Copper chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD)

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Combined Repeated Dose and Reproductive/ Developmental Toxicities of Copper Monochloride in Rats

Author:

Chung MK

Year:

2009

Bibliographic source:

Environ Toxicol. 24(4):315-26.

Reference 2

Reference Type:

publication

Title:

Copper Monochloride CAS No: 7758-89-6, Final 10/2006

Author:

OECD SIDS

Year:

2006

Bibliographic source:

INCHEM 2009

Report date:

2006

Reference 3

Reference Type:

other: Study cited in OECD SIDS

Title:

Unnamed

Year:

2004

Report date:

2004

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Copper chloride

EC Number:

231-842-9

EC Name:

Copper chloride

Cas Number:

7758-89-6

Molecular formula:

Cl Cu

IUPAC Name:

λ¹-copper(1+) chloride

Details on test material:

- Name of test material (as cited in study report): Copper monochloride
- Physical state: brown-gray powder
- Analytical purity: 97%
- Lot/batch No.: Sigma-Aldrich Corporation, St.Louis,MO); Lot No. 17119BO

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), specific pathogen-free colony
- Age at study initiation: 7-week-old animals
- Weight at study initiation: 297.0 - 369.6 g for males and 180.3 - 222.3 g for females
- Housing: Two animals were housed in a stainless wire cage (280 W 3 500 L 3 200 H mm) during the premating (each sex), mating (1
male and 1 female), and postmating periods (2 males). The mated females were housed individually in a polycarbonate cage (220 W 3 390 L 3 175 H mm) during the gestation period. The lactating animals with suckling pups were housed in the same polycarbonate cage.
- Diet: ad libitum with a commercial pelleted rodent chow with phytoestrogens (Jeil Feed, Daejeon, Korea).
- Water: ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 10 %
- Air changes (per hr): 10 to 20 times/h
- Light intensity of 150– 300 Lux
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance suspended in distilled water vehicle was freshly prepared daily before the treatment.

APPLICATION VOLUME
- The daily application volume (10 mL/kg) was calculated according to the most recent body weight. The vehicle control rats received an equivalent volume of distilled water alone.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males: 30 days;
Females: 39 - 51 days.

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1.3, 5, 20, 80 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: In a 14-day repeated oral dose toxicity study with dose levels of 1, 4, 16, and 64 mg/kg/day, no animals died. 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. Based on these results, 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.
- Post-exposure recovery period: no
- At scheduled termination, the animals were fasted overnight before the necropsy and blood collection.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes,
- Time schedule: Clinical signs including mortality, moribundity, general appearance, and behavior changes were observed once a day after dosing the animals during the study period. Detailed clinical observations were made in all animals outside the home cage once before the 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.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was measured once a week during pre-mating period. It was measured on day 1, 8, 15 and 21 of gestation and on day 1 and 4 of lactation.

HAEMATOLOGY: Yes,
- Time schedule for collection of blood: before necropsy, animals were anesthetized with ether and the abdomen was cut open to collect blood from the posterior vena cava using a syringe with a 24-gauge needle. A 3.2% sodium citrate and EDTA-2K were used as the anticoagulants for the prothrombin time test and other hematological test, respectively.
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes, one night before necropsy
- How many animals: five males and five females from each test group
- Parameters as following were examined: White blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), differential leukocyte count (neutrophil (NEU), lymphocyte (LYM), monocyte (Mono), eosinophil (EOS), basophil (BAS), and large unstained cells (LUC)), reticulocyte count (RET), prothrombin time (PT) and met-hemoglobin (Mhgb).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: see haematology
- How many animals: see haematology
- Parameters as following were examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphate (ALP), glucose (GLU), total protein (TP), albumin (ALB), Albumin/globulin ratio (A/G), blood urea nitrogen (BUN), creatinine (CREA), total cholesterol (TCHO), total bilirubin (TBIL), triglyceride (TG), phospholipid (PL), calcium (Ca), inorganic phosphorus (P), sodium (Na), potassium (K), and chloride (Cl) were measured by an ion autoanalyzer (644 Na/K/Cl Analyzer, Ciba-Corning Co., USA).

