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Repeated dose toxicity: via oral route - systemic effects

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Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006-11-01 to 2007-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996-03-22
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was selected for this study because it is a preferred species for reproductive toxicity testing as recommended by regulatory guidelines. The Crl:CD(SD) strain was chosen because of the consistently acceptable health status and extensive experience with this strain by the laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
Test ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Females nulliparous: yes
- Age at the start of treatment: males / females: approx. 65 days
- Weight at the start of treatment: males: 294.3 - 363.6 g; females: 217.8 - 267.7 g
-Housing:
males (pretest, premating period and postmating): housed individually in stainless, wire-mesh cages suspended above cageboards.
males (cohabitation period): housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboards.
females (premating): housed individually in stainless, wire-mesh cages suspended above cageboards.
females (cohabitation period): housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboards.
females (without evidence of copulation, postmating): housed individually in polycarbonate pans.
females (evidence of copulation; Days 0 - 19 of gestation): housed individually in stainless steel, wire-mesh cages suspended above cageboards.
females (evidence of copulation; Day 20 of gestation - Day 4 of lactation): housed individually in polycarbonate pans with bedding.
females (lactation period): housed with their litters in polycarbonate pans with bedding.
- Diet (ad libitum, except when fasted): pelleted PMI® Nutrition International, Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
- water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.
- certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 ºC
- Relative humidity: 30 % – 70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The test substance was administered once daily by oral intubation (gavage), the route recommended by test guidelines.
Vehicle:
corn oil
Remarks:
Mazola®
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- formulations of the test substance in the vehicle were prepared first daily until stability data were available that supported weekly preparation and then weekly and stored at room temperature until used.
- volume of test substance or vehicle given to each rat was based on the most recently recorded body weight.
- dosing volume: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation were collected as follows:
- one sample of the vehicle and 2 samples from each formulation concentration were collected near the beginning of the study. One sample was analyzed to verify the cobalt concentration. The second sample was held at room temp. and analyzed after 8 days for stability.
- one sample of the vehicle and one sample from each concentration was collected from formulation preparations near the middle and end of the dosing period and analysed to verify concentration.
Concentrations of the cobalt borate neodecanoate in separate dosing formulation samples were measured by inductively coupled plasma (ICO) spectroscopy.

Results:
Detailed analytical results from dosing formulation samples for concentration verification indicate that the test substance was at the targeted concentration in the samples (± 20 % of nominal) up to four weeks after formulation. Test substance was not found in the 0 mg/mL samples.
The test substance was stable over the period of used for the study.
Duration of treatment / exposure:
Males: 46 and 47 days
Non-pregnant females/pregnant females: 40-49 days
Frequency of treatment:
once daily
Dose / conc.:
0.5 mg/kg bw/day (actual dose received)
Dose / conc.:
1.5 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a two-week range finding study (DuPont Haskell (2006))*, male and female rats were dosed with 30, 300, or 600 mg/kg/day of the test substance (dose volume: 10 mL/kg). The 300 and 600 mg/kg/day groups were terminated due to mortality, clinical signs of toxicity, and body weight loss. At 30 mg/kg/day, mean final body weight and overall body weight gain were reduced in males (26 % and 84 % lower than the control group, respectively) and females (5% and 25% lower than the control group, respectively). Clinical signs of toxicity were observed in males only at 30 mg/kg/day.
Additional male rats were added to the study design and were dosed with 5, 15 or 30 mg/kg/day of the test substance (dose volume: 5 mL/kg). There were no clinical signs of toxicity at any dose level tested. Final body weights were 6 %, 12% and 11% lower than controls at 5, 15, and 30 mg/kg/day, respectively. Overall mean body weight gain was 18%, 40% and 40% lower than controls at 5, 15 and 30 mg/kg/day, respectively.

*References:
- DuPont Haskell (2006). Repeated Dose Oral Toxicity: 2-Week Gavage Study in Rats. Unpublished Report. DuPont HL- 27601
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
1) cage-site examinations: at least once daily during the quarantine/pretest period and twice daily during the dosing period
2) careful clinical observations: approx. 2-3 hours post-dosing; observations were performed daily the same time each morning.
- Cage side observations checked:
1) cage-site examinations: mortality/moribund and abnormal behavior and/or appearance
2) careful clinical observations: acute/systemic toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during pretest (baseline), and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
1) females
- premating period: once a week- gestation period: days 0, 7, 14, and 21 of gestation.
- lactation period: days 0 and 4 of lactation- females without evidence of copulation as well as those that copulated and did not deliver a litter: weekly schedule until sacrifice

2) males: weekly schedule until sacrifice

All rats were weighed at terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Each feeder was weighed at the beginning and end of the weekly food consumption interval and the final weight of the feeder and the amount of spillage from the feeder during the interval were subtracted from the initial feeder weight. From the food consumption and body weight data, the mean daily food efficiency was calculated.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Premating and cohabitation periods: individual food consumption was determined weekly throughout the period, ending on test day 14. Food consumption was not measured during cohabitation for males and females or after cohabitation for males or females without evidence of copulation.
Gestation and lactation periods: individual food consumption of pregnant P1 females was determined on gestation days 0, 7, 14, and 21, and for lactating females on lactation days 0 and 4. Food consumption was not measured for females that did not deliver a litter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes
- mean daily food efficiency (g weight gain/g food consumed)

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on test days 13-14 (haematology) and test days 47 - 48 or 41 - 50 (coagulation, males and females, respectively)
- Anaesthetic used for blood collection: Yes, carbon dioxide
- Animals fasted: Yes, at least 15 hours
- How many animals: first 5 surviving animals/sex/group
- Parameters examined: red blood cell count, hemoglobin, hematocrit, mean corpuscular (cell) volume, mean corpuscular (cell) hemoglobin, mean corpuscular (cell) hemoglobin concentration, red cell distribution width, absolute reticulocyte count, platelet count, white blood cell count, differential white blood cell count, microscopic blood smear examination, prothrombin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on test day 13 -14- Animals fasted: Yes, at least 15 hours
- How many animals: first 5 surviving animals/sex/group
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, cholesterol, triglycerides, glucose, total protein, albumin, globulin, calcium, inorganic phosphorus, sodium, potassium, and chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of test substance administration and prior to the end of the premating period
- Dose groups that were examined: all animals
- Abbreviated functional observational battery (approach/touch, sharp auditory stimulus, tail pinch, fore- and hindlimb grip strength, and pupillary constriction) and motor activity were conducted (4 replicates for each evaluation).

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Rats that survived to the scheduled sacrifice were euthanized on days 47–48 (males) or days 41– 50 (females).

Gross examinations were performed on all adult rats. The presence and number of uterine implantation sites and ovarian corpora lutea were evaluated for all cohabited females.

Following tissues were collected from all adults, including the one male that died during the study:
liver, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, urinary bladder, lungs, trachea, heart, bone marrow (collected with sternum), thymus, spleen, mandibular lymph node, mesenteric lymph node, Peyer’s patches (collected from sections of the digestive tract), thyroid gland, adrenal glands, brain (3 sections, including cerebrum, cerebellum, and medulla/pons), spinal cord (3 sections, including cervical, mid-thoracic, and lumbar), sciatic nerve, sternum, testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, vagina, and gross observations.

The following tissues were weighed wet from all adult rats:
liver, kidneys, heart, thymus, spleen, adrenal glands, brain, testes, epididymides, prostate, seminal vesicles (with coagulating glands and fluids), ovaries (with oviducts), and uterus (with cervix). Paired organs were weighed together. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated.

All tissues were fixed, processed, sectioned, stained, and examined microscopically.
Processing and microscopic evaluation of tissues were as follows:
The reproductive organs from all control and 5 mg/kg/day group rats were processed to slides and evaluated microscopically. The remaining collected tissues were processed to slides and evaluated microscopically from randomly selected individual (5/sex/dose) control and 5 mg/kg/day group rats. Gross observations were examined microscopically for all animals. All tissues collected from the control male that died during the study were also examined microscopically.
Statistics:
The following statistical tests were used
- Levene’s test for homogeneity
- Shapiro-Wilk test for normality
- One-way analysis of variance
- Dunnett’s test
- Kruskal-Wallis test
- Dunn's test
- Sequential application of Cochran-Armitage test for trend
- Repeated measures analysis of variance followed by Linear contrasts
- Sequential application of the Jonckheere-Terpstra trend test
- Fisher’s Exact test with a Bonferroni correction

Male and female data were evaluated separately. The level of significance selected was p < 0.05.

*Reference:
- Haseman, J.K. and Hogan, M.D. (1975). Selection of the experimental unit in teratology studies. Teratology, 12, 165-171.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Males / Females
- no substance related deaths in male or females at any dose level tested.
- one control male was accidently killed on day 13.
- one female in the 1.5 mg/kg/d group was sacrificed on lactation day 2 due to no surviving pups. This was not considered substance related effect.
- no test substance-related clinical observations at any dose level tested in either male or female rats.

BODY WEIGHT AND BODY GAIN
Males / Females
- no test substance related effects on body weight parameters at any dose level

FOOD CONSUMPTION AND FOOD EFFICIENCY
Males / Females
- no test substance related effects on food consumption at any dose level
- instances of statistically significant increases in food consumption were considered spurious and unrelated to the test substance (females only).

HAEMATOLOGY
- no treatment-related changes in mean hematology parameters in male or female rats on test day 13 (males) or test day 14 (females).
- no statistically significant or treatment-related changes in coagulation parameters in male or female rats at test days 47 - 48 (male) or test day 41–50 (female).
The following statistically significant changes in group mean hematology parameters were considered to be unrelated to treatment (and nonadverse) because the changes did not occur in a dose-related pattern:
- mean corpuscular hemoglobin was minimally increased (104% of control) in male rats dosed with 1.5 mg/kg/day.
- platelet count was minimally decreased (88% of control) in male rats dosed with 0.5 mg/kg/day.

CLINICAL CHEMISTRY
- no test substance related effects on clinical chemistry parameters at any dose level.
The following statistically significant change in a group mean clinical chemistry parameter at test day 13 in male rats was considered to be unrelated because the change did not occur in a dose-related pattern:
- alanine aminotransferase was minimally decreased (78% of control) in male rats dosed with 0.5 or 5 mg/kg/day.

NEUROBEHAVIOUR
1) Abbreviated functional observational battery
- 0, 0.5, 1.5, or 5 mg/kg/day: no test substance-related effects or statistically significant differences on forelimb and hindlimb grip strength in either males or females.
No test substance effects or statistically significant effects for any behavioral parameter evaluated in males or females.

2) Motor Activity
- 0, 0.5, 1.5, or 5 mg/kg/day: no test substance-related effects on duration of movement or number of movements in males or females.
- 5 mg/kg/day: a marginal decrease in total duration of movement observed in males was not considered treatment-related because a similar decrease was not evident in number of movements in males
- 5 mg/kg/day: no effects on either number of movements or duration of movements in females, and the values were within the range of historical controls and within the range of values observed during the baseline evaluations. The significantly lower trend in number of movements during the first 10-minute interval for females during the baseline evaluation was spurious since administration of the test substance had not been initiated.

ORGAN WEIGHTS
- no test substance-related organ weight effects in the males and females.
- 5 mg/kg/day: a small (11%), statistically significant, increase in the mean relative spleen weight (% body weight) of males, as compared to controls (spurious finding). A coincidentally slightly lower mean final body weight and slightly greater mean absolute spleen weight in the males, as compared to controls, resulted in the increased relative value. There was no associated gross or microscopic pathology in any spleens.
- 0.5, 1.5, and 5 mg/kg/day: female spleen weight parameters were similar in the groups receiving the test substance.
- all other individual and mean organ weight differences were considered to be spurious and unrelated to test substance administration.

