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Toxicological information

Genetic toxicity: in vitro

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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: see 'Remark'
Remarks:
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. These data have been read-across from a structurally similar compound.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Dicerium tricarbonate

Method

Species / strain
Species / strain / cell type:
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 heoparinized tubes. The tubes were sent to RCC-CCR to initate cell cultures within 24h after blood collection. If necessary, the blood was stored before use at 4 deg C.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 fraction of rats induced with phenobarbital/napthoflavone
Test concentrations with justification for top dose:
Experiment 1: 45.6 - 7020 ug/mL with and without metabolic activiation
Experiment 2: 87.0 - 2500 ug/mL iwth and without metabolic activation
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)
Controlsopen allclose all
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:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: 660 ug/mL (5.32 mM) (Expt 1 and II) and 800 ug/mL (6.45 mM) (Expt 1) without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: 45 ug/mL (0.396 mM) (Expt 1) and 37.5 ug/mL (0.132 mM) (Expt II) with metabolic activation
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 item 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 item 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:
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 item 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 item (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

Test item

Polyploid

Mitotic indices

Aberrant cells

interval

concentration

cells

in %

in %

in µg/mL

in %

of control

incl. gaps*

excl. gaps*

with exchanges

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control1

0.2

100.0

 1.0

 0.0

 0.0

Positive control2

0.0

81.4

 8.0

 8.0S

 1.0

 244.4

0.0

98.9

 2.5

 2.0S

 0.0

 427.7P

0.0

105.7

 1.0

 1.0

 0.0

 748.5P

0.2

105.4

 1.0

 1.0

 0.0

1309.9P

0.0

96.9

 2.5

 2.0S

 0.0

Exposure period 22 hrs without S9 mix

I

22 hrs

Solvent control1

0.0

100.0

 1.5

 1.5

 0.0

Positive control3

0.0

44.5

11.5

10.5S

 1.0

 244.4

0.0

92.3

 0.0

 0.0

 0.0

 427.7P

0.2

76.5

 0.0

 0.0

 0.0

 748.5P

0.0

89.0

 1.0

 1.0

 0.0

Exposure period 46 hrs without S9 mix

II

46 hrs

Solvent control1

0.0

100.0

 1.0

 1.0

 0.0

Positive control3

0.2

138.5

10.5

10.5S

 1.0

  87.0

0.0

118.9

 0.5

 0.5

 0.0

 152.3P

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

Test item

Polyploid

Mitotic indices

Aberrant cells

interval

concentration

cells

in %

in %

in µg/mL

in %

of control

incl. gaps*

excl. gaps*

with exchanges

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

0.2

100.0

 1.0

 0.0

 0.0

Positive control2

0.0

73.4

 8.0

 8.0S

 0.0

 244.4

0.0

89.0

 1.0

 1.0

 0.0

 427.7P

0.2

126.6

 0.5

 0.5

 0.0

 748.5P

0.0

113.2

 0.5

 0.5

 0.0

II

46 hrs

Solvent control1

0.0

100.0

 1.5

 1.0

 0.0

Positive control3

0.0

58.5

22.5

21.5S

7.5

 87.0

0.2

98.5

 2.0

 2.0

 0.5

 152.3P

0.0

72.9

 0.5

 0.5

 0.0

 266.6P

0.0

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     CPA45.0 µg/mL

3     CPA37.5 µg/mL

Applicant's summary and conclusion

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

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as deterimined by the chromosome aberration test in human lymphocytes in vitro.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration assay) (Schulz, 2006), primary lymphocyte cultures were exposed to Cerium carbonate (65.62% of purity), in distilled water, at concentrations of 45.6 - 7020 µg/mL or 87.0 - 2500 µg/mL with and without metabolic activation. The substance was tested up to precipitating concentrations. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background. Therefore, the substance is considered to be non-clastogenic in this chromosome aberration test when tested up to precipitating concentrations.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline EU Method B.10 for in vitro cytogenetic mutagenicity data.

These data have been read-across from the analogue substance cerium carbonate.