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Description of key information

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. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
16.7 mg/kg bw/day
Study duration:

Additional information

Repeated dose toxicity

In order to minimise animal testing, all further studies have utilised 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 BPD 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


Repeated Dose toxicity: Oral





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)

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)

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 toxicity: inhalation

The 28 day repeat dose inhalation study on Cu2O was used as a 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.

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. 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 conclusions 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.


Neutrophils 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: other routes

These studies are not required under REACH regulation data requirements.

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

Justification for classification or non-classification