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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

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
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Copper cyanide
EC Number:
208-883-6
EC Name:
Copper cyanide
Cas Number:
544-92-3
Molecular formula:
CCuN
IUPAC Name:
λ¹-copper(1+) iminomethanide

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on mating procedure:
Mating evaluation:
Following 2-week treatments for both sexes, one male was paired with one female for 2 weeks, and females (animal No. 60, 81) in which mating was not detected during the first mating period were given one additional week of re-mating with the males within the same group. The first 24 hour-period following mating was designated as day 0 of pregnancy, if sperms or vaginal plugs were detected. Successful copulation was decided by identifying parturition or implantation sites in uterus at caesarean section. Female (animal No.: 85) which did not deliver was sacrificed at 27 day following mating. Based on the results, following indices were calculated: mating index, fertility index, pregnancy index
Analytical verification of doses or concentrations:
no
Remarks:
see the details below
Details on analytical verification of doses or concentrations:
Analysis for the formulated test article was conducted under non GLP. As the result of analysis, it was impossible to analyze the formulations due to high specific gravity of test article and foam formation when the formulated test article was stirred with a magnetic bar.
Duration of treatment / exposure:
Male rats were treated 2 weeks before mating to the end of the mating period, for at least 28 or more days.
Female rats were treated 2 weeks before mating until day 4 of lactation including the mating and gestation periods.
Frequency of treatment:
Daily treatment
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
4 mg/kg bw/day (nominal)
Dose / conc.:
16 mg/kg bw/day (nominal)
Dose / conc.:
64 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 rats/sex/dose for the dose group 4 and 16 mg/kg/d
16 rats/sex/dose for the dose groups 0 and 64 mg/kg/d
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
1) Mortality and clinical observation
Clinical signs were checked once a day during the non-treatment period and before and after dosing the animals during the treatment period. Clinical signs including mortality, moribundity, general appearance, abortion, premature delivery, difficult or prolonged parturition, and behaviour changes, were observed and detailed clinical observation was conducted at group assignment and the days measuring body weights. At detailed observation, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). In addition, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were recorded. Clinical signs for each animal were recorded with date, time of finding and duration using Path/Tox system, and degree of clinical sign was also recorded if necessary.
On mating start day, additional clinical observation was conducted once after pairing each male and female in a cage, and for only females on the day of mating confirmation clinical signs were checked once before placing in a polycarbonate cage, and once before and after treatment. On parturition day, clinical signs were checked once before and after treatment, and, additionally, clinical observation was conducted twice for animal No. 57 and 60, and once for remnants.

2) Body weight
Individual body weights of both sexes were measured once a week during the pre¬mating, mating and recovery periods. During the gestation and lactation periods, body weights were measured as follows:
Pregnant animals : Days 0, 7, 14 and 20 of gestation

3) Food consumption
In the pre-mating period, pregnancy and lactation periods, food consumption was recorded by measuring full feeder weight on day of body weight measurement and empty feeder weight on the next day. It was not measured during the mating period. On mating start day, a diet was supplied on the day before mating start and food consumption was recorded on the following day. In addition, day 4 of lactation a diet was supplied on day 3 of lactation and food consumption was recorded on the following day. On fasting day before necropsy, a diet was supplied on the day before fasting and food consumption was recorded on the following day.

4) Mating evaluation
Following 2-week treatments for both sexes, one male was paired with one female for 2 weeks, and females (animal No. 60, 81) in which mating was not detected during the first mating period were given one additional week of re-mating with the males within the same group. The first 24 hour-period following mating was designated as day 0 of pregnancy, if sperms or vaginal plugs were detected. Successful copulation was decided by identifying parturition or implantation sites in uterus at caesarean section. Female (animal No.: 85) which did not deliver was sacrificed at 27 day following mating. Based on the results, following indices were calculated: mating index, fertility index, pregnancy index

5) Observation during the gestation and lactation periods
A) Observation on gestation and parturition: Abortion, premature delivery and dystocia or prolonged parturition were observed.
B) Observation on parturition date: Gestation length, delivery index were observed.
C) Observation during the lactation period: Nursing behaviours of dams was observed.

6) Animal selection
Functional observations, urinalysis for males, hematology, serum biochemistry and organ weights (all animals for reproductive organs, liver, spleen, thymus, heart, and salivary gland except a non-pregnancy) and histopathology were examined in six animals selected from each main group of each sex.

