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REACH

Copper

EC number: 231-159-6 | CAS number: 7440-50-8

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

ORAL : The pivotal repeat dose study was a 90-day study by the oral route with copper sulphate pentahydrate.  In rats and mice, ingestion of copper sulphate pentahydrate produced forestomach lesions that could be to the irritant effects of the compound.  The NOAEL for this effect was 16.7 mg Cu/kg bw/day in rats and 97 and 126 mg Cu/kg bw/day in male and female mice respectively. In rats inflammation of the liver was observed.  The NOAEL’s for liver and kidney damage were 16.7 mg Cu/kg bw/day in rats.  This is the pivotal study and the NOAEL of 16.7 mg Cu/kg bw/day will be used in the risk characterisation. 
INHALATION : the pivotal study in the 28 days rat inhalation study on Cu2O.  The main conclusion is observed effects at all doses (0.2-2 mg/m3) but no adverse effects at the highest dose tested (2 mg/m3).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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.

Qualifier:

equivalent or similar to guideline

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:

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system 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

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

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:

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: none

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.

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.

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Sex:

male/female

Basis for effect level:

other: absence of hyperplesia 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: presnce of hyperplesia and hyperkeratosis of the forestomach, inflmmation of th liver

Critical effects observed:

not specified

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 rat 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.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 16.7 mg/kg bw/day
Study duration:: subchronic
Species:: rat
Quality of whole database:: High quality study (Q1) with NOAEC

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with Good laboratory Practice and internationally accepted guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI.
- Age at study initiation: The animals were approximately 9.5 weeks old at the initiation of exposures.
- Weight at study initiation: Individual body weights ranged from 297 g to 359 g for males in the core study groups, and from 298 g to 360 g for males in the satellite study groups at randomization.
- Water (e.g. ad libitum): ad libitum.

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks:

Filtered air. Mean temperature and mean relative humidity between 20°C to 26°C and 30% to 70%, respectively.

Remarks on MMAD:

MMAD / GSD: 1.725 µm MMAD (mass median aerodynamic diameter) +/- 1.73 µm GSD (geometric standard deviation).

Details on inhalation exposure:

A dust aerosol atmosphere of the test substance was generated using a single generation system consisting of a jet mill air micronizer (model 00, Jet-O-Mizer, Fluid Energy Aljet, Hatfield, PA) operating as a particle size reduction and dispersion device. The test substance powder was delivered to the jet mill at a constant rate using an auger-type feeder (Schenck AccuRate, Inc., Whitewater, WI). The resulting aerosol was passed through a cyclone for removal of large particles and aggregates and then delivered to the primary distribution chamber. This chamber was used to distribute aerosol to cyclones and inlets for the 0.8 and 2.0 mg/m3 exposure chambers and to a dilution stage and the secondary distribution chamber. From the secondary chamber, diluted aerosol was distributed to cyclones and inlets for the 0.2 and 0.4 mg/m3 exposure chambers. Distribution was achieved using compressed air-powered transvector jets (VDF-100, Vaccon Company, Inc., Medway, MA) to draw and dilute controlled amounts of the aerosol from the distribution chambers. At each chamber inlet, chamber supply air was added to dilute the aerosol stream to the desired concentration.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days, 6 hours per day.

Frequency of treatment:

5 days per week.

Remarks:

Doses / Concentrations:
0.2, 0.4, 0.8, 2.0 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0.21, 0.41, 0.8, 2.0 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

For the core study, 20 males and 20 females per concentration (control and high) and 10 males and 10 females per concentration (low, med-low, med-high).

For the satellite study, 10 males and 10 females per exposure (control and high) and time-point (1, 2, or 3 weeks)

Control animals:

yes, sham-exposed

Details on study design:

Adaptation to test substance exposure was assessed by sacrificing animals at intermediate time-points (satellite group), at week 0, week 1, and week 2.

Observations and examinations performed and frequency:

MORTALITY:
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS:
Clinical examinations were performed twice daily on the days of exposure: prior to exposure and 0-1 hour following exposure (designated 1 hour post-exposure for report presentation purposes). On non-exposure days and throughout the recovery period, the animals were observed once daily.

BODY WEIGHT:
Individual body weights were recorded approximately weekly during the pretest period, beginning at least 1 week prior to test substance exposure, on the day of randomization, on study days 0, 4, 11, 18, and 25 (prior to the first, fifth, tenth, fifteenth, and twentieth exposures, respectively), and weekly during the recovery period. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the days of the interim, primary, and recovery necropsies.

FOOD CONSUMPTION:
Individual food consumption was recorded approximately weekly for all animals, beginning at least one week prior to test substance exposure and throughout the study. Food intake was calculated as g/animal/day for the corresponding body weight intervals. When food consumption could not be measured for a given interval (due to spillage, weighing error, obvious erroneous value, etc.), the appropriate interval was footnoted as "NA" (Not Applicable) on the individual tables.

