Basic copper zinc nitrate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-12-19 to 2003-09-18

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

1984-04-04

GLP compliance:

yes

Test material

Test material information

Test material form:

solid

Details on test material:

1) Copper hydroxide
- CAS No. / EC No.: 20427-59-2 / 243-815-9
- State of aggregation: light blue powder

2) Copper oxide
- CAS No. / EC No.: 1317-39-1 / 215-270-7
- State of aggregation: red-brown powder

3) Copper oxychloride
- CAS No. / EC No.: 1332-65-6 / 215-572-9
- State of aggregation: light green, fine homogenous powder

4) Tribasic copper sulfate
- CAS No. / EC No.: 1333-22-8 / ---
- State of aggregation: greenish-blue solid

5) Bordeaux mixture
- CAS No. / EC No.: 8011-63-0 / ---
- State of aggregation: green powder

6) Copper sulfate pentahydrate
- CAS No. / EC No.: 7758-99-8 / 616-477-9
- State of aggregation: blue crystals

Radiolabelling:

no

Test animals

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Details on species / strain selection:

The Crl:CD®(SD)IGS BR rat has been used extensively in chemical absorption, distribution, metabolism and elimination studies. The rat was the laboratory animal of choice for toxicity studies used to understand any potential adverse health effects associated with copper substances. Therefore, Crl:CD®(SD)IGS BR rats were also selected for the proposed absorption and biodistribution studies. Only male rats were tested in the current study because no difference in toxicity was observed between male and female rats in a 2-week drinking water or 2- and 13-week dietary feeding studies with copper sulfate (Hebert, 1993)*.

*Reference:
- Hebert C.D., Elwell M.R., Travlos G.S., Fitz C.J., Bucher J.R. (1993). Subchronic toxicity of cupric sulfate administered in drinking water and feed to rats and mice. Fundam Appl Toxicol. 21: 461-475.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
NOTE: rats were obtained with or without surgically implanted jugular vein or bile duct cannulas
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at the time of dosing: approx. 7 weeks of age
- Weight at at the time of dosing: did not exceed ± 20 % of the mean weight by dose group
- Housing (dosing period): housed individually; for experiments requiring excreta collection, animals were placed in plastic metabolism units (Experiments 1A, 2, and 3)
- Diet (ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 (note: animals of experiment 1A were fasted before and after dose administration with the purpose of minimizing variability in absorption)
- Water (ad libitum): tap water
- Quarantine period:
Non-cannulated animals: at least 6 days
Jugular vein or bile duct cannulated animals: less than 6 days

The jugular vein cannulas were checked by flushing with heparin:saline solution. Rats with patent cannulas were used on study. Bile-duct cannulated rats were selected for use based on measurable bile flow the evening before dose administration.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 1 °C
- Relative humidity: 40 - 60 %
- Photoperiod (hrs dark / hrs light): approximate 12-hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: ultra-pure water/diet slurry

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The copper substances were prepared in the vehicle for administration. The vehicle was chosen to account for matrix effects and biological aspects of endogenous copper uptake from foodstuffs. Pre-determined amounts of rodent diet and purified water were combined and homogenized. A specified amount of each copper substance was combined with the diet slurry and further homogenized to give a suspension necessary for the specified dose. The vehicle volume was approximately 4 mL/kg bw.
Dose suspension calculations were performed using the DEBRAS laboratory information management system (Lablogic LTD). Dose solutions were prepared and refrigerated or frozen until used. Dose suspensions were stirred continuously during administration.

DOSE VERIFICATION:
Dose verification was performed by analyzing at least three aliquots of the dose suspensions for chemical analysis of copper content (one aliquot before, one during, and one after dose administration).

