Reaction mass of copper oxide and manganese dioxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

year of publication: 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Subchronic toxicity of cupric sulfate administered in drinking water and feed to rats and mice

Author:

Hébert, C.D.; Elwell, M.R.; Travlos, G.S.; Fitz, C.J.; Bucher, J.R.

Year:

1993

Bibliographic source:

Fundamental and Applied Toxicology, 21, 461-475

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1993

Reference 3

Reference Type:

review article or handbook

Title:

European Union Risk Assessment Report COPPER, COPPER II SULPHATE PENTAHYDRATE, COPPER(I)OXIDE, COPPER(II)OXIDE, DICOPPER CHLORIDE TRIHYDROXIDE CAS No: 7440-50-8, 7758-99-8, 1317-39-1, 1317–38–0, 1332-65-6 - VOLUNTARY RISK ASSESSMENT

Author:

European Copper Institute

Year:

2007

Bibliographic source:

https://echa.europa.eu/de/copper-voluntary-risk-assessment-reports

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Copper (II) sulfate, pentahydrate

EC Number:

616-477-9

Cas Number:

7758-99-8

Molecular formula:

CuSO4.5H2O

IUPAC Name:

Copper (II) sulfate, pentahydrate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- purity: 99-100%
- Source: JT Baker, Phillipsburg, NJ, USA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: all dose formulations were within +/- 10% of theoretical concentrations

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dosed feed formulations were prepared by first making a premix of powdered cupric sulfate mixed with an equal volume of meal flour (NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners, PA, USA) sifted through a USS No. 80 sieve) and then mixing the premix into an appropriate amount of feed.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA, USA)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: yes
- Housing: 5 rats per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 - 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3 +/- 5
- Humidity (%): 52 +/- 21
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

92 days

Frequency of treatment:

continuous with feed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg diet

Remarks:

cupric sulphate pentahydrate

Doses / concentrations 2

Dose / conc.:

500 mg/kg diet

Remarks:

cupric sulphate pentahydrate

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg diet

Remarks:

cupric sulphate pentahydrate

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg diet

Remarks:

cupric sulphate pentahydrate

Doses / concentrations 5

Dose / conc.:

4 000 mg/kg diet

Remarks:

cupric sulphate pentahydrate

Doses / concentrations 6

Dose / conc.:

8 000 mg/kg diet

Remarks:

cupric sulphate pentahydrate

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

Additional dosing of 10 rats/sex/exposure group for special studies (intermediate time points for clinical pathology determinations).

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at least at end of study (day 92)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 5, 21 and end of study (day 92)
- Anaesthetic used for blood collection: Yes (Animals were anesthetized with 70% CO2:30% O2 and blood samples were collected from the retroorbital sinus.)
- Animals fasted: No
- Parameters examined are listed below in section other.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 5, 21 and end of study (day 92)
- Animals fasted: Not specified
- Parameters examined are listed below in section other.

URINALYSIS: Yes
- Time schedule for collection of urine: day 19 and end of study (day 92)
- Metabolism cages used for collection of urine: YES (overnight collection, urine samples were collected in chilled collection cups)
- Animals fasted: Not specified
- Parameters examined are listed below in section other.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Automated hematologic analyses were performed using an Ortho ELT-8 Laser Hematology Counter (Ortho Instruments, Westwood, MA). Differential leukocyte and nucleated red blood cell counts were determined by microscopic examination of blood smears stained with a modified Wright-Giemsa stain. Reticulocyte counts were determined by microscopic examination of blood smears stained with the supravital stain new methylene blue. Biochemical analyses for clinical chemistry and urinalysis were performed using a Hitachi 704 Automatic Chemistry Analyzer (Boehringer-Mannheim, Indianapolis, IN).

The following assays were performed on serum using reagents and methods provided by the manufacturer: urea nitrogen (UN), creatinine, alanine aminotransferase (ALT), alkaline phosphatase
(AP), sorbitol dehydrogenase (SDH), 5'-nucleotidase, bile salts, albumin, and total protein. Urine samples were evaluated for clarity, color,
volume, specific gravity, and the presence of formed elements. Total protein, glucose, creatinine, aspartate aminotransferase (AST), and A'-acetyl-
/3-glucosaminidase (NAG) were measured using a Hitachi chemistry analyzer.