7) Function observations
Neurobehavioral evaluation for six selected males of each group was conducted on a day before the final treatment and for six selected females was done after separating their pups on day 4 of lactation. According to SOP/ANI/059 ‘Functional Observation Battery’ following parameters were examined; startle response, approach response, tail pinch response, touch response, papillary constriction, Infra Mot test in which number of movement was evaluated in 6 consecutive blocks of 10 minutes each as well as for the total 60-min session, and grip strength. FOB tests for recovery groups were not conducted, since no treatment-related changes in FOB tests were observed in main groups.

8) Urinalysis
Volume (Vol), specific gravity (SG), pH, protein (PRO), ketone body (KET), occult blood (OB), glucose (GLU), bilirubin (BIL), nitrite (NIT), urobilinogen (URO), color (COL), clarity (CLA) and sediment [cast, epithelial cell (EPI), erythorocyte (RBC), leucocyte (WBC)J were analyzed by using automatic tester (CliniTek-500, Bayer) and urine stick (Multistix, Bayer) with collected urine for approximately 17 hours of selected six males per group and all males of recovery group at the last week of treatment. The sediment test was carried out with a microscopy, and urine volume was measured by reading a scale on a collection tube with naked eyes.

9) Hematology
Six selected animals per group were fasted overnight for scheduled collections. Blood samples were taken by venipuncture at the post vena cava under isoflurane anesthesia, and then were collected into CBC bottles containing EDTA-2IC. Samples for coagulation were collected at necropsy into tubes containing 3.2 % sodium citrate. All items except PT and APTT are presented in the following table and were measured by a hematological autoanalyzer (ADVIA120, Bayer, USA), and PT and APTT was carried out with blood samples treated with 3.2% sodium citrate using a blood clotting analyzer (ACL 300 plus, Instrumentation Laboratory, Italy).

10) Serum biochemistry
Blood samples collected on tubes without anticoagulant were kept at room temperature and were centrifuged at 3,000 rpm for 10 minutes on the days of necropsy for all selected animals used in hematology examination. Items are shown in the following table and were measured using an autoanalyzer (Toshiba 200FR NEO, Toshiba Co., Japan).
Litter observations:
1) Body weight
Individual body weights of both sexes were measured once a week during the pre-mating, mating and recovery periods. During the gestation and lactation periods, body weights were measured as follows:
Lactation animals : Days 0 and 4 post-partum.

2) Food consumption
In the pre-mating period, pregnancy and lactation periods, food consumption was recorded by measuring full feeder weight on day of body weight measurement and empty feeder weight on the next day. It was not measured during the mating period. On mating start day, a diet was supplied on the day before mating start and food consumption was recorded on the following day. In addition, day 4 of lactation a diet was supplied on day 3 of lactation and food consumption was recorded on the following day. On fasting day before necropsy, a diet was supplied on the day before fasting and food consumption was recorded on the following day.

3) Observation during the lactation period
A) Observation on parturition date: litter size, dead pups, sex ratio, external anomalies of live pups were observed.
B) Observation during the lactation period: viability of pups was observed.
D) Body weights of live pups on Day 0 and 4 of lactation.
Postmortem examinations (parental animals):
A) Necropsy : At scheduled termination, all live males and females were necropsied after repeated dosing for at least 28 or more days and at day 5 of lactation, respectively. In recovery groups, males and females were terminated at 15 days and 16 days, respectively, after the first scheduled sacrifice of selected animals of the main groups for examination of hematology and serum biochemistry. Non-pregnant female (animal No. 85) was sacrificed at day 27 after mating confirmation. Animals without blood collection were anesthetized with CO2 gas overdose following overnight fasting, and then terminated by exsanguinating the abdominal aorta. For all animals, gross necropsy consisted of a complete external and internal examination and identification of all abnormal findings. Organs with gross lesions were preserved at a proper fixative. The numbers of corpora lutea and implantation sites were counted at necropsy of females, and based on the results, followings were calculated: pre-implantation loss, post-implantation loss and viability index on day 4 of lactation.

