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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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
Vehicle:
corn oil
Remarks:
Mazola®
Details on 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
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: until evidence of copulation was observed or until 2 weeks had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): On the day copulation was confirmed, the female was transferred back to individual cage housing.
Days 0 - 19 of gestation: stainless steel, wire-mesh cages suspended above cageboards
Day 20 of gestation - Day 4 of lactation: polycarbonate pans with bedding
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
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
1) cage-side 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 toxicityStarting on day 20 of gestation for mated females or 7 days after the end of the cohabitation period for females without evidence of copulation, female rats were observed at least twice daily for signs of delivery and offspring.

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
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in male parental generations:
- organ weights: testis, epididymis, prostate, seminal vesicles (with coagulating glands and fluids)
Litter observations:
The day when delivery was complete was designated day 0 postpartum.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Day 0 postpartum:
- abnormality, abnormal behaviour, appearance, and mortality
- number and sex of live and dead pups of each litter
- body weight of live pups of each litter
Day 4 postpartum:
- abnormality, abnormal behaviour, appearance, and mortality
- number and sex of live pups of each litter
- body weight and gross external examination of live pups of each litter

GROSS EXAMINATION:
Gross and microscopic examination was not conducted on any F1 pups. Organs were not weighed.
Postmortem examinations (parental animals):
SACRIFICE
Rats that survived to the scheduled sacrifice were euthanized on days 47–48 (males) or days 41– 50 (females).

GROSS NECROPSY
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 degistive tract), thyroid gland, adrenal glands, brain (3 sections, including cerebrum, cerebellum, midbrain 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
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:
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.
Postmortem examinations (offspring):
GROSS NECROPSY / HISTOPATHOLOGY
Gross and microscopic examination was not conducted on any F1 pups. Organs were not weighed.
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
- Analysis of covariance
- Dunnett-Hsu- Non-parametric analysis of covariance
- 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. For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation (Haseman and Hogan, 1975).
* 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.
Reproductive indices:
Mating index (%) = (number mated*/number paired) x 100
Fertility index (%) = (number pregnant**/number mated*) x 100
Post-implantation Loss (%)*** = ((number of implantation sites - number of pups born)/number of implantation sites) x 100

* evidence of copulation = intravaginal copulatory plug, sperm in vaginal lavage, uterine implantation sites, or delivery of a litter.
** including those found dead pregnant during gestation.
*** determined for each litter.
Offspring viability indices:
Live born index (%)*= (number of pups born alive / number of pups born) x 100
Viability index (%)** = (number of pups alive PND 4 preculling / number of pups born alive) x 100

* determined for each litter.
** determined for each litter.; excluding litters sacrificed due to death of dam during lactation
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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).

REPRODUCTIVE PERFORMANCE
- no test substance related effects on mating, fertility, gestation length, number of implantation sites, post-implantation loss, or number of corpora lutea at any dose level
- failure of five adult pairs to produce litters was not test substance related. Three control pairs, one pair of the 1.5 mg/kg bw/day group, and one pair of the 5 mg/kg bw/day group had no gross or microscopic pathology to explain their reproductive failure.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
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
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
- no test substance-related effects on the number of pups born, born alive, and alive on day 4 as well as no effects on survival indices during lactation days 0-4 at any dose level.

CLINICAL SIGNS (OFFSPRING)
- no test substance-related clinical observations in the pups at any dose level tested.

BODY WEIGHT (OFFSPRING)
- no test substance-related effects on mean pup weight either at birth or on day 4 of lactation at any dose level tested.

