Registration Dossier

Administrative data

Description of key information

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:
2014-07-03 to 2014-11-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 1998-09-21
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
Limit test:
no
Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males and females: 52 days
- Weight at first dosing: males: 269.0 - 321.5 g; females: 183.3 - 227.8 g
- Housing: animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm; Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material.
- Diet (ad libitum): commercial diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 0.5% hydroxypropyl methylcellulose gel
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concnetrations. The administration formulations were freshly prepared daily.
Administration volume: 5 mL/kg bw/day

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

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

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

Results:
The results of the analysis show that the test item formulations were correctly prepared. The actual tricobalt tetraoxide concentrations in the respective test item formulations ranged from 95.6 % to 103.0 % of the nominal concentrations before administration to the last male animal of the group on test day 1, and from 87.5 % to 101.7 % of the nominal concentrations before administration to the last female animal of the group on test day 90 (last administration day of the study).
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
100, 300, and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Main study (per group): 10 males/10 females
Recovery group (control group and 1000 mg/kg bw/day dose group only; per group): 5 males/5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels for this study were selected taking into account any existing toxicity and toxicokinetic data available for the test item at the time of initiation of the study.
- Recovery groups were included in this study. One recovery group was included for the control group and other recovery group for the 1000 mg/kg bw/day dose group. These groups were kept for 28 days after the treatment period without receiving the test item.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (main study animals and recovery animals)
- Time schedule:
Clinical signs: before and after dosing at each time of dosing as well as regular daily
Mortality: twice daily
- Cage side observations (included): skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

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

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

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

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

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

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

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

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

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

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

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

2) Functional tests: grip strength and locomotor activity

NOTE: Besides the above described parameters the following parameters were also investigated in this study: hormone levels and stages if the oestrous cycle. Please refer for the results of these parameters to Section 7.8.1 Toxicity to reproduction: k_Hansen_2015_CoCl2
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (main study animals and recovery animals)
On test day 91, all main study animals were dissected following a randomisation scheme. Necropsy of all animals allocated to the recovery period was performed on test day 119.
The animals were euthanized by carbon dioxide, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

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

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

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

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

BONE MARROW (main study animals and recovery animals)
Fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of all main study and recovery animals stained according to
PAPPENHEIM. The myeloid : erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the control group and the 1000 mg/kg bw/day group.
Statistics:
Means and standard deviations were calculated on time-point specific data sets for each sex separately. Data taken from Provantis® (body weight, body weight gain, food consumption, haematology, clinical chemistry, and urinalysis) were analysed by ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons were made with the control group (reference item) by the DUNNETT test. Before preparing the ANOVA, the data were analyzed for homogeneity of variances (BARTLETT test) and normal distribution (SHAPIRO-WILKS test).
The test item-treated groups (100, 300, and 1000 mg/kg bw/day dose groups) were compared with the control group.

The following statistical methods were used:
- multiple t-test based on DUNNETT, C. W. New tables for multiple Comparisons with a control. Biometrics, 482-491 (Sept 1964): body weight, food consumption, haematology, clinical chemistry, urinalysis, absolute and relative organ weights (p ≤ 0.05 and p ≤ 0.01)
- exact test of R. A. FISHER: histology (p ≤ 0.05)
- STUDENT's t-test: all numerical functional tests: body temperature, hind leg splay, grip strength, spontaneous motility (p ≤ 0.05 and p ≤ 0.01)
- Chi² test: bone marrow (p ≤ 0.01)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1) Treatment period:
- 100, 300 or 1000 mg/kg bw/day: none of the male and female rats treated orally revealed any test item-related changes in behaviour or external appearance.
- 1000 mg/kg bw/day: two female animals revealed an exophthalmus from test day 79 onwards. This is considered to be a coincidental finding that is not test item-related.
- the faeces of all test item-treated male and female animals were dark stained from test day 48 onwards for the male animals and from test day 41 onwards for the female animals. The intensity of the staining increased with the dose level. The dark staining of the faeces was not recorded before test day 41/48 as only from these time points onwards a clear difference in the colour of the faeces compared to the control animals was evident for all test item-treated animals. The dark staining of the faeces is considered to be related to the black-grey colour of the test item and not to be a toxicological effect. The consistency of all animals' faeces was normal throughout the treatment period.
- no deaths were noted at any dose level.
- all main study animals survived until their scheduled terminal sacrifice.
- detailed clinical observations: none of the animals treated with 100, 300 or 1000 mg/kg bw/day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour as assessed in test weeks 1 to 13 (all groups).
- detailed clinical observations: all parameters of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination in test week -1. One male animal treated with 1000 mg/kg b.w./day revealed a haemorrhagic nose in test week 12. Furthermore, one female animal treated with 1000 mg/kg bw/day revealed a movable solid palpable mass with a diameter of approximately 8 to 20 mm near the second mammary gland from test week 9 onwards. Lastly, another female 1000 mg/kg bw/day dose animal revealed areas with thin fur (size approximately 10 mm × 30 mm) on both forelimbs in test weeks 10 to 13. All findings noted are considered as spontaneous changes that are not related to the test item due to the single occurrence in each case.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

BONE MARROW EXAMINATION
Treatment and recovery period (recovery restricted to the control group and 1000 mg/kg bw/day group):
- 1000 mg/kg bw/day: myeloid : erythroid ratio of the male and female animals was not influenced in comparison to the control group at the end of the treatment period and at the end of the recovery period.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
NOAEL (rat, 90 days): 300 mg tricobalt tetraoxide/kg bw/day