B) Organ weights : Absolute organ weights were measured and their relative organ weights (organ-to-body weight ratios) were calculated from the terminal body weight for the following organs of selected six animals when they were sacrificed. However, bold underlined organs were weighed in all animals except the non-pregnant.
Brain, pituitary gland, adrenal gland, liver, spleen, kidneys, heart, thymus, lungs, salivary gland, thyroid, testes, epididymis, seminal vesicle, prostate, ovary, uterus

C) Histopathological examination : On completion of the gross pathology examination, the following underlined organs of all animals except the non-pregnancy were retained. The other organs and tissue were also retained for only selected six animals per groups.
abnormal lesion, skin (included mammary gland in females), seminal vesicle, prostate, urinary bladder, testes, epididymides, ovaries, uterus, vagina, spleen, pancreas, mesenteric lymph node, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, adrenal glands, liver, salivary gland, mandibular lymph node, thyroid (included parathyroid), aorta, thymus, heart, lungs (bronchea), tongue, trachea, esophagus, sciatic nerve, skeletal muscle, sternum, femur, eye with optic nerve, haderian gland, brain, pituitary gland, spinal cord (thoracic)
Full examination for the retained organs was conducted. Neutral buffered 10% formalin was used for fixation and preservation, except testis, epididymides and eyeballs. Bouin’s fixative was used for testis and epididymides and Davidson’s solution for eyeball. Organs listed above were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and examined microscopically.
Histopathological examination was performed on above list of tissues from animals in the vehicle control and 64 mg/kg/day groups. Additional examination was conducted in liver, spleen, sternum and femur of the 4 and 16 mg/kg/day groups, since treatment-related findings were observed in these organs.
Postmortem examinations (offspring):
All live pups were sacrificed while anesthetized with CO2 gas and all pups including dead offspring were necropsied with special attention to all vital organs. Individual identification for Fl pups was not conducted and pups of each litter were numbered in the order of necropsy.
Statistics:
The data were analyzed for homogeneity of variance using Bartlett's test. Homogeneous data were analyzed using the Analysis of Variance and the significance of inter-group differences were analyzed using Dunnett's t test. Heterogeneous data were analyzed using Kruskal-Wallis test and the significance of inter-group differences between the control and treated groups was assessed using Dunn's Rank Sum test. The data of recovery groups were analyzed for homogeneity of variance using F test. Homogeneous data were analyzed using the Dunnett's t test and the significance of inter-group differences were analyzed using using Dunn's Rank Sum test. Heterogeneous data were analyzed using t test and the significance of inter-group differences between the control and treated group was assessed using Kruskal-Wallis test. Statistical analyses were performed by comparing the different dose groups with the vehicle control group using Path/Tox System. Results of comparisons are indicated only when significance at p<0.05 or p<0.01 was attained.
Reproductive indices:
Pregnancy index
Mating index
Fertility indexDelivery index
Pre-implantation loss
Post-implantation loss
Offspring viability indices:
Percentage of live pups to implantation
Percentage of dead pups to implantation
Viability index
Sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In males, soft feces were observed in all animals of the 64 mg/kg/day group, salivation was observed in one animal each, soiled perineal region was observed in 1 and 2 males during the premating and mating periods, respectively. Salivation was observed in 1 male of the 16 mg/kg/day group during the mating period. In females of the 64 mg/kg/day group, paleness, salivation, discoloured urine and soft feces were observed in 1, 2, 1 and 2 females during the premating period, respectively, soft feces was observed in 3 females during the mating period, salivation and soft feces were observed in 1 and 4 females during the gestation period, respectively, and paleness, salivation and liquid feces were observed in one animal each during the lactation period.
In recovery groups, soiled perineal region, discoloured urine and soft feces were observed in 1, 1 and 4 males in the 64 mg/kg/day group during treatment period, and no clinical sign was observed during recovery period. Paleness, salivation, soiled perineal region, discoloured urine and soft feces were observed in 1, 1, 1,2 and 2 females in the 64 mg/kg/day group during treatment period, and no treatment-related changes in clinical sign were observed during recovery period. Loss of fur was observed with a low incidence in all study groups.
Soft feces observed in both sexes of the 64 mg/kg/day group was considered to be treatment-related, since the incidence was high, and soiled perineal region in males and liquid feces in females observed in the 64 mg/kg/day group were considered to be treatment-related, since these signs were related to soft feces.
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, a statistically significant decrease in body weight was observed on days 7 and 14 of premating and days 7 and 14 of mating in the 64 mg/kg/day group, compared to the vehicle controls. In females, a statistically significant decrease in body weight was observed on day 14 of premating, day 7 of gestation and day 0 of lactation in the 64 mg/kg/day group. In body weights of recovery groups, a statistically significant decrease in body weight was observed on day 1 of treatment in males of the 64 mg/kg/day group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In males, a statistically significant decrease in food consumption was observed on day 2 of premating in the 16 and 64 mg/kg/day groups of both sexes, compared to the vehicle control group.
In recovery group, a statistically significant decrease in food consumption was observed in both sexes of the 64 mg/kg/day group on day 2 of treatment.
Decreased food consumption observed in both sexes of the 64 mg/kg/day group might have resulted from disorder in nutritional resorption from the gastrointestinal tracts, soft feces and liquid feces, which resulted in body weight reduction. A decrease in food consumption observed in both sexes of the 16 mg/kg/day group was considered to be incidental since it was transient during the study period and there was no body weight change.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In males, no statistically significant changes were observed in any of the treatment groups. In females, a statistically significant decrease in red blood cell count (RBC), hemoglobin (HGB) and hematocrit (HCT), and an increase in corpuscular hemoglobin (MCH) and reticulocyte percentage (RET%) were observed in the 64 mg/kg/day group.
In recovery group, white blood cell (WBC) count and reticulocyte percentage were statistically significantly decreased in males of the 64 mg/kg/day group. A statistically significant increase in mean corpuscular hemoglobin and a decrease in platelet and reticulocyte percentages were observed in females of the 64 mg/kg/day group.
The decrease in RBC count, hemoglobin and hematocrit, and an increase in MCH and reticulocyte percentage observed in females of the 64 mg/kg/day group were considered to be of toxicological significance, since histopathological changes were also observed in hematopoietic organs such as bone marrow.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in A/G ratio and alanine aminotransferase (ALT) was observed in males of the 64 mg/kg/day group, and a decrease in glucose was observed in 16 mg/kg/day group. A decrease in total cholesterol was observed in females of the 4 and 64 mg/kg/day groups.
In recovery group, a statistically significant increase in A/G ratio in males of 64 mg/kg/day group and a decrease in sodium were observed in females of the 64 mg/kg/day group.
Serum biochemical changes observed in the treatment groups were considered to be incidental, since these were not dose-related, within the normal ranges4), or no histopathological findings in related organs.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes in neurobehavioral tests were observed in any of the treatment group, except that a statistically significant decrease in HLGS was observed in males of the 4 mg/kg/day group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In histopathological examination of main animals, extramedullary hemopoiesis in liver and spleen and increased hemopoiesis in femur/marrow and sternum/marrow were observed in females of the 64 mg/kg/day group.
There were no treatment-related histopathological findings in reproductive organs of males and females in dosing and recovery group. Other microscopic findings observed in dosing and recovery animals were considered not to be treatment-related but incidental as these were spontaneously observed in rats of this age.

