Diphosphoric acid, copper salt

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Cu 1+ and Cu 2+ salts

Molecular formula:

Cu

IUPAC Name:

Cu 1+ and Cu 2+ salts

Specific details on test material used for the study:

Cu 2+ as copper hydroxide
Cu2+ as Copper Oxide
Cu2+ as Copper Oxychloride
Cu2+ as Tribasic Copper Sulphate
Cu2+ as Bordeaux Mixture
Cu2+ as copper sulfate pentahydrate

Lot/batch number:
Copper hydroxide: 380-71-05
Copper oxide: 200802
Copper oxychloride: 27003B
Tribasic copper sulphate: 471/2002
Bordeaux mixture: 1/170
Copper sulfate pentahydrate: 03415PU

Purity:
Copper hydroxide: 60.1% copper
Copper oxide: 87% copper
Copper oxychloride: 57.39% copper
Tribasic copper sulphate: 31.12% copper
Bordeaux mixture: 27.38% copper
Copper sulfate pentahydrate: 25.45% copper

Description:
Copper hydroxide: Light bue powder
Copper oxide: Red-brown powder
Copper oxychloride: Light green, fine, homogenous powder
Tribasic copper sulphate: greenish-blue solid
Bordeaux mixture: Green powder
Copper sulfate pentahydrate: Blue crystals

Stability:
No evidence of instability was observed for all test substance

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories Inc, Raleigh, North Carolina.
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: Variation did not exceed +/- 20% of the mean weight by dose group.

No additional information provided on test animals, environmental conditions or in-life dates.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: ultra-pure water/diet slurry

Details on exposure:

Approximately 4 ml/kg bw

No information on preparation of dosing solutions or homogeneity and stability of test material.

Duration and frequency of treatment / exposure:

See details on study design.

Doses / concentrations

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Main experiments:
Experiment 3: 20 mg Cu/kg bw for each of the six copper substances.
Experiment 4: 20 mg Cu/kg bw as copper hydroxide.

No. of animals per sex per dose / concentration:

Experiment 1A: 4 males
Experiment 1B: 4 males
Experiment 2: 1 male
Experiment 3: 5 males
Experiment 4: 5 males

Control animals:

yes, concurrent vehicle

Details on study design:

Pilot Experiments:
Experiment 1A: Plasma and liver pilot experiment with copper sulphate at concentrations of 0, 5, 20 or 65 mg Cu/kg bw to determine a single appropriate dose level for subsequent experiments. Serial blood samples were removed from the jugular vein cannula (pre-dose, 30 min and at 1, 2, 4, 6, 9, 13, 18, 24, 32, 40 and 48 hours after dose administration). Liver samples from one rat per dose group were also submitted to Cu analysis.
Experiment 1B: Plasma copper and surgical status pilot experiment to assess the potential impact of jugular vein and bile duct cannulation on plasma Cu concentrations of control animals. A water/diet slurry (0 mg Cu/kg bw) was administered to four rats unaltered by surgery, four rats with jugular vein cannulae and four rats with bile duct cannulae. Bile was collected approximately 17 to 0 hours before dosing and 0 - 24 hours and 24 - 48 hours after dosing and tail vein blood was collected approximately 17 and 0 hours before dosing and 24 and 48 hours after dosing.
Experiment 2: Absorption and disposition pilot experiment to test the feasibility of using excreta and tissue Cu burdens to compare bioequivalence from the different test substances. 65 mg Cu/kg as copper hydroxide, copper oxide, copper oxychloride, tribasic copper sulphate and bordeaux mixture or copper sulfate pentahydrate was administered to bile duct cannulated rats. Bile, liver, plasma, post-biliary intestinal content, post-biliary intestine wall, pancreas, urine, faeces and carcass were sampled at 24 hours after administration.

Main Experiments:
Experiment 3: Distribution and excretion experiment with biliary cannulation, to estimate bioavailability by summation of Cu recovery from tissues, carcass, excreted dose in bile and urine. Post exposure period, 24 hours.
Experiment 4: Quantification of the time course of Cu in liver and plasma after oral administration of one representative copper substance, copper hydroxide. Post exposure period, 48 hours.

