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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Already evaluated by the Competent Authorities for Biocides and Existing Substance Regulations.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
. Method developed by the US NTP specifically for the surposes of this study.
Deviations:
yes
Remarks:
. See below
Principles of method if other than guideline:
The study deviated from 'Directive 88/302/EEC B.26 Subchronic 90-Day Oral Toxicity Study in Rodents' as follows;

No additional top dose group or control animals group were included in the study for observation of recovery from toxic effects after the treatment period.

Ophthalomological examinations were only carried out where the eyes showed clinical signs of gross abnormalities. General eye examinations of the control and high dose group were not carried out.

Sensory activity and signs of neurotoxicity were not determined towards the end of the study. The study was conducted prior to this requirement being included in the guidelines. However, signs of reproductive toxicity were included in the test methodology.

Heamatological examinations did not include a measure of blood clotting time/potential.

It was not reported if animals were fasted overnight prior to blood sampling.

Determinations of plasma or serum did not include sodium, potassium or total cholesterol analysis.

Histopathological examinations did not include the aorta.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Lot/Batch number: 533344
Description: Blue, crystalline solid
Purity: Not reported
Stability: Stable at room temperature

Test animals

Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
Species: Rat
Strain: F344/N
Source: Simonsen Laboratories, Gilroy, California, USA
Sex: Male and Female
Age/weight at study initiation: Test animals were approximately 6 weeks old at study initiation. Male mean bodyweights ranged from 119-120 g, mean female bodyweights ranged from 105-107 g
Number of animals per group: In the base study, groups of 10 animals per sex were tested at each dose level. A supplementary study was carried out on 10 males and females per sex per dose for haematology and clinical chemistry evaluations on Days 5 and 21 (all surviving base-study rats
were also subject to the same examinations on test termination - Day 92).
Control animals: Yes

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: plain diet
Details on oral exposure:
Preparation of active ingredient in feed: Copper sulphate was mixed with NIH-07 Open Formula Diet in meal form.
Feed mix was available ad libitum throughout the study period.
Concentration in vehicle: 0 (control), 500, 1000, 2000, 4000 or 8000 ppm were administered to the test organisms in feed.
Doses were based on a preliminary 2-week feed study.
Controls: Yes -vehicle only
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity analysis were conducted on the copper sulphate feed mixture using inductively coupled plasma-atomic emission spectroscopy. Samples taken prior to study initiation and twice during the study, confirmed homogeneity between feed mixtures.
Duration of treatment / exposure:
92 days
Frequency of treatment:
7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000, 4000 or 8000 ppm in the feed (providing estimated intakes of 0, 8, 17, 34, 67 or 138 mg Cu/kg bw/day)
Basis:

No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: none

Examinations

Observations and examinations performed and frequency:
Clinical signs:

Test animals were observed weekly for clinical signs


Mortality:

Test animals were observed twice daily for mortality/morbidity.


Body weight:

Individual bodyweights were recorded prior to the start of the study, on Day 1 and weekly thereafter.


Food consumption:

Test animals were observed once weekly for food consumption.


Water consumption:

Not reported


Ophthalmoscopic examination:

See histological examinations


Haematology:

Blood samples were taken from all supplementary animals and base-study rats. Blood samples were collected from the retroorbital sinus.
Time points: Supplementary rats - Day 5 and 21, Base study rats - Day 92 and test termination
Parameters: hematocrit , haemoglobin concentration, erythrocyte count, reticulocytes, nucleated erythrocytes, mean cell volume and haemoglobin, concentration, platelets and leukocyte count and differential.

Clinical Chemistry:

Taken from all supplementary animals and base-study rats
time points: Supplementary rats - Day 5 and 21, Base study rats - Day 92 and test termination
Parameters: alanine aminotransferase , alkaline phosphatase, 5'-nucleotidase, sorbitol dehydrogenase, bile salts, total protein, albumin, creatinine and urea nitrogen.

