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Description of key information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening rats were exposed for 43 (males) and 63 (females) days with tripotassium hexacyanocobaltate. No adverse effect were observed up to the highest dose tested. Thus, the NOAEL for male and female animals is considered 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined repeated dose toxicity study with the reproduction/developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-18 to 2018-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Body weight: At the commencement of study (Day 1 of dosing phase), Animals R0201 and R0209 (Group 3 males) and Animal R0303 (Group 4 male) had body weights that exceeded the mean male body weight by up to 26%, which was outside the specified limit of 20%. Most of the remaining animals on the study were within 10% of the mean body weight for each sex on Day 1. No impact on body weight was noted throughout the dosing phase for either sex, and this deviation was considered to have no impact on study integrity.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a sealed original container, at 15 to 25 °C, protected from the light, kept dry in closed container
- Solubility of the test substance in the solvent/vehicle: formulations of 324.4 g/L were previously found to be homogenous (please also refer to Section 4.8 Water solubility: k_Smeykal_2017_Water solubility).
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories. Margate, United Kingdom
- Age at start of dosing: 10 to 12 weeks old
- Weight at start of dosing: males: 263.3 to 402.6 g; females: 174.5 to 228.7 g
- Housing: animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes. These include Arrowmight Type II (used for housing of animals during pairing or of females thereafter) and Type III (used for housing of animals during the remaining study) cages; bedding: provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom).
- Diet (ad libitum): VRF1 Diet (Special Diets Services Ltd. Witham, United Kingdom)
- Water (ad libitum): main tap supply water
- Acclimation period: at least 15 days
- Dietary/environmental enrichment: animals were provided with wooden Aspen chew blocks and rodent retreats. During gestation and lactation, nesting materials (i.e., paper wool) were provided as forms of environmental enrichment.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3 °C
- Relative humidity: 30 to 70%
- Air changes: minimum of 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
physiological saline
Remarks:
0.9 % saline
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepaired daily. The test article was formulated as a solution in 0.9% saline following dispensary SOPs and the formulation method (Method 8375590_O_01D), as maintained in the study data.
The formulations were stored at room temperature (15 to 25 °C) in a sealed container, protected from the light.
Dosing occurred within four hours of dose preparation.
A dose volume of 10 mL/kg was used. Dose volumes were calculated using the most recent recorded body weight for each animal.

VEHICLE
Source: supplied by Baxter, prepared by Covance
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
STABILITY DATA
It was not possible to demonstrate stability for the test article formulations due to the nature of the test article.

ACHIEVED CONCENTRATION AND HOMOGENEITY
Sample collection
Samples (3 x 5 mL aliquots [top, middle, and bottom] from all formulations) prepared for use during Week 1 were taken for analysis of homogeneity. The mean of the homogeneity results was taken as the achieved concentration.
Samples (1 x 5 mL aliquot [random] from all formulations) prepared for use during Week 6 were taken for analysis of achieved concentration.

SAMPLE ANALYSIS AND DISPOSITION
Samples were dispatched, frozen on dry ice and protected from light until analysis.

METHOD:
The analyses of the content of cobalt in the test solutions, in pure 0.9 % saline and in the test article itself were performed based on the analytical laboratory method LM-EAL-0237 and carried out using ICP-OES (Vista MPX ICP-OES, Varian). Measurements were realised after dilution with diluted nitric acid solution (each prepared analytical sample measured three times).
The test article potassium hexacyanocobaltate(III) itself (stored at room temperature) was weighed exactly into a 10 mL volumetric flask and filled up to the mark with purified water. 0.5 mL of this solution were diluted to 500 mL with diluted nitric acid solution and analysed by ICP-OES.

The test solution, which contained the test article potassium hexacyanocobaltate(III) in a concentration range of 10 to 100 mg/mL (0 mg/mL added for the control test solution) in 0.9 % saline, were defrosted at room temperature and homogenised by shaking. The pure 0.9 % saline, which was stored at room temperature, was also shaken before use. As the provided sample amounts of the test solutions were not enough to take 5 mL out, 2.5 mL were used and filled up to 5 mL with diluted nitric acid solution in order to achieve the same concentration as described in the laboratory method. The control test solutions, had to be diluted further than expected and described in the laboratory method with diluted nitric acid solution, since the matrix/salt amount led to a plugging of the analysis instrument. Instead of a dilution factor of 2 (1:1), a second dilution step was performed with a dilution factor of 20.
0 mg/mL= final dilution [v/v] 1:40
10 mg/mL= final dilution [v/v] 1:1000
30 mg/mL= final dilution [v/v] 1:5000
100 mg/mL= final dilution [v/v] 1:10000

