Registration Dossier

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:
2014-07-03 to 2014-11-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same 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:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Cobalt Oxide 72/73F
- Molecular formula: Co3O4
- Physical state: grey-black powder
- Storage condition of test material: cool and well-ventilated in a tightly closed container.
- Surface area (BET): 3.1 m²/g
- Particle size distribution: D10: 0.4 μm; D50: 1 μm; D90: 4 μm

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 and females: 52 days
- Weight at first dosing: males: 269.0 - 321.5 g; females: 183.3 - 227.8 g
- Housing: animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 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 diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): tap 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:
other: 0.5% hydroxypropyl methylcellulose gel
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concnetrations. The administration formulations were freshly prepared daily.
Administration volume: 5 mL/kg bw/day

The dose of the test item was adjusted to each animal's body weight daily up to and including test week 6, and once weekly thereafter.
The control animals received the vehicle orally at a constant volume in the same way once daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle mixtures, 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 concentration: immediately after preparation of the formulations as well as after 8 and 24 hours storage at room temperature (3 samples/test item group).
- homogeneity: at the start of administration, during administration (in the middle), and before administration to the last animal of each dose 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 tricobalt tetraoxide in samples was performed by analysis of cobalt with Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES).

Results:
The results of the analysis show that the test item formulations were correctly prepared. The actual tricobalt tetraoxide concentrations in the respective test item formulations ranged from 95.6 % to 103.0 % of the nominal concentrations before administration to the last male animal of the group on test day 1, and from 87.5 % to 101.7 % of the nominal concentrations before administration to the last female animal of the group on test day 90 (last administration day of the study).
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, and 1000 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 1000 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 were selected taking into account any existing toxicity and toxicokinetic data available for the test item at the time of initiation of the study.
- Recovery groups were included in this study. One recovery group was included for the control group and other recovery group for the 1000 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 first administration and once a week thereafter throughout the experimental period as well as on the last day of the treatment period and recovery period.

FOOD CONSUMPTION (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: 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)
- 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) and at the end of the recovery period (all recovery animals; test day 119)
- Anaesthetic used for blood collection: Yes, isoflurane 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 unclassified 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) and at the end of the recovery period (all recovery animals; test day 119)
- 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 the treatment period (all main study animals; test day 89) and at the end of the recovery period (all recovery animals; test day 117)
- Animals fasted: Yes
- 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, all main study animals were dissected following a randomisation scheme. Necropsy of all animals allocated to the recovery period was performed on test day 119.
The animals were euthanized by carbon dioxide, 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), 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 of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson’s solution and the testes in Bouin’s solution for optimum fixation.

Organs: adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (right and left ventricle, septum), large intestine (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 and oviducts (2), pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, sublingual and parotid gland), skin (left flank), spinal cord (3 sections), 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 1000 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 prepared and stained with Oil Red O and examined microscopically.
A 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 1000 mg/kg bw/day group following staining.

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 by cell differentiation (counting of 200 nuclei-containing cells) for the control group and the 1000 mg/kg bw/day group.
Statistics:
Means and standard deviations were calculated on time-point specific data sets for each sex separately. Data taken from Provantis® (body weight, body weight gain, food consumption, haematology, clinical chemistry, and urinalysis) were analysed by ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons were made with the control group (reference item) by the DUNNETT test. Before preparing the ANOVA, the data were analyzed for homogeneity of variances (BARTLETT test) and normal distribution (SHAPIRO-WILKS test).
The test item-treated groups (100, 300, and 1000 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 Comparisons with a control. Biometrics, 482-491 (Sept 1964): body weight, food consumption, haematology, clinical chemistry, urinalysis, absolute and relative organ weights (p ≤ 0.05 and p ≤ 0.01)
- exact test of R. A. FISHER: histology (p ≤ 0.05)
- STUDENT's t-test: all numerical functional tests: body temperature, hind leg splay, grip strength, spontaneous motility (p ≤ 0.05 and p ≤ 0.01)
- Chi² test: bone marrow (p ≤ 0.01)

