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EC number: 910-356-7 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Year of publication: 2009
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- no GLP status, deficiencies on test item reporting
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Remarks:
- OECD TG 422
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Year of publication: 2009
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- no GLP status, deficiencies on test item reporting
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 1996
- Deviations:
- not specified
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Orient Bio Co. Seoul, Korea
- Females (if applicable) nulliparous and non-pregnant: yes/no
- Age at study initiation: 9 weeks
- Weight at study initiation: not specified
- Housing: stainless steel, during lactation - polycarbonate cage
- Diet: ceommercial pelleted rodent chow with phytoestrogens (Jeil Feed, Daejeon, Korea) ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 50 +/-10
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- distilled
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: freshly prepared daily before the treatment
-daily application volume: 10 mL/kg - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- males: 30 days beginning 14 days before mating.
females: min 39 to max 52 days, throughout the mating and gestation period, from 2 weeks before
mating to day 3 of lactation - Frequency of treatment:
- once daily
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1.3 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 20 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 80 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 12
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on the results of a preliminary study (details see below), 80 mg/kg/
day was selected as the highest dose, and doses of 20, 5, and 1.3 mg/kg/day were selected as the
high, middle, and low doses, respectively, using a common ratio of x4.
- Other: preliminary study results
No animals died during the 14-day repeated oral dose toxicity study with dose levels of 1, 4, 16, and
64 mg/kg/day. The level of salivation increased at 16 and 64 mg/kg/day in a dose-dependent manne
r. There were no significant differences in body weight in the females but the body weight of males
decreased slightly at 64 mg/kg/day. - Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily after dosing
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before first administration and once a week thereafter
BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once a week during the premating period and on gestational days 0, 7, 14, and 20 as well as on lactational days 1 and 4.
OTHER:
- The level of food consumption was recorded once a week during the premating period and on gesta tional days 1, 8, 15, and 21 as well as on lactational days 1 and 4.
- During the final week of treatment, urinalysis was carried out in 5 males from each group with fresh urine using a CliniTek-100 urine chemistry analyzer (Ames Division, Miles Laboratory, USA) to
determine the specific gravity, color, pH, glucose, protein, ketone body, occult blood, bilirubin, urobilinogen, and nitrite.
- At scheduled termination, the animals were fasted overnight before the necropsy and blood collection. Blood samples were drawn from the posterior vena cava using a syringe with a 24-gauge ne
edle under ether anesthesia, and 3.2% sodium citrate and EDTA-2K were used as the anticoagulants for the prothrombin time test and other hematological test, respectively.
a) The following hematological parameters were examined in 5 males and 5 females selected randomly from each group: white blood cell (WBC), red blood cell (RBC), hemoglobin concentration
(HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration, platelet (PLT), differential leukocyte count, reticulocy
te count (ADVIA 120 hematology system, Bayer, USA), prothrombin time (ACL 300 Plus, Instru
mentation Laboratory, Italy), and methemoglobin (Spectrophotometer, Shimadzu, Japan).
b) The following serum biochemical parameters were evaluated in 5 males and 5 females selected randomly from each group using an autoanalyzer (Shimadzu CL-7200, Shimadzu Co, Japan): as
partate aminotransferase, alanine aminotransferase, alkaline phosphatase, glucose, total protein, albumin, albumin/globulin (A/G) ratio, blood urea nitrogen, creatinine, total cholesterol, total bilirubin, triglyceride, phospholipids, calcium, and inorganic phosphorus. Serum electrolytes such as chloride, sodium, and potassium were measured by an ion autoanalyzer (644 Na/K/Cl Analyzer,Ciba-Corning Co., USA). - Sacrifice and pathology:
- SACRIFICE
- Male animals: All surviving animals 30 days after beginning of treatment.
- Maternal animals: All surviving animals after day 3 of lactation.
GROSS NECROPSY
- Gross necropsy consisted of a careful examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Special attention was paid to the
reproductive organs. The implantation sites and corpora lutea were counted.
HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: brain, pituitary gland, thymus, lung, heart, liver, spleen, kidneys , adrenal glands, thyroid glands, salivary glands, testes, epididymides, prostates, seminal vesicles, ovaries, and uterus.
Full histopathology was carried out on the preserved organs and tissues of the selected animals from the control and highest dose groups.
