Re‐evaluation of the existing health‐based guidance values for copper and exposure assessment from all sources

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. 2023 Jan 17;21(1):e07728. doi: 10.2903/j.efsa.2023.7728

Reference	Study design and population	Duration of study	Form of copper doses	Safety‐related parameters investigated	Potential markers of Cu status	Liver enzymes	Markers of oxidative damage	Other adverse effects
(Olivares et al., 1998 )	Double‐blinded randomised placebo‐controlled trial 128 healthy infants	From 3 to 12 months of age	G1 (N = 48): < 0.1 mg/L water (27 FF; 21 BF) G2 (N = 80): 2 mg/L water (56 FF; 24 BF) FF infants in G1: 0.8 ± 0.5 mg/day Cu at months 4 to 6; 1.2 ± 0.7 mg/day Cu at months 6 to 9; 1.2 ± 0.7 mg/day Cu at months 9 to 12. FF infants in G2: 2.3 ± 0.8 mg/day Cu at months 4 to 6; 2.5 ± 0.7 mg/day Cu at months 6 to 9; 2.4 ± 0.7 mg/day Cu at months 9 to 12. Formula was fortified with Fe, Cu (7.87 μmol/L) and Zn	Serum Cu, CP eSOD, eMT Bilirubin; AST, GGT, SGPT (= ALT) at 6, 9 and 12 months of age Monthly examination for clinical and anthropometric evaluations	Serum Cu, CP: no effect eSOD, eMT: no effect	Bilirubin; AST, GGT, SGPT (= ALT): no effect	–	Growth and morbidity: no effect between groups
(Harvey et al., 2003 )	Metabolic study 12 M (20–59 years)	P1: 8 weeks P2: 8 weeks P3: 8 weeks ≥ 4 weeks washout between periods	P1: 1.6 mg/day total Cu P2: 0.7 mg/day total Cu P3: 6.0 mg/day total Cu During all periods, diet contained 0.7 mg/day Cu; additional Cu provided as CuSO₄ (liquid formula)	Plasma Cu, CP and eSOD Platelet aggregation, packed cell volume, Hb, mean cell Hb concentration Plasma lipoproteins and triacylglycerols, Apo A1 and B Plasma, erythrocyte and platelet GPx Plasma ferritin	Plasma Cu, CP, eSOD: no effect	–	Plasma, erythrocyte, platelet GPx: no effect	Platelet aggregation, plasma lipoproteins (total, HDL‐, LDL‐cholesterol), triacylglycerols, apo A1 and B: no effect Hb, mean cell haemoglobin concentration and packed cell volume: no effect
(Baker et al., 1999 ) (FOODCUE England)	Longitudinal intervention trial 11 healthy M (20–59 years)	8 weeks/period 4 weeks washout between periods	P1: 1.6 mg/day total Cu P2: 0.7 mg/day total Cu P3: 6 mg/day total Cu Controlled diet contained 0.7 mg/day; additional Cu provided as CuSO₄ (solution)	Serum Cu, CP Serum osteocalcin (bone formation) Urinary pyridinoline and deoxypyridinoline (bone resorption) Urinary creatinine Blood count (WBC, RBC, Hb, HTC, platelet) Clinical chemistry (Na, K, bicarbonate, urea, creatinine, total bilirubin, total protein, albumin, globulin, LDH, AST, ALT, ALP, GGT, Ca, P, total cholesterol, glucose)	Serum Cu, CP: no effect	NR	–	Biochemical markers of bone metabolism: no effect Urinary creatinine: no effect
