Cobalt bis(2-ethylhexanoate)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

; only 4 animals were tested (acc. to guideline: 5 test animals are required); samples for analysis were taken at only one time point (acc. to guideline. samples should be taken at two separate times).

GLP compliance:

no

Remarks:

As an investigative study, this study was conducted in accordance with applicable test facility SOPs and was fully documented but was not subjected to Quality Assurance audit or review.

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

1308-06-1

Cas Number:

1308-06-1

IUPAC Name:

1308-06-1

Constituent 2

Reference substance name:

tricobalt tetraoxide

IUPAC Name:

tricobalt tetraoxide

Details on test material:

- Name of test material (as cited in study report): Tricobalt tetraoxide, black cobalt (II, III) oxide- Molecular formula (if other than submission substance): Co3O4- Molecular weight (if other than submission substance): 250.80 g/mol- Physical state: solid No further details are given.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Harlan, USA - Age at study initiation: At treatment, the animals were approximately 21 days old. - Assigned to test groups randomly: yes, under following basis: Randomisation was done by stratification using body weight as the parameter. Males and females were randomised separately. Animals in poor health or at the extremes of the body weight range were not assigned to groups.- Fasting period before study: no - Housing: Each dam and litter were housed on corn cob bedding in solid bottomed cages equipped with an automatic watering valve. After randomization and until euthanasia, animals were housed in groups of 13 pups in the single dose phase study and 9 to 11 pups (including spare animals) in the multi-dose phase study, alongside their respective mother rats (dams).- Diet: All animals had free access to a standard certified pelleted commercial laboratory diet except during designated procedures. - Water: Municipal tap water, suitable for human consumption, was freely available to the animals except during designated procedures.- Acclimation period: 7 days; Following arrival, each animal was given a general physical examination by a member of the veterinary staff to ensure normal health status. ENVIRONMENTAL CONDITIONS- Temperature (°C): 22 +/- 3 - Humidity (%): 50 +/- 20 - Photoperiod: 12 hours dark/light cycle No further details are given.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: aqueous 1% v/v methylcellulose - Amount of vehicle (if gavage or dermal): 10 mL/kg body weight- Lot/batch no. (if required): 125K0196 (methylcellulose 400 cps)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: not applicableDIET PREPARATIONAll formulations were prepared once prior to each phase on the first day of use. The required amount of each compound was weighed into a pre weighed beaker and a small amount of vehicle was added to make a paste. The formulations were brought to final weight with the vehicle and homogenised until an apparently homogenous suspension was obtained.

Duration of treatment / exposure:

16 hours

Frequency of treatment:

single administration on day one

Post exposure period:

15 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 other: mg/kg bw (nominal conc.)

Doses / concentrations 2

Dose / conc.:

1 000 other: mg/kg bw (nominal conc.)

Doses / concentrations 3

Dose / conc.:

2 000 other: mg/kg bw (nominal conc.)

No. of animals per sex per dose:

groups of 2 male and 2 female rats were treated with the vehicle control or test substance

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide monohydrate (CAS: 6055-19-2) - Route of administration: oral, gavage (single dose) - Doses / concentrations: The control article was administered using a dose volume of 10 mL/kg bodyweight. 1,2-Dimethylhydrazine dihydrochloride (CAS: 306-37-6)- Route of administration: oral, gavage (single dose) - Doses / concentrations: The control article was administered using a dose volume of 10 mL/kg bodyweight. The positive control solution was freshly prepared on the day of use.

