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Key value for chemical safety assessment

Effects on fertility

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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
Reason / purpose:
reference to same study
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
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
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
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
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
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: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
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)
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.
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
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.
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'
Reproductive effects observed:
not specified
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.

Endpoint:
fertility, other
Remarks:
based on repeated dose 90-day oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-09 to 2015-02-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998-09-21
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: to be stored cool and well-ventilated in a closed container, preferably under inert atmosphere.
Species:
rat
Strain:
other: CD
Details on species / strain selection:
The rat was selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
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 first dosing: males: 52 days; females: 65 days
- Weight at first dosing: males: 244.9 - 295.5 g; females: 204.1 - 246.5 g
- Housing: animals 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, Germany) was used as bedding material for the cages.
- Diet (ad libitum): commercial ssniff®-R/M-H V1534 (ssniff® Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): drinking water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The administration formulations were freshly prepared every day by dissolving the test item in the vehicle to the appropriate concentrations.
Administration volume: 2 mL/kg bw/day
The dose of the test item was adapted to the animal's body weight daily up to and including test week 6, and weekly thereafter.
The control animals received the vehicle at a constant volume of 2 mL/kg bw/day orally once daily in the same way.
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the administration formulations, samples of approximately 10 mL were taken at the following times and stored at -20°C or colder until analyses:
1) At study initiation (on the first administration day of male animals):
- analysis of stability and concentration: immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature (3 samples/test item group).
- homogeneity: at the start of administration, during (middle) administration and before administration to the last animal of the test item group (3 samples/test item group).

2) At study termination (on the last administration day of female animals):
- analysis of concentration: during treatment always before administration to the last animal of the group (1 sample/test item group).
The determination of the content of the test item cobalt dichloride hexahydrate in samples was performed by analysis of cobalt with Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES).

Results:
The generated results verify the concentration, the homogeneity and the stability of the test item cobalt dichloride hexahydrate in application mixtures during the toxicology study. The actual cobalt dichloride hexahydrate concentrations ranged from 101.4% to 102.1% of the nominal concentrations.
Duration of treatment / exposure:
90 days (except male recovery animals: 91 days)
Frequency of treatment:
once daily
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main study (per group): 10 males/10 females
Recovery group (control group and 30 mg/kg bw/day dose group only; per group): 5 males/5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels for this study have been selected based on available data.
- Recovery groups were included in this study. One recovery group was included for the control group and other recovery group for the 30 mg/kg bw/day dose group. These groups were kept for 28 days after the treatment period without receiving the test item.
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (main study animals and recovery animals)
- Time schedule:
Clinical signs: before and after dosing at each time of dosing as well as regular daily
Mortality: twice daily
- Cage side observations (included): skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

DETAILED CLINICAL OBSERVATIONS: Yes (main study animals and recovery animals)
- Time schedule: once before the first exposure and once a week thereafter
- Observations (included): skin, fur, eyes, mucous membranes, occurrence of secretions, excretions, autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour.

BODY WEIGHT: Yes (main study animals and recovery animals)
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (main study animals and recovery animals):
- Food consumption for each animal determined and relative food consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes (main study animals and recovery animals)
- Time schedule for examinations: daily

HORMONE LEVELS:
Blood was preferably collected by puncture of the vena jugularis under light ether anaesthesia as follows (all main study and recovery animals):
- predose (before the first administration; all main study and recovery animals)
- during study conduct, at the end of test week 6 (all main study and recovery animals)
- at the end of test week 13 (before necropsy; all main study and recovery animals)
- at the end of the recovery period (before necropsy; all recovery animals)
The following parameters of all animals of the control group and the 30 mg/kg bw/day dose group were examined: testosterone, progesterone, and 17β-estradiol
Oestrous cyclicity (parental animals):
The stages of the oestrous cycle observed were recorded individually for each female rat (all females of the main study group and recovery group)
Time schedule:
- before the first administration (test week 0; monitoring duration: 7 days)
- during study conduct (test weeks 5/6; monitoring duration: 12 days)
- at the end of the treatment period (test weeks 12/12 before necrospy of main study animals; monitoring duration: 12 days)
- at the end of the recovery period (test weeks 16/17 before necropsy of recovery animals; monitoring duration: 12 days)
Sperm parameters (parental animals):
Detailed histopathological examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all male main study and recovery of the control group and the 30 mg/kg bw/day group following staining.
Litter observations:
not applicable
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes (main study animals and recovery animals)
On test day 91, the main study animals were dissected following a randomisation scheme. Animals not dissected on test day 91 were dosed until one day before sacrifice. Necropsy of all animals allocated to the recovery period was performed on test day 119.
The animals were euthanized under ether atmosphere, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof was 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 were 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, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS: Yes (main study animals and recovery animals)
The weights of the following organs of all animals were determined: adrenal gland (2), liver, thymus, brain, ovary (2), prostate and seminal vesicles with coagulating glands as a whole, epididymis (2), pancreas, heart, spleen, uterus (incl. cervix), kidney (2), and testicle (2).
Paired organs were weighed individually and identified as left or right.

HISTOPATHOLOGY: Yes (main study animals and recovery animals)
The following organs or parts of organs with the exception of the eyes, epididymides and testicles of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson’s solution for optimum fixation. The epididymides and testicles were preserved in Bouin’s fixative.
Organs: adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (3 levels: right and left ventricle, septum), intestine, large (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer´s patches; Swiss roll method), 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), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary (2), pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, sublingual and parotid gland), skin (left flank), spinal cord (3 levels: cervical, mid-thoracic, lumbar), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumours (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix), and vagina.

The afore-listed organs of all main study and recovery animals of the control group and the 30 mg/kg bw/day group were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
Detailed histopathological examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all male main study and recovery of the control group and the 30 mg/kg bw/day group following staining.
The organs and tissues listed above were examined.
Postmortem examinations (offspring):
not applicable
Statistics:
The test item-treated groups (3, 10, and 30 mg/kg bw/day dose groups) were compared with the control group.
The following statistical methods were used:
- Multiple t-test based on DUNNETT, C. W. New tables for multiple relative and absolute organ weights comparisons with a control Biometrics, 482 - 491 (September 1964): body weight, food consumption, relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)
- STUDENT's t-test: all numerical functional tests: body temperature; hormone levels (p ≤ 0.05 and p ≤ 0.01)
- Exact test of R. A. FISHER: histopathology (p ≤ 0.05)
These statistical procedures were used for all data.
Reproductive indices:
not applicable
Offspring viability indices:
not applicable
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1) Treatment period:
- 10 mg/kg bw/day: body weight of the male animals was reduced by 5 to 7% from test day 50 onwards (not statistically significant). Body weight gain changed accordingly. The body weight at autopsy was reduced by 6% (not statistically significant) for the male animals. No changes were noted for the female animals.
- 30 mg/kg bw/day: body weight of the animals was reduced by 5 to 14% from test day 8 onwards for the males (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 8, 22, and 43 to 90) and by 5 to 10% from test day 29 onwards for the females (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 50 to 90), respectively, compared to the control group. Body weight gain changed accordingly. The body weight at autopsy was reduced by 11% for the males (statistically significant at p ≤ 0.05) and by 9% for the females (not statistically significant), respectively.
- the reduced body weights at the 10 mg/kg bw/day and 30 mg/kg bw/day dose groups are considered as test item-related.

2) Recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- the differences in body weight between the animals previously treated with 30 mg/kg bw/day and the control group were still present at the end of the recovery period: The body weight of the male and female animals was still reduced by 17% or by 13%, respectively, on test day 118 (statistically significant reductions at p ≤ 0.05 on test day 97 for the males and at p ≤ 0.01 on test days 97 to 118 for the females) compared to the control group.
- the male animals revealed a slightly higher body weight gain than the control group during the recovery period indicating a trend towards recovery, while the body weight gain of the females was in the range of the control group. The body weight at autopsy was reduced by 17% for the males and by 13% for the females (statistically significant at p ≤ 0.01 for the females), respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Treatment period:
- 10 or 30 mg/kg bw/day: the following test item-related changes in haematological parameters were noted for the male and female animals on test day 91 (male and female main study and female recovery animals) and 92 (male recovery animals). In general, the male animals were affected to a higher degree.
10 mg/kg bw/day (test day 91/92 (combined)):
haemoglobin content (males: +11%; p≤0.01)
erythrocytes (males: +10%; p≤0.01)
haematocrit value (males: +12%; p≤0.01)
reticulocytes (males: -33%; p≤0.05)
platelets (males: -13%)
mean corpuscular volume (females: +4%; p≤0.05);

30 mg/kg bw/day (test day 91/92 (combined)):
haemoglobin content (males: +25%; females: +14%; p≤0.01)
erythrocytes (males: +19%; females: +11%; p≤0.01)
haematocrit value (males: +23%; females: +14%; p≤0.01)
thromboplastin time (males: +7%; p≤0.01)
activated partial thromboplastin time (males: +8%; p≤0.05)
mean corpuscular volume (males: +4%; females: +3%; p≤0.05 (males only))
mean corpuscular haemoglobin (males: +5%; p≤0.01)
reticulocytes (males: -24%)
platelets (males: -26%; females: -12%; p≤0.01 (males only))
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment period:
- 10 or 30 mg/kg bw/day: the following test item-related changes in biochemical parameters were noted for the male and female animals on test day 91 (male and female main study and female recovery animals) and 92 (male recovery animals):
10 mg/kg bw/day (test day 91):
bilirubin (males: +17%)

10 mg/kg bw/day (test day 91/92 (combined)):
bilirubin (males: +14%)

30 mg/kg bw/day (test day 91):
bilirubin (males: +34%; p≤0.01; females: +16%; p≤0.05)

10 mg/kg bw/day (test day 91/92 (combined)):
bilirubin (males: +29%; p≤0.01; females: +16%; p≤0.05)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment and recovery period (full histopathological evaluation restricted to the control group and 30 mg/kg bw/day group):
- microscopic evaluation revealed test item-related changes in the bone marrow (erythroid hyperplasia) of the femur. There was a significant and test item-related increase for erythroid hyperplasia in the bone marrow of the male and female animals treated with 10 or 30 mg/kg bw/day compared to the controls:
4 of 10 males and 7 of 10 females in the 10 mg/kg bw/day dose group and 7 of 10 animals for both sexes in the 10 mg/kg bw/day dose group versus 0 of 10 in the 3 mg/kg bw/day dose group and controls. The bone marrow change (erythroid hyperplasia) attained statistical significance in animals of the 10 or 30 mg/kg bw/day dose groups for both sexes. After cessation of treatment, no test item-related changes were observed for the recovery animals anymore.
- all other microscopic changes seen in all organs in all animals were either coincidental, or were considered to lie within the normal range of background alterations, which may be seen in untreated rats of this age and strain.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
CLINICAL SIGNS AND MORTALITY
1) Treatment period:
- no test item-related changes in behaviour or external appearance were noted for the male and female animals treated with 3, 10 or 30 mg/kg bw/day.
- 10 mg/kg bw/day: one male showed a haemorrhagic left canthus on test days 26 to 32 and one female showed a reddened right eyelid and/or a haemorrhagic canthus on test days 33 to 53.
- 30 mg/kg bw/day: pilo-erection was noted for 2 male recovery animals on test days 30 to 36. No changes in behaviour or external appearance were noted for the female animals.
- these findings are not considered to be test item-related due to the low number of animals affected.
- faeces of all animals were of normal consistency.
- no deaths were noted at any dose level. All main study animals survived until their scheduled terminal sacrifice.
- none of the animals treated with 3, 10 or 30 mg/kg bw/day revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour at the detailed clinical observations.

2) Recovery period (restricted to the control group and 30 mg/kg bw/day group):
- no abnormalities in behaviour, external appearance or faeces were observed for the male and female animals previously treated with 30 mg/kg bw/day.
- no deaths were noted.
All recovery animals survived until the scheduled recovery sacrifice.
- none of the animals revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities, or behaviour at the detailed clinical observations.