Details on dosing and sampling:

Experiment 3 samples: Blood, plasma, liver, combined stomach and contents, combined post-biliary intestines and contents, carcass, bile, urine, faeces, and cage wash (collection period: 24 hours).
Experiment 4 samples: Whole blood, plasma, liver (0, 1.5, 3, 6, 9 12, 18, 24, 30, 36, 48 hours).

Examinations: Clinical observations (daily), mortality (daily), body weights (at study initiation and at termination), Cu analysis of dose suspensions, representative food samples, whole blood, plasma, tissues, excreta and cage wash as appropriate.

Copper analysis: Microwave digestion was used to prepare non-aqueous samples. Copper was analysed by ICP-AES. The method detection limit (MDL) and limit of quantitation (LOQ) were 0.0054 and 0,012 ppm, respectively.

See details on study design for additional information.

Results and discussion

Preliminary studies:

Experiment 1A:
No consistent dose response in plasma Cu concentration was observed over the 48 hour collection period, which was attributed to the bioregulation of copper uptake. Elevated copper concentrations were determined in livers of high dose animals. Generally all dose groups including the vehicle control exhibited a drop in plasma concentrations after dose administration which was attributed to fasting prior to and after administration. For this reason fasting was discontinued in subsequent experiments.

Experiment 1B:
All animals appeared healthy. Plasma copper concentrations were higher in rats with cannulation surgery compared to control rats. This finding was attributed to a generalised inflammatory response which is known to raise levels of ceruloplasmin and thus the carrying capacity of Cu in plasma. Rats without surgery showed the greatest body weight gain during the study followed by jugular vein cannulated rats and then by bile cannulated rats.

Experiment 2:
Absorption for copper sulphate and the five other copper substances ranged from 4.67 to 6.86% of the administered dose, based on the amount of copper measured in bile, liver, pancreas, plasma, and urine. Some distention and fluid retention in the intestinal tract was noted during sample collection. Thus the dose was adjusted to 20 mg/kg bw for the main experiments.
The results of the pilot studies were used to adjust the experimental designs of the main experiments.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The results of the main experiments showed that five forms of copper were similarly absorbed to copper sulphate, following oral administration.

Main ADME results 2

Type:

distribution

Results:

The ranking of copper concentrations in excreta and tissue samples was generally faeces > GI tissue and contents (post-biliary) > stomach and contents > liver > bile > plasma ~ whole blood ~ carcass > urine.

Main ADME results 3

Type:

excretion

Results:

Complete clearence after termination of Cu administration.

Toxicokinetic / pharmacokinetic studies

Toxicokinetic parameters

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 10 hrs

Metabolite characterisation studies

Metabolites identified:

not measured

Any other information on results incl. tables

Experiment 3:

All rats appeared healthy with no signs of infection. The actual dose levels ranged from 21.8 to 24.3 mg Cu/kg bw for the six copper test substances. Control animals received 0.052 mg Cu/kg bw from copper present in the diet. The absorbed dose ranged from 10.7 to 12.9% based on percent of the dose measured in whole blood, liver, carcass, bile and urine. No statistically significant difference in absorption was observed between the six copper test substances. The ranking of copper concentrations in excreta and tissue samples was generally faeces > GI tissue and contents (post-biliary) > stomach and contents > liver > bile > plasma ~ whole blood ~ carcass > urine > cage wash. The total recovery ranged between 109 and 141%, the majority of which was measured in the faeces and in the GI tract tissue and contents. Recovery values in excess of 100% of the administered dose were explained with continued uptake of Cu from normal dietary uptake. Based on the results of this experiment, it was concluded that the six copper test substances have essentially the same relative bioavailability under the conditions of this experiment. The results are summarised in the attached Table 1.