Duration and frequency of treatment / exposure:

single dose; Study design included bile-cannulated rats, and blood/tissue analysis as well as excretion (urine/faeces).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.008 mg/kg bw/day (actual dose received)

Remarks:

based on copper (Experiment 1A)

Doses / concentrations 2

Dose / conc.:

5.5 mg/kg bw/day (actual dose received)

Remarks:

based on copper (Experiment 1A)

Doses / concentrations 3

Dose / conc.:

22 mg/kg bw/day (actual dose received)

Remarks:

based on copper (Experiment 1A)

Doses / concentrations 4

Dose / conc.:

72.4 mg/kg bw/day (actual dose received)

Remarks:

based on copper (Experiment 1A)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

based on copper (Experiment 2)

Doses / concentrations 6

Dose / conc.:

65 mg/kg bw/day (nominal)

Remarks:

based on copper (Experiment 2)

Doses / concentrations 7

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

based on copper (Experiment 3)

Doses / concentrations 8

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

based on copper (Experiment 3)

Doses / concentrations 9

Dose / conc.:

0.025 mg/kg bw/day (actual dose received)

Remarks:

based on copper (Experiment 4)

Doses / concentrations 10

Dose / conc.:

24.9 mg/kg bw/day (actual dose received)

Remarks:

based on copper (Experiment 4)

No. of animals per sex per dose / concentration:

Experiment 1A: 4 animals/group
Experiment 1B: 4 animals/group
Experiment 2: 1 animal/group
Experiment 3: 5 animals/group
Experiment 4: 5 animals/group/collection time

Control animals:

yes

Positive control reference chemical:

not applicable

Details on dosing and sampling:

GENERAL INFORMATION:
Four experiments with the following objectives were conducted in adult male rats:
Experiment 1A: objective was to evaluate the use of plasma and liver copper concentrations as a measure of absorbed dose in rats administered 0, 5, 20, or 65 mg Cu/kg bw using copper sulfate
Experiment 1B: objective was to determine the effect of surgical status on plasma copper levels of sham dosed rats with and without implantation of jugular vein or bile duct cannulas
Experiment 2: objective was to determine the feasibility of using bile-duct cannulated rats to quantify the relative bioavailability for each of the six copper test substances at a dose level of 65 mg Cu/kg bw.
Experiment 3: an experiment was conducted to quantify the disposition and bioavailability of the six test substances at a dose level of 20 mg Cu/kg bw using bile-duct cannulated rats
Experiment 4: objective was to demonstrate clearance of copper from the liver after oral gavage administration of one representative copper substance, copper hydroxide, at 20 mg Cu/kg bw

EXPERIMENT 1A - PLASMA AND LIVER PILOT (test substance: copper sulfate pentahydrate only):
- 4 animals with jugular vein cannulas/group were administered 0, 5, 20, or 65 mg Cu/kg bw with copper sulfate prepared in a water/diet slurry.
- following dosing, serial blood samples were removed from the jugular vein cannula and placed into tubes containing EDTA.
- blood was collected from the tail vein if the jugular cannula lost patency.
- time and frequency of sampling: pre-dose and 30 minutes as well as 1, 2, 4, 6, 9, 13, 18, 24, 32, 40, and 48 hours after dose administration (sample volume: ~0.2 mL/timepoint).
- liver samples from one rat/dose group were also submitted for copper analysis (sampling: 48 hours after dose administration)

EXPERIMENT 1B - PLASMA COPPER AND SURGICAL STATUS PILOT (test substance: none)
- 12 rats were obtained (4 unaltered by surgery, 4 with jugular vein cannulas, and 4 with bile duct cannulas).
- animals received sham oral gavage dose administration of a water:diet slurry with no additional copper added

Bile-duct cannulated rats:
- bile-duct cannulated rats arrived with the cannulas looped back to the small intestine.
- loop was cut on Day 6 (pm) and animals placed in metabolism units with access to a dextrose/essential salt (KCl, NaCl) solution prepared in deionized water to help maintain normal bile flow.
- time and frequency of sampling: bile was collected approx. 17 to 0 hours before dosing and 0 - 24 and 24 - 48 hours after dosing. Tail vein blood was collected at approximately 1 7 and 0 hours before dosing and 24 and 48 hours after dosing.
- urine, faeces, and carcasses from bile-duct cannulated rats were retained for copper analysis methods development.

- red blood cells from all rats and carcasses from normal and jugular vein cannulated rats were discarded.
- tail vein blood was collected from the normal and jugular vein cannulated animals for a longer period as indicated.

