Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Cobalt dichloride hexahydrate
- State of aggregation: solid, lilac crystals
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

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

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).

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

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.