlorine those group cLinear aTwo-way bTwo-way of comparisons. the regression control analysis analysis group. ofvariance analysis of variance indicated yielded yielded a group-time group-time strong probability interactions interactions of change (RG-values) (RG with values) respect S 0.05 S Chlorate Chlorine dioxide Chlorite Chlorate Chlorite Chlorite Chlorite Chlorite Chlorite 0.05 in comparisons of treatment group values to in both the omnibus and treatment group-control to time; p-values S 0.05. 
the for tive normal tistical A/G puscular globin during the ing to possibility caution confidence cannot origin them. four into absence linear magnitude ratio, nor values, analyses the say range in biochemical consideration hemoglobin can dealing regression 12-week of that T-4 only of we laboratory per a a of indicate (RIA), change these concurrent attribute that change, with week treatment parameters. concentration, analysis the trends trends the with free was a drift, the good physiological small results. control respect thyroxine, computed. were percent were period. were probability one group To We of gauge group indicated. and must less to change physiological However, can consequence size time These mean methemo- than the and say that, exercise and occurs of rela- with 0.05 tak- cor- sta- We the the for in Discussion logical agents, et methemoglobin et levels rite. tory destruction (6), Several al. al. hemolytic The animals (7,8), were (9) effects chlorinedioxide, oral researchers associated Abdel-Rahman of observed has administration hemoglobin anemia of formation. been oral in oral and have ingestion shown chlorite animals et and In chlorite suppressed addressed al. of studies to methemoglobin and chlorate (5) treated of induce and ingestion chlorate. the by the glutathione Couri Heffernan with to oxidative oxidizing physio- labora- Musil et chlo- with for- 
al. 61 mation high the and toxicity a decreased administration to gility, ity reported. ty, cal tify clinical attempt chlorine trolled massive urinalyses Heffernan dioxide ingestion levels of in impact ingestion slightly During Unfortunately, concentrations rats increase embryonic studies increased Fridlyand of any levels and and clinical (18). erythrocytes; (10, evaluation adversely of of volume dioxide significant to in yielded increased glucose-6-phosphate the In rate orally chlorine red were et (19,20) of mean 11). man elucidate the kidney/body al. toxicity chlorite and course of conditions. cell of of performed The consumed chlorite is (7). and corpuscular African affected of weight transient the Kagan have described raw count severely dioxide, exposure two-year sodium possibility Haller chlorate information of and morphologic the ingestion data failed (14-16) the (17). gain (13) thyroid Green weight the effects chlorine and changes chlorite was limited. chlorite, and in three-phase examined volume, mortality teratogenic were dehydrogenase to The related in a this of was to acquired. conclusively monkey, Northgraves meticulous ratio function; man renal available of maternal mice dioxide observed. changes report was in Epidemiologi- suggested the and effects. osmotic reported hemoglobin under the rate was toxicity evaluated potential study, chlorite, chlorate Routine chlorine chlorite chronic in was on shown exam-toxici- and activ- iden- were rats; Oral con- The (12) fra- the an by by at a a 

62 LUBBERS, CHAUAN AND BIANCHINE judged ination definitive was investigation oral treatment; achieve er, statistically One longer of lites, the oral within absence chlorine made cannot chlorite treatment ingestion to of proportions have finding the this in however, significant disinfectant of rule any of limits and immediate body detrimental the of of of period, out detrimental chlorate, chlorine relative the of of of the the none trends clinical information three ingestion. these physiological study, possibility physiological dioxide safety of was phases were importance. trends physiological these the demonstrated In and and associated was relative of several might consequence. that, trends tolerance its this made. response. metabo- Howev- impact human over indeed safety cases, were with No by of a REFERENCES 
