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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:
Confirmation of an acute no-observed-adverse-effect and low-observed-adverse-effect level for copper in bottled drinking water in a multisite international study.
Author:
Araya M., Chen B., Klevay L.M., Strain J.J., Johnson L-A., Robson P., Shi W., Nielsen F., Zhu H., Olivares M., Pizarro F., and Haber, L.T.
Year:
2003
Bibliographic source:
Regulatory Toxicology and Pharmacology 38 (2003) 389-399.

Materials and methods

Study type:
study with volunteers
Endpoint addressed:
acute toxicity: oral
Test guideline
Qualifier:
according to
Guideline:
other: US Department of Health and Human Services, Food and Drug Administration (1997).
Deviations:
no
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.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
CuSO4.5H2O
Distilled deionised water was prepared daily at each study site. 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:
Four 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]; the Northern Ireland Centre for Food and Health
(NICHE), University of Ulster, Coleraine, Northern Ireland [Coleraine] and the Department of Environmental Health, Fudan University (formerly
Shanghai Medical University), Shanghai, China [Shanghai].
Previous work had indicated a biological trend for female subjects to have a tendency to be more sensitive in reporting GI (gastrointestinal) symptoms, primarily nausea. Thus in order to define a more sensitive NOAEL, the investigators chose to recruit female only volunteers with 70 subjects
recruited at each site (total 280 subjects).
At each site; Coleraine, Santiago, Grand Forks and Shanghai; a group of 70 female adults (18 – 60 yrs old) 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% either side of 40 yrs old.

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:
The core study design was a 3x3 two-way factorial design (volume x dose) with doses of 0.4, 0.8 and 1.2 mg Cu in volumes of 100, 150 and 200 ml
bottled drinking water. An additional 2 doses (0 and 1.6 mg Cu) were added at the 200 ml volume in an attempt to corroborate the results from the
previous study (Araya et al,. 2001). The final concentrations of copper sulphate utilised in the study are presented in Table 1.

A single lot of bottled spring water was obtained from a water bottler (Naya, Quebec, Canada) and distributed in sufficient quantity to each of the 4
sites. Analysis of the spring water did not reveal any remarkable contamination or unusual physical factors. Cu contamination was not found at a
detection limit of 0.002 mg/l.

A daily stock of 80 mg Cu/l was prepared at each site and its concentration confirmed by analysis (AAS). The stock solution was then serially diluted
to the daily administered bolus of copper in bottled drinking water and again the concentration was confirmed by analysis. Chemical analysis
confirmed that the actual prepared copper solutions were ±3% of the target concentration for the duration of the study at all the 4 sites. Analysis of
the used cups from Grand Fork and Shanghai showed that between 0.6 and 0.4 % of the dose had been retained by the drinking cups respectively.

The protocol followed was very similar to Araya et al., (2001) with the subjects fasting overnight prior to attending each facility on one morning for 11 successive weeks until all 11 administrations were completed. Each subject was to complete ingestion of the bolus within 3 hours of rising. Additional dietary restrictions included no alcohol on the evening prior to the test and no medicines or dietary supplements on the morning of the test.
Immediately prior to then 15 and 60 mins after consumption of the test bolus (within 2 mins) each subject completed a written questionnaire on
symptoms. These included; nausea, abdominal pains, vomiting and diarrhoea were the primary outcome variables as well as several symptoms not
thought to be initiated by copper ingestion given to distract the subject (included; backache, sweating, palpitations, heartburn, headache, feeling of
anxiety, dizziness and salivation).
At time 0, once the subject was defined as asymptomatic, she was given one of the 11 test solutions assigned to each subject in random order and
blinded to both the subject and experimental supervisor. The solution was consumed as quickly as possible, typically within 2 minutes. After 15 mins, asymptomatic subjects were taken to a lounge and provided with magazines where they stayed for a further 45 mins.

Instructions were given to all subjects in an ordered, planned and consistent manner across the sites.
Examinations:
Other: GI symptoms collected via questionnaire.
Medical treatment:
Not applicable.

Results and discussion

Clinical signs:
Nausea, abdominal pains, vomiting and diarrhoea.
Results of examinations:
Other:
In order to corroborate the nausea results from the earlier study (Araya et al,. 2001) the results from the 200 ml volumes were considered first. A
200 ml volume us also a portion size frequently ingested when fluids are consumed. Consistent with the study by Araya et al,. (2001) nausea was
most frequently reported within the first 15 min after ingestion, and the prevalence of the symptom decreased with time after the dose.
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 269 individuals completed the study (Santiago = 70, Grand Forks = 68, Coleraine = 58, Shanghai = 73). The screened respondents were healthy before and throughout the study.

 

Nausea was the earliest and most prevalent symptom observed within 15 mins of the dose (24.3, 41.1, 25.9 and 50.0% of the subjects at the 4 sites; Santiago, Shanghai, Coleraine and Grand Forks respectively reported at least one occurrence of nausea at any dose. The average prevalence of nausea among all subjects of 35.7% at 15 mins. The overall prevalence for subjects reporting nausea decreased from 15.6% at 15 mins to 6.3% at 60 mins in the 6 mg Cu/l (1.2 mg Cu in 200 ml) group (see Table 2). Similarly for those consuming 8 mg Cu/l (1.6 mg Cu in 200 ml), the overall prevalence decreased from 26.0% at 15 mins to 7.8% at 60 mins.

