Resin acids and Rosin acids, cobalt salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2006-07-20 to 2006-08-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1996-09-30

Deviations:

no

Principles of method if other than guideline:

The study was also based on the following guideline for neurotoxicological investigations: EU Method B.43 (2004).

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2006-02-24

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, cobalt salts

EC Number:

273-321-9

EC Name:

Resin acids and Rosin acids, cobalt salts

Cas Number:

68956-82-1

IUPAC Name:

λ²-cobalt(2+) bis((1R,4aR,4bR,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxylate)

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Produit Y
- State of aggregation: purple powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and humidity, and under argon gas.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen because it is a rodent species commonly accepted by regulatory authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at the laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Rj Han:SD
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at start of treatment: approx. 6 weeks old
- Weight at start of treatment (mean body weight): males: 228 g (range: 209 g to 240 g); females: 181 g (range: 169 g to 192 g)
- Fasting period before study: food, but not water was removed.
- Housing: individually housed in suspended wire-mesh cages (43.0 X 21.5 X 18.0 cm). A metal tray, containing autoclaved sawdust, was placed under each cage.
- Diet (ad libitum, except when fasted): SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany; distributed weekly)
- Water (ad libitum): tap water (filtered with a 0.22 μm filter)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
- batches of diet were analyzed by the suppliers for composition and contaminant levels.
- bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
- no contaminants were present in the diet and drinking water at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Relative humidity: 50 ± 20 %
- Ventilation: approx. 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since it is a route of exposure which is requested by the authorities for this type of product.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- test item was administered as a solution in the vehicle. The mortar was pre-cooled and placed on melting ice, then a small quantity of vehicle was put into the mortar. The weighed powder was added to the mortar and the vehicle was then added drop by drop and gently mixed to obtain a homogeneous emulsion. The remaining quantity of vehicle was added in order to achieve the concentration of 3, 10 or 30 mg/mL.
- test item dosage forms were prepared daily.
- quanity of dosage form administered to each animal was adjusted according to the most recently recorded body weight.
- dosage volume: 5 mL/kg/day
- dosage forms were stirred continuously throughout the dosing procedure

VEHICLE
- Supplier: Sigma (Saint-Quentin-Fallavier, France)
- Batch No.: 015R0115

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dosage forms: concentration
The concentration of test item was determined in samples of each control and test item dosage form prepared for use in weeks 1 and 4. Analyses were carried out using ICP-OES.
Results of the chemical analysis of the dosage forms: concentration
A satisfactory agreement was observed between the nominal and actual concentrations of the test item in the administered dosage forms since the deviations from nominal concentration remained in an acceptable range of ± 10% in weeks 1 and 4.

Duration of treatment / exposure:

29 days (according to the necropsy schedule)

Frequency of treatment:

once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5 males / 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected on the basis of the results of a 7-day range-finding toxicity study by the oral route performed in Sprague-Dawley rats (CIT/Study No. 31592 TSR). Groups of three male and three female rats received the test item by gavage at the dose-level of 150, 450 or 1000 mg/kg/day for 7 days. A control group receiving the vehicle alone (corn oil) was run concurrently. The following parameters were examined: mortality (daily), clinical signs (daily), body weight (treatment days 1, 4 and 7), food consumption (3- or 4-day periods), and full macroscopic examination (macroscopic lesions were preserved).

Results:
1000 mg/kg/day:
Mortality:
- all animals died on day 4 or 5.
- prior the death, 2/3 animals from each sex showed excessive salivation from day 2.
- male and the female found dead on day 5 lost respectively 23 g or 11 g between days 1 and 4, and had a food consumption 50% lower from controls.
- at necropsy, no test item treatment-related changes were noted.

Body weight:
- animals lost weight between days 1 and 4, before the death (males: -23 g, and females: -11 g).

Food consumption:
- before the deaths, animals ate circa 50% less than controls.

450 mg/kg/day:
Mortality:
- one male and one female were found dead on day 6 or 7, and another female was prematurely sacrificed due to poor clinical condition.
- before the death, marked clinical observations were noted, namely: piloerection (3/3), ptyalism (2/3), hunched posture (2/3), cold to the touch (1/3), slight dehydration (1/3), dyspnea (1/3), soft feces (1/3), thin appearance (1/3) and soiled urogenital region (1/3).
- before the death or premature sacrifice, a body weight loss or very low body weight gain was associated with a low food consumption.
- at necropsy, no factors which could have contributed to the death were observed.

Clinical signs:
- two surviving males showed marked clinical signs: soft feces, associated with hunched posture, and thin appearance and piloerection in one animal.

