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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-09 to 2015-02-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 1998-09-21
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: crystals
Details on test material:
- Name of test material (as cited in study report): Cobalt dichloride hexahydrate
- Molecular formula: CoCl2·6 H2O
- Physical state: solid, lilac crystals
- Storage condition of test material: to be stored cool and well-ventilated in a closed container, preferably under inert atmosphere.

Test animals

Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males: 52 days; females: 65 days
- Weight at first dosing: males: 244.9 - 295.5 g; females: 204.1 - 246.5 g
- Housing: animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material.
- Diet (ad libitum): commercial ssniff®-R/M-H V1534 (ssniff® Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): drinking water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The administration formulations were freshly prepared every day by dissolving the test item in the vehicle to the appropriate concentrations.
Administration volume: 2 mL/kg bw/day

The dose of the test item was adapted to the animal's body weight daily up to and including test week 6, and weekly thereafter.
The control animals received the vehicle at a constant volume of 2 mL/kg bw/day orally once daily in the same way.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the administration formulations, samples of approximately 10 mL were taken at the following times and stored at -20°C or colder until analyses:
1) At study initiation (on the first administration day of male animals):
- analysis of stability and concentration: immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature (3 samples/test item group).
- homogeneity: at the start of administration, during (middle) administration and before administration to the last animal of the test item group (3 samples/test item group).

2) At study termination (on the last administration day of female animals):
- analysis of concentration: during treatment always before administration to the last animal of the group (1 sample/test item group).

The determination of the content of the test item cobalt dichloride hexahydrate in samples was performed by analysis of cobalt with Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES).

Results:
The generated results verify the concentration, the homogeneity and the stability of the test item cobalt dichloride hexahydrate in application mixtures during the toxicology study. The actual cobalt dichloride hexahydrate concentrations ranged from 101.4% to 102.1% of the nominal concentrations.
Duration of treatment / exposure:
90 days (except male recovery animals: 91 days)
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
3, 10, and 30 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Main study (per group): 10 males/10 females
Recovery group (control group and 30 mg/kg bw/day dose group only; per group): 5 males/5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels for this study have been selected based on available data.
- Recovery groups were included in this study. One recovery group was included for the control group and other recovery group for the 30 mg/kg bw/day dose group. These groups were kept for 28 days after the treatment period without receiving the test item.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (main study animals and recovery animals)
- Time schedule:
Clinical signs: before and after dosing at each time of dosing as well as regular daily
Mortality: twice daily
- Cage side observations (included): skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

DETAILED CLINICAL OBSERVATIONS: Yes (main study animals and recovery animals)
- Time schedule: once before the first exposure and once a week thereafter
- Observations (included): skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards).

BODY WEIGHT: Yes (main study animals and recovery animals)
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (main study animals and recovery animals):
- Food consumption for each animal determined and relative food consumption calculated as g food/kg body weight/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: Yes (main study animals and recovery animals)
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes (main study animals and recovery animals)
- Time schedule for examinations: prior to the start of administration and at main study termination (all main study and recovery animals), and at the end of the recovery period (all recovery animals)(before blood sampling for laboratory examinations at all time points)
- Parameters examined: adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, and fundus.
Prior to examination, mydriasis was produced after instillation of MYDRUM® eye drops into the conjunctival sacs.

HAEMATOLOGY: Yes (main study animals and recovery animals)
- Time schedule for collection of blood: at the end of the treatment period (test day 91, except male recovery animals test day 92; main study animals before necropsy) and at the end of the recovery period (all recovery animals)
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes
- How many animals: all main study animals and all recovery animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, absolute and relative differential blood count (neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes, monocytes and large unstained cells), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes (main study animals and recovery animals)
- Time schedule for collection of blood: at the end of the treatment period (test day 91, except male recovery animals test day 92; main study animals before necropsy) and at the end of the recovery period (all recovery animals)
- Animals fasted: Yes
- How many animals: all main study animals and all recovery animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), triglycerides, urea (in blood), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase

URINALYSIS: Yes (main study animals and recovery animals)
- Time schedule for collection of urine: at the end of test week 13 (all main study group and recovery animals; before necropsy) and at the end of the recovery period (all recovery animals; before necropsy)
- Animals fasted: No
- Parameters examined: colour, turbidity, volume, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite, and microscopic examinations of urine samples (epithelial cells, leucocytes, erythrocytes, organisms, crystalluria, and further constituents (i.e. sperm, casts))