The following general organ samples were taken and fixed in a 10% buffered formalin solution (pH 7.0): the brain, spinal cord, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, adrenal glands, spleen, heart, thymus, thyroid glands, trachea, lungs, pituitary gland, ovary, uterus, vagina, seminal vesicle, prostate gland, mammary gland, urinary bladder, intestinal and mandibular lymph nodes, sciatic nerve, skeletal muscle, bone marrow (femur), sternum, salivary gland, esophagus,
tongue, aorta, and other organs with abnormal findings from all animals. - Other examinations:
- details on examinationos concerning reproductive parameters are reported in section 7.8.1
- Statistics:
- The data are presented as the mean +/- SD. If the data were parametric, it was subjected to oneway analysis of variance (ANOVA). Otherwise, the data were analyzed using Kruskal- Wallis nonparametric ANOVA (1952). If either of these tests showed statistical significance, the data were analyzed using the multiple comparison procedure reported by Dunnett (1964) or Scheffe (1953). The clinical signs and gross findings are presented as the frequencies and were analyzed using a chi-square-test followed by a Fisher’s exact test (1970) where necessary. Statistical analyses were performed by comparing the treatment groups with the vehicle control group using the Path/Tox System (version 4.2.2. Xybion Medical System Co., NJ, USA) and Statistical Analysis Systems (SAS/STAT User’s Guide Version 6.12, NC, USA). P values < 0.05 and < 0.01 were considered significant.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - In the males, loss of fur was observed in 2, 1 and 2 males in the vehicle control, 20 and 80 mg/kg groups, respectively. Salivation was observed in 3, 5, 12, and 12 males in the 1.3, 5, 20, and 80 mg/kg groups, respectively. Soft stools were observed in 1 and 2 males in the 20 and 80 mg/kg groups, respectively. Diarrhea was observed in 1 and 2 males in the 5 and 80 mg/kg groups, respectively.
Blackish stools were observed in 8 males in the 80 mg/kg group.
- In females, loss of fur was observed in 2, 2, 1, 1, and 2 females in the vehicle control, 1.3, 5, 20, a nd 80 mg/kg groups, respectively. Pale color change of the skin was observed in 1 female in the 80 mg/kg group. Reddish tears from the eyes and lacrimation were observed in 1 female in the 1.3 mg/kg group. Crust formation of the skin was observed in 1 female in the 20 mg/kg group. Lacrimation was observed in 1 female each in the 1.3 and 20 mg/kg groups. Salivation was observed in 1, 8, and 11 females in the 5, 20, and 80 mg/kg groups, respectively. Blackish stools were observed in 2, 7, and 9
females in the 5, 20, and 80 mg/kg groups, respectively. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Total of 3 females in high dose group died
(One female each in the 80 mg/kg group died on days 1 and 13 of the premating period, and on day 20 of gestation. The dead animals did not show any remarkable clinical symptoms with regard to the parameters including body weight and food consumption before death. At necropsy, the animals exhibited a black discoloration of the stomach, dark-red discoloration of the lung, thoracic fluid foamy trachea, and lung, but no other gross changes. The histopathological examination of the dead females revealed squamous cell hyperplasia of the stomach and a congestion of the kidneys and lung in 3, 3, and 1 females, respectively.) - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- In males, although there was a tendency for a decrease in body weight in the highest dose group compared with the vehicle control group, there were no statistically significant changes in any of the treatment groups during the premating and mating periods. No statistically significant changes in the body weight of the females were observed in any treatment group during the premating, gestation, and lactation periods.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- In both males and females, there was significantly less food consumption on day 1 of treatment in the 80 mg/ kg group than in the vehicle control group. However, there
were no significant changes in food consumption observed in any of the treatment groups during the gestation and lactation periods. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- in high dose group only
(In males, there was a statistically significant decrease in the RBC, HGB, HCT, MCV, and MCH levels and a significant increase in the WBC, PLT, and neutrophil levels in the 80 mg/kg group when compared with the controls. In females, there was a statistically significant increase in PLT and a significant decrease inMCH in the 80 mg/kg group.) - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- - In males, there was a statistically significant decrease in the total protein and total bilirubin levels, a nd a significant increase in the A/G ratio in the 80 mg/kg group compared with the control group. The total bilirubin of the 1.3 mg/ kg group was significantly higher than the vehicle control group. The potassium levels of the 1.3 and 5 mg/kg groups decreased significantly.
