Naphthenic acids, cobalt salts

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Stearic acid, cobalt salt

EC Number:

237-016-4

EC Name:

Stearic acid, cobalt salt

Cas Number:

13586-84-0

IUPAC Name:

Stearic acid, cobalt salt

Test material form:

solid: pellets

Details on test material:

- Name of test material (as cited in study report): Cobalt (II) stearate
- State of aggregation: purple solid pastille

Test animals

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 or test system 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

males and females

Doses / concentrations 2

Dose / conc.:

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

Remarks:

males and females

Doses / concentrations 3

Dose / conc.:

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

Remarks:

males only

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

3

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

mortality

4

Dose descriptor:

NOAEL

Effect level:

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

Based on:

element

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

mortality

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

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.