Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cuprous oxide.- Analytical purity: 96.45%- Lot/batch No.: 73367- Storage condition of test material: The test substance was stored at room temperature and was considered stable under these conditions.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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.