OTHER FINDINGS (OFFSPRING)
Sex ratio: an apparent test substance-related statistically significant increase in mean sex ratio at 5 mg/kg/day as compared with the control group mean was found. The mean values for the control and 5 mg/kg/day groups, 39.39 and 64.96, respectively, were both outside of the range typically observed in control groups (historical control data of laboratory: 0.45 to 0.59). The low control group mean appears to be largely the result of data from one litter comprised mainly of females; whereas, the elevated 5 mg/kg/day group mean appears to be resulting from three litters with higher numbers of male pups. These data suggest a possible tendency towards increased masculinization of offspring at 5 mg/kg/day. This interpretation should be viewed as conservative and in the proper perspective. It is important to consider that these are screening study data and as such, the group sizes are relatively small. In addition, the lack of any other test substance-related and/or corroborative toxicity precludes drawing definitive conclusions from these data.
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable
Critical effects observed:
no
Reproductive effects observed:
not specified
Conclusions:
The controls had a sex ratio of 39.39% males (more females and outside the historical control range), and the 5.0 mg/kg/day group had a pup sex ratio of 64.96% (more males, and again outside the historical control range of 45-59% males). However, there is no evidence of increased preimplantation loss (the difference between the number of ovarian corpora lutea as a measure of eggs ovulated) or of postimplantation loss at 5.0 mg/kg/day (numbers of uterine implantation sites as a measure of conceptuses which implanted), expected if the female offspring preferentially died, and no evidence of reduced litter size (again expected if female offspring were lost).
There is also no evidence of reduced pup body weights per litter at any group, including the 5.0 mg/kg/day group on pnd 0 or 4. However, the report did not present pup body weights by sex by litter, so it is unknown whether the body weight at 5.0 mg/kg/day was accurate or from the excess males (males are heavier) at the top dose; there might have been a body weight effect.There are no endpoints (e.g., body weights, feed consumption, clinical signs, behavioral tests, clinical chemistry, organ weights, histopathology, etc.) which indicate any toxicity to the adults or offspring.
This study did not identify a dose with toxicity to either the adults or the offspring.
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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: approximately 65 days; females: approximately 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
Vehicle:
corn oil
Remarks:
Mazola®
Details on 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.
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: until evidence of copulation was observed or until a period of 2 weeks had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): On the day copulation was confirmed, the female was transferred back to individual cage housing.
Days 0 - 19 of gestation: stainless steel, wire-mesh cages suspended above cageboards
Day 20 of gestation - Day 4 of lactation: polycarbonate pans with bedding
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 were 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 (2006a))*, 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.
In a study with a similar cobalt containing compound (DuPont Haskell (2006b))*, decreasing the dose volume from 10 to 5 mL/kg resulted in significantly reduced toxicity at 30 mg/kg/day. Based on these results, the concentrations selected for the current study were 0, 5, 15, and 45 mg/kg/day.

*References:
- DuPont Haskell (2006a). Repeated Dose Oral Toxicity: 2-Week Gavage Study in Rats. Unpublished Report. DuPont Haskell HL- 27601
- DuPont Haskell (2006b). Repeated Dose Oral Toxicity: 2-Week Gavage Study in Rats. Unpublished Report. DuPont Haskell HL- 27601
Positive control:
none
Parental animals: Observations and examinations:
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
Starting on day 20 of gestation for mated females or 7 days after the end of the cohabitation period for females without evidence of copulation, female rats were observed at least twice daily for signs of delivery and offspring.

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
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in male parental generations:
- organ weights: testis, epididymis, prostate, seminal vesicles (with coagulating glands and fluids)
Litter observations:
The day when delivery was complete was designated day 0 postpartum.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Day 0 postpartum:
- abnormality, abnormal behaviour, appearance, and mortality
- number and sex of live and dead pups of each litter
- body weight of live pups of each litter
Day 4 postpartum:
- abnormality, abnormal behaviour, appearance, and mortality
- number and sex of live pups of each litter
- body weight and gross external examination of live pups of each litter

GROSS EXAMINATION OF DEAD PUPS:
A gross postmortem examination was conducted for pups that were moribund, found dead, or sacrificed because of death of the dam during lactation.
Postmortem examinations (parental animals):
SACRIFICE
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 NECROPSY
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.

