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Repeated dose toxicity: oral

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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.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-18 to 2018-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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. Estrous cycle: During the predose phase, a number of females exhibited estrous cycle length outside the regular 4- to 5-day, but it was not possible to exclude all such females from the study because only six spare females were available for replacement. As these females are ordered at an age when they are sexulaly mature, it is rare to receive females which are not cycling regularly, therefore only 6 spare females are ordered instead of at least 8-12, to reduce animal usage in compliance with animal welfare principles. In spite of inclusion on the study of a number of females with cycle length outside 4- to 5-day, most such females exhibited mean cycle length of 4- to 5-day during the first two weeks of dosing phase, i.e. before commencement of pairing for mating. An female with mean cycle lenght outise this limit during the pre-pairing dosing phase was also pregnant. No impact of test article was noted on female mating or fertility and, as such, 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 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
Details on mating procedure:
- M/F ratio per cage: 1 male/1 female
- Length of cohabitation: the female was placed with the same male until pregnancy had occured or 15 days had elapsed.
- Proof of pregnancy: mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. The day on which mating was confirmed was designated GD 0.
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for additional 5 days.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: following mating, females were housed individually during gestation or the post-pairing phase, and with their litter during the lactation phase.
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
Parental animals: Observations and examinations:
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.

PARTURITION:
Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered, or until a potential GD 25, whichever was soonest. Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, where possible, and marked the end of gestation; when not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made.

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
Oestrous cyclicity (parental animals):
Daily vaginal lavage (washings) was conducted for all females during acclimation (predose), and for 14 consecutive days prior to dosing. The stage of estrous was recorded, and females with regular 4- to 5-day cycles were included on study, where possible. Daily vaginal lavage was performed on females from the start of dosing until the confirmation of mating and on the morning of LD 14, prior to necropsy.
Sperm parameters (parental animals):
Parameters examined in parental generation:
spermatogenic staging, interstitial testicular cell structure, testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); culled pups had the sex determined at necropsy and blood for thyroid hormone quantification was selected and were then discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups (on PND 1,4,7 and 13), stillbirths, live births, postnatal mortality (daily), body weight (on PND 1,4,7, and 13), anogenital distance (AGD- on PND 4), presence of nipples/areolae in male pups (on PND 13), clinical observation (daily)

THYROID HORMONE (TSH & T4) DETERMINATION
- Time schedule for examinations: PND 4 (surplus pups); PND 13
- Animals fasted: No
- How many animals: PND 4- from all culled pups, to provide one pooled sample for each litter (where possible); PND 13- at least one pup/sex/litter (if possible)
Postmortem examinations (parental animals):
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

Additional sections of testes and epididymides were also stained with periodic acid Schiff (PAS) for spermatogenic staging for all Group 1 and 4 males.
Postmortem examinations (offspring):
SACRIFICE
Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 13 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, major blood vessels were severed to exsanguinate the animal (decapitation for PND 4 pups).

GROSS NECROPSY
Surplus pups culled on PND 4 were discarded following blood sample collection and sex determination. Full macroscopic examinations were conducted for all decedents and one pup/sex/litter sacrificed on PND 13. External examinations for macroscopic abnormalities, with particular attention to the external reproductive genitals, were conducted for all remaining pups sent to necropsy on PND 13.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands of pups culled on PND 13 (one pup/sex/litter across groups) were weighed approximately 24 hours post-fixation and preserved in 10% neutral-buffered formalin.
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 (adult) - Procedure I (ANOVA) - Tox Reporting
- Locomotor activity - Procedure I (ANOVA) - Tox Reporting

The following data were analyzed using Pristima.
- Body weights (adult) - Procedure I (ANOVA) - Pristima
- Body weight gains (adult) - Procedure I (ANOVA) - Pristima
- Food consumption (gestation and lactation) - Procedure I (ANOVA) - Pristima
- Adult absolute organ weights and organ:terminal body weight ratios for adults Procedure I (ANOVA)
- The mean number of estrous cycles and mean cycle length - Procedure III
- Male and female mating, fecundity, and fertility indices - Procedure IV (one-sided lower tail)
- Pup weights (male, female, and combined) - Procedure II (litter size as the covariate)