Urinalysis:

Taken from all supplementary animals and base-study rats
Time points: Supplementary rats - Day 5 and 21, Base study rats - Day 92 and test termination
Parameters: creatinine, glucose, protein, asparate aminotransferase, N-acetyl-ß-D-glucosaminidase, volume and specific gravity.

Tissue Metal Level Analysis:

Plasma and tissue samples (liver, kidney and testis) were collected from all surviving male base-study rats
Time Points: Day 92 - copper, zinc, magnesium and calcium analysis.

Blood samples (2 ml) were collected from the retroorbital sinus and placed into 3 ml Vacutainer® tubes containing EDTA. The samples were centrifuged and the separated plasma collected. To prepare for analysis, samples were weighed to the nearest 0.1 mg, digested in a nitric acid-perchloric
acid mixture and heated until evolution of nitric acid was complete. The residue was then dissolved in 10% perchloric acid solution and an aliquot removed for analysis by ICP-AES. Metal concentrations were determined by comparing the instrument response to the digested tissues to spiked
tissue standards.
Sacrifice and pathology:
Organ Weights:

Organ weight of the following organs were recorded; liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen, brain, heart

Gross and histopathology:

Complete necropsies were performed on all animals in the control and high dose groups and on all other animals that died early.
Parameters: adrenal glands, brain (three sections), esophagus, eyes (if grossly abnormal) femur with marrow, gross lesions, heart, intestines (large: cecum, colon, rectum: small: duodenum, jejunum. Ileum), kidneys, liver, lung/mainstream bronchi, lymph nodes (mandibular, mesenteric) mammary gland, nasal cavity and turbinates (three sections), ovaries, pancreas, parathyroid glands, pharynx (if grossly abnormal), pituitary gland, preputial or
clitoral glands, prostate gland, salivary glands, spinal cord/sciatic nerve (if neurological signs were present), spleen, stomach (forestomach, glandular stomach), testes (with epididymis) thymus, thyroid gland, trachea, urinary bladder and uterus.

Supplemental histological examination:

To characterise the distribution of copper in the liver and kidney, section of both organs from selected male and females were stained for copper using the rhodanine method. In order to determine the nature of the proteinaceous droplets (see in previous study on rats) sections from selected animals were stained for carbohydrate (PAS method), protein (Mallory-Heidenhain method), lipofuscin (AFIP method) and a-2-microglobulin (immunochemistry). Liver sections from the same rats were stained for lipofuscin, and kidney and liver sections from rats of both sections were examined by transmission electron microscopy. Perl's stain for iron was used to stain sections of spleen from rats in all groups.
Other examinations:
Sperm morphology and vaginal cytology:

Sperm morphology and vaginal cytology evaluations were performed on rats from the 0, 500, 200 and 4000 ppm groups (10 animals per sex and dose group). The method employed was as follows:

National Toxicology Program (NTP) 1987. Technical Protocol for Sperm Morphology and Vaginal Cytology Evaluations in Toxicity Testing for Rats and Mice, 10/31/82 version. Research Triangle Park, N.C.

Females: 12 days prior to sacrifice, the vaginal vaults of 10 individuals per dose group were lavaged and the aspirated lavage fluid and cells stained with Toluidine Blue. Relative numbers of leukocytes, nucleated epithelial cells and large squamous epithelial cells were determined and used to ascertain estrous cycle stage.

Males: Sperm motility was evaluated at necropsy. The left testis and epididymis were weighed, the tail of the epididymis was removed from the epididymis body and weighed. Test yolk was applied to slides and a small incision made in the cauda. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the number of motile and non-motile spermatozoa counted for five microscopic fields per slide. Following motility
determination, each left cauda were placed in phosphate buffered saline solution for sperm density determination with a hemacytometer.
Statistics:
The following statistical procedures were followed;

Dunnet, C.W. 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1095-1121

Williams, D. A. 1971. Biometrics, 27, 103-117

Williams, D.A. 1972. The comparison of several dose levels with a zero dose control. Biometrics 28, 519-531

Shirley, E. 1977. A nonparametric equivalent of William's test for contrasting increasing dose levels of a treatment. Biometrics 33, 386-389

Dun, O.J. 1964. Multiple comparisons using rank sums. Technometrics 6, 241-252

Jonckheere, A.R. 1954. A distribution free k-sample test against ordered alternatives. Biometrika, 41, 133-145

Dixon & Massay 1951 Introduction to Statistical Analysis, McGraw-Hill Book Co.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical signs:

No clinical signs of toxicity could be directly attributed to cupric sulphate consumption in any male or female group. For further details please refer to attached Table 5.