WATER CONSUMPTION:
Reverse osmosis-treated drinking water, delivered by an automatic watering system, was provided ad libitum throughout the study, except during exposure.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY:
All animals of core group and recovery group.
Time point: end of study (week 3), end of recovery (week 16).
Parameters: Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count, Reticulocyte count (percent and absolute), Mean platelet volume, Red cell distribution width, Hemoglobin distribution width, Platelet estimate, Red cell morphology.
Differential leukocyte count (percent and absolute of the following cell types) -Neutrophil, -Lymphocyte, -Monocyte, -Eosinophil, -Basophil, -Large unstained cell.

CLINICAL CHEMISTRY:
In bronchoalveolar lavage fluid of all study animals (core, satellite, recovery).
Time points: end of week 3 (core), end of week 0, 1, 2 (satellite), end of week 16 (recovery).
Total and differential cell counts for: Alveolar macrophages, Neutrophils, Lymphocytes, Eosinophils, Basophils, Epithelial cells.
Lactate dehydrogenase (LDH), Total Protein.

URINALYSIS: No.

Sacrifice and pathology:

GROSS PATHOLOGY:
All dose groups.
Complete necropsies were conducted for 5 animals/sex/group at the interim and primary necropsies and 5 animals/sex in the control and high concentration groups at the recovery necropsy.
The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

ORGAN WEIGHTS:
All dose groups.
organs: Adrenal glands, brain, epididymides, heart, kidneys, liver, lungs (prior to BAL and inflation with fixative), ovaries and oviducts, spleen, testes, thymus (paired organs were weighed together).

HISTOPATHOLOGY:
Microscopic examinations were performed on the left lung, nasal tissues, mediastinal and bronchial lymph nodes, liver, kidneys, brain, and gross lesions for all animals.

Other examinations:

Reversibility of effects was examined after a 13-week recovery period (no exposure to test substance), in the control and high-dose animals (recovery group).

Adaptation to test substance exposure was assessed by sacrificing animals at intermediate time-points (satellite group), at week 0, week 1, and week 2.

Copper levels in lung tissue, lung lavage fluid, liver, brain of all animals were measured by atomic absorption spectroscopy.

Wet/dry lung weight ratio to assess lung oedema in all animals.

Clinical chemistry and cytology of bronchoalveolar lavage fluid of all animals.

Statistics:

Statistical analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. For statistical evaluation of the satellite groups, each test substance-treated group was compared to the control group exposed for the same length of time (number of exposures). Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly.

Body weight, body weight change, food consumption, hematology, organ weight, lung wet weight/dry weight, and bronchoalveolar lavage fluid total protein, lactate dehydrogenase, total cell counts, and differential cell counts (percent) data were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group.

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:

not examined

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical signs and no mortalities.

BODY WEIGHT AND WEIGHT GAIN
In the core group, there were no test substance-related effects on mean body weights during or at the end of the 4-week exposure, with the exception of transient lower body weight gains in the 2 mg/m3 group males from study days 0 to 4 and 4 to 11. These were statistically significant when compared to the control group, and were considered to be related to test substance exposure (Table 1).

In the satellite group, there were no test substance-related effects on body weights following 1, 2, or 3 weeks of exposure at 2.0 mg/m3.

FOOD CONSUMPTION
Test substance-related slightly lower mean food consumption than the control group was noted for the 2 mg/m3 group males of the core group from study day 0 to 4. There were no other statistically significant differences in the core group when the control and test substance-treated groups were compared.

Food consumption was unaffected by test substance exposure during 1, 2, or 3 weeks of exposure at 2.0 mg/m3 in the satellite group.

FOOD EFFICIENCY
Not examined.

WATER CONSUMPTION
Not examined. Water was provided ad libitum.

OPHTHALMOSCOPIC EXAMINATION
Not examined.

HAEMATOLOGY
Higher mean neutrophil counts than the control group were observed in the 0.2, 0.4, 0.8, and 2.0 mg/m3 groups. Mean absolute neutrophil counts were higher for males (↑61.7%-112.1%) and females (↑52.3%-120.2%) at all exposure levels of the test substance. These higher mean values were statistically significant only for the 0.4 and 0.8 mg/m3 group males and the 0.8 and 2.0 mg/m3 group females. There were no other test substance-related hematology alterations.

At the study week 16 recovery blood collection, there were no test substance-related hematology alterations.

CLINICAL CHEMISTRY
BALF clinical chemistry. Concentration-dependent increases in lactate dehydrogenase (LDH) and total protein values were noted in males and/or females following 4 weeks of exposure at 0.2, 0.4, 0.8, and 2.0 mg/m3. These alterations resolved during the recovery period in the 2.0 mg/m3 group.

URINALYSIS
Not examined.

NEUROBEHAVIOUR
Not examined.

ORGAN WEIGHTS
Mean absolute and relative lung weights were statistically significantly higher in a dose-related manner at exposure levels ≥ 0.4 mg/m3. The magnitude of differences from control for absolute weights ranged from 28.3% to 81.4% for males and 30.6% to 89.8% for females. At the study week 16 recovery necropsy, the mean absolute and relative lung weights for the 2.0 mg/m3 group males and females remained slightly higher than mean control group values (10% and 9%, respectively, for absolute lung weights), but the differences were much less than at the primary necropsy, suggesting a return toward normal lung weights.