Looking at the treatment period the following test item-related changes were noted:
The body weight of the male animals treated with 1000 mg/kg bw/day was slightly reduced by up to 10% compared to the control group as of test day 22. Accordingly, the body weight gain was reduced by up to 17 percentage points in comparison to the control. The body weight at autopsy of the males of 1000 mg/kg bw/day dose group was reduced by 12% on test day 91. Furthermore, the body weight of the female animals treated with 1000 mg/kg bw/day was only marginally reduced by up to 5% compared to the control group as of test day 64. The body weight gain and the body weight at autopsy were similarly only slightly reduced compared to the control group. Overall, the reduced body weights at the 1000 mg/kg bw/day dose are considered as test item-related.

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

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

Overall, no further test-item related changes were observed.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
Study duration:
subchronic
Species:
rat
System:
haematopoietic

Repeated dose toxicity: inhalation - systemic effects

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

inorganic poorly soluble

poorly soluble in aqueous solutions with organic ligand

inhalation-local

reactive

non-reactive

 

 

Further details on the read-across approach are given in Appendix 1.1 of the CSR for the oral systemic effects and Appendix 1.2 of the CSR for the inhalation local effects.

 

Tricobalt tetraoxide is assigned to the read-across groups (i) oral-systemic: inorganic poorly soluble and (ii) inhalation-local: non-reactive

Toxicological relevance of the non-common compound

The toxicological relevance of the non-common compound in tricobalt tetraoxide for oral-systemic toxicity is discussed in Appendix 1.1 of the CSR.

 

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

 

Based on the read-across approach presented in Appendix 1.1 of the CSR the substances of the inorganic poorly soluble cobalt substances are grouped and assessed for their hazardous properties using information from the source substance tricobalt tetraoxide and supporting evidence form cobalt sulfide.The source substances tricobalt tetraoxide and cobalt sulfide, have been tested for repeated dose toxicity, resulting in comparable effect levels (28-day NOAEL Co3O4: 714mg Co/kg bw/day; 90-day NOAEL Co3O4: 214mg Co/kg bw/day; 28-day NOAEL CoS: 647mg Co/kg bw/day). The NOAEL of the 28-day studies are based on a complete absence of adverse effects up to the limit dose of 1000 mg/kg bw/day. The NOAEL of the 90-day study is based on increased red blood cell parameters in the peripheral blood. No histopathological correlate was found in and organ related to haematopoiesis. Further details on the findings are presented below.

 

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. 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 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.

 

No test item-related influence was observed on any of the haematological parameters for the treatment with 100 mg tricobalt tetraoxide/kg bw/day at the end of the treatment period. At 300 mg tricobalt tetraoxide/kg bw/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 haemoglobin concentration (HGB), number of erythrocytes (RBC), and haematocrit value (HCT), the number of platelets (PLT) was slightly reduced. The changes were still within the historical range of that rat strain. The identical findings were observed in the animals of the 1000 mg tricobalt tetraoxide/kg bw/day group, now exceeding the historical control range. At the intermediate and high dose, the male animals showed a higher susceptibility to the haematological changes. No test item-related effects were observed for the number of leucocytes (WBC), the relative reticulocyte count (Reti), the relative and absolute differential blood count, the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC) at the end of the treatment period (test day 91). All changes in haematological parameters previously observed after repeated treatment with 1000 mg tricobalt tetraoxide/kg bw/day had subsided after 4 weeks of recovery (test day 119, day 28 of the 4-week recovery period), indicating a complete recovery. 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. Under the test conditions of this 90-day repeated dose toxicity study with tricobalt tetraoxide, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic effects was 300 mg tricobalt tetraoxide/kg bw/day by oral administration based on increased levels of red blood cells number, decreased platelet number at the high dose group. 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 a repeated dose toxicity study with reprotox screening (according to OECD 422 and under GLP) cobalt sulphide 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). The NO(A)EL for systemic effects was above 1000 mg/kg b.w./day, based on a complete absence of adverse effects.

 

The NOAEL for systemic effects derived from the sub-chronic study with tricobalt tetraoxide is used as point of departure for the derivation of DNEL (oral, systemic effects) for the general population. Based on the DNEL for tricobalt tetraoxide, all further DNELs for the substances in the inorganic poorly 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.

 

 

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 31.9 μg Co/cm² loading) and 1.08% for the high exposure scenarios (ca 319 μg 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 substance of the inorganic poorly soluble cobalt substances group, show an almost complete absence of adverse effects in guideline compliant repeated dose toxicity studies via the oral route. The sole adversity is represented by 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) at the limit dose of 1000 mg/kg bw/day. Neither macroscopic, nor microscopic findings were noted. These findings were seen in a sub-chronic oral study in rats with the source substance tricobalt tetraoxide. There was an absence of adverse effects in two sub-acute oral study in rats with cobalt sulphide and tricobalt tetraoxide.

For risk assessment purposes, a read-across is applied by using the cobalt equivalent NOAEL derived from the sub-chronic oral study with tricobalt tetraoxide (i.e. 300 mg Co3O4/kg bw/day or 214 mg cobalt/kg bw/day) for the calculation of the substance-specific DNEL (oral, systemic, long-term) for all members of the inorganic poorly soluble 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

 

Justification for classification or non-classification