Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-06-17 to 2014-08-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2001-01-22
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14
Limit test:
no

Test material

Constituent 1
Reference substance name:
tricobalt tetraoxide
IUPAC Name:
tricobalt tetraoxide
Constituent 2
Chemical structure
Reference substance name:
Tricobalt tetraoxide
EC Number:
215-157-2
EC Name:
Tricobalt tetraoxide
Cas Number:
1308-06-1
Molecular formula:
Co3O4
IUPAC Name:
tricobalt tetraoxide
Constituent 3
Reference substance name:
1308-06-1
Cas Number:
1308-06-1
IUPAC Name:
1308-06-1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Cobalt Oxide 72/73F
- Molecular formula: Co3O4
- Physical state: grey-black powder
- Storage condition of test material: cool and well-ventilated in a tightly closed container.
- Surface area (BET): 3.1 m²/g
- Particle size distribution: D10: 0.4 μm; D50: 1 μm; D90: 4 μm

Test animals

Species:
rat
Strain:
other: CD
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at day 0 of gestation: 67 days
- Weight at day 0 of gestation: 189.1 - 271.6 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: 12 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:
other: 0.5% hydroxypropyl methylcellulose gel
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.

VEHICLE - 0.5% hydroxypropyl methylcellulose gel
- Source: Fagron, Barsbüttel, Germany
- Batch no.: 13D03-N03

Administration volume: 5 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; 100, 300, and 1000 mg/kg bw/day dose groups).
- homogeneity: at start of administration, during (middle) administration and before administration to the last animal of the dose group (3 samples / dose level group; 100, 300, and 1000 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 group (1 sample/dose level group; 100, 300, and 1000 mg/kg bw/day dose groups).

The determination of the content of the test item tricobalt tetraoxide in samples was performed by analysis of cobalt with Inductively Coupled Plasma Optical Emission Spectrometry (IPC-OES).

Results:
- concentration: 88.0% - 99.7%
- stability: 89.3% - 96.1%
- homogeneity: 89.6% - 97.2%
These results indicated correctly prepared and homogenised test item vehicle mixtures, which were stable for at least 24 hours.
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
No. of animals per sex per dose:
25 female rats (20 dams with litters were evaluated)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on available toxicological data.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily; 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.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 of gestation, followed by daily weighings
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
A dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues 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), large intestine (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
- Animals fasted: No
- How many animals: all dams (rats with litter and without litter)
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, relative differential blood count, absolute differential blood count, reticulocytes, platelets, haematocrit value, 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 examinated 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 of early resorptions: Yes
- Number 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 perfromed 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 the fetuses) 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 ≤ 0.01 (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

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- mortality: no test item-related premature death was noted in the control or in any of the test item treated groups (100, 300 or 1000 mg/kg bw/day).
One animal of the 1000 mg/kg bw/day dose group died on gestation day 15, twenty to 60 minutes after the administration of the test item. No pre-mortal signs of toxicity were noted. Necropsy revealed a thickened and black discoloured right lung lobe and a thorax filled with black liquid. Due to the findings at necropsy, a misgavage was considered as the cause of death. This animal was excluded from further evaluation.

- clinical observations: no changes in behaviour, the external appearance or the faeces were noted in the control group or in any of the test item treated groups (100, 300 or 1000 mg/kg bw/day).

- body weight and body weight gain: no test item-related changes in body weight in comparison to the control group were noted for the dams of all treatment groups (100, 300 or 1000 mg/kg bw/day. Body weight gain for the whole gestation period revealed no test item-related differences (control group: 59.7%, 100 mg/kg bw dose group: 61.8%, 300 mg/kg bw dose group: 59.1%; 1000 mg/kg bw dose group: 56.1%). Furthermore, the 3-day intervals of body weight change also revealed no test item-related changes, though a statistically not significant reduction in body weight gain between gestation days 6 and 9 by 32.6% was noted for the dams of the 1000 mg/kg bw dose group. This corresponds to a marginal difference of 4.2 g between the dams of the 1000 mg/kg bw dose group and the dams of the control group. This slight alteration is without biological relevance and not considered as an adverse effect of the test item. Lastly, no influence of the gravid uterus weight on the whole body weight was noted.