HISTOPATHOLOGY / ORGAN WEIGHTS
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).
Postmortem examinations (offspring):
SACRIFICE/GROSS NECROPSY
A gross postmortem examination was conducted for pups that were moribund, found dead, or sacrificed because of death of the dam during lactation.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organs were not weighed and no microscopic examinations were conducted on any F1 pups.
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
- Analysis of covariance
- Dunnett-Hsu- Non-parametric analysis of covariance
- 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. For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation (Haseman and Hogan, 1975).
* 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.
Reproductive indices:
Mating index (%) = (number mated*/number paired) x 100
Fertility index (%) = (number pregnant**/number mated*) x 100
Post-implantation Loss (%)*** = ((number of implantation sites - number of pups born)/number of implantation sites) x 100

*evidence of copulation = intravaginal copulatory plug, sperm in vaginal lavage, uterine implantation sites, or delivery of a litter.
** including those found dead pregnant during gestation.
*** determined for each litter.
Offspring viability indices:
Live born index (%)*= (number of pups born alive / number of pups born) x 100
Viability index (%)** = (number of pups alive PND 4 preculling / number of pups born alive) x 100

* determined for each litter.
** determined for each litter.; excluding litters sacrificed due to death of dam during lactation
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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).
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.
Dermal irritation (if dermal study):
not examined
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
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 evidence 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
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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 these 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 te 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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- no test substance-related effects on mating, fertility, pre-coital interval, or gestation length, or post-implantation loss at any dosage.
- 45 mg/kg/day: number of implantation sites and number of corpora lutea were slightly lower (not statistically significant).
- 5, 15, and 45 mg/kg/day: mortality observed in dams that occurred within the last few days of gestation (gestation days 21-22) and around the time of parturition (lactation days 0-1) is test substance related and appears to be related to that specific phase during pregnancy (mechanism involved could not be discerned)
- precoital interval was statistically higher in 5 mg/kg/day females compared to the control value, but not for the 15 and 45 mg/kg/day groups (no dose response).
- 0 and 15 mg/kg/day: two adult pairs (one pair each in the groups) failed to produce litters.
These incidences of reproduction failures were not dose related and thus was not considered to be test substance related. There were no gross or microscopic pathology findings to explain the reproductive failures.
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- 45 mg/kg/day: adverse, test substance-related effects on the number of offspring born, born alive, and/or surviving to lactation day 4 occurred. Only 3 litters were born, and the mean number of offspring per litter was 39% lower than the mean number of offspring per litter for the controls. Only one litter survived to postnatal day 4. These effects resulted in significantly lower number of offspring born alive, lower number of offspring alive on postnatal day 4, and lower live born index
- 5 mg/kg/day: 16 offspring from 2 litters were found dead during postnatal day 0-4 (test substance related and adverse effect, given the effects on maternal and offspring survival observed at dosages of 15 and 45 mg/kg/day)
- 45 mg/kg/day: 11 offspring from 2 litters were found dead during postnatal day 0-4
- 0 mg/kg/day: 4 offspring from 2 litters were found dead during postnatal day 0 - 4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 5, 15, and 45 mg/kg/day: adverse, test substance-related, statistically significant effects on offspring body weight were observed in the 5, 15, and 45 mg/kg/day groups.
- 45 mg/kg/day: of the surviving offspring, mean weights were 5% lower than the control means on postnatal day 0.
- 15 mg/kg/day: offspring body weight was significantly lower than the control value on postnatal day 0 and postnatal day 4 (15% and 13%, respectively).
- 5 mg/kg/day: offspring body weight was also 7% lower (statistically significant) than the control group on postnatal day 4.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Most pups submitted for necropsy were from females that were found dead or were sacrificed prior to delivery of their litters. Thus, incidences of lesions commonly seen in term foetus that die or are not delivered were increased in the treated groups. These included unexpanded lungs and absence of a milk spot in the stomach. There were no other test substance-related gross observations in pups.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
CLINICAL SIGNS (OFFSPRING)
- no test substance-related clinical observations in the surviving offspring at any dose level tested.