Sections of liver and kidney from selected rats in the 13-week feed study were stained for copper using the rhodanine method (Sheehan
and Hrapchak, 1980), and sections of spleen were stained for iron using Perl's stain. Sections from kidneys of selected rats in
the 13-week feed study were stained for carbohydrate (PAS method), protein (Mallory-Heidenhain method), lipofuscin (AFIP method), and alpha2-microglobulin (immunohistochemistry). Liver sections from the same rats
were stained for lipofuscin, and kidney and liver sections from rats of both sexes were examined by transmission electron microscopy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
A complete necropsy was performed on all early death animals and at the termination of each study on all treated and control animals. Body
weights and the weights of the liver, thymus, right kidney, right testis, heart, lungs, and brain were determined. Organs and tissues were examined for gross lesions and fixed in 10% neutral buffered formalin.

HISTOPATHOLOGY: Yes
A standard battery of 34 organs and tissues (NTP, 1992) were trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Complete histopathologic examinations were carried out on all control animals, all early death animals, all animals in the highest dose group with at least 60% survivors, and all animals in higher dose groups. Organs identified as target organs (liver, kidney, and forestomach) were examined to a no-effect level in lower exposure groups.

Other examinations:

Tissue metal level analyses.
In the 13-week feed study, samples of plasma, liver, kidney, and testis were collected on Day 92 for determination of copper, magnesium, zinc, and calcium content. Samples were weighed, digested in a nitric acid-perchloric acid mixture, and heated until
evolution of nitric acid was complete. The residue was dissolved in 10% perchloric acid, and an aliquot was analyzed by Inductively Coupled Plasma-Atomic Emission Spectroscopy.

Sperm morphology and vaginal cytology evaluations
were performed. The control and the three highest exposure groups for each species were evaluated. Epididymal sperm motility was evaluated at necropsy as previously described (NTP, 1991). Vaginal cytology was evaluated in animals during the week preceding necropsy, using procedures outlined in the National Toxicology Program's SM VCE protocol (Morrissey et ai, 1988). Vaginal saline lavage was performed on females for 12 consecutive days prior to scheduled termination, and the relative preponderance of leukocytes, nucleated epithelial cells, and large squamous epithelial cells in the lavage fluid were used to identify the stages of the estrual cycle.

Statistics:

Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which are approximately normally distributed, were analyzed using the parametric multiple comparisons procedures of Williams (1971, 1972) and Dunnett (1955). Clinical chemistry and hematology data, which typically have skewed distributions, were analyzed using the nonparametric multiple comparisons methods of Shirley (1977) and Dunn (1964). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams,Shirley) was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose response (Dunnett, Dunn).
The outlier test of Dixon and Massey (1951) was employed to detect extreme values. Because the vaginal cytology data are proportions, an arcsine transformation was used to bring the data into closer conformance with normality assumptions. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for the simultaneous equality of measurements across dose levels.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity that could be directly attributed to cupric sulfate toxicity were observed during the course of the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All rats survived to the end of the 13-week study, with the exception of one female rat in the 1000 ppm group that was accidentally killed during dosing.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weight gains were significantly depressed in male rats in the two highest dose groups (4000 and 8000 ppm) and in female rats in the highest dose group (8000 ppm).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Average feed consumption by male and female rats in the 8000 ppm dose groups was slightly depressed relative to controls; however, feed consumption in all other treatment groups of rats was similar to that of controls. Average daily compound consumption increased proportionally with increasing concentrations of cupric sulfate in the feed.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

for details see field "Details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

for details see field "Details on results"

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

for details see field "Details on results"

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In general, absolute organ weights of rats in the two highest dose groups were depressed, while relative organ weight to body weight ratios for treated groups were similar to those of the controls or increased with decreasing mean body weights. These
changes could be attributed to the lower final mean body weights in the higher dose groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Forestomach:
In the 13-week feed study, chemical-related gross lesions were present in the forestomachs of rats receiving cupric sulfate at concentrations of 2000 ppm or greater. In rats, this lesion was characterized by an enlargement of the
limiting ridge in all animals in the 4000 and 8000 ppm groups and in seven females and nine males in the 2000 ppm groups.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Forestomach:
Histopathologic findings consisted of minimal to moderate hyperplasia of the squamous mucosa at the site of the limiting ridge. This lesion was characterized by a thickening and increased folding of the squamous mucosa; hyperkeratosis was also a component of the squamous cell hyperplasia (Fig. 1). The incidence and severity of this lesion were dose related (Table 4). When this lesion was more severe (moderate grade), 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 of erosion/ ulceration in the forestomach, and no lesions were present in other areas of the forestomach mucosa.

