Cobalt lithium dioxide

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998-09-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system 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

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

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

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

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

IMMUNOLOGY: No

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.

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

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

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.

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

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subchronic

Species:

rat

System:

haematopoietic

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-19 to 2016-11-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

2009-09-07

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-08-14

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
1) Tricobalt tetraoxide (test item)
- Storage condition of test material: at room temperature, dry, protected from light

2) Cobalt sulfate heptahydrate (reference item)
- Storage condition of test material: at room temperature, dry, at a dark place

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use. In this study the specified Wistar strain is preferred to the Fischer strain because young Fischer rats available in Germany sporadically show a slight latent inflammation of lungs which might interfere with the scheduled examinations.

*Reference:
- Vu, V., Barrett, J.C., Roycroft, J., Schuman, L., Dankovic, D..Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212 (1996)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males: approx. 250 g; females: approx. 175 g
- Housing: housed in Makrolon® (polycarbonate) cages type III, two rats per cage; bedding material: absorbing softwood (Lignocel BK 8-15')
- Diet (ad libitum): commercial chow in pellet form (Ssniff "V1534"; ssniff Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 2 - 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
- food quality: a certificate of feed analysis is issued by the supplier periodically. The analysis is done at the LUFA-ITL in Kiel, Germany, i.e. in a certified lab for this kind of analysis (DIN EN ISO/IEC 17025:2000).
- water quality: a certificate of water analysis issued by the water supplier (Stadtwerke Hannover) is sent periodically to Fraunhofer ITEM.

ENVIRONMENTAL CONDITIONS
- Temperature: 22° + 2° C
- Relative humidity: 55% + 15 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.51 - <= 2.14 µm

Remarks on MMAD:

MMAD (tricobalt tetraoxide): 1.81 µm - 2.14 µm
MMAD (cobalt sulfate heptahydrate): 1.51 µm - 1.59 µm
GSD (tricobalt tetraoxide): 1.97 - 2.26
GSD (cobalt sulfate heptahydrate): 2.07 - 2.23

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus / Method of holding animals in test chamber : the aerosol is given to the rats by a flow-past nose-only inhalation exposure system. Aerosols are supplied to each rat individually. For exposure to the test item the rats were restrained in acrylic tubes with adjustable backstops. The exposure tubes are arranged around a cylinder capable to take up 16 tubes per platform. The rat nose is located at the front end of a tube being connected to a cylinder delivering the aerosol. Through the thin pipes, the aerosol is supplied to each rat nose individually and exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivering cylinder. The position of exposure tubes of rats at the cylinder is changed daily according to a rotation plan to minimize exposure differences due to geometry. The exposure units (1x clean air control, 3x treatment Co3O4, 2x treatment CoSO4 * 7H2O) are located each under a separate hood to prevent contamination among different dose groups.

For a period of 2 - 3 weeks prior to exposure animals were trained to become accustomed to nose-only tubes.

- Air flow rate: airflow to each rat is approx. 1 L/min which is calculated to be laminar (rat minute volume: 0.2 L). Therefore measurement of the oxygen concentration is not necessary.

- System of generating particulates/aerosols:
1) Tricobalt tetraoxide: the particulate sample aerosols were generated by dispersing the dry powder of Co3O4. Dispersion is achieved by a feeding system and a high-pressure, high-velocity pressurized air dispersion nozzle developed by the laboratory. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test substance under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer

2) Cobalt sulfate heptahydrate: the particulate sample aerosols were generated by nebulising an aqueous solution of CoSO4 x 7 H2O using pressurised air. Due to rapid evaporation of the droplets the rats were exposed to solid particles.

- Temperature, humidity, air flow: air flow, temperature and relative humidity were measured continuously and recorded by 20-minute means. The limits were set at 22° C + 2° C for temperature and 55 % + 15 % for relative humidity. Animal room lighting was on a 12 hour light/dark cycle controlled by an automatic timing device.

- Method of particle size determination: the MMAD was determined twice during the 28-day exposure period for each test/reference item exposure unit (6 units) by a cascade impactor (Marple impactor).

TEST ATMOSPHERE
- Brief description of analytical method used: the actual aerosol concentrations was measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration is determined throughout the study by comparing to gravimetric concentrations.
Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. These samples were collected at a port of the nose-only exposure unit, thus, under the same conditions the rats are inhaling the aerosol. The evaluation of filter samples was done by gravimetrical analysis. As a permanent control of the aerosol concentrations is guaranteed by photometers the scheduled filter sampling frequency is sufficient (in agreement with OECD guideline 412).
- Samples taken from breathing zone: yes
The measured mean concentrations were very close to the target concentration for all dose groups of the test and reference item, i.e. amounted to 102%, 103% and 101% in the Co3O4 and to 101% and 101% in the CoSO4 groups.

Prior to the 28-day exposure of rats, technical trials to adjust particle size distributions and exposure levels were conducted.

Tricobalt tetraoxide:
Nominal aerosol concentrations of 5, 20, and 80 mg/m³ were used for the test item in the low, mid and high dose groups, respectively. According to the MPPD model, in the low, mid and high dose groups deposited masses of approx. 0.37, 1.5 and 6 mg/lung could be expected for an aerosol with an MMAD of approx. 1.75 µm (5.2% deposition fraction), thus inducing a particle overload situation in the high dose group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentrations were very close to the target concentration for all dose groups of tricobalt tetraoxide, i.e. amounted to 102, 103 and 101 % in the low, mid and high dose groups.
The mean concentrations were very close to the target concentration for all dose groups of cobalt sulfate heptahydrate, i.e. amounted to 101 % for each dose group.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

5.12 mg/m³ air (analytical)

Remarks:

SD: 0.87; tricobalt tetraoxide (test item)

Dose / conc.:

20.5 mg/m³ air (analytical)

Remarks:

SD: 2.18; tricobalt tetraoxide (test item)

Dose / conc.:

80.78 mg/m³ air (analytical)

Remarks:

SD: 7.70; tricobalt tetraoxide (test item)

Dose / conc.:

2.04 mg/m³ air (analytical)

Remarks:

SD: 0.21; cobalt sulfate heptahydrate (reference item)

Dose / conc.:

10.19 mg/m³ air (analytical)

Remarks:

SD: 0.83; cobalt sulfate heptahydrate (reference item)

No. of animals per sex per dose:

main study: 10 males / 10 females
recovery group: 10 males / 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: a 14-day dose range finding study was conducted in male and female Crl: WI rats using nose-only exposure. The test substances were tricobalt tetraoxide (13.41, 46.14 and 219.16 mg/m³) and cobalt sulfate heptahydrate (1.81 and 8.94 mg/m³). Groups of eight males and eight females were used. A negative control group (clean air) was run concurrently. The following endpoints were investigated: clinical observation, body weight, gross pathology, histopathology, immunohistochemistry and bronchoalveolar lavage.

All male and female test animals survived treatment and effects indicating systemic toxicity were not observed. Sex-specific differences were not detected. Furthermore, body weight development did not show any statistically significant changes as compared to concurrent controls.
Lung weights showed statistically significant changes as compared to concurrent controls in the mid/high Co3O4 and the high CoSO4 dose groups. In addition, bronchoalveolar lavage analysis showed a statistically significant increase in the polymorphonuclear neutrophil (PMN) percentages in the Co3O4 mid and high dose groups of both sexes and in the female CoSO4 high dose group.
The histopathological examination did not show any adverse effects for Co3O4 in the respiratory tract. In lungs, a dose-dependent alveolar/interstitial accumulation of particle-laden macrophages was observed. However, CoSO4 exposed animals exhibited adverse changes in the larynx in both exposure groups.
Lastly, immunohistochemistry investigation did not showed any increase of 8-OH-dG-positive cells in lung parenchyma.

