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EC number: 215-572-9
CAS number: 1332-65-6
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
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
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.
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