BODY WEIGHT AND WEIGHT GAIN
1) Treatment period:
- 3 mg/kg bw/day: body weight, body weight gain and body weight at autopsy were not influenced in the male and female animals in a test item-related way compared to the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- no test item-related influence was noted on the relative food consumption of the male and female animals treated with 3, 10 or 30 mg/kg bw/day during the treatment period and of the male and female animals previously treated with 30 mg/kg bw/day during the recovery period compared to the control group.
- slight but statistically significant (at p ≤ 0.05) increases in food consumption noted for the 30 mg/kg bw/day dosed males and females in test week 4 and for the 30 mg/kg bw/day dosed male animals in test week 14 are considered to be due to the reduced body weight and to be without any biological relevance.

WATER CONSUMPTION AND COMPOUND INTAKE
- visual appraisal of the drinking water consumption did not reveal any test item-related differences between the test item-treated animals and the control animals throughout the treatment and the recovery period.

OPHTHALMOSCOPIC EXAMINATION
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- no changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus were noted in the male and female rats of the animals treated with 3, 10 or 30 mg/kg bw/day at the end of the treatment period.
- no changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.

HAEMATOLOGY
1) Treatment period:
- 3 mg/kg bw/day: no test item-related influence on haematological parameters was noted for the male and female animals at the end of the treatment period.
- no test item-related influence was noted for the number of leucocytes, the relative and absolute differential blood count, and the mean corpuscular haemoglobin concentration.
- statistically significant differences in haematological parameters compared to the control which are not considered to be test item-related were found in the following parameters: leucocytes, absolute lymphocytes, absolute eosinophilic granulocytes, absolute large unstained cells, and absolute basophilic granulocytes

2) Recovery period (restricted to the control group and 30 mg/kg bw/day group):
- all changes in haematological parameters previously observed after repeated treatment with 30 mg/kg bw/day had subsided after 4 weeks of recovery.
- no effects related to the previous treatment were observed on the haemoglobin content, the numbers of erythrocytes, leucocytes and platelets, the relative reticulocyte count), the haematocrit value, the relative and absolute differential blood count, the thromboplastin time, the activated partial thromboplastin time, the mean corpuscular volume, the mean corpuscular haemoglobin and the mean corpuscular haemoglobin concentration at the end of the recovery period.
- statistically significant differences in haematological parameters compared to the control which are not considered to be test item-related were found for the following parameters: absolute eosinophilic granulocytes

Please also refer for results about haematology to "Attached background material" below.

CLINICAL CHEMISTRY
1) Treatment period:
- 3 mg/kg bw/day: no test item-related influence on biochemical parameters was noted for the male and female animals at the end of the treatment period.
- 10 and 30 mg/kg bw/day:
- no test item-related influence was noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium and the serum level of bile acids. Further, the plasma activity of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and lactate dehydrogenase was not influenced.
- statistically significant differences in biochemical parameters compared to the control which are not considered to be test item-related were found in the following parameters: albumin, globulin, albumin/globulin ratio, cholesterol, creatinine, glucose, protein, triglycerides, calcium, chloride, and alkaline phosphatase

2) Recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- all changes in biochemical parameters previously observed after repeated treatment with 30 mg/kg bw/day had subsided after 4 weeks of recovery.
- no effects related to the previous treatment were noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, bilirubin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium, and the serum level of bile acids. Further, the plasma activity of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase was not influenced.

URINALYSIS
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg bw/day: no test item-related influence on the urinary status was noted for the male and female animals at the end of the treatment period.
- no test item-related influence on the urinary status was noted for the male and female animals previously treated with 30 mg/kg bw/day at the end of the recovery period.
- no test item-related changes were noted for the specific gravity of the urine, the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.
- statistically significant differences in urine parameters compared to the control which are not considered to be test item-related were found in the following parameter: pH

NEUROBEHAVIOUR
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- neurological screening performed at the end of the treatment period on test day 86 and at the end of the recovery period on test day 118 did not reveal any test item-related influence on the male and female rats treated with 3, 10 or 30 mg/kg bw/day, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.
- statistically significant differences in neurological parameters compared to the control which are not considered to be test item-related were found in the following parameters: body temperature, forelimb grip strength, and hindlimb grip strength

ORGAN WEIGHTS
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg bw/day: no test item-related changes in relative and absolute organ weights were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- statistically significant differences in relative and absolute organ weights compared to the control which are not considered to be test item-related were found in the following parameters: brain (relative), gonads (left testis, relative), spleen (relative), adrenal (left, absolute), brain (absolute), kidney (left, absolute), kidney (right, absolute), and gonads (right ovary, absolute)

GROSS PATHOLOGY
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group):
- 3, 10 or 30 mg/kg bw/day: no test item-related changes were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- there were no test item-related abnormalities (gross pathology, tissue masses or tumours) in any tissue, including the adrenal gland, kidney and pancreas, in any of the exposed animals at the end of the treatment period, nor at the end of the recovery period.
- macroscopic changes were noted in the kidney (cyst), spleen (rough surface, adhered to peritoneum), stomach (haemorrhagic foci), uterus (cystic, filled with clear liquid) and testis, epididymis, seminal vesicle and prostate (reduced in size) in individual animals of the control and test item-treated groups at terminal or recovery sacrifice.
- these changes are considered to be incidental findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment and recovery period (full histopathological evaluation restricted to the control group and 30 mg/kg bw/day group):
- there were no histopathological findings in any tissues, including the adrenal gland, kidney and pancreas, in any of the animals exposed p.o. to 30 mg/kg bw/day.

BONE MARROW EXAMINATION
Treatment and recovery period (recovery restricted to the control group and 30 mg/kg bw/day group).
- 30 mg/kg bw/day: no test item-related changes in the myeloid:erythroid ratio were noted for the male and female rats at the end of the treatment period.
- no test item-related changes were noted for the male and female rats previously treated with 30 mg/kg bw/day at the end of the recovery period.
- the slightly decreased myeloid:erythroid ratio (statistically significant at p ≤ 0.05) noted for the previously high dosed females at the end of the recovery period is considered to be in the normal range of variation and without any biological relevance.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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
not applicable
Generation:
F1
Remarks on result:
not measured/tested
Critical effects observed:
not specified
Key result
Reproductive effects observed:
no
Conclusions:
NOAEL (fertility): 30 mg/kg bw/day
No test item-related changes were noted on oestrous cycles. Furthermore, histopathological examination performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure), did not reveal any test item-related effects. Lastly, no effects on the hormone levels of the male and female rats were observed.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
Study duration:
subchronic
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 dichloride is assigned to the read-across group for oral-systemic: bioavailable cobalt substances group.

 

 

 

Effects on fertility

 

Cobalt dichloride is the source substance for the soluble inorganic cobalt substances group based on the read-across approach as outlined in Appendix 1.1 in the CSR. Two soluble inorganic cobalt substances, cobalt dichloride and cobalt powder, have been tested for effects on fertility, resulting in an absence of effects on the reproductive organs of male and female animals up to the maximum tolerated dose. In all studies, the effect levels were based on similar findings, predominantly body weight effects and haematological findings.

 

In the sub-chronic repeated dose toxicity study, cobalt dichloride was given male and female rats at doses of 0, 3, 10, 30 mg/kg bw/day. A total of 10 males and 10 females per group were given the test items suspended in 0.5% hydroxypropyl methylcellulose gel orally via gavage once daily for 90 days. Additional 2 groups of 5 male and 5 female animals, dosed with 0, 30 mg/kg bw/day were assigned as recovery animals, kept for 28 days after the treatment period without receiving the test item. Additional examinations were added to the study design:

 

(i) monitoring of the oestrus cycle pre-dose, during study conduct, at the end of test week 13 and at the end of the recovery period in all female animals

(ii) hormone level status (testosterone, progesterone, 17beta-estradiol) pre-dose, during study conduct, at the end of test week 13 and at the end of the recovery period in all animals

(iii) Detailed histopathologic examination on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure)

 

During the conduct of the study no deaths occurred and no test item-related changes in behaviour or external appearance were observed. The body weight of the male animals treated with 10 mg Cobalt dichloride hexahydrate/kg b.w./day and the body weight of the male and female animals treated with 30 mg Cobalt dichloride hexahydrate/kg b.w./day were slightly reduced. Body weight gain and body weight at autopsy changed accordingly.

 

No test-item related changes were observed during histopathological examination of the male and female reproductive organs. Histopathological examination performed on one testicle and one epididymis with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure, did not reveal any test item-related effects. No test-item related influence on the ovaries, oviducts, uterus (incl. cervix) and vagina were noted. No test item-related difference was noted in the mean number of oestrous cycles for the female animals. No test item-related influence was noted on the serum levels of the hormones testosterone, progesterone, and 17 beta-estradiol in the male and female animals treated with 30 mg Cobalt dichloride/kg bw/day compared to the control group during study conduct, at the end of the treatment period, and at the end of the recovery period.

 

The No-Observed-Effect-level (NOEL) for fertility/reproductive effects was above 30 mg cobalt dichloride/kg bw/day by oral administration based on a complete absence of effects on reproductive organs, oestrus cycle, qualitative sperm staging and hormone levels.

 

 

In a combined repeated dose toxicity studies with the reproduction/developmental toxicity screening test (according to OECD 422 and under GLP) cobalt was administered orally to rats at dose levels of 30, 100, 300 and 1000 mg/kg bw/day during the pre-mating, mating and post-mating periods to parental males as well as during the pre-mating, mating, gestation and lactation periods until day 3 post-partum (or shortly thereafter) to parental female animals.

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 /kg bw/day onwards. In addition, reductions of body weight were noted from 300 mg cobalt /kg bw/day onwards. Premature deaths occurred in five female rats at 100 mg cobalt /kg bw/day and eight female rats at 300 mg cobalt /kg bw/day. Treatment with 1000 mg cobalt /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/kg bw/day onwards and adrenal changes and pulmonal lesions at 1000 mg cobalt/kg bw/day.

 

Histopathological inspection did not reveal any pathological changes. No histopathological correlate could be found for the macroscopical lesions noted at necropsy. No test item-related influence was noted on the sperm staging or interstitial cell structure (qualitative examination). Treatment with 300 mg cobalt/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/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/kg bw/day. In order to estimate a possible correlation between maternal toxicity and F1-Generation (pups) findings, the litter weight of pups was compared in dams with clinical signs within each group. Dams were classified based on the severity and occurrence of clinical signs. As a result it appeared that the earlier the clinical signs occurred in the dams a more pronounced weight reduction was noted for the pups of the respective dams. An increased F1-Generation (pups) mortality rate and a slightly decreased viability index were noted from 100 mg cobalt/kg bw/day onwards.

 

The NO(A)EL for effects on the F0-generation was 30 mg cobalt/kg bw/day, based on mortality, clinical signs of toxicity, effects on food consumption and macroscopic pathological changes observed at and above 100 mg cobalt/kg bw/day and reduced body weight at and above 300 mg cobalt/kg bw/day.

 

The NO(A)EL for effects on the reproductive toxicity was 30 mg cobalt/kg bw/day, based on an increased F1-Generation (pups) mortality rate and a slightly decreased viability index at 100 mg cobalt/kg bw/day and on post-implantation losses, decreases in the live birth index and significantly reduced litter weights of pups observed at 300 mg cobalt/kg bw/day.

 

In addition to the above studies there is a substantial number of information on the toxicity of soluble cobalt compounds to organs of male reproduction. These effects on male reproduction have been the reason to classify several cobalt substances for impairment of fertility in the EU. However, the available studies on fertility, summarized in IUCLID section 7.8.1, are all rated 3 (not reliable) or 4 (not assigned); the studies themselves do not show a clear dose-response relationship and beyond that suffer from either one or several of the following shortcomings:

 

                   Studies were not conducted according to guideline

                   The size of animal groups used in the published studies is smaller than stipulated within the guidelines and therefore, the statistical sensitivity of these studies is compromised.

                   only one dose level was given to animals instead of three dose levels as foreseen in the guideline

                   crucial parameter not reported, such as body weight, food/water consumption, clinical observations, full necropsy/histopathology

                   due to missing food/water consumption data, the actual ingested dose in drinking water/feeding studies could not be determined

 

Furthermore, none of the available studies represent state-of-the-art, guideline-compliant, extended one-generation reproduction toxicity studies or other relevant study designs, from which robust no-effect levels for human risk assessment could be derived. Moreover, the above-mentioned studies have a primary focus on effects in males, whereas there are no adequate data whatsoever to assess the effects on female reproduction.