Mortality:

Except for one female that was accidentally killed, all rats survived to the end of the study. For further details please refer to attached Table 5.

Body weight gain:

Final mean bodyweights of test organisms were lower than those of the controls for male rats in the 500, 4000 and 8000 ppm groups and for female rats in the 8000 ppm group. These differences were most pronounced in males in the high dose (8000 ppm). For further details please refer to
attached Table 5.

Food consumption and compound intake:

For male and female rats in the 500, 1000, 2000 and 4000 ppm groups, average daily food consumption was similar to that of the controls. However, food consumption by both sexes in the 8000 ppm dose groups was below that of the controls. Despite this, the average daily compound consumption increased proportionally with increasing concentrations of copper sulphate in the feed. For further details please refer to attached Table 5.

Ophtalmoscopic examination:

Not reported.

Haematology:

Significant changes in haematology parameters were noted in both sexes at all time points. At Day 5, significant increases in hematocrit (HCT) and hemoglobin (HGB) concentrations were noted in high dose male and female rats. By Day 21, these parameters were significantly decreased for male rats in the two highest dose groups (4000 and 8000 ppm) and female rats in the three highest dose groups. At Day 92, HCT and HGB concentrations were significantly decreased in males in the two highest dose groups and in females in the highest dose group. At Day 5, significant increases in erythrocyte (RBC) counts were noted in males in the two highest dose groups and in the high dose females; on Day 92, the only significant increase in RBC count was noted in the high-dose males. In both sexes, in the two highest dose groups, significant decreases in reticulocytes counts were noted on Day 5. By Day 21, reticulocyte counts in males and females in the same dose groups were significantly greater than those of the controls; at Day 92, this
parameter was significantly increased in high dosed males. The only significant change noted in nucleated erythrocytes was a marginal decrease in high dose males at Day 5.

On Day 5, mean cell volume (MCV) values were significantly decreased in males in the two highest dose groups and in females in the highest dose group; mean cell hemoglobin (MCH) values were also significantly decreased for males in the two highest dose groups. At Days 21 and 92, decreases
in MCV and MCH were noted in both sexes in the three highest dose groups, and all decreases were significant with the exception of the Day 92 MCH values for females receiving 4000 ppm. The only significant changes in mean cell hemoglobin concentrations were increases noted on Day 21 in
high dose females and in males in the two highest dose groups.

At Days 5 and 21, significant increases in platelet counts were noted in males and females in the three highest dose groups; the Day 5 platelet count for males in the 1000 ppm group was also significantly increased compare to the controls. At Day 92, increases in platelet counts were noted for both sexes in the two highest dose groups, but this was only significant for males.

Leukocyte counts were increased at all time points in both sexes in the two highest dose groups, with significant increases occurring at Day 5 in high-dose males, at Day 21 in males in the 4000 ppm dose group, and at Day 92 in high-dose males and females: leukocyte count was also significantly increased at Day 21 in males receiving 2000 ppm copper sulphate. Significant increases in lymphocytes were noted at Day 5 in high dose males, at Day 21 in males receiving 2000 or 4000 ppm copper sulphate, and at Day 92 in high dose females. The only other significant change in haematology parameters was an increase in segmented neutrophils at Day 92 in high dose male rats. For further details please refer to the attached Table 1.

