Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-05-09 to 2011-07-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

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:
adopted 1996-03-22
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Chemical name: Cobalt Powder
- Product name: S-320 Cobalt Fine Powder
- Molecular formula: Co
- Molecular weight: 58.93
- Physical state: powder
- Storage condition of test material: kept in containers of same material as the original one. Product was stored in tightly closed container in a dry, cool and well ventilated place.
- Particle size:
D10=6.4 μm
D50=12.8 μm
D90=23.6 μm
D95=27.6 μm
- Melting point: 1493°C
- Water solubility: insoluble

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 58 and 60 days; females: 58 and 60 days
- Weight at study initiation: males: 214.1 to 252.6 g; females: 142.2 to 184.7 g
- Housing (except during the mating period): males and females (F0-generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material.
- Diet (ad libitum): commercial ssniff® R-Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 5 days

Health checks were performed on the day of delivery and at first administration. Each animal was clinically examined for signs of abnormality or disease.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous hydroxypropyl methylcellulose gel (Methocel)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concentrations. The test item formulations were freshly prepared on each administration day.

- Administration volume: 2 mL/kg bw/day
- The animals of the control group received the vehicle (Methocel) at a constant volume of 2 mL/kg bw orally once daily.
- The amount of the test item was adjusted to the animal's actual body weight daily.

VEHICLE - 0.5% aqueous hydroxypropyl methylcellulose gel (Methocel)
- FAGRON GmbH & Co. KG, 22885 Barsbüttel, Germany
- Batch no.: 09D14-N28
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: until pregnancy had occurred or 12 days had elapsed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered. This procedure was repeated until at least 8 pregnant dams were available for each group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle mixtures, two (2) samples of approx. 5 mL were taken at the following time points and stored at -20°C or colder:
1) Start of treatment period:
- Concentration and stability:
Immediately after preparation of the test item-vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature (2 x 3 samples/dose level group (except 1000 mg/kg bw/day group)).
- Homogeneity:
At start of administration, during (middle) administration and before administration to the last animal of each dose level group (2 x 3 samples/dose level group (except 1000 mg/kg bw/day group)).
Number of samples: 2 x 18
2) End of treatment period:
Concentration:
During treatment with the test item always before administration to the last animal/dose level group
(2 x 1 sample/group (except 1000 mg/kg bw/day group)).
Number of samples: 2 x 3
Sum of all samples: 2 x 21

The concentration of cobalt powder in the administration mixtures was determined by gravimetric analysis (ICP-OES).

Results:
The recoveries for the test item application mixtures with the nominal concentration 15 to 150 mg test item/mL found by gravimetric analysis of the cobalt
powder content in the range between 92.67% and 122.98%. These data verify the content, homogeneity and stability of the application mixtures during the
toxicological study.
The fact that the high concentration exceeded the admissible limit of 120% is of no safety relevance for the results of the study as the animals were not incompletely dosed but slightly overdosed.
Duration of treatment / exposure:
- Males: beginning 2 weeks before mating and continuing during the mating period and approx. 2 weeks post mating until the minimum total dosing period of 28 days was completed (up to and including the day before sacrifice)
- Females: beginning 2 weeks before mating and continuing up to and including day 3 postpartum or the day before sacrifice
Frequency of treatment:
Males and females: once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100, 300, and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected by the Sponsor based on available toxicological data and the 14-day dose range finding study of cobalt powder by oral administration to rats (LPT project no. 26279). In this dose-range finding study, the test item was applied orally by gavage at concentrations of 1.5, 4.5, 20, 60, 180, 500 or 1000 mg/kg bw/day to 3 males and 3 female rats per dose group, once daily for 14 days. A control group receiving the vehicle (0.5% aqueous hydroxypropyl methylcellulose gel (Methocel)) only was also used. The administration volume was 2 mL/kg bw/day.

Results:
None of the animals treated orally with 1.5, 4.5, 20, 60, 180, 500 or 1000 mg cobalt powder/kg bw/day revealed any changes in behaviour or external appearance. No deaths occurred. The faeces of all test item-treated animals were normally formed. No test item-related changes were noted for body weight or food and drinking water consumption. Macroscopic examination revealed no test item-related changes at any of the tested dose levels.
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs at least once daily and mortality twice daily. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
- Cage side observations: behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

Mortality: further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and once a week thereafter at the same time, each time.