(O'Connor et al., 2003 ) (FOODCUE Northern Ireland)	Double‐blind ed crossover trials 24 healthy M and F (22 completed, 11 M and 11 F) (22–45 years)	6 weeks/period	P1: 3 mg/day as CuSO₄ P2: placebo P3: 3 mg/day as CuGC P4: placebo P5: 6 mg/day as CuGC P6: placebo In addition to background diet; diet contained 1.43 ± 0.1 mg/day Cu in M and 1.03 ± 0.1 mg/day in F	eSOD, WBC SOD Platelet and WBC Cyt c oxidase activities Plasma CP Serum DO Liver enzymes (GGT, ALT) DNA damage (Comet assay, with and without endonuclease)	CP, eSOD: no effect Serum DO: ↑ following all three suppl. periods WBC Cyt c oxidase: ↑ following suppl. with 6 mg CuGC/day (data not shown)	GGT, ALT: no effect	DNA damage: no effect (samples lost for 4 to 6 subjects in each period)	–
(Rock et al., 2000 ) (FOODCUE France)	Double‐blinded crossover trials 13 healthy M and 13 healthy F (50–72 years)	6 weeks/period 6 weeks washout between periods	P1: 3 mg/day as CuSO₄ P2: placebo P3: 3 mg/day as CuGC P4: placebo P5: 6 mg/day as CuGC P6: placebo In addition to background diet; mean Cu intake was ~1.4 mg/day Cu in M and ~1.2 mg/day Cu in F	RBC resistance to oxidation (AAPH‐induced oxidation in vitro test) Plasma fat‐soluble vitamins eSOD	eSOD: no effect	–	RBC oxidation: ↑ half‐time (LT50) haemolysis after 3 mg/day CuSO₄ and 6 mg/day CuG Plasma tocopherols, retinol, lutein: no effect Plasma lycopene and plasma α and β carotene: ↓ after 3 mg/day CuGC	–
(Turley et al., 2000 ) (FOODCUE)	Double‐blinded crossover trials (4 centres, diff. doses) 24 healthy M and F (22–45 years) [Northern Ireland]	6 weeks/period	Up to 3 mg/day as CuSO₄ and up to 6 mg/day as CuGC, in addition to background diet Background diet contained 1.43 mg/day Cu in M and ~1.03 mg/day in F	Serum/plasma Cu (Denmark, France) Plasma CP (all centres) Susceptibility of LDL to in vitro induced oxidation (all centres)	Serum/plasma Cu: ↑ after 3 mg/day CuSO₄ and 6 mg/day CuSO₄ (Denmark); no effect (France) Plasma CP: no effect observed in any centre	–	LDL susceptibility to Cu‐ or peroxynitrite‐induced oxidation: no effect in any centre	–
Same study 11 healthy M (20–59 years) [England]	8 weeks/period	Up to 6 mg/day total Cu controlled diet contained 0.7 mg/day; additional Cu provided as CuSO₄ (solution)
Same study 16 healthy F (18–30 years) [Denmark]	4 weeks/period	Up to 6 mg/day as CuSO₄ In addition to background diet (Cu content ND)
Same study 14 M and 14 F (50–72 years) [France]	6 weeks/period	Up to 6 mg/day as CuGC In addition to background diet; mean Cu intake was ~1.4 mg/day Cu in M and ~1.2 mg/day Cu in F (Rock et al., 2000)