RESULTS:
For details on results please refer to the field "any other information on material and methods incl. tables".
Duration of treatment / exposure:
males: 43 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase].
females: 63 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, throughout gestation, and until necropsy [LD 14, or 26 days post-coitum for females that did not litter]).
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males/ 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
the dose levels were selected based on available toxicological data and a 21-day dose range finding study (please refer to section 7.8.1 Toxicity to reproduction: s_Rashid_2019). In a previous 3-week range-finding study (Covance Study 8375589), dose levels of up to 1000 mg/kg/day were well tolerated. Body weight development and associated food consumption in animals administered the test article remained unaffected. Additionally, no test article-related macroscopic observations were noted at necropsy, with no effect on organ weights.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and end (nominal) of the working day for signs of ill health or overt toxicity.
POSTDOSE OBSERVATION:
Animals were observed daily immediately after dosing and approximately 0.5, 1, 2, and 4 hours postdose. In the absence of any toxicologically significant postdose observations during the first 3 days of dosing, no further postdose observations were scheduled.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination on Day 9 of the predose phase (males) or Day 16 of the predose phase (females) and then daily from the first day of dosing to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: males: male body weights were recorded once during acclimation (Day 7 [Day 9 of the predose phase]), on the first day of dosing, at weekly intervals thereafter including the last day of the phase, and before necropsy.
females: female body weights were recorded once during acclimation (Day 7 [Day 16 of the predose phase]); on the first day of dosing; at weekly intervals prior to pairing and until confirmation of mating; on GD 0, 7, 14, and 20; and on LD 0 (if applicable), 1, 4, 7, 13, and 14 (prior to necropsy). Body weights for females with no confirmation of mating were recorded weekly during the post-pairing phase.

FOOD CONSUMPTION: Yes
The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 1 to 13. Consumption was calculated as g/animal/day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy on Study Day 44 (Post-Pairing Day 15 for males) or LD 14 for females.
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: 10 males/group; 7-8 females/group
- Parameters checked: Haemoglobin, Red Blood Cells, Packed Cell Volume, Mean Cell Volume, Mean Cell Haemoglobin, Mean Cell Haemoglobin Concentration, Reticulocytes, Absolute reticulocytes, Red Cell Distribution Width, Haemoglobin Distribution Width
White Blood Cells, Neutrophils, Lymphocytes,Monocytes, Eosinophils, Basophils, Large Unstained Cells, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Large Unstained Cells, Platelets, Platelet Crit, Mean Platelet Volume, Platelet Distribution Width
Coagulation tests: prothrombin time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy on Study Day 44 (Post-Pairing Day 15 for males) or LD 14 for females.
- Animals fasted: Yes, overnight
- How many animals: 10 males/group; 8-10 females/group
- Parameters checked:
Aspartate Aminotransferase, Alanine Aminotransferase, Alkaline Phosphatase, Total Cholesterol, Total Bilirubin, Total Protein, Albumin, Globulin, Albumin/Globulin Ratio, Sodium, Potassium, Chloride, Calcium Gen 2, Inorganic Phosphate, Enzymatic Creatinine, Urea, Glucose, Bile Acid

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations
Functional observation battery (FOB):
males were assessed once prior to dose initiation and once weekly thereafter. Females were assessed once prior to dose initiation; once weekly during the pre-pairing and pairing phases; on GD 0, 7, 14, and 20; and on LD 1, 7, and 13. Females with no confirmation of mating were assessed once weekly during the post-pairing phase.
Motor activity: during Week 6 of the dosing phase (Post-Pairing Day 7) for males and on LD 7 for females
Quantitative assessments: during Week 6 of the dosing phase (Post-Pairing Day 12) for males and on LD 7 for females.
- Dose groups that were examined:
Functional observation battery (FOB): all dose groups; males: 10/dose; females: 10/dose (during gestation 9-10/dose)
Motor activity and quantitative assessments: all dose groups; 5 animals/sex/dose (five males with the lowest identification numbers and the first five littered females/group)

IMMUNOLOGY: No

THYROID HORMONE DETERMINATION: Yes (T4 & TSH)
- Time schedule for examinations: were withdrawn from the jugular vein inlife (males) or abdominal aorta at necropsy (females) on Study Day 45 (Post-Pairing Day 15 for males) or LD 14 for females. Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00).
- Animals fasted: Yes, overnight
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- How many animals: all parental males
Sacrifice and pathology:
SACRIFICE
- Male animals: Males were sacrificed by isoflurane anesthesia on Post-Pairing Day 15 (Day 44) after the preliminary evaluation of female data and an overnight period without food. Sacrifices were carried out in a controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
- Maternal animals: Females were sacrificed by isoflurane anesthesia on LD 14 (those that littered) or Day 26 post coitum (those that did not litter), after an overnight period without food. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Females were sacrificed in a controlled randomization order, when possible.