NEUROBEHAVIOURAL EXAMINATION: Yes (main study animals & recovery animals)
- Time schedule for examinations: week 13 (main study groups) and week 17 (recovery groups)
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity

1) Observational screening:
Righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, and auditory function

2) Functional tests: grip strength and locomotor activity

NOTE: Besides the above described parameters the following parameters were also investigated in this study: hormone levels and stages if the oestrous cycle. Please refer for the results of these parameters to Section 7.8.1 Toxicity to reproduction: k_Hansen_2015_CoCl2
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (main study animals and recovery animals)
On test day 91, the main study animals were dissected following a randomisation scheme. Animals not dissected on test day 91 were dosed until one day before sacrifice. Necropsy of all animals allocated to the recovery period was performed on test day 119.
The animals were euthanized under ether atmosphere, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS: Yes (main study animals and recovery animals)
The weights of the following organs of all animals were determined: adrenal gland (2), liver, thymus, brain, ovary (2), prostate and seminal vesicles with coagulating glands as a whole, epididymis (2), pancreas, heart, spleen, uterus (incl. cervix), kidney (2), and testicle (2).
Paired organs were weighed individually and identified as left or right.

HISTOPATHOLOGY: Yes (main study animals and recovery animals)
The following organs or parts of organs with the exception of the eyes, epididymides and testicles of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson’s solution for optimum fixation. The epididymides and testicles were preserved in Bouin’s fixative.

Organs: adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (3 levels: right and left ventricle, septum), intestine, large (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer´s patches; Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary (2), pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, sublingual and parotid gland), skin (left flank), spinal cord (3 levels: cervical, mid-thoracic, lumbar), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumours (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix), and vagina.

The afore-listed organs of all main study and recovery animals of the control group and the 30 mg/kg bw/day group were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
Detailed histopathological examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all male main study and recovery animals of the control group and the 30 mg/kg bw/day group following staining.
The organs and tissues listed above were examined.

BONE MARROW (main study animals and recovery animals)
Fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of all main study and recovery animals stained according to
PAPPENHEIM. The myeloid : erythroid ratio was determined for the control group and the 30 mg/kg bw/day group by cell differentiation (counting of 200 nuclei-containing cells).
Statistics:
The test item-treated groups (3, 10, and 30 mg/kg bw/day dose groups) were compared with the control group.
The following statistical methods were used:
- Multiple t-test based on DUNNETT, C. W. New tables for multiple relative and absolute organ weights comparisons with a control Biometrics, 482 - 491
(September 1964): body weight, food consumption, haematology, clinical biochemistry, urinalysis, relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)

- STUDENT's t-test: all numerical functional tests: body temperature, hind leg splay, grip strength, spontaneous motility; hormone levels (p ≤ 0.05 and p ≤ 0.01)

- Chi²-test: bone marrow (p ≤ 0.05)

- Exact test of R. A. FISHER: histopathology (p ≤ 0.05)

These statistical procedures were used for all data.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1) Treatment period:
- no test item-related changes in behaviour or external appearance were noted for the male and female animals treated with 3, 10 or 30 mg/kg bw/day.
- 10 mg/kg bw/day: one male showed a haemorrhagic left canthus on test days 26 to 32 and one female showed a reddened right eyelid and/or a haemorrhagic canthus on test days 33 to 53.
- 30 mg/kg bw/day: pilo-erection was noted for 2 male recovery animals on test days 30 to 36. No changes in behaviour or external appearance were noted for the female animals.
- these findings are not considered to be test item-related due to the low number of animals affected.
- faeces of all animals were of normal consistency.
- no deaths were noted at any dose level. All main study animals survived until their scheduled terminal sacrifice.
- none of the animals treated with 3, 10 or 30 mg/kg bw/day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour at the detailed clinical observations.

2) Recovery period (restricted to the control group and 30 mg/kg bw/day group):
- no abnormalities in behaviour, external appearance or faeces were observed for the male and female animals previously treated with 30 mg/kg bw/day.
- no deaths were noted. All recovery animals survived until the scheduled recovery sacrifice.
- none of the animals revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour at the detailed clinical observations.