The bronchial and mediastinal lymph nodes exhibited higher mean absolute and relative weights at ≥ 0.4 mg/m3. The bronchial lymph nodes weights were statistically significant compared to the control group for the 0.8 and 2.0 mg/m3group males and females, and the mediastinal lymph nodes weights were statistically significant for the 2.0 mg/m3 group males. At the study week 16 recovery necropsy, mean absolute and relative weights for mediastinal and bronchial lymph nodes of the 2.0 mg/m3 group animals showed no statistical differences from control group, and were considered normal.

The lung wet/dry weight ratio did not increase at any exposure, indicating that there was no lung edema at any exposure level or duration. At the highest dose (2 mg/m3), a small but significant reduction of the lung wet/dry weight ratio was observed (↓14% in males, ↓8% in females) at the end of the 28-day exposure period, when compared to controls. There was no difference in the wet/dry lung weights ratio between control and 2 mg/m3 animals after the recovery period.

There were no other test substance-related effects on any other organ weights.

GROSS PATHOLOGY
At the primary necropsy, 2 males from the 0.8 mg/m3 group had enlarged bronchial lymph nodes. This finding correlated microscopically with lymphoid hyperplasia for 1 of these animals (male no. 7085) and was considered to be test substance-related. No other macroscopic observations were considered to be related to test substance exposure. At the study week 16 recovery necropsy, there were no macroscopic observations considered to be related to test substance exposure.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were histopathological findings in the following organs: lungs, lymph nodes and nasal cavity.

Lung: Alveolar histiocytosis was observed in an exposure-related pattern in all animals (from minimal at the lowest exposure to moderate in the high exposure group). Intermixed with the alveolar macrophages were minimal to mild numbers of neutrophils, indicating acute inflammation, and evident in all animals at ≥ 0.4 mg/m3. Eosinophilic cellular debris often accompanied the alveolar histiocytosis and acute inflammation. The cellular debris appeared to be degenerative alveolar macrophages. Alveolar septal epithelium appeared normal. Mononuclear perivascular infiltrates were increased and clearly test substance-related for males at ≥ 0.8 mg/m3 and for females at ≥ 0.4 mg/m3. The respiratory epithelium lining the airways and alveoli (ciliated epithelium and type I pneumocytes) did not appear (by routine light microscopy) to be adversely affected. There was no evidence of direct epithelial damage or subsequent regeneration. The cellular debris within alveoli appeared to be degenerative alveolar macrophages. Pulmonary edema fluid was not a histologic feature of the pulmonary response, as was confirmed by the wet/dry lung weight ratio.

In ancillary work, Masson Trichrome staining suggested a very slight increase in collagen in the high dose animals (2 mg/m3), with minimal and occasionally mild staining also in the control groups. The staining severity scores between treatment and control, as well as after recovery, did not differ significantly from each other. Consequently, all doses were re-assessed by computerized morphometric analyses of lung samples to more objectively quantify lung fibrosis. Mean collagen area percentages were higher for the 0.8 mg/m3 group males (↑33.8%) and for the 2.0 mg/m3 group males and females (↑23.9% and ↑16%, respectively). These differences were not statistically significant, and did not increase with dose. For the 0.2 and 0.4 mg/m3 group males, the mean collagen area percentages were slightly higher (↑10.1 %-12.5%; not statistically significant). Mean collagen area percentages for the 0.2, 0.4, and 0.8 mg/m3 group females were not remarkably altered by test substance exposure, yet lung dry weights were higher for the 0.4 and 0.8 mg/m3 group females. Since collagen staining and lung dry weight did not appear to be correlated, it was proposed that macrophages and/or neutrophils cells may contribute to the dry lung weight measurements.

Following the 13-week recovery period, the mean collagen area percentage for the 2.0 mg/m3 group females remained slightly higher (↑11.2%; 30% mean collagen area percentage in control females, and 33% in test article treated females). This difference was not statistically significant and was reduced from the higher primary necropsy value. For the 2.0 mg/m3 group males at the recovery evaluation, the mean collagen area percentage was negligibly different (↑1.9%) from the control group mean. However, the control group mean was higher than previously seen at the primary necropsy, with control animals displaying 38.7% mean collagen area percentage in lung and test article treated animals (high dose) displaying 39.5%. This increase in collagen staining in control animals after the recovery period is an unexplained finding, but may reflect the staining seen in the control groups in original examination (Masson Trichrome). Overall, the morphometric analysis shows that there is no dose-response in collagen staining, as well as some unexplained staining in control animals.

Taking together the outcome of the pathology reports and the computerized analysis, there is no significant effect on collagen content of the lung.

Lymph nodes: Lymphoid hyperplasia of the bronchial lymph node was present in the majority of males and females at ≥ 0.4 mg/m3. The affected lymph nodes were clearly larger in section on the glass slides and microscopically had expanded paracortical populations of lymphocytes.

