Copper hydroxide nitrate (Cu2(OH)3(NO3))

Exposure related observations in humans: other data

Administrative data

Endpoint:

exposure-related observations in humans: other data

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2004

Materials and methods

Type of study / information:

Exposure related observations in humans

Endpoint addressed:

other: gastrointestinal effects

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Randomized double-blind community intervention trial: controlled exposure study in a community whose members maintained living conditions as close to real life as possible.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Copper sulfate

IUPAC Name:

Copper sulfate

Test material form:

other: stock solution of Copper sulfate

Details on test material:

Copper sulfate in drinking water at concentrations of 2, 4 and 6 mg/L as Copper.

Method

Ethical approval:

not specified

Details on study design:

Randomized, double-blind community intervention trial was designed to evaluate differences in the report of gastrointestinal symptoms in subjects exposed during 2 months to Cu concentrations of < 0.01 mg/L [usual Cu concentration in tap water in Santiago, Chile (Troncoso et al. 1997)], 2 mg/L [WHO provisional-guidelinevalue set in 1998 (WHO 2003)], 4 mg/L [concentration at which gastrointestinal symptoms were significantly increased in previous controlled clinical trials (Araya et al. 2001; Olivares et al. 2001)], or 6 mg/L [concentration at which vomiting was first reported (Araya et al. 2001; Olivares et al. 2001)]. Nausea, vomiting, diarrhea, and abdominal pain were defined outcome variables. The 2-month exposure allowed for assessing the effect of time, and it was considered safe from 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 Cu consumption and appearance of symptoms was not controlled. One person per household (usually the mother) ensured 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.
Two communities were selected in southeastern Santiago on the basis of their sizes, and all houses were censused, including gathering general information and identifying potential candidates for the study. Houses in both communities were built as a group more than 13 years before; they shared the city water source and all had Cu pipes that had not been modified or changed in the last 5 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 of age. Exclusion criteria were defined a priori: a) severe chronic illnesses requiring multiple chronic medication; b) alcoholism (> 120 mL alcohol/day); c) smoking more than 40 cigarettes/day; and d ) consumption of drugs. Eight families met these criteria; also, 15 additional families moved to another area. After two meetings with the community in which we explained the protocol and invitited 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 1,365 individuals. No families dropped out of the study during the 2-month observation period.
Two persons knew the randomization list, the one who generated it and the one who prepared Cu stock solutions. Because of difficulties in masking Cu taste in water, some individuals were expected to taste the Cu at higher concentrations; participants were carefully instructed not to share their perceptions with field workers or other persons. During a 2-month pilot phase, families received placebo water (< 0.01 mg Cu/L), to validate procedures and forms. Data obtained during this period provided basal information for sample size calculation. The Committee of Ethics for Research in Humans of the Institute of Nutrition and Food Technology (INTA), University of Chile, approved this protocol. An International Technical Advisory Committee also reviewed ethical aspects of the protocol. All volunteers received written and oral information about the protocol and were free to refuse continuing in the study at any time.

Exposure assessment:

measured

Details on exposure:

Test waters:
Twice per week, a box containing eight 80-mL screw-cap bottles of similar external appearance, coded by color, was delivered to each family; bottles contained a stock solution of Cu sulfate (Merck, Darmstadt, Germany; pro analysis grade) in amounts to reach the concentration 2, 4, or 6 mg Cu/L when diluted to 10 L with tap water. Water for home consumption was prepared by pouring the stock solution into a graduated 20-L container
(provided by the researchers) and filling it to 10 L with tap water. Participants were instructed to agitate the container before drinking the water, drink the water when they were at home, and not share 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.
The actual Cu content in stock solutions was measured daily, whereas in home-prepared water this was performed once per week by means of unexpected visits to the households, either early in the morning, at mid-day, or in the evening. Cu was measured by atomic absorption spectrophotometry (model 2280; Perkin Elmer, Norwalk, CT, USA). 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. Quality of tap water was tested at the INTA once, following U.S. Environmental Protection Agency protocol (Troncoso et al. 1997).
Because water was prepared and maintained at home, potential bacteriologic contamination was investigated in a subsample of 179 randomly chosen households, once per household. Sampling was distributed along the 2-month survey. We used mesophilic aerobic counts (MAC), total coliform counts (TCC), and fecal coliforms as indicators of bacterial contamination. These parameters were determined by means of routine procedures (Downes et al. 2001; Eaton et al. 1995).

