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EC number: 215-572-9
CAS number: 1332-65-6
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.
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)
Test material(common name): Cu2+ as copper sulphate pentahydrate
Hébert, C.D. (1993)
mouse (B6C3F1) male/female
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)
Test material (common name): Cu2+ as copper sulphate pentahydrate
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
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
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
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).
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
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
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
Repeated dose toxicity: other routes
These studies are not required under REACH regulation data requirements.
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