Liver:
There was a dose-related increase in the incidence and severity of chronic-active inflammation in the livers of 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 characterized 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 1 to several hepatocytes was often seen adjacent to or within the foci of inflammation. Transmission electron microscopy (TEM) of the livers of male and female rats showed that within the cytoplasm of hepatocytes in the periportal area, there were degenerative changes consisting of increased numbers of secondary lysosomes, many of which were enlarged and contained clear, nonstaining crystalline structures and electron-dense material. Livers of rats in all dose groups were stained for the presence of copper, and positive staining was limited to the 4000 and 8000 ppm groups. At 8000 ppm, staining in the liver had a clear periportal to midzonal distribution and consisted of a few to numerous (10 to 20) red granules of 1 to 2 mm size in the cytoplasm of hepatocytes. In addition, there was minimal staining of some of the cells in the inflammatory foci. At 4000 ppm, staining was periportal, and there was a marked reduction in the number of cells stained and the number of granules per cell.

Kidney:
Microscopic and ultrastructural examinations revealed an increase in the size and number of cytoplasmic protein droplets present in the epithelium of proximal convoluted tubules of kidneys of rats at doses of 2000 ppm and higher. This lesion was morphologically similar in both sexes, but was less severe in females than in males. In treated male rats, the protein droplets were much larger and more numerous than those in control males or in treated females, and many large droplets were present in the tubule lumina. A few droplets were also present in the tubule lumina of female rats. These droplets stained strongly positive for protein but were negative by iron, PAS, and acid-fast (lipofuscin) staining methods. Kidney sections from male control and high-dose rats stained positive for alpha2-microglobulin, but there were no clear quantitative differences in staining between treated and control rats. In addition to changes in size and number, many of the protein droplets in the kidneys of male rats had irregular crystalline shapes; however, the large crystalline structures seen in the kidneys of males were not present in females. Also present in the kidneys from the high-dose groups was minimal nuclear enlargement (karyomegaly) in renal tubule cells. Degeneration of renal tubule epithelium was present in three females from the 8000 ppm dose group. Other ultrastructural changes in kidney epithelium were minimal; there was minimal degeneration evidenced by margination of nuclear chromatin in a few renal tubule cells.
Kidneys sections from male and female rats in all dose groups were stained for the presence of copper, and positive staining was seen only in the two highest dose groups (4000 and 8000 ppm). 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 protein droplets, especially in the 4000 ppm groups, did not stain positive for copper. Positive staining of kidney tubule cells was limited to the cortex, with no staining in the medullary rays, outer medulla, or inner medulla. In addition to kidney and liver, sections of heart and spleen were present on all copper-stained slides; however, no positive staining
was present in either of these tissues in any dose group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- effects on iron storage:
Sections of spleen from four rats per dose group were evaluated for the presence of iron (hemosiderin) with the Perl's iron stain. In the 8000 ppm groups of male and female rats there were only a few iron positive granules in the cytoplasm of macrophages in the red pulp. The reduction in iron-positive material in spleens of rats from the 2000 and 4000 ppm groups was much less prominent than that in the 8000 ppm group but a minimal decrease was evident when compared to the normal amount of iron-positive granules in the spleen of controls.

- tissue metal levels:
Samples of liver, kidney, testis, and plasma from all male rats were analyzed for copper, zinc, magnesium, and calcium. Copper accumulated in the liver and kidney in a dose-related manner and was accompanied in the three highest dose groups by an accumulation of zinc in these tissues (Table 7). Copper levels were also elevated in the plasma and testis of rats in the three highest dose groups. A significant dose-related decrease occurred in plasma calcium, accompanied by a similar, although not statistically significant, decrease in calcium in the kidney. A significant increase in magnesium was noted in the plasma of high dose rats.