Nasal cavity: The nasal cavity was less affected than the lung by cuprous oxide inhalation, and findings were seen only in some test substance-exposed males. Sporadic minimal focal olfactory epithelium degeneration was observed in one male each in the 0.8 and 2 mg/m3 exposure group. Minimal to mild subacute inflammation was evident in Nasal Levels II and III of several 2.0 mg/m3 group males from the core study. Similar to the lung, the ciliated respiratory epithelium in the nasal cavity appeared normal. No test substance-related nasal findings were observed in the females, and following the 13-week recovery period (males and females).

Recovery Necropsy: At the study week 16 recovery necropsy, there were no microscopic findings considered to be test substance-related.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not applicable

OTHER FINDINGS:
Bronchoalveolar Lavage Fluid (BALF) cytology. Test substance-related effects on BALF cytology were noted in males and females following 4 weeks of exposure at 0.2 mg/m3 or higher.

After 28 days of exposure, total bronchoalveolar lavage (BAL) fluid nucleated cell counts increased in an exposure-dependent manner. The increases were statistically significant in the 0.8 and 2 mg/m3 groups. The increase in total cell counts was associated with a higher proportion of neutrophils in all test substance-exposed groups. In the 2.0 mg/m3 males and females, the mean percentage of neutrophils was 73.3% and 69.6%, respectively, at study week 3, while neutrophils comprised < 2% of the controls. At the end of the 28-day exposure, the differences from control for the proportion of BALF neutrophils and macrophages were roughly exposure concentration-related with all differences reaching statistical significance. The changes in BALF cytology were overall not progressive with time, and peaked at day 12 of the satellite evaluation. At the study week 16 recovery evaluation, there were no test substance-related effects on BALF cytology.

Dose descriptor:

LOEL

Effect level:

0.2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Non-adverse effects were seen at this dose.

Dose descriptor:

NOAEL

Effect level:

>= 2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The highest dose level tested and based on the lack of findings in the lung weight ratio.

Critical effects observed:

not specified

Conclusions:

The LOEL is 0.2 mg cuprous oxide/m3, as (non-adverse) effects were seen at this dose. The NOAEL is >= 2 mg cuprous oxide/m3, the highest dose level tested and based on the lack of findings in the lung weight ratio. No STOT classification is proposed, as none of the observed effects were considered severe enough to merit classification by the inhalation route.

Executive summary:

A GLP-compliant 28 -day repeat-dose inhalation study was conducted in accordance with OECD Guideline 412, with the addition of a 13 -week recovery period and an evaluation of adaptation to test substance exposure (three intermediate time-points at week 0, week 1, and week 2). Further additional study endpoints were measurements of copper levels in lung tissue, lung lavage fluid, liver, brain, as well as wet/dry lung weight ratio and clinical chemistry and cytology of bronchoalveolar lavage fluid of all animals. The additional study endpoints were designed to aid in the interpretation of any test substance effects. Minor protocol deviations did not negatively impact the quality or integrity of the data nor the outcome of the study.

The overarching findings of this study were the exposure level-dependent appearance of macrophages in the lung, an increase in neutrophil number in BALF as well as in blood, and an increase in LDH and protein levels in the BALF. An increase in inflammation scores (neutrophil-dominated inflammation) was observed in the lung (the highest score being “mild”), and there was a decrease in the wet/dry lung weight ratio (highest exposure level only). Some nasal findings were reported for the high and medium-high exposures in the males.

Macrophages and neutrophils: The role of macrophages in the lung is to engulf and eliminate foreign bodies such as aerosol particles. Their appearance in the BALF upon exposure to cuprous oxide particles can be interpreted as a normal part of lung clearance. Macrophages in turn summon neutrophils. Neutrophils are highly motile and move quickly to a site of an event, such as the presence of particles. Neutrophils are attracted by various factors, including the presence of macrophages, and have a number of mechanisms for the attack of an insult, including phagocytosis, release of granule proteins, or "respiratory burst".

Based on the study results, an increase in neutrophil numbers (blood or BALF), in the absence of any immunotoxic endpoint or evidence of injury to lung epithelial cells should not be considered adverse. Neutrophil effects were seen at all exposure levels, including exposure level with no toxic endpoint. There was no "dose"-response relationship between the neutrophil levels in blood or BALF and the increasing exposure-levels. This indicates that these were secondary effects, it cannot be determined whether or not these effects are adverse.

LDH and Protein in BALF: There was an exposure-dependent increase in LDH and total protein levels in the BALF. LDH increased 11 -fold in both males and females at the highest exposure compared to control, and 6 -fold in both sexes at the medium-high exposure (0.8 mg cuprous oxide/m3) compared to control. The increase in total protein was slightly lower, with 7 -fold (males) and 8.5 -fold (females) at the highest exposure, and 5 -fold for both sexes at the medium high exposure. Neither LDH nor total protein levels increased with duration of exposure from 1 to 4 weeks (satellite group), and both parameters returned to control levels after the recovery period.