Clinical chemistry:

Significant changes in serum chemistry parameters occurred in male and female rats at all time point in the two highest groups. Alanine aminotransferase activities were significantly increased at all time points in both sexes in the two highest dose groups; and was significantly increased at Day 92 in males receiving 1000 or 2000 ppm. At Days 5 and 21, decreases in alkaline phosphate activities were noted in both sexes in the two highest dose groups; except for Day 21 in males in the 4000 ppm group, all these decreases were significant. Changes in sorbitol dehydrogenase (SDH) were limited to Days 21 and 92. At both of these time points, SDH activates were significantly elevated in males in the two highest dose groups and in high dose females; significant increases in SDH activities were also noted at Day 92 in males in the 2000 ppm group and females in the 4000 ppm group. When compared to the control values, 5'necleotidase was significantly decrease in high-dose females at Days 5 and 21 and in high dose males at Day 5; at Day 92, however, this parameter was significantly increased in males receiving 4000 and 8000 ppm cupric sulphate.

At Day 5, slight increases in bile salts were noted in males in the three highest dose groups; however, female bile salts were decreased for all treated groups, with significant decreases in the 1000 and 8000 ppm groups. By Day 21, no significant changes were noted in females, but significant increases were noted in males in the two highest dose groups. At Day 92, significant increases in bile salts were noted in high-dose males and in females receiving 2000 or 4000 ppm copper sulphate.

At all time points, total protein was significantly decreased in high dose males and in females in the 4000 and 8000 ppm dose groups; at Days 5 and 21, total protein was also significantly decreased in males and females receiving 4000 and 2000 ppm copper sulphate respectively. At Days 5 and 21, decreases in albumin concentrations were noted in both sexes at the three highest doses, all of these were significant, excluding the Day 21 for males receiving 2000 ppm. At Day 92, this parameter was significantly decreased in high dose males and females in the two highest groups.

Urea nitrogen (UN) was significantly increased for both sexes in the two highest groups at Day 5, and by Day 21, this was significantly increased in males in the three highest dose groups and females in the highest dose group. At Day 92, UN was significantly elevated in the high-dose males and females as well as females receiving 1000, 2000 or 4000 ppm copper sulphate. The only significant change in creatinine was an increased noted in high dose females on Day 92.For further information please refer to attached Table 2.

Urinalysis:

Significant changes in urinalysis parameters were noted in supplemental study rats at Days 19 and in base study Day 90. Significant increases in urinary aspirate aminotransferase (AST) activities, occurred at Days 19 and 90 in both sexes in the highest dose groups. Increases in this parameter also occurred at both time points in male and female rats in the 4000 ppm groups. A few significant increases in AST activities occurred in animals in the lower dose groups (500 to 2000 ppm). Significant increases in N-acetyl-ß-D-glucosaminidase activities were noted in both sexes in the highest dose
group on Day 90; at this time point, increases also occurred in males and females in the 4000 ppm groups. Glucose output was significantly increased at Day 19 in males in the 2000 ppm group and at Day 90, this parameter was significantly elevated in males in the two highest dose groups. A significant decrease in protein output was noted in the high dose males at Day 19, however, the Day 90 elevation in base study rats, this parameter was significantly increased relative to the controls in males in the two highest dose groups. No significant changes in glucose or protein output were noted in females at either time point. Please refer to attached Table 3 for further information.

Organ weights:

Significant changes in absolute organ weights were limited to males and females in the high dose groups and included decreases in absolute brain, heart, kidney, liver, lung and thymus weights in males and absolute kidney weight in females. Generally, relative organ weights for treated groups were similar to those of the controls or increased with decreasing mean body weights in the two highest dose groups (4000 and 8000 ppm). For further information please refer to attached Table 5.

Gross and histopathology:

Gross lesions were present in the forestomach of both sexes receiving copper sulphate at concentrations of 2000 ppm or greater. The limiting ridge that forms the junction of the forestomach squamous mucosa with the glandular gastic mucosa appeared enlarged in all rats in the 4000 and 8000 ppm dose groups.