GROSS PATHOLOGY
- no test substance-related gross observations in any of the males and females.
All gross observations, recorded at necropsy, were consistent with normal background lesions that occur in rats of this age and strain.
- control male found dead had dark discolouration of the lungs

HISTOPATHOLOGY: NON-NEOPLASTIC
- no test substance-related microscopic findings in any of the males and females.
All microscopic observations were consistent with normal background lesions in rats of this age and strain.
- minimal cardiomyopathy was increased in the 5 mg/kg/day males, as compared to control males (not test substance related). In the first 5 rats per group selected for histopathology, the incidence of cardiomyopathy was 0/5 and 3/5 in the control and 5 mg/kg/day dose groups, respectively. In all rats examined (excluding control rat found dead on day 13), the incidence of cardiomyopathy was 1/8 and 3/6 in the control and 5 mg/kg/day dose groups, respectively.The cardiomyopathy observed in all 4 affected hearts was morphologically consistent with the naturally occurring multifocal degenerative and inflammatory condition commonly seen in rats of this age. In each of the 4 hearts, the minimal cardiomyopathy consisted of 1 to 4 foci of mononuclear inflammatory cell infiltrates which were associated with the degeneration and/or loss of several cardiac myofibers. Since the overall incidence of cardiomyopathy (4/14 males) in this study was comparable to historical controls, the distribution of affected hearts (1/8 vs. 3/6) was considered spurious.
- cardiomyopathy, was not observed in any female rats in this study. Naturally occurring cardiomyopathy is also common in control female rats of this age, however the incidence and severity of lesions is less than observed in males.
- control male found dead had focal traumatic lesion observed in the esophageal tissue adjacent to the trachea (probably dosing trauma).
Dose descriptor:
NOAEL
Effect level:
5 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
Dose descriptor:
NOAEL
Effect level:
1.1 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
cobalt
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
Prior to this combined repeated dose toxicity study with a reproduction developmental toxicity screening test, a 14-day dose range-finder using male and female rats was performed. The rats were dosed by gavage with 30, 300, and 600 mg/kg/day of the test substance (dosing volume: 10 mL/kg).

The following findings were made for males as follows:
600 mg/kg bw/day: terminated due to mortality, clinical signs, and body weight loss
300 mg/kg bw/day: terminated due to mortality, clinical signs, and body weight loss
30 mg/kg bw/day: clinical signs, reduced mean final body weight (26 % lower), reduced overall body weight gain (84 % lower)

The findings for the females were as follows:
600 mg/kg bw/day: terminated due to mortality, clinical signs, and body weight loss
300 mg/kg bw/day: terminated due to mortality, clinical signs, and body weight loss
30 mg/kg bw/day: reduced mean final body weight (5 % lower) and reduced overall body weight gain (25 % lower)

After testing these animals, additional male rats were added to the study and were dosed with 5, 15 or 30 mg/kg/day (dosing volume: 5 mL/kg).

The following findings were made for these additional animals as follows:
Final body weights were 6%, 12% and 11% lower than controls at 5, 15, 30 mg/kg/day, respectively. Overall body weight gain was 18%, 40% and 40% lower than controls at 5, 15, and 30 mg/kg/day.
No test substance-related effects were observed for clinical observations, mortality, body weight, food consumption, haematology, clinical chemistry, neurobehaviour, organ weights, gross pathology or histopathology for males and females at any dose level. Minimal grade cardiomyopathy was increased in the males of the 5 mg/kg bw /day group (1/8 in control group and 3/6 in the 5 mg/kg/day group) as compared to control males, but was not interpreted to be test substance related. Based on the results, the no-observed-adverse-effect level (NOAEL) for repeated dose toxicity was 5 mg/kg bw/day (equivalent to 1.1 mg cobalt/kg bw/day).
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
Qualifier:
according to
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. certificate)
Remarks:
signed 2006-02-24
Limit test:
no
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.
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 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
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
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
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
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
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
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.
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
haematology
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
1.17 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
haematology
clinical biochemistry
histopathology: non-neoplastic
Critical effects observed:
no
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.
Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2007-04-27 to 2008-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996-03-22
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: CD(SD)
Details on species / strain selection:
The rat was selected for this study because it is a preferred species for toxicity testing as recommended by test guidelines. The Crl:CD(SD) strain was chosen because extensive background information is available from the literature, the supplier, and previous studies conducted by the laboratory. This strain is also considered suitable relative to hardiness and incidence of spontaneous disease.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Females nulliparous: yes
- Age at the start of treatment: males: approx. 65 days; females: approx. 71 days
- Weight at the start of treatment: males: 301.9 - 362.0 g; females: 204.4 - 260.5 g
- Housing:
males (pretest, premating period and postmating): housed individually in stainless, wire-mesh cages suspended above cageboards.
males (cohabitation period): housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboards.
females (cohabitation period): housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboards.
females (without evidence of copulation, postmating): housed individually in polycarbonate pans.
females (evidence of copulation; Days 0 - 19 of gestation): housed individually in stainless steel, wire-mesh cages suspended above cageboards.
females (evidence of copulation; Day 20 of gestation - Day 4 of lactation): housed individually in polycarbonate pans with bedding.
females (lactation period): housed with their litters in polycarbonate pans with bedding
- Diet (ad libitum, except when fasted): pelleted PMI® Nutrition International, Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: approximately 7 days

DETAILS OF FOOD AND WATER QUALITY:
- water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.
- certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study.

ENVIRONMENTAL CONDITIONS:
- Temperature: 19 - 25 ºC (target with an acceptable tolerance range of 18-26 ºC)
- Relative humidity: 30 % – 70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The test substance was administered orally (gavage) as it is the route recommended by test guidelines.
Vehicle:
corn oil
Remarks:
Mazola®
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- formulations of the test substance in the vehicle were prepared daily and stored at room temperature until used.
- volume of test substance or vehicle given to each rat was based on the most recently recorded body weight.
- dosing volume: 5 mL/kg

VEHICLE:
- corn oil was not expected to contain any contaminants that would have interfered with the conduct of the study.
- vehicle was assumed to be stable under the conditions of the study and was stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation were collected once near the beginning of the study from all the formulation concentrations, and again near the end of the study. One sample of the vehicle and 2 samples from each formulation concentration was collected. Samples from dosing formulations were submitted for concentration verification analysis. Concentrations of the cobalt neodecanoate in separate dosing formulation samples were measured by inductively coupled plasma (ICO) spectroscopy.
Results:
The recovery values obtained for these samples were 98 - 104 %
The data for samples collected indicate that the test substance was at the targeted concentrations in the samples (± 20 % of nominal) up to eight days after formulation. Cobalt neodecanoate was not detected in the blank.
Duration of treatment / exposure:
Males: 47 and 48 days
Non-pregnant females/pregnant females: 41-48 days
Frequency of treatment:
once daily
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
45 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a two-week range finding study (DuPont Haskell (2006))*, male and female rats were dosed with 30, 300, or 600 mg/kg/day of the test substance (dose volume: 10 mL/kg). The 300 and 600 mg/kg/day groups were terminated due to mortality, clinical signs of toxicity, and body weight loss. At 30 mg/kg/day, mean final body weight and overall body weight gain were reduced in males (20 % and 70 % lower than the control group, respectively) and females (3% and 31% lower than the control group, respectively). Clinical signs of toxicity were observed in males only at 30 mg/kg/day.
Additional male rats were added to the study design and were dosed with 5 or 15 mg/kg/day of the test substance (dose volume: 5 mL/kg). There were no clinical signs of toxicity at any dose level tested. Final body weights were 4 % lower than controls at 15 mg/kg/day. Overall mean body weight gain was 22 % lower than controls at 15 mg/kg/day. There were no reductions in final body weight or overall body weight gains at 5 mg/kg/day.

*References:
- DuPont Haskell (2006). Repeated Dose Oral Toxicity: 2-Week Gavage Study in Rats. Unpublished Report. DuPont Haskell HL- 27601
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
1) daily animal health observations: at least once daily during the quarantine/pretest period and once daily during the dosing period
2) careful clinical observations: within 3 hours post-dosing; on 2 separate occasions, observations were performed approximately 4 to 5 hours after dosing.
- Cage side observations checked:
1) daily animal health observations: mortality/moribund and abnormal behavior and/or appearance
2) careful clinical observations: acute/systemic toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before first dosing (baseline), and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
1) females- premating period: once a week
- gestation period: days 0, 7, 14, and 21 of gestation.
- lactation period: days 0 and 4 of lactation
- females without evidence of copulation as well as those that copulated and did not deliver a litter: weekly schedule until sacrifice

2) males: weekly schedule until sacrifice
All rats that survived to the scheduled terminal sacrifice were weighed prior to sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Each feeder was weighed at the beginning and end of the weekly food consumption interval and the final weight of the feeder and the amount of spillage from the feeder during the interval were subtracted from the initial feeder weight. From the food consumption and body weight data, the mean daily food efficiency was calculated.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Premating and cohabitation periods: individual food consumption was determined weekly throughout the period, ending on test day 14. Food consumption was not measured during cohabitation for males and females or after cohabitation for males or females without evidence of copulation.
Gestation and lactation periods: individual food consumption of presumed pregnant P1 females was determined on gestation days 0, 7, 14, and 21, and for lactating females on lactation days 0 and 4. Food consumption was not measured for females that did not deliver a litter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes
- mean daily food efficiency (g weight gain/g food consumed)

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on test day 14 (haematology) and test days 48 and 42 - 49 (coagulation, males and females, respectively)
- Anaesthetic used for blood collection: Yes, carbon dioxide
- Animals fasted: Yes, at least 15 hours
- How many animals: first 5 surviving animals/sex/group
- Parameters examined: red blood cell count, hemoglobin, hematocrit, mean corpuscular (cell) volume, mean corpuscular (cell) hemoglobin, mean corpuscular (cell) hemoglobin concentration, red cell distribution width, absolute reticulocyte count, platelet count, white blood cell count, differential white blood cell count, microscopic blood smear examination, prothrombin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on test day 14
- Animals fasted: Yes, at least 15 hours
- How many animals: first 5 surviving animals/sex/group
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, cholesterol, triglycerides, glucose, total protein, albumin, globulin, calcium, inorganic phosphorus, sodium, potassium, and chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of test substance administration and prior to the end of the premating period
- Dose groups that were examined: all animals
- Abbreviated functional observational battery (approach/touch, sharp auditory stimulus, tail pinch, fore- and hindlimb grip strength, and pupillary constriction) and motor activity were conducted (4 replicates for each evaluation).

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Rats that survived to the scheduled sacrifice were euthanized on days 48–49 (males) or days 42–49 (females). All animals were dosed until the day prior to sacrifice. Rats with poor health were submitted for euthanasia prior to the scheduled sacrifice.

Gross examinations were performed on all adult rats. The presence and number of uterine implantation sites and ovarian corpora lutea were evaluated for all cohabited females.

Following tissues were collected from all adults:
liver, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, urinary bladder, lungs, trachea, heart, bone marrow (collected with sternum), thymus, spleen, mandibular lymph node, mesenteric lymph node, Peyer’s patches (collected from sections of the intestine), thyroid gland, adrenal glands, brain (3 sections, including cerebrum, cerebellum, midbrain and medulla/pons), spinal cord (3 sections, including cervical, mid-thoracic, and lumbar spinal cord), sciatic nerve, sternum, testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, vagina, and gross observations.

The following tissues were weighed wet from all adult rats that survived to the scheduled sacrifice: liver, kidneys, heart, thymus, spleen, adrenal glands, brain, testes, epididymides, prostate, seminal vesicles (with coagulating glands and fluids), ovaries (with oviducts), and uterus (with cervix). Paired organs were weighed together. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated.