BODY WEIGHT AND WEIGHT GAIN
1) Treatment period:
- 3 mg/kg bw/day: body weight, body weight gain and body weight at autopsy were not influenced in the male and female animals in a test item-related way compared to the control group.
- 10 mg/kg bw/day: body weight of the male animals was reduced by 5 to 7% from test day 50 onwards (not statistically significant). Body weight gain changed accordingly. The body weight at autopsy was reduced by 6% (not statistically significant) for the male animals. No changes were noted for the female animals.
- 30 mg/kg bw/day: body weight of the animals was reduced by 5 to 14% from test day 8 onwards for the males (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 8, 22, and 43 to 90) and by 5 to 10% from test day 29 onwards for the females (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 50 to 90), respectively, compared to the control group. Body weight gain changed accordingly. The body weight at autopsy was reduced by 11% for the males (statistically significant at p ≤ 0.05) and by 9% for the females (not statistically significant), respectively.
- the reduced body weights at the 10 mg/kg bw/day and 30 mg/kg bw/day dose groups are considered as test item-related.

2) Recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- the differences in body weight between the animals previously treated with 30 mg/kg bw/day and the control group were still present at the end of the recovery period: The body weight of the male and female animals was still reduced by 17% or by 13%, respectively, on test day 118 (statistically significant reductions at p ≤ 0.05 on test day 97 for the males and at p ≤ 0.01 on test days 97 to 118 for the females) compared to the control group.
- the male animals revealed a slightly higher body weight gain than the control group during the recovery period indicating a trend towards recovery, while the body weight gain of the females was in the range of the control group. The body weight at autopsy was reduced by 17% for the males and by 13% for the females (statistically significant at p ≤ 0.01 for the females), respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- no test item-related influence was noted on the relative food consumption of the male and female animals treated with 3, 10 or 30 mg/kg bw/day during the treatment period and of the male and female animals previously treated with 30 mg/kg bw/day during the recovery period compared to the control group.
- slight but statistically significant (at p ≤ 0.05) increases in food consumption noted for the 30 mg/kg bw/day dosed males and females in test week 4 and for the 30 mg/kg bw/day dosed male animals in test week 14 are considered to be due to the reduced body weight and to be without any biological relevance.

WATER CONSUMPTION AND COMPOUND INTAKE
- visual appraisal of the drinking water consumption did not reveal any test item-related differences between the test item-treated animals and the control animals throughout the treatment and the recovery period.

OPHTHALMOSCOPIC EXAMINATION
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- no changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus were noted in the male and female rats of the animals treated with 3, 10 or 30 mg/kg bw/day at the end of the treatment period.
- no changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.

HAEMATOLOGY
1) Treatment period:
- 3 mg/kg bw/day: no test item-related influence on haematological parameters was noted for the male and female animals at the end of the treatment period.
- 10 or 30 mg/kg bw/day: the following test item-related changes in haematological parameters were noted for the male and female animals on test day 91 (male and female main study and female recovery animals) and 92 (male recovery animals). In general, the male animals were affected to a higher degree.

10 mg/kg bw/day (test day 91/92 (combined)):
haemoglobin content (males: +11%; p≤0.01)
erythrocytes (males: +10%; p≤0.01)
haematocrit value (males: +12%; p≤0.01)
reticulocytes (males: -33%; p≤0.05)
platelets (males: -13%)
mean corpuscular volume (females: +4%; p≤0.05);

30 mg/kg bw/day (test day 91/92 (combined)):
haemoglobin content (males: +25%; females: +14%; p≤0.01)
erythrocytes (males: +19%; females: +11%; p≤0.01)
haematocrit value (males: +23%; females: +14%; p≤0.01)
thromboplastin time (males: +7%; p≤0.01)
activated partial thromboplastin time (males: +8%; p≤0.05)
mean corpuscular volume (males: +4%; females: +3%; p≤0.05 (males only))
mean corpuscular haemoglobin (males: +5%; p≤0.01)
reticulocytes (males: -24%)
platelets (males: -26%; females: -12%; p≤0.01 (males only))

- no test item-related influence was noted for the number of leucocytes, the relative and absolute differential blood count, and the mean corpuscular haemoglobin concentration.