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:
no effects observed
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:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1) Treatment period:
- 100, 300 or 1000 mg/kg bw/day: none of the male and female rats treated orally revealed any test item-related changes in behaviour or external appearance.
- 1000 mg/kg bw/day: two female animals revealed an exophthalmus from test day 79 onwards. This is considered to be a coincidental finding that is not test item-related.
- the faeces of all test item-treated male and female animals were dark stained from test day 48 onwards for the male animals and from test day 41 onwards for the female animals. The intensity of the staining increased with the dose level. The dark staining of the faeces was not recorded before test day 41/48 as only from these time points onwards a clear difference in the colour of the faeces compared to the control animals was evident for all test item-treated animals. The dark staining of the faeces is considered to be related to the black-grey colour of the test item and not to be a toxicological effect. The consistency of all animals' faeces was normal throughout the treatment period.
- no deaths were noted at any dose level.
- all main study animals survived until their scheduled terminal sacrifice.
- detailed clinical observations: none of the animals treated with 100, 300 or 1000 mg/kg bw/day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour as assessed in test weeks 1 to 13 (all groups).
- detailed clinical observations: all parameters of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination in test week -1. One male animal treated with 1000 mg/kg b.w./day revealed a haemorrhagic nose in test week 12. Furthermore, one female animal treated with 1000 mg/kg bw/day revealed a movable solid palpable mass with a diameter of approximately 8 to 20 mm near the second mammary gland from test week 9 onwards. Lastly, another female 1000 mg/kg bw/day dose animal revealed areas with thin fur (size approximately 10 mm × 30 mm) on both forelimbs in test weeks 10 to 13. All findings noted are considered as spontaneous changes that are not related to the test item due to the single occurrence in each case.

2) Recovery period (restricted to the control group and 1000 mg/kg bw/day group)
- 1000 mg/kg bw/day: no abnormalities in behaviour or external appearance were observed for the treated male and female animals during the recovery period. Furthermore, the exophthalmus of the female recovery animal previously treated, that is considered not test item-related, was still present until the end of the recovery period. Lastly, the faeces of the male and female animal previously treated was still dark stained until test day 95. From test day 96 onwards, the dark staining of the faeces had subsided and the colour had returned to normal. The consistency of all animals' faeces was normal throughout the recovery period.
- no deaths were noted during the recovery period. All recovery animals survived until the scheduled recovery sacrifice.
- detailed clinical observations: none of the animals treated with 100, 300 or 1000 mg/kg bw/day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour as assessed in test weeks 14 to 17 (control group and 1000 mg/kg bw/day group only).

BODY WEIGHT AND WEIGHT GAIN
1) Treatment period
- 100 or 300 mg/kg bw/day: no test item-related changes were noted for the male and female animals.
- 1000 mg/kg bw/day (males only): body weight of the male animals was slightly reduced by up to 10% compared to the control group as of test day 22 (p ≤ 0.05 or p ≤ 0.01). The body weight gain was reduced by up to 17 percentage points in comparison to the control. The body weight at autopsy of the male animals was reduced by 12% on test day 91 (p ≤ 0.01).
- 1000 mg/kg bw/day (females only): body weight of the female animals was only marginally reduced by up to 5% compared to the control group as of test day 64 (not statistically significant). The body weight gain and the body weight at autopsy were similarly only slightly reduced compared to the control group.
- reduced body weights at the 1000 mg/kg bw/day dose are considered as test item-related.

2) Recovery period (restricted to the control group and 1000 mg/kg bw/day group)
- slight differences in body weight between the animals previously treated with 1000 mg/kg bw/day and the control group had nearly completely subsided at the end of the treatment period in both the male and female animals.
- animals previously treated with the 1000 mg/kg bw/day dose revealed a higher body weight gain than the control group during the recovery period.
- no noteworthy difference was noted for the body weight at autopsy between the animals previously treated with the 1000 mg/kg bw/day group dose and the control group at recovery sacrifice.

FOOD CONSUMPTION
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg b.w./day: no test item-related influence was observed on the food consumption of the male and female animals compared to the control animals throughout the treatment period and the recovery period.
- 1000 mg/kg bw/day: food consumption of the male animals appeared to be slightly increased by 5% compared to the control group in test week 5 (period of test days 29 to 36, p ≤ 0.05). This effect is considered to be due to the reduced body weight of these male animals.
- test item-treated female animals appeared to reveal a statistically significant (at p ≤ 0.05 or p ≤ 0.01) increase in the food consumption of up to 9% in test weeks 3, 4 and/or 5 at all dose levels. This is considered to be a coincidental effect as the food intake of the female animals was generally slightly higher in all test item-treated groups than in the control group during the first six test weeks, but no dose-response relationship was noted.
- statistically significant differences in food consumption compared to the control group that are not considered to be test item-related are as follows: increased relative food consumption
- relatively low food consumption noted for the control and the high dose group in test week 17 of the recovery period (test day 111 to test day 118) is due to the overnight fasting of the animals before urine collection on test day 117.

