Reaction mass of cobalt olivine and crystalline silicon dioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

experimental testing initiated on 2010-03-12 and was completed on 2010-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt bis(2-ethylhexanoate)

EC Number:

205-250-6

EC Name:

Cobalt bis(2-ethylhexanoate)

Cas Number:

136-52-7

Molecular formula:

C8H16O2.1/2Co

IUPAC Name:

cobalt(2+) bis(2-ethylhexanoate)

Details on test material:

- Name of test material (as cited in study report): Cobalt 2-ethyl hexanoate - Physical state: was received as a blue liquid, but soon after arrival was identified as a purple/blue waxy solid- Storage condition of test material: stored at 15-25°C in the dark

Method

Target gene:

hprt locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media, containing 100 units/mL Penicillin, 100 µg/mL Streptomycin, 2.5 µg/mL Amphotericin B, 0.5 mg/mL Pluronic (except for RPMI 20) and heat inactivated horse serum (0% v/v for RPMI A; 10% v/v for RPMI 10 and 20% v/v for RPMI 20).The master stock of L5178Y tk+/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen.- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yesFor each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Range Finder (with and without S9-mix): 62.5, 125, 250, 500, 1000 and 2000 µg/mL. Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment.Experiment I (with and without S9-mix): 15, 30, 50, 65, 80, 95, 110, 125, 150 and 200 µg/mL; Experiment II (with and without S9-mix): 10, 20, 40, 55, 70, 80, 90, 100, 120 and 150 µg/mL.Cultures selected for mutation assessment: - Experiment I (with and without S9-mix): 0, 15, 30, 50, 65, 80 and 95 µg/mL; - Experiment II (with S9-mix): 0, 10, 20, 40, 55, 70, 80, 90, 100 and 120 µg/mL; - Experiment II (without S9-mix): 0, 20, 40, 55, 70, 80, 90 and 100 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Cobalt 2-ethyl hexanoate was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 300.5 mg/mL. The solubility limit in culture medium was approximately 751.3 to 1503 μg/mL, as indicated by precipitation at the higher concentration which persisted for 3 hours after test article addition.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline 1-oxide; 0.1 and 0.15 µg/mL (dissolved in DMSO)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activationMigrated to IUCLID6: 2 and 3 µg/mL (dissolved in DMSO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium- Exposure duration: 3 hours at 37°C- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed.- Selection time (if incubation with a selection agent): At the end of the expression period, cell were plated for 6TG resistance. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (12 to 13 days) and wells containing clones were identified and counted.SELECTION AGENT (mutation assays): 6-thioguanine (6TG)NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9, was performed in duplicate cultures (single cultures only used for positive control treatments).DETERMINATION OF CYTOTOXICITY- Method: relative survival:Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described for the Mutation Experiments. Single cultures only were used and positive controls were not included. Following treatment, cells were centrifuged washed with tissue culture medium and resuspended in 20 mL RPMI 10 and plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by microscope and counted.OTHER:Analysis of results: All calculations were performed either manually or by computer using validated software.- Plating efficiency (PE) in any given culture is therefore: PE = P/No of cells plated per well;- Percentage relative survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [PE (test)/PE (control)] x 100;- Mutant frequency (MF) is usually expressed as "mutants per 10^6 viable cells". In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated: MF = [PE (mutant)/PE (viable)] x 10^6

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05). 2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05). 3. The effects described above were reproducible.Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No statistically significant increases in mutant frequency were observed following treatment with Cobalt 2-ethyl hexanoate at any concentration analysed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORSOsmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment. - Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration selected (1000 μg/mL), compared to the concurrent vehicle controls (individual data not reported).- Precipitation: Following the 3 hour treatment in the Range Finder Experiment, precipitate was observed at the highest two concentrations in the absence and presence of S9 (1000 and 2000 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and higher concentrations were discarded.RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S9, ranging from 62.5 to 2000 μg/mL (limited by solubility in culture medium). The highest concentration to provide >10% RTG was 125 μg/mL, which gave 10% and 18% RS in the absence and presence of S-9, respectively.COMPARISON WITH HISTORICAL CONTROL DATA: Comparison of controls with historical means. ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment I concentrations, ranging from 15 to 200 μg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest four concentrations tested in the absence and presence of S9 (110 to 200 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. The highest concentrations selected was 95 μg/mL in the absence and presence of S9, which gave 11% and 13% RS, respectively. In Experiment II concentrations, ranging from 10 to 150 μg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest two concentrations in the absence of S9 (120 and 150 μg/mL) and the highest concentration in the presence of S9 (150 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest concentration tested in the absence of S9 (10 μg/mL) was not selected as there were sufficient non-toxic concentrations. The highest concentrations selected were 100 μg/mL in the absence of S9 and 120 μg/mL in the presence of S9, which gave 14% and 13% RS, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Cobalt 2-ethyl hexanoate: summary of results

 

Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		%RS	MF§					%RS	MF§
0		100	1.34			0		100	3.60
15		97	2.98		NS	15		101	1.99		NS
30		85	2.07		NS	30		91	2.03		NS
50		56	1.34		NS	50		76	5.11		NS
65		31	1.54		NS	65		38	3.38		NS
80		15	0.99!		NS	80		19	2.60		NS
95		11	4.05		NS	95		13	6.10		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		82	18.02			2		30	25.22
0.15		67	36.80			3		31	58.24

Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		%RS		MF§				%RS		MF§
0		100		5.21		0		100		4.32
20		102		3.94	NS	10		86		3.26	NS
40		83		3.57	NS	20		69		5.02	NS
55		61		5.71	NS	40		92		3.60	NS
70		38		4.36	NS	55		90		3.92	NS
80		35		5.21	NS	70		71		3.70	NS
90		24		6.25	NS	80		53		5.11	NS
100		14		6.16	NS	90		42		6.23	NS
						100		27		3.53	NS
						120		13		6.11	NS
Linear trend				NS		Linear trend				NS
NQO						B[a]P
0.1		86		27.60		2		68		37.09
0.15		96		27.51		3		60		52.10

 

§                            6‑TG resistant mutants/10⁶viable cells 7 days after treatment

%RS                      Percent relative survival adjusted by post treatment cell counts

NS                         Not significant

!                              Based on one replicate only

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeIt is concluded that Cobalt 2-ethyl hexanoate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).

Executive summary:

Cobalt 2-ethyl hexanoate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9). A 3 -hour treatment incubation period was used for all experiments. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S9, ranging from 6.25 to 2000 μg/mL (limited by solubility

in culture medium). The highest concentration to provide >10% RTG was 125 μg/mL, which gave 10% and 18% RS in the absence and presence of S9, respectively.

Accordingly, for Experiment I ten concentrations, ranging from 15 to 200 μg/mL, were tested in the absence and presence of S9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance was 95 μg/mL in the absence and presence of S9, which gave 11% and 13% RS, respectively. In Experiment II ten concentrations, ranging from 10 to 150 μg/mL μg/mL, were tested in the absence and presence of S9. Seven days after treatment, the highest concentrations selected to determine viability and 6TG resistance were 100 μg/mL in the absence of S9 and 120 μg/mL in the presence of S9, which gave 14% and 13% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S9. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals.

In Experiments I and II, no statistically significant increases in mutant frequency were observed following treatment with Cobalt 2-ethyl hexanoate at any concentration analysed in the absence and presence of S9 and there were no significant linear trends.