- statistically significant differences in haematological parameters compared to the control which are not considered to be test item-related were found in the following parameters: leucocytes, absolute lymphocytes, absolute eosinophilic granulocytes, absolute large unstained cells, and absolute basophilic granulocytes

2) Recovery period (restricted to the control group and 30 mg/kg bw/day group):
- all changes in haematological parameters previously observed after repeated treatment with 30 mg/kg bw/day had subsided after 4 weeks of recovery.
- no effects related to the previous treatment were observed on the haemoglobin content, the numbers of erythrocytes, leucocytes and platelets, the relative reticulocyte count), the haematocrit value, the relative and absolute differential blood count, the thromboplastin time, the activated partial thromboplastin time, the mean corpuscular volume, the mean corpuscular haemoglobin and the mean corpuscular haemoglobin concentration at the end of the recovery period.
- statistically significant differences in haematological parameters compared to the control which are not considered to be test item-related were found for the following parameters: absolute eosinophilic granulocytes

Please also refer for results about haematology to "Attached background material" below.

CLINICAL CHEMISTRY
1) Treatment period:
- 3 mg/kg bw/day: no test item-related influence on biochemical parameters was noted for the male and female animals at the end of the treatment period.
- 10 or 30 mg/kg bw/day: the following test item-related changes in biochemical parameters were noted for the male and female animals on test day 91 (male and female main study and female recovery animals) and 92 (male recovery animals):

10 mg/kg bw/day (test day 91):
bilirubin (males: +17%)

10 mg/kg bw/day (test day 91/92 (combined)):
bilirubin (males: +14%)

30 mg/kg bw/day (test day 91):
bilirubin (males: +34%; p≤0.01; females: +16%; p≤0.05)

10 mg/kg bw/day (test day 91/92 (combined)):
bilirubin (males: +29%; p≤0.01; females: +16%; p≤0.05)

- no test item-related influence was noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium and the serum level of bile acids. Further, the plasma activity of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and lactate dehydrogenase was not influenced.
- statistically significant differences in biochemical parameters compared to the control which are not considered to be test item-related were found in the following parameters: albumin, globulin, albumin/globulin ratio, cholesterol, creatinine, glucose, protein, triglycerides, calcium, chloride, and alkaline phosphatase

2) Recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- all changes in biochemical parameters previously observed after repeated treatment with 30 mg/kg bw/day had subsided after 4 weeks of recovery.
- no effects related to the previous treatment were noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, bilirubin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium, and the serum level of bile acids. Further, the plasma activity of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase was not influenced.

URINALYSIS
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg bw/day: no test item-related influence on the urinary status was noted for the male and female animals at the end of the treatment period.
- no test item-related influence on the urinary status was noted for the male and female animals previously treated with 30 mg/kg b.w./day at the end of the recovery period.
- no test item-related changes were noted for the specific gravity of the urine, the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.
- statistically significant differences in urine parameters compared to the control which are not considered to be test item-related were found in the following parameter: pH

NEUROBEHAVIOUR
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- neurological screening performed at the end of the treatment period on test day 86 and at the end of the recovery period on test day 118 did not reveal any test item-related influence on the male and female rats treated with 3, 10 or 30 mg/kg bw/day, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.
- statistically significant differences in neurological parameters compared to the control which are not considered to be test item-related were found in the following parameters: body temperature, forelimb grip strength, and hindlimb grip strength

ORGAN WEIGHTS
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg bw/day: no test item-related changes in relative and absolute organ weights were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- statistically significant differences in relative and absolute organ weights compared to the control which are not considered to be test item-related were found in the following parameters: brain (relative), gonads (left testis, relative), spleen (relative), adrenal (left, absolute), brain (absolute), kidney (left, absolute), kidney (right, absolute), and gonads (right ovary, absolute)

GROSS PATHOLOGY
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg b.w./day: no test item-related changes were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- there were no test item-related abnormalities (gross pathology, tissue masses or tumours) in any tissue, including the adrenal gland, kidney and pancreas, in any of the exposed animals at the end of the treatment period, nor at the end of the recovery period.
- macroscopic changes were noted in the kidney (cyst), spleen (rough surface, adhered to peritoneum), stomach (haemorrhagic foci), uterus (cystic, filled with clear liquid) and testis, epididymis, seminal vesicle and prostate (reduced in size) in individual animals of the control and test item-treated groups at terminal or recovery sacrifice.
- these changes are considered to be incidental findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment and recovery period (full histopathological evaluation restricted to the control group and 30 mg/kg bw/day group):
- microscopic evaluation revealed test item-related changes in the bone marrow (erythroid hyperplasia) of the femur. There was a significant and test item-related increase for erythroid hyperplasia in the bone marrow of the male and female animals treated with 10 or 30 mg/kg bw/day compared to the controls: 4 of 10 males and 7 of 10 females in the 10 mg/kg bw/day dose group and 7 of 10 animals for both sexes in the 10 mg/kg bw/day dose group versus 0 of 10 in the 3 mg/kg bw/day dose group and controls. The bone marrow change (erythroid hyperplasia) attained statistical significance in animals of the 10 or 30 mg/kg bw/day dose groups for both sexes. After cessation of treatment, no test item-related changes were observed for the recovery animals anymore.
- there were no histopathological findings in any tissues, including the adrenal gland, kidney and pancreas, in any of the animals exposed p.o. to 30 mg/kg bw/day.
- all other microscopic changes seen in all organs in all animals were either coincidental, or were considered to lie within the normal range of background alterations, which may be seen in untreated rats of this age and strain.