All tissues were fixed, processed, sectioned, stained, and examined microscopically.
Processing and microscopic evaluation of tissues were as follows:
- 45 mg/kg/day dose group females: only one female rat in the 45 mg/kg/day group survived to the scheduled sacrifice. Tissues from animals in this group were not evaluated microscopically.
- scheduled sacrifice: all tissues were evaluated microscopically from up to 5 rats/sex that were sacrificed by design in the control and 45 mg/kg/day dose group (males only) and 15 mg/kg/day dose group (females only).
- Decedents: all protocol tissues were evaluated microscopically from rats that were submitted for an unscheduled sacrifice due to deteriorating health and for rats that were found dead.
- Reproductive failures: all reproductive tissues were evaluated microscopically from the male and female mated pairs that failed to produce a litter.
- Target organs: available target organs were evaluated microscopically from all adult rats in all groups (males: spleen and bone marrow; females: intestinal tract (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum), spleen, thymus, adrenal glands, and kidneys).
Statistics:
The following statistical tests were used
- Levene’s test for homogeneity
- Shapiro-Wilk test for normality
- One-way analysis of variance
- Dunnett’s test
- Kruskal-Wallis test
- Dunn's test
- Sequential application of Cochran
-Armitage test for trend
- Repeated measures analysis of variance followed by Linear contrasts
- Sequential application of the Jonckheere-Terpstra trend test
- Fisher’s Exact test with a Bonferroni correction
Male and female data were evaluated separately. The level of significance selected was p < 0.05.

*Reference:
- Haseman, J.K. and Hogan, M.D. (1975). Selection of the experimental unit in teratology studies. Teratology, 12, 165-171.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 15 and 45 mg/kg/day: test substance-related clinical signs of toxicity occurred which included wet fur in the perioral/chin region following the daily dosage administration (not considered to be adverse).

2) Females:
Gestation:
- 15 and 45 mg/kg/day: test-substance-related clinical signs of toxicity were observed. Females had increased incidences of labored breathing, dehydration, diarrhea, dystocia, and hunched over posture during the daily post-dosing observations.
Lactation
- 5, 15, and 45 mg/kg/day: test-substance-related clinical signs of toxicity were observed, as follows:
- 45 mg/kg/day group: lethargy, stained fur/skin (yellow, red, and/or brown), ataxia, and slow breathing rate, during the daily post-dosing observations or weekly detailed clinical observations.
- 15 mg/kg/day group: yellow stained fur/skin.
- 5 mg/kg/day group: lethargy and yellow stained fur/skin.
Mortality:
mortality observed, treatment-related
Description (incidence):
Females:
Gestation:
- 15 and 45 mg/kg/day: test substance-related mortality occurred. Seven rats in the 45 mg/kg/day group were found dead during gestation days 21-22, and 2 were sacrificed in extremis on gestation day 22. Two rats in the 15 mg/kg/day group were found dead during gestation days 22-23, and 3 were sacrificed in extremis on gestation days 22-23.
Lactation:
-5 or 45 mg/kg/day: test substance-related mortality occurred. One female in the 5 mg/kg/day group was found dead on lactation day 1. One female in the 45 mg/kg/day group was found dead on lactation day 1, and one was sacrificed in extremis on lactation day 1. Number of dams surviving to lactation day 4 was 11, 11, 6, and 1 for the 0, 5, 15, and 45 mg/kg/day groups, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 5, 15, or 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in body weight and/or weight gain occurred.
- 45 mg/kg/day: body weight was significantly decreased during test days 27, 34, 41, and 48, and weight gain was decreased over the intervals of days 0-7, 13-20, 20-27, 27-34, 34-41, 41-48, 0-13, 13-48, and 0-48. By test day 48, males had 18% lower body weight, and weight gain over days 0-48 was 50% lower than the control value.
- 15 mg/kg/day: body weight was slightly lower (8%) on test day 48, and weight gain was significantly lower over the interval of test days 34-41. Body weight gain values over the intervals of test days 13-48, and 0-48 were significantly lower (37% and 25%, respectively) compared to the control value.
- 5 mg/kg/day: body weight gain was significantly lower for the interval of test days 13-48 (18%) compared to the control value; weight gain for the interval of test days 0-48 was 12% lower than the control value (not statistically significant).

2) Females:
Gestation:
- 15 and 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in body weight and weight gain occurred.
- 45 mg/kg/day: on gestation day 21, body weight was 21% lower and weight gain values for gestation days 14-21 and 0-21 were 68% and 49% lower than the control values, respectively.
- 15 mg/kg/day: body weight was 12% lower on gestation day 21, and weight gain values for gestation days 14-21 and 0-21 were 46% and 29% lower than the control values, respectively.
Lactation:
- 45 mg/kg/day: adverse, test substance-related decreased body weight was observed on lactation day 0; weight gain during lactation days 0-4 in the one surviving dam was similar to control.

Please also refer for results to the field "Attached background material" below.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 45 mg/kg/day: test substance-related, statistically significant decreases in food consumption occurred. Food consumption was significantly decreased during test days 0-7.

2) Females:
Gestation:
- 15 and 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in food consumption and food efficiency occurred.
- 45 mg/kg/day: food consumption values were significantly lower on gestation days 7-14, 14-21, and 0-21, and were 20% lower than the control value for gestation days 0-21.
- 15 mg/kg/day: food consumption and/or food efficiency was significantly lower for gestation days 14-21 and gestation days 0-21, and was 21% and 10% lower than the control values for gestation days 14-21 and 0-21, respectively.
Lactation:
- 15 and 45 mg/kg/day: adverse, test substance-related, decreases in food consumption occurred. Food consumption was 21% and 35% lower than the control value during lactation days 0-4 in 15 and 45 mg/kg/day females, respectively.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 45 mg/kg/day: test substance-related, statistically significant decreases in food efficiency occurred. Food efficiency was significantly decreased during test days 0-7, and food efficiency was decreased during test days 0-13. Since the food efficiency value over test days 0-13 was only 7% lower than the control value, this minimal difference was not considered to be adverse.

2) Females:
Gestation:
- 15 and 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in food efficiency occurred.
- 45 mg/kg/day: food efficiency values were significantly lower for gestation days 7-14, 14-21, and 0-21, and were 38% lower than the control value for the gestation days 0-21 period.
- 15 mg/kg/day: food consumption and/or food efficiency was significantly lower for gestation days 14-21 and gestation days 0-21, and was 21% and 10% lower than the control values for gestation days 14-21 and 0-21, respectively.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Males:
- 45 mg/kg/day: adverse, test substance-related, statistically significant decreases in mean duration of movement and number of movements occurred. Total duration of movement was 22% lower compared to the control value, and mean number of movements was 12% lower compared to the control value. Mean duration of movement and number of movements were also significantly lower during the sixth 10-minute interval. In the absence of other neurobehavioral changes, the decreased motor activity was considered to be secondary to systemic toxicity (gastroenteropathy, dehydration, reduced body weight and body weight gain, reduced food consumption).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Females:
Thymus:
- 15 mg/kg/day group: thymic weight parameters were decreased. Thymus weight relative to body weight was decreased to 75% of the control group mean. Thymic weight changes correlated with microscopic changes of lymphoid atrophy and/or necrosis in the thymus of some rats.

Adrenal:
- 15 mg/kg/day: adrenal weight parameters were increased. Adrenal weight relative to body weight was increased to 126% of the control group mean. Adrenal weight increases were associated with microvesiculation of the adrenal cortex.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Females:
- test substance-related enteropathy was considered to be the cause of death in all decedents.
- 15 and/or 45 mg/kg/day: a few gross lesions observed were likely related to exposure to the test material, as follows:
15 mg/kg/day: small spleens (2/12 rats), stomach discoloration (1/12 rats), ulcer/erosion of the stomach mucosa (1/12 rats)
45 mg/kg/day: small spleens (1/12 rats), stomach discoloration (2/12 rats), ulcer/erosion of the stomach mucosa (1/12 rats)
- all other gross observations occurred in low incidences and/or were consistent with normal background lesions that occur in rats of this age and strain.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1) Males:
- 45 mg/kg/day: test substance-related microscopic findings were limited to increased erythropoiesis in the spleen and bone marrow. Minimally increased extramedullary hematopoiesis of the spleen was present in 4/12 rats and minimal erythroid hyperplasia of the bone marrow was present in 9/12 rats. Neither findings were present in the remaining treated groups or in the control groups.
- increased erythropoiesis in bone marrow and spleen was correlative to the slight increase in red cell mass parameters, was not associated with evidience of toxicity in these organs, and thus was not considered adverse.

2) Females (tissues from females in the 45 mg/kg/day group were not evaluated microscopically):
- 5 and 15 mg/kg/day: test substance-related microscopic findings were present, as summarized below:
Gastrointestinal tract:
- several degenerative and regenerative changes, as well as mucosal atrophy, were present in the gastrointestinal tract of 8/12 rats in the 15 mg/kg/day, and in one dead rat in the 5 mg/kg/day group. These gastrointestinal changes were interpreted to be the cause of death in all female decedents.
- 3/7 rats in the 15 mg/kg/day group that survived to the terminal sacrifice had test substance-related microscopic changes in the gastrointestinal tract, which were generally minimal to mild and limited to only one section of the gastrointestinal tract.
- changes observed in the intestines included the following diagnoses: degeneration/necrosis of mucosal epithelium (villous and crypt epithelium); atrophy of villi and crypts; regeneration of mucosal epithelium, and mucosal inflammation. Lesions were observed in all segments of the small intestine and in the cecum and colon. Lesions were graded as minimal to severe and were most severe in the jejunum, ileum, and cecum.
- further changes:
15 mg/kg/day: low incidences of erosion or ulceration of the gastric mucosa were present. Ulcer/erosion was present in the glandular mucosa of 3/12 rats and in the nonglandular mucosa of 2/12 rats. Goblet cell depletion of the rectum was present in 4/12 rats.
5 mg/kg/day: goblet cell depletion of the rectum was present in one rat (the decedent).

Lymphoid organs:
- 15 mg/kg/day: test substance-related necrosis of lymphocytes and/or atrophy of lymphoid regions were present in the thymus and/or spleen in 9/12 rats. - 5 mg/kg/day: lymphoid atrophy was present in 4/12 rats. Lymphoid atrophy was limited to the thymus, except for one animal which also had splenic involvement.
- lymphoid changes were graded as minimal to severe and were generally most consistently present and most severe in the thymus.

Adrenal glands:
- 15 mg/kg/day: minimally increased microvesiculation of adrenal cortical cells was present in 5/12 rats. The change was diffuse within the cortex and was characterized by multiple, small, clear vacuoles within the cytoplasm of adrenal cortical cells. These changes in the current study were considered to be test substance related, but they were not considered adverse, as they were not associated with morphologic evidence of cytotoxicity to affected cells.

Kidneys:
- 15 mg/kg/day: minimal vacuolation of renal tubular epithelium was present in renal cortical tubules of 5/12 rats. Tubular vacuolation was characterized by small, discrete, clear granules in the basilar cytoplasm of renal cortical epithelial cells. This change was not associated with other microscopic findings or with clinical pathology changes indicative of renal injury and vacuolation of renal tubules was not considered to be adverse.
- 5 mg/kg/day: minimal vacuolation of renal tubular epithelium was present in renal cortical tubules of the one decedent. Tubular vacuolation was characterized by small, discrete, clear granules in the basilar cytoplasm of renal cortical epithelial cells. This change was not associated with other microscopic findings or with clinical pathology changes indicative of renal injury and vacuolation of renal tubules was not considered to be adverse.

