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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Cobalt borate neodecanoate complex
- State of aggregation: blue viscous liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was selected for this study because it is a preferred species for 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.