URINALYSIS: Yes
- Time schedule for collection of urine: 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).
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- How many animals: Five males and five females were selected from each test group.
- Parameters as following were examined: color, specific gravity, pH, glucose, protein, ketone body, occult blood, bilirubin, urobilinogen, nitrite, and urine sediment.

OTHER:
- Functional observation tests: Functional observation tests such as right reflex, traction test, pupil reflex, auditory reflex, and negative geotaxis were performed on 5 animals of each group at the end of dosing date.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All adult animals were subjected to a full, detailed gross necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities including their contents. The following organs were weighed: brain, pituitary gland, thymus, lung, heart, liver, spleen, kidneys, adrenal glands, thyroid glands, and salivary gland.

HISTOPATHOLOGY: Yes
Full histopathology was carried out on the preserved organs and tissues of the selected animals from the control and highest dose groups.
The following organs of the selected animals in the high dose groups and the controls were preserved in 10 % buffered formalin solution: brain, spinal cord, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, adrenal glands, spleen, heart, thymus, thyroid glands, trachea, lungs, pituitary gland, intestinal lymph node, mandibular lymph node, sciatic nerve, skeletal muscle, bone marrow (femur), sternum, salivary gland, esophagus, tongue, aorta, and pancreas.
The tissues from the vehicle control and highest dose groups were routinely processed, embedded in paraffin, and sectioned at 3 ~ 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.

Statistics:

If the variance was homogenous, the data were subjected to one-way ANOVA. Otherwise, they were analyzed by the Kruskal-Wallis nonparametric ANOVA. If either of these tests showed statistical significance, the data were analyzed by the multiple comparison procedure of Dunnett of Scheffe to compare the treated groups with the controls. Clinical signs and gross findings were presented as frequencies, and they were analyzed by χ2-test followed by the Fisher’s exact test where necessary. A statistical difference was observed at p < 0.05 or p < 0.01.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
- Mortality: No death was observed for male rats. Deaths (3/12) observed only in the female 80 mg/kg group are considered to be treatment-related effects because the number of deaths was relatively high, which is uncommon in rat toxicological studies.
Although the dead animals showed some gross and histopathological changes in stomach, lung, trachea, and kidneys (see below), they were devoid of any remarkable clinical symptoms with regard to the parameters including body weight and food consumption before death. Therefore, these deaths are considered to be due to shock, renal complications, or cardiac origin, but the precise cause of deaths could not be determined.
- Clinical signs:
In the 80 mg/kg bw/day group blackish stool was observed in males (67 %) and females during pre- and mating period (33 %) as well as in females during gestation and lactation period (100 %).
• 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.
• Females:
- Anemia was observed in one female of the 80 mg/kg bw/day group.
- 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.
- 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.

Of the treatment-related clinical signs observed, the increased incidence of salivation, soft stool, diarrhea, and blackish stool probably resulted from the gastrointestinal toxicity of copper mono chloride. It was previously reported that copper salts act as irritants if they reach the gastrointestinal tract, and may produce increased salivation, nausea, vomiting, gastric pain, hemorrhagic gastritis, and diarrhea (ACGIH, 1986. Copper. In: Documentation of the Threshold Limit Values and Biological Exposure Indices, 5th ed. Cincinnati, OH; American Conference of Governmental Industrial Hygienists, 146 p.).
The other sings observed each sex were not considered to be affected by the test substance due to low incidence and no abnormal findings in histopathological examination.

BODY WEIGHT AND WEIGHT GAIN
In males, although there was a tendency for a decrease in body weight in the highest dose group compared with the vehicle control group, but there were no dose-related changes in males and females of all treatment groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The significant decrease in food consumption observed in the 80 mg/kg group on day 1 is believed to be related to the treatment because it was observed in both genders and was associated with the higher incidence of clinical signs. However, it was considered to be toxicologically insignificant because the decrease was transient and was not accompanied by changes in body weight. There were no other dose-related changes in all other treatment groups.