EXPERIMENT 2 - ABSORPTION AND DISPOSITION PILOT (test substances: copper sulfate pentahydrate, copper hydroxide, copper oxychloride, Bordeaux mixture, tribasic copper sulfate, and copper oxide)
- 7 rats with bile duct cannulas were obtained.
- 1 animal each was administered control diet slurry, copper sulfate pentahydrate, copper hydroxide, copper oxychloride, Bordeaux mixture, tribasic copper sulfate, or copper oxide (test item concentration: 65 mg Cu/kg based on the results of liver copper concentrations from Experiment 1A).
- time and frequency of sampling: rats were housed in metabolism units for collection of excreta (urine, faeces, and bile) overnight prior to dosing and for 0 to 24 hours after dose administration. During the collection period, the drinking water was substituted with a solution of dextrose:NaCl:KCl salt prepared in deionized water. At 24 hours after dose administration, samples collected for copper analysis included bile, liver, plasma, post-biliary intestinal content, post-biliary intestine wall, pancreas, urine, faeces, and carcass.

EXPERIMENT 3 - ABSORPTION AND DISPOSITION (test substances: copper sulfate pentahydrate, copper hydroxide, copper oxychloride, Bordeaux mixture, tribasic copper sulfate, and copper oxide)
- 35 rats were used (5 rats per vehicle control, and 5 for each of the six copper substances).
- rats were received with biliary cannulas and tether buttons and used as previously described in Experiments 1B and 2 (test item concentration: 20 mg Cu/kg based on the results of Experiment 2).
- time and frequency of sampling: collection period was 24 hours, at which time the animals were sacrificed. Samples for collection and analysis of copper included dose (pre-dose, mid-dose, and post-dose aliquots), blood, plasma, liver, combined stomach and contents, combined post-biliary intestines and contents, carcass, bile, urine, faeces, and cage wash.
- for collection of the stomach and post-biliary tract samples and contents, the appropriate sections were ligated. Ligatures were applied in the following locations: one above the stomach where the oesophagus meets the diaphragm, two below the stomach just before the biliary tract, and one at the rectum. This procedure prevented contamination of the carcass with unabsorbed dose from the intestine.
- feed residue was included as part of the cage wash. Cage wash was collected using water and dilute nitric acid (3 %) as a rinse solution.
- copper measured in the cage wash was not considered part of the absorbed dose because it contained feed residue powder.

EXPERIMENT 4 - LIVER CONCENTRATION TIME COURSE WITH COPPER HYDROXIDE
- total of 110 rats were used according to the design given below.
- one test item group (20 mg Cu/kg) and one control group were used
- samples for collection include dose (pre-dose, mid-dose, and post-dose aliquots), whole blood, plasma, and liver.
- dose aliquots, plasma and liver sample were submitted for copper analysis.
- percent of administered dose in the liver was calculated for the 20 mg Cu/kg group.
- a non-compartimental kinetic analysis of the liver copper concentration data was performed to determine area-under-the-curve (AUC) for the 0 and 20 mg Cu/kg bw groups and Tmax, Cmax, and the apparent elimination half-life for the 20 mg Cu/kg bw group.

OBSERVATIONS:
- clinical observations and mortality: cage-site examinations to detect moribund or dead rats and abnormal behaviour and/or appearance among rats were conducted at least once daily. Moribund rats were sacrificed.
- body weight: immediately before or after sacrifice

SAMPLE COLLECTION:
- time course experiments (Experiments 1A and 1B): whole blood was collected from the cannula, tail vein, or cardiac puncture into EDTA tubes, and placed on wet ice or refrigerated at 4 - 10 °C. Plasma was separated from red blood cells by centrifugation and stored frozen at <-10 °C until analysis. The plasma was analyzed for copper.

- terminal sacrifice experiments (Experiments 2, 3, and 4): whole blood was collected by cardiac puncture and placed into EDT A tubes. Processing was similar to that described above, except that a portion of whole blood was saved and refrigerated at 4 - 10 °C and a portion was used to prepare plasma. Whole blood and plasma were analyzed for copper for Experiment 3. Plasma was analyzed for copper for Experiments 2 and 4.