GROSS NECROPSY/ ORGAN WEIGHTS
Organ weights were recorded at each scheduled sacrifice excluding non-littered females. Organ weights were obtained from last 5 males with the highest identification number and the first 5 littered females (that survided to scheduled necropsy) within control and high dose group, and all decedents. The number of implantation sites was recorded in the female animals.
Weights of the following organs were recorded:
adrenal, brain, epididymis, heart, kidney, liver, ovary, pituitary, prostate, seminal vesicle with coagulating gland, spleen, testis, thymus, thyroid with parathyroid, uterus with cervix

HISTOPATHOLOGY
All tissues from control and high dose group (last 5 males with the highest identification number and the first 5 littered females (that survided to scheduled necropsy)) were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin (H/E).

The following tissues were prepared for microscopic examination:
adrenal, animal identification, aorta, brain (including cerebrum, cerebellum, and pons), cecum, colon, duodenum, epididymis, eye, esophagus, femur with bone marrow and femorotibial joint, gut associated lymphoid tissue (GALT)/Peyer’s patch, gross lesions, heart, ileum, jejunum, kidney, liver, lungs with main stem bronchi and bronchioles, lymph node (mandibular &mesenteric), mammary gland, muscle (biceps femoris), nerve (optic & sciatic), ovary, oviduct, pituitary, prostate, rectum, seminal vesicle with coagulating glands, spinal cord (cervical, lumbar & thoracic), spleen, sternum with bone marrow, stomach, testis, thymus, thyroid with parathyroid, trachea, ureter, urinary bladder, uterus with cervix, vagina
Other examinations:
For other examinations please refer to Toxicity to reproduction, section 7.8.1- k_Barraclough_2019
Statistics:
Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P < 0.05. By default, significant results were reported as * = P < 0.05, + = P < 0.01, and/or # = P < 0.001.

The following data were analyzed using Tox Reporting.
- Continuous behavioral (FOB) data - latency to first step, number of rears, hind limb foot splay, forelimb grip strength, and hind limb grip strength - Procedure I (ANOVA) - Tox Reporting
- Clinical pathology and thyroid hormones - Procedure I (ANOVA) - Tox Reporting
- Locomotor activity - Procedure I (ANOVA) - Tox Reporting

The following data were analyzed using Pristima.
- Body weights - Procedure I (ANOVA) - Pristima
- Body weight gains - Procedure I (ANOVA) - Pristima
- Food consumption (gestation and lactation) - Procedure I (ANOVA) - Pristima
- Absolute organ weights and organ:terminal body weight ratios - Procedure I (ANOVA)

For further details on statistics please refer to the field "any other information on materials and methods incl. tables".
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS:
No test article-related clinical observations were noted.
Incidental clinical observations included skin- and fur-related observations (hair loss, sores/lesions, thinning fur, and/or staining), with isolated incidences of vocalization, broken/maloccluded teeth, red ear, or damaged ear or tail. These observations were mostly limited to a small number of animals across different dose groups, were not dose related, were transient, and/or were with comparable incidences as controls; therefore, they were considered not test article related.

POSTDOSE OBSERVATION:
Postdose observations (monitored over the first 3 days of the dosing phase) were limited to one female administered 1000 mg/kg/day (Animal R0709) noted with mouth rubbing upon return to home cage on Day 1. This incident was considered not test article related due to its isolated nature.

MORTALITY:
No unscheduled adult deaths occurred.

BODY WEIGHT AND WEIGHT CHANGES:
No test article-related changes in body weight were evident.
Any minor differences from controls, including those achieving statistical significance, were considered incidental.

FOOD CONSUMPTION:
No test article-related changes in food consumption were evident.

HEMATOLOGY:
Hematology findings were limited to minimal to mild decreases in absolute and percentage reticulocyte counts for males administered 300 or 1000 mg/kg/day and minimal to mild increases in hemoglobin and packed cell volume for females administered 300 or 1000 mg/kg/day; females administered 100 mg/kg/day were also observed with a minimal increase in packed cell volume. These differences from controls were generally dose-dependent; however, they were minor, specific to one sex only with most individual values within the historical data ranges, had no histopathology correlates, and were considered of no toxicological significance.