- all other microscopic findings in this study were consistent with normal background lesions in rats of this age and strain.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1) Males:
- no unscheduled deaths in any male group.
- the remainder of the clinical observations recorded besides the ones stated above was common for this age and strain of rat, and was not considered to be test substance related.

2) Females:
Premating:
- test substance-related mortality or clinical observations did not occur

BODY WEIGHT AND WEIGHT GAIN
2) Females:
Premating:
- 45 mg/kg/day: body weight gain was slightly decreased (31% lower compared to the control value, not statistically significant) over the interval of test days 0-13.
- 5 or 15 mg/kg/day: no test substance-related or statistically significant differences.
Gestation:
- 5 mg/kg/day: no test substance related effects or statistically significant differences on body weight parameters.
Lactation:
- 5 or 15 mg/kg/day: no effects on weight or weight gain during lactation days 0-4.

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

FOOD CONSUMPTION AND COMPOUND INTAKE/FOOD EFFICIENCY
1) Males:
- 5 and 15 mg/kg/day: no test substance-related or statistically significant effects on food consumption or food efficiency

2) Females
Premating:
- no test substance-related or statistically significant effects on food consumption parameters at any level tested.
Gestation:
- 5 mg/kg/day: no test substance-related or statistically significant effects on food consumption or food efficiency.
Lactation:
- 5 mg/kg/day: no test substance-related or statistically significant effects on food consumption or food efficiency.

HAEMATOLOGY
- no adverse changes in mean hematology parameters in male or female rats at test day 14.

The following statistically significant changes were considered non-adverse or not treatment related:
- red blood cell, hemoglobin, hematocrit were minimally increased in male and female rats dosed with 45 mg/kg/day (variable statistical significance; <108% of control). Associated with these minimal increases were minimal but statistically significant increases in absolute reticulocyte counts (180% and 172% of control, male and female) and a minimal increase in red cell distribution width (116% of control, males only). These hematology changes are likely treatment related, but due to the minimal nature of theses changes (increases in red cell mass parameters were less than 10% above the control group means), they were not considered to be adverse.
- platelet count was minimally increased in male rats dosed with 15 or 45 mg/kg/day (124% and 135% of control, respectively) (uncertain relationship to treatment; no toxicological significance).
- absolute eosinophil count was minimally decreased in male rats dosed with 5 mg/kg/day (54% of control; not dose related)
- prothrombin time was minimally prolonged in male rats dosed with 45 mg/kg/day (106% of control; no clinical significance).

CLINICAL CHEMISTRY
- no adverse changes in mean clinical chemistry parameters in male or female rats at test day 14.
The following statistically significant changes were considered non-adverse:
- cholesterol was mildly decreased in male rats dosed with 15 or 45 mg/kg/day (variable significance, 70% and 57% of control). Since minimal decreases in cholesterol are not known to be associated with adverse effects in male rats, these changes in cholesterol at 15 and 45 mg/kg/day were not considered to be adverse.
- inorganic phosphorus was minimally increased in male rats dosed with 45 mg/kg/day (110% of control). Values in individual animals were similar to controls, no changes in other parameters that are often associated with increases in inorganic phosphorus, and no treatment-related changes in inorganic phosphorus were present in females. Therefore, the minimal increase in inorganic phosphorus was not considered to be treatment related.
- chloride was minimally increased in male and female rats dosed with 15 or 45 mg/kg/day (103-105% of control). Mean chloride concentrations were similar across all groups, reflecting the lack of relevant change across the range of concentrations of the test substance. The changes were considered to be non-adverse based on their minimal nature.

The following statistically significant changes in group mean clinical chemistry parameters at test day 14 were considered to be unrelated to treatment (and non-adverse) because the change did not occur in a dose-related pattern:
Females:
- alkaline phosphatase was minimally increased and calcium was minimally decreased in rats dosed with 5 mg/kg/day (148% and 95% of control, respectively).
- creatinine was minimally increased in rats dosed with 5 or 15 mg/kg/day (111% and 117% of control, respectively).

NEUROBEHAVIOUR
1) Abbreviated functional observational battery
- 0, 5, 15, 45 mg/kg/day: no test substance-related effects or statistically significant differences on forelimb and hindlimb grip strength in either males or females. No statistically significant effects for any behavioral parameter evaluated in males or females.
- dark colored urine was observed in 3 males administered 45 mg/kg/day and one female in the 15 mg/kg/day group (no adverse effects associated with this observation).

2) Motor activity
Females:
- no test substance-related or statistically significant differences in duration of movement or number of movements at any dosage of the test substance.

ORGAN WEIGHTS
1) Males:
Liver:
- weight parameters (absolute weight, and weight relative to both body and brain weight) had statistically significant decreases relative to controls in all treated groups.
- final body weights taken at terminal sacrifice were also decreased in these groups (statistically significant in the 45 mg/kg/day group).
- since liver weight decreases in association with decreases in body weight, the decreases in liver weight parameters in treated male groups were due, at least in part, to the decreased body weight in these groups.
- conclusion: decreases in liver weights relative to body weight were less severe (compared to control) than were changes in absolute liver weight and liver weight relative to brain weight. Furthermore, liver weight changes were not associated with correlative, treatment-related changes in liver-related clinical pathology parameters or with microscopic changes in the liver. These liver weight changes were not considered to be adverse findings.

2) Females:
- organ weights of the 45 mg/kg/day group were not included in the evaluation, since organ weight data were available for only one female. The organ weights of the early decedents in that group were not collected.
- no other primary test substance-related organ weight changes in male or female rats at any of the dose levels were evaluated.
- several statistically significant organ weight changes in male rats in the 45 mg/kg/day group were considered secondary to mean final body weight change (taken at necropsy) in this group (decreased to 82% of control). No treatment-related microscopic findings were present in affected organs.
- for organs whose weight tends to decrease with decreases in body weight (heart, seminal vesicles), statistically significant changes were characterized by decreased absolute weight and organ weight relative to brain weight.
- for organs whose weight is less affected by decreases in body weight (spleen, testes, brain, kidney) statistically significant weight changes were limited to increases in organ weight relative to body weight.
- a statistically significant decrease in brain weight relative to body weight in the 15 mg/kg/day male group was considered to be due to decreased final body weight in this group (body weight decrease not statistically significant).
- a statistically significant increase in heart weight relative to brain weight in the 15 mg/kg/day male group was not considered test substance related as it did not occur in a dose-related manner.
- a statistically significant increase in liver weight relative to body weight in the 15 mg/kg/day female group was not associated with changes in other liver weight parameters or with test substance-related clinical pathology or microscopic changes in the liver. Therefore, this change was not considered to be test substance related.

GROSS PATHOLOGY
1) Males:
- no test substance-related gross observations.

2) Females:
- 45 mg/kg/day: one female had evidence of dystocia, which may have been contributory to its early death.
Dose descriptor:
LOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
0.7 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
cobalt
Sex:
male/female
Remarks on result:
other: no NOAEL identified
Critical effects observed:
no
Conclusions:
Prior to this combined repeated dose toxicity study with a reproduction developmental toxicity screening test, a 14-day dose range-finder using male and female rats was performed. The rats were dosed by gavage with 30, 300, and 600 mg/kg/day of the test substance (dosing volume: 10 mL/kg). The following findings were made for males as follows:
600 mg/kg bw/day: mortality, clinical signs, and body weight loss
300 mg/kg bw/day: mortality, clinical signs, and body weight loss
30 mg/kg bw/day: mean final body weight (20 % lower), overall body weight gain (70 % lower), and clinical signs
The findings for the females were as follows:
600 mg/kg bw/day: mortality
300 mg/kg bw/day: mortality and clinical signs
30 mg/kg bw/day: mean final body weight (3 % lower) and overall body weight gain (31 % lower)
After testing these animals, additional male rats were added to the study and were dosed with 5 and 15 mg/kg/day (dosing volume: 5 mL/kg). The following findings were made for these additional animals as follows:
15 mg/kg bw/day: mean final body weihgt (4 % lower), overall body weight gain (22 % lower), and clinical signs
5 mg/kg bw/day: no effects

No treatment related effects were observed for haematology and clinical chemistry in males and females at any dose level. No test substance-related mortality was observed for males, whereas 1/12, 5/12, and 11/12 female rats were found dead or were sacrificed prior to scheduled termination in the 5, 15, and 45 mg/kg/day groups, respectively.
At the 5 mg/kg bw/day dose level, males showed a decrease in body weight gain (5/12; days 0 - 48). At the 15 mg/kg bw/day dose level, clinical signs as well as decreased body weight (6/12; day 48) and body weight gain (8/12; days 0 - 48) were observed. In the highest dose level of 45 mg/kg bw/day, clinical signs, decreased body weight (10/12; day 48), decreased body weight gain (12/12; days 0 - 48), decreased food consumption, decreased food efficiency, and decreased motor activity (secondary to systemic toxicity), and dark coloured urine (3/12) were observed. Lastly, increased erythropoiesis in the spleen and bone marrow as well as minimally increased extramedullary haematopoiesis of the spleen (4/12) and minimal erythroid hyperplasia of the bone marrow were recorded for male rats.

In male rats, pathological findings were only made at the 45 mg/kg bw/day dose level. At this dose level, increased erythropoiesis in the spleen and bone marrow, minimally increased extramedullary haematopoiesis of the spleen (4/12), and minimal erythroid hyperplasia of the bone marrow (9/12) were recorded.

In female rats, test-substance-related clinical signs of toxicity during lactation (lethargy and yellow stained fur/skin) were observed at the 5 mg/kg bw/day dose level. Furthermore, clinical signs (increased incidences of laboured breathing, dehydration, diarrhea, dystocia, and hunched over posture), decreased body weight (8/11; day 21), decreased body weight gain (9/11; days 0 - 21 days), and decreased food consumption were recorded. The same findings found for the mid dose were found for the 45 mg/kg bw/day dose level, where body weight and body weight gain were decreased in all females.

Pathology of the females at the 5 mg/kg bw/day dose level, gastroenteropathy and goblet cell depletion of the rectum (1/12; considered to be the cause of death), lymphoid necrosis and/or atrophy in the thymus (4/12), including (1/12) with splenic involvement, minimal vacuolation of renal tubules (1/12), and minimal microvesiculation of the adrenal cortex (1/12) were recorded. The pathological findings increased in severity when 15 mg/kg bw/day were administered to female rats. At this dose level decrease in thymus weight, increase in adrenal weight, small spleens (2/12), stomach discolouration (1/12), stomach ulcer/erosion (1/12), gastroenteropathy (8/12) including, ulcer/erosion in the glandular mucosa (3/12), and in the nonglandular mucosa (2/12), and goblet cell depletion of the rectum (4/12), lymphoid necrosis and/or atrophy in the thymus and spleen (9/12), minimal vacuolation of renal tubules (5/12), and minimal microvesiculation of the adrenal cortex (5/12) were found. Lastly, a small spleen (1/12), stomach discolouration (2/12) and stomach ulcer/erosion (1/12) were recorded at the 45 mg/kg bw/day dose level.