LDH- and protein increases in BALF can be a consequence of damage and leakage of the lung epithelium, which may remain invisible to standard light microscopy (in this study, no indications of epithelial damage or irritation was observed microscopically in the lung parenchyma). LDH and protein can also be released by macrophages upon activation, or by neutrophils. The LDH increases seen in this study at the 0.8 mg/mg3 exposure can be explained by LDH release from degenerative alveolar macrophages (the appearance of degenerative alveolar macrophages was reported in the histopathology report). In the absence of any microscopically-visible epithelial damage, it is conceivable that the observed increase in LDH and protein was a consequence of leakage from activated macrophages and/or neutrophils in the lung, and was a result of macrophages engulfing large amounts of copper or a large number of particles during the process of clearance. The LDH and protein levels at the high exposure level (2 mg/m3) exceed those seen in the literature for macrophage-only release. A contribution of LDH and protein from epithelial leakage cannot be excluded, however there was an absence of lung epithelial damage.

Lung weights: The lung weights (both wet and dry) increased as a function of exposure concentration. This could be a result of cellular content (macrophages, neutrophils) rising within lungs as a consequence of exposure. There was no increase in the wet/dry ratio, indicating that there was no edema at any exposure level. There was a small but significant decrease in the wet/dry ratio at the highest exposure level only. This can only be accounted for by the rise in copper levels, as Masson Trichrome staning of lung tissue samples, supported by quantitative computerized morphometric analysis confirmed that there was no significant effect on collagen content that would have contributed to an increase in lung weight.

Nasal findings: Some nasal findings were observed at the medium-high and high exposure levels. There was sporadic minimal focal olfactory epithelium degeneration affecting mostly the ethmoturbinates in Nasal Levels IV, V, and VI. Minimal to mild subacute inflammation was seen in Nasal Levels II and III of several 2.0 mg/m3 group males from the core study. Similar to the lung, the ciliated respiratory epithelium in the nasal cavity appeared normal. No test substance-related nasal findings were observed following the 13 -week recovery period. In the light of the full recovery of the findings and the fact that the rat is an obligate nose-only breathing animal with a high proportion of olfactory epithelium, these findings are not considered adverse.

Copper levels: No test substance-related effects on copper levels in the brain were observed, indicating that there is no transport of copper by the olfactory nerve. Copper levels in the liver rose slightly from 6.6 to 7.6 µg copper/g tissue, but remained within the normal range without the appearance of liver pathology. This indicates good clearance of copper from the body. In the lung and BALF, copper levels were detectable only in the 0.8 and 2 mg/m3 exposure groups, reaching a maximum of 231 ng Cu/ml fluid in the males and 347 ng Cu/ml in the females. Copper levels did not increase with longer exposure duration from 1 to 4 weeks (satellite group), indicating rapid clearance of copper from the lungs. Levels of copper in lung tissue and BALF were similar to control levels after the recovery period.

Time-course and recovery: When determining the potential adversity of the effects seen in this study, two general observations need to be kept in mind:

1. The time-course indicates that none of the measured endpoints showed an increase with longer duration of exposure. This could be indicative of some adaptation, and can be interpreted as a lack of progressive damage in this study.

2. The full reversibility of all effects (except as noted below) indicates acute, transient responses. The only exception is the lung weights in the males at the highest exposure level (remained 10% higher than controls).

The study LOEL is 0.2 mg cuprous oxide/m3, as (non-adverse) effects were seen at this dose. The study NOAEL is >= 2 mg/kg cuprous oxide/m3, the highest dose level tested and based on the lack of findings in the lung weight ratio. No STOT classification is proposed from this study as none of the observed effects were considered severe enough to merit classification by the inhalation route.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEC
Study duration:: subacute
Species:: rat
Quality of whole database:: Q1 study with no adverse effects at highest dose tested (>2 mg/m3)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with Good laboratory Practice and internationally accepted guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks:

Filtered air. Mean temperature and mean relative humidity between 20°C to 26°C and 30% to 70%, respectively.

Remarks on MMAD:

MMAD / GSD: 1.725 µm MMAD (mass median aerodynamic diameter) +/- 1.73 µm GSD (geometric standard deviation).

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days, 6 hours per day.

Frequency of treatment:

5 days per week.

Remarks:

Doses / Concentrations:
0.2, 0.4, 0.8, 2.0 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0.21, 0.41, 0.8, 2.0 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

Control animals:

yes, sham-exposed

Details on study design:

Adaptation to test substance exposure was assessed by sacrificing animals at intermediate time-points (satellite group), at week 0, week 1, and week 2.

Observations and examinations performed and frequency:

Sacrifice and pathology:

Other examinations:

Statistics:

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:

not examined

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:

not examined

Details on results:

Dose descriptor:

LOEL

Effect level:

0.2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Non-adverse effects were seen at this dose.

Dose descriptor:

NOAEL

Effect level:

>= 2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The highest dose level tested and based on the lack of findings in the lung weight ratio.