 

Conclusion

 

The current test results (sub-acute and sub-chronic repeated dose toxicity study with cobalt and cobalt dichloride in male and female rats with additional examinations on fertility impairment) does not support the identification of any adverse effects towards male reproductive organs for the bioavailable cobalt substances group. However, these is still existing information in the public domain which identified a hazard with regard to male reproduction. Consequently all members of the bioavailable cobalt substances group will be self-classified as toxic for reproduction category 1B (H360F). The available data on the repeated dose toxicity of the bioavailable cobalt substances group is adequate to support a robust risk assessment, by using the point of departure for the most sensitive systemic effect on the haematopoietic system.

 

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-02-09 - 2015-05-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001-01-22
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (+10°C to +25°C) in a closed container under N2 atmosphere
Species:
rat
Strain:
other: CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age on day 0 of gestation: 61 days
- Weight on day 0 of gestation: 194.3 - 248.5 g
- Housing (except during the mating period): dams 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 released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages.
- Diet (ad libitum): commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared every day. The test item was suspended in the vehicle to the appropriate concentration and was administered orally at a constant volume. The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way. Administration volume: 2 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item formulations, samples of 10 mL were taken at the following times and stored at -20°C or colder until analysis:
1) At study initiation
- analysis of stability and concentration: immediately after preparation of the formulation as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples/dose level group; 25, 50, and 100 mg/kg bw/day dose groups).
2) At study termination (at a time when the majority of animals was dosed)
- analysis of concentration: during treatment with the test item always before administration to the last animal of the dose group (1 sample/dose level group; 25, 50, and 100 mg/kg bw/day dose groups).
The determination of the content of the test item cobalt dichloride hexahydrate in samples was performed by analysis of cobalt with Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES).

Results:
The generated results verify the concentration, the homogeneity and the stability of the test item cobalt dichloride hexahydrate in application mixtures during the study. The actual cobalt dichloride hexahydrate concentrations ranged from 99.8% to 100.9% of the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male/1 female animal (sexually mature male rats of the same breed served as partners. The female breeding partners were randomly chosen.)
- Proof of pregnancy: each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy).
Duration of treatment / exposure:
Day 6 to 19 of gestation
Frequency of treatment:
once daily
Duration of test:
20 gestation days
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 female rats (20 dams with litters were only evaluated)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
the dose levels have been selected based on the results of a dose-range-finding study in rats (LPT Report No. 31175). In this dose-range finding study, cobalt dichloride hexahydrate was applied orally by gavage at concentrations of 30, 60, and 100 mg/kg bw/day to 5 pregnant female rats per dose group, once daily from gestation day 6 until gestation day 19. A control group receiving the vehicle (tap water) only was also used. The administration volume was 2 mL/kg bw/day. Four litters per group were evaluated.

Results:
1) Examination of the dams:
No test item-related premature death was noted as well as no test item-related changes in behaviour, the external appearance or the consistency of the faeces. Furthermore, at 60 and 100 mg /kg bw/day, slight and statistically not significant reductions in body weight (max. by 14% on gestation day 20) and body weight gain (max. by 90% between gestation days 18 and 20) were noted at the end of the study. The carcass weights of the dams of the 60 and 100 mg /kg bw/day dose groups were accordingly reduced by 10% (statistically not significant; 60 mg /kg bw/day dose group) and by 17% (p ≤ 0.05; 100 mg /kg bw/day dose group). Looking at the food consumption, at 60 and 100 mg/kg bw/day, moderate (statistically not significant) reductions in food consumption were noted for the dams of the 60 and 100 mg /kg bw/day dose groups by 23.6% and 43.5% between gestation days 19 and 20.
No test item-related changes were noted for drinking water consumption. In addition, no test item related findings were noted during the internal macroscopic inspection at necropsy. No differences on the gravid uterus weights were noted between the control groups and the treatment groups.
Lastly, at 60 and 100 mg/kg bw/day, the net weight change during the treatment period was statistically significantly reduced by 92% (p ≤ 0.05; 60 mg/kg bw/day dose group) and by 123% (p ≤ 0.01; 100 mg/kg bw/day dose group).

2) Examination of the foetuses:
No test item-related changes were noted for the number of resorptions and the post-implantation loss, respectively. Furthermore, no dead foetuses were noted in the study as well as no test item-related differences in sex distribution. Also, no test item-related differences were noted in foetal weight. No runts were noted. In addition, no test item-related differences were noted in placental weight.
Lastly, no external malformation was noted in the treatment groups as well as no external variation.

Under the present test conditions, the no-observed-effect level (NOEL) was 30 mg cobalt dichloride hexahydrate/kg bw/day for the dams and above 100 mg cobalt dichloride hexahydrate/kg bw/day for the foetal organism.
Based on the data obtained in this dose-range-finding study, the following dose levels were suggested for the main study:
Group 1: Control
Group 2: 30 mg cobalt dichloride hexahydrate /kg bw/day
Group 3: 60 mg cobalt dichloride hexahydrate /kg bw/day
Group 4: 100 mg cobalt dichloride hexahydrate /kg bw/day
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for any signs of behavioural changes, reaction to treatment, or illness. Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day as well as on the weekend
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Foetuses obtained this way were examined for abnormal development, whenever possible.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 of gestation, followed by daily weighing
The body weight gain was calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20).
Furthermore, the net weight change from day 6 is given.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
All rats are euthanized, exanguinated and laparotomised. A dissection with macroscopic examination of the internal organs of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals was found dead. In case of macroscopical findings, the affected organs were preserved in 7% buffered formalin for possible future histopathological examinations.

The following target organs or parts thereof of all laparotomised female animals (including non-pregnant females, females with a total resorption of all implants and prematurely deceased animals) were fixed in 7% buffered formalin:
adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), eye with optic nerve (2), gross lesions observed, heart (3 levels: right and left ventricle, septum), intestine, large (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer's patches; Swiss roll method), 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), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary (2) (and oviducts), pancreas, pituitary, salivary glands (mandibular, parotid, sublingual), skin (left flank), spinal cord (3 sections), spleen, stomach, thymus, thyroid (2) (incl. parathyroids), tissues masses or tumours (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix), and vagina.
Paired organs were marked as left or right.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: gestation day 20
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Parameters examined: relative differential blood count, absolute differential blood count, erythrocytes, leucocytes, haematocrit value, haemoglobin content, platelets, reticulocytes, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes, the ovaries and the uteri of the dams were removed and the uteri (in toto) were weighed. Uteri without foetuses were examined for possible implantation sites according to SALEWSKI to confirm their pregnancy status.

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number and size of early resorptions: Yes
- Number and size of late resorptions: Yes
- Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
- The number of foetuses (alive and dead) and placentae (location in uterus and assignment to the foetus) was determined.
- Location of foetuses in the uterus were determined.
- Weights of the placentae were determined.
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No
- Sex and weights of foetuses were determined (foetuses were considered as runts if their weight was less than 70% of the mean litter weight).
- Viability of foetuses were determined.
Statistics:
Statistical analyses of the parametrical values were done by Provantis using the following settings:
Analysis of normal distribution and homogeneity of variances was performed by using the SHAPIRO-WILKS test and the BARTLETT test.
Data not normally distributed or with heterogenous variances between the groups were stepwise log- or rank-transformed.
One-way analysis of variance (ANOVA) was performed with non-transformed or log-transformed data.
The KRUSKAL-WALLIS test was used for rank-transformed data.
In case of significant differences (found by ANOVA or KRUSKAL-WALLIS test), intergroup comparisons with the control group were made by parametric or nonparametric DUNNETT multiple comparison tests (p ≤ 0.05 and p ≤ 0.01).
Parametrical values not captured by Provantis (e.g. number and weight of foetuses) were analysed by the DUNNETT test (p ≤ 0.05 and p ≤ 0.01).
Prior to the DUNNETT test homogeneity of variances was tested using the BARTLETT test.
In case of heterogeneity of variances, the STUDENT's t-test was carried out (p ≤ 0.05 and p ≤ 0.01).

Statistical analyses of non-parametrical data (e.g. resorption-, malformation-, variation and retardation rates) were performed using the following settings:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01); or Chi² test, n ≤ 100 (p ≤ 0.05 and p ≤ 0.01).
Indices:
Total malformation rate [%] = (malformed foetuses per group/foetuses per group) x 100
Total variation rate [%] = (foetuses per group with variations/foetuses per group) x 100
Total retardation rate [%] = (foetuses per group with retardations/foetuses per group) x 100
Pre-implantation loss [%] = (corpora lutea - implantations/corpora lutea) x 100
Post-implantation loss [%] = (Implantations - living foetuses/Implantations) x 100
Mean post-implantation loss per group [%] = sum of post-implantation loss [%] per litter/number of litters per group
Historical control data:
Background data summarising results of the last 55 embryotoxicity studies in Sprague-Dawley rats performed by the laboratory in the years 2000 to July 2014.
Data included were as follows: general reproductive indices, malformations, skeletal retardations, skeletal variations, visceral variations, and external variations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Scattered occurrences of piloerection, reduced motility and salivation were noted for several dams of the 50 mg/kg bw/day and the 100 mg/kg bw/day dose group:
50 mg/kg bw/day: piloerection was noted for 7/20 dams on 1 or 2 test days between gestation days 15 and 20. Furthermore, reduced motility (slight to moderate) was noted for 11/20 dams on 1 up to 3 test days between gestation days 6 and 19. Lastly, salivation (slight to moderate) was noted for 18/20 dams on 1 up to 6 test days between gestation days 6 and 19.
100 mg/kg bw/day: piloerection was noted for 8/20 dams on 1 up to 3 test days between gestation days 17 and 20. Furthermore, reduced motility (slight to moderate) was noted for 3/20 dams on 1 up to 3 test days between gestation days 13 and 18. Lastly, salivation (slight to moderate) was noted for 15/20 dams on 1 up to 5 test days between gestation days 6 to 19.

