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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:
Determination of an acute no-observed-adverse-effect level (NOAEL) for copper in water
Author:
Araya M., McGoldrick M. C., Klevay L.M., Strain J.J., Robson P., Nielsen F., Olivares M., Pizarro F., Johnson L., and Poirier, K.
Year:
2001
Bibliographic source:
Regulatory Toxicology and Pharmacology 34, 137-145 (2001)

Materials and methods

Study type:
study with volunteers
Endpoint addressed:
acute toxicity: oral
Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: Guidelines to conduct such studies published by the US Department of Health and Human Services, Food and Drug Administration (1997)
Deviations:
not specified
Principles of method if other than guideline:
The study protocols, questionnaires, data sheets to record the results and operational definitions of the outcome variables were standardised over
the 3 test sites in large part, on the guidelines to conduct such studies published by the US Department of Health and Human Services, Food and Drug Administration (1997).
The World Health Organisation (WHO, 1993) has identified nausea as the main symptom to evaluate acute effects associated with ingestion of copper containing waters. Because the prevalence of nausea in the general population is not known, the sample size was calculated on the basis of
preliminary data obtained in Santiago, Chile; that revealed a basal prevalence of gastrointestinal (GI) symptoms of 5% (unpublished) to detect
significant differences for a ≥15% change in GI morbidity rate (Zacarias et al., 2001). Using an α error of 0.05 and a β error of 0.2 (power = 80%) the
number of subjects for each testing dose group was calculated as 47. As previous work demonstrated a dropout rate of 20%, the group number was
set as 60, resulting in a targeted total of 180 recruited subjects for the 3 sites.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
CuSO4.5H2O: A single, identical lot of copper sulphate pentahydrate (USP) pro analysis grade (98.5-100.5%, dry basis) was obtained (Fisher Scientific, Springerfield, NJ, Lot No. 974491) and distributed among these 3 sites.

Method

Type of population:
general
Subjects:
3 sites were selected to carry out this protocol: the Institute of Nutrition and Food Technology, University of Chile, in Santiago, Chile [Santiago]; the
Grand Forks Human Nutrition Research Center, North Dakota, United States [Grand Forks]; and the Northern Ireland Centre for Diet and Health,
University of Ulster, Coleraine, Northern Ireland [Coleraine].
At each site; Coleraine, Santiago and Grand Forks; a group of 60 adults were recruited by local advertisements.
Each individual completed a lifestyle questionnaire so that subjects who were heavy drinkers, smokers, taking prescription drugs or were pregnant
were excluded from the study. The sample was then stratified to include approximately 50% of each sex with 50% either side of 40.
All subjects received monetary compensation at the completion of study.
Ethical approval:
other: Ethical approval for testing of human subjects was provided by each site’s Institutional Review Board or Ethics Committee. All subjects gave their informed consent before inclusion into the study.
Route of exposure:
oral
Reason of exposure:
intentional
Exposure assessment:
measured
Details on exposure:
Distilled deionised water was prepared daily at each study site. Concentrations of the test solutions (0, 2, 4, 6 and 8 mg/l of copper as CuSO4) were
prepared daily and confirmed by atomic absorption spectrophotometry at each site. The administered bolus amount of water was 200 ml. Providing a target dose of 0, 0.4, 0.8, 1.2 and 1.6 mg of elemental copper per trial, respectively. Chemical analysis confirmed that the actual prepared copper
solutions were ±3% of the target concentration for the duration of the study for the 4 copper concentrations at the 3 sites.

The copper containing solutions were given unmasked even though copper in drinking water can have a distinct metallic taste to some individuals.
This was following preliminary work which showed that taste masking was highly variable between and within the sites tested. Also the addition of
another substance would introduce an additional testing variable.

Subjects fasted over night and came to the test facility one morning a week for 5 successive weeks. On arrival (time 0), individuals were asked to
complete a written questionnaire containing a list of symptoms and signs (Table 1), indicating whether these were present or absent at that time. The
list of symptoms included the primary outcome variables, nausea, abdominal pain, vomiting and diarrhoea as well as several distractor symptoms;
backache, sweating, palpitations, heartburn, headache, feeling of anxiety, dizziness and salvation. When it was confirmed that the subject was
asymptomatic he or she was given one 200 ml test solution of 0, 2, 4, 6 or 8 mg Cu/l; assigned to each subject in a random order; and blinded to the experimental subject and laboratory supervisor. After 15 mins subjects completed a questionnaire again, and remained on site for one hour in a
lounge with magazines and TV.

At the end of that hour of direct observation the individuals completed the same questionnaire before leaving the study facilities. The following
morning subjects were contacted and the same questionnaire completed at 24 h post dosing. At each evaluation time, instructions given and
language used during the contact with the subjects was carefully planned and reviewed prior to the beginning of data collection in order to make them comparable at each of the 3 test sites.
Examinations:
GI symptoms collected via questionnaire.
Medical treatment:
None.

Results and discussion

Clinical signs:
Nausea, abdominal pains, vomiting and diarrhoea
Results of examinations:
Other: Nausea was most frequently reported within the first 15 min after ingestion, and was of a transient nature. Table 2 lists the results for each GI
outcome reported by location and sex. Distractor symptoms were infrequently reported and unrelated to copper dose; this not included in the
summary analysis.