- In females, there was no statistically significant change in the serum biochemical parameters compared with the vehicle control group at any dose tested. - Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- - In males, there were significant decreases in the absolute organ weight of the salivary gland in the 20 and 80 mg/kg groups, and the seminal vesicles in the 20 mg/kg group (data not shown). Significant decreases in absolute organ weight were also observed for the thyroid gland in the 1.3 and 20 mg/kg groups.
- In females, there were no statistically significant differences in the absolute and relative organ weights of the treatment groups compared with the vehicle control group. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- onyl effects which occured in single animals were observed
(1 male in high dose group: paleness of the testis, epididymis,kidney, prostate gland, and seminal vesicle,
1 male in high dose group: Luminal gas and changes in the contents of the ileum, cecum, and colon
1 female in low dose group: changes in the content of the uterus) - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - In males, squamous cell hyperplasia of the stomach was observed in 1, 2, 8, and 11 males in the 1.
3, 5, 20, and 80 mg/kg groups, respectively. Increased hematopoiesis of the femur was observed in 8
males in the 80 mg/kg group . Extramedullary hematopoiesis of the spleen was observed in 3 males i
n the 80 mg/kg group. Lymphoid cell infiltration of the kidney was found in 2 and 1 male in the vehicle
control and 80 mg/kg groups, respectively. Tubular basophilia of the kidney was observed in 4 males
each in the vehicle control and 80 mg/kg groups. The incidence of squamous cell hyperplasia of the
stomach in the >=20 mg/kg groups and bone marrow hyperplasia of the femur in the 80 mg/kg group
were significantly higher than those in the vehicle control group, respectively.
- In females, squamous cell hyperplasia of the stomach was observed in 2, 5, 6, and 9 females in
the 1.3, 5, 20, and 80 mg/ kg groups, respectively. Extramedullary hematopoiesis of the spleen wa
s noted in 1 female in the 5 mg/kg group. Lymphoid cell infiltration of the kidney was observed in 1
and 4 females in the vehicle control and 80 mg/kg groups, respectively. Tubular basophilia of the
kidney was observed in 3 and 6 females in the vehicle control and 80 mg/kg groups, respectively. C
ongestion of the kidney was observed in 3 females in the 80 mg/kg group. The incidence of squamous
cell hyperplasia of the stomach observed in the 5 mg/kg groups was significantly higher than that in
the vehicle control group. - Histopathological findings: neoplastic:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 1.3 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- histopathology: non-neoplastic
- Dose descriptor:
- NOAEL
- Effect level:
- 5 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- histopathology: non-neoplastic
- Conclusions:
- A combined repeated dose and reproductive/developmental toxicity study (OECD TG 422) with copper monochloride was performed with rats.
The most critical effects in parental animals was the increase in squamous cell hyperplasia of the stomach. Based on these findings, the NOAELs were concluded to be 5 mg/kg bw and day in male rats and 1.3 mg/kg bw and day (low dose group) in female rats. - Executive summary:
This study investigated the combined repeated dose and reproductive/developmental toxicity of copper monochloride in rats according OECD test guideline 422. The test substance was administered once daily by gavage at 0, 1.3, 5, 20, or 80 mg/kg/day. Male rats were dosed for a total of 30 days beginning 14 days before mating. Female rats were dosed from 2 weeks before mating to day 3 of lactation throughout the mating and gestation period. At 80 mg/kg/day, deaths were observed in 3 out of 12 females. There was a dose-dependent increase in the incidence of clinical signs and a reduction in the food consumption. Hematological and serum biochemical investigations revealed a decrease in the red blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (MCH), and serum total protein levels and an increase in the white blood cell and platelets in males, and a decrease in the MCH and an increase in the platelets in females. Histopathological examination showed an increased incidence of squamous cell hyperplasia of the stomach in both genders as well as increased hematopoiesis of the femur in males. There was an increase in the number of icteric and runt pups at birth. At 20 mg/kg/day, there was an increase in the incidence of clinical signs and squamous cell hyperplasia of the stomach in both genders. At 5 mg/kg/day, an increase in the incidence of squamous cell hyperplasia of the stomach was observed in females. There were no adverse effects in the lowest group in both genders. Based on these findings, the no observed adverse- effect levels of copper monochloride were concluded to be 5 mg/kg/day in male rats and 1.3 mg/kg/day in female rats for general toxicity and 20 mg/kg/day for reproductive/developmental toxicity.