The following data were analyzed using Statistical Analysis System (SAS).
- The duration of gestation; the number of implantation sites; the number of pups born; the number of pups alive on PND 1 and 4 (before culling), % male pups on PND 1, percent post implantation loss, and live birth and survival indices, where appropriate - Procedure III
- Ano-genital distance (males only) - Procedure II (cube root of mean pup weight as the covariate)
- Clinical pathology (male and female pups), where appropriate - Procedure I (ANOVA) - Tox Reporting
- Pup absolute organ weights and organ:terminal body weight ratios - Procedure I (ANOVA) - Pristima

For further details on statistics please refer to the field "any other information on materials and methods incl. tables".
Reproductive indices:
For reproductive indices please refer to the field "any other information on materials and methods incl. tables".
Offspring viability indices:
For offspring viability indices please refer to the field "any other information on materials and methods incl. tables".
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Of the pregnant females (10, 9, 9, and 10 administered control article [vehicle], 100, 300, or 1000 mg/kg/day, respectively), all littered and maintained their pups to LD 13 except two females administered 1000 mg/kg/day (Animals R0707 and R0799). Histopathology assessment of the uteri from these two females confirmed presence of implantation, with total post-implantation loss presumed to be due to early resorptions. In the absence of similar observations in other groups, this change was considered test article related and adverse based on the occurrence in high-dose dams.
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.

ESTROUS CYCLES:
The number and length of estrus cycles was unaffected by the test article. Minor differences were not dose related and were considered incidental.

REPRODUCTIVE INDICES:
Mating performance was unaffected by the test article, with most animals mating within 5 days of pairing.
Two males administered 100 mg/kg/day (Animals R0104 and R0109), two males administered 300 mg/kg/day (Animals R0204 and R0210), and one male administered 1000 mg/kg/day (Animal R0310) did not mate with their respective female partners within the allotted 10 days of pairing. These unmated females were re-paired with previously proven males for up to an additional 5 days, which had successfully mated with other females, from the corresponding groups. Following the second pairing, signs of mating were still not detected for one female administered 100 mg/kg/day (Animal R0504) and one female administered 300 mg/kg (Animal R0604), but the remaining females had mated. It is noted that Female R0604 (300 mg/kg/day) was in pseudopregnancy (a phenomenon often observed in this type of study) during pairing, which may explain the lack of mating for these animals. In absence of a dose relationship, these observations were considered incidental.

Mating and fertility indices for males administered the test article at all dose levels and females administered 100 or 300 mg/kg/day were lower than controls. With the exception of one male administered 1000 mg/kg/day (Animal R0310), the remaining males and all females from this group had mated, while all females from this group were pregnant. In absence of a dose relationship, this observation was considered unrelated to the test article.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
CLINICAL OBSERVATIONS:
No test article-related clinical observations were noted for offspring from test article treated dams at all dose levels.
The clinical observations recorded were low-incidence findings, without dose dependence or only noted in controls, and were considered incidental.

BODY WEIGHT:
No effect on pup body weights was evident following maternal administration of the test article at all dose levels.

ANO-GENITAL DISTANCE:
No effect on ano-genital distance was observed in litters from dams administered the test article at any dose level.

NIPPLE/AREOLAE COUNT:
No nipples/areolae were present for male offspring that survived to PND 13.

PUP THYROID HORMONE:
Thyroid hormone levels were unaffected in offspring of dams administered up to 1000 mg/kg/day.

ORGAN WEIGHT:
No thyroid weight or thyroid:body weight ratio changes considered Potassium Hexacyanocobaltate(III) related were recorded for pups sacrificed on PND 13.

MACROSCOPIC OBSERVATION:
No macroscopic findings considered Potassium Hexacyanocobaltate(III) related were recorded on PND 13 for pups from dams administered 100, 300, or 1000 mg/kg/day.
Most tissues were macroscopically unremarkable, and/or the findings observed were generally consistent with the usual pattern of findings in rats of this strain, age, and stage of development.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
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 (pre-mating, throughout gestation, and during the first 2 weeks of lactation) 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.
The number and length of oestrus cycles, mating, and fertility were unaffected by the test article.
Male reproductive performance was not affected at any dose level, and the NOAEL for reproductive toxicity in males is considered 1000 mg/kg/day.
Ten, nine, nine, and ten females administered control article (vehicle) or 100, 300, or 1000 mg/kg/day, respectively, were pregnant. Of those, ten, nine, nine, and eight dams, respectively, littered and maintained a live litter to LD 13. Early resorption was the presumed cause of in utero litter losses for two females administered 1000 mg/kg/day, and in the absence of similar observations in other groups, this change was considered test article related and adverse based on its occurrence in high dose dams. However, post implantation loss manifested as early resorptions is an occasional observation in rodent developmental toxicity studies and occurs in 4.6% per litter (2.0-8.4% per litter). Due to the limited data on post-implantation losses available for this study design (only qualitative data available) and the low number of pregnant animals, no definitive conclusion can be drawn whether the effect is to be considered a true substance related response or an incidental finding. The NOAEL for reproductive toxicity in females is considered 300 mg/kg/day.
Offspring development was not affected following administration of the test article at any dose level, and the NOAEL for offspring development is considered 1000 mg/kg/day.
Minor changes in haematology or clinical parameters observed in one or both sexes were considered of no toxicological significance. Decreases in seminal vesicle weights in males and ovary weights in females were observed at all dose levels, but without 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.

