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Endpoint:
direct observations: clinical cases, poisoning incidents and other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Already evaluated by the Competent Authorities for Biocides and Existing Substance Regulations.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Community Based Randomized Double Blind Study of Gastrointestinal Effects and Copper Exposure in Drinking Water
Author:
M. Araya, M. Olivares, F. Pizarro, A. Llanos, G. Figueroa, and R. Uauy
Year:
2004
Bibliographic source:
Environmental Health Perspectives Volume 112, Number 10, July 2004

Materials and methods

Study type:
study with volunteers
Endpoint addressed:
acute toxicity: oral
Test guideline
Qualifier:
no guideline followed
Deviations:
not applicable
Principles of method if other than guideline:
The Committee of Ethics for Research in Humans, INTA, University of Chile, approved this protocol. An International Technical Advisory Committee
(ITAG) also reviewed ethical aspects of the protocol.
Quality assurance of field operation and data entry: In addition to field workers a field supervisor worked every day in the community. Data
collected was taken to INTA daily and reviewed for detecting missing values. Three times per week, data were reviewed again with a computational
supervisor, detecting missing data and errors, correcting them whenever possible. Data were fed to the computer at the end of the collection period, corrected and validated prior to analysis.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Copper sulphate (Merck, Darmstadt, Germany) pro analysis grade,

Method

Type of population:
general
Subjects:
Families in the general population.
Sample size: Pilot phase data showed a basal prevalence of "total gastrointestinal symptoms" (the four outcomes defined for this study) of 5%. Using
power 80% and probability 5%, sample size to detect a change in frequency from 5 to 15% was 141 families per group (EPI INFO 6.0). Estimating 5%
drop out the final number of families per group was set at 150.
Two communities were selected in south-eastern Santiago on the basis of their sizes and all houses were censed, gathering general information and
identifying potential candidates for the study. Houses in both communities were built as a group more than 13 years before, sharing the city water
source, all having copper pipes which were not modified or changed in the last five years.Because the intervention consisted of preparing at home a
“test water” to be consumed by all family members, sample selection was by family, including population ≥18 years (Figure 1). Exclusion criteria were
defined a priori:
1) severe chronic illnesses requiring multiple chronic medication,
2) alcoholism (>120 ml alcohol/day),
3) smoking more than 40 cigarettes per day and
4) consumption of drugs.
Eight families met these criteria and in addition 15 moved to another area; after two meetings with the community explaining the protocol and inviting them to participate, 441 families signed an informed consent (one per participant) and were randomized (using a computer generated random list) to receive <0.01, 2, 4 or 6 mg Cu/L, representing a total of 1365 individuals. There were no dropouts during the two-month observation period.
Ethical approval:
confirmed and informed consent free of coercion received
Remarks:
All volunteers received written and oral information about the protocol and were free to refuse continuing in the study at any time.
Route of exposure:
oral
Reason of exposure:
intentional
Exposure assessment:
measured
Details on exposure:
Study design. This randomized double blind community intervention trial was designed to evaluate differences in the report of gastrointestinal
symptoms in subjects exposed during 2 months to <0.01 (customary copper concentration in tap water in Santiago) (Troncoso et al., 1997), or to 2
(WHO provisional-guideline-value set in 1998), 4 (copper concentration at which gastrointestinal symptoms report significantly increased in
previous controlled clinical trials (Araya et al., 2001, Olivares et al., 2001) or 6 mg of copper per litre (concentration at which vomiting was first reported) (Araya et al., 2001, Olivares et al., 2001). Nausea, vomiting, diarrhoea and abdominal pain were defined outcome variables. The two-month
exposure allowed assessing the effect of time and it was considered safe for potential chronic adverse effects. Participating families continued living