(Turnlund et al., 2004 )	Metabolic study 9 M (26–49 years)	P1: 18 days P2: 129 days P3: 18 days	P1: 1.6 mg/day total Cu P2: usual diet + 7 mg/day Cu supplement P3: 7.8 mg/day total Cu During periods 1 & 3, diet contained 1.6 mg/day Cu; additional Cu provided as CuSO₄ (liquid formula) During P2, subjects consumed supplements that provided 7 mg Cu/day as CuSO₄	Plasma Cu, CP and eSOD, plasma benzylamine oxidase Plasma MDA, urinary TBARS Plasma ascorbate and dehydroascorbate after 1 g vitamin C dose given on day 14 of each period Complete and differential blood cell count Serum IL‐2R, IL‐6, IgG and C3 Delayed hypersensitivity skin response test (battery of seven recall antigens) All study subjects, along with a control group of 10 subjects, were immunised with a trivalent influenza vaccine 2 weeks before the end of the high copper intake period and antibody titres were measured 14 days after immunisation	eSOD: ↑ P3 vs P1 Plasma benzylamine oxidase activity: ↑ P3 vs. P1 CP activity: ↑ P3 vs. P1 Urinary Cu: ↑ P3 vs. P1 Hair copper ↑ P3 vs P1 Plasma Cu: no effect	–	Plasma MDA: no effect Urinary TBARS: ↑ P3 vs. P1 Plasma ascorbate and dehydroascorbate: no effect	Polymorphonuclear cells: ↓ P3 vs. P1 (total count, % WBC) Lymphocytes: ↑ P3 vs. P1 (total count) IL‐2R: ↓ P3 vs. P1 Serum influenza antibody titres: ↓ experimental vs control subjects WBC count, IL‐6, IgG and C3: no effect Delayed hypersensitivity skin response test: no effect
(Pizarro et al., 1999 )	Randomised controlled crossover trial (Latin‐square design) 60 healthy adult women	2 weeks/period 1 week washout with tap water between periods	Mean ± SD Cu intake from water: P1: 0.04 ± 0.02 (0 mg/L water) P2: 1.74 ± 0.66 (1 mg/L water) P3: 4.68 ± 2.24 (3 mg/L water) P4: 7.94 ± 2.69 mg/day (5 mg/L water) As CuSO₄ in water in addition to background diet; average background intake of Cu: 1.7 mg/day	GI symptoms Serum Cu, CP and GGT, ALT, AST Hb	Serum Cu, CP: no effect	GGT, ALT, AST: no effect	–	Hb: no effect Nausea, vomiting, abdominal pain: ↑ for Cu ≥ 3 mg/L Headache, salivation: non‐significant ↑
(Kessler et al., 2008a )	Prospective, double‐blinded phase 2 clinical trial 68 patients with mild Alzheimer's disease; n = 33 in G1 (age 69.5 ± 1.4); n = 35 in G2 (age 70.4 ± 1.1)	12 months	G1: placebo G2: 8 mg/day as Cu‐(II)‐orotate‐dihydrate In addition to background diet (Cu content ND)	Plasma Cu, Zn, CP, liver enzymes. Aβ42, Tau and Phospho‐Tau in CSF Cognition test Adverse event recorded	Plasma Cu, CP: no effect	Liver enzymes: no effect	–	Haematological parameters: no effect Alzheimer's disease Assessment Scale (Cognitive subscale) or the Mini Mental Status Examination: no diff between groups
(Pratt et al., 1985 )	Double‐blinded placebo‐controlled trial G1: 7 adults (3 M and 4 F) (mean age 42 years) G2: 7 adults	12 weeks	G1: 10 mg/day CuGC G2: Placebo In addition to background diet (Cu content ND)	Serum Cu; hair Cu HTC, MCV and serum chemistry (triglyceride, cholesterol, AST, GGT, LDH, ALP, K, mg, Zn) Side effects reported	Serum Cu: no effect Hair Cu: no effect	AST, GGT, LDH, ALP: no effect	–	HTC, MCV and serum chemistry: no effect Nausea, diarrhoea, heartburn: no diff between groups