Due to a number of minor deviations in the execution of the experiments as detailed below, the study is considered a guideline study with acceptable restrictions (RL 2):
- 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.
- Estrous cycle: During the predose phase, a number of females exhibited estrous cycle length outside the regular 4- to 5-day, but it was not possible to exclude all such females from the study because only six spare females were available for replacement. As these females are ordered at an age when they are sexulaly mature, it is rare to receive females which are not cycling regularly, therefore only 6 spare females are ordered instead of at least 8-12, to reduce animal usage in compliance with animal welfare principles. In spite of inclusion on the study of a number of females with cycle length outside 4- to 5-day, most such females exhibited mean cycle length of 4- to 5-day during the first two weeks of dosing phase, i.e. before commencement of pairing for mating. An female with mean cycle length outside this limit during the pre-pairing dosing phase was also pregnant. No impact of test article was noted on female mating or fertility and, as such, this deviation was considered to have no impact on study integrity.
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
range-finding study for an OECD 422 study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-12-20 to 2018-01-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of the study was to determine the toxicity of potassium hexacyanocobaltate(III) following daily oral (gavage) administration to the rat for 3 weeks and to provide the basis for the selection of dose levels for subsequent studies. Groups of five male and female Crl:WI(Han) rats were treated at dose levels of 0, 100, 300 and 1000 mg/kg bw/day once daily for at least 3 weeks. The control article (vehicle) was 0.9% saline. Assessment of toxicity was based on mortality, clinical and postdose observations, body weights, food consumption, organ weights, and macroscopic observations.
GLP compliance:
no
Remarks:
this study is a non-regulatory study for which a claim of GLP compliance was not made. However, the laboratory procedures used were fully commensurate with international standards of GLP.
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 rodent species known to be accepted by various regulatory authorities.
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: 6 to 7 weeks old
- Weight at start of dosing: males: 162.9 to 202.4 g; females: 119.4 to 171.2 g
- Housing: housed in groups of two or three animales in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014); bedding: provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom).
- Diet (ad libitum): 5LF2 EU Rodent Diet (International Product Supplies Ltd, London, United Kingdom)
- Water (ad libitum): main supply water
- Acclimation period: 13 days
- Dietary/environmental enrichment: animals were provided with wooden Aspen chew blocks, amber tunnels, and dried pasta shells as forms of dietary and environmental enrichment.

DETAILS OF FOOD AND WATER QUALITY:
- water quality: no contaminants were present in the water at levels which might have interfered with achieving the objective of the study.
- food quality: no contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.

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
Details on route of administration:
The oral route of administration was chosen because it is an acceptable and commonly used route of exposure for studies of this type.
Vehicle:
physiological saline
Remarks:
0.9 % saline
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was formulated as a solution in the vehicle and the formulations were prepared daily.
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. Individual dose volumes were calculated from the most recent body weights for each animal.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
21 days
Frequency of treatment:
once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the OECD 423 acute oral (gavage) toxicity study, the LD50 (lethal dose 50) was determined to be greater than 2000 mg/kg body weight.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at the beginning and the end of the working day as well as upon return to the home cage after detailed clinical observations and at 0.5, 1, 2, and 4 hours postdose daily for the first 3 days of the dosing phase (also on Day 4 of the dosing phase upon return to home cage postdose observations were recorded)
- Cage side observations checked: signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals during the predose phase and daily during the dosing phase, including the day of terminal necropsy;

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 of the predose phase as well as on Days 1, 4, 8, 12, 15, 19, and 20 of the dosing phase; terminal body weight was recorded before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No