at home, carrying out customary activities. Daily water consumption and symptoms were recorded in diaries once a day, therefore strict relation
between time of copper consumption and appearance of symptoms was not controlled. One person per household (usually the mother) reviewed that diaries were filled every night. Twelve trained field workers visited each family every second day, reviewing data recorded and delivering bottles
containing the stock solution to be used for water preparation (see below). Because the intervention consisted of preparing at home a “test water” to
be consumed by all family members, sample selection was by family, including population ≥18 years (Figure 1). After two meetings with the
community explaining the protocol and inviting them to participate, 441 families signed an informed consent (one per participant) and were
randomized (using a computer generated random list) to receive <0.01, 2, 4 or 6 mg Cu/L, representing a total of 1365 individuals. There were no
dropouts during the two-month observation period. Two persons, the one that generated it and the one that prepared copper stock solutions knew
the randomization list. Due to difficulties in masking copper taste in water, some individuals were expected to taste higher concentrations;
participants were carefully instructed not to share their appreciations with field workers or other persons.
During a 2-month pilot phase, families received placebo water (<0.01 mg Cu/L) and procedures and forms were validated. Data obtained during this period provided basal information for sample size calculation.
Test waters. Twice per week a box with eight 80 ml screw cap bottles with similar external appearance, coded by colour, containing a stock solution of copper sulphate, in amounts to reach the concentration 2, 4 or 6 mg Cu/L when diluted to 10 L with tap water, was delivered to each family. Water for home consumption was prepared pouring the stock solution in a graduated 20L container (provided by the researchers) and filling it up to 10 L with
tap water. Instructions included using the water after agitating the container, while participants were at home, not sharing it with visitors. The same
person that prepared the test water was also responsible for maintaining stock solutions out of reach of children and adults.
Actual copper content in stock solutions was measured daily while in home-prepared water this was done once per week, by means of unexpected
visits to the households, either early in the morning, at mid-day or in the evening. Copper was measured by atomic absorption spectrophotometry
(Perkin Elmer, model 2280, Norwalk, CT). The national system responsible for tap water in Santiago (Empresa Metropolitana de Obras Sanitarias
(EMOS), provided tap waters used at INTA laboratories and in the community. Its quality was tested at INTA once, following EPA protocol (Troncoso et al., 1997). Because water was prepared and maintained at home, potential bacteriological contamination was investigated in a sub-sample of 179
randomly chosen households, once per household. Sampling was distributed along the two-month survey. Mesophilic Aerobic Counts (MAC), Total
Coli form Counts (TCC) and faecal coli forms were chosen as indicators of bacterial contamination. These parameters were determined by means of
routine procedures (APHA/AWWA/WEF 1995; Pouches et al., 1999).
Examinations:
Health survey. For symptom recording participants filled the dairy choosing from a list based on previous studies, validated during the pilot phase,
that included the four symptoms defined as outcomes and the following symptoms that blinded the subject as to the variables of interest (being
energetic -a positive effects assigned to copper-, lack of energy, cough, headache, backache, chest pain, others: explain…). Operational definitions of symptoms were provided during the initial meeting and again during home visits. Participants were instructed to discontinue the test water and have plain tap water for 48 hours when they experienced any symptom included in the list. Then they were asked to continue drinking the assigned water.
Should symptoms appear again subjects were instructed to discontinue the assigned test water permanently and consult the full time (research)
physician located in the community. If symptoms were to persist they should seek help at the local emergency service. Two physicians belonging to
the research team were always available on call during the study period.
Medical treatment:
Not applicable.