BODY WEIGHT: Yes
Males and females: first day of dosing, weekly thereafter and at termination.
During gestation (females): days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 postpartum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes, the quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week. From these data the food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg bw/day) = (Total food given (g) - Total food left (g))/(Number of animals days# x Body weight (kg))
# = The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily (visual appraisal)

REPRODUCTIVE PERFORMANCE:
- the duration of gestation was recorded and calculated from day 0 of pregnancy.
- after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
- live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1 postpartum) and on day 4 postpartum. Any abnormal behaviour of the pups was recorded.

Evaluation/parameters:
Number of pregnant females
Pre-coital time
Gestation length calculated from day 0 of pregnancy

Number of pups absolute
- at birth (alive and dead)
- after 4 days of life

Number of pups per dam
- at birth
- after 4 days of life

Number of male and female pups
- at birth
- after 4 days of life

Number of stillbirths
- absolute
- per dam

Number of pups with malformations
- absolute
- per dam
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in male parental generations:
one epididymis and one testicle were used for the sperm count; sperm viability was determined and the sperm morphology was examined in all adult male animals of all groups, except in the 1000 mg/kg bw/day group, according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis of the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all adult males of all groups except the males of the 1000 mg/kg bw/day group.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- any abnormal behaviour of pups was recorded.
- as soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
- live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1 postpartum) and on day 4 postpartum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: male animals were sacrificed after a minimum total dosing period of 28 days if no longer needed for further mating.
- Maternal animals: dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females showing no evidence of copulation were sacrificed 24 days after the last day of the mating period.

GROSS NECROPSY
At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to Salewski (1964)*.
The number of corpora lutea and implantation sites were recorded in the female adult animals.

The adult animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera was examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS:
The weights of the following organs of all adult animals were determined before fixation: epididymis (2), heart, ovary (2), testicle (2), thyroid (including parathyroids), uterus, and vagina.
The weights of the following organs of the selected 20 adult males and 20 adult females (5 animals/sex/group) were determined before fixation: adrenal gland (2), brain, kidney (2), liver, spleen, and thymus.
Adrenal glands, gonads and kidneys were weighed individually and identified as left or right.

HISTOPATHOLOGY: Yes
The following organs or parts of organs of all adult animals were fixed: epididymis (1), gross lesions, heart (left and right ventricle, septum), mammary gland, ovary (2), prostate, seminal vesicle, testicle (1), thyroid (incl. parathyroids), uterus (incl. cervix and oviducts), and vagina.
In addition, the following organs or parts of organs of the selected 20 adult males and 20 adult females (5 animals/sex/group) were fixed: adrenal gland (2), bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), small intestine (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method), large intestine (colon, rectum), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles) preserved by inflation with fixative and then immersion, lymph node (1, cervical), lymph node (1, mesenteric), nerve (sciatic), oesophagus, spinal cord (3 sections), spleen, stomach, thymus, tissue masses or tumours (including regional lymph nodes), tongue (including base), trachea (including larynx), and urinary bladder.
Any other organs displaying macroscopic changes were also preserved.
The organs listed below were examined histologically after preparation. Parathyroids were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained and examined histologically.
- selected adult animals of the control group and the 300 mg/kg bw/day group (5 animals/sex/group): adrenal gland (2), bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), gross lesions, small intestine (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method), large intestine (colon, rectum), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles) preserved by inflation with fixative and then immersion, lymph node (1, cervical), lymph node (1, mesenteric), nerve (sciatic), oesophagus, seminal vesicle, spinal cord (3 sections), spleen, stomach, thymus, tissue masses or tumours (including regional lymph nodes), tongue (including base), trachea (including larynx), and urinary bladder.
- all adult animals (except the animals of the 1000 mg/kg bw/day group; target organs): heart (left and right ventricle, septum), ovary (2), thyroid (including parathyroids), uterus (including cervix and right and left oviducts), and vagina.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis of the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all adult males of all groups except the males of the 1000 mg/kg bw/day group.

