Tricarbonyl(methylcyclopentadienyl)manganese

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:

experimental study

Adequacy of study:

key study

Study period:

Not mentioned

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed similar to OECD 473 wih minor deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

No detailed information about test substance.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Tricarbonyl(methylcyclopentadienyl)manganese

EC Number:

235-166-5

EC Name:

Tricarbonyl(methylcyclopentadienyl)manganese

Cas Number:

12108-13-3

Molecular formula:

C9H7MnO3

IUPAC Name:

tricarbonyl(methyl-η5-cyclopentadienyl)manganese

Details on test material:

- Name of test material (as cited in study report): mmt
- Stability under test conditions: Stable, cells treated in dark incubators
- Test material handling: When dilutions were being prepared and aftewards added to the cells, the compound was exposed to light

Method

Target gene:

The target is not a specific gene but the structural integrity of the genetic material expressed as chromosome aberrations visible with the light microscope.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle MEM supplementedwith 1% sodium pyruvate, 1% non-essential amino acids and 10% fetal calf serum (Gibco)
- Properly maintained: yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Exogenous Metabolic activation medium: 5.4% 20 mM HEPES buffer pH7.2, 0.2% 0.5 M MgCl2, 0.2% 3.3 KCl, 2% 40mM NADP, 2% 50 Mm Glucose-6-Phosphate and 7% Aroclor 1254-induced rat liver homogenate (S9)

Test concentrations with justification for top dose:

0, 0.01, 0.02 and 0.04 µl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: the test chemical was diluted in either serum-free complete medium or an exogenous metabolic activation medium containing: 5.4% 20 mM HEPES buffer pH 7.2 (Sigma, St. Louis, MO); 0.2% 0.5 M MgCl2 (Sigma); 0.2% 3.3 M KCl; 2% 40 mM NADP; 2% 50 mM glucose-6-phosphatase and 7% Aroclor 1254-induced rat liver homogenate (S9).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: No clear information available on the negative control, it is assumed to have been the vehicle control.

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Test without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

No clear information available on the negative control, it is assumed to have been the vehicle control.

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Test with metabolic activation

Details on test system and experimental conditions:

DURATION
- Exposure duration:
-Trial 1 (absence of metabolic activation) 3 hours; Trial 2 (absence of metabolic activation) 16 hours
-Trial 1 and 2 (presence of metabolic activation) 3 hours

NUMBER OF CELLS EVALUATED: One hundred metaphase cells were analysed from each of two cultures for each treatment. Only 50 cells were scored when the frequency of chromosomal aberrations was very high.

Evaluation criteria:

Chromosomal aberrations.

Statistics:

Data was analysed using the chromosomal aberration data management and analysis system software developed under contact to the U.S.Environmental Protection Agency.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No additional information.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 - Induction of chromosomal aberrations by mmt in the absence of metabolic activation

Dose (µl/ml)	Number of cells	Chromatid			Chromosome			Aberrations/cell (± Standard Error)	% Cells with Aberrations (± Standard Error)
		Gaps	Breaks	Exch	Gaps	Breaks	Exch
Trial 1, 3 hr exposure
0	200	0	3	0	0	2	3	0.040±0.000	4±0
0.01	200	1	2	0	2	2	5	0.045±0.005	4.5±0.5
0.02	200	0	1	0	3	1	3	0.025±0.015	2.5±1.5
0.04	90	2	0	0	0	2	2	0.156±0.122	5.56±1.5
MMS	60	0	22	24	0	20	1	**8.583±0.483	**100±0
ρ value for trend analysis                                                                                          0.088                          0.412
Trial 2, continuous (16 hr) exposure
0	191	4	0	0	4	4	9	0.063±0.008	6.28±0.75
0.01	200	1	2	2	2	3	5	0.060±0.020	6.00±2.00
0.02	200	1	4	0	3	7	13	*0.115±0.045	10.00±1.00
0.04	150	1	4	0	9	5	3	0.080±0.030	8.00±3.00
MMS	45	1	45	32	2	31	4	**8.220±0.430	**100.00±0.00
ρ value for trend analysis                                                                                         0.404                         0.178

 

Table 2 - Induction of chromosomal aberrations by mmt in the presence of metabolic activation

Dose (µl/ml)	Number of cells	Chromatid			Chromosome			Aberrations/cell (± Standard Error)	% Cells with Aberrations (± Standard Error)
		Gaps	Breaks	Exch	Gaps	Breaks	Exch
Trial 1, 3 hr exposure
0	200	3	3	0	0	9	3	0.055±0.015	4.50±0.50
0.01	75	1	5	0	1	0	1	0.080±0.030	4.00±0.00
0.02	200	4	7	7	1	3	12	**0.135±0.025	*10.50±0.50
0.04	100	4	34	17	4	14	10	**1.080±0.220	**45.00±7.00
CP	100	1	44	34	10	39	20	**1.450±0.270	**65.00±5.00
ρ value for trend analysis                                                                                         0.000                          0.000
Trial 2, 3 hr exposure
0	200	1	1	1	0	2	11	0.065±0.045	5.00±3.00
0.01	200	0	1	0	1	3	5	0.045±0.005	4.00±1.00
0.02	200	1	11	7	2	13	8	*0.295±0.128	14.50±3.65
0.04	100	4	33	30	6	22	13	**1.760±0.060	**51.00±3.00
CP	100	0	52	45	1	42	9	**3.110±0.190	**76.00±0.00
ρ value for trend analysis                                                           0.000                          0.000

Applicant's summary and conclusion

Conclusions:

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

In the presence of metabolic activation, mmt induced structural chromosomal aberrations in Chinese Hamster Ovary Cells.. There was significant (p<0.0114), reproducible increase in chromosomal aberrations at concentrations as low as 0.02 µl/ml (0.12 mM). Without metabolic activation, mmt failed to induce a significant increase in chromosomal aberrations following either a 3 hr (p = 0.412) or continuous (p = 0.178) exposure.

Executive summary:

A chromosome aberration assay was conducted similar to OECD 473 using wild-type Chinese hamster ovary (CHO) cells exposed to mmt at concentrations of 0, 0.01, 0.02 and 0.04 µl/ml with and without metabolic activation.

mmt was tested up to 0.04 µl/ml, which is within the solubility range. Positive controls induced, as expected, an increase in chromosomal aberrations both with metabolic activation (cyclophosphamide) and without metabolic activation (methylmethanesulfonate). For mmt, in absence of metabolic activation, there was no evidence of an induction of chromosomal aberration, while in the presence of metabolic activation, there was a concentration dependent positive response for the two highest concentrations tested (0.02 and 0.04 µl/ml mmt).