Data source
Reference
- Reference Type:
- publication
- Title:
- Combined repeated dose and reproductive/developmental toxicities of copper monochloride in rats
- Author:
- Chung, M.K.; Baek, S.S.; Lee, S.H.; Kim, H.; Choi, K.; Kim, J.C.
- Year:
- 2 009
- Bibliographic source:
- Environmental Toxicology, 24, 315-326
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 1996
- Deviations:
- not specified
- GLP compliance:
- not specified
- Limit test:
- no
Test material
- Reference substance name:
- Copper chloride
- EC Number:
- 231-842-9
- EC Name:
- Copper chloride
- Cas Number:
- 7758-89-6
- Molecular formula:
- ClCu
- Test material form:
- solid: particulate/powder
- Remarks:
- brown-gray
- Details on test material:
- SOURCE: Sigma-Aldrich Co., St. Louis, MO
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Orient Bio Co. Seoul, Korea
- Females (if applicable) nulliparous and non-pregnant: yes/no
- Age at study initiation: 9 weeks
- Weight at study initiation: not specified
- Housing: stainless steel, during lactation - polycarbonate cage
- Diet: ceommercial pelleted rodent chow with phytoestrogens (Jeil Feed, Daejeon, Korea) ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 50 +/-10
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- distilled
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: freshly prepared daily before the treatment
-daily application volume: 10 mL/kg - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear]referred to as day 0
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: not specified - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- males: 30 days beginning 14 days before mating.
females: min 39 to max 52 days, throughout the mating and gestation period, from 2 weeks before mating to day 3 of lactation - Frequency of treatment:
- once daily
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1.3 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 20 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 80 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 12
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Based on the results of a preliminary study (details see below), 80 mg/kg/day was selected as the highest dose, and doses of 20, 5, and 1.3 mg/kg/day were selected as the high, middle, and low doses, respectively, using a common ratio of x4.
- Other: preliminary study results
No animals died during the 14-day repeated oral dose toxicity study with dose levels of 1, 4, 16, and 64 mg/kg/day. The level of salivation increased at 16 and 64 mg/kg/day in a dose-dependent manner. There were no significant differences in body weight in the females but the body weight of males decreased slightly at 64 mg/kg/day. - Positive control:
- no
Examinations
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily after dosing
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before first administration and once a week thereafter
BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once a week during the premating period and on gestational days 0, 7, 14, and 20 as well as on lactational days 1 and 4.
OTHER:
- The level of food consumption was recorded once a week during the premating period and on gestational days 1, 8, 15, and 21 as well as on lactational days 1 and 4.
- During the final week of treatment, urinalysis was carried out in 5 males from each group with fresh urine using a CliniTek-100 urine chemistry analyzer (Ames Division, Miles Laboratory, USA) to determine the specific gravity, color, pH, glucose, protein, ketone body, occult blood, bilirubin, urobilinogen, and nitrite.
- At scheduled termination, the animals were fasted overnight before the necropsy and blood collection. Blood samples were drawn from the posterior vena cava using a syringe with a 24-gauge needle under ether anesthesia, and 3.2% sodium citrate and EDTA-2K were used as the anticoagulants for the prothrombin time test and other hematological test, respectively.
a) The following hematological parameters were examined in 5 males and 5 females selected randomly from each group: white blood cell (WBC), red blood cell (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration, platelet (PLT), differential leukocyte count, reticulocyte count (ADVIA 120 hematology system, Bayer, USA), prothrombin time (ACL 300 Plus, Instrumentation Laboratory, Italy), and methemoglobin (Spectrophotometer, Shimadzu, Japan).