Critical effects observed:

not specified

Conclusions:

Executive summary:

Time-course and recovery: When determining the potential adversity of the effects seen in this study, two general observations need to be kept in mind:

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEC
Study duration:: subacute
Species:: rat
Quality of whole database:: Q1 study with no adverse effects at highest dose tested (2 mg/m3).

Additional information

Repeated dose toxicity - oral

In order to minimise animal testing, the assessment is based on available studies on copper sulphate. Extensive studies have shown that copper and copper compounds are considered equally or less bioavailable to a number of animal species when compared to copper sulphate, therefore the use of copper sulphate studies in determining the DNEL’s is justified on scientific grounds.

There are many studies in the public domain dealing with the repeat and chronic toxicity of copper compounds to several animal species. However, these studies did not meet the higher quality criteria (1 or 2) under the REACH quality criterion selection and will therefore not be used in the risk assessment and will not be described in this document. However, the VRAR, 2008 provides a full review of these studies and the discussion on the unsuitability/unacceptability of these studies, risk assessment. The studies summarised below have been identified as the pivotal studies in this Section

Non human information

Method

Results

Remarks

Reference

rat (F344/N) male/female

subchronic (oral: feed)

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)

Exposure: 92 days (7 days per week)

equivalent or similar to EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents) (. Method developed by the US NTP specifically for the surposes of this study.)

NOAEL: 1000 ppm (male/female)

LOAEL: 2000 ppm (male/female)

1 (reliable without restriction)

key study

experimental result

Test material(common name): Cu2+ as copper sulphate pentahydrate

Hébert, C.D. (1993)

mouse (B6C3F1) male/female

subchronic (oral: feed)

0, 1000, 2000, 4000, 8000 or 16000 ppm in the feed (providing estimated intakes of 0, 44, 97, 187, 398 and 815 mg Cu/kg bw/day in males and 0, 52, 126, 267, 536 and 1058 mg Cu/kg bw/day in females). (nominal in diet)

Exposure: 92 days (7 days per week)

NOAEL: 1000 ppm (male/female)

LOAEL: 2000 ppm (male/female)

1 (reliable without restriction)

key study

experimental result

Test material (common name): Cu2+ as copper sulphate pentahydrate

Hébert, C.D. (1993)

The NTP study summarised above is considered to be the pivotal study for Cu2+ presented as copper sulphate pentahydrate and results in an NOAEL of 16.7 mgCu/kg/bw/day in the rat. This study will be used in the subsequent calculation of an oral and systemic DNEL.

A chronic study (>= 1 year) is not considered appropriate, as no serious or severe toxicity effects of particular concern were observed in the 90-day study for which the available evidence is adequate for toxicological evaluation and risk characterisation.

Repeated dose inhalation

The 28 days repeated dose inhalation study on Cu2O was used as highly reliable study and read-across to copper. The study was carried out according to OECD Guideline 412. Further additional study endpoints were measurements of copper levels in lung tissue, lung lavage fluid, liver, brain, as well as wet/dry lung weight ratio and clinical chemistry and cytology of bronchoalveolar lavage fluid of all animals. The additional study endpoints were designed to aid in the interpretation of any test substance effects.

Macrophages and Neutrophils

The role of macrophages in the lung is to engulf and eliminate foreign bodies such as aerosol particles. It is possible to interpret their appearance in the BALF upon exposure to cuprous oxide particles as a normal part of lung clearance. Macrophages in turn summon neutrophils. Neutrophils are highly motile and attracted by various factors, including the presence of macrophages, and have a number of mechanisms to defend the host, such as phagocytosis, release of granule proteins, or "respiratory burst".

An increase in neutrophil numbers (blood or BALF), in the absence of an immunotoxic endpoint or evidence of injury to lung epithelium, is not necessarily adverse. Neutrophil effects were seen at all exposure levels, and based on the current study endpoints, it cannot be determined whether or not these effects are adverse (See neutrophil evaluation below (by Gary R. Burleson, Ph.D.). It is therefore concluded that the NOAEL based on neutrophil effects is above 2 mg cuprous oxide/m3),

LDH and Protein in BALF

There was an exposure-dependent increase in LDH11 and total protein levels in the BALF. LDH increased 11-fold in both males and females at the highest exposure compared to control, and 6-fold in both sexes at the medium-high exposure (0.8 mg cuprous oxide/m3) compared to control. The increase in total protein was slightly lower, with 7-fold (males) and 8.5-fold (females) at the highest exposure, and 5-fold for both sexes at the medium high exposure. Neither LDH nor total protein levels increased with duration of exposure from 1 to 4 weeks (satellite group), and both parameters returned to control levels after the recovery period.

LDH- and protein increases in BALF can be a consequence of damage and leakage of the lung epithelium, however, in this study no indication of epithelial damage or irritation was observed microscopically in the lung parenchyma. LDH and protein can also be released by macrophages upon activation, or by neutrophils.