The observations for the dams of the 50 mg/kg bw/day and the 100 mg/kg bw/day dose group listed above are considered as test item-related.
Test item-related observations that were only noted in dams of the 100 mg/kg bw/day dose group were a haemorrhagic nose or snout noted for 3/20 dams on one day each on gestation day 19 or 20.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- body weight:
- 25 mg/kg bw/day: slight and statistically not significant reductions in body weight in comparison to the control group (max. by 5.3% on gestation day 20) were noted for the dams. As the carcass weight and the net body weight change between the start of treatment and the end of the study revealed statistically significant reductions in comparison to the control group, the slight reductions in body weight are also considered as test item-related but not regarded to be adverse cause of a reduced food consumption between gestation days 18 amd 20.
- 50 mg/kg bw/day: the body weight of the dams was statistically significantly reduced (p ≤ 0.05 /0.01) from gestation day 8 or 9 up to the end of the study on gestation day 20. In detail, the body weight of the dams was 6.2 % (p ≤ 0.01) below the value of the control group on gestation day 8 (two days after the start of dosing). In the further course of the study the differences in body weight between the treated dams and the control group slightly increased, leading to a body weight for the treated dams on gestation day 19 that was 10.6% below the value of the control group. Thereafter, the difference in body weight between the treated dams and the control group increased to a greater extent, leading to a body weight for the treated dams that was 14.2% below the value of the control group on gestation day 20.
- 100 mg/kg bw/day: the body weight of the dams was statistically significantly reduced (p ≤ 0.05 /0.01) from gestation day 8 or 9 up to the end of the study on gestation day 20. In detail, body weight development that was noted for the treated dams was very similar to those of the 50 mg/kg bw/day dose group. On gestation day 9 the body weight of the treated dams was 5.5% (p ≤ 0.05), on gestation day nineteen 10.2% (p ≤ 0.01) and on gestation day twenty 15.1% (p ≤ 0.01) below the value of the control group.
- body weight gain (whole study): body weight gain was statistically significantly (p ≤ 0.01) reduced in the 50 and 100 mg/kg bw/day dose group in comparison to the control group.
- 3 day intervals of body weight gain: the most pronounced differences in body weight gain for the dams of the 25, 50, and 100 mg/kg bw/day dose group in comparison to the control group were noted after the start of dosing between gestation days 6 and 9 and at the end of the study between gestation days 18 and 20. In detail, between gestation days 6 and 9 no increase in body weight was noted for the dams of the 100 mg/kg bw/day dose group and the dams of the 50 mg/kg bw/day dose group even showed a reduction in body weight by 1.3 g. In contrast, the dams of the 25 mg/kg bw/day dose group showed an increase in body weight by 9.2 g and the dams of the control group by 13.2 g.
Between gestation days 18 and 20 the body weight of the dams of the control group increased by 29.1 g in comparison to 23.5 g in the in 25 mg/kg bw/day dose group, 13.0 g in the 50 mg/kg bw/day dose group and only 0.3 g in the 100 mg/kg bw/day dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
50 or 100 mg/kg bw/day: statistically significant ( p ≤ 0.05 / 0.01) reductions in food consumption in comparison to the control group were noted on several days between gestation days 7 and the end of the study. In detail, the reductions in food consumption in comparison to the control group varies between 2.6% (not significant) and 19.3% (p ≤ 0.01) for the dams of the 50 mg/kg bw/day dose group and 1.0% (not significant) and 17.1% (p ≤ 0.01) for the dams of the 100 mg/kg bw/day dose group between gestation days 7 and 18. Thereafter, between gestation days 18 and 19 and / or gestation days 19 and 20, a further distinct decrease in food consumption was noted for all test groups in comparison to the control group. The last evaluated food consumption (between the morning of gestation day 19 and the morning of gestation day 20) was 19.9% (p ≤ 0.01) below the control group for the dams of the 25 mg/kg bw/day dose group, 44.5% p ≤ 0.01) below the control group for the dams of the 50 mg/kg bw/day dose group and 59.1% (p ≤ 0.01) below the control group for the dams of the 100 mg/kg bw/day dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted on several of the investigated haematological parameter, indicating a test item-related influence on the haematological parameters:
- 50 mg/kg bw/day:
increased haemoglobin (p≤0.01);
increased erythrocytes (p≤0.01);
increased platelets (p≤0.05);
increased haematocrit value (p≤0.01);
decreased absolute lymphocytes (p≤0.05);
decreased eosinophilic granulocytes (p≤0.05);
decreased absolute basophilic granulocytes (p≤0.01);
decreased mean corpuscular haemoglobin concentration (p≤0.01)

- 100 mg/kg bw/day:
increased haemoglobin (p≤0.01);
increased erythrocytes (p≤0.01);
increased reticulocytes (p≤0.05);
increased platelets (p≤0.05);
increased haematocrit value (p≤0.01);
decreased absolute lymphocytes (p≤0.01);
increased absolute monocytes (p≤0.01);
decreased eosinophilic granulocytes (p≤0.01);
decreased absolute basophilic granulocytes (p≤0.05);
decreased mean corpuscular haemoglobin concentration (p≤0.01)
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The following findings were considered test item-related:
- 25 mg/kg bw/day: one dam with haemorrhagic focus in the stomach (concluded to be not adverse)
- 50 mg/kg bw/day: three dams with a few haemorrhagic foci in the stomach
- 100 mg/kg bw/day: seven dams with a few or several haemorrhagic foci in the stomach (one dam had additionally several ulcers in the stomach); four dams had dark (-brown content in the intestines; two dams had a small caecum with no content; one dam with a thickened mucosa in the caecum and a whitish layer on mucosa in the duodenum. The gastro-intestinal lesions were accompanied by pathological findings in the form of a pale liver (one dam), a spleen reduced in size (three dams), enlarged adrenals (two dams), light-brown to dark discoloured urine in the urinary bladder (one dam) as well as external changes in the form of pale eyes and an anal region soiled with faeces (one dam) or a haemorrhagic snout (one dam).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant change are not test item-related:
- 50 mg/kg bw/day: the preimplantation loss was statistically significantly reduced.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
- mortality:
no test item-related premature death was noted, neither in any of the test item treated groups (25, 50 or 100 mg/kg bw/day), nor in the control.

- clinical observations:
no test item-related changes in behaviour, the external appearance, and the faeces were noted for the dams of the 25 mg/kg bw/day dose group. The following observations were only sporadically noted for 1 or 2 dams and not considered as test item-related:
- 25 mg/kg bw/day: a slightly reduced motility was noted for 2/20 dams on one day each on gestation day 10 or 12.
- 100 mg/kg bw/day: a decreased body temperature (gestation days 17 and 18) and pale eyes (gestation day 20) were noted for one dam. In addition, a dark discoloured urine was noted for another dam on gestation day 20.

- no influence of the gravid uterus weight on the whole body weight was noted.

- food consumption:
- 25 mg/kg bw/day: as the slight, but statistically significant reductions in food consumption noted for the dams of the 25 mg/kg bw/day dose group were only noted at the end of the study between gestation days 18 and 20 and were not considered as test item-related.

- water consumption:
- 25, 50 or 100 mg/kg bw/day: no test item-related changes in the consumption of drinking water was noted for the dams.

- Macroscopic examinations:
The following sporadic observations in individual control and test item-treated animals are considered as spontaneous as no dose-response-relationship was noted:
- control: upper pole yellowish discoloured kidney (left; one dam); marbled kidney (bilateral; one dam); reddened lungs25 mg/kg bw/day: dilatation of renal pelvis of kidney (right; one dam); dilated ureter (one dam); dilated aorta (abdominal region; one dam); brown focus in lungs (two dams)
- 50 mg/kg bw/day: few/several grey/haemorrhagic/dar-red foci in the lungs (three dams); spleen reduced in size (one dam); multiple red foci in thymus (one dam)
- 100 mg/kg bw/day: agenesis of the right uterus and ovary duct (one dam); yellowish discoloured placenta (one foetus); few/multiple haemorrhagic/dark-red foci (two dams); enlarged, partly pale right kidney (one dam)
The changes in the lungs were possibly due to a regurgitation of the test item.In addition, rough fur was noted in two 50 mg/kg bw/day dose animals with gastric lesions and in individual 100 mg/kg bw/day dose animals with gastric lesions (three dams) or without gastric lesions (three dams) indicating a slightly poor condition.

- uterus weight and net body weight change:
- 25, 50 or 100 mg test item/kg bw/day: no test item-related changes in the gravid uterus weight were noted for the dams of all treatment groups. In parallel to the reductions in body weight statistically significant (p ≤ 0.01) reductions were noted for the carcass weights of the dams, which were considered as test item-related. In detail, the carcass weights of the dams of the 25 mg/kg bw/day dose group were reduced by 8.7% in comparison to the control group, whereas the carcass weights of the dams of the 50 and 100 mg/kg bw/day dose groups were reduced by nearly the same value (18.2% or 18.1%) in comparison to the control group. Furthermore, in all treatment groups reductions in the net body weight (body weight without gravid uterus) were noted during the treatment period, leading to statistically significantly (p ≤ 0.01) reduced values of net body weight change in comparison to the control group. In detail, the net body weights for the dams of the 50 and 100 mg/kg bw/day dose groups were reduced in the period between gestation days 6 to 20 by 10.6 g or 14.1 g, whereas the net body weight of the dams of the 25 mg/kg bw/day dose group increased by 18.1 g and those of the dams of the control group increased by 38.4 g during the period between gestation days 6 and 20.

- Haematology:
- 25 mg/kg bw/day: no test-item related influence was noted for the dams.

- Reproduction data: No abortion occurred in the study.
- 25, 50 or 100 mg/kg bw/day: no test item-related differences for the pre-implantation loss ratio, resorptions/implantation ratio, and post-implantation loss ratio were noted between the dams of the control group and the treated dams.
The following statistically significant changes are not test item-related:
- 25 and 50 mg/kg bw/day: the ratios of early and/or total resorptions to implantation sites was statistically significantly (p ≤ 0.05) decreased. In detail, 4 resorptions (all early) were noted in three 25 mg/kg bw/day dose dams as well as 3 early resorptions in three 50 mg/kg bw/day dose dams while 11 early resorptions were noted in seven control dams.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
50 or 100 mg/kg bw/day: slight but statistically significant (p ≤ 0.01 or p ≤ 0.05) reductions for the mean foetal weights (by 8% for the male and female pups together) were noted. As the reductions were only slight and the foetal body weights were still within the range of the laboratory background data, they are not considered as test item-related.
The effects on the foetal weights are related to an indirect effect of the severely reduced body weight of the dams, and hence, no direct effect of the test item per se.

Please also refer to the field "Attached background material" below
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes in the foetal incidences of skeletal variations, which are not considered to be test item-related are as follows (all values are inside the range of laboratory background data):
rib(s) wavy (25, 50 or 100 mg/kg bw/day; p ≤ 0.05 / p ≤ 0.01)
sternebrae bipartite (25 mg/kg bw/day; p ≤ 0.05)
total foetal skeletal variations (100 mg/kg bw/day; p ≤ 0.05)

Please also refer to the field "Attached background material" below
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- mortality: No test item-related increase was noted for the incidence of dead foetuses in the litters of the dams of the control group and in the litters of the 25, 50 or 100 mg/kg bw/day dose group dams. As a spontanous finding one dead foetus was noted in the litter of one dam of the 25 mg/kg bw/day dose group.

- sex distribution:
- 25, 50 or 100 mg/kg bw/day: no test item-related influence on the ratio of live male foetuses to live female foetuses was noted for all treatment groups.

All values of the treatment groups were within the range of the laboratory background data.

- weight of placentae:
- 25, 50 or 100 mg/kg bw/day: no test item-related differences of the placental weights were noted between the control group and the treatment groups.
- 50 mg/kg bw/day: the slight but statistically significant reduction noted for the mean placental weights (p ≤ 0.05) by 8% was still in the range of the laboratory background data and is not considered as test item-related.
- weight of foetuses:
- 25, 50 or 100 mg/kg bw/day: no test item-related differences of the foetal weights were noted between the control group and the treatment groups.
- 50 or 100 mg/kg bw/day: slight but statistically significant (p ≤ 0.01 or p ≤ 0.05) reductions for the mean foetal weights (by 8% for the male and female pups together) were noted. As the reductions were only slight and the foetal body weights were still within the range of the laboratory background data, they are not considered as test item-related.
The effects on the foetal weights are possibly related to an indirect effect of the severely reduced body weight of the dams, and hence, no direct effect of the test item per se.
- number of runts:
- control: one runt
- 25 mg/kg bw/day: two runts in two litters
- 50 mg/kg bw/day: two runts in one litter
- 100 mg/kg bw/day: one runt

- external macroscopic examination (twins): No increase in the incidence of twins in the test item groups or the control group.
- 100 mg/kg bw/day: a set of one male twin and one late resorption was noted in one litter. This finding is within the normal range of variability.
- external macroscopic examination (malformations):
- 25, 50 or 100 mg/kg bw/day: no test item-related macroscopically visible malformations were noted for the foetuses of the treatment groups.