Results and discussion

Clinical signs:
Abdominal pain and nausea were the most frequently reported symptoms.
Results of examinations:
Health survey: The number of families enrolled were fewer than the estimated sample size, however, the greater frequency of symptom report found
in the survey (in comparison with the prevalence obtained in the pilot phase) allowed statistical analysis and detection of significant differences.
Traditional analysis of this type of data consists of determining the proportion of responders in the study group, because persons that repeat their
responses cannot be treated as independent observations. In turn, analysis of responses does not take into consideration the fact that one individualmay report more than one symptom, and symptoms more than once. Characteristics of the individuals, copper concentration in test waters and
copper and water consumption is shown in Table 1, while the proportion of responding individuals appears in Table 2. 222/1365 individuals
reported at least one symptom in 665 occasions, providing a total of 794 symptoms. 16.3% of surveyed individuals were “responders”, i.e., reported
at least one symptom at least once. Individuals of group 0 represented 2.9% of these responders, providing 18% of the total number of symptoms
reported during the study. “Responders” were more frequently women (64.2%, x2= 70.84, p= 0.0000). Only 2.6% of responses were obtained from
individuals ≥60 years; although there were no significant differences by age interval, comparison of responses obtained in the first and third terciles for age gave an Odd Ratio of 0.41 (CI ). Individuals that ingested less than 500 ml of test waters per day represented less than 1% in all four study
groups. Using chi square analysis symptom report increased significantly over the basal prevalence at 4 and 6 mg Cu/L while there were no
differences between 4 and 6 mg Cu/L (Table 2). Abdominal pain and nausea were the most frequently reported symptoms. Distribution of the total
number of responses reported during the 2 month controlled copper exposure expressed as percentage of total symptom report per group is shownin Table 3. Symptoms reported yielded “combinations” throughout the survey (individuals tended to repeat their pattern of report, 182/222
responders reporting the same “combination” of symptoms throughout the survey. Symptom report significantly decreased along the 9 weeks (x2 = 486.909, p= 0.0000), yielding an Odd Ratio for symptom report of 0.06 and 0.09 in the last two weeks of study, respectively. This did not coincide
with decreasing fluid consumption along time. Reports of total “unrelated symptoms” (used to distract the participants) were similar in the four study groups (x2 = 2.32, p=0.5083). Counting process analysis using the stepwise method yielded a model that included sex (p< 0.0001) and copper
concentration received on the day of event (p< 0.0001). Covariates age, volume of fluid intake, and (waters consumed as) total, “plain” and “mixed”
waters did not have a significant effect on the model. Cumulative hazard curves for increasing copper concentrations in test water by sex and
stratified by weeks showed a progressive decrease of risk along time (Figure 2). Counting process analysis confirmed results of the pre-hoc analysis, which found an increased risk of symptoms associated with increasing copper concentration and with female gender. Analysis of risk differences
between genders using the 95% CI of the cumulative hazard ratio showed that the risk remains significantly higher from week 1 to week 4 for all
copper concentrations (except basal 0). Figure 3 shows the relative risk (RR) of symptoms against exposure to increasing copper concentration in
test water (compared to no exposure); on week 1 the RR became significant at concentration 4 mg/L [RR (95% CI) = 1.53 (1.02-2.05) in women, and at6 mg/L in men [RR (95% CI) = 1.9 (1.02-2.79) ]. With advancing time, the significance shifted to higher copper exposure, such that on week 2 for men and week 4 in women, copper concentration required to obtain significant differences on symptom report was higher than 6 mg Cu/L. Mistakes made while preparing test waters at home created the opportunity to evaluate the effect of copper exposure higher than 6 mg/L (actual range 6 to 13 mg/L)using the same risk analysis with counting process; this analysis involves a low number of families (n= 126) and individuals (n= 417). The resulting
cumulative hazard curves suggest an exponential increment of symptoms associated to rising copper concentration in test water (Figure 3).
Effectivity of medical treatment:
Not applicable
Outcome of incidence:
See other information on results.

Any other information on results incl. tables

Results:

1.     Comparability of enrolled and censed families:

Families and individuals that fulfilled entry criteria were assigned to the intervention groups as shown in Figure 1. There were no significant differences in socio-demographic indicators measured between participating and not participating families. All enrolled families and subjects (n= 1365) provided data throughout the study period. There were not individual withdraws because of violations to the protocol.

2.     Comparability of intervention groups:

Randomization resulted in similar groups for the analyzed variables. Baseline demographic characteristics and behaviours that would modify patterns of drinking water were similarly distributed in the four groups (Table 1).

3.     Bacteriologic study:

Water was clean by EMOS contemporary data for the study area. Faecal coli forms were not detected. 22/179 (12.3%) of home water samples had either MAC or TCC positive; in 5/179 (2.8%) both indicators were positive (2, 2 and 1 families in groups 0, 2 and 6 mg Cu/L). Because bacteriological evaluations were done once per family and symptom report had low frequency it was not possible to analyze results using data obtained during the week of bacteriological sampling. There was no association between the proportion of individuals that reported symptoms or total number of symptoms reported and at least one positive parameter in the bacteriological study. In families in whom both parameters were positive, TCC was significantly associated with total symptom report (p= 0.0012, Fisher’s test).

4.     Adherence to the assigned group:

Mean copper concentration measured in test waters prepared at home, fluid intake and mean daily dose of copper received by the study subjects appear in Table 1. Individual daily water consumption had significant day-to-day variability (ranging from 0 to 6 litres/day, in one person), but inter group differences were not significant, expressed either as “total”, “plain water” or “mixed waters”. Week to week fluid intake along the 9 study-weeks did not reveal significant differences (ANOVA). In one occasion weekly measurement of copper concentration in home prepared waters detected two families whose waters were well off the expected values: one had 2 instead of <0.01 mg Cu/L and the other had 2 instead of 4 mg Cu/L. Information obtained from these families revealed that they decided to interchange one stock solution bottle in order to share the potential “benefits” of having more copper in their drinking water. Cu concentration measured in test waters at home was >6 mg Cu/L (range 7-13 mg/L) in 8043 (out of 85.996) occasions, affecting 417/1365 participants.