Under the conditions of this study, a no-observed-adverse-effect level (NOAEL) for systemic toxicity was not achieved due to effects on mortality, clinical signs of toxicity, body weight parameters, and/or food consumption parameters at all dosages in males and females. A LOAEL of 5 mg/kg bw/day (equivalent to 0.7 mg cobalt/kg bw) could be determined for male and female rats. Adverse effects were observed in males and female animals already at the lowest dose, manifested as decreased weight gain, lethargy, gastroenteropathy and subsequent secondary effects.
Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2007-04-19 to 2008-09-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996-03-22
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD)
Details on species / strain selection:
The rat was selected for this study because it is a preferred species for toxicity testing as recommended by test guidelines. The Crl:CD(SD) strain was chosen because extensive background information is available from the literature, the supplier, and previous studies conducted by the laboratory. This strain is also considered suitable relative to hardiness and incidence of spontaneous disease.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Females nulliparous: yes
- Age at the start of treatment: males: approx. 71 days; females: approx. 77 days
- Weight at the start of treatment: males: 350.0 g - 426.8 g; females: 221.7 - 280.0 g
- Housing:
Males (pretest, premating and postmating period): housed individually in stainless steel, wire-mesh cages suspended above cageboards
Males (cohabitation period): housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboards
Females (pretest, premating and postmating period): housed individually in stainless steel, wire-mesh cages suspended above cageboards
Females (cohabitation period): housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboards
Females (without evidence of copulation, postmating): housed individually in polycarbonate pans
Females (evidence of copulation; Days 0-19 of gestation): housed individually in stainless steel, wire-mesh cages suspended above cageboards
Females (evidence of copulation; Day 20 of gestation - Delivery, Day 4 of lactation): housed individually in polycarbonate pans with bedding
Females (lactation period): housed with their litters in polycarbonate pans with bedding
- Diet (ad libitum, except when fasted): pelleted PMI® Nutrition International, Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Quarantine period: approximately 15 days

DETAILS OF FOOD AND WATER QUALITY:
- water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.
- certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C (target with an acceptable tolerance range of 18 - 26 °C)
- Relative humidity: 30 % - 70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The test substance was administered orally (gavage) as it is the route recommended by test guidelines.
Vehicle:
other: 0.1 % Tween-80 in 0.5 % aqueous methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- formulations of the test substance in the vehicle were prepared daily and stored at room temperature until used.
- volume of the test substance or vehicle given to each rat was based on the most recently recorded body weight.
- dosing volume: 5 mL/kg

VEHICLE:
- vehicle mixture was not expected to contain any contaminants that would interfere with the conduct of the study.
- vehicle was assumed to be stable under the conditions of the study and was stored refrigerated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation to analyse uniformity of mixing, concentration, and stability were taken 2 times, near the beginning and middle of the study. One sample of vehicle and 4 independent samples from each formulation concentration were collected near the beginning of the study. The vehicle sample and 3 samples (top, middle, and bottom samplings) from each formulation were analysed after submission to verify concentration and uniformity of mixing. The fourth sample was held for 5 hours at room temperature and then analysed for stability of the test substance in the formulation.
One sample of the vehicle and one sample from each concentration were collected from formulation preparations near the middle of the dosing period and analysed to verify the concentration of elemental cobalt.
Concentrations of the cobalt neodecanoate in separate dosing formulation samples were measured by inductively coupled plasma (ICO) spectroscopy.

Results:
The recovery values obtained for these samples were 99 - 102 %.
The test substance was at the targeted levels (± 20 % of nominal); up to eight days after formulation), mixed uniformly, and was stable under the conditions of the study. Test substance was not found in 0 mg/mL samples.
Duration of treatment / exposure:
Males: 45 - 46 days
Non-pregnant females/pregnant females: 40 - 45 days
Frequency of treatment:
once daily
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
males and females
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
males and females
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
males only
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
females only
No. of animals per sex per dose:
12 males and /or 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a two-week range finding study (DuPont Haskell (2006))*, male and female rats were dosed with 50, 500, or 1000 mg/kg/day of the test substance at a dose volume of 10 mL/kg. The males at 500 and 1000 mg/kg/day were terminated due to mortality, clinical signs of toxicity, and body weight loss. At 50 mg/kg/day, mean final body weight and overall body weight gain were reduced in males (8% and 31% lower than the control group, respectively). There were no clinical signs of toxicity observed in males at 50 mg/kg/day.
The females at 1000 mg/kg/day were terminated due to mortality, clinical signs of toxicity, and body weight loss. There was test substance-related mortality in females at 500 mg/kg/day. At 500 mg/kg/day, mean final body weight and overall body weight gain were reduced in females (6% and 41% lower than the control group, respectively). Clinical signs of toxicity were observed in females at 500 mg/kg/day. There were no test substance-related effects on final body weight, overall body weight gain, or clinical signs of toxicity in females at 50 mg/kg/day.

* References:
DuPont Haskell Laboratory. (2006). Repeated Dose Oral Toxicity: 2-Week Gavage Study in Rats. Unpublished Report. DuPont-20764
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
1) daily animal health observations: at least once daily during the quarantine/pretest period and once daily during the dosing period
2) careful clinical observations: within 3 hours post-dosing

- Cage side observations checked:
1) daily animal health observations: mortality/moribund and abnormal behaviour and/or appearance
2) careful clinical observations: acute/systemic toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during pretest (baseline), and weekly (at least) thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
1) Females
- premating period: once a week
- gestation period: days 0, 7, 14 and 21 of gestation
- lactation period: days 0 and 4 of lactation
- females without evidence of copulation as well as those that copulated and did not deliver a litter: weekly schedule until sacrifice

2) Males: weekly schedule until sacrificeAll rats were weighed at terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:

Each feeder was weighed at the beginning and end of the weekly food consumption interval and the final weight of the feeder and the amount of spillage from the feeder during the interval were subtracted from the initial feeder weight. From the food consumption and body weight data, the mean daily food efficiency was calculated.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Premating and cohabitation periods: individual food consumption was determined weekly throughout the period, ending on test day 14. Food consumption was not measured during cohabitation for males and females or after cohabitation for males or females without evidence of copulation.
Gestation and lactation periods: individual food consumption of pregnant P1 females was determined on gestation days 0, 7, 14, and 21, and for lactating females on lactation days 0 and 4. Food consumption was not measured for females that did not deliver a litter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes
- Mean daily food efficiency (g weight gain/g food consumed)

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on test day 14 (hematology and clinical chemistry) and test days 46 and 41-46 (coagulation, males and females, respectively)
- Anaesthetic used for blood collection: yes (carbon dioxide)
- Animals fasted: Yes, at least 15 hours
- How many animals: first 5 surviving animals/sex/group
- Parameters examined: red blood cell count, red cell distribution width, hemoglobin, absolute reticulocyte count, hematocrit, platelet count, mean corpuscular (cell) volume, white blood cell count, mean corpuscular (cell) hemoglobin, differential white blood cell count, mean corpuscular (cell) hemoglobin concentration, microscopic blood smear examination, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on test day 14
- Animals fasted: Yes, at least 15 hours
- How many animals: first 5 surviving animals/sex/group
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, total protein, sorbitol dehydrogenase, albumin, alkaline phosphatase, globulin, total bilirubin, calcium, urea nitrogen, inorganic phosphorus, creatinine, sodium, cholesterol, potassium, triglycerides, chloride, and glucose

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of test substance administration and prior to the end of the premating period
- Dose groups that were examined: all animals
- Abbreviated functional observational battery (approach/touch, sharp auditory stimulus, and tail pinch, fore- and hindlimb grip strength, pupillary constriction) and motor activity were conducted (4 replicates for each evaluation)

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Rats that survived until the scheduled sacrifice were euthanized on days 46 – 47 (males) or days 41 – 46 (females). All animals were dosed until the day prior to sacrifice. Rats with poor health were submitted for euthanasia prior to the scheduled sacrifice.
Gross examinations were performed on all adult rats. The presence and number of uterine implantation sites and ovarian corpora lutea were evaluated for all cohabited females.

Following tissues were collected from all adults:
liver, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, urinary bladder, lung, trachea, heart, bone marrow (collected with sternum), thymus, spleen, mandibular lymph node, mesenteric lymph node, Peyer's patches (collected from sections of the intestine), thyroid gland, adrenal glands, brain (3 sections, including cerebrum, cerebellum, midbrain, and medulla/pons), spinal cord (3 sections, including cervical, mid-thoracic, and lumbar spinal cord), sciatic nerve, sternum, testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, vagina, and gross observations.

The following tissues were weighed wet from all adult rats:
liver, kidneys, heart, thymus, spleen, adrenal glands, brain, testes, epididymides, prostate, seminal vesicles (with coagulating glands and fluids), ovaries (with oviducts), and uterus (with cervix). Paired organs were weighed together. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated.

All tissues were fixed, processed, sectioned, stained and examined microscopically.

Processing and microscopic evaluation of tissues were as follows:
Scheduled sacrifice: all tissues were processed to slides and evaluated microscopically from up to 5 rats per sex that were sacrificed by design in the control as well as 40 mg/kg/day and 100 mg/kg/day groups (males and females, respectively). There was only 1 female of the 100 mg/kg/day group that survived to the scheduled sacrifice.

Decedents: all protocol tissues were processed to slides and evaluated microscopically from rats that were submitted for an unscheduled sacrifice due to deteriorating health and for rats that were found dead.
Reproductive failures: all reproductive tissues were processed to slides and evaluated microscopically from the male and female mated pairs that failed to produce a litter.

Target organs: target organ effects were limited to female rats.
The following target organs were evaluated microscopically from all adult female rats:
intestinal tract (duodenum, jejunum, ileum, cecum, colon, and rectum), Peyer’s patches, spleen, thymus, adrenal glands, and kidneys.
Statistics:
The following statistical tests were used:
- Levene's test for homogeneity
- Saphiro-Wilk test for normality
- One-way analysis of variance
- Dunnett's test
- Kruskal-Wallis test
- Dunn's test
- Sequential application of Cochran-Armitage test for trend
- Repeated measures analysis of variance followed by Linear contrasts
- Sequential application of the Jonckheere-Terpstra trend test
- Fisher’s Exact test with a Bonferroni correction

Male and female data were evaluated separately. The level of significance selected was p < 0.05.

*References:
Haseman, J.K. and Hogan, M.D. (1975). Selection of the experimental unit in teratology studies. Teratology, 12, 165-171
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Females (gestation period):
- 15 mg/kg/day: one female had red stained skin/fur on gestation day 21 in the daily post-dosing observation. Although the incidence of this observation was low, and red stained fur/skin is occasionally observed in reproduction studies, this sign was also observed at 100 mg/kg/day during the same time frame, and, therefore, is suggestive of a test substance-related effect on the 15 mg/kg/day dose group.
- 100 mg/kg/day: test substance-related clinical signs of toxicity occurred. Post-dosing clinical signs of dehydration, hunched-over posture, red stained skin/fur, laboured breathing, dystocia, wet fur, and/or diarrhea were observed beginning gestation day 20 in 10 of the 12 females. One female was removed from the study on gestation day 13 due to excessive toxicity. Entropathy was considered to be the cause of death in all decedents and was considered to be test substance related. One female had evidence of dystocia, which may have also been contributory to its early death.
Mortality:
mortality observed, treatment-related
Description (incidence):
Females (gestation period):
- 100 mg/kg/day: test substance-related mortality occurred. Eight dams were found dead and 2 were sacrificed in extremis during gestation days 21-22.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females (gestation period):
- 100 mg/kg/day: adverse, test substance-related, statistically significant decreases in body weight an d weight gain occurred. Body weight was 14% lower on gestation day 21 compared to controls. In addition, weight gain during gestation days 14-21 and 0-21 was 53% and 33% lower than control values, respectively

Please also refer to the field "Attached background material" below.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females:
Gestation:
- 15 mg/kg/day: test substance-related effects on food consumption parameters occurred. Food consumption during gestation days 0-21 was significantly decreased (10% lower than control group), resulting in slightly decreased (not statistically significant) weight gain for the same interval.
- 100 mg/kg/day: test substance-related effects on food consumption parameters occurred. Food consumption and food efficiency were significantly decreased (17%, 43%, and 12%, 24% lower than control group, respectively) on gestation days 14-21 and 0-21, and were considered to be adverse since they resulted in decreased body weight and weight gain during this period.