The following observation is considered as spontaneous:
omphalocele (diameter approx. 2 mm) with prolapse of liver, stomach, spleen and intestines in one foetus of the 50 mg/kg bw/day dose group. This malformation is well within the laboratory background data.
- external macroscopic examination (variations):
- 25, 50 or 100 mg/kg bw/day: no macroscopically visible variations were noted for the foetuses of the control group and the treatment groups.
- internal macroscopic examination:
- 25, 50 or 100 mg/kg bw/day: no malformations or variations were noted during the examination for the foetuses of the control group and the treatment groups.
- skeletal examination (malformations):
- 25, 50 or 100 mg/kg bw/day: no malformations were noted during skeletal examinations of the foetuses according to DAWSON in the control group and in any of the treatment groups.
- skeletal examination (variations):
- 25, 50 or 100 mg/kg bw/day: no test item-related differences for the incidences of the observed variations were noted between the control group and the test item treated groups.
The skeletal variations observed were related to the ribs (less than 13 rib(s), rib(s) shortened, rib(s) wavy) and the sternum (sternebra(e) bipartite, fused (severity slight), misaligned (severity: slight), misshapen).
- skeletal examination (retardations):
- 25, 50 or 100 mg/kg bw/day: no test item related differences in incidences of the observed skeletal retardations were noted between the foetuses of the control group and the treated foetuses.
The observed skeletal retardations were related as follows:skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas) hyoid (unossified) sternum (sternebra(e) incompletely ossified, reduced in size or unossified)thoracic vertebral bodies (bipartite or dumbbell-shaped) lumbar vertebral bodies (dumbbell-shaped) caudal vertebral bodies (only one body ossified or all bodies unossified) os ischii (incompletely ossified, reduced in size or unossified) os pubis (incompletely ossified or unossified) metacarpalia (absence of ossification in metacarpalia 2 to 5) metatarsalia (absence of ossification in metatarsalia 2 to 5)
All observed incidences with statistically significant changes were within the range of the laboratory background data and are not considered as test item-related.
- soft tissue examination (malformations):
- 25, 50 or 100 mg/kg bw/day: no test item-related malformations were noted during soft tissue examinations of the foetuses according to WILSON in any of the treatment groups.
The following sporadic observations are considered as spontaneous: one foetus with an omphalocele, noted in the 50 mg/kg bw/day dose group.
- soft tissue examination (variations):
- 25, 50 or 100 mg/kg bw/day: no test item-related differences for the incidences of the observed soft tissue variations were noted between the control group and the test item treated groups.
Soft tissue variations were noted in the 4th cerebral ventricle (dilatation), the kidneys (uni- or bilateral dilatation of the renal pelvis, misplaced), the ureter (hydroureter), the liver (haemorrhagic focus/foci) and the brain (subdural haemorrhage(s)in the meninx).
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
NOAEL (maternal toxicity): 25 mg cobalt dichloride hexahydrate/kg bw/day
NOAEL (developmental toxicity): 100 mg cobalt dichloride hexahydrate/kg bw/day
Maternal toxicity (behavior, external appearance and reduction of body weight and body weight gain, food consumption, changes of hematological parameters as well as gastro-intestinal lesions) was observed at dose levels of 50 and 100 mg/kg bw/day.
The maternal NOAEL is based on the lack of significant general toxicity (body weights, food consumption) and lack of significant haematological changes at this dose level. Further, in this dose group, there was a stable body weight gain throughout the study, whereas body weight development and final body weights were severely affected in the 50 and 100 mg/kg bw dose groups. Although some changes (i.e. net body weight change and haematological parameters) were seen at the low dose (25 mg/kg b.w./day), these changes were not considered adverse as they did not reach statistical significance (except MCHC), and were less pronounced than in the higher exposure groups.
No test item-related changes were noted for the number of resorptions, postimplantation loss, and the foetal body weight. No test item-related dead foetuses, malformations, variations or retardations were noted at any of the tested dose levels. Furtherhmore, no test item-related sex distribution were noted in the study. Lastly, no test item-related differences in placental weight were noted.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-15 to 2018-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001-01-22
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2017-05-08
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: well ventilated at +10 °C to +25 °C, in a closed container, preferably under inert atmosphere
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Age at gestation day 0: 5 months
- Weight at gestation day 6: 3.72 to 5.07 kg
- Fasting period before study:
- Housing: kept separately in breeding cages with wire floors (with an area of approx. 0.45 m²)
- Diet (ad libitum): commercial ssniff® K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 15 % (maximum range)
- Air changes: 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- test item formulations were freshly prepared every day.
- test item was suspended in the vehicle to the appropriate concentration and was administered orally at a constant volume.
- during administration, the formulations were constantly stirred until the last animal per group was dosed.
- amount of the test item was daily adjusted to the current body weight of the animal.
- control animals received the vehicle at the same administration volume daily in the same way.

ADMINISTRATION VOLUME: 2 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item formulations using ICP-OES, samples of approximately 2 x 7.5 mL each were taken at the following times and stored at -20°C or colder:
1) At start of dosing (gestation day 6):
- analysis of stability and concentration: immediately after preparation of the formulation as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples / test item group).
- homogeneity: at start of administration, during (middle) administration and before administration to the last animal of the dose group (3 samples / test item group).

2) At end of the dosing period (at a time when the majority of animals was dosed)
- analysis of concentration: during treatment with the test item always before administration to the last animal of the group (1 sample / test item group).

Results:
The actual concentrations of the test item in the test item vehicle-mixtures for the 5 and 13 mg/kg bw/day dose groups ranged between 83.4% and 96.8% of the nominal concentrations.
At 32 or 80 mg Cobalt dichloride hexahydrate/kg b.w./day, test item formulation analysis revealed test item concentrations of 92.4% to 100.5% of the nominal
concentration which were within the acceptance criteria of 100% ± 10%. The marginal lower than expected concentrations in some samples are possibly
caused by the inhomogeneity of the test item delivered. Variances of up to 9.7% were noted between the aliquots taken from the same formulation. The delivered test item was slightly inhomogeneous with respect to particle size and colour, in particular, slightly larger, dark coloured particles could not be dissolved.
The test item formulation samples of the high intermediate and high dose group met the generally reported acceptance criteria. Both high dose levels showed signs of toxicity whereas the low and low intermediate dose group did not show any signs of toxicity. The slightly lower than expected test item concentrations in the test item formulations for the low and low intermediate dose group were, therefore, considered to be of no toxicological relevance.

Concentration:
- immediately after preparation: 83.4 % - 93.5 %
- before administration to the last animal at the end of study: 91.1 % - 98.0 %
Stability:
- 8 hours after preparation: 88.5 % - 99.0 %
- 24 hours after preparation: 86.3 % - 98.4 %
Homogeneity:
- before administration to the first animal: 86.4 % - 99.8 %
- during administration to the animals: 84.5 % - 100.5 %
- before administration to the last animal: 87.3 % - 97.5 %
Details on mating procedure:
- Impregnation procedure: artificial insemination (day of mating is day 0 of pregnancy)
Duration of treatment / exposure:
Day 6 to 28 of gestation
Frequency of treatment:
once daily
Duration of test:
29 days
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
13 mg/kg bw/day (actual dose received)
Dose / conc.:
32 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 female rabbits
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels for the main study were selected based on the results of a dose-range-finding study for a study of prenatal developmental toxicity in pregnant rabbits. In this dose-range-finding study, groups of 6 female New Zealand White rabbits were treated with doses of 1, 5 or 25 mg Cobalt dichloride hexahydrate/kg bw/day in tap water by oral gavage once daily from the 6th to 28th day of pregnancy.

Results:
- Mortality: no premature death was noted for the control group and the treatment groups.
- Clinical signs: no changes in behaviour, external appearance or the faeces were noted in the control group and the test item-treated groups.
- Body weight and body weight gain: no test item-related differences for the body weight gain was noted for the treatment groups. However, in the 25 mg/kg bw/day dose group, a tendency towards a decreased net body weight gain (-0.235 g compared to -0.107 g for the control group, not significant) was noted.
- Food consumption: at 25 mg/kg bw/day, a reduced food consumption was noted at the end of the study (at maximum 66.4% below the value of the control group on GD 28 to 29, not significant).
- Drinking water consumption: no test item-related influence was noted for the drinking water consumption at any of the tested dose levels (visual assessment).
Necropsy findings: no test item-related observation was noted for the treatment groups during necropsy.
- Uterus and carcass weights: no test item-related influence on the uterus and carcass weight was noted for the treatment groups.
- Examination of the foetuses: No test item-related influence was detected on the prenatal foetal development at any dose level with respect to the incidence of resorptions, number of live foetuses and the values calculated for the post-implantation loss. No test item-related differences on sex distribution were noted. At 25 mg/kg bw/day, a decrease was noted for the body weight of the foetuses (at maximum 24.2% below the value of the control group, p ≤ 0.01). No test item-related differences on placental weights were noted.
- Foetal alterations: no test item-related malformation was noted during the external examination or the gross inspection of the internal organs. The external examination at laparotomy and the gross inspection of the internal organs revealed no variations.

Based on the results, dose levels of 5, 13, 32 or 80 mg Cobalt dichloride hexahydrate/kg bw/day (administered by oral gavage from the 6th to 28th day of pregnancy) were selected for the main study of prenatal developmental toxicity rabbits.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily for any signs of behavioural changes, reaction to treatment, or illness. Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day as well as on the weekend.
Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday. Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development whenever possible.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day of delivery as well as daily on gestation days 6 to 29.
Body weight gain was calculated in intervals (i.e. day 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29).
The carcass weight (terminal body weight minus uterine weight) and the net weight gain from day 6 (carcass weight minus body weight on day 6) are given.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily (by visual appraisal)

HAEMATOLOGY: Yes
- Time schedule for collection of blood:on the day of laparotomy (before necropsy)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all dams with terminal sacrifice; non-pregnant animals and animals with abortion were excluded from evaluation.
- Parameters examined: erythrocytes, haematocrit value and haemoglobin content

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
A dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead.
The ovaries and uteri were removed; the gravid uteri (in toto) were weighed. A macroscopic examination of all subcutaneous tissues and internal organs of the dams was carried out. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were 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. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin. Organs of the dams to be preserved: sternum with bone marrow and macroscopic lesions.
Haematoxylin-eosin stained paraffin sections of the bone marrow of the dams of the dose groups 0, 13 and 32 mg/kg bw/day (at maximum n = 20 per group) were prepared and evaluated.
Furthermore, samples from tissues noted with pathologic changes (liver, lungs, stomach, small intestine and large intestine) of in total 41 animals of the treatment groups (5, 13, 32 and 80 mg/kg bw/day) were examined microscopically:
- 5 mg/kg bw/day (total: 4 animals):
large intestine: 2 animals
stomach: 3 animals

- 13 mg/kg bw/day (total: 3 animals):
large intestine: 1 animal
stomach: 3 animals

- 32 mg/kg bw/day (total: 15 animals):
large intestine: 6 animals
small intestine: 1 animal
liver: 3 animals
lung: 1 animal
stomach: 13 animals

- 80 mg/kg bw/day (total: 19 animals):
large intestine: 10 animals
small intestine: 2 animals
liver: 4 animals
lung: 2 animals
stomach: 17 animals
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- number and size of resorptions were determined
- location of foetuses in the uterus were determined
Fetal examinations:
- External examinations: Yes, all per litter (dead or alive)
- Soft tissue examinations: Yes, all per litter; thorax and peritoneal cavity were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver, discolouration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue. Kidneys were removed and incised to check for damages. The abdominal organs were removed. The diaphragm was removed to check the position of the heart (left -right) and to check for damage to the heart.
- Head examinations: Yes; head was removed from 50% of the foetuses and fixed in BOUIN'S solution. An examination according to WILSON was carried out, inspecting the internal head
structures. The cranium was opened for the remaining 50% of the foetuses and the brain was removed for external inspection in toto.
- Skeletal examinations: Yes; the eviscerated foetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).