-pancreas was collected for Experiment 2 only.
- livers were collected and weighed at sacrifice, followed by the post-biliary intestinal contents and intestinal wall to avoid cross contamination.
- during collection of the intestinal contents for Experiment 2, the intestinal walls were rinsed with ultra pure water. The water rinse was added back to the intestinal contents. The stomach and its contents was left with the carcass.
- for Experiment 3, the stomach and contents and post-biliary tract and contents were collected for analysis as described above.
- except for whole blood and red blood cells, all the tissues samples and the remaining carcass were stored frozen at <-10 °C. The carcass, stomach and contents, and post-biliary tissues and contents were removed from the freezer, added to liquid nitrogen then homogenized in a blender.
- urine and faeces were collected and stored frozen at <-10 °C. The faeces were manually homogenizing in purified water.
- cage wash was obtained after terminal sacrifice by rinsing the collector with water and diluted nitric acid (3 %). Cage wash samples were stored at room temperature.
- representative samples (n = 2 replicates) of rodent chow were analyzed for copper content.
- observations from the preliminary studies (Experiments 1A and 2) were used to assess the potential for cross contamination between food residue and excreta.
- no contamination of feed in the cage feeder was observed. Feed residue powder rinsed off the urine and faeces separation cone and collector was included in the cage wash.

COPPER ANALYSIS:
- dose suspensions, representative samples of food, whole blood, plasma and other tissues, excreta and cage wash were analysed for total copper. Microwave digestion was used to prepare non-aqueous samples. Copper was analyzed by ICP-AES
- drinking water provided to rats was analysed for copper content
- sub-samples of each test substance were analysed for copper content.

Statistics:

Group data are summarized using descriptive statistics for mean, standard deviation, standard error, and percent coefficient of variation also referred to percent relative standards deviation. Values were calculated in a standard spreadsheet program (Microsoft® Excel 2000). Percent copper absorbed data for the five copper substance in Experiment 3 were analyzed for statistical significance relative to the copper sulfate group. The analysis was performed using SAS®. A preliminary analysis showed homogeneous normally distributed data using Levene's test for homogeneity and Shapiro-Wilk test for normality (Shapiro & Wilk, 1965; Snedecor & Cochran, 1967)*. The data were analyzed by one-way analysis of variance followed by Dunnett's test for multiple comparisons at a significant level of p < 0.05 (Dunnett, 1964; Dunnett, 1980; Tamhane, 1979)*.
The kinetic (non-compartmental) analysis to obtain AUC and the apparent elimination half-life for the Experiment 4 liver time course data was performed using commercially available pharmacokinetic software (WinNonlin™ Professional, Version 3.3).

*References:
- Shapiro, S.S. and Wilk, M.B. (1965). An analysis of variance test for normality (complete samples). Biometrika 52, 591-611.
- Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition, pp 246 - 248 and 349 - 352. The Iowa State University Press, Iowa.
- Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482 - 491.
- Dunnett, C.W. (1980). Pairwise multiple comparisons in the unequal variance case. J. Amer. Statist. Assoc. 75, 796 - 800.
- Tamhane, A.C. (1979). A comparison of procedures for multiple comparison of means with unequal variances. J. Amer. Statist. Assoc. 74, 471 - 480.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Cu concentration (blood plasma) at 20 mg/kg bw (experiment 3) nominal dosing: control: 2.76 µg Cu/g plasma; in treated rats the blood concentrations ranged from 2.40 to 3.18 μg Cu/g across all post-dose time points.

Main ADME results 2

Type:

absorption

Results:

At a nominal target dose of 20 mg Cu/kg body weight (experiment 3), absorption (plasma, liver, carcass, bile, urine) for 5 Cu substances was statistically similar relative to CuSO4 and ranged from 10.7% to 12.9% of the administered dose.

Main ADME results 3

Type:

distribution

Results:

Cu concentrations (liver): Mean concentration in the control rats was 5.23 μg Cu/g. Mean conc. in treated rats at 24 hours after dose administration (dose: 20 mg/kg bw, experiment 3) were approximately 3-4 times higher and ranged from 15.8 - 20.6 μg Cu/g.

Main ADME results 4

Type:

excretion

Results:

Cu concentration (faeces): In control rats, a mean of 31.5 μg Cu/g faeces was determined; after dosing of 20 mg/kg bw: 694 to 1099 μg Cu/g faeces were observed.