b) The following serum biochemical parameters were evaluated in 5 males and 5 females selected randomly from each group using an autoanalyzer (Shimadzu CL-7200, Shimadzu Co, Japan): aspartate aminotransferase, alanine aminotransferase,
alkaline phosphatase, glucose, total protein, albumin, albumin/globulin (A/G) ratio, blood urea nitrogen, creatinine, total cholesterol, total bilirubin, triglyceride, phospholipids, calcium, and inorganic phosphorus. Serum electrolytes such as chloride, sodium, and potassium were measured by an ion autoanalyzer (644 Na/K/Cl Analyzer,Ciba-Corning Co., USA). - Oestrous cyclicity (parental animals):
- not specified
- Sperm parameters (parental animals):
- Parameters examined in male parental generations:
testis weight, epididymis weight (During the necropsy of the males, the testes and epididymides of 5 animals selected from each group were removed and weighed. )
other:
- The left testis was homogenized and sonicated with 12 mL distilled water for sperm head counts. The sperm suspension was placed into a hemacytometer (Neubauer, Germany) and the number of homogenizationresistant sperm heads was counted under an optical microscope.
a) To determine the sperm motility, the left cauda epididymis was minced in 10 mL of Hank’s balanced salt solution (Sigma-Aldrich Co., St. Louis, MO) adjusted to pH 7.2 containing 10 mg/mL bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO) and incubated at 37°C for 5 min. The motility was observed using a microscope with a microwarm plate (Microwarm, Japan).
b) The sperm morphology was also examined under an optical microscope using sperm smears (sperm suspension containing 1% eosin Y) that were collected from the left cauda epididymis. The sperm in the cauda epididymis was counted by placing the
remnant of the sperm suspension of the cauda epididymis into a 50 mL tube. This suspension was homogenized for about 2 min. The sperm counts of the cauda epididymis were examined using the same procedures used for the testis.
c) In addition, the serum testosterone levels were measured using a RIA (Radio Immuno Assay) method. - Litter observations:
- The number of live, dead, and runt pups were counted on the day of delivery.
Live pups weighing at least one-third less than the control mean were designated as runts (Kelich et al., 1995; Byrd and Francis, 1998).
The litter size, gender ratio, body weights, and external abnormalities were also recorded within 24 h of parturition (day 0) and on day 4 of lactation. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals 30 days after beginning of treatment.
- Maternal animals: All surviving animals after day 3 of lactation.
GROSS NECROPSY
- Gross necropsy consisted of a careful examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Special attention was paid to the reproductive organs. The implantation sites and corpora lutea were counted.
HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: brain, pituitary gland, thymus, lung, heart, liver, spleen, kidneys, adrenal glands, thyroid glands, salivary glands, testes, epididymides, prostates, seminal vesicles, ovaries, and uterus.
Full histopathology was carried out on the preserved organs and tissues of the selected animals from the control and highest dose groups.
The following general organ samples were taken and fixed in a 10% buffered formalin solution (pH 7.0):
the brain, spinal cord, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, adrenal glands, spleen, heart, thymus, thyroid glands, trachea, lungs, pituitary gland, ovary, uterus, vagina, seminal vesicle, prostate gland, mammary gland, urinary bladder, intestinal and mandibular lymph nodes, sciatic nerve, skeletal muscle, bone marrow (femur), sternum, salivary gland, esophagus, tongue, aorta, and other organs with abnormal findings from all animals.
The testes and epididymides were preserved in Bouin’s fixative. The tissues from the vehicle control and highest dose groups were routinely processed, embedded in paraffin, and sectioned at 3 to 5 µm. The sections were stained with Hematoxylin-Eosin for the microscopic examination. The examination of the spleen, stomach, and femur was extended to the animals in the other dose groups because histopathological changes were observed in the aforementioned organs of the highest dose group. - Postmortem examinations (offspring):
- SACRIFICE - on day 4 of lactation
GROSS NECROPSY - yes - Statistics:
- The data are presented as the mean +/- SD. If the data were parametric, it was subjected to one-way analysis of variance (ANOVA). Otherwise, the data were analyzed using Kruskal- Wallis nonparametric ANOVA (1952). If either of these tests showed statistical significance, the data were analyzed using the multiple comparison procedure reported by Dunnett (1964) or Scheffe (1953). The clinical signs and gross findings are presented as the frequencies and were analyzed using a chi-square-test followed by a Fisher’s exact test (1970) where necessary. Statistical analyses were performed by comparing the treatment groups with the vehicle control group using the Path/Tox System (version 4.2.2. Xybion Medical System Co., NJ, USA) and Statistical Analysis Systems (SAS/STAT User’s Guide Version 6.12, NC, USA). P values < 0.05 and < 0.01 were considered significant.