- sex and number of foetuses alive were determined
- weight of foetuses was determined
- macroscopic inspection (gross evaluation) of the placentae
- weight of placenta was determined
- number of foetuses (alive and dead) and placentae (location in the uterus and the assignment of the foetuses was determined)
Statistics:
Statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi² test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi² test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi² test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the
external macroscopic examination of the foetuses) or an internal computer program (e.g. findings during the foetal skeletal or soft tissue examination).
The statistical evaluation of the pre- and post-implantation index per group using the number of corpora lutea, implantation sites and/ or foetuses per group was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.
Indices:
Total malformation rate [%] = (malformed foetuses per group/foetuses per group) x 100
Total variation rate [%] = (foetuses per group with variations/foetuses per group) x 100
Total retardation rate [%] = (foetuses per group with retardations/foetuses per group) x 100
Pre-implantation loss [%] = (corpora lutea - implantations/corpora lutea) x 100
Post-implantation loss [%] = (Implantations - living foetuses/Implantations) x 100
Historical control data:
Background data summarising results of the last 14 embryotoxicity studies in rabbits (New Zealand White) performed by the laboratory in the years 2013 to July 2017.
Data included were as follows: general reproductive indices, skeletal retardations, skeletal variations, external/internal variations, soft tissue variations of the foetal head, external/internal malformations and visceral malformations of the foetal head.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 32 mg/kg bw/day: 5/21 evaluated dams were noted with low amounts of excreted faeces on 2 to 7 test days. As nearly a fourth of the evaluated dams were affected and a dose response-relationship with regard to the incidence and the duration was present (please also refer to the 80 mg/kg bw/day group below), low amounts of excreted faeces were considered to be test item-related. Furthermore, diarrhoea was noted for 2/21 evaluated animals on 1 or 4 test days. This observation is related to the test item-related observation of low amounts of faeces and therefore, was also considered to be test item-related. All observations were made between GD 12 and GD 28.

- 80 mg/kg bw/day: 17/20 evaluated dams were noted with reduced amounts of excreted faeces on up to 22 days. As nearly all animals were affected and a dose response-relationship was present the observation of reduced amounts of excreted faeces was considered to be test item-related. Additionally, diarrhoea was noted for 2/20 evaluated dams on 1 or 4 test days. As diarrhoea was related to the observation of reduced amounts of excreted faeces, diarrhoea was also considered to be test item-related. All observations were made between GD 8 and GD 23.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
MORTALITY:
- 32 mg/kg bw/day: 4/21 pregnant dams were found dead between GD 16 and GD 24.
- 80 mg/kg bw/day: 1/20 pregnant dam was found dead on GD 14, two non-pregnant dams were found dead/sacrificed moribund on GDs 15 and 21.

As these evaluated dams were found dead and all were noted with similar pathological changes (changes in the gastro-intestinal tract or reduced amount of excreted faeces) the premature deaths noted for the dams treated with 32 and 80 mg/kg bw/day were considered to be test item-related.

MORIBUND/SACRIFICED PREMATURELY
- 32 mg/kg bw/day:
Premature scarifice after abortion: 9/21 pregnant females
- 80 mg/kg bw/day:
Premature scarifice after abortion: 4/20 pregnant females
Termination due to animal-welfare reasons: 10/20 pregnant femals

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1) Body weight:
- 80 mg/kg bw/day: a statistically significantly lower body weight compared to the control group was noted between GD 11 and GD 29 (at maximum 28.2% below the value of the control group on GD 29, p ≤ 0.01). The gravid uterus weight was noted to be test item-related reduced. As also the net body weight gain and the carcass weight were reduced, the distinctly and constantly decreased body weight was considered to be test item-related.

2) Body weight gain:
- 32 mg/kg bw/day: a decreased body weight gain in comparison to the control group was noted for the period from GD 6 to GD 29 (p ≤ 0.05; considered to be test item-related).
- 80 mg/kg bw/day: strongly and constantly decreased body weight gain compared to the control group was noted for period from GD 6 to GD 29 (p ≤ 0.01; considered to be test item-related).

3) Net body weight gain from day 6:
32 or 80 mg/kg bw/day: the net body weight gain was distinctly reduced (407 g below the body weight on GD 6 for the 32 mg/kg bw/day dose group, not significant or 773 g below the body weight on GD 6 for the 80 mg/kg bw/day group, p ≤ 0.01). These distinct reductions correspond to the reduced food consumption and therefore, were considered to be test item-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 32 or 80 mg/kg bw/day: a markedly, constantly and dose dependent reduced food consumption (statistically significant at p ≤ 0.05/0.01 or not) was noted for the whole dosing period (at maximum 87.7 % below the value of the control group in the 80 mg/kg bw/day dose group on GD 15 to GD 16, p ≤ 0.01). Therefore, the reduced food consumption for the two dose groups was considered to be test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 80 mg/kg bw/day: distinctly increased values were noted for the haemoglobin concentration (35.2% above the value of the control group, p ≤ 0.01), the number of erythrocytes (43.8% above the values of the control group, p ≤ 0.01) and the haematocrit (38.1% above the value of the control group, p ≤ 0.01). These distinct differences were considered to be test item-related.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1) Carcass weight:
- 32 mg/kg bw/day: a slight but test item-related reduction in the carcass weight was noted (5.3% below the value of the control group, not statistically significant). Although statistically not significant it was considered to be test item-related.
- 80 mg/kg bw/day: a reduced carcass weight was noted for the 5 animals that were examined at laparotomy (19.1% below the value of the control group, p ≤ 0.01). This reduction was considered to be test item-related.

2) Gravid uterus weight:
- 80 mg/kg bw/day: markedly and statistically significant reduction in the gravid uterus weight that was noted was due to the high number of resorptions and a reduction of the foetal body weight and therefore, was considered to test item-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Gastro-intestinal changes:
- 5, 13, 32 and 80 mg/kg bw/day: gastro-intestinal changes were noted from a dose level of 5 mg/kg bw/day onwards. These findings are considered to reflect signs of local toxicity related to the irritating properties of the test item. A dose response in the gastro-intestinal changes was observed from 14% (3 of 21) or 18% (4 of 22) in the low and low intermediate group, to 67% (14 of 21) or 85% (17 of 21) at 32 or 80 mg/kg bw/day. Based on the dose-response and consistency of this finding, this is considered to be a test-item related effect.

Stomach:
- 5 mg/kg bw/day: discolouration, red (mucosa)(2/21 dams)
- 13 mg/kg bw/day: discolouration, red (mucosa)(4/21 dams)
- 32 mg/kg bw/day: discolouration, red (mucosa)(9/21 dams; p ≤ 0.01)
- 80 mg/kg bw/day: discolouration, red (mucosa)(13/20 dams; p ≤ 0.01); black contents (2/20 dams; p≤ 0.05)

Intestines:
- 5 mg/kg bw/day: filled with dark brown watery liquid (2/21 dams)
- 13 mg/kg bw/day: filled with dark brown watery liquid (1/22 dams)
- 32 mg/kg bw/day: filled with dark brown watery liquid (6/21 dams; p ≤ 0.05)
- 80 mg/kg bw/day: filled with dark brown watery liquid (5/20 dams; p ≤ 0.05); dilatation (6/20 dams; p≤ 0.05)

2) Liver
- 32 and 80 mg/kg bw/day: changes of the liver (thick, porous, pale and/or dark/red discolouration) were noted in 3 dams at 32 mg/kg bw/day (one dam found dead and two dams were terminal sacrificed on gestation day 29) and in 4 dams at 80 mg/kg bw/day (all sacrificed during the termination of the group). The changes in the liver are considered to be probably test item-related.

3) Lungs:
- 32 and 80 mg/kg bw/day: changes in the lungs (dark red/red mottled discoloration, induration or inflation) were observed in one dam at 32 mg/kg bw/day (found dead) and in 2 dams at 80 mg /kg bw/day (both sacrificed during the termination of group 5). The changes in the lungs are considered to be probably test item-related.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 32 and 80 mg/kg bw/day: mild to moderate focal necrosis of hepatocytes surrounded by mild to moderate vacuolization were noted in the livers.
- 5, 13, 32 and 80 mg/kg bw/day: test item-related local findings were noted for the stomach and the large intestine mucosa: examinations revealed minimal to moderate bleeding/congestions of the gastric mucosa, isolated ulcerations and vacuolization of epithelial cells in the stomach as well as moderate subepithelial lympho-histiocytic infiltrations in the colon and the caecum. These findings were caused by the corrosive properties of the test item and were dose-related.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS:
- 5 and 13 mg/kg bw/day: no or no test item-related changes in behaviour, the external appearance or faeces were noted. In the 5 mg/kg bw/day dose group 2 dams were noted with reduced amounts of excreted faeces and/or diarrhoea. 2 animals were transiently affected on a few test days (2 and 5 test days, respectively)
- 32 mg/kg bw/day: no or no test item-related changes in behaviour or the external appearance were noted.

MORTALITY:
- 0, 5 and 13 mg/kg bw/day: no test item-related premature death of pregnant females was noted. Premature sacrifice after abortion was conducted in one pregnant female of both test item treated groups, respectively.

BODY WEIGHT AND WEIGHT CHANGES:
1) Body weight
- 5 and 13 mg/kg bw/day: no test item-related changes in body weight were noted in the dams after oral treatment.
- 32 mg/kg bw/day: the body weight was significantly lower compared to the control group in the period from GD 16 until study termination on GD 29 (at maximum 8.1% below the value of the control group on GD 28, not significant). This corresponds to a reduction in the whole body weight gain and slight reduction in the carcass weight and net body weight gain in comparison to the control group

2) Body weight gain:
- 0, 5 and 13 mg/kg bw/day: no test item-related differences between the control group and 5 or 13 mg/kg bw/day dose groups were noted for the body weight gain.

3) Net body weight gain from day 6:
- 5 and 13 mg/kg bw/day: no test item-related changes between the control group and 5 or 13 mg/kg bw/day dose groups were noted for the net body weight gain (without gravid uterus) between gestation day 6 and gestation day 29.

FOOD CONSUMPTION:
- 5 and 13 mg/kg bw/day: no test item-related changes in food consumption were noted between the dams of the control group and the dams treated with the test item. Only transiently, statistically no significant differences in comparison to the control group were noted for the food consumption (at maximum 19.9% below the value of the control group for the 5 mg/kg bw/day dose group on GD 19 to GD 20, statistically not significant). Therefore, these differences were considered to be not test item-related.

WATER CONSUMPTION:
- 5, 13, 32 and 80 mg/kg bw/day: no test item-related changes in the consumption of drinking water were noted for the dams treated with the test item.

HAEMATOLOGICAL FINDINGS:
- 5, 13 and 32 mg/kg bw/day: no test item-related changes were noted for the haemoglobin concentration, the number of erythrocytes and the haematocrit for the dams treated with the test item.

ORGAN WEIGHTS:
1) Carcass weight:
- 5 and 13 mg/kg bw/day: no influence on the carcass weight was noted.

2) Gravid uterus weight:
- 5 mg/kg bw/day: no test item-related influence on the gravid uterus weight was noted.
- 13 and 32 mg/kg bw/day: a slight reduction in the gravid uterus weight were noted. The reduction was due to the occurrence of dams with a total implantation loss and was considered to be not test item-related.

GROSS PATHOLOGICAL FINDINGS:
- 32 mg/kg bw/day (dams found dead between GD 16 and GD 24): macroscopic inspection revealed no observations for one dam, but the animal was noted to have a low amount of excreted faeces and diarrhoea. The three remaining dams were noted with macroscopic changes related to the gastro-intestinal tract as well as macroscopic observations related to the lungs (one dam) or the liver (another dam) but no change in behaviour, external appearance or in the faeces.
- 80 mg/kg bw/day: macroscopic inspection revealed a thin stomach wall and a severely reddened stomach mucosa. Furthermore, low amounts of excreted faeces were noted on GD 8 to GD 12. For the dam but no changes of behaviour or external appearance.

1) Placenta:
- 5, 13 and 32 mg/kg bw/day: changes of the placenta (pale, white/beige foci, indurations or beige discolorations) were noted for one dam at 5 mg/kg bw/day, for 3 dams at 13 mg/kg bw/day and for one dam at 32 mg/kg bw/day (with a haemorrhagic vagina). These observations were considered to be not test item-related as only individual dams were affected by placental changes and no corresponding placental changes were noted in 80 mg/kg bw/day dose group.

2) Vagina:
- 5 and 13 mg/kg bw/day: a haemorrhagic vagina was noted in one dam each at 5 and 32 mg/kg bw/day as a result of abortion (not considered to be test item-related).