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary toxicity test was performed, partly to determine an estimated maximum tolerated dose (MTD) of the test article given the uncertainty and variability of published values. Dose levels for each phase of the preliminary test were determined at the time of the test and were based on the clinical signs/mortalities obtained in the previous phase.TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Colchicine was administered to all animals (except those in Group 1 (preliminary study) in the single dose phase) at 4 μg/kg 1 hour prior to euthanasia by intraperitoneal injection (10 mL/kg bodyweight) to arrest bone marrow cells in metaphase. The appropriate animals were euthanised 16 hours after the last or only treatment by exsanguination from the abdominal aorta under isofluorane anaesthesia.DETAILS OF SLIDE PREPARATION: Both femurs were dissected from each animal and the proximal heads removed. The proximal head was removed from each femur while keeping the distal head intact, and as much tissue as possible was removed from the bones. The bone marrow from both femurs of each animal was pooled/eluted in 5 mL Hanks’ Balance Salts Solution by aspiration through an appropriate size needle fitted to a plastic syringe. The resulting cell suspensions were centrifuged and the resulting pellets were resuspended in 10 mL aqueous 0.075M potassium chloride (hypotonic solution) and incubated for approximately 12 minutes at ca. 37°C, before addition of 2 mL of fixative (3 vol methanol:1 vol acetic acid) with mixing. Following centrifugation, the supernatant was discarded and the cells treated with 3 changes of neat fixative. After the third change of fixative, the cell pellet was collected by centrifugation and resuspended in fixative at an appropriate density for slide preparation.The fixed cells were dropped onto clean slides in a humid atmosphere and air-dried before staining. At least two slides were prepared from each animal. Slides were washed with 3 changes of purified water then stained with 10% (v/v) Giemsa for 15 minutes. METHOD OF ANALYSIS:Mitotic Index (MI): Slides were randomised and examined by light microscopy, and the mitotic index was determined by examination of at least 1000 cells per animal. The relative mitotic index (RMI) was calculated as a percentage ratio compared with the concurrent vehicle control group.Detailed Examination for Chromosome Aberrations: A total of 100 readable metaphases per animal were examined for the presence of chromosome aberrations using oil-immersion optics.Readable metaphases were identified by the following criteria:- chromosome number between 40 and 44 in a single stage of condensation, - well-spread with minimal overlap of chromosomes and chromosome arms, - chromatids separate with centromere intact and - structure of chromosomes clear and well-defined. The International System for Chromosome Aberration Nomenclature (ISCN 1985) was followed to designate the observed aberrations. Since the nature of chromosomal and chromatid gaps is uncertain (they may or may not represent true breaks in chromatid structure), these two types of aberration were recorded but not included in statistical analysis of aberrations. A gap is defined as an unstained region within the chromatid, where the width of this region is shorter than the width of a chromatid. The incidences of numerical types of aberration, such as polyploidy and endoreduplication, were recorded and reported here, as required by OECD guideline 475. The location (Vernier reading) of observed aberrant metaphases was recorded for potential peer review.OTHER: Unscheduled Euthanasia: Animals euthanized for humane reasons pre- or post-treatment underwent exsanguination from the abdominal aorta following isoflurane anaesthesia. No gross pathology examination was performed and animal carcasses were discarded without examination or tissue retention. No tissues were retained for any animal.

Evaluation criteria:

no data

Statistics:

Due to the limited group size, no formal statistical analysis was performed on results from the single dose experiment. Exact two-sided p-values were calculated for MI, and one-sided values for other parameters using permutation tests (Gibbons 1985, Agresti et al 1990) based on absolute values and rank dose level with the individual animal being considered as the unit of variance.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

No substantial increase in the bone marrow metaphases showing chromosome aberrations was obtained.

Toxicity:

no effects

Remarks:

No adverse clinical signs were observed and no mortalities were obtained.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY- Dose range: up to the standrad limit dose of 2000 mg/kg- Clinical signs of toxicity in test animals: No clinical signs and no mortalities were observed in any treatment group in the preliminary phase.RESULTS OF DEFINITIVE STUDYNo treatment had any apparent effect on mitotic index in the bone marrow. As expected, animals in the first group, which did not receive colchicine, had a relatively low mitotic rate in bone marrow.- Evidence of cytotoxicity in tissue analysed: In a multi-dose phase Cobalt sulphate, Cobalt monoxide and Tricobalt tetraoxide were additionally tested. Animals were dosed once daily for up to 5 days. High multiple doses of cobalt compounds depress erythropiesis, as indicated by a reduction in bone marrow mitotic index with Cobalt monoxide and an associated general reduction in reticulocyte counts with all three compounds.No further details.

Any other information on results incl. tables

Single dose of tricobalt tetraoxide – Summary Results for Bone Marrow

Treatment	Dose (mg/kg)	MI	RMI	No. cells examined(M+F)	% Aberrant Male	% Aberrant Female	% Aberrant (M+F)
Vehicle^†	-	1.6	66	400	1.5	6.0	3.8
Vehicle	-	2.4	100	400	0.0	3.0	1.5
Tetraoxide	500	2.9	121	400	1.5	0.5	1.0
Tetraoxide	1000	3.0	125	400	2.5	0.0	1.3
Tetraoxide	2000	2.6	107	400	0.0	1.0	0.5
CP	10	3.6	150	400	19.5	7.5	13.5
DMH	10	3.6	142	400	4.5	8.0	6.3

† Not treated with colchicines; All other animals were given colchicine at 4 mg/kg by I.P. injection 1 hour prior to euthanasia.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Tricobalt tetraoxide has no mutagenic effects in the chromosome aberration tests. Evident for the validity of the exposure route is the results from a multi-dose phase test conducted with three cobalt compounds. The results show that under given experimental conditions cobalt ions reaches the bone marrow .