All animals were subjected to necropsy and the scheduled necropsies were performed on Day 22 of the dosing phase after an overnight period without food.
The epididymis and testis from all male animals were dissected, freed from fat and other contiguous tissue, and weighed before fixation. Left and right organs were weighed together.
A full macroscopic examination was performed and all lesions were recorded.
The following tissues from each animal were preserved: epididymis, gross lesions, ovary, oviduct, prostate, seminal vesicle (incl. coagulating glands), testis, uterus with cervix and vagina. Tissues were retained in fixative.
Statistics:
Data from treated animals were compared with control data.
The following data were analyzed statistically: body weight changes, terminal body weights, absolute organ weights and organ:body weight percentages
Data for each sex were analyzed separately; only data collected on or after the first day of dosing were analyzed statistically.
The following statistical test were used, if applicable, for data analysis: Levene’s test, rank transformation, two-sample t-test, ANOVA, pairwise comparisons, Dunnett’s test and t-tests,
All pairwise comparisons were evaluated at the 5.0, 1.0, and 0.1%, two tailed probability levels.
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS.:
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on clinical observations was noted. All clinical observations were considered incidental as they occurred in controls, showed no dose relationship, and/or are commonly observed background findings for this species and strain.
No postdose observations were observed.

MORTALTY.
0, 100, 300 and 1000 mg/kg/day: no unscheduled mortality occurred.

BODY WEIGHT AND WEIGHT CHANGES:
0, 100, 300 and 1000 mg/kg/day: throughout the dosing phase, mean body weight gain for test article treated animals tended to be greater than controls; however, the increase did not show a dose relationship. The changes were, therefore, considered to have an uncertain relationship to test article and of no toxicological significance.

FOOD CONSUMPTION:
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on food consumption was noted. Food consumption values were similar between all groups, including controls.

ORGAN WEIGHTS:
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on organ weight was noted. Variation in organ weights were considered incidental as they showed no relationship to dose and were within the expected background ranges for this species and strain.

GROSS PATHOLOGICAL FINDINGS:
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on macroscopic observations was noted. All observations were considered incidental as they showed no dose relationship and/or are commonly observed background findings for this species and strain.

Remarks on result:
other: This dose range finder showed that a daily oral gavage administration of 100, 300, or 1000 mg/kg/day to rats for 3 weeks was well tolerated, with no adverse findings.
Critical effects observed:
not specified
Conclusions:
Daily exposure of male and female Crl:WI(Han) rats to potassium hexacyanocobaltate (III) (100, 300 and 1000 mg/kg/day) via gavage for a 21 day period caused no treatment-related effects on clinical signs, mortality, body weight, food consumption, organ weights and macroscopic observations.

Based on the findings from this dose-range finder, it was concluded that doses up to 1000 mg/kg/day may be tolerated in studies of longer duration.
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
reproductive toxicity, other
Remarks:
range-finding study for an OECD 422 study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-12-20 to 2018-01-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of the study was to determine the toxicity of potassium hexacyanocobaltate(III) following daily oral (gavage) administration to the rat for 3 weeks and to provide the basis for the selection of dose levels for subsequent studies. Groups of five male and female Crl:WI(Han) rats were treated at dose levels of 0, 100, 300 and 1000 mg/kg bw/day once daily for at least 3 weeks. The control article (vehicle) was 0.9% saline. Assessment of toxicity was based on mortality, clinical and postdose observations, body weights, food consumption, organ weights, and macroscopic observations.
GLP compliance:
no
Remarks:
this study is a non-regulatory study for which a claim of GLP compliance was not made. However, the laboratory procedures used were fully commensurate with international standards of GLP.
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 rodent species known to be accepted by various regulatory authorities.
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: 6 to 7 weeks old
- Weight at start of dosing: males: 162.9 to 202.4 g; females: 119.4 to 171.2 g
- Housing: housed in groups of two or three animales in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014); bedding: provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom).
- Diet (ad libitum): 5LF2 EU Rodent Diet (International Product Supplies Ltd, London, United Kingdom)
- Water (ad libitum): main supply water
- Acclimation period: 13 days
- Dietary/environmental enrichment: animals were provided with wooden Aspen chew blocks, amber tunnels, and dried pasta shells as forms of dietary and 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 exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was formulated as a solution in the vehicle and the formulations were prepared daily.
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. Individual dose volumes were calculated from the most recent body weights for each animal.
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
21 days
Frequency of treatment:
once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the OECD 423 acute oral (gavage) toxicity study, the LD50 (lethal dose 50) was determined to be greater than 2000 mg/kg body weight.
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at the beginning and the end of the working day as well as upon return to the home cage after detailed clinical observations and at 0.5, 1, 2, and 4 hours postdose daily for the first 3 days of the dosing phase (also on Day 4 of the dosing phase upon return to home cage postdose observations were recorded)
- Cage side observations checked: signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals during the predose phase and daily during the dosing phase, including the day of terminal necropsy;