Body weight:
- body weight loss was noted in both sexes from days 1 to 4, and in females during interval days 4 to 7. During this last interval, males had a very low body weight gain. This resulted in no body weight gain in males and in an overall body weight loss in females (-15 g).

Food consumption:
- males had a food consumption approximately 50% lower than controls (p<0.05 days 1 to 4), while in females, it was -16% (days 1 to 4), and -42% (days 4 to 7) lower from controls.

Organ weights:
- a marked increase in relative liver weight were noted in both males and females, respectively, +56% and +37%- size reduction of prostate and seminal vesicles was observed in one male

150 mg/kg/day
Body weight:
- males gained circa 45% less weight than controls during the whole dosing period, while a similar body weight evolution was noted between control and test item-treated females.

Food consumption:
- males ate 21% to 26% less food than controls whereas similar food consumption was noted between test item-treated and control females.

The other organ weights differences between test-treated and control animals were considered of no toxicological importance. Consequently, under the experimental conditions of this study, 150 mg/kg/day could be selected as the high dose-level in the further 4-week study to be conducted in the same species.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked / Time schedule: mortality and signs of morbidity (at least twice a day during the treatment period, including weekends and public holidays) and clinical signs (once a day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study. One observation was done once at the end of the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the first day of treatment and then once a week until the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The quantity of food consumed by the animals in each cage was recorded once a week until the end of the study.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- Animals fasted: Yes, animals were deprived of food for an overnight period of at least 14 hours.
- How many animals: all animals
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leukocytes, differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes and monocytes), and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes; prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours.
- How many animals: all animals
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period.
- Dose groups that were examined: all animals
- Battery of functions tested: measurement of reactivity to manipulation or to different stimuli and motor activity.
The following parameter measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature (at the end of observations), and motor activity.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

On completion of the treatment period, after at least 14 hours of fasting, animals were sacrificed by carbon dioxide inhalation followed by exsanguination.
The body weight of each animal was recorded before sacrifice at the end of the treatment period.

The following organs were weighed wet as soon as possible after dissection:
adrenals, brain (including medulla/pons, cerebellar and cerebral cortex), epididimydes, heart, kidneys, liver, ovaries, spleen, testes, thymus, and thyroids with parathyroids. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

For all study animals, the following tissues were preserved:
macroscopic lesions, adrenals, aorta, brain (including medulla/pons, cerebellar and cerebral cortex), cecum, colon, duodenum, epididimydes, esophagus, eyes with Harderian glands, femoral bone with articulation, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area, optic nerves, ovaries, pancreas, pituitary gland, prostate, rectum, salivary glands (sublingual and submaxillary), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus (horns and cervix), and vagina.

All tissues required for microscopic examination were embedded in paraffin wax, sectioned at approximately four microns in thickness and stained with hematoxylin-eosin.
A microscopic examination was performed on:
- on all of the following perserved tissue for animals in the control and 150 mg/kg/day groups sacrificed at the end of the treatment period:
macroscoic lesions, adrenals, brain (including medulla/pons, cerebellar and cerebral cortex), cecum, colon, duodenum, epididimydes, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), ovaries, prostate, sciatic nerve, seminal vesicles, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus (horns and cervix), and vagina.
- all macroscopic lesions from the animals in the 15 and 50 mg/kg/day groups sacrificed on completion of the treatment period.

Based upon the microscopic results of the 150 mg/kg/day dose group and after agreement of the Sponsor, other tissues from the 15 and 50 mg/kg /day dose groups may be examined.
Following microscopic findings noted at 150 mg/kg/day in the spleen and in the lungs, these organs were microscopically examined for all animals from the 50 and 15 mg/kg/day dose groups.

Statistics:

For the statistical analyses of body weight, food consumption, hematology, blood biochemistry and organ weight data the following tests were used:
- Kolmogorov-Lilliefors test
- Bartlett test
- Fisher test
- Mann-Whitney / Wilcoxon test
- Dunn test
- Fisher test
- Student test
- Dunnett test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males and Females:
- 15, 50, and 150 mg/kg/day: ptyalism (excessive salivation) with an increasing frequency and duration (from 3 to 17 days) in both sexes (treatment related effect). Ptyalism was not present in the control group (except in 1/5 females for 3 days only) and was dose-related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
- 50 and 150 mg/kg/day: body weights of males treated at 50 and 150 mg/kg/day were significantly lower than in controls, respectively from day 22 or day 8 and until the end of the treatment period. These low body weights were consecutive to low (and statistically significant) body weight gains during the dosing period, resulting in low mean body weight gain over the whole treatment period in males at 50 and 150 mg/kg/day (respectively -15%, p<0.05 and -28%, p<0.01).