The distribution of outcomes combined across sites and reported at each concentration tested is shown in Table 3.
Effectivity of medical treatment:
Not applicable
Outcome of incidence:
See any other information on results.

Any other information on results incl. tables

A total of 179 individuals completed the study (= 60,= 61and Coleraine = 58). Nausea was the earliest and most prevalent symptom observed (16.7, 36.1 and 29.3% of the subjects at the 3 sites respectively reported at least one occurrence of nausea) with an average prevalence of nausea among all subjects of 27.3%.

 

Of the 49 subjects who reported nausea, 29 reported it at only one concentration, 17 at 2 concentrations of copper and 2 reported nausea at 3 concentrations tested. Only 1 subject reported nausea after all 4 concentrations of copper and did not report a positive response at 0 mg Cu/l. Three individuals reported nausea after consuming water containing no copper. One of these 3 individuals reported no further incidence of nausea; the remaining 2 reported nausea after consuming 8 mg Cu/l.

 

Vomiting only occurred in only one female at thesite and was not considered to be treatment related. Five subjects (2.8%; 2 women and 3 men) developed diarrhoea at 0, 2, 4, 8 and 8 mg Cu/l respectively. Diarrhoea occurred between 1 and 24 h after administration of the copper. Whilst nausea and vomiting papered clearly related, diarrhoea was not associated with the other 2 symptoms (only one individual with diarrhoea presented with nausea, which occurred after consuming 8 mg Cu/l).

 

The final reduced models obtained from the repeated measure logistic regression analysis for nausea and GI symptoms included gender, test site and copper dose as significant outcome predictors (see Table 4). Order of dose administration and age did not significantly affect the experiment outcomes (P>0.05). Subjects tested inreported significantly fewer occurrences of nausea and GI symptoms than did subjects in, while subjects in Coleraine were similar to those in. Copper concentration was the strongest predictor of both nausea and GI symptoms, with incidence increasing (significantly P <0.0001) as the administered dose increased. Incidence of abdominal pain and diarrhoea were not related to copper concentration (P>0.05).

 

The predicted frequencies of occurrences of nausea and GI symptoms at the concentrations tested are given for men, women and all subjects in Table 5. Despite there being a gender specific response effect, the purpose of the study was to define the minimum dose effect across all subjects regardless of gender, age and cultural background. A 2 stage polynomial regression model was used to generate a single dose-response curve for nausea (Figure 1) and GI symptoms. The minimum LOAEL for nausea and GI symptoms across all subjects was 6 mg Cu/l and the NOAEL was 4 mg Cu/l for acute exposure to copper. These data were determined from the logistic regression analysis presented in Table 4.

 

Factors that may have affected the study were site, sex and age. However, data in Tables 4 and 5 show no clear statistical significance in the total responses for these factors.

Calculation of the threshold of the appearance of GI symptoms from the pooled data set of the 3 sites showed that for female adults this value was 8 mg Cu/l.

Applicant's summary and conclusion

Conclusions:
As shown in Figure 1; there is an increase in the slope of the curve for nausea between 4 and 8 mg Cu/l suggesting a threshold level. Upper
confidence levels showed that the first 3 to 5% of the population would respond at 2.5-3 and 3.5-4 mg Cu/l respectively; which supports the
provisional guideline value of 2 mg Cu/l set by WHO (1993) as a safe value. The determination of an upper safe limit and gastric tolerance observed inthis study will also help to define copper doses in supplements as well.
Executive summary:

Background:

Copper is both an essential element and toxic at high doses. A principal consequence of high copper overload in the gut is the appearance of nausea, cramps, emesis and diarrhoea. The purpose of this study was to determine the threshold for acute gastrointestinal effects associated with drinking water containing random sequence of copper concentrations (as CuSO4.5H2O), in an experimentally controlled situation.

 

Materials and Methods:

A prospective double blind control study was designed to determine the acute NOAEL of nausea in an apparently healthy population of 179 individuals who drank copper containing water as the sulphate salt. Subjects were recruited at 3 different international sites and given a blind randomly selected dose; 0, 2, 4, 6 or 8 mg Cu/l in a bolus of 200 ml (final total copper dose was equivalent to 0, 0.4, 0.8, 1.2 and 1.6 mg) once weekly over a consecutive 5-week period. Gastrointestinal (GI) symptoms of nausea, abdominal pains, vomiting or diarrhoea were screened for a period of up to 24 hr.

 

Results and Discussion: Nausea was the most frequently reported effect and was reported within the first within the first 15 mins of ingestion. For the combined tri-site population (n=179), 8, 9, 14, 25 and 44 subjects responded positively to one or more GI symptoms at 0, 2, 4, 6 and 8 mg Cu/l, respectively. Analysis of the data clearly demonstrated a clear dose response to the combined positive GI effects and to the nausea alone. Statistically significant greater reporting of effects occurred at 6 and 8 mg Cu/l. Therefore, an acute NOAEL and lowest-observed-adverse-effect level of 4 and 6 mg Cu/l (0.8 and 1.2 mg Cu), respectively, were determined in drinking water for a combined international human population.