Lactation:
- 15 mg/kg/day: test substance-related effects on food consumption occurred. Food consumption was 18% lower compared to the control value.
- 100 mg/kg/day: test substance-related effects on food consumption occurred. Food consumption was 45 % lower than the control value for the one surviving 100 mg/kg/day female.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Females (gestation period):
- 100 mg/kg/day: food efficiency was significantly decreased on gestation days 14-21 and 0-21, and was considered to be adverse since it resulted in decreased body weight and weight gain during this period.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Females:
- 15 or 100 mg/kg/day: test substance-related microscopic findings were present,as stated below:

Intestinal tract:
- 100 mg/kg/day: several degenerative and regenerative changes were present in the small and large intestines of 10/11 females (test substance-related). These intestinal changes were interpreted to be thecause of death in all 10 female decedents.
- The following diagnoses were included: Degeneration/necrosis of mucosal epithelium (villous and crypt epithelium); atrophy of villi and crypts; regeneration of mucosal epithelium, and mucosal inflammation. Lesions were observed in all segments of the small intestine and in the cecum and colon. Lesions weregraded as minimal to severe (grades 1 to 4) and were most severe in the jejunum, ileum, and cecum. In the rectum, changes were limited to minimal depletion of mucous in goblet cells.

Lymphoid organs:
- 100 mg/kg/day: necrosis of lymphocytes and/or atrophy of lymphoid regions were present in the thymus, spleen, and/or Peyer’s patches in all female rats (11/11) (test substance-related).
- 15 mg/kg/day: thymic lymphoid atrophy was present in 6/12 females (test substance-related).
- lymphoid changes were graded as minimal to severe and were generally most consistent and most severe in the thymus.

Many rats with test substance-related enteropathy also had lymphoid necrosis or atrophy. However, several rats in the 15 mg/kg/day group had lymphoid atrophy in the thymus without microscopic evidence of enteropathy. Therefore, the lymphoid changes observed in female rats administered 15 mg/kg/day and above cannot be attributed solely to secondary effects of the severe systemic stress that occurred in 100 mg/kg/day females.

- 100 mg/kg/day: Minimal to mild increased microvesiculation of adrenal cortical cells was present in 10/11 females (test substance-related). Mild to severe focal adrenal cortical necrosis was also present in 3/11 females (test substance-related).
- 15 mg/kg/day: Minimal to mild increased microvesiculation of adrenal cortical cells was present in 1/12 females (test substance-related). Mild to severe focal adrenal cortical necrosis was also present in 1/12 females (test substance-related).
- 15 and 100 mg/kg/day: The change was diffuse within the cortex, and was characterized by multiple, small, clear vacuoles within the cytoplasm of adrenal cortical cells.
Some degree of microvesiculation is a normal finding in rats of this strain and age, and the diagnosis in this study represents an increase beyond that typically seen in controls. While these changes in the current study were considered to be test substance related, they were not considered adverse, as they were not associated with morphologic evidence of cytotoxicity to affected cells. Adrenal cortical necrosis may occur in control rats; however, a test substance-related effect for the single occurrence in the 15 mg/kg/day female group cannot be ruled out. The underlying pathogenesis of the adrenal cortical necrosis was not determined, but there was no evidence of an association between adrenal cortical necrosis and the microvesiculation noted above.

Kidneys:
- 100 mg/kg/day: Vacuolation of renal tubular epithelium was present in renal cortical tubules of 10/11 females (test substance-related).
- The lesions were graded as minimal in all but one animal and were characterized by small, discrete, clear granules in the basilar cytoplasm of renal cortical epithelial cells. This change was not associated with other microscopic findings or with clinical pathology changes indicative of renal injury. Therefore, vacuolation of renal tubules was not considered to be adverse.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1) Males:
- no test substance-related deaths
- no test substance-related clinical signs of toxicity
- all clinical observations were common for this age and strain of rats.

2) Females
:Premating:
- test substance-related mortality or clinical observations did not occur
- all clinical observations were common for this age and strain of rats.
Gestation:
- no test substance-related clinical signs of toxicity were observed during the evaluation of detailed clinical observations in the gestation period for any dosage.
- 5 mg/kg/day: test substance-related mortality or clinical observations did not occur.
- 15 mg/kg/day: no test substance-related mortality was observed.
Lactation:
- test substance-related mortality or clinical observations did not occur.

BODY WEIGHT AND WEIGHT GAIN
1) Males
- test substance-related or statistically significant effects on body weight parameters did not occur.

2) Females
Premating:
- test substance-related or statistically significant effects on body weight parameters did not occur.
Gestation:
- 5 mg/kg/day: weight gain was 24% and 18% lower on gestation days 14-21 and 0-21, respectively (not test item related effect).
- 15 mg/kg/day: weight gain for 15 mg/kg/day females was only 9% lower on gestation days 14-21 and 0-21.
Lactation:
- 5 and 15 mg/kg/day: test substance-related or statistically significant effects on body weight parameters did not occur.
- 100 mg/kg/day: only 1 dam survived to lactation, and this animal’s body weights on lactation days 0 and 4 were lower relative to the control group; however, this animal’s weight gain was comparable to the control group.

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

FOOD CONSUMPTION AND COMPOUND INTAKE/ FOOD EFFICIENCY
1) Males
- test substance-related effects on food consumption parameters did not occur.

2) Females
Premating:
- test substance-related effects on food consumption parameters did not occur.
Lactation:
- no statistically significant effects on food efficiency at any dosage.

HAEMATOLOGY
- no treatment-related changes in mean hematology parameters in male or female rats at test day 14.
- no statistically significant or treatment-related changes in coagulation parameters in male or female rats evaluated at test day 46 (male) or test days 41-46 (female).
The following statistically significant change in a group mean hematology parameter was considered to be unrelated to treatment (and non-adverse) because the change did not occur in a dose-related pattern:
- absolute reticulocyte count was minimally increased (131% of control) in male rats dosed with 15 mg/kg/day.

CLINICAL CHEMISTRY
- no statistically significant or treatment-related changes in clinical chemistry parameters in male or female rats evaluated at test day 14.

NEUROBEHAVIOUR
1) Abbreviated functional observational battery
- no test substance-related effects or statistically significant differences on forelimb and hindlimb grip strength in either males or females at any dose level. No test substance-related effects or statistically significant effects for any behavioural parameter evaluated in males and females at any dose level.
- 2 of the 12 males in the 40 mg/kg/day group and 8 of the 12 females in the 100 mg/kg/day group had dark coloured urine (no adverse effects associated with this observation).

2) Motor activity
- no test substance-related effects on duration of movement or number of movements in males and females at any dose level.

ORGAN WEIGHTS
1) Males
- no test substance-related organ weight changes at any dose level occurred.
The following statistically significant organ weight changes were considered unrelated to treatment or nonadverse:
- Thymus: statistically significant decreases in absolute thymus weight and thymus weight relative to brain weight were present in males in the 40 mg/kg/day group. However, there were no statistically significant changes in thymus weight relative to body weight and no microscopic changes in the thymus of male rats at this dose.
- Seminal vesicle: a statistically significant increase in absolute seminal vesicle weight was present in males in the 15 mg/kg/day group (change was not dose related).
- Liver: a statistically significant increase in liver weight relative to brain weight was present in males administered 40 mg/kg/day. This change was not associated with changes in other liver weight parameters or with microscopic changes in the liver.

2) Females
- no test substance-related organ weight changes occurred at any dose level.
- organ weight data were available for only one P1 female in the 100 mg/kg/day group, as organ weights were not collected on the 10 early decedents in that group.

GROSS PATHOLOGY/NEUROPATHOLOGY
- no test substance-related gross observations in adult male and female rats occurred.
- gross observations occurred in low incidences and/or were consistent with normal background lesions that occur in rats of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
1) Males
- no test substance-related findings in male rats at any dose level were observed.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
3.8 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
0.5 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Critical effects observed:
no
Conclusions:
Prior to this combined repeated dose toxicity study with a reproduction developmental toxicity screening test, a 14-day dose range-finder using male and female rats was performed. The rats were dosed by gavage with 50, 500, and 1000 mg/kg/day of the test substance (dosing volume: 10 mL/kg).

The following findings were made for males as follows:
1000 mg/kg bw/day: mortality, clinical signs of toxicity, and body weight loss
500 mg/kg bw/day: mortality, clinical signs of toxicity, and body weight loss
50 mg/kg bw/ day: reduced mean final body weight (8 % lower than control), reduced overall body weight gain (31% lower than control)

The findings for the females were as follows:
1000 mg/kg bw/day: mortality, clinical signs of toxicity, and body weight loss
500 mg/kg bw/day: mortality, clinical signs of toxicity, reduced mean final body weight (6 % lower than control), reduced overall body weight gain (41 % lower than control)
50 mg/kg bw/ day: no test substance-related effects occurred. No treatment related effects were observed for haematology and clinical chemistry in males and females at any dose level.

In the males of all dose levels no test substance related effects were observed during pathology, and clinical observations. No test substance-related mortality was observed for males and females in the 5 and 15 mg/kg/day group, whereas test substance-related mortality occurred in the females of the 100 mg/kg/day group during the gestation period (8/10 female rats were found dead and 2/12 female rats were sacrificed prior to scheduled termination). Enteropathy was considered to be the cause of death in all decedents and was considered to be test substance related.

Test item related clinical signs of toxicity occured in the females of the 15 and 100 mg/kg/day dose groups. Cage-side observations revealed red-stained skin/fur on gestation day 21 in 1/12 female rat from the 15 mg/kg/day dose group and additionally dehydration, hunched-over posture, laboured breathing, dystocia, wet fur and/or diarrhea were observed beginning gestation day 20 in 10/12 females in the females of the 100 mg/kg/day dose group.
Test item-related effects were observed in females on body weight, body weight gain, food consumption and food efficiency. In the females of the 100 mg/kg/day dose group, body weight decreased about 14 % on gestation day 21 compared to control group and body weight gains decreased about 53 % during gestation days 14 – 21 as well as about 33 % during gestation days 0 – 21 compared to the control group. In the females of the 15 mg/kg/day group, the food consumption during gestation days 0 - 21 was significantly decreased (10 % lower than control group), resulting in slightly decreased (not statistically significant) weight gain for the same interval. Also, food consumtpion of this dose group was 18 % lower compared to the control value during the lactation period. Furthermore, in the females of the 100 mg/kg/day group food consumption decreased about 17 % and 12 % during gestation days 14 – 21 and gestation days 0 – 21, respectively, and were considered to be adverse since they resulted in decreased body weight and weight gain during this period. Also, food efficiency was signficantly decreased on gestation days 14 - 21 and 0 - 21 in the 100 mg/kg/day dose group females, and was considered to be adverse sine it resulted in decreased body weight and weight gain during this period. Lastly, during the lactation period food consumption was 45 % lower than the control value for the one surviving 100 mg/kg/day female.
In female rats, several test substance-related non-neoplastic changes were recorded in the 15 and 100 mg/kg/day dose groups. In the 15 mg/kg/day group, thymic lymphoid atrophy (6/12), ninimal to mild increased microvesiculation of adrenal cortical cells (1/12; not considered adverse) and focal adrenal cortical necrosis (1/12) were observed. In the females of the 100 mg/kg/day dose group several degenerative and regenerative changes in small and large intestines were observed in 10/11 rats, leading to enteropathy and contributed to death prior to scheduled sacrifice. Additionally, necrosis of lymphocytes and/or atrophy of lymphoid regions (thymus, spleen, and/or Peyer's patches; 11/11), ninimal to mild increased microvesiculation of adrenal cortical cells (10/11; not considered adverse), mild to severe focal adrenal cortical necrosis (3/11) and vacuolation of renal tubular epithelium in renal cortical tubules (10/11; not considered adverse) were observed.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males was determined to be 40 mg/kg/day (equivalent to 3.8 mg cobalt/kg bw/day). A NOAEL of 5 mg/kg/day (equivalent to 0.5 mg cobalt/kg bw/day) was determined for females based on clinical findings, mortality, histopathological findings as well as on effects on body weight, body weight gain, and food consumption.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
Study duration:
subacute
Species:
rat
System:
gastrointestinal tract

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.