Please also refer to the field "Attached background material" below

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
- 50 and 150 mg/kg/day: mean erythrocyte and hemoglobin concentrations, and the hematocrit in males were higher at 50 and 150 mg/kg/day, (p<0.01 except for erythrocytes at 50 mg/kg/day) (test item treatment-related; probably due to hemoconcentration).

Females:
- 150 mg/kg/day: same tendency in mean erythrocyte and hemoglobin concentrations, and the hematocrit as in males was noted in the females, but reached a statistically significant level for hemoglobin only (p<0.05) (test item treatment-related; probably due to hemoconcentration).

Please also refer to the field "Attached background material" below.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
- 150 mg/kg/day: compared to control mean values, a statistically significant, low (-26%) plasma glucose level was noted in males (relationship with the test item-treatment could not be ruled out).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Spleen:
- 15, 50, and 150 mg/kg/day: increased extramedullary hematopoiesis (mainly erythropoiesis) was seen in the spleen of males and females given 50 or 150 mg/kg/day (test compound-related effect), but not in the animals treated at 15 mg/kg/day (at this dose-level, the difference in severity from controls was not significant).

Cecum and colon:
- 150 mg/kg/day: moderate epithelial degeneration/necrosis was noted in the cecum and colon of 1/5 males (relationship to treatment with the test item cannot be ruled out).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS
Males:
- 150 mg/kg/day: 1/5 male have shown chromodacryorrhea or had scabs (not treatment related). These findings were either noted with a similar incidence in controls (chromodacryorrhea) or are common in laboratory rats of this strain and age.

MORTALITY
Males and Females:
- no premature deaths occured during the study.

BODY WEIGHT AND WEIGHT GAIN
Males:
- 15 mg/kg/day: mean body weight and body weight change were similar to controls.
Females:
- 15, 50, and 150 mg/kg/day: mean body weight and body weight change were similar to controls.

FOOD CONSUMPTION AND COMPOUND INTAKE
Males:
- 150 mg/kg/day: a non statistically significant slightly lower (approx. 10%) food consumption compared to the controls (week 1 to 3) was osberved.
- 50 mg/kg/day: a slightly lower food consumptions in weeks 3 and 4 (respectively -10% and -8% compared to controls) was obsered.
- 15 mg/kg/day: food consumption was equivalent to control values.Females:15, 50, and 150 mg/kg/day: food consumption was equivalent to control values.

HAEMATOLOGY
Males:
- 50 and 150 mg/kg/day: a decrease in eosinophils was noted (values were within the physiological range and were due only to a relatively high control mean value; not to be toxicologically relevant).
Females:
- 150 mg/kg/day: decreased prothrombin time (-7%, p<0.05), although statistically significant, was due to a high control mean value (considered not to be of toxicological importance).
Males and females:
- all the other hematology findings were slight, not dose-related, or were the contribution of only a few individuals, and as such, were considered not to be of toxicological importance (i.e., increases in mean cell volume and mean cell hemoglobin at 50 mg/kg/day in males, decrease in mean cell hemoglobin concentration at 15 mg/kg/day in females).

CLINICAL CHEMISTRY
Males and females:
- a statistically significant, high mean chloride concentration was noted in the males at 50 and 150 mg/kg/day (+4% and +5% respectively). In females, the same tendency was noted, and mean values were statistically significant at the all dose-levels.
- in the absence of other electrolyte disorders, these modifications were considered to be of minimal toxicological importance and most probably related to the low number of animals per group.
- 50 and 150 mg/kg/day: compared to control mean values, statistically significant, low plasma cholesterol levels were noted in males treated at 50 and 150 mg/kg/day (p<0.05 and p<0.01, respectively; not to be toxicologically relevant).
- other statistically significant variations noted in the blood biochemistry were slight, not dose-related or were the contribution of only a few individuals, and were thus considered not to be toxicologically relevant (i.e., increase in alanine aminotransferase activity and plasma creatinine levels, respectively in males and in females treated at 150 mg/kg/day, decrease in plasma urea level in males and females treated at 50 mg/kg/day, decrease in calcium concentration in females treated at 15 and 50 mg/kg/day, and decrease in total proteins in females treated at 15 mg/kg/day).