Histopathological findings that correspond to the gross lesions consisted of minimal to moderate hyperplasia of the squamous mucosa at the site of the limiting ridge. This lesion was characterised by a thickening and increased folding of the squamous mucosa; hyperkeratosis was also a component of the squamous cell hyperplasia. The increased incidence and severity of this lesion were dose related. When this lesion was more severe, there was often an increase in the number of inflammatory cells and/or edema in the lamina propria of the limiting ridge. There was no evidence or erosion/ulceration and no lesions were present in other areas of the squamous mucosa.

Other histopathological findings were present in the liver and kidney in both sexes. There was a dose related increase in the incidence and severity of chronic-active inflammation in the liver of male and female rats. This lesion was present in most rats in the 4000 and 8000 ppm groups and in one male in the 2000 ppm group and was characterised by multiple foci of a mixture of mononuclear inflammatory cells, primarily macrophages. These foci of inflammation occurred primarily in the periportal portion of the hepatic lobules. Necrosis of one to several hepatocytes was often observed adjacent to or within the foci of inflammation.

Chemical related cytoplasmic alteration was present in the kidneys of male and female rats at doses of 2000 ppm and greater. This lesion was morphologically similar in both sexes but was less severe in females. A few droplets were also present in the tubule lumina of female rats. In treated male rats, the protein droplets were much larger and more numerous than those in the control males or in the treated females, and many large droplets were present in the tubule lumina. These droplets stained strongly positive for protein but were negative by iron, PAS and acid-fast staining methods. Results of a-2-microglobulin staining of kidney sections from male and female control and high dose rats were inconclusive. While the kidneys of male rats stained positive for a-2-microglobulin, there were no clear qualitative differences in staining between treated and control rats. Also present in the kidneys of rats in the high dose groups was minimal nuclear enlargement in renal tubule cells. Degeneration of the renal tubule epithelium was present in three females in the 8000 ppm group.

Tissue Metal Level Analysis:

The results of the analysis indicated that copper accumulated in the liver and kidney in a dose related manner and was accompanied by an accumulation of zinc in these tissues. Copper concentrations were significantly increased in the kidney and liver of rats in all treated groups. Copper levels were also significantly elevated in the plasma and testis of rats in the three highest dose groups. Significant increases in zinc concentration in the kidney
and liver were noted in animals in the three highest dose groups, and concentrations of calcium in plasma were significantly decreased in the 4000 and 8000 ppm groups. Significant increases in magnesium were noted in the kidney and plasma of rats receiving 2000 ppm copper sulphate as well as in the plasma of rats receiving 8000 ppm copper sulphate. For further information please refer to attached Table 4.

Nonneoploastic lesions:

A summary of nonneoplastic lesions is presented in the attached document Table 6.

Supplemental histological examination:

Liver and kidneys of rats were stained for the presence of copper. Positive staining in liver sections was limited to 4000 and 8000 ppm. At 8000 ppm, staining in the liver had a clear periportal to midzonal distribution and consisted of a few to numerous (10-20) red granules of 1-2 mm in the cytoplasm of hepatocytes. In addition there was minimal staining of the cytoplasm in some of the cells in the inflammatory foci. At 4000 ppm, staining of the hepatocytes was limited to the periportal area and there was a marked reduction in the number of cells stained and the number of granules per cell.

Kidney sections also stained positive for copper only in the two highest dose groups. Staining consisted of red granules in the cytoplasm of the renal tubule epithelium and a diffuse or stippled red staining of the protein droplets in the cytoplasm and the tubule lumen. However, many of these (especially in the 4000 ppm group) did not stain positive for copper. Positive staining of the kidney tubule cells was limited to the cortex; there was not staining in the medullary rays outer and inner medulla. Sections of heart and spleen showed no positive stained in any dose group.

Sections of spleen from 4 rats per dose group were evaluated for iron. In the 8000 ppm groups there was only a few iron-positive granules in the cytoplasm of macrophages in the red pulp. The reduction in iron-positive material in the spleens from the 2000 and 4000 ppm groups was much less
prominent than the 8000 ppm group, but a minimal decrease was evident compared to the controls.

