Reaction mass of copper oxide and manganese dioxide

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:

2009

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

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:

ClCu

Test material form:

solid: particulate/powder

Remarks:

brown-gray

Details on test material:

SOURCE: Sigma-Aldrich Co., St. Louis, MO

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 / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Doses / concentrations 5

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)

1

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

2

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.