WATER CONSUMPTION
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- no 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 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related changes of the eyes and the optic region were observed in any animal neither at the end of treatment nor at the end of the 4-week recovery.

HAEMATOLOGY
1) Treatment period
- 100 mg/kg bw/day: no test item-related influence was observed on any of the haematological parameters at the end of the treatment period.

- 300 mg/kg bw/day (males only):
haemoglobin (+9%; p≤0.01)
erythrocytes (+10%; p≤0.01)
platelets (-10%)
haematocrit (+9%; p≤0.01)

- 1000 mg/kg bw/day:
haemoglobin (males: +26%; females: +14%; p≤0.01)
erythrocytes (males: +23%; females: +11%; p≤0.01)
platelets (males: -32%; females: -13%; p≤0.01 (males only))
haematocrit (males: +25%; females: +13%; p≤0.01)

- no test item-related effects were observed for the number of leucocytes, the relative reticulocyte count, 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 treatment period (test day 91).

2) Recovery period (restricted to the control group and 1000 mg/kg bw/day group)
- 1000 mg/kg bw/day: all changes in haematological parameters previously observed after repeated treatment had subsided after 4 weeks of recovery (test day 119, day 28 of the 4-week recovery period).
- no test item-related effects were observed on the haemoglobin content, numbers of erythrocytes, leukocytes and platelets, relative reticulocyte count, haematocrit value, relative and absolute differential blood count, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration at the end of the recovery period (test day 119).
- statistically significant differences in haematological parameters compared to the control animals on test day 91 (end of treatment) or test day 119 (end of recovery) that are not considered to be test item-related are as follows:
Treatment period (1000 mg/kg bw/day):
decreased absolute eosinophilic granulocytes (males; p ≤ 0.05)
decreased absolute large unclassified cells (males; p ≤ 0.05)
increased mean corpuscular haemoglobin (females; p ≤ 0.05)

Recovery period (1000 mg/kg bw/day):
decreased reticulocytes (males; p ≤ 0.01)
decreased absolute large unclassified cells (males; p ≤ 0.05)

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

CLINICAL CHEMISTRY
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related influence was noted on the biochemical parameters of the male and female animals at the end of the treatment period and at the end of the recovery period. All data are considered to be within the limits of normal biological variability.
- statistically significant changes in biochemical parameters in comparison to the control group listed as follows are considered to be coincidental and not related to the test item:
Treatment period:
decreased total cholesterol (1000 mg/kg bw/day; males; p ≤ 0.01)
decreased urea (in blood)(100, 300, and 1000 mg/kg bw/day; males; p ≤ 0.05 or p ≤ 0.01)
decreased calcium (1000 mg/kg bw/day; males; p ≤ 0.05)
increased chloride (300 mg/kg bw/day; males; p ≤ 0.05)
increased chloride (300 and 1000 mg/kg bw/day; females; p ≤ 0.01)
increased sodium (1000 mg/kg bw/day; females; p ≤ 0.05)

URINALYSIS
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: daily treatment did not lead to any test item-related changes of the urinary parameters of the male and female animals compared to the control group at the end of the treatment period and at the end of the recovery period.
- statistically significant differences in the urinary parameters compared to the control animals on test day 89 or 117 that are not considered to be test item related are as follows:
Treatment period:
increased pH value (1000 mg/kg bw/day; females; p ≤ 0.05)
increased relative urine volume (1000 mg/kg bw/day; females; p ≤ 0.05)

Recovery period:
decreased specific gravity (1000 mg/kg bw/day; females; p ≤ 0.05)

NEUROBEHAVIOUR
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals in test week 13 and in test week 17.
- individual male and female animals of the test item-treated and/or control groups revealed a score different from the normal score for the parameters urination, toe pinch, tail pinch, wire maneuver, and/or positive geotropism in test week 13 and/or in test week 17. No general difference in the frequency and degree was noted for these parameters between the test item-treated groups and the control group. All occurrences of scores different from the normal score for the examined parameters are considered to be within the normal range of biological variation.
- the following statistically significant changes in comparison to the control animals observed for numerical neurological parameters in test week 13 or 17 are considered to be coincidental effects and not to be related to the treatment with the test item: decreased body temperature, decreased hind leg splay, increased/decreased hind limb (grip strength), and increased spontaneous motility