- food consumption: no test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 100, 300 or 1000 mg/kg bw/day. Differences in food consumption with significant differences in comparison to the control group which were considered as not test item-related were as follows: females of the 1000 mg/kg bw/day dose group showed a decreased in food consumption on gestation days 19 -20 (p≤0.05). The slight alteration in comparison to control animals is without biological relevance.

- water consumption: no test item-related changes in the consumption of drinking water was noted for the dams treated with 100, 300 or 1000 mg/kg bw/day by visual appraisal.

- macroscopic examinations: macroscopic inspection of the internal organs of the dams revealed no changes in the control and the test item-treated groups (100, 300 or 1000 mg/kg bw/day). The following sporadic observations are considered as spontaneous:
100 mg/kg bw/day: a few dark red foci (diameter up to approx. 0.5 mm) were noted in the lungs of one dam
1000 mg/kg bw/day: the observations made for the prematurely deceased animal

- Uterus weight and net body weight change: no test item-related changes in the gravid uterus weight and the carcass weight in comparison to the control group were noted for the dams of all treatment groups (100, 300 or 1000 mg/kg bw/day). Furthermore, no test item-related changes in the absolute and the net (without uterus) body weight change in comparison to the control group were noted for the dams of all treatment groups (100, 300 or 1000 mg/kg bw/day) between gestation day 6 and 20. A statistically significantly (p≤0.05) decreased net weight change from day 6 until laparotomy by 27.2% was noted for the dams of the 1000 mg/kg bw/day dose group. This corresponds to a slight difference of 8.7 g between the dams of the 1000 mg/kg bw/day dose group and the dams of the control group (32.0 g for the dams of the control group in comparison to 23.3 g for the dams of the 1000 mg/kg bw/day dose group). This slight alteration is without biological relevance and not considered as an adverse effect of the test item.

- reproduction data of the dams: no test item-related differences for pre-implantation loss, post-implantation loss and resorptions/implantation site ratios were noted between the dams of the control group and the dams treated with 100, 300 or 1000 mg/kg bw/day. The following 2 statistically significant changes are not test item-related: in the 100 mg/kg bw/day dose group the ratio of corpora lutea to implantation sites (preimplantation loss) was statistically significantly reduced. However, a reduced preimplantation loss in comparison to the control group is not an adverse effect, furthermore, implantation had occurred before the start of treatment. Additionally, in the 100 mg/kg bw/day dose group the ratio of late resorptions to implantation sites was statistically significantly (p≤0.05) increased. In detail, 6 late resorptions were noted in 6 different dams in comparison to one late resorption in the control group. As no dose-response relationship was noted (only one late resorption each was found in the dams of the 300 mg/kg bw/day and the 1000 mg/kg bw/day dose group), this observation is considered as spontaneous and not test item-related.

- haematology: no test-item related influence was noted for the dams treated with 100, 300 or 1000 mg/kg bw/day.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- mortality: no dead foetuses were noted in the litters of the dams of the control group and in the litters of the dams treated with 100, 300 or 1000 mg/kg bw/day.

- sex distribution of foetuses: no test item-related influence on the ratio of live male foetuses to live female foetuses were noted for all treatment groups (100, 300 or 1000 mg/kg bw/day). All values of the treatment groups were within the range of laboratory background data. However, an increased male to female ratio was noted in the control group, leading to statistically significantly (p≤0.05) reduced male to female ratios in all treatment groups in comparison to the control group. As the male to female ratio of the control group was above the background range, this observation is considered as spontaneous and the statistically significant decreases in the male to female ratios of the treatment groups are not test item-related.

- weight of placentae: no test item-related differences of the placental weights were noted between the control group and the treatment groups (100, 300 or 1000 mg/kg bw/day).

- weight of foetuses: no test item-related differences of the foetal weights were noted between the control group and the treatment groups (100, 300 or 1000 mg/kg bw/day). Only one runt was noted in the 1000 mg/kg bw/day dose group. No runts were noted in the control, the 100 and 300 mg/kg bw/day dose groups.