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 of the predose phase as well as on Days 1, 4, 8, 12, 15, 19, and 20 of the dosing phase; terminal body weight was recorded before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
not applicable
Sperm parameters (parental animals):
Parameters examined in all males: testis weight and epididymis weight
Litter observations:
not applicable
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No

All animals were subjected to necropsy and the scheduled necropsies were performed on Day 22 of the dosing phase after an overnight period without food.
The epididymis and testis from all male animals were dissected, freed from fat and other contiguous tissue, and weighed before fixation. Left and right organs were weighed together.
A full macroscopic examination was performed and all lesions were recorded.
The following tissues from each animal were preserved: epididymis, gross lesions, ovary, oviduct, prostate, seminal vesicle (incl. coagulating glands), testis, uterus with cervix and vagina. Tissues were retained in fixative.
Postmortem examinations (offspring):
not applicable
Statistics:
Data from treated animals were compared with control data.
The following data were analyzed statistically: body weight changes, terminal body weights, absolute organ weights and organ:body weight percentages
Data for each sex were analyzed separately; only data collected on or after the first day of dosing were analyzed statistically.
The following statistical test were used, if applicable, for data analysis: Levene’s test, rank transformation, two-sample t-test, ANOVA, pairwise comparisons, Dunnett’s test and t-tests,
All pairwise comparisons were evaluated at the 5.0, 1.0, and 0.1%, two tailed probability levels.
Reproductive indices:
not applicable
Offspring viability indices:
not applicable
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
CLINICAL SIGNS.:
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on clinical observations was noted. All clinical observations were considered incidental as they occurred in controls, showed no dose relationship, and/or are commonly observed background findings for this species and strain.
No postdose observations were observed.

MORTALTY.
0, 100, 300 and 1000 mg/kg/day: no unscheduled mortality occurred.

BODY WEIGHT AND WEIGHT CHANGES:
0, 100, 300 and 1000 mg/kg/day: throughout the dosing phase, mean body weight gain for test article treated animals tended to be greater than controls; however, the increase did not show a dose relationship. The changes were, therefore, considered to have an uncertain relationship to test article and of no toxicological significance.

FOOD CONSUMPTION:
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on food consumption was noted. Food consumption values were similar between all groups, including controls.

ORGAN WEIGHTS:
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on organ weight was noted. Variation in organ weights were considered incidental as they showed no relationship to dose and were within the expected background ranges for this species and strain.

GROSS PATHOLOGICAL FINDINGS:
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on macroscopic observations was noted. All observations were considered incidental as they showed no dose relationship and/or are commonly observed background findings for this species and strain.

REPRODUCTIVE FUNCTION: SPERM MEASURES
0, 100, 300 and 1000 mg/kg/day: no test article-related effect on epididymis and testis weights was noted.
Remarks on result:
other: This dose range finder showed that a daily oral gavage administration of 100, 300, or 1000 mg/kg/day to rats for 3 weeks was well tolerated, with no adverse findings.
Critical effects observed:
not specified
Generation:
F1
Remarks on result:
not measured/tested
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Daily exposure of male and female Crl:WI(Han) rats to potassium hexacyanocobaltate (III) (100, 300 and 1000 mg/kg/day) via gavage for a 21 day period caused no treatment-related effects on clinical signs, mortality, body weight, food consumption, organ weights and macroscopic observations.

Based on the findings from this dose-range finder, it was concluded that doses up to 1000 mg/kg/day may be tolerated in studies of longer duration.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

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:
2016-07-29
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tripotassium hexacyanocobaltate
EC Number:
237-742-1
EC Name:
Tripotassium hexacyanocobaltate
Cas Number:
13963-58-1
Molecular formula:
C6CoN6.3K
IUPAC Name:
tripotassium hexacyanocobalttriuide
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: light yellow powder
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).

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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".

Results and discussion

Results of examinations

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.

Effect levels

Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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.