CLINICAL BIOCHEMISTRY:
Compared with controls, a minimal non-dose-related decrease in inorganic phosphate was observed for males administered 300 or 1000 mg/kg/day. While individual values in most control males were above the high end of historical control data ranges (1.0 – 2.1), most males administered 300 or 1000 mg/kg/day had these values within these ranges. The corresponding values in females were comparable with controls, and in absence of any histopathology correlates, these changes were considered of no toxicological significance.

THYROID HORMONES:
Thyroid hormone levels were unaffected by the test article.

BEHAVIOUR:
Neurobehavioral evaluations, including weekly functional observations or detailed clinical assessment, and grip strength or foot splay assessment, performed towards the end of the dosing phase, did not identify any effect of test article in males or females.
Locomotor activity was unaffected by test article administration.
During Trial 5, total activity count for males administered 1000 mg/kg/day was statistically significantly lower (p≤0.05) than controls.Overall total activity count for both sexes administered the test article was neither dose-dependent nor statistically significant, and this observation was considered incidental.
Any other statistically significant differences were not observed for the high dose animals and were also considered incidental.

ORGAN WEIGHTS:
Decreased absolute seminal vesicle weights and body weight ratios were recorded for males, though not in a strictly dose-related manner. No histopathology correlates were evident, and in the absence of any effect on mating, fecundity, or fertility indices, this observation was considered of no toxicological significance.
Other organ weight and organ weight ratio changes, including those considered statistically significant, were attributed to normal biological variation and were considered not Potassium Hexacyanocobaltate(III)-related as they were small in magnitude, not dose-dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.

GROSS PATHOLOGY:
No macroscopic findings considered Potassium Hexacyanocobaltate(III) related were recorded.
Most tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.

HISTOPATHOLOGY:
No microscopic findings considered Potassium Hexacyanocobaltate(III) related were recorded.
Microscopic findings were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Conclusions:
Once daily oral gavage administration of 100, 300, or 1000 mg/kg/day potassium hexacyanocobaltate(III) to male rats for 43 consecutive days and to female rats for up to 63 days was well tolerated. No unscheduled adult deaths occurred. No test article-related clinical or postdose observations were noted in adults. No effect of test article on body weight or food consumption was evident in adults. No macroscopic or microscopic findings considered test article related were recorded for adults. No test article-related thyroid hormone or thyroid weight changes were noted for adult males, adult females, or offspring at any dose level.
Minor changes in haematology or clinical parameters observed in one or both sexes were considered of no toxicological significance. Decreases in seminal vesicle weights were also observed at all dose levels, but without any dose relationship or histopathology correlates, and were therefore considered of no toxicological significance. In view of these results, the no observed adverse effect level (NOAEL) for males and females for systemic toxicity is considered 1000 mg/kg/day.

No deviations in execution or reporting were recorded, thus the study is considered a guideline study being reliable without restrictions (RL 1):
Guideline study (RL 1) with minor restrictions which have no impact on the study integrity:
- Body weight: At the commencement of study (Day 1 of dosing phase), Animals R0201 and R0209 (Group 3 males) and Animal R0303 (Group 4 male) had body weights that exceeded the mean male body weight by up to 26%, which was outside the specified limit of 20%. Most of the remaining animals on the study were within 10% of the mean body weight for each sex on Day 1. No impact on body weight was noted throughout the dosing phase for either sex, and this deviation was considered to have no impact on study integrity.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
key study available

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Once daily oral gavage administration of 100, 300, or 1000 mg/kg/day potassium hexacyanocobaltate(III) to male rats for 43 consecutive days and to female rats for up to 63 days was well tolerated. No unscheduled adult deaths occurred. No test article-related clinical or postdose observations were noted in adults. No effect of test article on body weight or food consumption was evident in adults. No macroscopic or microscopic findings considered test article related were recorded for adults. No test article-related thyroid hormone or thyroid weight changes were noted for adult males, adult females, or offspring at any dose level.

Minor changes in haematology or clinical parameters observed in one or both sexes were considered of no toxicological significance. Decreases in seminal vesicle weights were also observed at all dose levels, but without any dose relationship or histopathology correlates, and were therefore considered of no toxicological significance. In view of these results, the no observed adverse effect level (NOAEL) for males and females for systemic toxicity is considered 1000 mg/kg/day.

Justification for classification or non-classification

Repeated dose toxicity, oral

Since no adverse effect could be observed in a repeated dose toxicity study in which animals were dosed up to 1000 mg/kg bw/day, no classification for tripotassium hexacyanocobaltate is warranted.

According to the criteria of regulation (EC) No 1272/2008 tripotassium hexacyanocobaltate does not fulfil the classification criteria for specific target organ toxicity by repeated exposure (STOT-RE).