- external examination (malformations): no test item-related macroscopically visible malformations were noted for the foetuses of the treatment groups (100, 300 or 1000 mg/kg bw/day) during the external examination of the foetuses at laparotomy.
The following sporadic observations are considered as spontaneous:
control group: one foetus with spina bifida.
300 mg/kg bw/day: one foetus with a stumpy tail (brachycaudia) and one foetus with multiple malformations (hydrocephalus, an abnormal shortness of the upper jaw (micrognathia), oedematous soft tissue in the abdomen).
The findings noted in the 300 mg/kg bw/day dose group are not considered to be related to the treatment with tricobalt tetraoxide as these findings are known to occur spontaneously in this rat strain. Further, the incidences are within the range of laboratory background data.

- external examination (variations): no macroscopically visible variations were noted for the foetuses of the control group and the treatment groups (100, 300 or 1000 mg/kg bw/day) during the external examination of the foetuses at laparotomy.

- internal examination: no malformations or variations were noted during the examination for the foetuses of the control group and the treatment groups (100, 300 or 1000 mg/kg bw/day).

- skeletal examination (malformations): no malformations were noted during skeletal examinations of the foetuses according to DAWSON in the control group and in any of the treatment groups (100, 300 or 1000 mg/kg bw/day).

- skeletal examination (variations): the skeletal variations observed were related to the ribs (wavy ribs) the sternum (sternebra(e) bipartite, misaligned (severity: slight)) and the os pubis (bipartite).
No test item-related differences for the incidences of the observed variations were noted between the control group and the test item treated groups (100, 300 or 1000 mg/kg bw/day).

- skeletal examination (retardations): the observed skeletal retardations were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite, dumbbell-shaped or reduced in size), the lumbar vertebral bodies (dumbbell-shaped), the sacral vertebral bodies (unossified), the caudal vertebral bodies (only one body ossified or all bodies unossified), the os ischii (incompletely ossified or unossified), the os pubis (incompletely ossified or unossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5).
No test item related differences in incidences of the observed skeletal retardations were noted between the foetuses of the control group and the foetuses treated with 100, 300 or 1000 mg/kg bw/day.
All observed incidences with statistically significant changes were within the range of laboratory background data and are not considered as test item-related.
Statistically significant changes in the foetal incidences of skeletal retardations, which are not considered to be test item-related:
absence of ossification in metacarpalia 2 to 5 (100, 300 or 1000 mg/kg bw/day; p ≤ 0.05/p ≤ 0.01)
caudal vertebral bodies (all bodies unossified)(300 mg/kg bw/day; p ≤ 0.01)
os ischii unossified (300 mg/kg bw/day; p ≤ 0.05)
sacral vertebral body/bodies unossified (300 mg/kg bw/day; p ≤ 0.05)
sternebra(e) reduced in size (1000 mg/kg bw/day; p ≤ 0.05)
sternebra(e) unossified (300 or 1000 mg/kg bw/day; p ≤ 0.01)
thoracic vertebral body/bodies dumbbell-shaped (300 mg/kg bw/day; p ≤ 0.05)
total foetal skeletal retardations

- soft tissue examination (malformations): no test item-related malformations were noted during soft tissue examinations of the foetuses according to WILSON in any of the treatment groups (100, 300 or 1000 mg/kg bw/day).
The following sporadic observations are considered as spontaneous:
300 mg/kg bw/day: one foetus with a hydrocephalus (confirming the afore-mentioned external malformation).
1000 mg/kg bw/day: one foetus with an abnormal smallness of the eyes (microphthalmia).

- soft tissue examination (variations): soft tissue variations were noted in the 3rd or 4th cerebral ventricle (dilatation), the kidneys (uni- or bilateral dilatation of the renal pelvis) and the liver (haemorrhagic focus/foci).
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 (100, 300 or 1000 mg/kg bw/day).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 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:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (maternal toxicity) > 1000 mg tricobalt tetraoxide/kg bw/day
NOAEL (developmental toxicity) > 1000 mg tricobalt tetraoxide/kg bw/day
Looking at the results of the dams, no test item-related premature deaths, signs of clinical toxicity or findings at necropsy were noted (including uterus and carcass weights). Furthermore, body weight, food consumption, drinking water consumption and the haematological parameters were not influenced by the test item.
Also, the results of the foetuses showed no test item-related changes for the number of resorptions and the foetal body weight as well as no dead fetuses, no test item-related malformations, variations or retardations at any of the tested dose levels.