Main ADME results 5

Type:

excretion

Results:

Cu concentration (urine): In control rats, a mean of 0.534 μg Cu/g urine was determined; after dosing a level of 0.882 to 1.34 μg Cu/g urine could be determined.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

EXPERIMENT 2: ABSORPTION AND DISPOSITION PILOT
- absorption for copper substances of the administered dose based on the amount of copper measured in bile, liver, pancreas, plasma, and urine as follows:
control: NA
copper sulfate pentahydrate: 6.87 %
copper hydroxide: 6.12 %
copper oxychloride: 4.71 %
Bordeaux mix: 5.70 %
tribasic copper sulfate: 4.68 %
copper (I) oxide: 4.94 %

- copper in carcass was not added to the absorbed dose in this experiment because it contained the stomach and contents.

EXPERIMENT 3 - ABSORPTION AND DISPOSITION (test substances: copper sulfate pentahydrate, copper hydroxide, copper oxychloride, Bordeaux mixture, tribasic copper sulfate, and copper oxide)
- absorbed dose ranged based on the percent of the dose measured in whole blood, liver, carcass, bile, and urine as follows:
control: NA
copper sulfate pentahydrate: 12.03 %
copper hydroxide: 12.50 %
copper oxychloride: 11.18 %
Bordeaux mix: 12.91 %
tribasic copper sulfate: 12.17 %
copper (I) oxide: 10.69 %
- no statistically significant difference in absorption was observed between the six copper test substances.

EXPERIMENT 4 - LIVER CONCENTRATION TIME COURSE WITH COPPER HYDROXIDE
- The mean concentrations of Cu in plasma were essentially the same in the control and copper hydroxide groups. The mean values ranged from 0.737 to 1.14 µg Cu/g plasma.
- plasma concentrations were consistent with those measured in normal rats not subjected to surgical implantation of jugular or bile duct cannulas in Experiment 1B.
- plasma copper concentrations appeared to show a slight diurnal variation.

Details on distribution in tissues:

EXPERIMENT 1A - PLASMA AND LIVER PILOT (test substance: copper sulfate pentahydrate only):
- copper analysis of liver: results showed an elevated copper concentration in the 65 mg/kg dose group (21.9 µg/g tissue), but essentially the same concentration in the 0, 5, and 20 mg/kg groups (4.29, 3.93, and 4.94 µg/g tissue, respectively).
- results of the experiment indicated that the copper concentration in liver, but not plasma, was responsive to dose. Liver copper concentration could be used for further kinetic evaluation. A dose level of 65 mg Cu/kg bw for each copper test substance was selected based on this result for use in Experiment 2.

EXPERIMENT 2: ABSORPTION AND DISPOSITION PILOT
- total copper recovery based on the sum of the copper in all excreta and tissue samples were as follows:
control: NA
copper sulfate pentahydrate: 77.92 %
copper hydroxide: 98.36 %
copper oxychloride: 94.58 %
Bordeaux mix: 82.13 %
tribasic copper sulfate: 93.23 %
copper (I) oxide: 92.52 %

- ranking of copper concentrations in excreta and tissue samples of rats administered the copper test substances was generally faeces > GI contents (post-biliary) > liver> GI tract tissue (post-biliary)> carcass (including stomach contents)> bile> urine~ plasma> pancreas.

EXPERIMENT 3 - ABSORPTION AND DISPOSITION (test substances: copper sulfate pentahydrate, copper hydroxide, copper oxychloride, Bordeaux mixture, tribasic copper sulfate, and copper oxide)
- ranking of copper concentrations in excreta and tissue samples of rats administered the copper test substance was generally faeces> GI tissue+ contents (post-biliary)>stomach + contents > liver > bile > plasma ~ whole blood ~ carcass > urine > cage wash.
- total recovery for the six test substances ranged (majority of which was measured in the faeces and GI tract tissue + contents) as follows:
control: NA
copper sulfate pentahydrate: 108.96 %
copper hydroxide: 121.19 %
copper oxychloride: 122.87 %
Bordeaux mix: 141.11 %
tribasic copper sulfate: 120.87 %
copper (I) oxide: 128.73 %

- recovery greater than 100 % was consistent with continued uptake of copper from normal dietary intake.