BONE MARROW EXAMINATION
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group).
- 30 mg/kg bw/day: no test item-related changes in the myeloid:erythroid ratio were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- the slightly decreased myeloid:erythroid ratio (statistically significant at p ≤ 0.05) noted for the previously high dosed females at the end of the recovery period is considered to be in the normal range of variation and without any biological relevance.

Effect levels

Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effect level is based on the following parameters: haematology and body weight

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (rat, 90 days): 3 mg cobalt dichloride hexahydrate/kg bw/day

Looking at the treatment period the following test item-related changes were noted:
The body weight of the male animals treated with 10 mg/kg bw/day was reduced by 5 to 7% from test day 50 onwards. Body weight gain changed accordingly. The body weight at autopsy was reduced by 6% for the male animals. No changes were noted for the 10 mg/kg bw/day dosed female animals. Furthermore, the body weight of the male and female animals treated with 30 mg/kg bw/day was reduced by 5 to 14% from test day 8 onwards and by 5 to 10% from test day 29 onwards, respectively, compared to the control group. Body weight gain changed accordingly. The body weight at autopsy was reduced by 11% (males) and 9% (females), respectively.

Next, test-item related changes were seen in the haematological parameters and, in general, the male animals were affected to a higher degree. The haemoglobin content, erythrocytes, and haematocrit value were increased by ≥ 10% in males treated with 10 or 30 mg/kg bw/day. On the other hand, reticulocytes and platelets were decreased by ≥ 10% in males treated with 10 or 30 mg/kg bw/day. Thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, and mean corpuscular haemoglobin were increased up to 8% in males receiving 30 mg/kg bw/day. Lastly, haemoglobin content, erythrocytes, haematocrit value, and mean corpuscular volume were increased between 3 and 14% in females receiving 30 mg/kg bw/day. The latter parameter was also increased by 4% in females receiving 10 mg/kg bw/day.

In addition, clinical chemistry parameters were also affected be the test item. The plasma levels of bilirubin were increased by 14% to 17% for the male animals treated with 10 mg/kg bw/day and by 29% to 34% for the male and 16% for the female animals treated with 30 mg/kg bw/day on test day 91/92 (combined) or 91.

Furthermore, microscopic evaluation revealed test item-related changes in the bone marrow (erythroid hyperplasia) of the femur. There was a significant and test item-related increase for erythroid hyperplasia in the bone marrow of the male and female animals treated with 30 mg/kg bw/day compared to the controls: 7 of 10 animals for both sexes in the 30 mg/kg bw/day dose group versus 0 of 10 in controls. Bone marrow of animals treated with 10 mg/kg bw/day displayed significant erythroid hyperplasia, although of marginal to slight severity when compared to controls. All animals at 3 mg/kg bw/day did not
have any relevant changes when compared to the controls for both sexes. These changes correlated with the haematological changes in the 30 mg/kg bw/day dose group for both sexes.

Following the treatment period, a recovery period followed that was conducted with the control group and the 30 mg/kg bw/day dose group. During the recovery period the body weight of the male and female animals previously treated with 30 mg/kg bw/day was still reduced by 17% or by 13%, respectively, on test day 118 compared to the control group. The male animals revealed a slightly higher body weight gain than the control group during the recovery period indicating a trend towards recovery, while the body weight gain of the females was in the range of the control group. The body weight at autopsy was reduced by 17% (males) and 13% (females), respectively. Furthermore, all changes previously observed in haematological and biochemical parameters and at histological examination after repeated treatment with 30 mg/kg bw/day had subsided after 4 weeks of recovery.

Overall, no further test-item related changes were observed.