- Post-exposure recovery period in recovery groups: 3 months
Upon cessation of exposure the animals of the recovery groups were kept in Makrolon® cages.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (main study and recovery group)
- Time schedule: at least once a day (cages); at least before and after exposure and sometimes also during exposure
- Cage side observations checked: clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes (main study and recovery group)
- Time schedule: once a week

BODY WEIGHT: Yes (main study and recovery group)
- Time schedule for examinations: twice a week in the first 4 weeks and once a week thereafter throughout the study for all animals

FOOD CONSUMPTION: Yes (main study and recovery group):
- Time schedule for examinations: weekly during the study period (including post-exposure observation period) using 10 and after day 1 5 animals per dose and sex group

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 and recovery group)
- Time schedule for examinations: weekly during the study period (including post-exposure observation period) using 10 and after day 1 five animals per dose and sex group

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (main study and recovery group)
- Time schedule for collection of blood: day 1 after exposure (exception: blood for prothrombin and thromboplastin times were taken only before sacrifice) an after additional 91 days after exposure
- Anaesthetic used for blood collection: Yes, slight isoflurane anesthesia
- Animals fasted: Yes, 16-hour fasting period (tap water ad libitum)
- How many animals: 5 animals/group/sex
- Parameters checked: red blood cells, hemoglobin, hematocrit, reticulocytes, mean cell volume, mean hemoglobin/erythrocyte, mean hemoglobin concentration/erythrocyte, prothrombin time, total white blood cells, differential white cell count, platelets,

CLINICAL CHEMISTRY: Yes (main study and recovery group)
- Time schedule for collection of blood: day 1 after exposure (exception: blood for prothrombin and thromboplastin times were taken only before sacrifice)
- Animals fasted: Yes 16-hour fasting period (tap water ad libitum)
- How many animals: 5 animals/group/sex
- Parameters checked: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-Glutamyl transpeptidase, urea, triglyceride, total bilirubin, creatinine, total protein, albumin, globulin, ALB/GLB, glucose, cholesterol, sodium, calcium, potassium, phosphorus and chloride

URINALYSIS: Yes (main study and recovery group; 5 males / 5 females/dose group))
- Time schedule for collection of urine: day 1 after exposure
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, and leukocytes

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: Yes
Oxidative damage of the lung tissue can result in genotoxicity in situ effects, e.g. the occurrence of 8-hydroxy-deoxyguanosine - 8-OH-dG. The immunohistochemically stained cells were quantified in the lung tissue and the epithelium of the terminal bronchioles. Examination was done in the left lung lobe of 5 animals per time-point and group in the control groupand high dose groups (80.78 mg Co3O4/m³ and 10.19 mg CoSO4 *H2O/m³ groups) 1 day upon cessation of exposure.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (main study and recovery group)
All animals were subjected to a complete necropsy, which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
The rats used for bronchoalveolar lavage investigations (BAL) were anesthetized with an overdose pentobarbital sodium and killed. For rats used for histopathological investigations an overdose of carbon dioxide was used.
The abdominal cavity was opened and the diaphragm was cut allowing the lungs to collapse. Heart, esophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN; mediastinal and tracheobronchial) were removed from the lung convolution.
The lung and the lower half of the trachea were weighed, and used for BAL or histopathology. For histopathology the lung was inflated under a pressure of about 20 cm water with formalin and was fixed by immersion, and used for histopathology.
The following organs were trimmed and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, lung, and heart. The respiratory tract was preserved as follows: nasal passages (including nasal -associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD Guideline no. 412 were prepared for histopathology.


HISTOPATHOLOGY: Yes (main study and recovery group)
The histopathology of all organs (respiratory tract, adrenal glands, bone marrow of the femur, brain (cerebrum and cerebellum), esophagus, femur with knee joint, heart, kidneys, liver, ovaries, seminal vesicles, spinal cord (cervical, thoracic and lumbar cords), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands and uterus) was performed in all animals per group and sex at days 1 and day 91 post-exposure (with the exception of the 5.12 mg Co3O4/m³, 20.50 mg Co3O4/m³ and 2.04 mg CoSO4 * 7H2O/m³ groups); particular attention was paid to:
• Histopathology of respiratory tract including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) (respiratory tract will be examined in groups 5.12 mg Co3O4/m³, 20.50 mg Co3O4/m³ and 2.04 mg CoSO4 + H2O/m³ groups, too);
• Trimming: lungs 5 sections; nose 4 sections; larynx: 3 sections; trachea: 1 section
Tissues for histological examination will be fixed for at least one week in buffered formalin (10%). Bones will be decalcified prior to embedding.
Lung lobes will be fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H & E). In addition, masson trichrome staining will be used for detection of connective tissue production (lower half).

Other examinations:

BRONCHOALVEOLAR LAVAGE (main study and recovery group)
Bronchoalveolar lavage was performed in 5 male and 5 female rats per group after end of exposure (day 1) and following end of recovery (day 91). The method of Henderson et al. (1987)* was used with minor modifications.
Following preparation, the lungs were lavaged with saline using two lavages each. The lavage fluid was collected in calibrated tubes and the harvested volume was recorded. The leukocyte concentration of the lavagate was determined using a counting chamber and two cytoslides were prepared with a cytocentrifuge for differential cell count (macrophages, neutrophils, eosinophils, lymphocytes).
After centrifugation of the lavage fluid, biochemical indicators relevant for diagnosis of lung damage were determined in the supernatant (lactic dehydrogenase - LDH, β-glucuronidase, total protein). These parameters were analyzed according to routine clinical chemistry protocols using a Cobas Fara device.

The justification of the parameters is given below:
Cytological parameters
• total cell count (recruitment of lung leukocytes)
• differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions; a total of 400 leukocytes per rat will be evaluated)
Biochemical parameters
• lactic dehydrogenase (LDH = cytosolic marker enzyme; increased permeability of membranes, cell damage and lysis)
• β-glucuronidase (measure of phagocytic activity of macrophages; lysis of macrophages)
• total protein (marker of transsudation; damage of epithelial cells)
Cytokine levels (MCP-1, IL-8 alias CINC-1, HIF-1α) were analysed using ELISA. Along with the samples to be analysed, a standard curve of the respective cytokine was included on each plate. The plates were read on a microplate reader, i.e. a Tecan InfiniteF200Pro. Cytokine levels were determined using a four-parameter logistic curve fit to the standard curve.
• Monocyte chemoattractant protein-1 (MCP-1)
• Interleukin-8 (IL-8 alias CINC-1)
• Hypoxia-inducible factor 1-α (HIF-1α)
The HIF1-α analysis was expanded to the BALF cell sediments (gained by centrifugation) to juxtapose the two compartments (BALF supernatant and BALF cell sediment).

*Reference:
- Henderson, R.F., Mauderly, J.L. Pickrell, J.A., Hahn, R.F., Muhle, H., and Rebar, A.H. Comparative study of bronchoalveolar lavage fluid: Effect of species, age and method of lavage. Exp. Lung Res. 13, 329 342 (1987)

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test (Dunnett, 1955; 1964)*.
The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the PROVANTIS system.

*References
- Dunnett C. W. A multiple comparison procedure for comparing several treatments with a control, J Am Stat Assoc, 50, 1096–1121 (1955)
- Dunnett C. W. New tables for multiple comparisons with a control, Biometrics, 20, 482–491 (1964)

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Tricobalt tetraoxide
80.78 mg/m³: one day after end of exposure, in females, the relative lung weights were statistically significantly increased (treatment group (lung/bw g/kg): 8.68; control group (lung/bw g/kg): 6.41; p < 0.01). At day 91 of the recovery period absolute and relative lung weights in the male and female Co3O4 high dose groups came out with statistically significant increases as compared to controls.
Males:
Absolute lung weight (g): treatment group: 3.07; control group: 2.00; p< 0.01
Relative lung weight (lung/bw g/kg): treatment group: 7.47; control group: 4.95; p< 0.01
Females:
Absolute lung weight (g): treatment group: 2.08; control group: 1.19; p< 0.01
Relative lung weight (lung/bw g/kg): treatment group: 9.43; control group: 5.36; p< 0.01

Absolute and relative lung weights of rats used for bronchoalveolar lavage showed a statistically significant increase at day 1 in the 80.78 mg Co3O4/m³ and 10.19 mg CoSO4 * 7H2O/m³ dose groups (males and females). At day 91 of the post-exposure observation period the findings in the Co3O4 groups persisted whereas in the CoSO4 * 7H2O groups all values had returned to normal values.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1) Tricobalt tetraoxide
5.12, 20.50 and 80.78 mg/m³:
a) Lung (after 28 days of exposure; 5 animals/sex7dose):
Within the lung, there was a dose-dependent multifocal very slight to slight alveolar accumulation of particle-laden macrophages in the low, mid and high dose exposed male (all very slight in low and mid dose and all slight in the high dose group). In addition, there was a very slight multifocal interstitial accumulation of particle-laden macrophages in 2/5 mid dose males and females and in all high dose males and females. Furthermore, a very slight accumulation of particle-laden macrophages in the bronchus-associated lymphoid tissue (BALT) was seen in 2/5 mid dose males and females and in all high dose males and females. Particles free in the alveoli were observed dose-dependent in all mid dose males and females (all very slight) and all high dose males and females (all slight). The free particles were not within cells but attached to eosinophilic granular material, which was interpreted as proteinaceous material. A very slight lipoproteinosis was visible in 2/5 mid dose males and females and in all high dose males and females. The lipoproteinosis is most likely cellular detritus from alveolar macrophages but might also represent exudate from pulmonary vessels. In 1/5 high dose males and females a very slight multifocal alveolar granulocytic cell infiltration was found. There was a very slight multifocal mononuclear cell infiltration in the lung parenchyma at the terminal bronchus in 3/5 high dose males and in 1/5 mid dose females and in 4/5 high dose females.

b) Lung (after additional 90 days of recovery; 5 animals/sex/dose)
Within the lung, there was a dose-dependent multifocal very slight to slight alveolar accumulation of particle-laden macrophages in the low, mid and high dose exposed male (all very slight in low and mid dose and all slight in the high dose group). In addition, there was a very slight multifocal interstitial accumulation of particle-laden macrophages in 3/5 mid dose males and females and in all high dose males and females. Furthermore, an accumulation of particle-laden macrophages in the bronchus-associated lymphoid tissue (BALT) was seen in 2/5 low dose males and females (very slight), in all mid dose males and females (all very slight) and in all high dose males and females (4/5 very slight and 1/5 slight in males and in females, respectively). Particles free in the alveoli were observed dose-dependent in all mid dose males and females (all very slight) and all high dose males and females (all slight). A lipoproteinosis was visible in 3/5 mid dose males and females (males 2/5 very slight and 1/5 slight; females 3/5 very slight) and in all high dose males and females (slight in 1/5 and moderate in 4/5 males and females, respectively). The lipoproteinosis is most likely cellular detritus from alveolar macrophages but might also represent exudate from pulmonary vessels. A multifocal alveolar granulocytic cell infiltration was found very slight in 2/5 mid dose males and in 1/5 mid dose females and, in addition, in 4/5 high dose males (2/5 very slight and 2/5 slight) and in 3/5 high dose females (all three very slight). There was a very slight multifocal mononuclear cell infiltration in the lung parenchyma at the terminal bronchus in 2/5 mid dose males and females (all very slight) and in 5/5 high dose males and females (in 4/5 males very slight and in 1/5 males slight as well as in all females very slight). A very slight (multi)focal bronchio-alveolar hyperplasia of the bronchiolar type was visible in 4/5 high dose males and in 1/5 low dose, 2/5 mid dose and 5/5 high dose females. A very slight (multi)focal interstitial fibrosis was observed in 1/5 mid dose males and females as well as in 4/5 high dose males and in 5/5 high dose females. There was a focal slight alveolar macrophage aggregation in 1/5 males and a multifocal moderate in 1/5 males (both high dose group).

c) Lung-associated lymph nodes (after 28 days of exposure; 5 animals/sex/dose):
After 28 days of exposure, in the lung-associated lymph nodes (LALN) a very slight to slight accumulation of particle-laden macrophages was visible in mid and high dose exposed animals (male mid dose: 4/5 very slight, male high dose: 2/5 very slight and 2/5 slight; female mid dose: 4/5 very slight, female high dose: 1/5 very slight and 3/5 slight).

d) Lung-associated lymph nodes (after additional 90 days of recovery; 5 animals/sex/dose):
After additional 90 days recovery following exposure, in the lung-associated lymph nodes (LALN) a very slight to moderate accumulation of particle-laden macrophages was visible in low, mid and high dose exposed animals (male low dose: 2/5 very slight, male mid dose: 2/5 very slight, 2/5 slight and 1/5 moderate, male high dose: 1/5 very slight, 2/5 slight and 2/5 moderate; female low dose: 2/5 very slight, female mid dose: 1/5 very slight and 2/5 slight, female high dose: 2/5 very slight, 1/5 slight and 1/5 moderate).
The diffuse sinusoidal dilatation represented a single event and was interpreted as unrelated to the treatment.

e) Larynx (after 28 days of exposure and after additional 90 days of recovery; 5 animals/sex/dose/time point):
A very slight focal subepithelial accumulation of particle-laden macrophages was visible in one mid dose male and one high dose female after 28 days of exposure as well as in one high dose male after additional 90 days recovery following exposure.

f) Remaining organs of the respiratory tract (after 28 days of exposure and after additional 90 days of recovery; 5 animals/sex/time point):
There was a dose-dependent very slight accumulation of particle-laden macrophages in the nasal associated lymphoid tissue in the animals after 28 days exposure (2/5 low dose males and females; 4/5 mid dose males and females and 5/5 high dose males and females) as well as after additional 90 days recovery (2/5 low dose males and females; 3/5 mid dose males and 4/5 mid dose females as well as 5/5 high dose males and females).
Single animals of the exposed groups showed focally a very slight accumulation of particle-laden macrophages at the tracheal bifurcation.
The mononuclear cell infiltrations in different organs of the respiratory tract in some animals of all groups are commonly found background lesions and were interpreted as unrelated to the treatment. Other detected lesions such as dilatation of submucosal glands, focal granulomatous inflammation and inflammatory cell infiltration represented single events and were interpreted as unrelated to the treatment.

2) Cobalt sulfate heptahydrate:
2.04 and 10.19 mg/m³:
a) Lung (after 28 days of exposure; 5 animals/sex/dose):
Within the lung, there was a dose-dependent multifocal very slight lipoproteinosis in all high dose animals (males and females).

b) Lung (after additional 90 days of recovery; 5 animals/sex/dose)
Within the lung, there was very slight focal cholesterol granuloma in 1/5 high dose males and females. Furthermore, there was a very slight focal alveolar macrophage aggregation in 1/5 low dose males as well as in 1/5 males and in 2/5 females of the high dose group.
A perivascular granulocytic cell infiltration was visible in some animals of all groups, including the control animals. The perivascular granulocytic cell infiltration is a commonly found background lesion, which is unrelated to the treatment. Other detected lesions such as neuroendocrine cell hyperplasia, inflammatory cell infiltration, pulmonary hemorrhage and osseous metaplasia represented single events and were interpreted as unrelated to the treatment.

c) Lung-associated lymph nodes (after 28 days of exposure; 5 animals/sex/dose):
No treatment related findings were detected in the lung-associated lymph nodes after exposure.

d) Larynx (after 28 days of exposure; 5 animals/sex/dose):
In the larynx accentuated at the base of the epiglottis, a focal ulcerative inflammation was visible in 1/5 low dose males (slight) and in 5/5 high dose males (1/5 slight, 3/5 moderate and 1/5 severe) as well as in 5/5 high dose females (3/5 moderate and 2/5 severe). At the same location a focal chronic-active inflammation was seen in 3/5 low dose males (2/5 very slight and 1/5 slight) as well as in 2/5 low dose females (very slight). In addition, at the base of the epiglottis was a focal squamous metaplasia in the low and high dose exposed animals (male low dose: 2/5 very slight and 2/5 slight, male high dose: 1/5 very slight and 4/5 slight; female low dose: 4/5 very slight, female high dose: 5/5 slight). At the same location a very slight focal epithelial alteration was observed in 1/5 low dose males and females.

e) Larynx (after additional 90 days of recovery; 5 animals/sex/dose):
Accentuated at the base of the laryngeal epiglottis, there was a focal squamous metaplasia in the high dose exposed animals (male high dose: 1/5 very slight and 1/5 slight; female high dose: 2/5 very slight). Furthermore, at the same location a very slight focal epithelial alteration was observed in 2/5 high dose males and a very slight focal erosion was seen in 1/5 high dose males. In addition, a focal chronic inflammation was found in the low and high dose exposed animals (male low dose: 2/5 very slight and 1/5 slight, male high dose: 1/5 very slight and 1/5 slight; female high dose: 3/5 very slight). The tissue at the base of the laryngeal epiglottis showed focally chronic granulation tissue in the high dose exposed animals (male high dose: 3/5 very slight and 2/5 slight; female high dose: 1/5 very slight and 2/5 slight). The chronic granulation tissue is part of a reparation process, which might finally end in fibrosis. Furthermore, two high dose exposed females exhibited a degeneration of the cartilage at this location.

f) Remaining organs of the respiratory tract
No treatment related findings were detected in the remaining organs of the respiratory tract after exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: one day after exposure, statistically significant increases of polymorphonuclear neutrophils (PMN) were detected in the mid (19%/13%; M/F; p < 0.001 or p < 0.05) and high dose groups (31%/35%; M/F; p < 0.001) of both sexes; in the low dose groups, PMN showed the same levels as clean air controls (<1%). Following 91 days of recovery, the PMN levels had increased moderately in the mid dose groups (27%/18%; M/F; p < 0.001) and persisted at similar levels in the high dose groups (34%/34%; M/F; p < 0.001).

Also, for lactic dehydrogenase (LDH), ß-glucuronidase and total protein statistically significant increases were detected in the mid (LDH and total protein; M) and high dose groups (LDH, β-glucuronidase and total protein; M+F) at day 1 after end of exposure. Overall, a clear dose-dependency of observed effects was found.
At day 91 of the post-exposure observation period, the statistically significant level of LDH persisted in the mid dose group (M) and was detected also in females (mid dose). LDH, β-glucuronidase and total protein levels persisted in the high dose groups (M+F).

At day 1 after end of exposure, MCP-1 and IL-8 concentrations in the BALF supernatant showed a dose-dependent increase (statistically significant in the mid and high dose groups; p < 001 or p < 0.05); persisting after recovery).

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: one day after exposure, statistically significant increases of the polymorphonuclear neutrophils (PMN) percentages were detected in both doses and sexes (low dose: 12%/12%; M/F; p < 0.05 (males only) - high dose: 25%/25%; M/F; p < 0.001). Following 91 days of recovery, all the effects observed in the CoSO4 high groups (M+F) had returned to normal.

One day after exposure, in the low dose group lactic dehydrogenase (LDH) (males) and in the high dose groups (males and females) LDH, ß-glucuronidase and total protein were statistically significantly increased. Overall, a clear dose-dependency of observed effects was found.
At day 91 of the post-exposure observation period, all the effects observed in the high groups (M+F) had returned to normal.

At day 1 after end of exposure, MCP-1 and IL-8 concentrations in the BALF supernatant showed statistically significant increases in the high dose group (p < 0.001 and p < 0.05, respectively) and partially low dose groups (only IL-8; p < 0.05); in the recovery periods these effects returned to normal.

Details on results:

CLINICAL SIGNS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: effects indicating systemic toxicity were not observed. Sex differences were not detected.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: effects indicating systemic toxicity were not observed. Sex differences were not detected.

MORTALITY
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: no unscheduled deaths occurred during the study period.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no unscheduled deaths occurred during the study period.

BODY WEIGHT AND WEIGHT CHANGES
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: relevant statistically significant changes were not observed in the treatment groups as compared to controls.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: relevant statistically significant changes were not observed in the treatment groups as compared to controls.

FOOD CONSUMPTION
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12 and, 20.50 mg/m³: no relevant statistically significant changes were observed
- 80.78 mg/m³: relevant statistically significant changes were observed only in the female Co3O4 high dose group (decrease as compared to controls; findings not considered as having no toxic relevance).

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no relevant statistically significant changes were observed

WATER CONSUMPTION
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12 and, 20.50 mg/m³: no relevant statistically significant changes were observed
- 80.78 mg/m³: relevant statistically significant changes were observed only in the female Co3O4 high dose group (decrease as compared to controls; findings not considered as having no toxic relevance).

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no relevant statistically significant changes were observed

HAEMATOLOGICAL FINDINGS
1) Tricobalt tetraoxide (main study and recovery group)
- 80.78 mg/m³: one day after exposure, the mean cell volumes were statistically significantly increased in males and females (males: treatment group: 57.20 fl; control group: 59.74 fl; p < 0.05) (females: treatment group: 57.00 fl; control group fl: 59.78; p < 0.05).
- 5.12 and 80.78 mg/m³: one day after eyposure, lymphocytes (calc.) showed statistically significant decreases in males (low dose group: 4.554 G/L; control group: 5.904 G/L; p < 0.05)(high dose group: 4.396 G/L; control group: 5.904 G/L; p < 0.05).
- 20.50 mg/m³: at day 91 of the recovery period, lymphocytes (%) were statistically significantly decrease in males (treatment group: 62.2 %; control group: 81.8 %; p < 0.05)(not considered a toxicological relevant finding)

Changes in the other parameters did not reach statistical significance.
Overall, no clear dose-dependency was observed; neither showed both sexes conclusive results.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 mg/m³: one day after exposure, lymphocytes (calc.) showed statistically significant decreases in males (treatment group: 3.926; control group: 5.904 G/L; p < 0.01). Correspondingly, also lymphocytes (%) showed significant effects in males (treatment group: 72.6 %; control group: 85.6 %; p < 0.01). Furthermore, one day after exposure, segmented neutrophils (calc.) were statistically significantly increased in females (treatment group: 0.918 G/L; control group: 0.420 G/L; p < 0.05). Correspondingly, also segmented neutrophils (%) showed significant effects in in males and females (males: treatment group: 24.6 %; control group: 11.0 %; p < 0.01)(females: treatment group: 18.8 %; control group: 10.4 %; p < 0.05).
- 2.04 and 10.19 mg/m³: one day after exposure, the thromboplastin time was statistically significantly decreased in males (low dose group: 14.68 seconds; control group: 17.24 seconds; p < 0.01)(high dose group: 14.84 seconds; control group: 17.24 seconds; p < 0.05). This finding persisted until day 91 of the recovery period, the thromboplastin time values were still statistically significantly decreased in the low and high groups (males; low dose group: 17.70 seconds; high dose group: 17.70 seconds; control group: 20.64 seconds; p < 0.05) as well as in the in the high group of females (high dose group: 17.38 seconds; control group: 19.86 seconds; p < 0.05).

Changes in the other parameters did not reach statistical significance.
Overall, no clear dose-dependency was observed; neither showed both sexes conclusive results.

CLINICAL CHEMISTRY
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: no statistically significantly increases/decreases were detected for any parameter, except for albumin that was statistically significantly reduced in males of the mid dose group on day 91 of the recovery period (mid dose group: 34.04 g/L; control group: 36.54 g/L; p < 0.05)( considered not to be a relevant finding).

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: one day after exposure, urea was statistically significantly decreased in males (low dose group: 4.293 mmol/L; control group: 5.834 mmol/L; p < 0.01)(high dose group: 4.600 mmol/L; control group: 5834 mmol/L; p < 0.05). Furthermore, one day after exposure, total bilirubin was statistically significantly increased in females of the high dose (high dose group: 1.88 µmol/L; control: 1.14 µmol/L; p< 0.01).

Overall, no clear dose-dependency was observed.

URINALYSIS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: no statistically significantly increases/decreases were detected.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no statistically significantly increases/decreases were detected.

ORGAN WEIGHTS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: one day after end of exposure lung weights (absolute and relative) of the male Co3O4 groups did not show statistically significant increases as compared to concurrent controls. In addition, females of the low and mid doses did not show statistically significant increases in absolute and relative lung weights as compared to concurrent controls.
Furthermore, no statistically significant increases or decreases were observed for any other organ.

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: one day after end of exposure lung weights (absolute and relative) of the male and females did not show statistically significant increases as compared to concurrent controls.
Furthermore, no statistically significant increases or decreases were observed for any other organ.

GROSS PATHOLOGICAL FINDINGS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: all animals were sacrificed at scheduled dates. Upon necropsy, test item- or dose-related macroscopical findings were not observed. Only some incidental macroscopical observations were obtained

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: all animals were sacrificed at scheduled dates. Upon necropsy, test item- or dose-related macroscopical findings were not observed.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
Tricobalt tetraoxide and cobalt sulfate heptahydrate (main study and recovery group; 5 animals/per sex /dose/time point)
Within the other investigated organs, only incidental findings were detected. These represented a mononuclear cell infiltration in different organs. Furthermore, there were a focal hypertrophy and a focal fatty degeneration in the cortex of the adrenal gland, a focal mineralization in the brain, a submucosal inflammatory cell infiltration in the glandular stomach, an inflammatory cell infiltration accompanied by myofiber degeneration in the heart, a lymphoid hyperplasia in the spleen, a tubules and cords hyperplasia and a congenital cyst in the thymus and an interstitial fibrosis and ectopic thymic tissue in the thyroid gland. In the kidney, a focal cortical tubular basophilia, a cortical cysts, cortical and medullary cysts, unilateral hydronephrosis, interstitial nephritis, pyelonephritis and reactive hyperplasia of the pelvic epithelium was visible in single animals. The liver exhibited in single animals a granulocytic cell infiltration and a periportal inflammatory cell infiltration. An estrous related luminal dilatation of the uterus was visible in some animals.

One animal (5.12 mg Co3O4/m³ dose group) showed macroscopically a 2 mm in diameter sized nodule in the epididymis. This nodule represented histologically a focal spermatic granuloma.
One animal (5.12 mg Co3O4/m³ dose group) exhibited macroscopically alopecic areas at the head and front paws. These areas showed histologically a mixed inflammatory cell infiltration in the hair follicles with accompanying hair cycle arrest.
One animal (10.19 mg CoSO4 * 7H2O/m³ dose group) showed macroscopically an attachment of pancreas, liver stomach and omentum with 3mm in diameter sized brown nodules. These areas showed histologically chronic granulation tissue with hemosiderosis consistent with a previous hemorrhage.
One animal (10.19 mg CoSO4 * 7H2O/m³ dose group) exhibited macroscopically a slight herniation of the liver, which represented histologically a hepatodiaphragmatic nodule.
All lesions occur occasionally in rats and are considered to be unrelated to the exposure.

BRONCHOALVEOLAR LAVAGE
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: the females of the high dose group showed a statistically significant increase in lymphocytes. This finding is considered as incidental because all other groups remained at control levels.

Variation in the HIF-1α results observed in both the BALF supernatant and BALF cell sediment for the tricobalt tetraoxide groups cannot be fully determined, resulting in difficulty in concluding on the significance of the results. A study with a longer duration of exposure (i.e. 90-day repeated-dose) may provide further clarification on this endpoint.

IMMUNOHISTOCHEMISTRY - 8-OH-dG ANALYSIS
1) Tricobalt tetraoxide (main study and recovery group)
- 5.12, 20.50 and 80.78 mg/m³: no statistically significant changes compared to concurrent controls were observed

2) Cobalt sulfate heptahydrate (main study and recovery group)
- 2.04 and 10.19 mg/m³: no statistically significant changes compared to concurrent controls were observed

Key result

Dose descriptor:

NOAEC

Effect level:

5.12 mg/m³ air (analytical)

Based on:

test mat.

Remarks:

Cobalt tetraoxide

Sex:

male/female

Basis for effect level:

other: bronchoalveolar lavage

Key result

Dose descriptor:

LOAEC

Effect level:

20.5 mg/m³ air (analytical)

Based on:

test mat.

Remarks:

Cobalt tetraoxide

Sex:

male/female

Basis for effect level:

other: bronchoalveolar lavage

Key result

Dose descriptor:

LOAEC

Effect level:

2.04 mg/m³ air (analytical)

Based on:

test mat.

Remarks:

Cobalt sulfate heptahydrate

Sex:

male/female

Basis for effect level:

other: bronchoalveolar lavage

Remarks on result:

other: equivalent to 1.13 mg cobalt sulfate/m³

Critical effects observed:

no

Conclusions:

In this repeated dose inhalation toxicity study, the administration of cobalt tetraoxide and cobalt sulfate heptahydrate to male and female Wistar rats at doses of 5.12, 20.5 and 80.78 mg/m³ and 2.04 and 10.19 mg/m³, respectively, for a duration of 28 days produced no unscheduled deaths or signs of systemic toxicity. Furthermore, no effects on body weight, food consumption, water consumption, haematology, clinical chemistry, urinanalysis, immunohistochemistry (8-OH-dG) or macroscopical findings were observed.

After administration of 80.78 mg Co3O4/m³, the relative lung weights of females were statistically significantly increased compared to controls. Following a recovery period of 90 days, it was observed that the absolute and relative lung weights in the males and females were statistically significantly increased compared to the controls. One day after exposure, no effects were observed for the 5.12 and 20.50 mg/m³ dose groups regarding lung weights.Lastly, after administration of 2.04 and 10.19 mg cobalt sulfate heptahydrate no effects were observed for organ weights.

One day after the end of cobalt tetraoxide exposure, histopathological evaluation revealed exposure-related findings in lungs. Interpreted as adverse findings were: the alveolar infiltration of granulocytic cells, the interstitial mononuclear cell infiltration at the terminal bronchus, the lipoproteinosis and the interstitial fibrosis, which were seen in the 80.78 mg/m³ and partially 20.50 mg/m³ dose exposed animals (males and females). After additional 91 days of recovery the following findings occurred statistically significantly: alveolar infiltration of granulocytic cells, the interstitial mononuclear cell infiltration at the terminal bronchus, the lipoproteinosis and the interstitial fibrosis in the 80.78 mg/m³ dose exposed males and females.

In addition, for the cobalt sulfate heptahydrate treatment groups exposure-related findings were found in the lung as well as in the larynx. In the 10.19 mg/m³ group (males and females), lipoproteinosis occurred statistically significantly. Mononuclear cell infiltration was detected in the 10.19 mg/m³ group (females). In the larynx the squamous cells metaplasia, the ulcerative inflammation, the chronic-active inflammation, the chronic inflammation, the erosion and chronic granulation tissue and the degeneration of the cartilage were interpreted as adverse findings, which were seen in the 10.19 mg/m³ and partially 2.04 mg/m³ dose exposed animals. The squamous cells metaplasia and the ulcerative inflammation occurred statistically significantly in the 10.19 mg/m³ group (males and females). After additional 91 days of recovery the chronic granulation tissue occurred statistically significantly in the 10.19 mg/m³ group (males).

In this study a bronchoalveolar lavage was carried out. At day 1 after end of exposure statistically significant increases of polymorphonuclear neutrophils (PMN) were detected in the 20.50 (19%/13%; males/females) and 80.78 mg Co3O4/m³ dose groups (31%/35%; males/females) of both sexes; in the low dose groups, PMN showed the same levels as clean air controls (<1%). The PMN influx persisted after 91 days of recovery. In the two CoSO4 7H2O groups statistically significant increases of the PMN percentages were detected in both sexes (2.04 mg/m³ dose: 12%/12%; males/females - 10.19 mg/m³ dose: 25%/25%; males/females). After 91 days of recovery the effect disappeared. For lactic dehydrogenase, ß-glucuronidase and total protein statistically significant increases were detected in the 20.50 mg Co3O4/m³ (LDH and total protein; males) and 80.78 mg Co3O4/m³ dose groups (LDH, β-glucuronidase and total protein; males and females) at day 1 after end of exposure. In the 2.04 mg CoSO4 7H2O/m³ dose group (LDH; males) and in the 10.19 mg CoSO4 7H2O/m³ dose group all three parameters were statistically significantly increased (males and females). Cytokine concentrations (MCP-1, IL-8 alias CINC-1) at day 1 after end of exposure showed a dose-dependent increase in the Co3O4 groups (statistically significant in the mid and high dose groups; persisting after recovery); in the CoSO4 groups statistically significant increases were observed in the high dose group and partially low dose groups (only IL-8); in the recovery periods these effects returned to normal. Overall, a clear dose-dependency of observed effects was found.

Absolute and relative lung weights of rats used for bronchoalveolar lavage showed a statistically significant increase at day 1 in the 80.78 mg Co3O4/m³ and 10.19 mg CoSO4 7H2O/m³ dose groups (males and females). At day 91 of the post-exposure observation period the findings in the Co3O4 groups persisted whereas in the CoSO4 groups all values had returned to normal values.

The HIF1-α analysis was measured in BALF and the BALF cell sediments (gained by centrifugation) Variation in the HIF-1α results observed in both the BALF supernatant and BALF cell sediment for the tricobalt tetraoxide groups cannot be fully determined, resulting in difficulty in concluding on the significance of the results. A study with a longer duration of exposure (i.e. 90-day repeated-dose) may provide further clarification on this endpoint.

Based on the finding of an increase of polymorphpnuclear neutrophils, the NOAEC and LOAEC of cobalt tetraoxide in the Wistar rat for both sexes are considered to be 5.12 mg/m³ and 20.5 mg/m³, respectively. In addition, it has to be mentioned that after 28 days of exposure to 5 mg/m³, the cobalt tetraoxide low dose group behaves toxicologically very similar as the well-characterised TiO2 inert dust (“Bayertitan T”; rutile modification; after a 90-day exposure of rats to 5 mg/m3; historical data).

Based on the finding of an increase of polymorphpnuclear neutrophils, the LOAEC of cobalt sulfate heptahydrate in the Wistar rat for both sexes is considered to be 2.04 mg/m³ (equivalent to 1.13 mg cobalt sulfate/m³).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

Study duration:

chronic

Species:

other: human data

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Introductory remark – read-across

 

Read-across entails the use of relevant information from analogous substances (the ‘source’ information) to predict properties for the ‘target’ substance(s) under consideration. Substances whose physicochemical or toxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a category of substances. Structural similarity is a pre-requisite for any read-across approach under REACH (ECHA Read-Across Assessment Framework, 2015).

 

In accordance with Annex XI, 1.5 of the REACH regulation and the ECHA Guidance Read-Across Assessment Framework (ECHA, 2017), the similarities may be based on:

 

1) A common functional group (i.e. chemical similarity within the group);

2) Common precursors and/or likelihood of same breakdown products through physical and/or biological processes which result in structurally-similar degradation products (i.e. similarity through (bio) transformation); or

3) A constant pattern in the changing of the potency of the properties across the group (i.e. of physical-chemical and/or biological properties).

 

Due to the absence of substance specific information for the majority of substances within the cobalt category, the approach will read-across data from representative source substances to all other members of the read-across group.

 

Due to the route-specific toxicological properties of the cobalt category substances, several read-across groups are formed as shown in the table below:

 

	Route	Read-across group
Cobalt category	oral-systemic	bioavailable cobalt substances group
Cobalt category	oral-systemic	inorganic poorly soluble
Cobalt category	oral-systemic	poorly soluble in aqueous solutions with organic ligand
Cobalt category	inhalation-local	reactive
Cobalt category	inhalation-local	non-reactive

 

Further details on the read-across approach for the dermal sensitisation, oral systemic effects and the inhalation local effects are given in in IUCLID section 13.2.

 

Lithium cobalt dioxide is assigned to the read-across groups (i) oral-systemic: bioavailable cobalt substances group, (ii) inhalation-local: non-reactive and (iii) Low release of cobalt ion in artificial sweat – no skin sensitisation.

 

 

Toxicological relevance of the non-common compound

The toxicological relevance of the non-common compound in lithium cobalt dioxide for oral-systemic toxicity is discussed in IUCLID section 13.2.

 

Human data - inhalation

 

Repeated dose toxicity - local effects

The comprehensive discussion of the available human data can be found at the beginning of chapter 5 of the CSR and in section 7.10 of the IUCLID.

 

The overall outcome was that, based on the findings of the epidemiological studies in workers by Swennen et al. (1993) and Verougstraete et al. (2004), Roto (1980) and Sauni et al. (2010) a cobalt concentration of 0.12 mg Co/m³ will be used as NOAEC for setting a DNEL (inhalation, chronic, local effects).

 

Repeated dose toxicity - systemic effects

An investigation on the effects of cobalt exposure in a Finnish cobalt plant in Kokkola on the cardiovascular system of workers was published by Linna et al. (2004). The cross-sectional study population comprised 203 male workers with at least one year of exposure to cobalt at the end of 1999. The average exposure time was 15.0 years with a mean cumulative exposure to cobalt of 0.40 mg-year (median 0.18 mg-year, range 0.02-2.52). The control group consisted of an age-stratified sample of 94 male workers in a zinc plant that had not been exposed to cobalt, arsenic or lead. The zinc exposure level was 0.1-0.2 mg/m³ for four fifths of the workers, and for one fifth it was about 1 mg/m³. No significant differences in the electrocardiography findings and conduction parameters, heart rate, blood pressure and laboratory tests (inter alia serum T4 and TSH levels) were found between the cobalt exposed and control workers. There were no significant differences between the exposed group and the control group in the prevalence of reported cardiovascular diseases, diabetes mellitus, or pulmonary diseases, except asthma, diagnosed by a physician. Echocardiography was performed on a subset of 122 cumulatively most exposed workers, of which 109 was analysed, and 60 controls with same age distribution, of which 57 were analysed. The average exposure time was 21.2 years with a mean exposure to cobalt of 0.58 mg-year (median 0.47 mg-year, range 0.03-2.52). The echocardiographic data were studied using a regression analysis and an analysis of covariance (ANCOVA). In the final analyses high and low exposure was determined on the basis of being above or below the median mg-years of cobalt exposure. Two of the echocardiography parameters measured was associated with cobalt exposure. In the higher exposure group the left ventricular isovolumic relaxation time (mean 53.3, 49.1, and 49.7 ms in the high exposure (>0.47 mg-year), low exposure (<0.47 mg-year), and control groups, respectively) and the deceleration time of the velocity of the early rapid filling wave (mean 194.3, 180.5, and 171.7 ms for those in the high exposure, low exposure, and control groups, respectively) were prolonged, indicating altered left ventricular relaxation and early filling. The clinical significance of these changes, however, remains to be evaluated. Minor increases in the left ventricular wall thickness concurred with these observations. No signs of systolic cardiac dysfunction were found. The ejection fraction, fractional shortening, and the left ventricular end diastolic diameter were similar in the exposed and control groups.

 

No clinically significant cardiac dysfunction, no evidence of polycythaemia and only equivocal indications of interferences with thyroid metabolism were observed in workers occupationally exposed to inorganic cobalt compounds. Therefore, it can be concluded that systemic effects following inhalation exposure are expected at higher dose levels compared to the dose levels for local effects. Thus, no DNEL(inhalation, systemic) will be derived for workers and the general population.

 

 

Animal data - inhalation

 

Creutzenberg (2017) conducted a 28-day repeated dose inhalation toxicity study with tricobalt tetraoxide according to OECD guideline 412. The substance was administered to groups of 10 male and 10 female Wistar rats via nose-only inhalation at concentrations of 5.12, 20.5 and 80.78 mg/m³ (analytical concentrations). A negative control group (clean air) and recovery groups was run concurrently (recovery period: 90 days). Cobalt sulfate heptahydrate was tested as reference item. The following endpoints were investigated: clinical signs, mortality, body weights, food/water consumption, haematology, clinical chemistry, urinalysis, macroscopical examination, organ weights, histopathology, and immunohistochemistry (8-OH-dG only). In addition to the aforementioned parameters, a bronchoalveolar lavage was performed measuring leukocyte, macrophages, neutrophiles, eosinophils, lymphocytes, lactic dehydrogenase,β-glucuronidase and total protein in the lavage fluid. Lastly, cytokine levels (HIF-1α, IL-8 and MCP-1) were analysed in the lavage fluid using ELISA.

Tricobalt tetraoxide produced no unscheduled deaths or signs of systemic toxicity. No effects on body weight, food consumption, water consumption, haematology, clinical chemistry, urinalysis, immunohistochemistry or macroscopical findings were observed. After administration of 80.78 mg/m³, the relative lung weights of females were statistically significantly increased compared to controls. Following a recovery period of 90 days, the absolute and relative lung weights in the males and females were statistically significantly increased compared to the controls. One day after the end of exposure, histopathological evaluation revealed adverse exposure-related findings in the lungs (alveolar infiltration of granulocytic cells, interstitial mononuclear cell infiltration at the terminal bronchus, lipoproteinosis; interstitial fibrosis) for the 80.78 mg/m³ and partially 20.50 mg/m³ exposed animals (both sexes). After recovery alveolar infiltration of granulocytic cells, interstitial mononuclear cell infiltration at the terminal bronchus, lipoproteinosis and the interstitial fibrosis in the 80.78 mg/m³ dose exposed males and females was observed.

In the bronchoalveolar lavage, day 1 after end of exposure statistically significant increases of polymorphonuclear neutrophils (PMN) were detected in the 20.50 (19%/13%; males/females) and 80.78 mg/m³ dose groups (31%/35%; males/females) of both sexes, which persisted after 91 days of recovery. For lactic dehydrogenase, ß-glucuronidase and total protein statistically significant increases were detected in the 20.50 mg/m³ (LDH and total protein; males) and 80.78 mg/m³ dose groups (LDH, β-glucuronidase and total protein; males and females) on day 1 after end of exposure.

The NOAEC of cobalt tetraoxide in the Wistar rat for both sexes is 5.12 mg/m³. Based on the significant but weak increase of polymorphonuclear neutrophils in BAL the LOAEC is 20.5 mg/m³.

 

 

Conclusions - inhalation

 

In human epidemiological studies following prolonged inhalation exposure, no clinically significant cardiac dysfunction due to cobalt exposure was found. Also no further adverse systemic effects were reported in humans. Therefore, it can be concluded that systemic effects following inhalation exposure are expected at higher dose levels compared to the dose levels for local effects. Thus, a DNEL for systemic effects will not be derived based on these data.

 

Human epidemiological data will be used for the hazard assessment of repeated dose toxicity via inhalation, non-neoplastic lesions. Changes in lung function were the predominant findings in the studies by Swennen et al. (1993) and Verougstraete et al. (2004), Roto (1980) and Sauni et al. (2010). A cobalt concentration of 0.12 mg Co/m³ will be used as NOAEC for the setting of a DNEL (inhalation, local, chronic).

 

The existing 28-day inhalation study with tricobalt tetraoxide in rats does not show any substance-related adverse effects. The effects of tricobalt tetraoxide after 28-day inhalation is best compared with the effects seen with other poorly-soluble low-toxicity particles (PSLT), leading to a minimal or mild inflammatory response only at the maximum tolerated concentration in repeated dose toxicity studies via inhalation. In order to conclusively state that the inhalation effects associated with tricobalt tetraoxide exposure are due to a non-test-item related PSLT effect, a longer duration RDT study needs to be conducted.

 

 

Animal data- oral

 

Repeated dose toxicity

Based on the read-across approach presented in IUCLID section 13.2 the substances of the bioavailable cobalt substances are grouped and assessed for their hazardous properties using information from the source substance cobalt dichloride and supporting evidence form cobalt metal and cobalt acetylacetonate.The source substances cobalt dichloride and cobalt metal, have been tested for repeated dose toxicity, resulting in comparable effect levels (28-day NOAEL CoCl2: 2.5mg Co/kg bw/day; 90-day NOAEL CoCl2: 0.74 mg Co/kg bw/day; 28-day NOAEL Co metal: 30 mg Co/kg bw/day). In all studies, the effect levels were based on similar findings, predominantly body weight effects and haematological findings. Further details on the findings are presented below.

 

 

In the sub-chronic repeated dose toxicity study, cobalt dichloride was given male and female rats at doses of 0, 3, 10, 30 mg/kg bw/day. A total of 10 males and 10 females per group were given the test items suspended in 0.5% hydroxypropyl methylcellulose gel orally via gavage once daily for 90 days. Additional 2 groups of 5 male and 5 female animals, dosed with 0, 30 mg/kg bw/day were assigned as recovery animals, kept for 28 days after the treatment period without receiving the test item. Additional examinations were added to the study design: (i) monitoring of the oestrus cycle pre-dose, during study conduct, at the end of test week 13 and at the end of the recovery period in all female animals, (ii) hormone level status (testosterone, progesterone, 17beta-estradiol) pre-dose, during study conduct, at the end of test week 13 and at the end of the recovery period in all animals, (iii) Detailed histopathologic examination on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure). During the conduct of the study, no deaths occurred and no test item-related changes in behaviour or external appearance were observed. The body weight of the male animals treated with 10 mg Cobalt dichloride hexahydrate/kg b.w./day and the body weight of the male and female animals treated with 30 mg Cobalt dichloride hexahydrate/kg b.w./day were slightly reduced. Body weight gain and body weight at autopsy changed accordingly. No test item-related influence was observed on the food and drinking water consumption, biochemical parameters, urinary parameters, the eyes and optic region at any of the tested dose levels. Observational screening and functional tests did not reveal any test item-related neurological effects. No test item-related macroscopic changes in organs or tissues were noted at necropsy. The histopathological examination of the high-dosed animals did not reveal any test item-related morphological lesions. At the end of the 4-week recovery period (restricted to the high dose group), the body weight in the previously high-dosed animals were within the range of the control group, indicating a complete recovery. Repeated oral treatment of male and female rats with 10 or 30 mg Cobalt dichloride /kg b.w./day led to a few test item-related changes in haematological parameters in comparison to the control animals. In particular, slightly increased values were noted for red blood cell-related parameters as the haemoglobin content, the number of erythrocytes, the haematocrit value, the mean corpuscular volume and the mean corpuscular haemoglobin. The number of reticulocytes and platelets was decreased and the thromboplastin time and activated partial thromboplastin time were increased. In general, the male animals were affected to a higher degree than the females. Correspondingly, the 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 Cobalt dichloride /kg b.w./day compared to the controls: 7 of 10 animals for both sexes in the high dose group versus 0 of 10 in controls. No test-item related changes were observed during histopathological examination of the male and female reproductive organs. 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. No test-item related influence on the ovaries, oviducts, uterus (incl. cervix) and vagina were noted. No test item-related difference was noted in the mean number of oestrous cycles for the female animals. No test item-related influence was noted on the serum levels of the hormones testosterone, progesterone, and 17 beta-estradiol in the male and female animals treated with 1000 mg Cobalt dichloride/kg bw/day compared to the control group during study conduct, at the end of the treatment period, and at the end of the recovery period. All changes previously observed in haematological and biochemical parameters and at histological examination after repeated treatment with 30 mg Cobalt dichloride/kg b.w./day had subsided after 4 weeks of recovery. Under the test conditions of this 90-day repeated dose toxicity study with Cobalt dichloride, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic effects was 3 mg cobalt dichloride/kg bw/day by oral administration based on findings related to the haematopoiesis and reduced body weight and body weight gain at the mid and high dose group. The No-Observed-Effect-level (NOEL) for fertility/reproductive effects was above 30 mg cobalt dichloride/kg bw/day by oral administration based on a complete absence of effects on reproductive organs, oestrus cycle, qualitative sperm staging and hormone levels.

 

In a 28-days repeated dose toxicity study with reprotox screening (according to OECD 422 and under GLP), cobalt metal powder was administered orally to rats at dose levels of 30, 100, 300 and 1000 mg/kg b.w./day during the pre-mating, mating and post-mating periods to parental males as well as during the pre-mating, mating, gestation and lactation periods until day 3 post-partum (or shortly thereafter) to parental female animals. Piloerection, reduced motility, soft faeces/diarrhoea and reduced food consumption were noted - in relation to the dose - from a dose level of 100 mg Cobalt Powder/kg b.w./day onwards. In addition, reductions of body weight were noted from 300 mg Co-balt Powder/kg b.w./day onwards. Premature deaths occurred in five female rats at 100 mg Cobalt Powder/kg b.w./day and eight female rats at 300 mg Cobalt Powder/kg b.w./day. Treatment with 1000 mg Cobalt Powder/kg b.w./day caused the premature death of nine of ten males and all ten females. Macroscopic inspection revealed changes of the gastro-intestinal tract - mainly in the prematurely deceased animals - from a dose level of 100 mg Cobalt Powder/kg b.w./day onwards and adrenal changes and pulmonal lesions at 1000 mg Cobalt Pow-der/kg b.w./day. Histopathological inspection did not reveal any pathological changes. No histopathological correlate could be found for the macroscopic lesions noted at necropsy. No test item-related influence was noted on the sperm staging or interstitial cell structure (qualitative examination). The NO(A)EL for systemic effects was 30 mg/kg b.w./day, based on mortality, clinical signs of toxicity, effects on food consumption and macroscopic pathological changes observed at and above 100 mg Cobalt Powder/ kg b.w./day and reduced body weight at and above 300 mg Cobalt Powder/kg b.w./day.

 

In a 28-day repeated dose toxicity study, rats were given oral doses of cobalt acetylacetonate of 0, 15, 50, 150 mg/kg bw/day. In the 50 mg/kg/day dose group, males displayed lower mean body weight gains and a lower food consumption. Additionally, haemoglobin concentration was increased in males of this dose group. Whereas in the 150 mg/kg/day dose group, males and females both displayed reduced body weight gain, lower food consumption (except females had a higher food consumption during weeks 2 and 3) and an increase in some blood parameters (red blood cell count, haemoglobin, haematocrit). Lastly, ptyalism was observed in 4 of 5 females treated at 150 mg/kg/day. One of these females also had soft feces for 7 days during week 2. Under the conditions of this study, the no observed adverse effect level (NOAEL) for males was determined to be 15 mg/kg/day (equivalent to 3.05 mg cobalt/kg/day) based on clinical signs, body weight and weight gain, food consumption, and haematology. The NOAEL for females was determined to be 50 mg/kg/day (equivalent to 10.6 mg cobalt/kg/day) based on haematological findings.

 

The NOAEL for systemic effects derived from the sub-chronic study with cobalt dichloride is used as point of departure for the derivation of DNEL (oral, systemic effects) for the general population. Based on the DNEL for cobalt dichloride of, all further DNELs for the substances in this group will be calculated taking into account the cobalt mass fraction of total molecular mass of the respective substance. Details on the substance specific derivation of DNELs-oral, systemic for the general population are given in the report, which can be found as attachment to the endpoint summary in section 7 of the IUCLID.

 

In addition to the above, several other (published) studies on bioavailable cobalt substances were identified which however do not fulfil the relevance, reliability and adequacy criteria as foreseen by the ECHA Guidance for information requirements. The most prominent deficiencies are: single dose studies, targeted studies examining isolated organ systems, incomplete or unclear description of the experimental procedures, several shortcomings in execution and reporting (e.g. test item insufficiently described, animal strain, age, weight or source not reported, dosing unclear, route of administration unclear, exposure period unclear or too short). Therefore, these studies are discussed below only briefly for information purposes (further information is provided in the IUCLID).

·        In a 30-day study, male and female rats were fed an iron-sufficient diet mixed with cobalt dichloride at 5 doses (10-300 ppm). A NOAEL of 50ppm was identified based on thymus weight difference (Chetty, 1979).

·        In a 90-day drinking water study male rats were given cobalt dichloride at 500 ppm (Domingo, 1984). Treated animals showed a reduced body weight gain and development of polycythaemia. It is unclear whether reduced water consumption was caused by impaired palatability of the cobalt dichloride supplemented drinking water. No NOAEL/LOAEL for systemic toxicity could be established.

·        Female rats given cobalt dichloride during GD 6-15 showed signs of maternal toxicity by reduced body weight gain, decreased food consumption, haematological changes. A NOAEL of 50 mg/kg bw/day was established (Paternain, 1988).

·        Cobalt sulfate administered to pregnant mice, rabbits and rats at doses of 20, 100, 200 mg/kg bw/day produced dose-dependent maternal toxicity (Szakmáry, 2001). No NOAEL/LOAEL for maternal toxicity could be established.

·        In short term and subchronic studies on cobalt dichloride in rats, polycythaemia and increased haemoglobin were induced at doses of 0.5 mg Co/kg bw/day and above (Stanley et al., 1947; Murdock, 1959; Krasovskii and Fridlyand, 1971).

·        Male rats were dosed with 100, 200 and 400 ppm of cobalt chloride hexahydrate in drinking water for at least 12 weeks (Pedigo, 1988).

·        Thyroid necrosis was observed in mice dosed by the oral route for 15 to 45 days with cobalt chloride at 400 ppm (Shrivastava et al., 1996).

·        In rats given cobalt sulfate in the diet for 24 weeks at 40 mg/kg bw/day, cardiac enzyme activity and mitrochondrial ATP production were significantly reduced. The hearts of treated animals were isolated and were found to have left ventricular hypertrophy and impaired ventricular function (Haga et al., 1996; Clyne et al., 2001). In contrast, the same working group did not find any myocardial dysfunction in male rats administered cobalt sulfate for 8 weeks (Pehrsson, 1991).

·        Rats treated with cobalt sulfate at 26 mg Co/kg/bw per day for 8 weeks or cobalt chloride at 50 mg/kg bw/day for 3 weeks had cardiac degeneration (Grice et al., 1969; Morvai et al., 1993).

·        Guinea pigs given cobalt sulfate at 20 mg Co/kg bw per day for 5 weeks had abnormal EKGs, increased heart weight, and cardiac lesions (Mohiuddin et al., 1970).

·        In a study series of one working group, the authors investigated the influence of cobalt dichloride on male rats via oral route for 57-98 days at varying single doses. Testicular atrophy, degeneration and necrosis, induction of polycythaemia were reported. The authors concluded that observed testicular degeneration was not a primary response to cobalt, but instead suggested that the testes become hypoxic due to both a blockage of veins and arteries by red blood cells as well as changes in permeability caused by a thickening of basal lamina and basement membranes (Corrier, 1985, Nation, 1983, Bourg, 1985, Mollenhauer, 1985).

·        In an additional study series the authors investigated the potential toxicity of cobalt dichloride in male and female mice and rats. Cobalt dichloride was administered via the oral route in varying concentrations for 7-60 days. They reported changes in blood parameters, increase in oxidative stress markers and changes in hepatic, cardiac as well as renal enzyme expression/activity (Zaksas (2013), Akinrinde (2016), Ajibade (2017), Akinrinde (2016b), Awoyemi (2016)).

 

 

Repeated dose toxicity: dermal

The submission of a repeated dose toxicity study via dermal route is considered unjustified, since:

 

(a) Lung function impairment is the predominant finding in human epidemiological data by Swennen et al. (1993) and Verougstraete et al. (2004), Roto (1980) and Sauni et al. (2010), whereas no significant systemic toxicity due to prolonged inhalation exposure towards cobalt substances was found. It can be concluded that systemic effects following inhalation exposure are expected at higher dose levels compared to the dose levels for local effects. In order to be protective against local effects after repeated dose toxicity via inhalation, the human NOAEC derived from the above mentioned human epidemiological data will be used to derive a DNEL for all cobalt substances. Consequently, the inhalation route is considered as the route of exposure showing the highest concern for which safety levels for workers and consumers are to be implemented

 

(b) In total 22 out of 26 cobalt substances prepared by the Cobalt REACH Consortium (CoRC) are legally and/or self-classified for dermal sensitisation properties. The risk management measures for such substances foresee to minimise dermal exposure to as low as reasonably achievable. Protective gloves according to EN 374 have to be worn at all workplaces unless any exposure to the substance can be excluded when taking into account the nature of the conducted process, applied exposure prevention measures and physical appearance of the substance of concern in the specific type of application (e.g. protecting from splashes by containment of emission source).

 

(c) Based on the physico-chemical properties of all inorganic cobalt substances and results of a dermal absorption study with a cobalt salt of high in vitro bioaccessibility in artificial sweat suggest a potential for a negligible rate of absorption through the skin. A dermal absorption rate of 0.38% for the low exposure scenarios (ca 31.9μg Co/cm² loading) and 1.08% for the high exposure scenarios (ca 319μg Co/cm² loading) was determined. These values also account for part of the material associated with the stratum corneum and the test was conducted with a highly water soluble form of Cobalt in an aqueous solution. Thus, these values are considered to represent a conservative estimate.

In conclusion, the dermal absorption of cobalt has been shown to be low in a guideline-conform in-vitro percutaneous absorption study conducted under GLP with the highly soluble substance cobalt dichloride (Roper, 2010). This renders percutaneous uptake a negligible route of entry into the body, which is why this route is not further considered in risk characterisation

 

Conclusions - oral

 

The substances of the bioavailable cobalt substances group exert adverse findings in test animals, such as

(i) reduced body weight and body weight gain

(ii) induction of adverse gastro-intestinal effects upon macroscopic inspection, manifested as soft faeces/diarrhoea in mid-dose animals and reddened intestines, caecum or stomach in high-dose and above

(iii) microscopically visible induction of erythroid hyperplasia in the bone marrow after sub-chronic exposure with correlating changes in haematological parameters, such as increased values in red blood cell parameters (haemoglobin content, the number of erythrocytes, the haematocrit value, the mean corpuscular volume and the mean corpuscular haemoglobin).

These findings were seen in a sub-chronic oral study in rats with cobalt dichloride and in a sub-acute oral study in rats with cobalt metal.

 

For risk assessment purposes, a read-across is applied by using the cobalt equivalent NOAEL derived from the sub-chronic oral study with cobalt dichloride (i.e. 3 mg CoCl2 6H20/kg bw/day or 0.744 mg cobalt/kg bw/day) for the calculation of substance-specific DNEL (oral, systemic, long-term) for all members of the bioavailable cobalt substances group.

 Details on the substance specific derivation of DNELs-oral, systemic for the general population are given in the report, which can be found as attachment to the endpoint summary in section 7 of the IUCLID.

 

 

Statement on the preferential use of human data in risk assessments for human health

 

(I) In almost 20 years of practical conduct of risk assessments under the “Existing Substances Regulation (793/93), human data has been given preference over animal studies. This is documented in the Technical Guidance Document in chapter 3.1 as follows: „Generally human data will only be available for existing substances. If both animal data and human data are available, as a general rule, well reported relevant human data for any given endpoint is to be given preference for the risk assessment.“ (ECB, 2003).

 

(II) Similarly, the US Environmental Protection Agency (EPA) in their guidance have stated that they look to human data whenever possible in completing human risk assessments: "If adequate human studies (confirmed for validity and applicability) exist, these studies are given first priority in the dose-response assessment, and animal toxicity studies are used as supportive evidence" (EPA, 1989). Often, such data can be obtained from epidemiological studies, which do not involve the intentional dosing of research participants, but rather evaluate the effects of exposures that have occurred in an occupational setting or because of the peculiarities of a specific geographical setting. Regardless of the origins of such human data, risk assessments based on human data have the advantage of avoiding the problems inherent in interspecies extrapolation" (EPA, 1993). In the same document, EPA also states: “The default assumptions that are of particular relevance to the issues raised by third-party intentional human dosing studies are those that bridge gaps between animal results and estimates of effects in humans. In the context of FIFRA, for example, EPA has routinely divided the calculated "safe" dose for animals by a factor of 10, to account for the possibility that humans are more sensitive to the substance being tested than are the animal species. Third-party submitters of human dosing studies have been particularly interested in modifying this default assumption by introducing data obtained directly from human studies.”

 

(III) When addressing the relevance and use of human data, ECHA guidance specifies the requirements for such studies as follows in section B.4.3.3 (human data) of their guidance, for the following four types of human data (ECHA, 2008):

 

Analytical epidemiology studies on exposed populations (case-control, cohort and cross-sectional studies) are useful for identifying a relationship between human exposure and effects and may provide the best data for risk assessment.

 

Descriptive or correlation epidemiology studies are useful for identifying areas for further research but are not very useful for risk assessment since they often can only identify patterns or trends but cannot ascertain the causal agent or degree of human exposure.

 

Case reports may demonstrate effects which cannot be observed in experimental animals. Thorough assessment of the reliability and relevance of case reports is needed because they often lack critical information on e.g. substance purity, human exposure, and effects.

 

Controlled studies in human volunteers are acceptable in very rare cases. Testing with human volunteers is strongly discouraged but when good quality data are already available, they should be used as appropriate in well justified cases.

 

In the case of cobalt and cobalt substances, the human studies that were used for the derivation of DNELs were assessed for their reliability and relevance, and were found to be of acceptable quality for the purpose envisaged.

 

(IV) Finally, the use of human data in risk assessment largely avoids a need for the application of assessment or extrapolation factors to account for differences in toxicokinetics, toxicodynamics, metabolic capacity and species sensitivity.

 

 

References

ECB (2003) Technical Guidance Document on Risk Assessment in support of Commission Directive 93/67/EEC on Risk Assessment for new notified substances, Commission Regulation (EC) No 1488/94 on Risk Assessment for existing substances, Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market, Part I, EUR 20418 EN/1

 

ECHA (2008) Guidance on information requirements and chemical safety assessment, Guidance on information requirements and chemical safety assessment, Part B: Hazard Assessment, European Chemicals Agency, 2008

 

EPA (1989) Risk Assessment Guidance for Superfund, Vol. 1: Human Evaluation Manual, EPA/540-1-89/002, US Environmental Protection Agency. available at:www.epa.gov/cgi-bin/claritgw?op-Display&document=clserv:OSWER:1175;&rank=4&template=epa

 

EPA (1993) Reference Dose (RfD): Description and Use in Health Risk Assessments, § 1.3.2.2.1, US Environmental Protection Agency, background document, available at:www.epa.gov/IRIS/rfd.htm.

 

IGHRC (2006) Guidelines on route-to-route extrapolation of toxicity data when assessing health risks of chemicals. The Interdepartmental Group on Health Risks from Chemicals, http://www.silsoe.cranfield.ac.uk /ieh/ighrc/ighrc.htm