EXPERIMENT 4 - LIVER CONCENTRATION TIME COURSE WITH COPPER HYDROXIDE
- liver copper concentration was clearly elevated after administration of 20 mg Cu/kg bw compared with the control group.
- peak concentration (Cmax) was 10.2 µg Cu/g tissue at 12 hours (Tmax) after dose administration.
- time course in the control group was essentially unchanged from 0 to 48 hours.
- background copper concentrations in the liver of control rats were approximately one-half the Cmax in rats dosed with 20 mg Cu/kg body weight at 12 hours (Tmax) after dose administration.
- mean percent dose in liver ranged from 0.96% to 1.85% in rats administered 20 mg Cu/kg bw.
- 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 dose administration of 20 mg Cu/kg bw. The AUC for the 20 mg Cu/kg bw group (343 hr*µg/g) was 1.4-fold greater than the AUC for the control group (239 hr* µg/g).
- apparent elimination half-life for the liver copper concentration to retum to normal was calculated on the time course data for the 20 mg Cu/kg bw group after subtraction of the pre-dose (0 hour) mean concentration of 4.16 µg Cu/g liver. This background correction gave an apparent half-life of 10 hours.
- liver copper concentration in the copper hydroxide group was equivalent to the control concentration at 48 hours after dose administration, indicating complete clearance of the 20 mg Cu/kg bw dose by this time.
- elimination half-life of 31 hours for the liver

Metabolite characterisation studies

Metabolites identified:

not measured

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

EXPERIMENT 1A - PLASMA AND LIVER PILOT (test substance: copper sulfate pentahydrate only):
- no consistent dose response in plasma copper concentration was observed over the 48 hour collection period (concentrations ranged from 1.2 to 3.3 µg copper/g plasma). The lack of dose response is likely attributable to the bioregulation of copper uptake.
- mean time 0 plasma copper concentrations in the 0 and 5 mg/kg groups (1.6 µg/g plasma) were lower than the time 0 concentrations in the 20 and 65 mg/kg groups (2.9 µg/g plasma). The time 0 difference in plasma copper concentrations suggests a temporal effect related to jugular vein cannula implantation.
- all dose groups including the 0 mg/kg group exhibited a drop in plasma concentrations after dose administration. This is most likely the result of fasting overnight and up to 4 hours after dose administration. Fasting was discontinued in subsequent experiments for this reason.

Any other information on results incl. tables

COPPER ANALYSIS:

- method of copper analysis implemented for this study was ICP-AES.

- method detection limit and limit of quantitation were 0.0054 and 0.021 ppm (mg/L), respectively.

- method had sufficient sensitivity, precision, accuracy, and linearity to analyze all sample matrices submitted for this study.

- range of recovery of samples spiked with known amounts of copper ranged from 97% to 108%.

- copper content of the dose preparation was consistently slightly higher than anticipated from analyses. It was suspected that a weighing error had occurred when the milligram samples were taken for analysis, and that this was reflected in numbers used to calculate dose content.

- reanalysis confirmed that the copper content of the technical materials was essentially similar to that declared by the manufacturer, and it was concluded that an error (probably a weighing error) had occuned in the initial copper content analysis. The slight overdosing that resulted from this error was considered to not adversely affect the outcome of the study.

COPPER FROM LABORATORY WATER AND RODENT DIET

- concentrations of copper in drinking water and rodent diets were evaluated from available information to determine their contribution to the background intake and body burden.

- copper concentration in laboratory drinking water: 0.027 ppm (daily intake of copper: estimated as 0.0027 mg Cu/kg bw, assuming water consumption of 25 mL per day for a 250 mg rat).

- copper concentration in the breeder's diet was 20 ppm.

- at the laboratory conducting the study the diet contained a concentration of approx. 11 ppm copper.

- daily dietary intake of copper can be estimated at 0.9 mg Cu/kg day assuming a 250 gram rat consumes 20 grams of diet per day.

- more copper is derived from the diet than water.

- daily estimated intake of 0.9 mg Cu/kg body weight from the normal diet is at most five time less than the lowest dose of 5 mg/kg selected for single oral gavage administration in this study.