ORGAN WEIGHTS
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related influence was noted on the relative or absolute organ weights of the male and female animals at the end of treatment (test day 91) and at the end of the recovery period (test day 119).
- statistically significant differences in relative and absolute organ weights compared to the control animals on test day 91 or test day 119 that are not considered to be test item-related are as follows:
Treatment period:
decreased adrenal gland weight (right; absolute)(300 mg/kg bw/day; females; p ≤ 0.05)
increased brain weight (relative)(1000 mg/kg bw/day; males; p ≤ 0.01)
decreased liver weight (absolute)(1000 mg/kg bw/day; males; p ≤ 0.05)
decreased ovary weight (right, relative)(300 mg/kg bw/day; females; p ≤ 0.05)
decreased ovary weight (right, absolute)(300 mg/kg bw/day; females; p ≤ 0.05)

Recovery period:
- 1000 mg/kg bw/day: increased relative weight of prostate and seminal vesicles with coagulating glands (p ≤ 0.05)

GROSS PATHOLOGY
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related changes in the organs and tissues of the animals were found after terminal sacrifice at the end of the treatment period (test day 91). No abnormal findings were noted at recovery sacrifice at the end of the 4-week recovery period (test day 119).

- a few minor macroscopic findings were noted which are considered to be not test item-related but to be of spontaneous nature in various organs of individual test item-treated and control animals, which were as follows:
liver (medial lobe): dark-brown focus or a yellow focus in the
thymus (left side): dark-red discoloured side
cardiac stomach: haemorrhagic focus
intestines: green-brown content
testes/epididymides: reduced in size and revealed a soft consistency
ovary (right): cystic and enlarged, filled with a dark-red liquid
uterus: dilated, filled with a clear liquid
adrenal gland (right): enlarged
axilla (left): subcutaneous, solid and red-yellow coloured tissue enlargement

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- no treatment-related microscopic changes were noted in the male and female animals of the control group and the 1000 mg/kg bw/day dose group.
- all microscopic changes seen in any organ of any animal are considered to be coincidental, or to be within the normal range of background alterations, which may be seen in untreated rats of this age and strain.
- histopathological examination performed on one testicle and one epididymis with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure, did not reveal any test item-related effects.

BONE MARROW EXAMINATION
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 1000 mg/kg bw/day: myeloid : erythroid ratio of the male and female animals was not influenced in comparison to the control group at the end of the treatment period and at the end of the recovery period.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (rat, 90 days): 300 mg tricobalt tetraoxide/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 1000 mg/kg bw/day was slightly reduced by up to 10% compared to the control group as of test day 22. Accordingly, the body weight gain was reduced by up to 17 percentage points in comparison to the control. The body weight at autopsy of the males of 1000 mg/kg bw/day dose group was reduced by 12% on test day 91. Furthermore, the body weight of the female animals treated with 1000 mg/kg bw/day was only marginally reduced by up to 5% compared to the control group as of test day 64. The body weight gain and the body weight at autopsy were similarly only slightly reduced compared to the control group. Overall, the reduced body weights at the 1000 mg/kg bw/day dose are considered as test item-related.

Next, repeated oral treatment of male and female rats with 300 or 1000 mg/kg bw/day led to a few test item-related changes in haematological parameters in comparison to the control animals. The haemoglobin, erythrocytes and haematocrit values of the males treated with 300 mg/kg bw/day were statistically significant increased between 9 and 10%. Also, in the 1000 mg/kg bw/day dose group the values of haemoglobin, erythrocytes and haematocrit of the males were statistically significant increased (between 23 - 26%) as well as in the females (between 11 - 14%). The platetes were statistically significant decreased (32%) in the males.

Following the treatment period, a recovery period followed that was conducted with the control group and the 1000 mg/kg bw/day dose group. No changes related to the previous 1000 mg/kg bw/day treatment with tricobalt tetraoxide were noted at the end of the 4-week recovery period. The slightly reduced body weight of the treated male animals had recovered to a level comparable to the control group at the end of the recovery period. The slightly increased values of red blood cell-related parameters (haemoglobin content, number of erythrocytes, haematocrit value) and the slightly decreased number of platelets in the previously dosed male and female animals had returned to values comparable to the control group at the end of the recovery period

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