*Reference:
- SALEWSKI, E. Färbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Naunyn-Schmiedebergs Archive Exp. Pathol. and Pharmacol. 247, 367 (1964)
Postmortem examinations (offspring):
GROSS NECROPSY
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
Statistics:
The data were evaluated as stated below.
1) body weight, food consumption, relative organ weights, reproduction data:
- 100 mg/kg bw/day group and 300 mg/kg bw/day group vs. control group:
Multiple t-test based on DUNNETT, C. W. New tables for multiple Comparisons with a control Biometrics, 482-491 (Sept 1964) p ≤ 0.01
- 30 mg/kg bw/day group vs. control group:
STUDENT's t-test p ≤ 0.01

2) Histopathology:
Exact test of R. A. FISHER (p ≤ 0.05)

3) For the comparison of classification measurements (for example the fertility index):
FISHER's exact test, n < 100 or chi²-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01) were employed.

Selection of DUNNETT test: for all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance is p ≤ 0.01.
These statistical procedures were used for all data.
Reproductive indices:
The following indices were calculated for each group:
- Fertility index female [%] = (number of pregnant rats / number of females used) x 100
- Gestation Index = (number of litters with live pups / number pregnant) x 100

For each litter and group the following indices were determined:
- Birth Index = (Total number of pups born (live +dead) / Number of implantation scars) x 100
- Pre-implantation loss [%] = (corpora lutea - implantations / corpora lutea) x 100
- Post-implantation loss [%] = (implantations - number of pups born alive / implantations) x 100
Offspring viability indices:
The following indices were calculated for each litter and group:
- Live Birth Index = (Number of pups born alive on day 0/1 / Total number born (live + dead)) x 100
- Viability Index = (number of pups alive on day 4 / number of pups live on day 0/1) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality:
- 30 mg/kg bw/day: no deaths
- 100 mg/kg bw/day: 5/10 females died (one female died prematurely on gestation day 21 and four females died between lactation days 2 to 4)
- 300 mg/kg bw/day: 8/10 females died (two female rats died during the mating period on test days 15 or 27, two further females died on gestation day 20 or 21 and four females died on the first lactation day)
- 1000 mg/kg bw/day: 9/10 males died (death on test day 12 to 24); 10/10 females died (four of them during the mating period, one during littering and five of them during the gestation period)

Clinical signs:
- males (pre-mating, mating and post-mating period): none of the male animals treated with 30 mg cobalt powder/kg bw/day revealed any test item-related signs of systemic toxicity. Piloerection was noted in two of ten male rats treated with 100 mg cobalt powder/kg bw/day and in three male rats of ten treated with 300 mg cobalt powder/kg bw/day for one to three test days. Laboured breathing was noted in one further male treated with 300 mg cobalt powder/kg bw/day on one test day. Pilo-erection, reduced motility and soft faeces were noted in the only surviving high dose male rat treated with 1000 mg cobalt powder/kg bw/day on one or two test days before sacrifice on test day 39. The nine prematurely deceased male rats revealed the following symptoms on one or a few days before death: piloerection, reduced motility, a haemorrhagic nose and/or soft faeces (soiled anus) were noted in all or several deceased males. In addition, hunched posture or abdominal position, slightly decreased respiratory rate, reduced body temperature (animal cold at touch), laboured breathing, ptosis and/or a pale skin (ears or whole body) were observed in one or a few males.
- females (pre-mating and mating period): no test item-related signs of systemic toxicity were noted in the female animals treated with 30 or 100 mg cobalt powder/kg bw/day. Soft faeces were noted in one low dose dam (treated with 30 mg cobalt powder/kg bw/day) on test day 17, this incidence is considered to be still within the spontaneous range as no corresponding findings were observed at 100 mg cobalt powder/kg bw/day. From 300 mg cobalt powder/kg bw/day onwards, pilo-erection and soft faeces were noted in a few to several females on a few test days.
- females (gestation period): none of the females treated with 30 mg cobalt powder/kg bw/day revealed any test item-related signs of systemic toxicity during the gestation period. Piloerection was noted from 100 mg cobalt powder/kg bw/day onwards and reduced motility and soft faeces or diarrhoea were noted from 300 mg cobalt powder/kg bw/day onwards in individual to nearly all females in relation to the dose.
- females (lactation period): none of the females treated with 30 mg cobalt powder/kg bw/day revealed any test item-related signs of systemic toxicity during the lactation period. From 100 mg cobalt powder/kg bw/day onwards, piloerection, reduced motility, soft faeces or diarrhoea were noted in nearly all females during the lactation period.
- prematurely deceased female rats (pre-mortal symptoms): piloerection and/or reduced motility were noted in all or several females. In addition, hunched posture, decreased respiratory rate, reduced body temperature (animal cold at touch), dyspnoea, tremor, miosis, ptosis, a haemorrhagic nose,
haemorrhagic urine, soft faeces or diarrhoea were observed. In addition, a pale skin was observed in some animals.

