Copper chloride

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

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)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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:

publication

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 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.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: If sperm or vaginal plugs were detected the first 24 h-period after mating was designated as day 0 of pregnancy.
- Based on these results, the precoital interval, copulation index, fertility index, and delivery index were calculated.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males: 30 days, beginning 14 days before mating;
Females: 39 - 51 days, throughout the mating and gestation period, from 2 weeks before mating to day 3 of lactation.

Frequency of treatment:

daily

Details on study schedule:

no data

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 vehicle

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.
- At scheduled termination, the animals were fasted overnight before the necropsy and blood collection.

Positive control:

no data

Examinations

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Parameters examined in male parental generations:
During the necropsy of the males, testis and epididymis of 5 animals selected from each group were removed and then weighed. Also sperm head counts, motility, and sperm morphology were examined.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
The number and sex of pups, stillbirths, live births as well as 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.
- Maternal animals: All animals, surviving and dead.

GROSS NECROPSY
- Gross findings: All adult animals were subject to 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. Special attention was paid to the reproductive organs. The implantation
sites and corpora lutea were counted. 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.

- During the necropsy of the males, the testes and epididymides of 5 animals selected from each group were removed and weighed. 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 homogenization resistant sperm heads was counted under an optical microscope. 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). 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 ~2 min. The sperm counts of the cauda epididymis were examined using the same procedures used for the testis.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathological examination: 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 ~ 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.

OTHER
- Hormone measurement: Ten males were selected from each test group at necropsy. Blood was collected from abdominal artery and centrifuged to
obtain serum. The serum testosterone levels were measured using a RIA (Radio Immuno Assay) method.

Postmortem examinations (offspring):

no data

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.

Reproductive indices:

- Copulation index = (No. of animals with successful copulation / No. of mated animals) × 100
- Fertility index (male) = (No. of impregnating animals / No. of animals with successful copulation) × 100
- Pregnancy index (female) = (No. of pregnant animals / No. of animals with successful copulation) × 100

Offspring viability indices:

delivery index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
- Mortality: No death was observed for male rats. Three females were dead and treatment-related death was found in one of the 80 mg/kg bw/day female group on day 1 of administration.
- Clinical signs: Treatment-related clinical sign such as anemia was observed in one female of the 80 mg/kg bw/day group because the symptom was consistent with anemic signs of hematological examination. Additionally, in the 80 mg/kg bw/day 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 %). 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.
- The dead animals did not show any remarkable clinical symptoms with regard to the parameters including body weight and food consumption before death.

For more detail see repeated dose toxicity: oral, rat; key.Chung 2009.Repeated dose toxicity:oral.rat

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)
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. Apart from this, there were no dose-related changes in all other treatment groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No statistical significances were observed except that the number of sperms in cauda epididymis was statistically, but not dose-dependent increased in the 1.3 and 5 mg/kg bw/day groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No data

OTHER REPRODUCTIVE DATA (PARENTAL ANIMALS)
- Precoital time, copulation index, fertility index, delivery index, gestation length, copora lutea, implantations, and mating data: There were no statistically significant differences compared with the controls. The results in detail were shown in the table attached.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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.
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 PATHOLOGY (PARENTAL ANIMALS)
Paleness of testies, epididymis, prostate gland and seminal vesicle as well as 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 female groups.
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.
At necropsy, the dead 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.

HISTOPATHOLOGY (PARENTAL ANIMALS)
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 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.

HAEMATOLOGY (PARENTAL ANIMALS)
In males, there was a statistically significant decreases in the RBC, HGB, HCT, MCV, and MCH levels, and a significant increases on WBC and PLT in the 80 mg/kg bw/day male groups. In the same 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. For females, a statistically significant decrease on MCH and an increase on PLT were observed in the 80 mg/kg bw/day group.

CLINICAL CHEMISTRY (PARENTAL ANIMALS)
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. In females, there was no statistically significant change in the serum biochemical parameters compared with the vehicle control group at any dose tested.

URINALYSIS (PARENTAL ANIMALS)
There were no treatment-related changes at any dose tested.

OTHER FINDINGS (PARENTAL ANIMALS)
- Hormone measurement: There were no statistically significant differences in the serum testosterone levels of all male treatment groups.
- 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 (P0)

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed on the reproductive organs and the fertility parameters assessed. The NOAEL of 80 mg/kg bw/day represents the highest test dose.

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
There were no treatment-related effects on the stillborns, live young at birth, gender ratio and viability index of offspring.

CLINICAL SIGNS (OFFSPRING)
- No data

BODY WEIGHT (OFFSPRING)
- There were no treatment-related effects on the body weights of offspring.

SEXUAL MATURATION (OFFSPRING)
- No data

ORGAN WEIGHTS (OFFSPRING)
- No data

GROSS PATHOLOGY (OFFSPRING)
- 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.

HISTOPATHOLOGY (OFFSPRING)
- No data

OTHER FINDINGS (OFFSPRING)
The number of runt pups was also significantly higher in the 80 mg/kg group than the vehicle control group.

Overall reproductive toxicity

Reproductive effects observed:

not specified