Generally, dams may show increased hemopoiesis due to pregnancy, but extramedullary hemopoiesis in liver and spleen and increased hemopoiesis of bone marrow of femur and sternum observed in the 64 mg/kg/d group were considered to be treatment-related, since it was shown a higher incidence when compared to control and its severity was from moderate to severe.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
Time course of mating: There were no statistically significant differences in the mean time taken to mate at any dose level tested.
Fertility and mating data: No treatment-related changes were observed in the copulation, fertility and pregnancy indices in any of the treatment groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
64 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
no
System:
male reproductive system
Key result
Critical effects observed:
no
System:
female reproductive system

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At examination of external malformations, dwarfism was observed in 1 pup of the 16 mg/kg/day group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
64 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
mortality
body weight and weight gain
gross pathology

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

There were no treatment-related changes in precoital time, copulation index, fertility index and pregnancy index. In addition, no treatment-related changes were observed in gestation length, delivery index, the numbers of corpora lutea and implantations, the numbers of live and dead pups, the percentage of live and dead pups to implantations, pre-implantation loss, post-implantation loss, sex ratio, viability index, external anomalies of neonates, body weights of pups on post-natal day 0 and 4, and gross findings of pups.

Applicant's summary and conclusion

Conclusions:
Based on these results, it was concluded that the oral administration of copper cyanide to rats resulted in soft feces, a decrease in body weight and food consumption in both sexes, soled perineal region in males and liquid feces in females, and a decrease in RBC count, hemoglobin and hematocrit, an increase in mean corpuscular hemoglobin and reticulocyte (%), enlarged spleen, an increase in spleen weight, exramedullary hemopoiesis in liver and spleen, and increased hemopoiesis in femur/marrow and sternum/marrow in females at 64 mg/kg/day. In addition, these findings were recovered or alleviated during the recovery period and target organs of the test article were considered to be spleen and bone marrow of femur and sternum. Therefore, under the present experimental conditions, no observed adverse effect levels (NOAEL) of the test article are considered to be 16 mg/kg/day for general toxicity in both sexes and over 64 mg/kg/day for both reproductive toxicity of parent animals and for Fl pups, and lowest observed adverse effect level (LOAEL) is considered to be 64 mg/kg/day for general toxicity in both sexes.