Detailed clinical observations:
- males: no further changes in the male rats as already stated above.
- females: findings stated above were confirmed. Thin hair was observed for one low dose female from test week 5 onwards.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)(1000 mg/kg bw/day group was excluded from statistical comparison due to a high mortality rate)
Body weight:
- males: the body weight in 300 and 1000 mg cobalt powder/kg bw/day groups was reduced from test week 2 onwards, being 13% (statistically significant at p ≤ 0.01) or 16% below the control value in test week 3 (mating period). The body weight at autopsy was reduced as well (12% below the control value) in the 300 mg cobalt powder/kg bw/day group. The body weight of the only surviving high dose male rat (1000 mg cobalt powder/kg bw/day) was slightly below the mean control value.
- females: the body weight of the female rats in the 300 mg cobalt powder/kg bw/day group was below the control on gestation day 20 (by 11%) and on lactation day 1 (by 21%, statistically significant at p ≤ 0.01). The body weight of the two surviving females was still reduced on lactation day 4 and at autopsy (20% or 19% below the control value). The body weight of the female rats in the 1000 mg cobalt powder/kg bw/day group was distinctly below the control (by 17%, no statistical comparison) on gestation day 14.

Food consumption:
- males: no test item-related influence was noted during the premating period at any tested dose level.
- females: the relative food intake of the animals treated with 100 or 300 mg cobalt powder/kg bw/day was distinctly or severely below that of the
control group by minus 37% or minus 68% on lactation day 1, being statistically significant at p ≤ 0.01 at 300 mg cobalt powder/kg bw/day on lactation day 1. On lactation day 4 the food intake of the five surviving females of the 100 mg/kg bw/day group or the two surviving females of the 300 mg cobalt powder/kg bw/day was still reduced by minus 40% or minus 65% compared to the corresponding food consumption of the control animals. A slight reduction of food intake (19% below the control value) was noted for the two high dose rats (1000 mg cobalt powder/kg bw/day) which were still alive on gestation day 14. No further data on food intake was available as both dams died shortly thereafter.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)(1000 mg/kg bw/day group was excluded due to a high mortality rate)
- no test item-related influence was noted on the sperm number, viability and morphology at any of the tested dose levels.
- qualitative sperm staging: no effect was noted on the sperm stages or interstitial testicle cell structure.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)(1000 mg/kg bw/day group was excluded due to a high mortality rate)
- fertility of the female rats was not influenced.
- no test item-related influence was noted on gestation length.
- no test item-related influence was noted on pre-coital time.
- there were no test item-related differences in the number of corpora lutea, implantation sites, in the number and sex of pups, runts or malformed pups. No test item-related influence was noted in the values calculated for the gestation length, the birth index and the live birth index between the control group and the animals treated with 30 or 100 mg cobalt powder/kg bw/day.
- treatment with 300 mg cobalt powder/kg bw/day resulted in a statistically significant (at p ≤ 0.01) increase of the post-implantation loss (30.9%, control 12.6%) and significant decrease (at p ≤ 0.01) in the live birth index (75.9%, control: 100 %).

ORGAN WEIGHTS (PARENTAL ANIMALS)(300 and 1000 mg/kg bw/day groups were excluded from statistical comparison)
- males and females: no test item-related findings were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- males: at 1000 mg cobalt powder/kg bw/day, a reddened stomach was noted in the only survivor of ten males of the high dose level.
Macroscopic inspection of the prematurely deceased nine males revealed pathological changes of the adrenals (enlarged and / or reddened) and the gastro-intestinal region (reddened intestines, caecum or stomach) in nearly all animals. In addition, lesions of the lungs (oedematous) were noted in some animals, changes of thymus (reddened) were seen in two of nine animals. All lesions are regarded to be test item-related.
- females: from a dose level of 100 mg cobalt powder/kg bw/day onwards, changes of the gastrointestinal tract (reddened, haemorrhagic foci, filled with fluid) were noted - in relation to the dose - in a few to several animals. In addition, a reddened thymus was noted in a few females treated with 100 mg cobalt powder/kg bw/day. These lesions are regarded to be test item related.
Further changes were noted at 1000 mg cobalt powder/kg bw/day in the form of enlarged and / or reddened adrenals in nearly all animals and oedematous lungs in some animals. These lesions are regarded to be test item-related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- males and females: histomorphological examination of rat organs after treatment with either 30, 100, 300 or 1000 mg cobalt powder /kg bw/day did not reveal any morphological lesions which are considered to be related to the test item.
For the macroscopical lesions noted at necropsy no histological correlate could be found.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on mortality, clinical signs of toxicity, effects on food consumption and macroscopic pathological changes observed at and above 100 mg cobalt powder/kg bw/day and reduced body weight at and above 300 mg cobalt powder/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on mortality, clinical signs of toxicity, effects on food consumption and macroscopic pathological changes observed at and above 100 mg cobalt powder/kg bw/day and reduced body weight at and above 300 mg cobalt powder/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