There is a wealth of studies demonstrating that macrophages can release significant amounts of LDH and protein when challenged. It has been shown that non-cytotoxic doses of metals (including copper) can stimulate release of LDH and protein from macrophages in the oral cavity (Wataha, Hanks, and Sun 1995). Increases of 4.5-fold in LDH release from macrophages were observed in chromium exposed macrophages in vitro (Vandana et al. 2006). A doubling of LDH release from alveolar macrophages during a moderate iron challenge (40 µM iron in medium) versus control medium (3 µM iron) has been observed (Wesselius et al. 1999). For copper, 5-fold increases in LDH release from rabbit alveolar macrophages have been observed after 24 hours of exposure (0.1 µM copper in control medium, versus 1000 µM in copper exposed cells) (Labedzka et al. 1989).

In WIL 708003, the increase in LDH and protein observed in the BALF could be a result of macrophages engulfing large amounts of copper or a large number of particles during the process of clearance, especially in the absence of epithelial damage. This is supported by the fact that the number of macrophages in the lung (histiocytosis) increases linearly with exposure, in parallel to the increase in LDH and protein. BALF LDH and -protein levels and lung histiocytosis are the only measured study endpoints that exhibit a linear exposure-response across the exposure concentrations.

Based on the data, it is reasonable to correlate LDH and protein levels with number of macrophages, rather than with tissue damage in the lung (not observed at any dose; no dose response).

Lung Weights

In WIL-708003, the lung weights (both wet and dry) increased as a function of exposure concentration. There was no increase in the wet/dry ratio, indicating that there was no edema at any exposure level. There was a significant decrease in the wet/dry ratio at the highest exposure level only, suggesting an increase in dry components within the lung at the highest exposure level. Since there were no histopathological findings in the lung, the occurrence of increased collagen staining as an indication of fibrosis was studied with several approaches, see below.

Masson Trichrome Staining for Collagen

The WIL study pathologist defined a very slight increase in collagen in the high dose animals (2 mg/m3) as not toxicologically relevant, as there was minimal and occasionally mild staining also in the control groups. The staining severity scores between treatment and control, as well as after recovery did not differ significantly from each other, and did not allow a conclusion.

The histopathology slides underwent a re-examination for a qualitative histopathological peer-review by German pathologists (Or. Ernst and Or. Rittinghausen, Fraunhofer Institute and Or. Böttner, Histovia). The findings were "very slight" (Fraunhofer) and "mild to moderate" (Histovia) increases in collagen in the high dose animals (2 mg/m3), with full reversal of the findings after the recovery period. Of the 4 reports (WIL, 2x Fraunhofer, Histovia), only the Histovia report concluded that these findings were statistically significant.

Further discussions with the WIL study director and study pathologist resulted in the conclusion that all doses should be re-assessed by a quantitative computer-based analysis for increases in collagen as a dose-response.

Morphometric Analysis of Lung Fibrosis

Computerized morphometric analyses of lung samples were conducted to more objectively quantitate lung fibrosis. Mean collagen area percentages were higher for the 0.8 mg/m3 group males (↑33.8%) and for the 2.0 mg/m3 group males and females (↑23.9% and ↑16%, respectively). These differences were not statistically significant, and did not increase with dose. For the 0.2 and 0.4 mg/m3 group males, the mean collagen area percentages were slightly higher (↑10.1 %-12.5%; not statistically significant). Mean collagen area percentages for the 0.2, 0.4, and 0.8 mg/m3 group females were not remarkably altered by test substance exposure, yet lung dry weights were higher for the 0.4 and 0.8 mg/m3 group females. Since collagen staining and lung dry weight do not appear to be correlated, it was proposed that macrophages and/or neutrophils cells may contribute to the dry lung weight measurements.

Following the 13-week recovery period, the mean collagen area percentage for the 2.0 mg/m3 group females remained slightly higher (↑11.2%; 30% mean collagen area percentage in control females, and 33% in test article treated females). This difference was not statistically significant and was reduced from the higher primary necropsy value. For the 2.0 mg/m3 group males at the recovery evaluation, the mean collagen area percentage was negligibly different (↑1.9%) from the control group mean. However, the control group mean was higher than previously seen at the primary necropsy, with control animals displaying 38.7% mean collagen area percentage in lung and test article treated animals (high dose) displaying 39.5%. This increase in collagen staining in control animals after the recovery period is an unexplained finding.

This is perhaps reflective of the staining seen in the control groups in original examination (Masson Trichrome), and, overall, the morphometric analysis shows that there is no dose-response in collagen staining, as well as some unexplained staining in control animals.

Taking together the outcome of the pathology reports and the computerized analysis, there is no significant effect on collagen content of the lung.

Neutrophil evaluation and conclusion of the 28-day inhalation toxicty study (Kirkpatrick, 2010)

At 0.2 mg/m3, higher blood neutrophil counts were observed following 4 weeks of exposure to cuprous oxide. Inhalation exposure also resulted in a higher proportion of neutrophils in the BALF of rats on study days 5, 12, and 19 (2.0 mq/m3) and at study week 3 (0.2 mg/m3 or higher).