 

Cobalt, borate neodecanoate complexes is assigned to the read-across groups (i) oral-systemic: poorly soluble in aqueous solutions with organic ligand

As discussed in the chapter on inhalation read-across (Appendix 1.2 of the CSR), the substance cobalt, borate neodecanoate complexes has not yet been assigned to either inhalation-local read-across group due to ongoing testing. The registrant will update the dossier immediately upon availability of the test data.

 

Toxicological relevance of the non-common compound

The toxicological relevance of the non-common compound in cobalt, borate neodecanoate complexes 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

 

Repeated dose toxicity

 

Cobalt sulfate

In 13-week inhalation toxicity studies, groups of 10 F344 rats and 10 B6C3F1 mice of each sex were exposed to aerosols of cobalt sulfate heptahydrate at concentrations of 0, 0.3, 1.0, 3.0, 10 or 30 mg/m³ (0, 0.063, 0.21, 0.63, 2.1 or 6.3 mg Co/m³), 6 hours/day, 5 days/week (Bucher et al., 1990). The MMAD of the aerosol was in the range of 0.83 to 1.10 µm. Mean body weights of mice exposed to 30 mg/m³ were lower than those of the controls throughout the study, and two of 10 males in this group died before the end of the study. At the end of the studies, lung weights were generally increased in rats and mice exposed to 1 mg/m³ and higher. The above described exposure of rats and mice to aerosols of cobalt sulfate heptahydrate resulted primarily in necrotising injury to the respiratory tract. The larynx appeared to be the most sensitive tissue, showing squamous metaplasia lesions after exposure at concentrations as low as 0.3 mg/m³ cobalt sulfate heptahydrate (equivalent to 0.063 mg cobalt/m³). Rats developed chronic inflammation of the larynx at concentrations of 1 mg/m³ and more severe effects in the nose, larynx, and lung at higher concentrations. Mice exhibited acute inflammation of the nose at concentrations of 1 mg/m³ and more severe effects in the nose, larynx, and lung at higher exposures. A NOAEC for local effects in the respiratory was not reached in these studies, as lesions, particularly in the larynx, were observed at the lowest concentration of 0.3 mg/m³ cobalt sulfate thus representing a LOAEC.

 

Thyroid function as indicated by serum T3, T4, and thyrotropin concentrations did not appear to be consistently affected in rats. Polycythemia was seen at 10 and 30 mg/m³ for female rats and at concentrations of 3 mg/m³ for male rats. No consistent significant haematological effects were seen in mice. At 6.3 mg Co/m³, mice showed hyperplasia of the mediastinal lymph nodes.

 

Reproductive system effects were more prominent in mice than in rats.Decreases of testicular and epididymal weights and of sperm counts and increased numbers of abnormal sperm occurred in mice exposed to 30 mg/m³. The NOAEC for testicular weight decrease in mice is 10 mg/m³. Sperm motility was significantly reduced in mice at 0.63 mg Co/m³ (lower concentrations were not evaluated) and at higher concentrations. In female mice, the oestrous cycle was significantly longer in the highest dose group. However, it is unclear to what extent the changes of these reproductive parameters were associated with a decline in fertility because effects on fertility were not studied. No statistically significant effects on sperm motility, sperm counts, or the incidence of abnormal sperm were observed in F344 rats exposed under identical dosing conditions.

 

Shortcomings of the study: The study report presents data on the effect on the respiratory tract in mice and rats in all dose groups comprehensively, and therefore allows the derivation of a NOAEC/LOAEC for local effects. However, since only selected species and/or dose groups were chosen for (i) histopathological examination (and where presented, the severity of such lesions is not indicated), (ii) investigation of reproductive system data, (iii) serum and thyroid function values, neither target organs for systemic toxicity nor any dose-response relationship for systemic effects can be determined. The body weight and clinical data which are available for both species and dose groups allow only the establishment of a NOAEC for general toxicity, but do not address non-lethal organ toxicity of minimal or moderate severity.

 

In a subsequent combined chronic inhalation toxicity/carcinogenicity studies, groups of male and female F344/N rats and B6C3F1 mice were exposed to aerosols of cobalt sulfate hexahydrate at concentrations of 0, 0.3, 1.0, or 3.0 mg/m³ for 6 hours/day, 5 days/week for 105 weeks (Bucher et al.). The MMAD (µm) ± GSD of the aerosol was in the range from 1.4 ± 2.1 to 1.6 ± 2.2. The study represents a highly reliable study without restrictions (RL 1). The respiratory tract was the primary site of non-neoplastic lesions and neoplasms.

 

In rats, proteinosis, alveolar epithelial metaplasia, granulatomous alveolar inflammation, and interstitial fibrosis were observed in the lung of all exposed groups. The incidence of hyperplasia of the respiratory epithelium of the lateral wall of the nose and atrophy of the olfactory epithelium in all exposed groups was significantly greater than those in controls, and the severity of these lesions increased with increasing exposure concentration. Nasal lesions in mice were less severe than in rats, but olfactory epithelial atrophy was observed at 1.0 mg/m³. The incidence of squamous metaplasia of the epiglottis in all exposed groups of rats and mice was significantly increased, and the severity of this lesion increased in rats with higher concentrations as well.

 

Taken together, 2-year exposure of rats and mice to cobalt sulfate resulted in non-neoplastic lesions of the nose, larynx and lung at all concentrations studied. Taking into account the lack of a NOAEC in the concentration-response assessment of cobalt sulfate a benchmark dose (BMD) was calculated using the US EPA BMD software (Version 2.0) with the Gamma Model (Version 2.13). The numbers of alveolar/bronchiolar adenoma or carcinoma in the lung of rats and mice were selected as benchmark response. The 95% lower confidence limit of the BMD for a treatment-related increase in response of 10% was calculated (BMDL10). The lowest BMDL10 value was that for female rat tumours with 0.414 mg/m³ cobalt sulfate which is equivalent to a cobalt concentration of 0.093 mg/m³.

 

 

Cobalt metal

Groups of five male and five female core study rats were exposed to cobalt metal particulate aerosol by inhalation at concentrations of 0, 2.5, 5, 10, 20, or 40 mg/m³, 6 hours per day, 5 days per week for 16 days. Additional groups of five female rats were exposed to the same concentrations for 16 days for tissue burden studies. All rats exposed to 40 mg/m³ and all male and three female rats exposed to 20 mg/m³ died before the end of the study. Mean body weights of males exposed to 10 mg/m³ and of females exposed to 10 or 20 mg/m³ were significantly decreased. Females exposed to 20 mg/m³ lost weight during the study.

Exposure-related clinical findings included abnormal breathing, lethargy, and thinness in male rats exposed to 20 or 40 mg/m³, and in females exposed to 40 mg/m³. Dark lungs were observed at necropsy in all rats exposed to 40 mg/m³ and most rats exposed to 20 mg/m³ that died early.

Increased incidences of nonneoplastic lesions of the lung occurred in exposed male and female rats and included haemorrhage, acute inflammation, alveolar epithelium hyperplasia, histiocytic cellular infiltration of the alveolus, cytoplasmic vacuolization of bronchiolar epithelium, necrosis of the bronchiolar epithelium, and interstitial fibrosis of the alveolar epithelium. Increased incidences of nonneoplastic lesions of the nose occurred in exposed male and female rats and included olfactory epithelium necrosis, olfactory epithelium atrophy, respiratory epithelium necrosis, and respiratory epithelium squamous metaplasia. Tissue concentrations of cobalt increased with increasing exposure concentration in all tissues examined.

 

Groups of 10 male and 10 female core study rats were exposed to particulate aerosols of cobalt metal by inhalation at concentrations of 0, 0.625, 1.25, 2.5, or 5 mg/m³, 6 hours per day, 5 days per week for 14 weeks. Additional groups of 10 male rats (clinical pathology study) were exposed to the same concentrations for 14 weeks. All male and female rats survived to the end of the study. Final mean body weights of males and females exposed to 5 mg/m³ were significantly less than those of the chamber controls, and the mean body weight gain of 5 mg/m³ males was significantly less than that of the chamber controls. At necropsy, pale foci were noted in the lungs of most exposed male and female rats. In male rats, exposure concentration-related increases in the haemoglobin concentration, erythrocyte count, haematocrit value, and manual packed cell volume occurred in the 2.5 and 5 mg/m³ groups on days 3 and 23 and in all exposed groups by week 14; at week 14, female rats also had increases in these parameters. Exposure concentration-related decreases in cholesterol concentrations were observed at all three time points in male and female rats. While this change was not always observed in the lower exposure groups, decreases were consistently observed in the 2.5 and 5 mg/m³ groups of both sexes on day 23 and at week 14. In addition, glucose concentration was decreased in males exposed to 1.25 mg/m³ or greater at week 14.

In the lung, chronic active inflammation and alveolar proteinosis occurred in all exposed males and females, and bronchiole epithelium hyperplasia occurred in all males and females exposed to 1.25 mg/m³ or greater. In the nose, incidences of olfactory epithelium degeneration and respiratory epithelium hyperplasia were significantly increased in males and females exposed to 2.5 or 5 mg/m³. The incidences of olfactory epithelium hyperplasia were significantly increased in 2.5 and 5 mg/m³ males and in 5 mg/m³ females. Significantly increased incidences of turbinate atrophy occurred in 2.5 mg/m³ females and 5 mg/m³ males and females. Tissue concentrations of cobalt increased with increasing exposure concentration in all tissues examined.,

 

Groups of five male and five female mice were exposed to cobalt metal particulate aerosol by inhalation at concentrations of 0, 2.5, 5, 10, 20, or 40 mg/m³, 6 hours per day, 5 days per week for 17 days. Three male and three female mice exposed to 40 mg/m³ died before the end of the study. Final mean body weights were significantly decreased in male and female mice exposed to 20 or 40 mg/m³, and mean body weight gains of 20 and 40 mg/m³ males and all exposed groups of females were significantly less than those of the chamber controls. Females exposed to 20 mg/m³ and males and females exposed to 40 mg/m³ lost weight during the study. Exposure-related clinical findings included abnormal breathing, lethargy, and thinness in male mice exposed to 20 or 40 mg/m³ and females exposed to 10 mg/m³ or greater. At necropsy, tan lungs were observed in most males and females exposed to 20 or 40 mg/m³. Lung weights of both sexes exposed to 10 mg/m³ or greater were significantly greater than those of the chamber controls. Liver weights of exposed male and female mice were significantly less than those of the chamber controls (except relative weight at 40 mg/m³). Increased incidences of nonneoplastic lesions of the lung occurred in exposed male and female mice and included alveolar histiocytic cellular infiltration, cytoplasmic vacuolization of the bronchiolar epithelium, alveolar/bronchiolar epithelium karyomegaly, interstitial fibrosis, and acute inflammation. Increased incidences of nonneoplastic lesions of the nose occurred in exposed groups of male and female mice and included acute inflammation, olfactory epithelium atrophy, olfactory epithelium necrosis, cytoplasmic vacuolization of the respiratory epithelium, and squamous metaplasia of the respiratory epithelium. Tissue concentrations of cobalt increased with increasing exposure concentration in all tissues examined.