 

Discussion:

This study assessed for the first time the effect of controlled copper exposure in individuals maintaining conditions close to real daily life, providing information both on the acute gastrointestinal responses and the effect of time in a two-month interval. Systematic review of the copper effects on human health recently performed by the National Research Council (1980) did not reveal enough information to conclude on the acute and sub acute effects of copper. Previous studies intending to evaluate copper effects on the population failed to clarify the relation of exposure copper in tap water and digestive symptoms (Buchanan et al., 1994; Knobeloch et al., 1994; Petterson and Rasmussen 1999; U.S. Department of Health & Human Services. 2000; Vergara et al., 1999; Zietz et al., 2003). In this study two different approaches for statistical analysis, changing copper concentration in drinking water from 0 to 4 results in a significant increment of gastrointestinal symptoms report. This figure also agrees with others previously obtained using controlled clinical trials as designs (Araya et al., 2001; Olivares et al., 2001).

 

Counting process analysis represented a significant improvement in the analysis because it allowed maintaining all participants present every occasion they registered data; this represented a major concern because participating families did not follow instructions strictly, leaving days without registering information when they went out of home for 2-3 days. A main limitation in this study is the lack of control of the exact timing of exposure and appearance of symptom. This was indeed done in the previous clinical trials mentioned that led to calculate the dose response curve (Araya et al., 2001; Gotteland et al., 2001; Olivares et al., 2001; Pizarro et al., 1999). Instead, in this study the main objective was to assess to what extent this dose response curve is applicable when individuals are exposed to copper in a more realistic fashion. Only copper concentration and sex were chosen for the model while total volume, daily copper dose and quality of the test waters ingested (“plain” or “mixed infusions”) were left out of the model. This result is most relevant because it indicates that copper concentration and volume are the main determinants of response, and dose and vehicle for copper ingestion are less important. Nausea has proved to be a good marker of early response to acute copper exposure; however, the high frequency of abdominal pain observed in this study was unexpected because abdominal pain was infrequently reported in previous studies. It is difficult to explain this finding; it is possible that repeated acute copper exposure may be part of the explanation, but this cannot be established by this study. Predominance of women in the reporting group is another relevant finding of the study (Figure 2); this difference was suspected in previous studies (Araya et al., 2001; Olivares et al.2001) and here was obtained both using the chi square analysis and by the counting process. Acute response to copper exposure is triggered very specifically in the stomach through mechanisms that result in vagal stimulation. Gender differences for vagal phenomena are not described. In gastric physiology another sex related observation is that females have lower gastric alcoholic dehydrogenase activity (Frezza et al., 1990); this has been interpreted as responsible for the different response to alcohol of women in comparison to men (Frezza et al., 1990). Whether other gender differences occur and how they may relate to response to copper remains to be clarified. Symptom report clearly decreased along time both in men and women (Figure 3). Although misreporting of symptoms due to decreasing motivation cannot be ruled out, several controls performed to detect this failed to reveal suggesting information; therefore, we interpret these result as suggesting an adaptive response to repeated copper exposure.

Applicant's summary and conclusion

Conclusions:
This study has established the symptoms, range of responses and relevant variables associated with repeated acute copper exposure in human
adults; even considering the findings in women (who appeared more sensitive to copper exposure), and result obtained during the first two weeks of exposure (that yielded the highest incidence of effects) the current provisional guideline for drinking water (2 mg Cu/L) set by WHO is safe (WHO
1993, WHO 2003); these data represent relevant information for regulators that must decide on the relation of copper exposure and the safety of
drinking water from human health.
Executive summary:

Materials and Methods:

Gastrointestinal effects were assessed in 1365 adults exposed to either <0.01 (controls), 2, 4 or 6 mg Cu/L of drinking water for 2 months in a randomized, double blind community based study. Risk of symptoms increased with increasing copper exposure and decreased with time. The best model by counting process analysis included copper concentration and sex.

Results and Discussion:

The Risk-of-symptoms remained significantly higher in women than in men from week 1-4 for all concentrations tested; on week 1 comparison with group <0.01 mg/L showed that differences became significant at 4 mg/L[RR (95% CI) = 1.53 (1.02-2.05) in women, and at 6 mg/L in men[1.9 (1.02-2.79)]. On week 2 for men and week 4 in women, copper concentration required to obtain significant differences on symptom report was higher than 6 mg Cu/. We conclude that exposure to copper in drinking water results in gastrointestinal symptoms, which are modulated by copper concentration, time and sex.