- Reproductive indices:
- - the precoital interval,
- copulation index,
- fertility index, and
- delivery index - Offspring viability indices:
- viability index
gender ratios
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - In the males, loss of fur was observed in 2, 1 and 2 males in the vehicle control, 20 and 80 mg/kg groups, respectively. Salivation was observed in 3, 5, 12, and 12 males in the 1.3, 5, 20, and 80 mg/kg groups, respectively. Soft stools were observed in 1 and 2 males in the 20 and 80 mg/kg groups, respectively. Diarrhea was observed in 1 and 2 males in the 5 and 80 mg/kg groups, respectively. Blackish stools were observed in 8 males in the 80 mg/kg group.
- In females, loss of fur was observed in 2, 2, 1, 1, and 2 females in the vehicle control, 1.3, 5, 20, and 80 mg/kg groups, respectively. Pale color change of the skin was observed in 1 female in the 80 mg/kg group. Reddish tears from the eyes and lacrimation were observed in 1 female in the 1.3 mg/kg group. Crust formation of the skin was observed in 1 female in the 20 mg/kg group. Lacrimation was observed in 1 female each in the 1.3 and 20 mg/kg groups. Salivation was observed in 1, 8, and 11 females in the 5, 20, and 80 mg/kg groups, respectively. Blackish stools were observed in 2, 7, and 9 females in the 5, 20, and 80 mg/kg groups, respectively. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Total of 3 females in high dose group died
(One female each in the 80 mg/kg group died on days 1 and 13 of the premating period, and on day 20 of gestation. The dead animals did not show any remarkable clinical symptoms with regard to the parameters including body weight and food consumption before death. At necropsy, the animals exhibited a black discoloration of the stomach, dark-red discoloration of the lung, thoracic fluid foamy trachea, and lung, but no other gross changes. The histopathological examination of the dead females revealed squamous cell hyperplasia of the stomach and a congestion of the kidneys and lung in 3, 3, and 1 females, respectively.) - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- In males, although there was a tendency for a decrease in body weight in the highest dose group compared with the vehicle control group, there were no statistically significant changes in any of the treatment groups during the premating and mating periods. No statistically significant changes in the body weight of the females were observed in any treatment group during the premating, gestation, and lactation periods.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- In both males and females, there was significantly less food consumption on day 1 of treatment in the 80 mg/ kg group than in the vehicle control group. However, there
were no significant changes in food consumption observed in any of the treatment groups during the gestation and lactation periods. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- in high dose group only
(In males, there was a statistically significant decrease in the RBC, HGB, HCT, MCV, and MCH levels and a significant increase in the WBC, PLT, and neutrophil levels in the 80 mg/kg group when compared with the controls. In females, there was a statistically significant increase in PLT and a significant decrease inMCH in the 80 mg/kg group.) - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- - In males, there was a statistically significant decrease in the total protein and total bilirubin levels, and a significant
increase in the A/G ratio in the 80 mg/kg group compared with the control group. The total bilirubin of the 1.3 mg/ kg group was significantly higher than the vehicle control group. The potassium levels of the 1.3 and 5 mg/kg groups decreased significantly.
- In females, there was no statistically significant change in the serum biochemical parameters compared with the vehicle control group at any dose tested. - Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - In males, squamous cell hyperplasia of the stomach was observed in 1, 2, 8, and 11 males in the 1.3, 5, 20, and 80 mg/kg groups, respectively. Increased hematopoiesis of the femur was observed in 8 males in the 80 mg/kg group . Extramedullary hematopoiesis of the spleen was observed in 3 males in the 80 mg/kg group. Lymphoid cell infiltration of the kidney was found in 2 and 1 male in the vehicle control and 80 mg/kg groups, respectively. Tubular basophilia of the kidney was observed in 4 males each in the vehicle control and 80 mg/kg groups. The incidence of squamous cell hyperplasia of the stomach in the >=20 mg/kg groups and bone marrow hyperplasia of the femur in the 80 mg/kg group were significantly higher than those in the vehicle control group, respectively.