1. 2. 3. 4. 5. 6. J. chlorite ron. diamine teers. glucose-6-phosphate male 889-892 rising Palin, and rite, chlorite Abdel-Rhaman, C102 water Lubbers, Lubbers, R. Lubbers, R. Couri, dioxide Environ. The The combined Pathol. bromine, and volunteers. on A. J. dose effects D., and (1982). to and (DPD). Environ. effects blood T. J. J. J. effects normal administration metabolites R., R., R., Path. Methods and Toxicol. chlorate available of iodine glutathione Chauhan, and Chauhan, J. M. of Abdel-Rahman, chronic of Toxicol. J. Pathol. healthy Inst. S., Bianchine, C102, chronic dehydrogenase 5 for and to Environ. (2, Couri, on chlorine, Water the normal administration S., S., ozone, 3): and Toxicol. and of 3(1,2): glutathione adult determination, Miller, administration Miller, chlorine 865-878 D., J. hemolysis C102, Engr. Pathol. healthy male using R. chlorine 431-449 and M.S. 5 J. The J. deficient (2, (1982). and dioxide, 21: Bull, K., dependent volunteers. K. diethyl-p-phenylene of 3): Effect effects Toxicol. in adult 537-549 and dioxide and C103 chlorine (1979). rat 879-888 R.J. in of healthy water Bianchine, Bianchine, chlorate and male of in of chlorite Kinetics system the and 5 (1976). J. drinking chicken. dioxide, chlorine (1982). (2, of volun- Envi- acute adult chlo- free and 3): J. J. to of in 
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Jung, rat, 3(1,2): vitro. logical ment damage Heffernan, damage vitro. Musil, 327-345 administration Pharmacol. makol. dioxide potential potentially Heffernan, Richardson, Bluter. Haller, safety. Fridyland, of Hygiene Moore, C-57L-J (1980). Moore, Environ. Moore, Environ. Berez, Boston, water ology. Haring, Contam. Michael, Greathouse, Chlorine related Ammar, icity Health Couri, Dawley standards mouse and of J. J. J., and 451-460 F., Arch. aspects J. 216: TAPPI D., S. Perspect. Naunyn-Schmiedebergs G. G. G. J. water B. and to (1964). compounds rats. to Environ. mice. Environ. E. Sanitation teratogenic high-risk Toxicol. G. dioxide Pathol. Pathol. Kontek, P., F., Subchronic J., cardiovascular and S., S. high-risk the S. S., and the Miller, W. W. Exptl. 103-111 M. E., for sodium Environ. D. A., A. and Environ. and DiBiasi, and J. and containing of 33: Calabrese, Assessment (1979). erythrocyte erythrocyte chicken Kuon, P., P., of residual Miday, water G., Environ. Toxicol. Toxicol. and chlorine 25: 46: Northgraves, Pathol. Zoetman, Pathol. P. Z., group Therap. 199-202 Calabrese, Calabrese, C. 36: chlorate Guion, Guion, from in (1951). potential chlorite 47-55 658-662 Kagan, Kraemer, toxicity Chalupa, H., Toxic Health R. D. 18-21 Health disinfection: the blood. R. chlorine to phenol. chlorine diesase 4(2, 4(2, Toxicol. Toxicol. dioxide L., Bull, Zum Pathol. E. copper K., 59: (1955). of C., C., B. induced induced non-human (1982). G. for (1971). on maternal J. of of 36(1): potentialities 3): 3): (1981). E. Jones, J. E. Perspect. C. 101-103, Bercz, J., and and W.W. Arch. Z. R. sodium D. inaktiven alternate and dioxide Environ. 271-279 J. erythrocytes 465-470 Chem. Corrosiveness mortality. J. application dioxide 2(6): and 2(6): Toxicol. blood and Experimental J., F., The 20-27 a Bull, Bull, by by G-6-PD-deficiency-a Ho, L., prospective Exptl. Chlorine J. chlorite Schmidt, Delphia, toxicity, 1487-1499 1501-1510 chlorite (1937). sodium sodium primate. and Technol. in effects 46: P., Murray, and treated (1980). disinfectants S. (1980). R. R. (1981). Pathol. hemoglobin 4(2, drinking Bull. 25-29 Miller, of C. Pathol. for Lucas, J. J. ingestion. of tissues. dioxide 3): in embryotox- chlorite of chlorite of J. P. Groups Oxidative the validation Oxidative continued water. Praze. Environ. (1979). Environ. Sprague- A-J drinking epidemi- Toxicol. 513-524 (1979). chlorine D., (1982). M., Toxico- water. R. treat- Phar- J. and and and and das and G., in in B. J. J. at J. 8: