Tricobalt tetraoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

fertility, other

Remarks:

based on a 90 day repeated dose toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-03 to 2014-11-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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 and females: 52 days
- Weight at first dosing: males: 269.0 - 321.5 g; females: 183.3 - 227.8 g
- Housing: animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm; Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material.
- Diet (ad libitum): commercial diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): tap water
- Acclimation period: 8 days

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

Route of administration:

oral: gavage

Vehicle:

other: 0.5% hydroxypropyl methylcellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concnetrations. The administration formulations were freshly prepared daily.
Administration volume: 5 mL/kg bw/day

The dose of the test item was adjusted to each animal's body weight daily up to and including test week 6, and once weekly thereafter.
The control animals received the vehicle orally at a constant volume in the same way once daily.

Details on mating procedure:

not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures, samples of approximately 10 mL were taken at the following times and stored at -20°C or colder until analyses:
1) At study initiation (on the first administration day of male animals):
- analysis of concentration: immediately after preparation of the formulations as well as after 8 and 24 hours at room temperature (3 samples/test item group).
- homogeneity: at the start of administration, during administration (in the middle), and before administration to the last animal of each dose group (3 samples/test item group).

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

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

Results:
The results of the analysis show that the test item formulations were correctly prepared. The actual tricobalt tetraoxide concentrations in the respective test item formulations ranged from 95.6 % to 103.0 % of the nominal concentrations before administration to the last male animal of the group on test day 1, and from 87.5 % to 101.7 % of the nominal concentrations before administration to the last female animal of the group on test day 90 (last administration day of the study).

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Main study (per group): 10 males/10 females
Recovery group (control group and 1000 mg/kg bw/day dose group only; per group): 5 males/5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels for this study were selected taking into account any existing toxicity and toxicokinetic data available for the test item at the time of initiation of the study.
- Recovery groups were included in this study. One recovery group was included for the control group and other recovery group for the 1000 mg/kg bw/day dose group. These groups were kept for 28 days after the treatment period without receiving the test item.

Positive control:

none

Parental animals: Observations and examinations:

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

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

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

FOOD CONSUMPTION (main study animals and recovery animals):
- Food consumption for each animal determined and relative food consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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

WATER CONSUMPTION: Yes (main study animals and recovery animals)
- Time schedule for examinations: daily

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

HAEMATOLOGY: Yes (main study animals and recovery animals)
- Time schedule for collection of blood: at the end of the treatment period (test day 91) and at the end of the recovery period (all recovery animals; test day 119)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes
- How many animals: all main study animals and all recovery animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, absolute and relative differential blood count (neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes, monocytes and large unclassified cells), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes (main study animals and recovery animals)
- Time schedule for collection of blood: at the end of the treatment period (test day 91) and at the end of the recovery period (all recovery animals; test day 119)
- Animals fasted: Yes
- How many animals: all main study animals and all recovery animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), triglycerides, urea (in blood), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase

URINALYSIS: Yes (main study animals and recovery animals)
- Time schedule for collection of urine: at the end of the treatment period (all main study animals; test day 89) and at the end of the recovery period (all recovery animals; test day 117)
- Animals fasted: Yes
- Parameters examined: colour, turbidity, volume, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite, and microscopic examinations of urine samples (epithelial cells, leucocytes, erythrocytes, organisms, crystalluria, and further constituents (i.e. sperm, casts))

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

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

2) Functional tests: grip strength and locomotor activity

HORMONE LEVELS:
Blood was collected by puncture of the vena jugularis under light isoflurane anaesthesia as follows (all main study and recovery animals):
- predose (before the first administration; all main study and recovery animals)
- during study conduct, at the end of test week 6, test day 42 (all main study and recovery animals)
- at the end of test week 13 (before necropsy, test day 91; all main study and recovery animals)
- at the end of the recovery period (before necropsy, test day 119; all recovery animals)
The following parameters of all animals of the control group and the 1000 mg/kg bw/day dose group were examined: testosterone, progesterone, and 17 beta-estradiol

Oestrous cyclicity (parental animals):

The stages of the oestrous cycle observed were recorded individually for each female rat (all females of the main study group and recovery group)
Time schedule:
- pre-dose (before the first administration) (monitoring duration: 7 days)
- during study conduct (test weeks 5/6; monitoring duration: 12 days)
- at the end of the treatment period (test weeks 12/13 before necrospy of main study animals; monitoring duration: 12 days)
- at the end of the recovery period (test weeks 16/17 before necropsy of recovery animals; monitoring duration: 12 days)

Sperm parameters (parental animals):

Detailed histopathological examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all male main study and recovery animals of the control group and the 1000 mg/kg bw/day group following staining.

Litter observations:

not applicable

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes (main study animals and recovery animals)
On test day 91, all main study animals were dissected following a randomisation scheme. Necropsy of all animals allocated to the recovery period was performed on test day 119.
The animals were euthanized by carbon dioxide, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

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

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

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

The afore-listed organs of all main study and recovery animals of the control group and the 1000 mg/kg bw/day group were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were prepared and stained with Oil Red O and examined microscopically.
A detailed histopathological examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all male main study and recovery animals of the control group and the 1000 mg/kg bw/day group following staining.

Postmortem examinations (offspring):

not applicable

Statistics:

Means and standard deviations were calculated on time-point specific data sets for each sex separately.
The test item-treated groups (100, 300, and 1000 mg/kg bw/day dose groups) were compared with the control group.

The following statistical methods were used:
- multiple t-test based on DUNNETT, C. W. New tables for multiple Comparisons with a control. Biometrics, 482-491 (Sept 1964): body weight, food consumption, absolute and relative organ weights (p ≤ 0.05 and p ≤ 0.01)
- exact test of R. A. FISHER: histology (p ≤ 0.05)
- STUDENT's t-test: all numerical functional tests: body temperature, hormone levels (p ≤ 0.05 and p ≤ 0.01)

Reproductive indices:

not applicable

Offspring viability indices:

not applicable

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

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

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

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

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

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

WATER CONSUMPTION (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- no test item-related differences between the test item-treated animals and the control animals throughout the treatment and the recovery period

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

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

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

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

- no test item-related effects were observed for the number of leucocytes, the relative reticulocyte count, the relative and absolute differential blood count, the thromboplastin time, the activated partial thromboplastin time, the mean corpuscular volume, the mean corpuscular haemoglobin and the mean corpuscular haemoglobin concentration at the end of the treatment period (test day 91).

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

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

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

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

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

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

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

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

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

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

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

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


REPRODUCTIVE FUNCTION: OESTROUS CYCLE (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 100, 300 or 1000 mg/kg bw/day: no test item-related difference was noted in the mean number of oestrous cycles of the female animals in the week before start of treatment (test week -1), in test weeks 5 and 6 during the treatment period, at the end of the treatment (test weeks 12/13), and at the end of the recovery period (test weeks 16/17) between the treated groups and the control group.
- 300 or 1000 mg/kg bw/day: the average number of oestrous cycles appeared to be slightly higher in the treated female animals than in the control animals in test weeks 5 and 6 during the treatment period. However, the slight differences are considered to be coincidental and not related to the test item treatment.

Please also refer for results about oestrous cycle to "Attached background material" below

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 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.


HORMONE LEVELS (PARENTAL ANIMALS)
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 1000 mg/kg bw/day: no test item-related influence was noted on the serum levels of the hormones testosterone, progesterone, and 17 beta-estradiol in the treated male and female animals compared to the control group during study conduct, at the end of the treatment period, and at the end of the recovery period.
- the individual data partly vary over a wide range, resulting in a large scatter.
- differences in hormone levels between the 1000 mg/kg bw/day dose and the control group were already noted at pre-dose examination. Therefore, all data obtained are considered to be within the normal range of biological variation. All differences between the test item-treated animals and the control group are considered as coincidental, despite of being statistically significant (at p ≤ 0.05 or p ≤ 0.01).
- The statistically significant changes in hormone levels in comparison to the control group that are not considered to be related to the test item treatment are as follows:
Treatment period:
decreased 17 beta-estradiol (1000 mg/kg bw/day; males; p ≤ 0.01)
increased progesterone (1000 mg/kg bw/day; males; p ≤ 0.01)
decreased testosterone (1000 mg/kg bw/day; males and females; p ≤ 0.05 or p ≤ 0.01)
Recovery period:
decreased progesterone (1000 mg/kg bw/day; males; p ≤ 0.05)

Please also refer for results about hormone levels to "Attached background material" below

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

not applicable

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Conclusions:

NOAEL (fertility): 1000 mg/kg bw/day
No test item-related influence on the oestrous cycle was noted. Furthermore, histopathological examination of one testicle and one epididymis with special emphasis on the qualitative stages of spermatogenesis and of the interstitial testicular structure, did not reveal any test item-related effects. Lastly, no test item-related influence on hormone levels was noted.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

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.

Tricobalt tetraoxide is assigned to the read-across group oral-systemic: inorganic poorly soluble.

Effects on fertility

 

Tricobalt tetraoxide and cobalt sulfide are the source substances for the inorganic poorly soluble cobalt substances group based on the read-across approach as outlined in Appendix 1.1 in the CSR. Two inorganic poorly soluble cobalt substances, tricobalt tetraoxide and cobalt sulfide, have been tested for effects on fertility, resulting in an absence of effects on the reproductive organs of male and female animals up to the limit dose of 1000mg/kg bw/day.

 

In a sub-chronic repeated dose toxicity study, tricobalt tetraoxide was given male and female rats at doses of 0, 100, 300, 1000 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, 1000 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. At the high dose of 1000 mg Tricobalt tetraoxide/kg bw/day, a slightly reduced body weight by up to 10 %, body weight gain, and body weight at autopsy were observed. This is considered to be a test item-related effect. The female animals were only marginally affected by up to 5 %, lacking statistical significance.

 

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 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 tricobalt tetraoxide/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.

 

The No-Observed-Effect-level (NOEL) for fertility/reproductive effects was 1000 mg tricobalt tetraoxide/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 acombined repeated dose toxicity studies with the reproduction/developmental toxicity screening test (according to OECD 422 and under GLP), cobalt sulfide was administered orally to rats at dose levels of 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. None of the parental animals died prematurely. The only treatment-related finding, not regarded as adverse, was piloerection noted in few male or female rats from a dose level of 100 mg Cobalt sulfide/kg b.w./day onwards. Macroscopic inspection did not reveal any test item-related changes at necropsy. Histopathological inspection did not reveal any pathological changes. No test item-related influence was noted on the sperm staging or interstitial cell structure (qualitative examination). No test item-related influence was noted on mating behaviour, fertility, implantation, the gestation length or the birth index. No test item-related influence was noted at 100, 300 or 1000 mg Cobalt sulfide/ kg b.w./day on the growth and development of the offspring from conception until sacrifice on day 4 post-partum or shortly thereafter.

The NO(A)EL for effects on the F0-generation and reproductive toxicity was above 1000 mg/kg b.w./day, based on a complete absence of adverse effects.

 

Conclusion

 

There is a substantial data base on the effects of soluble cobalt compounds to organs of male reproduction. However, the studies in themselves do not show a clear dose-response relationship and beyond that suffer from several shortcomings in experimental design and reporting. Furthermore, none of the available studies represent state-of-the-art, guideline-compliant, extended one-generation reproduction toxicity studies or other relevant study designs, from which robust no-effect levels for human risk assessment could be derived as specifically required under the REACH regulation. For this reason, DNELs for reproductive toxicity cannot be established with adequate reliability. Moreover, the above mentioned studies have a primary focus on effects in males, whereas there are no adequate data which would allow a hazard or risk assessment for effects on female reproduction. Existing studies show a hazard for male reproductive organs at high doses of cobalt dichloride. These findings have led to an existing harmonised classification as Cat 1B (H360F) for the substances cobalt dichloride, cobalt sulfate, cobalt diacetate, cobalt carbonate and cobalt dinitrate as well as to a self-classification of all remaining members of the bioavailable cobalt substances group.

 

No data was identified for the inorganic poorly soluble substance group. The screening studies (according OECD 422) with tricobalt tetraoxide and cobalt sulfide and the additional investigations on fertility impairment in a 90-day RDT oral toxicity study (according to OECD 408) did not show any effect on male or female fertility impairment. The proposed testing for an extended one-generation reproductive toxicity study with cobalt sulfide represents a major element in a weight of evidence approach needed to address the endpoint reproductive toxicity for the inorganic poorly soluble substance group.

 

Due to an absence of adverse effects for the inorganic poorly soluble cobalt substances group in toxicity studies on fertility impairment, no DNEL for reproductive toxicity will be derived.

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-17 to 2014-08-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001-01-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Limit test:

no

Species:

rat

Strain:

other: CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at day 0 of gestation: 67 days
- Weight at day 0 of gestation: 189.1 - 271.6 g
- Housing (except during the mating period): dams 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 released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages.
- Diet (ad libitum): commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 12 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:

other: 0.5% hydroxypropyl methylcellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared every day.
The test item was suspended in the vehicle to the appropriate concentration and was administered orally at a constant volume. The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.

VEHICLE - 0.5% hydroxypropyl methylcellulose gel
- Source: Fagron, Barsbüttel, Germany
- Batch no.: 13D03-N03

Administration volume: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item formulations, samples of 10 mL were taken at the following times and stored at -20°C or colder until analysis:
1) At study initiation
- analysis of stability and concentration: immediately after preparation of the formulation as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples/dose level group; 100, 300, and 1000 mg/kg bw/day dose groups).
- homogeneity: at start of administration, during (middle) administration and before administration to the last animal of the dose group (3 samples / dose level group; 100, 300, and 1000 mg/kg bw/day dose groups).

2) At study termination (at a time when the majority of animals was dosed)
- analysis of concentration: during treatment with the test item always before administration to the last animal of the group (1 sample/dose level group; 100, 300, and 1000 mg/kg bw/day dose groups).

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

Results:
- concentration: 88.0% - 99.7%
- stability: 89.3% - 96.1%
- homogeneity: 89.6% - 97.2%
These results indicated correctly prepared and homogenised test item vehicle mixtures, which were stable for at least 24 hours.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male/1 female animal (sexually mature male rats of the same breed served as partners. The female breeding partners were randomly chosen.)
- Proof of pregnancy: each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with
the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy).

Duration of treatment / exposure:

Day 6 to 19 of gestation

Frequency of treatment:

once daily

Duration of test:

20 gestation days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 female rats (20 dams with litters were evaluated)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected based on available toxicological data.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily; immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day as well as on the weekend.
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 of gestation, followed by daily weighings
The body weight gain was calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20).
Furthermore the net weight change from day 6 is given.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet 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

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
A dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.
The following target organs or parts thereof of all laparotomised female animals (including non-pregnant females, females with a total resorption of all implants and prematurely deceased animals) were fixed in 7% buffered formalin: adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), eye with optic nerve (2), gross lesions observed, heart (3 levels: right and left ventricle, septum), large intestine (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer's patches; Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary (2) (and oviducts), pancreas, pituitary, salivary glands (mandibular, parotid, sublingual), skin (left flank), spinal cord (3 sections), spleen, stomach, thymus, thyroid (2) (incl. parathyroids), tissues masses or tumours (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix), and vagina.
Paired organs were marked as left or right.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: gestation day 20
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: No
- How many animals: all dams (rats with litter and without litter)
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, relative differential blood count, absolute differential blood count, reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, the ovaries and the uteri of the dams were removed and the uteri (in toto) were weighed. Uteri without foetuses were examinated for possible implantation sites according to SALEWSKI to confirm their pregnancy status.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
- The number of foetuses (alive and dead) and placentae (location in uterus and assignment to the foetus) was determined.
- Location of foetuses in the uterus were determined.
- Weights of the placentae were determined.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No
- Sex and weights of foetuses were determined (foetuses were considered as runts if their weight was less than 70% of the mean litter weight).
- Viability of foetuses were determined.

Statistics:

Statistical analyses of the parametrical values were done by Provantis using the following settings:
Analysis of normal distribution and homogeneity of variances was perfromed by using the SHAPIRO-WILKS test and the BARTLETT test. Data not normally distributed or with heterogenous variances between the groups were stepwise log- or rank-transformed.
One-way analysis of variance (ANOVA) was performed with non-transformed or log-transformed data. The KRUSKAL-WALLIS test was used for rank-transformed data.
In case of significant differences (found by ANOVA or KRUSKAL-WALLIS test), intergroup comparisons with the control group were made by parametric or nonparametric DUNNETT multiple comparison tests (p ≤ 0.05 and p ≤ 0.01).
Parametrical values not captured by Provantis (e.g. number and weight of the fetuses) were analysed by the DUNNETT test (p ≤ 0.05 and p ≤ 0.01). Prior to the DUNNETT test homogeneity of variances was tested using the BARTLETT test. In case of heterogeneity of variances, the STUDENT's t-test was carried out (p ≤ 0.05 and p ≤ 0.01).
Statistical analyses of non-parametrical data (e.g. resorption-, malformation-, variation and retardation rates) were performed using the following settings:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi² test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01).

Indices:

Total malformation rate [%] = (malformed foetuses per group/foetuses per group) x 100
Total variation rate [%] = (foetuses per group with variations/foetuses per group) x 100
Total retardation rate [%] = (foetuses per group with retardations/foetuses per group) x 100
Pre-implantation loss [%] = (corpora lutea - implantations/corpora lutea) x 100
Post-implantation loss [%] = (Implantations - living foetuses/Implantations) x 100
Mean post-implantation loss per group [%] = sum of post-implantation loss [%] per litter/number of litters per group

Historical control data:

Background data summarising results of the last 55 embryotoxicity studies in Sprague-Dawley rats performed by the laboratory in the years 2000 to July 2014.
Data included were as follows: general reproductive indices, malformations, skeletal retardations, skeletal variations, visceral variations, and external variations.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Dead fetuses:

no effects observed

Details on maternal toxic effects:

- abortion: no abortion was observed.
- mortality: no test item-related premature death was noted in the control or in any of the test item treated groups (100, 300 or 1000 mg/kg bw/day).
One animal of the 1000 mg/kg bw/day dose group died on gestation day 15, twenty to 60 minutes after the administration of the test item. No pre-mortal signs of toxicity were noted. Necropsy revealed a thickened and black discoloured right lung lobe and a thorax filled with black liquid. Due to the findings at necropsy, a misgavage was considered as the cause of death. This animal was excluded from further evaluation.

- clinical observations: no changes in behaviour, the external appearance or the faeces were noted in the control group or in any of the test item treated groups (100, 300 or 1000 mg/kg bw/day).

- body weight and body weight gain: no test item-related changes in body weight in comparison to the control group were noted for the dams of all treatment groups (100, 300 or 1000 mg/kg bw/day. Body weight gain for the whole gestation period revealed no test item-related differences (control group: 59.7%, 100 mg/kg bw dose group: 61.8%, 300 mg/kg bw dose group: 59.1%; 1000 mg/kg bw dose group: 56.1%). Furthermore, the 3-day intervals of body weight change also revealed no test item-related changes, though a statistically not significant reduction in body weight gain between gestation days 6 and 9 by 32.6% was noted for the dams of the 1000 mg/kg bw dose group. This corresponds to a marginal difference of 4.2 g between the dams of the 1000 mg/kg bw dose group and the dams of the control group. This slight alteration is without biological relevance and not considered as an adverse effect of the test item. Lastly, no influence of the gravid uterus weight on the whole body weight was noted.

- food consumption: no test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 100, 300 or 1000 mg/kg bw/day. Differences in food consumption with significant differences in comparison to the control group which were considered as not test item-related were as follows: females of the 1000 mg/kg bw/day dose group showed a decreased in food consumption on gestation days 19 -20 (p≤0.05). The slight alteration in comparison to control animals is without biological relevance.

- water consumption: no test item-related changes in the consumption of drinking water was noted for the dams treated with 100, 300 or 1000 mg/kg bw/day by visual appraisal.

- macroscopic examinations: macroscopic inspection of the internal organs of the dams revealed no changes in the control and the test item-treated groups (100, 300 or 1000 mg/kg bw/day). The following sporadic observations are considered as spontaneous:
100 mg/kg bw/day: a few dark red foci (diameter up to approx. 0.5 mm) were noted in the lungs of one dam
1000 mg/kg bw/day: the observations made for the prematurely deceased animal

- Uterus weight and net body weight change: no test item-related changes in the gravid uterus weight and the carcass weight in comparison to the control group were noted for the dams of all treatment groups (100, 300 or 1000 mg/kg bw/day). Furthermore, no test item-related changes in the absolute and the net (without uterus) body weight change in comparison to the control group were noted for the dams of all treatment groups (100, 300 or 1000 mg/kg bw/day) between gestation day 6 and 20. A statistically significantly (p≤0.05) decreased net weight change from day 6 until laparotomy by 27.2% was noted for the dams of the 1000 mg/kg bw/day dose group. This corresponds to a slight difference of 8.7 g between the dams of the 1000 mg/kg bw/day dose group and the dams of the control group (32.0 g for the dams of the control group in comparison to 23.3 g for the dams of the 1000 mg/kg bw/day dose group). This slight alteration is without biological relevance and not considered as an adverse effect of the test item.

- reproduction data of the dams: no test item-related differences for pre-implantation loss, post-implantation loss and resorptions/implantation site ratios were noted between the dams of the control group and the dams treated with 100, 300 or 1000 mg/kg bw/day. The following 2 statistically significant changes are not test item-related: in the 100 mg/kg bw/day dose group the ratio of corpora lutea to implantation sites (preimplantation loss) was statistically significantly reduced. However, a reduced preimplantation loss in comparison to the control group is not an adverse effect, furthermore, implantation had occurred before the start of treatment. Additionally, in the 100 mg/kg bw/day dose group the ratio of late resorptions to implantation sites was statistically significantly (p≤0.05) increased. In detail, 6 late resorptions were noted in 6 different dams in comparison to one late resorption in the control group. As no dose-response relationship was noted (only one late resorption each was found in the dams of the 300 mg/kg bw/day and the 1000 mg/kg bw/day dose group), this observation is considered as spontaneous and not test item-related.

- haematology: no test-item related influence was noted for the dams treated with 100, 300 or 1000 mg/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- mortality: no dead foetuses were noted in the litters of the dams of the control group and in the litters of the dams treated with 100, 300 or 1000 mg/kg bw/day.

- sex distribution of foetuses: no test item-related influence on the ratio of live male foetuses to live female foetuses were noted for all treatment groups (100, 300 or 1000 mg/kg bw/day). All values of the treatment groups were within the range of laboratory background data. However, an increased male to female ratio was noted in the control group, leading to statistically significantly (p≤0.05) reduced male to female ratios in all treatment groups in comparison to the control group. As the male to female ratio of the control group was above the background range, this observation is considered as spontaneous and the statistically significant decreases in the male to female ratios of the treatment groups are not test item-related.

- weight of placentae: no test item-related differences of the placental weights were noted between the control group and the treatment groups (100, 300 or 1000 mg/kg bw/day).

- weight of foetuses: no test item-related differences of the foetal weights were noted between the control group and the treatment groups (100, 300 or 1000 mg/kg bw/day). Only one runt was noted in the 1000 mg/kg bw/day dose group. No runts were noted in the control, the 100 and 300 mg/kg bw/day dose groups.

- external examination (malformations): no test item-related macroscopically visible malformations were noted for the foetuses of the treatment groups (100, 300 or 1000 mg/kg bw/day) during the external examination of the foetuses at laparotomy.
The following sporadic observations are considered as spontaneous:
control group: one foetus with spina bifida.
300 mg/kg bw/day: one foetus with a stumpy tail (brachycaudia) and one foetus with multiple malformations (hydrocephalus, an abnormal shortness of the upper jaw (micrognathia), oedematous soft tissue in the abdomen).
The findings noted in the 300 mg/kg bw/day dose group are not considered to be related to the treatment with tricobalt tetraoxide as these findings are known to occur spontaneously in this rat strain. Further, the incidences are within the range of laboratory background data.

- external examination (variations): no macroscopically visible variations were noted for the foetuses of the control group and the treatment groups (100, 300 or 1000 mg/kg bw/day) during the external examination of the foetuses at laparotomy.

- internal examination: no malformations or variations were noted during the examination for the foetuses of the control group and the treatment groups (100, 300 or 1000 mg/kg bw/day).

- skeletal examination (malformations): no malformations were noted during skeletal examinations of the foetuses according to DAWSON in the control group and in any of the treatment groups (100, 300 or 1000 mg/kg bw/day).

- skeletal examination (variations): the skeletal variations observed were related to the ribs (wavy ribs) the sternum (sternebra(e) bipartite, misaligned (severity: slight)) and the os pubis (bipartite).
No test item-related differences for the incidences of the observed variations were noted between the control group and the test item treated groups (100, 300 or 1000 mg/kg bw/day).

- skeletal examination (retardations): the observed skeletal retardations were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite, dumbbell-shaped or reduced in size), the lumbar vertebral bodies (dumbbell-shaped), the sacral vertebral bodies (unossified), the caudal vertebral bodies (only one body ossified or all bodies unossified), the os ischii (incompletely ossified or unossified), the os pubis (incompletely ossified or unossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5).
No test item related differences in incidences of the observed skeletal retardations were noted between the foetuses of the control group and the foetuses treated with 100, 300 or 1000 mg/kg bw/day.
All observed incidences with statistically significant changes were within the range of laboratory background data and are not considered as test item-related.
Statistically significant changes in the foetal incidences of skeletal retardations, which are not considered to be test item-related:
absence of ossification in metacarpalia 2 to 5 (100, 300 or 1000 mg/kg bw/day; p ≤ 0.05/p ≤ 0.01)
caudal vertebral bodies (all bodies unossified)(300 mg/kg bw/day; p ≤ 0.01)
os ischii unossified (300 mg/kg bw/day; p ≤ 0.05)
sacral vertebral body/bodies unossified (300 mg/kg bw/day; p ≤ 0.05)
sternebra(e) reduced in size (1000 mg/kg bw/day; p ≤ 0.05)
sternebra(e) unossified (300 or 1000 mg/kg bw/day; p ≤ 0.01)
thoracic vertebral body/bodies dumbbell-shaped (300 mg/kg bw/day; p ≤ 0.05)
total foetal skeletal retardations

- soft tissue examination (malformations): no test item-related malformations were noted during soft tissue examinations of the foetuses according to WILSON in any of the treatment groups (100, 300 or 1000 mg/kg bw/day).
The following sporadic observations are considered as spontaneous:
300 mg/kg bw/day: one foetus with a hydrocephalus (confirming the afore-mentioned external malformation).
1000 mg/kg bw/day: one foetus with an abnormal smallness of the eyes (microphthalmia).

- soft tissue examination (variations): soft tissue variations were noted in the 3rd or 4th cerebral ventricle (dilatation), the kidneys (uni- or bilateral dilatation of the renal pelvis) and the liver (haemorrhagic focus/foci).
No test item-related differences for the incidences of the observed soft tissue variations were noted between the control group and the test item treated groups (100, 300 or 1000 mg/kg bw/day).

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

NOAEL (maternal toxicity) > 1000 mg tricobalt tetraoxide/kg bw/day
NOAEL (developmental toxicity) > 1000 mg tricobalt tetraoxide/kg bw/day
Looking at the results of the dams, no test item-related premature deaths, signs of clinical toxicity or findings at necropsy were noted (including uterus and carcass weights). Furthermore, body weight, food consumption, drinking water consumption and the haematological parameters were not influenced by the test item.
Also, the results of the foetuses showed no test item-related changes for the number of resorptions and the foetal body weight as well as no dead fetuses, no test item-related malformations, variations or retardations at any of the tested dose levels.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity

 

Tricobalt tetraoxide and cobalt sulfide are the source substances for the inorganic poorly soluble cobalt substances group based on the read-across approach as outlined in Appendix 1.1 in the CSR. Two inorganic poorly soluble cobalt substances, tricobalt tetraoxide and cobalt sulfide, have been tested for developmental toxicity, resulting in an absence of effects on the developing organism up to the limit dose of 1000mg/kg bw/day.

 

In a pre-natal developmental toxicity study (according to OECD 414 and under GLP), tricobalt tetraoxide was given to pregnant rats at doses of 0, 100, 300, 1000 mg/kg bw/day. A total of 25 females per group were given the test items suspended in 0.5% hydroxypropyl methylcellulose gel orally via gavage once daily from gestation day 6 until gestation day 19.

 

No test item-related premature death was noted in the control or in any of the test item treated groups. No changes in behaviour, the external appearance or the faeces were noted in the control group or in any of the test item treated groups. No test item-related changes in body weight in comparison to the control group were noted for the dams of all treatment groups. Body weight gain for the whole gestation period revealed no test item-related differences (control group: 59.7%, 100 mg/kg bw dose group: 61.8%, 300 mg/kg bw dose group: 59.1%; 1000 mg/kg bw dose group: 56.1%). Furthermore, the 3-day intervals of body weight change also revealed no test item-related changes.

 

Macroscopic inspection of the internal organs of the dams revealed no changes in the control and the test item-treated groups. No test item-related changes in the gravid uterus weight and the carcass weight in comparison to the control group were noted for the dams of all treatment groups. Furthermore, no test item-related changes in the absolute and the net (without uterus) body weight change in comparison to the control group were noted for the dams of all treatment groups (100, 300 or 1000 mg/kg bw/day) between gestation day 6 and 20.

The reproduction data of the dams revealed no test item-related differences for pre-implantation loss, post-implantation loss and resorptions/implantation site ratios between the dams of the control group and the dams treated with 100, 300 or 1000 mg/kg bw/day.

 

No dead foetuses were noted in the litters of the dams of the control group and in the litters of the dams treated with 100, 300 or 1000 mg/kg bw/day. No test item-related influence on the ratio of live male foetuses to live female foetuses were noted for all treatment groups. All values of the treatment groups were within the range of laboratory background data.

No test item-related differences of the placental or foetal weights were noted between the control group and the treatment groups.

No test item-related macroscopically visible malformations or variations were noted for the foetuses of the treatment groups during the external examination of the foetuses at laparotomy. The internal examination revealed no malformation or variations in the control or any of the treatment groups. Skeletal examination showed no test-item related malformations, variations or retardations in the control or any of the treatment groups. No test item-related malformations were noted during soft tissue examinations of the foetuses according to WILSON in any of the treatment groups. No test item-related differences for the incidences of the observed soft tissue variations were noted between the control group and the test item treated groups.

 

Under the test conditions of the pre-natal developmental toxicity study with tricobalt tetraoxide, the no-observed-adverse-effect level (NOAEL) was above 1000 mg Tricobalt tetraoxide/kg bw/day for the dams. No test item-related premature deaths, signs of clinical toxicity or findings at necropsy were noted. Body weight, food consumption and the haematological parameters were not influenced by the test item.

 

The no-observed-adverse effect level (NOAEL) for the foetal organism was above 1,000 mg Tricobalt tetraoxide/kg bw/day. No test item-related changes were noted for the number of resorptions and the foetal body weight. No dead foetuses, no test item-related malformations, variations or retardations were noted at any of the tested dose levels.

 

In acombined repeated dose toxicity studies with the reproduction/developmental toxicity screening test (according to OECD 422 and under GLP), cobalt sulfide was administered orally to rats at dose levels of 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. None of the parental animals died prematurely. The only treatment-related finding, not regarded as adverse, was piloerection noted in few male or female rats from a dose level of 100 mg Cobalt sulfide/kg b.w./day onwards. Macroscopic inspection did not reveal any test item-related changes at necropsy. Histopathological inspection did not reveal any pathological changes. No test item-related influence was noted on the sperm staging or interstitial cell structure (qualitative examination). No test item-related influence was noted on mating behaviour, fertility, implantation, the gestation length or the birth index. No test item-related influence was noted at 100, 300 or 1000 mg Cobalt sulfide/ kg b.w./day on the growth and development of the offspring from conception until sacrifice on day 4 post-partum or shortly thereafter.

 

The NO(A)EL for effects on the F0-generation and reproductive toxicity was above 1000 mg/kg b.w./day, based on a complete absence of adverse effects.

 

 

Conclusion

 

Due to an absence of adverse effects for developmental toxicity for the inorganic poorly soluble inorganic cobalt substances group in pre-natal developmental toxicity studies and reproductive toxicity screening tests, no DNEL for reproductive toxicity will be derived.

 

Since the two source substances of the inorganic poorly soluble cobalt substance group do not exert any developmental toxicity when administered via oral route up to the limit dose of 1000 mg/kg bw/day, further experimental testing for this endpoint is considered dispensable. A detailed justification for non-submission is presented in the IUCLID section 7.8.2 and in the CSR chapter 5.9.2 for the inorganic poorly soluble cobalt substance group members. In brief, the standard testing regime of Annexes VII-X may be adopted in case testing does not appear scientifically necessary. Specifically, a weight of evidence evaluation can be performed to demonstrate that evidence from several independent sources of information leading to the assumption/conclusion that a substance has or has not a particular dangerous property, while the information from each single source alone is regarded insufficient to support this notion. The registrant is of the opinion that sufficient weight of evidence is available to demonstrate an absence of pre-natal developmental toxicity in a second (non-rodent) species for the inorganic poorly soluble cobalt substance group and that testing on vertebrate animals shall be omitted.

 

In general, more and more scientific evidence is emerging that suggests that non-rodent species such as rabbits are not a priori more sensitive that rats with respect to developmental toxicity. However, particularly in the case of metals, the absence of metabolic conversion and similarities in toxicokinetic profiles render testing in a second, non-rodent species as questionable.

 

In the case of inorganic poorly soluble cobalt substances, the weight of evidence information demonstrates that tricobalt tetraoxide and cobalt sulfide are extremely poorly soluble in water, shows very low in vitro bioaccessibility over a whole range of surrogate biological media, is poorly bioavailable in vivo in several mammalian species (tricobalt tetraoxide) and is of low toxicological activity in

(i) a repeated dose toxicity study with reproductive toxicity screening with no adverse effects on reproductive organs or the developing foetus (cobalt sulfide) and,

(i) a sub-chronic repeated dose toxicity study via oral administration with no adverse effects on reproductive organs, hormone levels or the female cycle (tricobalt tetraoxide) and,

(ii) a pre-natal developmental toxicity study in rats, showing no adverse effects against the dams of the developing foetus (tricobalt tetraoxide).

 

Most importantly, highly reliable toxicokinetic investigations in five different species (baboons, guinea-pigs, rats, mice and hamsters) demonstrated no relevant species-differences in absorption and excretion of orally administered tricobalt tetraoxide. The low order of reproduction toxicity is manifested in an absence of concern in a developmental toxicity study in the rat and an absence of any adverse effects on organs of reproduction in any repeated dose toxicity studies reported to date. Detailed toxicokinetic investigations did not reveal any significant absorption and excretion differences between five species. Tricobalt tetraoxide showed very little gastrointestinal absorption.

 

Overall, the conduct of a developmental toxicity in a second species with a source substance of the inorganic poorly soluble cobalt substances read-across group cannot be expected to contribute any relevant information to the assessment of (otherwise negative) information on reproduction toxicity.

 

Justification for classification or non-classification

No proposal on classification and labelling for reproductive toxicity-fertility impairment can be made, due to lack of data.

The existing data for the inorganic poorly soluble cobalt substance group did not show any effect on male or female fertility impairment. The proposed testing for an extended one-generation reproductive toxicity study with the source substance cobalt sulfide represents a major element in a weight of evidence approach needed to address the endpoint reproductive toxicity for the inorganic poorly soluble substance group.

 

Due to an absence of adverse effects for developmental toxicity for the inorganic poorly soluble inorganic cobalt substances group in pre-natal developmental toxicity studies and reproductive toxicity screening tests, the classification criteria as developmental toxicant are not met, hence no classification is proposed.