Most test substance-related effects at 2.0 mg/m3 appeared to show a peak in the effect prior to completion of 4 weeks of exposure and therefore, the results were consistent with a possible plateau.

The immune system consists of three (3) arms: (1) the innate immunity arm, (2) the cell¬mediated immunity arm, and (3) the humoral-mediated immunity arm. Neutrophils are an important component of innate immunity. In immunotoxicity testing, there may be three areas of concern related to neutrophils: (1) decreased neutrophil numbers leading to increased susceptibility to encapsulated bacteria resulting in bacterial pneumonia, (2) decreased neutrophil function leading to increased susceptibility to encapsulated bacteria resulting in bacterial pneumonia, and (3) increased neutrophil numbers/function which may result in persistent, chronic inflammation. In this study, no indication of persistent, chronic inflammation was found (based on plateau for most effects during the exposure period, and full recovery of all effects indicative of inflammation after 13-weeks post-exposure). The pattern of responses in the lung and lung-draining lymph nodes in this study following cuprous oxide exposure is typical for inhalation exposure to aerosol particles. Inhalation exposure with cuprous oxide markedly affected neutrophil numbers at all exposure levels in this study (0.2, 004, 0.8, and 2.0 mq/m3). However, the effects were reversible and there were no observed test substance-related effects on hematology parameters, BALF parameters, or lung histopathology following the 13-week recovery period. The No-Observed-Adverse-Effect-Level (NOAEL) for the neutrophil effects is therefore considered> 2.0 mg/m3.

It is therefore concluded that the overall NOAEL for this study is >2 mg/m³.

Neutrophil and copper - additional considerations

When interpreting studies of essential trace elements, it needs to be remembered that these elements also play a role in many biological functions, have tight homeostatic control, and are closely linked to physiology with effects caused by excess exposure as well as deficiency.

Copper deficiency has many effects, including hematological and immune deficiencies. A decrease in white cells is a well-established and sensitive marker of a beginning copper deficiency (see e.g., (Oanzeisen et al. 2007)). Accordingly, in many human copper exposure studies, increases in copper dependent endpoints can be observed (e.g., an increase or restoration in activity of S001).

There is currently little direct evidence for copper causing an increase in neutrophil numbers in a copper replete individual, but there are individual reports indicating that copper supplementation does increase white cell activity and counts. A recent study from non-copper deficient cows reports an increase in the in vitro phagocytic activity of neutrophils upon copper supplementation (20 ppm/cow/day) (Oang et al. 2012). Similarly, exposure of freshwater fish Channa punctatus to copper sulphate (0.36 mg/L) caused an increase in blood white cell count, while all other hematological parameters were decrease (e.g. red cells, hemoglobin) (Singh et al. 2008).

When interpreting the increase of neutrophils in BALF and blood of the study WIL 708003, the strong relationship of these cells with copper needs to kept in mind.

The study will be used to calculate the DNEL inhalation

Repeated dose toxicity: dermal

This study is usually required when the dermal route of exposure is significant and the compound is known to be toxic by the dermal route and can penetrate through intact skin. The need to conduct this study with copper or copper compounds must therefore be considered not necessary as although the dermal route of exposure is the most significant route there is no evidence to indicate that copper or copper compounds can cause toxicity or indeed pass through intact skin at significant levels. Acute dermal toxicity studies showed no toxic effects up to and including the highest dose tested. Therefore an accurate and realistic determination of dermal toxicity can be derived from available sub-chronic oral exposure studies, permissible systemic copper levels and in vitro dermal penetration studies on copper and copper compounds.

Repeated dose toxicity: oral

These studies are not required under REACH regulation data requirements.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Read-across from high quality studies on Cu2+, administered as CuSO4, to rat and mice, retained by competent authorities on existing substances and biocides

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Read across from a high quality study on Cu2O with endpoints from the OECD Guideline 412.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Read across from a high quality study on Cu2O with endpoints from the OECD Guideline 412..

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; digestive: stomach; urogenital: kidneys

Repeated dose toxicity: dermal - systemic effects (target organ) respiratory: lung

Justification for classification or non-classification

Justification relevant to Specific target organ toxicity – repeated exposure

Chronic toxicity, oral:

The liver is the critical organ for copper. The high quality repeated dose study in rats (Hebert (1993) - rat ) is retained for assessing classification according to regulation (EC) 1272/2008 as specific target organ toxicant (STOT-RE) –, oral. Classification criteria are not met since no severe adverse effects were observed at the guidance value, oral for a Category 1 classification of 10 mg/kg bw .day and at the guidance value for a Category 2 classification of 100 mg/kg bw.day. No classification required.

Acute toxicity, inhalation:

In the 4-weeks study by inhalation in rat (Kirkpatrick 2010), no serious adverse effects were observed at the maximum tested concentration (2 mg/m3). Therefore, no STOT-RE classification is warranted.

Conclusions on classification and labelling

No classification as STOT-RE under regulation (EC) 1272/2008 is proposed. No classification or SCLs are considered necessary.

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