Experiment 4:

Animals of the control and test group received mean measured doses of 0.025 or 24.9 mg Cu/kg bw, respectively. Similar copper concentrations were determined in plasma of the vehicle control and the treatment group. Mean values ranged from 0.7 to 1.1 µg Cu/g plasma and were consistent with values obtained for control animals without surgery in the pilot experiment 1B. Administration with 20 mg Cu/kg bw as copper hydroxide caused clearly elevated copper concentrations in liver. While the time course in the control was essentially unchanged during the 48 hours after dosing, a peak concentration of 10.2 µg Cu/g tissue was reached at 12 hours after administration of the test substance. Background copper concentrations measured in livers of the control rats were approximately one half of the peak concentration found in rats of the treatment group. Overall, 0.96 to 1.85% of the administered dose of 20 mg Cu/kg bw was recovered in the liver. Non-compartmental kinetic analysis of the liver concentration data gave an apparent elimination half-life of 31 hours, estimated from the mean data 12 to 48 hours after administration of the test substance. However, the estimation of the linear elimination half-life ignores the fact that continuing exposure to copper occurs from dietary intake and that systemic uptake is subject to bio-regulatory control. It also ignores thhe natural background level of copper already present, and is therefore not an accurate representation. By subtracting the initial T₀ value, the apparent half-life of the dosed copper is 10.134 hours. The AUC for the 20 mg Cu/kg bw group (343 hr* µg/g) was was 1.4 -fold greater than the AUC for the control group (239 hr* µg/g). Upon study termination, copper concentrations in the liver were equivalent to the control, indicating complete clearance of the administered test substance. The results are presented in the attached Table 2 and Figure 1.

Applicant's summary and conclusion

Conclusions:

A relative terminal half-life of 10.134 hours was calculated.

Executive summary:

Materials and Methods:

Based on the results of three pilot experiments two main experiments were conducted, a distribution and excretion experiment with biliary cannulation to estimate the bioavailability of copper from six different test substances, and an experiment investigating the time course of copper in liver and plasma after oral administration of one representative copper substance, i.e. copper hydroxide. The sudy was conducted accoring to EC method B.36 (87/302/EEC) and OECD 417 (1984).

Results and Discussion:

The results of the main experiments showed that five forms of copper were similarly absorbed to copper sulphate, following oral administration to bile cannulated rats. The absorbed dose ranged from 10.7 to 12.9% based on percent of the dose measured in whole blood, liver, carcass, bile and urine. The ranking of copper concentrations in excreta and tissue samples was generally faeces > GI tissue and contents (post-biliary) > stomach and contents > liver > bile > plasma ~ whole blood ~ carcass > urine > cage wash. Administration of 20 mg Cu/kg bw as copper hydroxide had no effect on copper plasma levels. In contrast, increased concentrations of copper in liver were observed with a peak of 10.2 µg Cu/g tissue at 12 hours after administration. Thereafter copper concentrations decreased to control levels upon study termination. A relative terminal half-life of 10.134 hours was calculated.

Summary of absorption data for dietary copper

Reference	Animal model	Identity of copper	Dietary Cu			Duration of treatment/ measurement	Analytical method	Absorption of Cu (% of intake)		Other information	Study rating
			mg/kg feed	μg/d	mg/kgBW/da			Apparent	True
Himmelstein (2003)^d	Sprague Dawley rat (male)	Copper (I) oxide Copper oxychloride Copper sulphate Copper hydroxide	20 mg/kg BW (single oral dose – by gavage)			Measurement of Cu in tissues and excreta for 24h after dosing	ICP	10.7 – 12.9% of intake (absorption calculated as sum of Cu in whole blood, liver, carcass, bile and urine)		Cu levels in tissues and excreta similar for all substances.	3
Himmelstein (2003)^d	Sprague Dawley rat (male)	Copper hydroxide	20 mg/kg BW (single oral dose)			Measurement of Cu in whole blood, plasma and liver for 48h after dosing	ICP			Apparent half-life 10h	3

Values reported are mean values ± (if specified) standard deviation

NR – not reported