Reference

Study design and population

Duration of study

Form of copper doses

Safety‐related parameters investigated

Potential markers of Cu status

Liver enzymes

Markers of oxidative damage

Other adverse effects

(Olivares et al., 1998 )

Double‐blinded randomised placebo‐controlled trial

128 healthy infants

From 3 to 12 months of age

G1 (N = 48): < 0.1 mg/L water (27 FF; 21 BF)

G2 (N = 80): 2 mg/L water (56 FF; 24 BF)

FF infants in G1: 0.8 ± 0.5 mg/day Cu at months 4 to 6; 1.2 ± 0.7 mg/day Cu at months 6 to 9; 1.2 ± 0.7 mg/day Cu at months 9 to 12.

FF infants in G2: 2.3 ± 0.8 mg/day Cu at months 4 to 6; 2.5 ± 0.7 mg/day Cu at months 6 to 9; 2.4 ± 0.7 mg/day Cu at months 9 to 12.

Formula was fortified with Fe, Cu (7.87 μmol/L) and Zn

Serum Cu, CP

eSOD, eMT

Bilirubin; AST, GGT, SGPT (= ALT) at 6, 9 and 12 months of age

Monthly examination for clinical and anthropometric evaluations

Serum Cu, CP: no effect

eSOD, eMT: no effect

Bilirubin; AST, GGT, SGPT (= ALT): no effect

–

Growth and morbidity: no effect between groups

(Harvey et al., 2003 )

Metabolic study

12 M (20–59 years)

P1: 8 weeks

P2: 8 weeks

P3: 8 weeks

≥ 4 weeks washout between periods

P1: 1.6 mg/day total Cu

P2: 0.7 mg/day total Cu

P3: 6.0 mg/day total Cu

During all periods, diet contained 0.7 mg/day Cu; additional Cu provided as CuSO₄ (liquid formula)

Plasma Cu, CP and eSOD

Platelet aggregation, packed cell volume, Hb, mean cell Hb concentration

Plasma lipoproteins and triacylglycerols, Apo A1 and B

Plasma, erythrocyte and platelet GPx

Plasma ferritin

Plasma Cu, CP, eSOD: no effect

–

Plasma, erythrocyte, platelet GPx: no effect

Platelet aggregation, plasma lipoproteins (total, HDL‐, LDL‐cholesterol), triacylglycerols, apo A1 and B: no effect

Hb, mean cell haemoglobin concentration and packed cell volume: no effect

(Baker et al., 1999 )

(FOODCUE England)

Longitudinal intervention trial

11 healthy M (20–59 years)

8 weeks/period

4 weeks washout between periods

P1: 1.6 mg/day total Cu

P2: 0.7 mg/day total Cu

P3: 6 mg/day total Cu

Controlled diet contained 0.7 mg/day; additional Cu provided as CuSO₄ (solution)

Serum Cu, CP

Serum osteocalcin (bone formation)

Urinary pyridinoline and deoxypyridinoline (bone resorption)

Urinary creatinine

Blood count (WBC, RBC, Hb, HTC, platelet)

Clinical chemistry (Na, K, bicarbonate, urea, creatinine, total bilirubin, total protein, albumin, globulin, LDH, AST, ALT, ALP, GGT, Ca, P, total cholesterol, glucose)

Serum Cu, CP: no effect

–

Biochemical markers of bone metabolism: no effect

Urinary creatinine: no effect

(O'Connor et al., 2003 ) (FOODCUE Northern Ireland)

Double‐blind ed crossover trials

24 healthy M and F (22 completed, 11 M and 11 F)

(22–45 years)

6 weeks/period

P1: 3 mg/day as CuSO₄

P2: placebo

P3: 3 mg/day as CuGC

P4: placebo

P5: 6 mg/day as CuGC

P6: placebo

In addition to background diet; diet contained 1.43 ± 0.1 mg/day Cu in M and 1.03 ± 0.1 mg/day in F

eSOD, WBC SOD

Platelet and WBC Cyt c oxidase activities

Plasma CP

Serum DO

Liver enzymes (GGT, ALT)

DNA damage (Comet assay, with and without endonuclease)

CP, eSOD: no effect

Serum DO: ↑ following all three suppl. periods

WBC Cyt c oxidase: ↑ following suppl. with 6 mg CuGC/day (data not shown)

GGT, ALT: no effect

DNA damage: no effect (samples lost for 4 to 6 subjects in each period)

–

(Rock et al., 2000 )

(FOODCUE France)

Double‐blinded crossover trials

13 healthy M and 13 healthy F (50–72 years)