Water consumption:
Recording of consumption of fluids indicated the number and size of glasses, cups, and soup bowls, including the approximate amount left over;
300-mL mugs were given to participants, and soup bowls and cups used were measured at the beginning of the survey.

Results and discussion

Results:

Comparability of enrolled and censused families:
There were no significant differences in sociodemographic indicators measured between families who participated and those who did not. All enrolled families and subjects (n = 1,365) provided data throughout the study period. No individuals were withdrawn because of violations to the protocol.

Comparability of intervention groups:
Randomization resulted in similar groups for the analyzed variables. Baseline demographic characteristics and behaviors that would modify patterns of drinking water were similarly distributed in the four groups.
.
Bacteriologic study:
Water was clean by EMOS contemporary data for the study area. Fecal coliforms were not detected; 22 of 179 (12.3%) home water samples had either MAC or TCC positive, and in 5 of 179 (2.8%) both indicators were positive (2, 2, and 1 families in 0, 2, and 6 mg Cu/L groups, respectively). Because bacteriologic 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 bacteriologic sampling. No association was found between the proportion of individuals that reported symptoms or the total number of symptoms reported and at least one positive parameter in the bacteriologic study. In families in whom both parameters were positive, TCC was significantly associated with total symptom report (p = 0.0012, Fisher’s test).

Adherence to the assigned group:
Mean Cu concentration measured in test waters prepared at home, fluid intake, and mean daily dose of Cu received by the study subjects were reported. Individual daily water consumption had significant day-to-day variability (ranging from 0 to 6 L/day, in one person), but intergroup 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). On one occasion, weekly measurement of Cu concentration in homeprepared waters showed two families whose water Cu concentrations were different from the expected values: one had 2 mg Cu/L instead of < 0.01 mg Cu/L, and the other had 2 mg Cu/L 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 Cu in their drinking water. The Cu concentration measured in test waters at home was > 6 mg Cu/L (range, 7–13 mg/L) in 8,043 (out of 85,996) occasions, affecting 417 of 1,365 participants.

Health survey:
The number of families enrolled were fewer than the estimated sample size; however, the greater frequency of symptoms reported 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 individual may report more than one symptom, and symptoms more than once. Characteristics of the individuals, Cu concentration in test waters, and Cu and water consumption are shown in Table 1, whereas the proportion of responding individuals appear in Table 2. Of the 1,365 individuals, 222 reported at least one symptom in 665 occasions, providing a total of 794 symptoms; 16.3% of surveyed individuals were 'responders' that is, they 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%, ƒÔ2 = 70.84, p = 0.0000). Only 2.6% of responses were obtained from individuals . 60 years of age; although there were no significant differences by age interval, comparison of responses obtained in the first and third terciles for age gave an odds ratio of 0.41. Individuals who ingested < 500 mL test waters/day represented < 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, whereas there were no differences between 4 and 6 mg Cu/L . Abdominal pain and nausea were the most frequently reported symptoms. Symptoms reported yielded eight 'combinations' throughout the survey (individuals tended to repeat their pattern of report, with 182 of 222 responders reporting the same 'combination' of symptoms throughout the survey). Symptom report significantly decreased during the 9 weeks (ƒÔ2 = 486.909, p = 0.0000), yielding an odds ratio for symptom report of 0.06 and 0.09 in the last 2 weeks of study, respectively. This did not coincide with decreasing fluid consumption over time. Reports of total 'unrelated symptoms' (used to distract the participants) were similar in the four study groups (ƒÔ2 = 2.32, p = 0.5083).
Counting-process analysis using the stepwise method yielded a model that included sex (p < 0.0001) and Cu concentration received on the day of the event (p < 0.0001). The covariates of age, volume of fluid intake, and water consumed as total, plain, and mixed did not have a significant effect on the model. Cumulative hazard curves for increasing Cu concentrations in test water by sex and stratified by weeks showed a progressive decrease of risk over time (Figure 2). Counting-process analysis confirmed results of the preliminary analysis, which showed an increased risk of symptoms associated with increasing Cu concentration and with female sex. Analysis of risk differences between sexes using the 95% CI of the cumulative hazard ratio showed that the risk remains significantly higher from week 1 to week 4 for all Cu concentrations (except basal 0). Symptoms against exposure increased with Cu concentration in test water (compared with no exposure); on week 1 the RR became significant at 4 mg/L in women (RR = 1.53; 95% CI, 1.02–2.05)] and at 6 mg/L in men (RR = 1.9; 95% CI, 1.02–2.79). With advancing time, the significance shifted to higher Cu exposure, such that in week 2 for men and week 4 for women, the Cu concentration required to obtain significant differences on symptom report was > 6 mg Cu/L. Mistakes made while preparing test waters at home created the opportunity to evaluate the effect of Cu exposure > 6 mg/L (actual range, 6–13 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 Cu concentration in test water.