 

The difference in the occurrence of nausea at 15 and 60 mins is also illustrated by the difference in the probability of a positive response at the 2 highest bolus doses using the 200 ml volume, as estimated using the linear regression model. The probability of nausea was elevated over control at both time points, but the incidence rates at 60 mins were considerably lower than at 15 mins (see Table 3). The results obtained for the analysis of all 4 GI symptoms (nausea, abdominal pain, vomiting and diarrhoea) were similar to the analysis for nausea alone. Fig 1. Presents the incidence of nausea at each copper dose at 15 and 60 mins, for the 300 ml volume.

 

Table 2 also presents the total number of subjects reporting nausea (at either 15 or 60 min time points). The total number reporting nausea is close to the number of those reporting nausea at 15 mins. This means that most of the subjects reporting nausea at 60 mins had also reported nausea at 15 mins but some new respondents were reported.

 

Using the statistical significant increased incidence compared with control as the criterion to ascertain an adverse effect level and the 200 ml linear dose-response data at 15 mins for nausea effects at all sites; the generalised linear model analysis data indicated that for this dosing volume the LOAEL occurred at 1.2 mg Cu (6 mg Cu/l; p = 0.0004) and the NOAEL occurred at 0.8 mg Cu (4 mg Cu/l; p = 0.06). These results are comparable with those in Araya et al,. (2001) which was a 3 site, mixed sex study.

 

In addition a benchmark dose (BMD) was calculated for the 200 ml bolus linear dose-response data at 15 min for nausea effects at a benchmark response (BMR) of 0.10. The US EPA BMD software version 1.3 was used to evaluate these dichotomous data, using a multistage model with extra risk. The goodness of fit p-value was p = 0.94 with a BMD of 0.94 mg copper (4.8 mg Cu/l). The corresponding 95% lower confidence limit on the BMD (BMDL) was 0.84 mg copper (4.2 mg Cu/l).

 

As found with the 200 ml bolus, nausea was also the earliest and most prevalent symptom recorded following exposure to copper in a 150 and 100 ml bolus solution. Estimates of NOAEL/LOAEL volumes were made for comparison purposes as the study was not designed for this purpose having no control groups. The NOAEL/LOAEL for these volumes was estimated by comparison of the responses to those seen in the 200 ml control group. For 150 ml volume; the LOAEL occurred at a dose of 1.2 mg Cu (8 mg Cu/l) and a NOAEL of 0.8 mg Cu (5.3 mg/l). For the 100 ml volume; the LOAEL occurred at a dose of 0.8 mg Cu and a NOAEL of 0.4 mg Cu corresponding to concentrations of 8 and 4 mg Cu/l. These data are only approximations but show a consistent picture with the NOAEL consistently in the range of 4-5 mg Cu/l over a 2-fold range of total dose (100 – 200 ml doses).

 

Table 5 summarises the probability outcome of the 3 x 3 dosing for nausea at both 15 and 60 min by test site, water volume, copper dose and water volume x copper interaction. The 3 x 3 factorial ANOVA demonstrated that at 15 mins there was a significant effect of water volume (p = 0.032) and copper dose (p = 0.0001). As copper dose increases or as water volume decreases (thereby increasing the concentration) there was an increased probability of a female experiencing nausea (Figure 2). Likewise the highest incidence of symptoms was reported within the first 15 min. As with the linear 200 ml dose-response analysis, inclusion of all GI symptoms did not appreciably change the response incidence from nausea alone.

 

The interaction of volume by dose was not statistically significant (p = 0.97). This means that the effects of volume and dose were additive i.e. volume and dose acted independently, not synergistically or antagonistically. The probability of a positive response increased significantly as the copper dose increased, regardless of water volume. Similarly, the probability of a positive response decreased significantly as the water volume increased, regardless of copper dose. The lack of interaction of these terms is not informative regarding whether concentration is an appropriate explanatory variable. This study was designed to investigate dose-response and not concentration-response; so formal analyses based on concentration are not possible. However, as shown in Figure 3, there is a visually apparent concentration-response, overlaid on top of the dose-response. Nonetheless, based on the results of Figs 2 and 3, neither dose nor concentration fully predicts the response; both units of exposure are relevant in determining the response.

Applicant's summary and conclusion

Conclusions:
The results of this study and information generated in recent years in controlled trials conducted in asymptomatic adult populations permit better
understanding of the early effects induced by acute copper exposure. The LOAEL occurred at 1.2 mg Cu (6 mg Cu/l; p = 0.0004) and the NOAEL
occurred at 0.8 mg Cu (4 mg Cu/l; p = 0.06). These results are comparable with those in Araya et al,. (2001) which was a 3 site, mixed sex study.
Executive summary:

Materials and Methods:

In a double blind, 3x3 factorial (volume x dose) study, 70 adult females (18-60 yrs of age) at 4 different international sites (total pooledn= 269) were given 100, 150 or 200 ml of bottled drinking water with 0.4, 0.8 or 1.2 mg of Cu as the sulphate salt once a week. Two additional doses (0 and 1.6 mg Cu) were added at the 200 ml to determine a dose-response relationship and corroborate previously reported results. All subjects completed a questionnaire ay 0, 0.25 and 1 h post-dosing that screened for positive GI effects (nausea, abdominal pain, vomiting and diarrhoea).

Results and Discussion:

Nausea was the most prevalent symptom reported and was generally reported within the first 15 min (water volume, p<0.032; copper dose p<0.0001; and water volume x copper interaction p<0.97). As the volume was increased, the effects of Cu-induced nausea decreased; as Cu dose increased, the incidence of nausea increased. At 200 ml, a significant increase in reported incidence of nausea at 0.25 h occurred at 1.2 mg Cu (6 mg Cu/l), indicating a NOAEL of 0.8 mg Cu (4 mg Cu/l) for adult females. These data confirm previously determined human acute NOAEL study for Cu added to distilled water, and provide additional, controlled human data for determining safe concentrations of Cu in drinking water.