Tris[oxalate(2-)]dicerium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

14 October 2005 to 12 May 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Cited as Directive 84/449/EEC, B.10

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Dicerium tricarbonate

EC Number:

208-655-6

EC Name:

Dicerium tricarbonate

Cas Number:

537-01-9

IUPAC Name:

dicerium tricarbonate

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to RCC-CCR to initiate cell cultures within 24 hrs after blood collection. If necessary, the blood was stored before use at 4 °C.

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction of rats induced with Phenobarbital/ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1: 45.6 - 7020 µg/mL with and without metabolic activation
Experiment 2: 87.0 - 2500 µg/mL with and without metabolic activation
(see detailed table 1 below)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures
- Vehicle controls tested: medium with solvent or vehicle alone
- volume of vehicle/solvent in the medium: 10 % (v/v)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium with solvent or vehicle alone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
DURATION: (see detailed table 2 below)
- Exposure duration: 4, 22 or 46 hours
- Expression time: 0, 18 or 46 hours
- Selection time : 3 hours before harvesting
- Fixation time: 22 or 46 hours

* SPINDLE INHIBITOR: colcemid
* STAIN: stained with Giemsa
* NUMBER OF REPLICATES: two
* NUMBER OF CELLS EVALUATED: 100 well spread metaphase plates per culture were scored for cytogenetic damage.

DETERMINATION OF CYTOTOXICITY:
- Method: To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined in 1000 cells/culture.

OTHER: SCORING METHOD: The slides were evaluated using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates

Evaluation criteria:

A test material is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.

A test material is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps), and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05)

Results and discussion

Test results

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: In experiment I visible precipitation of the test material in the culture medium was observed at 427.7 µg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in experiment II in the absence and presence of S9 mix at 152.3 µg/mL and above
- No relevant increase in the osmolarity or pH values was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The aberration rates of the cells after treatment with the test material (0.0 - 2.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, exclusive gaps) and clearly within the range of the historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

See detailed results in tables 3 and 4 below.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 3: Results of chromosome analysis without metabolic activation

Exp.	Preparation interval	Test material conc. (µg/mL)	Polyploid cells (%)	Mitotic indices (% of control)	Aberrant cells (%)
					incl. gaps*	excl. gaps*	with exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1	0.2	100	1.0	0.0	0.0
		Positive control^2	0	81.4	8.0	8.0^S	1.0
		244.4	0	98.9	2.5	2.0^S	0.0
		427.7^P	0	105.7	1.0	1.0	0.0
		748.5^P	0.2	105.4	1.0	1.0	0.0
		1309.9^P	0	96.9	2.5	2.0^S	0.0
Exposure period 22 hrs without S9 mix
I	22 hrs	Solvent control^1	0	100	1.5	1.5	0.0
		Positive control^3	0	44.5	11.5	10.5^S	1.0
		244.4	0	92.3	0.0	0.0	0.0
		427.7^P	0.2	76.5	0.0	0.0	0.0
		748.5^P	0	89	1.0	1.0	0.0
Exposure period 46 hrs without S9 mix
II	46 hrs	Solvent control^1	0	100	1.0	1.0	0.0
		Positive control^3	0.2	138.5	10.5	10.5^S	1.0
		87.0	0	118.9	0.5	0.5	0.0
		152.3^P	0.2	153.7	1.0	1.0	0.0

 

*     Inclusive cells carrying exchanges

^P    Precipitation occurred

^S     Aberration frequency statistically significant higher than corresponding control values

¹     Deionised water 10 % (v/v)

²       EMS           800.0µg/mL

³     EMS           660.0µg/mL

Table 4: Results of chromosome analysis with metabolic activation

Exp.	Preparation interval	Test material conc. (µg/mL)	Polyploid cells (%)	Mitotic indices (% of control)	Aberrant cells (%)
					incl. gaps*	excl. gaps*	with exchanges
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1	0.2	100	1.0	0.0	0.0
		Positive control^2	0	73.4	8.0	8.0^S	0.0
		244.4	0	89	1.0	1.0	0.0
		427.7^P	0.2	126.6	0.5	0.5	0.0
		748.5^P	0	113.2	0.5	0.5	0.0
II	46 hrs	Solvent control^1	0	100	1.5	1.0	0.0
		Positive control^3	0	58.5	22.5	21.5^S	7.5
		87.0	0.2	98.5	2.0	2.0	0.5
		152.3^P	0	72.9	0.5	0.5	0.0
		266.6^P	0	92.8	2.5	2.0	0.0

 

*      Inclusive cells carrying exchanges

P      Precipitation occurred

S     Aberration frequency statistically significant higher than corresponding control values

1      Deionised water 10 % (v/v)

2      CPA 45.0 µg/mL

3      CPA 37.5 µg/mL

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that under the experimental conditions reported, the test material did not induce structural chomosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro.

Executive summary:

The potential of the test material to induce structural chromosomal aberrations was determined in a study performed in accordance with the standardised guideline EU Method B.10. During the study primary lymphocyte cultures were exposed to test material, in distilled water, at concentrations of 45.6 - 7020 µg/mL or  87.0 - 2500 µg/mL with and without metabolic activation, respectively. The test material was tested up to precipitating concentrations.
Under the conditions of the study there was no evidence of chromosome aberration induced over background. Positive controls induced the appropriate response.

Therefore, the test material is considered to be non-clastogenic in this chromosome aberration test when tested up to precipitating concentrations.