- Sperm morphology and vaginal cytology
Evaluations were performed on base-study rats in the 0, 500, 2000, and 4000 ppm groups. No change in testis, epididymis, or cauda epididymis weight, spermatid counts, or sperm motility were seen in males of either species at any dose of cupric sulfate.
Similarly, evaluation of vaginal cytology revealed no cupric sulfate-induced changes in estrous cycle length or in the relative amount of time spent in each phase of the cycle in females

Details on results:

Hematology and clinical chemistry evaluations were conducted in rats on Days 5, 21, and 92, and urinalysis was conducted on Days 19 and 90.
Significant changes in these clinical pathology parameters were noted in rats of both sexes at all time points, and these changes were, for the most part, limited to the 2000, 4000, and 8000 ppm dose groups. There is one table that shows the clinical pathology data for the day of termination (Day 92) only. On Day 5, significant increases in hematocrit (HCT), hemoglobin (HGB), platelet count, and erythrocytes (RBC), were seen in the 8000 ppm dose group of both sexes. These increases are consistent with a relative polycythemia related to dehydration. Also on Day 5, significant decreases in reticulocyte count, mean cell volume (MCV), and mean cell hemoglobin (MCH) were noted in high-dose animals. By Day 21, however, HCT and HGB levels 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 (2000,4000, and 8000 ppm). Decreases in MCV and MCH occurred on Day 21 in the three highest dose groups. These decreases in HCT, HGB, MCV, and MCH persisted until the end of the study. On Day 92, significant increases in RBCs and reticulocytes were noted in high dose males.
Significant elevations in ALT activity on Days 5, 21, and 92 and in sorbitol dehydrogenase (SDH) activity on Days 21 and 92 were indicative of hepatic injury that occurred early and persisted throughout the study. Decreases in AP activity were noted on Days 5 and 21 in both sexes in the two highest dose groups, but AP activity had returned to control levels by Day 92. At early time points, significant decreases in 5'-nucleotidase (5-NT) activity occurred in high-dose rats. However, by Day 92, this trend had reversed, and 5-NT activity was significantly increased in males receiving 4000 or 8000 ppm cupric sulfate. In the two highest dose groups at all time points, total protein and serum albumin concentrations were significantly decreased and UN was significantly increased relative to controls. Significant changes in urinalysis parameters included an increase in AST activity on Days 19 and 90, and an increase in NAG activity on Day 90 in rats in the two
highest dose groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Histopathological changes in forestomach, liver and kidney. Changes in clinical chemistry and urine analysis.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg diet

System:

other: Histopathological changes in forestomach, liver and kidney. Changes in clinical chemistry and urine analysis.

Organ:

kidney

liver

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

In this 13-week repeated dose toxicity study male and female Fisher F334/N rats received 0, 500, 1000, 2000, 4000, and 8000 ppm of cupric sulfate pentahydrate (CASRN 7758-99-8) via feed. Based on histopathological changes in forestomach, liver and kidney and changes in clinical chemistry and urine analysis, the NOAEL of this study was determined to be 1000 ppm (mg/kg diet).

Executive summary:

Within this subchronic toxicity study similar to a guideline study (OECD TG 408) male and female F344/N rats were dosed with cupric sulfate pentahydrate via feed at concentrations of 0, 500, 1000, 2000, 4000, and 8000 ppm (mg/ kg diet). Animals were evaluated for histopathology, clinical pathology, reproductive toxicity, and tissue metal accumulation, and target organs were examined by a variety of special stains and by electron microscopy to characterize the observed lesions.

Cupric sulfate concentrations of 4000 ppm and higher caused significant reductions in body weight gain. Hyperplasia and hyperkeratosis of the limiting ridge of the forestomach were present (starting at concentration of 2000 ppm). A dose-related increase in inflammation in the liver and changes in clinical chemistry parameters which were indicative of hepatocellular damage and cholestasis were seen. Histologic changes in the kidneys of rats consisted of a dose-related increase in the number and size of eosinophilic protein droplets in the epithelial cytoplasm and the lumina of the proximal convoluted tubules. Droplets were larger and more numerous in males than in females. Urinalysis results were suggestive of renal tubular epithelial damage. The present study confirms the liver and kidney toxicity of cupric sulfate in rats and extends these results to describe the occurrence of forestomach lesions in rats.

In addition, iron staining of spleens from treated animals indicated a marked depletion of iron stores in both male and female rats, while hematologic and clinical chemistry alterations were indicative of a microcytic anemia.

Cupric sulfate produced no adverse effects on any of the reproductive parameters measured in rats of either sex.

The NOAEL identified in this study thus is determined to be 1000 ppm (mg cupric sulfate pentahydrate/ kg diet).