3) Kidney:
- 80 mg/kg bw/day: a malpositioned left kidney was noted in one dam as a spontaneous finding.

4) Urinary bladder:
- 80 mg/kg bw/day: changes were noted in the urinary bladder of one dam (filled with red liquid; terminal sacrificed on gestation day 29)(not considered to be test-item related).

HISTOPATHOLOGICAL FINDINGS:
- 13 and 32 mg/kg bw/day: no test item-related microscopically changes of the sternal bone marrow were noted.
- 0, 13 and 32 mg/kg bw/day: the bone marrow was of a normal appearance in the control and treated rabbits. There was no morphological difference between the control and treated animals. The cells of the bone marrow were large, round myeloblasts, neutrophilic, eosinophilic and basophilic cells or nucleated erythroblasts. A different number of megakaryocytes in the bone marrow were noted between the animals in the control group and the dose groups 13 and 32 mg/kg bw/day). However, there were no test item related findings. The increase of fatty tissue between the bone marrow cells caused a reduction of cellularity. There was no difference between the groups.

- haemorrhages, dark pigments and mild pericholangitis were observed in the liver of individual animals. These changes are interpreted as incidental findings.
The severe bronchopneumonia purulenta as well as the alveolar oedema in the lungs are interpreted as incidental findings.
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
- 32 and 80 mg/kg bw/day: 9/21 and 4/20 evaluated dams, respectively; were noted with an abortion (considered to be test-item related findings).
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
- 13 mg/kg bw/day: 3 animals were noted with a total implantation loss. The increased post-implantation loss (p ≤ 0.01) were considered to be test item-related.
- 32 and 80 mg/kg bw/day: a statistically significantly higher post-implantation loss was noted (p ≤ 0.01) (considered to be test item-related).
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
- 13 mg/kg bw/day: 7 animals with up to 3 resorptions leading to 35 resorptions in total (p ≤ 0.05; considered to be test item-related).
- 32 and 80 mg/kg bw/day: in the 32 mg/kg bw/day dose group, 17 resorptions and in the 80 mg/kg bw/day dose group 42 resorptions were noted (p ≤ 0.01; considered to be test item-related).
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
NUMBER OF ABORTIONS:
- 5 and 13 mg/kg bw/day: no test item-related abortions were noted. At 5 mg/kg bw/day, one pregnant rabbit was noted with an abortion in the night between GD 20 and GD 21. This single occurrence was considered to be not test item-related, as abortions are known to occur spontaneously in rabbits of this strain and age and one abortion was also noted for a dam of the control group. At 13 mg/kg bw/day, no abortions were noted.

PRE- AND POSTIMPLANTATION LOSS:
- 5 mg/kg bw/day: a statistically significantly increased post-implantation loss was noted (p ≤ 0.01). As this was due to a very low number of resorptions in the control group and the post-implantation loss was within the laboratory background data range, the increased post-implantation loss was considered to be not test item-related.

EARLY AND LATE RESORPTION:
- 5 mg/kg bw/day: 7 resorptions were noted.

DEAD FOETUSES:
- 0, 32 and 80 mg/kg bw/day: no dead foetus was noted
- 5 mg/kg bw/day: one dam was noted with 5 dead foetuses (considered to be not test item-related).
- 13 mg/kg bw/day: one dam was noted with one dead foetus (considered to be not test item-related).
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
13 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
(local toxicity)
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Abnormalities:
not specified
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 80 mg/kg bw/day: strongly decreased weights were noted for the male and female foetuses (males: 35.9 g (mean; -23.9 %) and females: 38.58 g (mean; -19.5 %) as well as for the male and female foetuses combined (37.68 g (mean; - 21.1 %). As the weights were also outside the laboratory background data range, the decreased foetal weights were considered to be test item-related.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
FOETAL BODY WEIGHT CHANGES.
- 5, 13 and 32 mg/kg bw/day: no test item-related influence was noted on the mean foetal weights after administration.
- 32 mg/kg bw/day: a slightly lower but not statistically significant body weight was noted for the male and female foetuses as well as for male and female foetuses combined (at maximum 11.2% below the value of the control group for the male and female foetuses combined). As the weights were still well within the laboratory background data range, the slightly reduced foetal weights were considered to be not test item-related.

- 0, 32 and 80 mg/kg bw/day: no runts were observed.
- 5 mg/kg bw/day: 4 runts were observed.
- 13 mg/kg bw/day: 1 runt were observed.

CHANGES IN SEX RATIO:
- 5 and 80 mg/kg bw/day: the sex distribution of the foetuses in the dose groups (5 mg/kg bw/day: 0.58; 80 mg/kg bw/day: 0.50) were out of the laboratory background data range (ratio of male to female foetuses: 0.63 to 1.33). However, as the sex ratio was comparable to the control group, the increased number of female foetuses compared to the number of male foetuses was considered to be spontaneous and not test item-related.
- 13 and 32 mg/kg bw/day: the sex ratio was 0.83 and 1.03, respectively.

EXTERNAL MALFORMATIONS:
- 0, 5, 13, 32 and 80 mg/kg bw/day: no visible gross alteration (malformation or variation) was noted for the foetuses of the control group and the foetuses of the treatment groups.

SKELETAL MALFORMATIONS:
1) Malformation:
- 0, 5, 13, 32 and 80 mg/kg bw/day: no alterations in the form of malformations were noted during skeletal examinations of the foetuses according to DAWSON at any tested dose level.

2) Variations:
The skeletal variations observed during examination according to DAWSON were related to the skull (fontanelle enlarged or distinct areas not ossified), the sternum (sternebra(e) bipartite, fused to a slight degree, misaligned or misshapen) or the rib(s) (accessory 13th ribs (uni- or bilateral) or thickened), the thoracic vertebral bodies (misaligned).
No test item-related increase in the incidence of the above mentioned variations were noted in comparison to the control group.
Statistically significantly increased incidences of skeletal variations were noted for the observations of an enlarged fontanelle for the high intermediate dose group (32 mg/kg bw/day) and the sternebrae being bipartite for the low dose group (5 mg/kg bw/day). However, the incidence of the bipartite sternebrae was within the laboratory background data range of the test groups. The incidence of the enlarged fontanelle was outside the laboratory background data range but all observations of the fontanelle being enlarged were noted only for foetuses of one litter and the high incidence was due to the low number of evaluable litters. Therefore, both variations were considered to be not test item-related.

3) Retardations:
The retardations observed during skeletal examination (according to DAWSON) were related to the skull (hyoid unossified), the sternum (sternebra(e) unossified, incompletely ossified or reduced in size) or the hind limbs (absence of ossification in metatarsalia 2 to 5).
No test item-related influence was noted for the incidence of skeletal retardations in the low and low intermediate dose groups (5 or 13 mg/kg bw/day).
- 32 mg/kg bw/day: a statistically significantly increased incidence for unossified sternebrae and the total foetal skeletal retardations was noted. As the incidence was outside the laboratory background range and a dose response relationship was present, the increased incidences were considered to be test item-related. However as the literature reports that a reduced food consumption of the dams leads to a retarded ossification in the foetuses (Cappon et al., 2005)*, the increased incidence for unossified sternebrae and the total number of skeletal retardations were considered to be secondary effects caused by the pronounced maternotoxicity.
An assessment of the retardations found at 80 mg/kg bw/day was not possible as only 5 foetuses from one litter were available.

VISCERAL MALFORMATIONS:
- 0, 5, 13, 32 and 80 mg/kg bw/day: no test item-related malformations or variations were noted during the internal examination of the foetuses of the control group and the treatment groups.
The external inspection of the brain in 50% of the foetuses revealed no changes for any of the foetuses after opening of the cranium and removal of the brain.
- 5 and 13 mg/kg bw/day: one foetus each was noted with a variation in form of a malpositioned left kidney (considered to be not test item related).

- 0, 5, 13, 32 and 80 mg/kg bw/day: no alterations in the form of malformations were noted during soft tissue examinations of the foetal head according to WILSON at any tested dose level. No test item-related influence was noted in the number of soft tissue variations of the foetal head compared to the control at any tested dose level.
The observed and classified soft tissue variations that were noted during the examination of the foetal head according to WILSON were in the form of dilatations of the 4th cerebral ventricle, subdural haemorrhages in the meninges and haemorrhages of the cerebrum or the cerebral hemisphere. No test item-related differences in the incidences of the observed variations of the foetal head were noted between the control group and the test groups.
The observed cystic areas in the cerebral hemisphere noted in a total of 9 foetuses of the test item groups and in 1 control foetuses are known to be fixation-induced artefacts

PLACENTAE WEIGHT:
- 5, 13, 32 and 80 mg/kg bw/day: no test item-related differences for the placental weights were noted for any of the dose groups.
- 32 mg/kg bw/day: slightly lower weights were noted for the male and female placental weights as well as for the weight of male and female placentae combined (at maximum 13.4% below the value of the control group for the male placentae, statistically not significant). However, no statistically significant difference was noted and the weights were well within the range of the laboratory background data. Therefore, the decreased placental weights were considered to be not test item-related.

*References:
- CAPPON, G.D.: Effects of feed restriction during organogenesis on embryo-fetal development in rabbit. Birth Defects Res B Dev Reprod Toxicol. 74 (5), 424 - 430 (2005).
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: resorption
Key result
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In a prenatal developmental toxicity study, cobalt dichloride was administered orally to female rabbits at dose levels of 5, 13, 32 or 80 mg cobalt dichloride hexahydrate /kg bw/day from the 6th to 28th day of pregnancy. The 80mg/kg bw/daygroup was terminated prematurely due to the poor general condition of the animals, abortions and mortality.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) for systemic effects was 13 mg/kg bw/day for the dams based on significant decreases in body weight gain and food consumption at 32 mg/kg bw/day. There was increased mortality (19% loss of test animals) at this dose level. Low amounts of excreted faeces and/or diarrhoea were noted for the dams treated with 32 mg/kg bw/day. Histopathological changes were noted for the lungs and the liver at 32 mg/kg bw/day (necrosis and vacuolisation of hepatocytes). Furthermore, histopathological examination revealed test item-related local changes for the stomach mucosa (bleeding/congestions of gastric mucosa, isolated ulcerations and vacuolization of epithelial cells) and the large intestine mucosa (subepithelial lympho-histiocytic infiltration and vacuolization of epithelial cells) in all treatment groups (5, 13, 32 or 80 mg/kg bw/day) in a dose-related way. Based on the histopathological findings in the GI tract, the NOAEL for local effects in maternal animals is at 5 mg/kg bw/day, based on the onset of effects on the stomach mucosa (discoloration, red, white deposits, filled with liquid) at 13 mg/kg bw/day. No test item-related changes were noted during microscopic inspection of the sternal bone marrow of the control group and the low intermediate and high intermediate dose group (13 or 32 mg/kg bw/day).

The no-observed-adverse effect level (NOAEL) for the foetal organism was 5 mg/kg bw/day:
An increased number of resorptions was noted at 13 mg/kg bw/day, which was materno-toxic (local effects). At the materno-toxic (systemic effects) dose levels of 32 or 80 mg/kg bw/day an increased number of resorptions and/or abortions were noted. At 80 mg/kg bw/day, a decreased body weight was noted for the 5 available foetuses. No test item-related prematurely deceased foetuses, no malformations and no test item-related variations were noted. For the high intermediate dose group (32 mg/kg bw/day), an increase was noted for the incidence of unossified sternebrae (skeletal retardation) and the total number of skeletal retardations. However, as a reduced food consumption of the dams leads to a retarded ossification in the foetuses (Cappon et al., 2005), the increased incidences for unossified sternebrae and for the total number of skeletal retardations in the high intermediate dose group were considered to be secondary effects caused by the pronounced maternotoxicity. Under the conditions of the study, cobalt dichloride hexahydrate had no teratogenic potential.