Transmission electron microscopy of the livers of both sexes showed that within the cytoplasm of hepatocytes in the periportal area, there was degenerative changes consisting of increased numbers of secondary lysosomes, many of which were enlarged and contained clear, non-staining crystalline
structures and electron-dense material. Kidneys had mild to marked increases in the number and size of electron dense protein droplets in the cytoplasm of the proximal convoluted tubule epithelium. In addition to changes in the size and number, many droplets in the kidneys of male rats had irregular crystalline shapes.

Sperm Morphology and Vaginal Cytology:

There were no significant findings in males or females. See attached Table 7.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
other: Absence of hyperplasia and hyperkeratosis of the forestomach, inflammation of the liver
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
other: Presence of hyperplasia and hyperkeratosis of the forestomach, inflammation of the liver

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The LOAEL for forestomach lesions was 2000 ppm for both males and females.

The LO(A)EL for liver damage was 2000 ppm for males and 4000 ppm for females.

The LO(A)EL for kidney damage was 2000 ppm for males and 1000 ppm for females. This finding was considered not to be toxicologically significant, as the effect is specific to the rat.

The NO(A)EL for forestomach lesions was 1000 ppm for both males and females.

The NO(A)EL for liver damage was 1000 ppm for males and 2000 ppm for females.
Executive summary:

Materials and Methods:

The aim of the study was to examine the effect of copper sulphate (0, 500, 1000, 2000, 4000 or 8000 ppm) administered to male and female B6C3F1mice in feed for 13 weeks.  The test organisms were observed throughout the study for signs of clinical toxicity, mortality, bodyweight changes and food consumption.  Throughout the study blood and urine samples were collected to determine haematology, clinical chemistry and urinalysis parameters and tissue metal level. At the end of the study period all animals were sacrificed and subject to pathological examinations to determine any histological, sperm morphology or vaginal cytology abnormalities. The study was conducted to a methodology developed by the US National Toxicology Programme specifically for the test.  The study was conducted in accordance with GLP.

Results and Discussion

Haematological, clinical chemistry and urinalysis evaluations of rats revealed variable chemical-related changes that were, for the most part, restricted to the 4000 and 8000 ppm groups.  Increases in serum alanine aminotransferase and sorbitol dehydrogenase activities in both sexes were indicative of hepatocellular damage, as were increases in 5’-nucleotidase and bile salts in males.  Decreases in mean cell volume, hematocrit and haemoglobin indicated the development of a microcytic anaemia, while increases in reticulocyte numbers at the same time points suggested a compensatory response to the anaemia by the bone marrow.  Increases in urinary glucose and N-acetyl-β-D-glucosaminidase (a lysosome enzyme) and asparate aminotransferase (a cytosolic enzyme) were suggestive of renal tubule epithelial damage.

Dose related increases in copper occurred in all male rat tissues examined.  These increases were accompanied by increases in zinc in the liver and kidney.  Plasma calcium was significantly reduced in the 4000 and 8000 ppm groups, and there was a trend towards reduction in calcium in the kidney and testis as well.  In the 8000 ppm group, plasma magnesium was significantly increased relative to the controls.

 

Rats in the three highest dose groups had hyperplasia and hyperkeratosis of the forestomach, inflammation of the liver and increases in the number and size of protein droplets in the epithelial cytoplasm and the lumina of the proximal convoluted tubules.  Many of the droplets in the male kidneys were large and had irregular crystalline shapes.  These droplets stained strongly positive for protein but were negative for iron, PAS, and acid-fast (lipofuscin) staining methods.  Α-2-microglobulin was present in the droplets of male rats, but there was no dose-related qualitative difference in the content of this protein.  In the 4000 and 8000 ppm groups, copper was distributed in a periportal to midzonal pattern in the liver and was restricted to the cytoplasm of the proximal convoluted tubule epithelium in the kidney.  Copper was present in some, but not all, of the protein droplets.  Transmission electron microscopy of the livers of rats of each sex revealed increases in the number of secondary lysosomes in hepatocytes in the periportal area.