EXPERIMENT 1A - PLASMA AND LIVER PILOT (test substance: copper sulfate pentahydrate only):

- dose suspension homogeneity was achieved based on relative standard deviations (RSD) ranging from 7.6, 1.1, and 0.8 % for the 5, 20, and 65 mg/kg dose suspensions, respectively. The RSD% in the control dose was 39.9 %. The copper concentrations in the control dose suspension were above the instrument detection limit (0.0008 mg/g dose suspension), but below the method detection limit (0.0032 mg/g dose suspension). The background concentration of copper in the control dose (0.0018 mg Cu/g dose suspension) was similar to the expected concentration (0.0022 mg/g suspension) based on the approximately 1-to-5 dilution of ground chow reported to have 11 ppm copper. The actual mean doses were 0.0081, 5.5, 22.0, and 72.4 mg Cu/kg bw.

- 1 rat each from the 20 and 65 mg/kg dose groups exhibited signs of infection upon terminal sacrifice based on gross observation of lung tissue. Lungs from other rats appeared normal. Tissues were collected and histological evaluation indicated a localized acute infection of the lung. The infection was most likely due to contamination of the cannulas. The infection was not attributed to copper administration and did not impact the validity of the study.

EXPERIMENT 1B - PLASMA COPPER AND SURGICAL STATUS PILOT (test substance: none)

- all animals appeared healthy during this experiment and at the time of sacrifice.

- body weights of the animals were recorded prior to surgery and up to the termination of the study. Rats without surgery had the greatest weight gain followed by jugular vein cannulated rats then by bile cannulated rats.

- plasma copper concentrations were higher in rats with cannulation surgery compared with control rats.

- results of the experiment helped explain that the range of concentrations measured in jugular vein cannulated rats in Experiment 1A were elevated by the surgical status of the animals. This experiment also influenced the decision to limit the duration that bile-duct cannulated rats are used in subsequent experiments to no longer than 24 hours.

EXPERIMENT 2: ABSORPTION AND DISPOSITION PILOT (test substances: copper sulfate pentahydrate, copper hydroxide, copper oxychloride, Bordeaux mixture, tribasic copper sulfate, and copper oxide)

- target dose was achieved for each test substance.

- dose verification values were 64.3, 67.7, 59.1, 69.5, 80.6, and 67.1 mg Cu/kg bw for copper sulfate pentahydrate, copper hydroxide, copper oxychloride, Bordeaux mix, tribasic copper sulfate, and copper (I) oxide, respectively.

- control dose was 0.0115 mg Cu/kg bodyweight.

- pre-, mid- and post-dose samples indicated that the preparations were homogeneous.

- at the dose level used for this experiment (65 mg Cu/kg dose), some distention and fluid retention in the intestinal tract was noted during sample collection. Oligourea (reduced urine output) was also observed. The dose was adjusted to 20 mg/kg in subsequent experiments (Experiments 3 and 4) based on this observation.

- Experiment 2 demonstrated the feasibility of using excreta and tissues to calculate the relative bio-availability of copper from each test substance. Because the carcass burden of copper was not included in the estimate of absorbed dose for Experiment 2, adjustments were made in the tissue collection procedure to collect the stomach+ contents and post-biliary tissue + contents for analysis separate from the carcass in Experiment 3.

EXPERIMENT 3 - ABSORPTION AND DISPOSITION (test substances: copper sulfate pentahydrate, copper hydroxide, copper oxychloride, Bordeaux mixture, tribasic copper sulfate, and copper oxide)

- all rats appeared healthy with no signs of infection.

- gastrointestinal distention and fluid retention observed at the 65 mg Cu/kg dose level in Experiment 2 was absent.

- oligourea also appeared to be absent at the 20 mg Cu/kg bw dose.

- dose verification indicated mean dose levels that were marginally above the target dose of 20 mg Cu/kg body weight.

- actual dose levels were confirmed experimentally and ranged for the six copper test substances:

control: 0.052 mg Cu/kg bw

copper sulfate pentahydrate: 23.7 mg Cu/kg bw

copper hydroxide: 24.3 mg Cu/kg bw

copper oxychloride: 21.8 mg Cu/kg bw

Bordeaux mix: 23.2 mg Cu/kg bw

tribasic copper sulfate: 24.1 mg Cu/kg bw

copper (I) oxide: 22.4 mg Cu/kg bw

EXPERIMENT 4 - LIVER CONCENTRATION TIME COURSE WITH COPPER HYDROXIDE

- dose verification confirmed that the actual doses of copper were 0.025 and 24.9 mg Cu/kg bw for the control and copper hydroxide dose groups, respectively.