In summary, the NOAELs determined in this study are as follows:
Maternal NOAEL (systemic) = 13 mg/kg bw/day, based on significant effects on body weight gain and food con-sumption and increased mortality at 32 mg/kg bw/day
Maternal NOAEL (local) = 5 mg/kg bw/day, based on onset of GI irritation 13 mg/kg bw/day
Foetal NOAEL = 5 mg/kg bw/day, based on an increase in resorptions 13 mg/kg bw/day
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
Study duration:
subacute
Species:
rabbit
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

 

Cobalt dichloride is the source substance for the bioavailable cobalt substances group based on the read-across approach as outlined in Appendix 1.1 in the CSR. Two soluble inorganic cobalt substances, cobalt dichloride and cobalt powder, have been tested for developmental toxicity, resulting in an absence of effects on the developing organism up to the maximum tolerated dose.

 

In a pre-natal developmental toxicity study (according to OECD 414 and under GLP), cobalt dichloride was given to pregnant rats at doses of 0, 25, 50, 100 mg/kg bw/day. A total of 25 females per group were given the test items suspended in 0.5% hydroxypropyl methylcellulose gel orally via gavage once daily from gestation day 6 until gestation day 19.

 

No test item-related premature death was noted in the control or in any of the test item treated groups.

At 50 or 100 mg/kg bw/day piloerection, a slight to moderate reduced motility and slight to moderate salivation were noted for several dams on one or up to 6 test days.

At 100 mg/kg bw/day a haemorrhagic nose/snout was noted for 3 of 20 dams on gestation days 19 or 20. At 50 or 100 mg/kg bw/day statistically significantly reduced body weights in comparison to the control group were noted after the start of treatment (about 5 to 6% below the control group on gestation day 8 or 9). The reductions in body weight for both dose groups compared to the control group slightly increased during the further course of the study, leading to body weights on gestation day 20, that were 14.2% (intermediate dose group) or 15.1% (high dose group) below the value of the control group. The net weight change (body weight without gravid uterus) after the start of treatment until laparotomy (gestation day 6 to gestation day 20) was statistically significantly reduced for the dams treated with 50 or 100 mg/kg bw/day: control: + 38. 4 g, intermediate dose: -10.6 g, high dose: - 14.1g. Gastro-intestinal lesions in form of haemorrhagic foci in the stomach and intestines with a dark(-brown) content were noted in a dose related way for the dams dosed with 25, 50 or 100 mg/kg bw/day (low dose: 1 of 20 dams, intermediate dose: 3 of 20 dams, high dose: 9 of 20 dams). No test item related influence was noted on the gravid uterus weight. The carcass weights were statistically significantly reduced in comparison to the control group by 8.7, 18.2 and 18.1% for the dams treated with 25, 50 or 100 mg/kg bw/day.

 

The reproduction data of the dams revealed no test item-related differences for pre-implantation loss, post-implantation loss and resorptions/implantation site ratios between the dams of the control group and the dams treated with 25, 50 or 100 mg/kg bw/day.

 

No dead foetuses were noted in the litters of the dams of the control group and in the litters of the dams treated with 25, 50 or 100 mg/kg bw/day. No test item-related influence on the ratio of live male foetuses to live female foetuses were noted for all treatment groups. All values of the treatment groups were within the range of laboratory background data.

No test item-related differences of the placental or foetal weights were noted between the control group and the treatment groups.

 

No test item-related macroscopically visible malformations or variations were noted for the foetuses of the treatment groups during the external examination of the foetuses at laparotomy. The internal examination revealed no malformation or variations in the control or any of the treatment groups. Skeletal examination showed no test-item related malformations, variations or retardations in the control or any of the treatment groups. No test item-related malformations were noted during soft tissue examinations of the foetuses according to WILSON in any of the treatment groups. No test item-related differences for the incidences of the observed soft tissue variations were noted between the control group and the test item treated groups.

 

Under the test conditions of the pre-natal developmental toxicity study with cobalt dichloride, the no-observed-adverse-effect level (NOAEL) was 25 mg cobalt dichloride/kg bw/day for the dams, based on reduction of body weight and body weight gain, food consumption, changes of haematological parameters as well as gastro-intestinal lesions

 

The no-observed-adverse effect level (NOAEL) for the foetal organism was above 100 mg cobalt dichloride/kg bw/day. No test item-related changes were noted for the number of resorptions and the foetal body weight. No dead foetuses, no test item-related malformations, variations or retardations were noted at any of the tested dose levels.

 

In a prenatal developmental toxicity study, cobalt dichloride was administered orally to female rabbits at dose levels of 5, 13, 32 or 80 mg cobalt dichloride hexahydrate /kg bw/day from the 6th to 28th day of pregnancy. The 80mg/kg bw/daygroup was terminated prematurely due to the poor general condition of the animals, abortions and mortality.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) for systemic effects was 13 mg/kg bw/day for the dams based on significant decreases in body weight gain and food consumption at 32 mg/kg bw/day. There was increased mortality (19% loss of test animals) at this dose level. Low amounts of excreted faeces and/or diarrhoea were noted for the dams treated with 32 mg/kg bw/day. Histopathological changes were noted for the lungs and the liver at 32 mg/kg bw/day (necrosis and vacuolisation of hepatocytes). Furthermore, histopathological examination revealed test item-related local changes for the stomach mucosa (bleeding/congestions of gastric mucosa, isolated ulcerations and vacuolization of epithelial cells) and the large intestine mucosa (subepithelial lympho-histiocytic infiltration and vacuolization of epithelial cells) in all treatment groups (5, 13, 32 or 80 mg/kg bw/day) in a dose-related way. Based on the histopathological findings in the GI tract, the NOAEL for local effects in maternal animals is at 5 mg/kg bw/day, based on the onset of effects on the stomach mucosa (discoloration, red, white deposits, filled with liquid) at 13 mg/kg bw/day. No test item-related changes were noted during microscopic inspection of the sternal bone marrow of the control group and the low intermediate and high intermediate dose group (13 or 32 mg/kg bw/day).

The no-observed-adverse effect level (NOAEL) for the foetal organism was 5 mg/kg bw/day:

An increased number of resorptions was noted at 13 mg/kg bw/day, which was materno-toxic (local effects). At the materno-toxic (systemic effects) dose levels of 32 or 80 mg/kg bw/day an increased number of resorptions and/or abortions were noted. At 80 mg/kg bw/day, a decreased body weight was noted for the 5 available foetuses. No test item-related prematurely deceased foetuses, no malformations and no test item-related variations were noted. For the high intermediate dose group (32 mg/kg bw/day), an increase was noted for the incidence of unossified sternebrae (skeletal retardation) and the total number of skeletal retardations. However, as a reduced food consumption of the dams leads to a retarded ossification in the foetuses (Cappon et al., 2005), the increased incidences for unossified sternebrae and for the total number of skeletal retardations in the high intermediate dose group were considered to be secondary effects caused by the pronounced maternotoxicity. Under the conditions of the study, cobalt dichloride hexahydrate had no teratogenic potential.

In summary, the NOAELs determined in this study are as follows:

·        Maternal NOAEL (systemic) = 13 mg/kg bw/day, based on significant effects on body weight gain and food con-sumption and increased mortality at 32 mg/kg bw/day

·        Maternal NOAEL (local) = mg/kg bw/day, based on onset of GI irritation 13 mg/kg bw/day

·        Foetal NOAEL = 5 mg/kg bw/day, based on an increase in resorptions 13 mg/kg bw/day

 

 

In a combined repeated dose toxicity studies with the reproduction/developmental toxicity screening test (according to OECD 422 and under GLP) cobalt was administered orally to rats at dose levels of 30, 100, 300 and 1000 mg/kg bw/day during the pre-mating, mating and post-mating periods to parental males as well as during the pre-mating, mating, gestation and lactation periods until day 3 post-partum (or shortly thereafter) to parental female animals.

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 /kg bw/day onwards. In addition, reductions of body weight were noted from 300 mg cobalt /kg bw/day onwards. Premature deaths occurred in five female rats at 100 mg cobalt /kg bw/day and eight female rats at 300 mg cobalt /kg bw/day. Treatment with 1000 mg cobalt /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/kg bw/day onwards and adrenal changes and pulmonal lesions at 1000 mg cobalt/kg bw/day.

Histopathological inspection did not reveal any pathological changes. No histopathological correlate could be found for the macroscopical lesions noted at necropsy. No test item-related influence was noted on the sperm staging or interstitial cell structure (qualitative examination). Treatment with 300 mg cobalt/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/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/kg bw/day. In order to estimate a possible correlation between maternal toxicity and F1-Generation (pups) findings, the litter weight of pups was compared in dams with clinical signs within each group. Dams were classified based on the severity and occurrence of clinical signs. As a result it appeared that the earlier the clinical signs occurred in the dams a more pronounced weight reduction was noted for the pups of the respective dams. An increased F1-Generation (pups) mortality rate and a slightly decreased viability index were noted from 100 mg cobalt/kg bw/day onwards.

 

The NO(A)EL for effects on the F0-generation was 30 mg cobalt/kg bw/day, based on mortality, clinical signs of toxicity, effects on food consumption and macroscopic pathological changes observed at and above 100 mg cobalt/kg bw/day and reduced body weight at and above 300 mg cobalt/kg bw/day.

 

The NO(A)EL for effects on the reproductive toxicity was 30 mg cobalt/kg bw/day, based on an increased F1-Generation (pups) mortality rate and a slightly decreased viability index at 100 mg cobalt/kg bw/day and on post-implantation losses, decreases in the live birth index and significantly reduced litter weights of pups observed at 300 mg cobalt/kg bw/day.

 

 

In addition to the above studies there is a limited number of information on developmental toxicity of soluble cobalt compounds in several species, which suffers from several defects:

 

                   several studies (in particular those of Wide, 1984; Domingo et al., 1985) report effects on foetuses and neonates, but all studies suffer from several methodical and reporting deficiencies, thus rendering them of very limited use in a regulatory context. In addition, the reference by Gluhcheva et al. (2014) suffers from several methodological and reporting deficiencies, therefore was not relevant for human health risk assessment.

                   one study is apparently void of effects (Paternain et al., 1988) with regard to foetal effects, but maternal toxicity was observed at all dose levels, so that this study does not allow the derivation of a maternal no-effect level.

                   another study (Szakmary et al., 2001) provides a multitude of information, but which is partly contradictory in nature and also has reporting and methodological shortcomings.

 

In conclusion, these and other available studies show several deficiencies, thus rendering them of limited use in a regulatory context. Since the results are somewhat contradictory, they are considered unsuitable for a human health risk assessment and DNEL-setting under REACH.

Furthermore, none of the available studies represent state-of-the-art, guideline-compliant, extended one-generation reproduction toxicity studies or other relevant study designs, from which robust no-effect levels for human risk assessment could be derived.

 

 

Conclusion

The test results with the source substances of the bioavailable cobalt substances group (screening test with cobalt, pre-natal developmental toxicity study with cobalt dichloride in rats and rabbits) does not support the clear identification of adverse effects towards developmental toxicity. However, findings in the pre-natal developmental toxicity study in rabbits manifested as increased early resorptions at presence of some maternal toxicity does not allow to unequivocally conclude on an absence of developmental effects in rabbits. Consequently, cobalt dichloride and subsequently all members of the bioavailable cobalt substances group are self-classified for developmental toxicity Category 2 (H361d).

 

 

Justification for classification or non-classification

Based on the existing published data on fertility impairment of some members of the bioavailable cobalt substances group all members of the bioavailable cobalt substances group will be self-classified as toxic for reproduction category 1B (H360F). This is supported by the findings in the pre-natal developmental toxicity study showing a decreased ability of the dams to maintain pregnancy.

 

The test results with the source substances of the bioavailable cobalt substances group (screening test with cobalt, pre-natal developmental toxicity study with cobalt dichloride in rats and rabbits) does not support the clear identification of adverse effects towards developmental toxicity. However, findings in the pre-natal developmental toxicity study in rabbits manifested as increased early resorptions at presence of some maternal toxicity does not allow to unequivocally conclude on an absence of developmental effects in rabbits. Consequently, cobalt dichloride and subsequently all members of the bioavailable cobalt substances group are self-classified for developmental toxicity Category 2 (H361d).