6 weeks/period

6 weeks washout between periods

P1: 3 mg/day as CuSO₄

P2: placebo

P3: 3 mg/day as CuGC

P4: placebo

P5: 6 mg/day as CuGC

P6: placebo

In addition to background diet; mean Cu intake was ~1.4 mg/day Cu in M and ~1.2 mg/day Cu in F

RBC resistance to oxidation (AAPH‐induced oxidation in vitro test)

Plasma fat‐soluble vitamins

eSOD

eSOD: no effect

–

RBC oxidation: ↑ half‐time (LT50) haemolysis after 3 mg/day CuSO₄ and 6 mg/day CuG

Plasma tocopherols, retinol, lutein: no effect

Plasma lycopene and plasma α and β carotene: ↓ after 3 mg/day CuGC

–

(Turley et al., 2000 ) (FOODCUE)

Double‐blinded crossover trials

(4 centres, diff. doses)

24 healthy M and F (22–45 years) [Northern Ireland]

6 weeks/period

Up to 3 mg/day as CuSO₄ and up to 6 mg/day as CuGC, in addition to background diet

Background diet contained 1.43 mg/day Cu in M and ~1.03 mg/day in F

Serum/plasma Cu (Denmark, France)

Plasma CP (all centres)

Susceptibility of LDL to in vitro induced oxidation (all centres)

Serum/plasma Cu: ↑ after 3 mg/day CuSO₄ and 6 mg/day CuSO₄ (Denmark); no effect (France)

Plasma CP: no effect observed in any centre

–

LDL susceptibility to Cu‐ or peroxynitrite‐induced oxidation: no effect in any centre

–

Same study

11 healthy M (20–59 years)

[England]

8 weeks/period

Up to 6 mg/day total Cu controlled diet contained 0.7 mg/day; additional Cu provided as CuSO₄ (solution)

Same study

16 healthy F (18–30 years) [Denmark]

4 weeks/period

Up to 6 mg/day as CuSO₄

In addition to background diet (Cu content ND)

Same study

14 M and 14 F (50–72 years) [France]

6 weeks/period

Up to 6 mg/day as CuGC

In addition to background diet; mean Cu intake was ~1.4 mg/day Cu in M and ~1.2 mg/day Cu in F (Rock et al., 2000)

(Turnlund et al., 2004 )

Metabolic study

9 M

(26–49 years)

P1: 18 days

P2: 129 days

P3: 18 days

P1: 1.6 mg/day total Cu

P2: usual diet + 7 mg/day Cu supplement

P3: 7.8 mg/day total Cu

During periods 1 & 3, diet contained 1.6 mg/day Cu; additional Cu provided as CuSO₄ (liquid formula)

During P2, subjects consumed supplements that provided 7 mg Cu/day as CuSO₄

Plasma Cu, CP and eSOD, plasma benzylamine oxidase

Plasma MDA, urinary TBARS

Plasma ascorbate and dehydroascorbate after 1 g vitamin C dose given on day 14 of each period

Complete and differential blood cell count

Serum IL‐2R, IL‐6, IgG and C3

Delayed hypersensitivity skin response test (battery of seven recall antigens)

All study subjects, along with a control group of 10 subjects, were immunised with a trivalent influenza vaccine 2 weeks before the end of the high copper intake period and antibody titres were measured 14 days after immunisation

eSOD: ↑ P3 vs P1

Plasma benzylamine oxidase activity: ↑ P3 vs. P1

CP activity: ↑ P3 vs. P1

Urinary Cu: ↑ P3 vs. P1

Hair copper ↑ P3 vs P1

Plasma Cu: no effect

–

Plasma MDA: no effect

Urinary TBARS: ↑ P3 vs. P1

Plasma ascorbate and dehydroascorbate: no effect

Polymorphonuclear cells: ↓ P3 vs. P1 (total count, % WBC)

Lymphocytes: ↑ P3 vs. P1 (total count)