NOTE: the examinations on F1-offspring were restricted to the 30, 100 and 300 mg Cobalt Powder/kg bw/day groups as no offspring was available in the 1000 mg/kg bw/day group) due to the premature death of all females before littering.

VIABILITY (OFFSPRING)
- no test item-related deaths occurred in pups of the 30 mg cobalt powder/kg bw/day group until sacrifice on lactation day 4.
- from 100 mg cobalt powder/kg bw/day onwards, an increased F1-offspring mortality rate (stillbirths, prematurely deceased and cannibalised pups) was noted due to complete loss of litters of prematurely deceased dams. The mean viability index (300 mg/kg bw/day group: 95.4%, 1000 mg/kg bw/day group: 95.5%) was slightly decreased (control: 100).

CLINICAL SIGNS (OFFSPRING)
- no abnormal behaviour of pups was noted.

BODY WEIGHT (OFFSPRING)
- the mean litter weight of pups was slightly (not significantly) below the control weights on lactation day 0/1 (by up to 11%) and on lactation day 4 (by up to 18%) in groups of the 30 or 100 mg cobalt powder/kg bw/day groups.
- distinct reductions were noted for the mean litter weight of pups in the 300 mg cobalt powder/kg bw/day group (statistically significant at p ≤ 0.01 in male and total pups) on lactation day 0/1 (up to 20% below the control) and on lactation day 4 (up to 27% below the control).
- the total litter weight of pups was below that of the control in the 100 mg Cobalt Powder/kg bw/day group (female animals and total pups) and in the 300 mg cobalt powder/kg bw/day group due to the lower number of pups.

GROSS PATHOLOGY (OFFSPRING)
- external examinations at dissection revealed no external abnormalities in any of the pups examined.

Effect levels (F1)

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Dose descriptor:
NOEL
Generation:
F1
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the present test conditions the following findings were made:

Effects on the parental generation:
Piloerection, reduced motility, soft faeces/diarrhoea and reduced food consumption were noted - in relation to the dose - from a dose level of 100 mg cobalt
powder/kg bw/day onwards. In addition, reductions of body weight were noted from 300 mg cobalt powder/kg bw/day onwards.
Premature deaths occurred in five female rats at 100 mg cobalt powder/kg bw/day and eight female rats at 300 mg cobalt powder/kg bw/day. Treatment with 1000 mg cobalt powder/kg bw/day caused the premature death of nine of ten males and all ten females.
Macroscopic inspection revealed changes of the gastro-intestinal tract - mainly in the prematurely deceased animals - from a dose level of 100 mg cobalt powder/kg bw/day onwards and adrenal changes and pulmonal lesions at 1000 mg cobalt powder/kg bw/day.
Histopathological inspection did not reveal any pathological changes. For the macroscopical lesions noted at necropsy no histopathological correlate could be found.
No test item-related influence was noted on the sperm staging or interstitial cell structure (qualitative examination).
The NO(A)EL were determined to be 30 mg/kg bw/day.

Effects on the reproduction / Effects on the development of the conceptus and the F1-offspring (pups):
Treatment with 300 mg cobalt powder/kg bw/day resulted in an increase of the post-implantation loss and a decrease in the live birth index. No test item-related influence was noted on mating behaviour, fertility and the gestation length.
From 30 mg cobalt powder/kg bw/day onwards, the mean litter weight of pups was slightly reduced in a dose-related way (not significant at p ≤ 0.01), significant only at 300 mg cobalt powder/kg bw/day. An increased F1-offspring mortality rate and a slightly decreased viability index were noted from 100 mg cobalt powder/kg bw/day onwards.
The NO(A)EL were determined to be 30 mg/kg bw/day.