- In females, squamous cell hyperplasia of the stomach was observed in 2, 5, 6, and 9 females in the 1.3, 5, 20, and 80 mg/ kg groups, respectively. Extramedullary hematopoiesis of the spleen was noted in 1 female in the 5 mg/kg group. Lymphoid cell infiltration of the kidney was observed in 1 and 4 females in the vehicle control and 80 mg/kg groups, respectively. Tubular basophilia of the kidney was observed in 3 and 6 females in the vehicle control and 80 mg/kg groups, respectively. Congestion of the kidney was observed in 3 females in the 80 mg/kg group. The incidence of squamous cell hyperplasia of the stomach observed in the 5 mg/kg groups was significantly higher than that in the vehicle control group. - Histopathological findings: neoplastic:
- no effects observed
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- there were no treatment-related effects on the precoital interval, copulation index, fertility index, delivery index, gestation length, copora lutea, implantations, stillborns, live young at birth, gender ratio
Effect levels (P0)
open allclose all
- Dose descriptor:
- NOAEL
- Effect level:
- 5 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- copper monochloride
- Sex:
- male
- Basis for effect level:
- histopathology: non-neoplastic
- Dose descriptor:
- NOAEL
- Effect level:
- 1.3 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- copper monochloride
- Sex:
- female
- Basis for effect level:
- histopathology: non-neoplastic
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- there were no treatment-related effects on the viability index
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The number of runt pups was also significantly higher in the 80 mg/kg group than the vehicle control group.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- there was a significantly higher number of pups with gross lesions, namely, icterus, in the 80 mg/kg group than in the vehicle control group.
- Histopathological findings:
- not examined
Developmental neurotoxicity (F1)
- Behaviour (functional findings):
- not examined
Developmental immunotoxicity (F1)
- Developmental immunotoxicity:
- not examined
Effect levels (F1)
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 20 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- copper monochloride
- Sex:
- male/female
- Basis for effect level:
- gross pathology
Overall reproductive toxicity
- Key result
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 20 mg/kg bw/day (actual dose received)
- Treatment related:
- yes
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- yes
- Relevant for humans:
- yes
Applicant's summary and conclusion
- Conclusions:
- In this combined repeated dose and reproductive/developmental toxicity study (OECD TG 422) with copper monochloride in rats, there was an increase in the number of icteric and runt pups at birth in the high dose group, ledaing to a NOAEL for developmental effects of 20 mg/kg bw/day. At this dose level there were already several effects present in parental animals as well, however the most critical effects in parental animals was the increase in squamous cell hyperplasia of the stomach. Based on these findings, the NOAELs were concluded to be 5 mg/kg bw and day in male rats and 1.3 mg/kg bw and day (low dose group) in female rats.
- Executive summary:
This study investigated the combined repeated dose and reproductive/developmental toxicity of copper monochloride in rats according OECD test guideline 422. The test substance was administered once daily by gavage at 0, 1.3, 5, 20, or 80 mg/kg/day. Male rats were dosed for a total of 30 days beginning 14 days before mating. Female rats were dosed from 2 weeks before mating to day 3 of lactation throughout the mating and gestation period.
At 80 mg/kg/day, deaths were observed in 3 out of 12 females. There was a dose-dependent increase in the incidence of clinical signs and a reduction in the food consumption. Hematological and serum biochemical investigations revealed a decrease in the red blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (MCH), and serum total protein levels and an increase in the white blood cell and platelets in males, and a decrease in the MCH and an increase in the platelets in females. Histopathological examination showed an increased incidence of squamous cell hyperplasia of the stomach in both genders as well as increased hematopoiesis of the femur in males. There was an
increase in the number of icteric and runt pups at birth. At 20 mg/kg/day, there was an increase in the incidence
of clinical signs and squamous cell hyperplasia of the stomach in both genders. At 5 mg/kg/day, an increase in the incidence of squamous cell hyperplasia of the stomach was observed in females. There were no adverse effects in the lowest group in both genders. Based on these findings, the noobserved-adverse-effect levels of copper monochloride were concluded to be 5 mg/kg/day in male rats and 1.3 mg/kg/day in female rats for general toxicity and 20 mg/kg/day for reproductive/developmental toxicity.
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