IL‐2R: ↓ P3 vs. P1

Serum influenza antibody titres: ↓ experimental vs control subjects

WBC count, IL‐6, IgG and C3: no effect

Delayed hypersensitivity skin response test: no effect

(Pizarro et al., 1999 )

Randomised controlled crossover trial (Latin‐square design)

60 healthy adult women

2 weeks/period

1 week washout with tap water between periods

Mean ± SD Cu intake from water:

P1: 0.04 ± 0.02 (0 mg/L water)

P2: 1.74 ± 0.66 (1 mg/L water)

P3: 4.68 ± 2.24 (3 mg/L water)

P4: 7.94 ± 2.69 mg/day (5 mg/L water)

As CuSO₄ in water in addition to background diet; average background intake of Cu: 1.7 mg/day

GI symptoms

Serum Cu, CP and GGT, ALT, AST

Serum Cu, CP: no effect

GGT, ALT, AST: no effect

–

Hb: no effect

Nausea, vomiting, abdominal pain: ↑ for Cu ≥ 3 mg/L

Headache, salivation: non‐significant ↑

(Kessler et al., 2008a )

Prospective,

double‐blinded phase 2 clinical trial

68 patients with mild Alzheimer's disease; n = 33 in G1 (age 69.5 ± 1.4); n = 35 in G2 (age 70.4 ± 1.1)

12 months

G1: placebo

G2: 8 mg/day as Cu‐(II)‐orotate‐dihydrate

In addition to background diet (Cu content ND)

Plasma Cu, Zn, CP, liver enzymes.

Aβ42, Tau and Phospho‐Tau in CSF

Cognition test

Adverse event recorded

Plasma Cu, CP: no effect

Liver enzymes: no effect

–

Haematological parameters: no effect

Alzheimer's disease Assessment Scale (Cognitive subscale) or the Mini Mental Status Examination: no diff between groups

(Pratt et al., 1985 )

Double‐blinded placebo‐controlled trial

G1: 7 adults (3 M and 4 F)

(mean age 42 years)

G2: 7 adults

12 weeks

G1: 10 mg/day CuGC

G2: Placebo

In addition to background diet (Cu content ND)

Serum Cu; hair Cu

HTC, MCV and serum chemistry (triglyceride, cholesterol, AST, GGT, LDH, ALP, K, mg, Zn)

Side effects reported

Serum Cu: no effect

Hair Cu: no effect

AST, GGT, LDH, ALP: no effect

–

HTC, MCV and serum chemistry: no effect

Nausea, diarrhoea, heartburn: no diff between groups

↑: significant increase; ↓: significant decrease; AAPH: 2,29‐azo‐bis(2‐amidinopropane) hydrochloride; ALP: alkaline phosphatase; ALT: alanine transaminase; Apo: apolipoprotein; BF: breast‐fed; Ca: calcium; CP: caeruloplasmin; CSF: cerebrospinal fluid; Cyt c: cytochrome c; CuGC: copper glycine chelate; DNA: deoxyribonucleic acid; DO: diamine oxidase; eMT: erythrocyte metallothionein; eSOD: erythrocyte superoxide dismutase; F: females; FF: formula fed; GGT: γ‐glutamyl transferase; Hb: haemoglobin; GPx: glutathione peroxidase; HDL: high density lipoprotein; HTC: haematocrit; IgG: Immunoglobulin G; IL: interleukin; K: potassium; LDH: lactate dehydrogenase; LDL: low‐density lipoprotein; M: males; MCV: mean corpuscular volume; MDA: malondialdehyde; Na: sodium; ND: not determined; NR: not reported; P: potassium; RBC: red blood cell; SD: standard deviation; SGOT: serum glutamic‐oxaloacetic transaminase (= AST: aspartate aminotransferase); SGPT: serum glutamic‐pyruvic transaminase (ALT: alanine aminotransferase); SOD: superoxide dismutase; suppl.: supplementation; TBARS: thiobarbituric acid‐reactive substances; WBC: white blood cell; Zn: zinc.