Copper chloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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:

Unnamed

Year:

2004

Report date:

2004

Reference 3

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

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD TG 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, LOT No. – 17119BO

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Male and nulliparous female Sprague-Dawley rats
- Source: Orient Bio Co. (Seoul, Korea)
- Age at study initiation: 7 weeks
- 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: rodent chow with phytoestrogens (Jeil Feed, Daejeon, Korea), ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks
- Fasting period after study: At scheduled termination, the animals were fasted overnight before the necropsy and blood collection.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C,
- Humidity (%): 50 +/- 10 %,
- Air changes (per hr): 10 to 20 times/h
- Photoperiod (hrs dark / hrs light): A 12-h light/dark cycle (08:00 on, 20:00 off)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

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

TREATMENT
- The males were dosed through gastric intubation each morning for a total of 30 days beginning 14 days before mating.
- The females were dosed throughout the mating and gestation period, from 2 weeks before mating to day 3 of lactation.
- The daily application volume (10 mL/kg) was calculated according to the most recent body weight.

VEHICLE
- The vehicle control rats received an equivalent volume of distilled water alone.




- Premating exposure period: 2 weeks

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Proof of pregnancy: sperm in a vaginal rinse was referred to as day 0 of pregnancy.
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in a vaginal rinse was referred to as day 0 of pregnancy.
- Based on these results, the precoital interval, copulation index, fertility index, and delivery index were calculated.

Duration of treatment / exposure:

30 days for males and 39 - 51 days for females

Frequency of treatment:

daily

Duration of test:

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 no treatment

Details on study design:

Dose selection rationale:
- No animals died during a 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.
- 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.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS AND CHECK FOR CLINICAL SIGNS: Yes
- Time schedule: every day after dosing

DETAILED CLINICAL OBSERVATIONS: Yes,
- Time schedule: once before the first administration and once a week thereafter

BODY WEIGHT: Yes,
- Time schedule for examinations: 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): Yes
- 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.

OTHER:
- 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.
- 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.
- 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).
- 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).

POST-MORTEM EXAMINATIONS: Yes
- All the males and females were sacrificed at the end of study period.
- Organs examined:
- The animals were subjected to a full, detailed gross necropsy, which included 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.
- 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.
- Histopathology:
- 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.

Ovaries and uterine content:

Observation of pregnancy and delivery:
- Pregnancy period
- Delivery index = (No. of dams with live newborns / No. of pregnant dams) × 100

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- 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.
- Viability index at day 4 of postpartum = (No. of live pups at day 4 / No. of live pups at birth) × 100
- Body weights of pups on day 0 and 4 at postpartum

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.

Indices:

- Delivery index = (No. of dams with live newborns / No. of pregnant dams) × 100
- Viability index at day 4 of postpartum = (No. of live pups at day 4 / No. of live pups at birth) × 100

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical Signs and Mortality:
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.

Hematology:
In females, there was a statistically significant increase in PLT and a significant decrease in MCH in the 80 mg/kg group.

Gross Findings:
In females, there were changes in the content of the uterus in 1 female in the 1.3 mg/kg group.

Histopathological Findings
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.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- There were no treatment-related effects on copora lutea, implantations, stillborns, live young at birth, gender ratio, viability index, and the body weights of 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.
- The incidence of runts was statistically increased in the 80 mg/kg bw/day.
- There were no pups with gross lesions in all treatment groups on day 4 of postpartum.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: fetotoxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

As for the developmental toxic effects, there were a significantly higher number of pups with gross lesions at birth, namely, icterus and runt, detected in the highest dose group.