NEUROBEHAVIOUR
Males:
- 150 mg/kg/day: slightly less motor activity (horizontal movements and rearings) was noted (considered to be without toxicological importance as all values were within the range of normal control values; probably related to the low body weights). Furthermore, abnormal fur appearance in one male correlated with the presence of scabs.
- within the other autonomic and physiological functions and at the 15 and 50 mg/kg/day dose-levels, only minor and limited findings were noted in the males (presence of grooming in 1/5 males in the control group, defecation and/or urination in 2/5 males at 15 mg/kg/day and in one male at 150 mg/kg/day, moderate decrease in forelimb grip strength in one male in the 15 and 150 mg/kg/day groups). All these findings were considered to be without toxicological significance.

Females:
- motor activity of females was unaffected by the test item-treatment.
- no perturbations of the autonomic or physiological functions in any treated females, except in fur appearance for one female given 150 mg/kg/day due to the presence of scabs, in the forelimb grip strength of one control female and one female at 15 mg/kg/day (without toxicological significance).

ORGAN WEIGHTS
- few organ weight changes that were statistically significant were slight, not dose-related, often related to decreased body weights and did not correlate with any histological lesions (considered to be of no toxicological importance).

GROSS PATHOLOGY
- no treatment-related gross findings were noted at the end of the treatment period.
Males:
- 15, 50, and 150 mg/kg/day: spleen enlargement seen in all groups, including controls, did not correlate with higher weight or histopathological changes (considered not to be of toxicological importance).

HISTOPATHOLOGY: NON-NEOPLASTIC
Lungs:
- pulmonary lesions were noted in both treated and control rats and consisted of multiple foci of minimal to moderate subacute alveolitis, widespread in the lungs, characterized by alveolar foamy/vacuolated histiocytes, admixed with a few lymphoid cells and neutrophils, obliterating the alveolar architecture. Perivascular lymphoid cell cuffing was also frequently noted. This lesion was found with a similar incidence and severity in controls and treated animals. Several foci of interstitial fibrosis were noted in these alveolitis foci in some treated rats, especially at 150 mg/kg/day. These alterations could be considered as spontaneous changes. Aspiration of the test formulation may have contributed to the lesion. The higher incidence and severity seen in males at 150 mg/kg/day may reflect an irritant effect of aspirated compound.
- all the other microscopic findings reported were those which commonly occur in rats of this strain and age kept under laboratory conditions, and were considered to be of no toxicological importance.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

haematology

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

element

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

haematology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Prior to this repeated dose toxicity study, a 7-day range-finding toxicity study using male and female Sprague-Dawley rats was performed. The rats were dosed by gavage with dose-levels of 150, 450 or 1000 mg/kg/day.

The following findings were made for males as follows:
1000 mg/kg/day: mortality
450 mg/kg/day: mortality, clinical signs, soft faeces
150 mg/kg/day: low body weight gain and low food consumption

The following findings were made for females as follows:
1000 mg/kg/day: mortality
450 mg/kg/day: mortality, clinical signs, soft faeces
150 mg/kg/day: no effects

No treatment-related effects were observed for mortality, food consumption, organ weights. gross pathology, and neurobehavioral examinations in males and females at any dose level.
Both sexes in the 15, 50 and 150 mg/kg bw/d groups showed ptyalism with an increased duration and frequency (from 3 to 17 days) (males: 2/5, 4/5, and 5/5, respectivley; females: 2/5, 4/5, and 5/5, respectivley). The control group (except 1/5 females for 3 days) showed no presence of ptyalism. The occurence of ptyalism was dose-related and, thus, considered to be a treatment-related effect. Furthermore, the body weights at 50 and 150 mg/kg/d males were significantly lower than controls (from day 22 or day 8 until the end of treatment period) and were consecutive to low body weight gains in that treatment group (-15%,p<0.05 and -28%,p<0.01). At the 50 and 150 mg/kg/day dose levels, in the males increased mean erythrocyte, hemoglobin concentrations and hematorcrit values were found (p<0.01, except erythrocytes at 50 mg/kg bw/d). At the 150 mg/kg/day dose level, in the female animals a statistically significant higher level of hemoglobin was determined.
These findings are substance-related adverse effects. Also, decreased statistically significant (-26%) plasma glucose value were found in males at the 150 mg/kg dose level. A relationsip with the test item treatment could not be ruled out.

Lastly, microscopic examination showed an increased extramedullary hematopoesis (mainly erythropoiesis) in the spleen of males and females given 50 or 150 mg/kg/d. This was considered to be compound-related. Furthermore one 150 mg/kg bw/d male animal showed a moderate epithelial degeneration/necrosis in the cecum and colon, which could be a possible result of the test substance administration.

Under the conditions of this study, a no observed adverse effect level (NOAEL) for males and females was determined to be 15 mg/kg/day (equivalent to 1.17 mg cobalt/kg/day) based on clinical signs, body weight and weight gain, haematology, clinical biochemistry and histopathology.