OTHER FINDINGS (OFFSPRING)
- Sex ratio: ratio was statistically higher in the 45 mg/kg/day group, perhaps secondary to the lower offspring survival in that group. There were no test substance-related or statistically significant effects on sex ratio for 15 mg/kg/day and below.
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEL for offspring was not achieved due to decreased pup body weights at all doses and mortality at 5 and 45 (but not 15) mg/kg/day.
Remarks on result:
not determinable
Critical effects observed:
no
Reproductive effects observed:
not specified
Conclusions:
The embryofoetal and postnatal offspring toxicity at all doses was most likely caused by the profound maternal toxicity (including mortality) at all doses, accompanied by small spleens, stomach ulceration/erosion, reduced thymus weights, and associated histopathology in the spleen, thymus, kidneys, and adrenal cortex in dams at all doses. The maternal effects caused offspring in utero deaths at 45 mg/kg/day, postnatal deaths at 5 and 45 mg/kg/day, and reduced pup weights at all doses. The decreased offspring body weights at all doses and the increased pup mortalities at 5 and 45 mg/kg/day are not developmental toxicity but indicate primary effects on the maternal animals (systemic toxicity to reproductive toxicity, leading to developmental toxicity), resulting in offspring consequences. There were no reproductive or developmental toxicity NOAELs achieved in this study.
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 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
Vehicle:
other: 0.1 % Tween-80 in 0.5 % aqueous methylcellulose
Details on 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.
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: until evidence of copulation was observed or until 2 weeks had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): On the day copulation was confirmed, the female was transferred back to individual cage housing.
Days 0 - 19 of gestation: stainless steel, wire-mesh cages suspended above cageboards
Day 20 of gestation - Day 4 of lactation: polycarbonate pans with bedding
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
Parental animals: Observations and examinations:
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
Starting on day 20 of gestation for mated females or 7 days after the end of the cohabitation period for females without evidence of copulation, female rats were observed at least twice daily for signs of delivery and offspring.

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 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/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

WATER CONSUMPTION AND COMPOUND INTAKE: No
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in male parental generations:
- Organ weights: testes, epididymides, prostate, seminal vesicles (with coagulating glands and fluids)
Litter observations:
The day when delivery was complete was designated day 0 postpartum.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Day 0 postpartum:
- abnormality, abnormal behaviour, appearance, and mortality
- number and sex of live and dead pups of each litter
- body weight of live pups of each litter
Day 4 postpartum:
- abnormality, abnormal behaviour, appearance, and mortality
- number and sex of live pups of each litter
- body weight and gross external examination of live pups of each litter

GROSS EXAMINATION OF DEAD PUPS:
A gross postmortem examination was conducted for pups that were moribund, found dead, or sacrificed because of death of the dam during lactation.
Postmortem examinations (parental animals):
SACRIFICE
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 NECROPSY
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.

HISTOPATHOLOGY / ORGAN WEIGHTS
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.
Postmortem examinations (offspring):
SACRIFICE
A gross postmortem examination was conducted for pups that were moribund, found dead, or sacrificed because of death of the dam during lactation.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organs were not weighed and no microscopic examinations were conducted on any F1 pups.
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
- Analysis of covariance and Dunnett-Hsu
- Non-parametric analysis of covariance
- 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. For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation (Haseman and Hogan, 1975).
* 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.
Reproductive indices:
Mating index (%) = (number mated*/number paired) x 100
Fertility index (%) = (number pregnant**/number mated*) x 100
Post-implantation Loss (%)*** = ((number of implantation sites - number of pups born)/number of implantation sites) x 100

*evidence of copulation = intravaginal copulatory plug, sperm in vaginal lavage, uterine implantation sites, or delivery of a litter.
** including those found dead pregnant during gestation.
*** determined for each litter.
Offspring viability indices:
Live born index (%)*= (number of pups born alive / number of pups born) x 100
Viability index (%)** = (number of pups alive PND 4 preculling / number of pups born alive) x 100

* determined for each litter.
** determined for each litter.; excluding litters sacrificed due to death of dam during lactation
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. Enteropathy 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.
Dermal irritation (if dermal study):
not examined
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
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
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 the cause 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 were graded 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
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.

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.