 

Groups of 10 male and 10 female core study mice were exposed to particulate aerosols of cobalt metal by inhalation at concentrations of 0, 0.625, 1.25, 2.5, 5, or 10 mg/m³, 6 hours per day, 5 days per week for 14 weeks. One 2.5 mg/m³ female mouse was accidentally killed during the first week of the study; all other mice survived to the end of the study. The mean body weights of males and females exposed to 10 mg/m³ were significantly less than those of the chamber controls. Abnormal breathing was noted in approximately 50% of males and females exposed to 10 mg/m³. At necropsy, tan lungs were noted in mice exposed to 5 or 10 mg/m³. Lung weights of males exposed to 2.5 mg/m³ or greater and females exposed to 5 or 10 mg/m³ were significantly greater than those of the chamber controls. Liver weights of males exposed to 10 mg/m³ and females exposed to 2.5 mg/m³ or greater were significantly less than those of the chamber controls. Kidney weights of males and females exposed to 5 or 10 mg/m³ were significantly less than those of the chamber controls. Testes weights of males exposed to 5 or 10 mg/m³ were significantly less than those of the chamber controls.

In the lung, alveolar histiocytic cellular infiltration and bronchiole epithelium cytoplasmic vacuolization occurred in the lung of all exposed male and female mice. Bronchiole epithelium hyperplasia occurred in all mice exposed to 2.5 mg/m³ or greater. Alveolar proteinosis and alveolar/bronchiolar epithelium karyomegaly occurred in all males and females exposed to 5 or 10 mg/m³. The incidences of haemorrhage were significantly increased in 5 mg/m³ females and in 5 and 10 mg/m³ males. In the nose, the incidences of olfactory epithelium degeneration were significantly increased in males and females exposed to 1.25 mg/m³ or greater. Incidences of respiratory epithelium degeneration were significantly increased in males exposed to 1.25 mg/m³ or greater and females exposed to 2.5 mg/m³ or greater. Incidences of respiratory epithelium squamous metaplasia were significantly increased in males and females exposed to 2.5 mg/m³ or greater, and incidences of turbinate atrophy and chronic active inflammation were significantly increased in the 5 and 10 mg/m³ groups of males and females. The incidences of squamous metaplasia were significantly increased in the larynx of all exposed groups of males and females. Tissue concentrations of cobalt increased with increasing exposure concentration in all tissues examined.

 

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

·        Miniature swine were exposed to an inhalation of pure cobalt metal powder concentrations of 0.1 and 1.0 mg/m³ (Kerfoot, 1973 and 1975). Early detection of pulmonary disease is apparent from the pulmonary function tests showing a mark decrease in lung compliance, and from electron microscopy showing an increase in the amount of septal collagen.

·        A group of sensitised and non-sensitised guinea pigs were exposed by inhalation to 2.4 mg/m³ cobalt dichloride for six hours a day for two weeks. For the sensitised group, much more lavage liquid was retained in the lungs than in the other groups, and the percentage of neutrophils and eosinophils tended to be higher than in the non-sensitised exposed group.

·        In a publication series by one working group (Johansson, 1980, 1983, 1984, 1986, 1987, 1991, 1992, Berghem, 1987, Johansson and Camner, 1986, Camner and Johansson, 1992), male rabbits were exposed for 4-16 weeks to cobalt dichloride and concentrations of 0.5-2.0 mg/m³. Exposed animals had hyperplasia of type II cells in the lung with small nodules formed, interstitial inflammation, and increased number and activity of alveolar macrophages.

 

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).

 

Although two carcinogenicity studies with cobalt metal and cobalt sulfate are available, the cobalt metal study will not be considered for risk assessment purposes. The dose descriptor derived from this study (NOAEC: 1.25mg Co/m³) is above the BMDL10 derived from the carcinogenicity study with cobalt sulfate. Following the chronic inhalation exposure of cobalt sulfate in rats and mice, the 95% lower confidence limit of the BMD for a treatment-related increase in response of 10% was calculated (BMDL10), in which the numbers of alveolar/bronchiolar adenoma or carcinoma in the lung of rats and mice were selected as benchmark response (BMDL10: 92.7µg Co/m³). Since the carcinogenic mode of action is identical for both substances, both studies could be used for risk assessment purposes interchangeably. However, the point of departure derived from the cobalt sulfate study is ensuring a higher level of protection for humans, as it provides a significantly lower point of departure and a subsequent lower DNEL value for workers and consumers.

 

Animal data - oral

 

Based on the read-across approach presented in Appendix 1.1 of the CSR the substances of the poorly soluble cobalt in aqueous solution with organic ligand cobalt substances are grouped and assessed for their hazardous properties using weight of evidence information from all available repeated dose toxicity studies of that group as summarised below.

 

In addition to the findings for the systemic toxicity following oral administration, additional findings for the cobalt carboxylates need to be taken into account for hazard and risk assessment. Four repeated dose toxicity studies in rats via oral application are available for different members of the poorly soluble cobalt in aqueous solution with organic ligand cobalt substances. The lowest dose descriptor derived from the existing oral repeated dose toxicity studies will be used as point of departure for the DNEL derivation of all members of the poorly soluble cobalt in aqueous solution with organic ligand cobalt substances. The predominant findings in the studies were as follows:

 

Cobalt stearate: The dose level of 5 mg/kg bw/d represents the NOAEL for toxicity in female rats based on decreased body weight and food consumption, clinical signs of toxicity, mortality, and microscopic pathology effects (degeneration/necrosis of mucosal epithelium (villous and crypt epithelium); atrophy of villi and crypts; regeneration of mucosal epithelium, and mucosal inflammation. Lesions were observed in all segments of the small intestine and in the cecum and colon. Lesions were graded as minimal to severe (grades 1 to 4) and were most severe in the jejunum, ileum, and cecum.) at 15 and/or 100 mg/kg bw/d. The no-observed-adverse-effect level (NOAEL) for systemic toxicity in males was 40 mg/kg bw/d, the highest dose level tested.

 

Cobalt Borate Neodecanoate: There were no test substance-related effects on mortality, body and organ weights, food consumption, haematology, clinical chemistry, neurobehavioral parameters, pathology and histopathology at any dose level. The no-observed-adverse-effect level (NOAEL) for systemic toxicity in males and females was 5 mg/kg bw/d, the highest dose level tested.

 

Cobalt neodecanoate: Under the conditions of this study, a no-observed-adverse-effect level (NOAEL) for systemic toxicity was not achieved due to effects on mortality, clinical signs of toxicity, body weight parameters, and food consumption parameters at all dosages in males and females. A LOAEL of 5 mg/kg bw/day (equivalent to 0.7 mg cobalt/kg bw) was determined for male and female rats. Adverse effects were observed in males and female animals already at the lowest dose, manifested as decreased weight gain, lethargy, gastro-enteropathy and subsequent secondary effects.

 

Resin acids and Rosin acids, cobalt salts: The lowest dose of 15 mg/kg bw/d Resin acids and Rosin acids, cobalt salts was derived as no-observed-adverse-effect level (NOAEL), based on reduced body weight gain, mean body weights, changes in some haematology factors and histopathology finding in the spleen in male rats at 50 mg/kg bw/d. Moderate epithelial degeneration/necrosis was noted in the cecum and colon at high dose.

 

Table. Overview on repeated dose toxicity studies via oral application

 

 

Study type

Most relevant quantitative dose descriptor

Cobalt stearate

Co content: 9.5 %

Doses:

M 0, 5, 15, 40 mg/kg/day;

F 0, 5, 15, 100 mg/kg/day

12/sex/dose level

Exposure duration:
M d 46-47, F d 41-46

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

NOAEL(male)=
40 mg/kg bw/day
3.8 mg Co/kg bw/day

NOAEL(female, pregnant)=
5 mg/kg bw/day
0.5 mg Co/kg bw/day

Cobalt borate neodecanoate

Co content: 22.15%

Doses 0, 0.5, 1.5, and 5 mg/kg/day

12/sex/dose level

Exposure duration:
M d 47-48, F d 39-50

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

NOAEL(male)=
5 mg/kg bw/day
1.1 mg Co/kg bw/day

NOAEL(female, pregnant)=
5 mg/kg bw/day
1.1 mg Co/kg bw/day

Cobalt neodecanoate

Co content: 14.32%

Doses 0, 5, 15, or 45 mg/kg/day

12/sex/dose level

Exposure duration:
M d 48-49, F d 42-49

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

LOAEL(male)=
5 mg/kg bw/day
0.7 mg Co/kg bw/day

LOAEL(female, pregnant)=
5 mg/kg bw/day
0.7 mg Co/kg bw/day

Resin acids and Rosin acids, cobalt salts
Co content: 7.77%
Purity: 83.7%

Doses 15, 50, 150 mg/kg/day

5/sex/dose level

Exposure duration: 29 d

OECD 407 (Repeated Dose (28 Days) Toxicity (Oral))

NOAEL(male)=
15 mg/kg bw/day
1.17 mg Co/kg bw/day

NOAEL(female)=
15 mg/kg bw/day
1.17 mg Co/kg bw/day

The LOAEL derived for cobalt neodecanoate is used as point of departure for the derivation of DNELs-oral for the general population. The substance specific DNELs were converted in equivalent cobalt concentrations. The lowest equivalent cobalt concentrations will be used further in the hazard assessment of the poorly soluble in aqueous solution with organic ligand cobalt substances group.

 

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 being registered by the Cobalt Consortia and the results of a dermal absorption study with a cobalt salt of high in vitro bioaccessibility in artificial sweat, it is reasonable to conclude that the inorganic cobalt substances have a negligible rate of absorption through the skin. A dermal absorption rate of 0.38% for the low exposure scenarios (ca 31.9μg Co/cm² loading) and 1.08% for the high exposure scenarios (ca 319μg Co/cm² loading) was determined. These values also account for part of the material associated with the stratum corneum and the test was conducted with a highly water soluble form of Cobalt in an aqueous solution. Thus, these values are considered to represent a conservative estimate.

 

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

 

Conclusions - oral

 

Findings of local gastrointestinal toxicity for the poorly soluble in aqueous solution with organic ligand cobalt substances group need to be taken into account for hazard and risk assessment.

 

In a number of oral repeated dose toxicity studies, the cobalt carboxylates show adverse effects as follows:

·        Decreased body weight and food consumption, clinical signs of toxicity

·        Intestinal effects including the following diagnoses: degeneration/necrosis of mucosal epithelium (villous and crypt epithelium); atrophy of villi and crypts; regeneration of mucosal epithelium, and mucosal inflammation

·        Effects on the haematopoietic system were apparent in high doses, already showing significant adverse effects in the digestive tract.

 

It is assumed that these local effects are caused by the surface active/amphiphilic organic anions, exerting an enhancing/promoting effect of the irritating properties of the cobalt cation in the gastro-intestinal tract.

 

For risk assessment purposes, a read-across is applied by using the lowest cobalt equivalent dose descriptor derived from the oral repeated dose toxicity study oral study with cobalt neodecanoate (i.e.LOAEL = 5 mg cobalt neodecanoate/kg bw/day or 0.7 mg Co/kg bw/day) for the calculation of the substance-specific DNEL (oral, systemic, long-term) for all members of the poorly soluble in aqueous solution with organic ligand cobalt substances group.

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

 

References

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

 

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

 

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

 

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

 

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

Justification for classification or non-classification

The above discussed repeated dose toxicity studies are considered in a weight of evidence with regard to the conclusion for hazard assessment. The severity of the gastrointestinal irritation lead to the decision to self-classify all the poorly soluble in aqueous solutions with organic ligand cobalt substances with specific target organ toxicity after repeated exposure, category 1 (STOT-RE 1, H372- GI tract).