HAEMATOLOGY
The statistically significant decreases on RBC, HGB, HCT, MCV, and MCH, and increases on WBC and PLT in the 80 mg/kg bw/day male groups are expected to be treatment-related effects due to the dose-response relationship. Additionally, in the high dose group, neutrophil was increased compared with the controls. These findings were consistent with the hematological results observed in microcytic hypochromic anemia, also considered to be the primary cause of bone marrow hyperplasia of femur. Therefore, repeated oral dose of copper monochloride at 80 mg/kg is suggested to cause microcytic hypochromic anemia in rats.
For females, a statistically significant decrease on MCH and an increase on PLT were observed in the 80 mg/kg bw/day group. The incidence of skin pallor observed in the 80 mg/kg group (reported above) is probably due to a decrease in the number of RBCs, hematocrit, and hemoglobin concentration induced by the copper monochloride treatment. Copper compounds are known to be hematotoxic in humans and experimental animals.

CLINICAL CHEMISTRY
In males, there was a statistically significant decrease on TP and TBIL, and a significant increase in the A/G ratio in the 80 mg/kg bw/day group. In contrast, the TBIL of the 1.3 mg/kg bw/day group was statistically increased compared with the controls. There was a statistically significant decrease on potassium levels of the 1.3 and 5 mg/kg bw/day groups. These serum biochemical changes observed in the treatment groups were suggested of no toxicological significance due to the weak effects and/or the missing dose-response relationship.
In females, there was no statistically significant change in the serum biochemical parameters compared with the vehicle control group at any dose tested.

URINALYSIS
There were no treatment-related changes at any dose tested.

ORGAN WEIGHTS
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. Significant decreases in absolute organ weight were also observed for the thyroid gland in the 1.3 and 20 mg/kg groups. There were no differences in relative organ weights for males. 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.
The weight changes in the salivary gland, seminal vesicles, and thyroid gland observed in the male treatment groups are not considered to be treatment-related due to the weak effects and the failure of a dose-response relationship.

GROSS PATHOLOGY
Paleness of kidney was found in a single male of the 80 mg/kg bw/day group. Luminal gas and changes in the contents of the ileum, cecum, and colon were observed in 1 male each in the 80 mg/kg group. No abnormal organs were observed in all females.
Dead females of the 80 mg/kg bw/day group showed dark-red discoloration of lung, thoracic fluid, foamy trachea and lung, and black discoloration of stomach. They were observed in 2, 2, 1 and 1 females, respectively.

HISTOPATHOLOGY: NON-NEOPLASTIC
Males:
- Increased hematopoiesis of the femur was observed in 8 (66.7 %) males in the 80 mg/kg group
- Squamous cell hyperplasia of the stomach was observed in 1/12, 2/12, 8/11 and 11/12 males in the 1.3, 5, 20 and 80 mg/kg bw/day groups, respectively. The frequency of these findings was statistically higher in the 20 and 80 mg/kg bw/day groups than in the controls. These findings were considered to be caused by the test compound.
- 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.

Females:
- Squamous cell hyperplasia of stomach was observed in 2, 5, 6 and 9 females in the 1.3, 5, 20 and 80 mg/kg bw/day groups, respectively. These findings of high frequency were statistically significant compared to the controls in 5 mg/kg bw/day groups and above.
- 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.
- Congestion of the lung was observed in 1 female in the 80 mg/kg group.

The main histopathological findings, squamous cell hyperplasia of the stomach and increased hematopoiesis of the femoral marrow are considered to be treatment-related because these changes showed a clear dose response relationship.
This may largely be due to a direct irritating effect of copper monochloride on the mucous membrane of the stomach and is consistent with the increased incidence of clinical signs and decreased food consumption observed in the group. The increased incidence of femoral marrow hyperplasia observed in the male high-dose group is suggested to be a compensatory response against severe anemia due to the hematotoxic effects of copper monochloride.

OTHER FINDINGS
- Functional observation tests: No specific results were observed in righting reflex, papillary reflex and acoustic startle response. In the negative geotaxis test, 2 males and 1 female of the control and 20 mg/kg bw/day group, respectively, did not pass the test. Two animals failed in the traction test in each treatment group except for the female control.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

5 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: Squamous cell hyperplasia of the stomach

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

1.3 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: Squamous cell hyperplasia of the stomach

3

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

5 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: effects in haematology, clinical signs and organ weights.

4

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

20 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: mortality

Target system / organ toxicity

Critical effects observed:

not specified