REPRODUCTIVE PERFORMANCE
- no test substance-related effects on mating, fertility, pre-coital interval, gestation length, number of implantation sites, post-implantation loss, or number of corpora lutea at any dosage.
- 100 mg/kg/day: although there were no effects on fertility, only one animal delivered a litter due to mortality of 10 dams during the last 2 days of gestation. Ten of 11 high dose level P1 females required Caesarean to demonstrate their reproductive success.
- 5 and 15 mg/kg/day: the failure of 2 P1 adult pairs (one pair each in the 5 and 15 mg/kg/day groups) to produce litters was not test substance related. There were no gross or microscopic pathology findings to explain their reproductive failure.
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
other: decreased number of pups born alive at 15 and/or 100 mg/kg/day.
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
other: decreased number of pups born alive at 15 and/or 100 mg/kg/day.
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- 15 mg/kg/day: number of pups born alive and the number alive on lactation day 4 were significantly lower. The significantly lower number of pups alive on lactation day 4 is a reflection of the lower number born alive, and the viability index was similar to the control value.
- 100 mg/kg/day: only one litter was available for evaluation, in which the number of pups born alive was also decreased relative to the control group, and there was no effect on viability during lactation days 0-4.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- 100 mg/kg/day: gross changes, consistent with Cesarean delivery or still birth, were present in F1 offspring, where most dams did not survive to the scheduled sacrifice. Thus, incidences of lesions commonly seen in term foetus that die or are not delivered at term were increased in pups. These included unexpanded lungs and absence of a milk spot in the stomach.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
- 5 mg/kg/day: no test substance-related effects were observed on the number of pups born, born alive, and alive on day 4, nor were there any effects on survival indices during lactation days 0-4.

CLINICAL SIGNS (OFFSPRING)
- no test substance-related clinical observations in the pups at any dosage tested were observed.
- 15 mg/kg/day: all the offspring from one litter had sores located on the back (lactation days 4). While no other litters in this group were affected, it cannot be determined whether these observations were a test substance-related effect on either the offspring or on behaviour of the dam.

BODY WEIGHT (OFFSPRING)
- no test substance-related effects on mean pup weights either at birth or on lactation day 4 at any dosage tested were observed.

GROSS PATHOLOGY (OFFSPRING)
- no further test substance-related gross findings were found

OTHER EFFECTS
Sex ratio:
- 5 mg/kg/day: no test substance-related effects were observed on sex ratio during lactation days 0-4.
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable
Reproductive effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
In this study, the parental males were unaffected at 5 through 40 mg/kg/day. The parental females were adversely affected at 15 and 100 mg/kg/day. Their offspring were also affected at these doses. The greater maternal toxicity at 100 mg/kg/day, included mortality, clinical toxicity, decreased body weights and weight gains, decreased feed consumption, and adverse histopathology (including lymphoid and adrenal gland adverse histopathology, enteropathy, and renal tubular damage) and was mirrored by (and likely caused) the greater offspring toxicity at this dose (decreased numbers of live born pups; only one litter survived at this dose).
At 15 mg/kg/day, maternal toxicity was lesser (clinical signs of toxicity, reduced feed consumption, and lymphoid and adrenal gland histopathology), and the offspring effects were lesser: decreased number of pups on postnatal day 0 and 4 (there was no effect on viability index; the reduced numbers on postnatal day 0 were the reason for the reduced numbers [same as on postnatal day 0] on postnatal day 4). The most appropriate (likely and conservative) conclusion is that the profound maternal toxicity at 100 mg/kg/day resulted in the profound effects on the offspring (only one litter), and that the lesser maternal toxicity at 15 mg/kg/day resulted in the lesser offspring toxicity (reduced litter sizes).

Overall, cobalt stearate is not a primary reproductive or developmental toxicant. It is considered that the continuing and dose-related adult maternal toxicity at 15 and 100 mg/kg/day was responsible for the developmental offspring toxicity also at 15 and 100 mg/kg/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Introductory remark – read-across

 

Read-across entails the use of relevant information from analogous substances (the ‘source’ information) to predict properties for the ‘target’ substance(s) under consideration. Substances whose physicochemical or toxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a category of substances. Structural similarity is a pre-requisite for any read-across approach under REACH (ECHA Read-Across Assessment Framework, 2015).