Applicant's summary and conclusion

Conclusions:

This study assessed for the first time the effect of controlled Cu 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 2-month interval.
In the present study, two different approaches for statistical analysis, changing Cu concentration in drinking water from 0 to 4 mg/L, resulted in a significant increment of report of gastrointestinal symptoms. Counting-process analysis represented a significant improvement in the analysis because it treated all participants as present on every occasion that they registered data; this represented a major concern because participating families did not follow instructions strictly, leaving days without registering information when they were out of the home for 2–3 days. A main limitation of this study is the lack of control of the exact timing of exposure and appearance of symptoms.
The main objective was to assess to what extent this dose–response curve is applicable when individuals are exposed to Cu in a more realistic fashion. Only Cu concentration and sex were chosen for the model, whereas total volume, daily Cu 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 Cu concentration and volume are the main determinants of response, and dose and vehicle for Cu ingestion are less important. Nausea has proved to be a good marker of early response to acute Cu 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 Cu exposure may be part of the explanation, but this cannot be established by the present study. Predominance of women in the reporting group is another relevant finding of the study; this difference was suspected in previous studies and was obtained in the present study by using both chi-square analysis and the counting process. Acute response to Cu exposure is triggered very specifically in the stomach through mechanisms that result in vagal stimulation. Sex differences for vagal phenomena have not been 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 being responsible for the different response to alcohol of women compared to men (Frezza et al. 1990). Whether other sex differences occur and how they may relate to response to Cu remain to be clarified. Symptom reporting clearly decreased over time both in men and women. Although misreporting of symptoms due to decreasing motivation cannot be ruled out, no proof was found of this using several controls.
Therefore, results were interpretating as suggesting an adaptive response to repeated Cu exposure. In this study the symptoms, range of responses, and relevant variables associated with repeated acute Cu exposure in human adults were established. Even considering the findings in women (who appeared to be more sensitive to Cu exposure) and results obtained during the first 2 weeks of exposure (which yielded the highest incidence of effects), the current provisional guideline for drinking water (2 mg Cu/L) set by the WHO (WHO 1993, 2003) is safe; these data represent relevant information for regulators who must decide on the relationship of Cu exposure and the safety of drinking water for human health.

Executive summary:

Gastrointestinal effects were assessed in 1,365 adults exposed to either < 0.01 (controls), 2, 4, or 6 mg copper/L of drinking water for 2 months in a randomized, double-blind community-based study. The risk of symptoms increased with increasing Cu exposure and decreased with time.

The best model by counting-process analysis included Cu concentration and sex. The risk of symptoms remained significantly higher in women than in men during weeks 1–4 for all concentrations tested; at week 1 comparison with the < 0.01-mg/L group showed that differences became significant in women at 4 mg/L [relative risk (RR) = 1.53; 95% confidence interval (CI), 1.02–2.05),

and in men at 6 mg/L (RR = 1.9; 95% CI, 1.02–2.79). At week 2 for men and week 4 in women, the Cu concentration required to obtain significant differences on symptom report was > 6 mg Cu/L.

Conclusion is that exposure to Cu in drinking water results in gastrointestinal symptoms, which are modulated by Cu concentration, time, and sex.