 

In accordance with Annex XI, 1.5 of the REACH regulation and the ECHA Guidance Read-Across Assessment Framework (ECHA, 2017), the similarities may be based on:

 

1) A common functional group (i.e. chemical similarity within the group);

2) Common precursors and/or likelihood of same breakdown products through physical and/or biological processes which result in structurally-similar degradation products (i.e. similarity through (bio) transformation); or

3) A constant pattern in the changing of the potency of the properties across the group (i.e. of physical-chemical and/or biological properties).

 

Due to the absence of substance specific information for the majority of substances within the cobalt category, the approach will read-across data from representative source substances to all other members of the read-across group.

 

Due to the route-specific toxicological properties of the cobalt category substances, several read-across groups are formed as shown in the table below:

 

 

Route

Read-across group

Cobalt category

oral-systemic

bioavailable cobalt substances group

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 group for oral-systemic: poorly soluble in aqueous solutions with organic ligand.

 

 

Effects on fertility

 

Three guideline-compliant OECD 422 reproductive screening studies were conducted with members of the poorly soluble in aqueous solutions with organic ligand cobalt substances read-across group in the CD(SD) rat. The compounds tested were cobalt neodecanoate, cobalt stearate and cobalt borate neodecanoate. These studies were conducted under the US HPV program, and can be accessed at (EPA website, link to HPV program http://www.epa.gov/hpv/). 

 

While general toxicity was observed in the studies with cobalt neodecanoate and cobalt stearate (such as decreased food consumption, decreased body weight gain, and reduced weight of non-reproductive organs), there was no evidence suggesting that these substances are primary reproductive or developmental toxicants. Effects on reproductive or developmental endpoints occurred only in the presence of considerable general toxicity, suggesting that the reproductive or developmental effects were secondary to overall toxicity of the substances.

 

It is concluded that substances of the poorly soluble in aqueous solutions with organic ligand read-across group are not primary reproductive or developmental toxins, and that the DNELs based on local irritation (gastro-intestinal tract) are sufficient to prevent systemic effects.

 

Due to a lack of adverse effects on fertility, it is concluded that the conditions for proposing an extended one generation reproductive toxicity study for the read-across group “poorly soluble in aqueous solutions with organic ligand” cobalt substances are not met. Experimental testing is therefore scientifically not justified and therefore waived in accordance with the conditions laid down in Section 8.7.3 Column 1, Annex IX of regulation (EC) 1907/2006.

 

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity

Three screening for reproductive / developmental toxicity studies (in accordance with OECD 422) in rats via oral application are available for different poorly soluble in aqueous solutions 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.

In summary all available studies for the read-across group poorly soluble in aqueous solutions with organic ligand cobalt substances show adverse effects as follows: (i) Decreased body weight and food consumption, clinical signs of toxicity (ii) 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 (iii) Effects on the haematopoietic system, being the hallmark for the bioavailable Co substances group were apparent only in high doses, already showing significant adverse effects in the digestive tract.

There was no evidence suggesting that these substances are primary reproductive or developmental toxicants. Effects on reproductive or developmental endpoints occurred only in the presence of considerable general toxicity, suggesting that the reproductive or developmental effects were secondary to overall toxicity of the substances.

The above discussed studies are considered in a weight of evidence that the poorly soluble in aqueous solutions with organic ligand cobalt substances are not primary developmental toxins, and that the DNELs based on local irritation (gastro-intestinal tract) are sufficient to prevent systemic effects. 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). Further developmental toxicity testing is not considered to add any relevant information to the hereto negative data and is therefore waived in accordance with regulation 1907/2006 (EC), Annex XI, Section 1.2.

Justification for classification or non-classification

Based on the above discussed data, there was no evidence suggesting that the poorly soluble in aqueous solutions with organic ligand cobalt substances are primary reproductive or developmental toxicants. Effects on reproductive or developmental endpoints occurred only in the presence of considerable general toxicity, suggesting that the reproductive or developmental effects were secondary to overall toxicity of the substances. Based on the weight of evidence the classification criteria as reproductive or developmental toxicants are not met, hence no classification is proposed.