Tricarbonyl(methylcyclopentadienyl)manganese

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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 474 with minor restrictions. Sufficient informarion on methods and results for assessment.

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

No detailed information on the test material

GLP compliance:

not specified

Remarks:

Test conducted at the laboratories of Health and Welfare Canada

Type of assay:

micronucleus assay

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

Test animals

Species:

mouse

Strain:

C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 9 weeks old (Three daily treatment protocol) and 47-59 days old (single treatment protocol)
- Weight at study initiation: No data
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: No data
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
No data

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: USP grade olive oil (Life Brand)
- Justification for choice of solvent/vehicle: mmt is poorly soluble in water
-Amount injected: 0.1 ml/10 g body weight

Details on exposure:

The exposure was done through intraperitoneal injection.

Duration of treatment / exposure:

Trial 1 - Three days
Trial 2 - Single dose (1 day)

Frequency of treatment:

1 single dosage per day.

Post exposure period:

Trial 1 - 24 hours
Trial 2 - Samples were collected 24 and 48 hours after exposure.

Doses / concentrations

Remarks:

Doses / Concentrations:
Trial 1 (3 days study) - 12.5, 25 and 50 mg/kg bw/day; Trial 2 (1 day study) - 50, 75 and 100 mg/kg bw/day.
Basis:
other: injected

No. of animals per sex per dose:

Trial 1 - 5 males and 5 females per dose
Trial 2 - 5 males and 5 females per dose

Control animals:

yes

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): known inducer of chromosomal aberrations upon metabolic activation
- Route of administration: intraperitoneal
- Doses / concentrations:
Trial 1 (3 days treatment) - 45 mg/kg cyclophosphamide dissolved in phosphate buffered saline
Trial 2 (1 day treatment) - 25 mg/kg cyclosphosphamide in phosphate buffered saline

Examinations

Tissues and cell types examined:

Polychromatic (PE) (orange) and normochromatic (NE) (very pale green) erytrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
-Trial 1 (3 days treatment)- A preliminary range-finding study using 2 animals of each sex and the same treatment regimen to be used in the micronucleus test, indicated that doses above 50 mg/kg could cause animal deaths or distress.
-Trial 2 (1 day treatment) - In order to determine the Maximum Tolerable Dose (MTD) for single i.p. injections, 2 mice of each sex were injected with the test material and monitored for 48 hours for signs of toxicity. The dose was administered in a volume of 0.1 ml/10 g bw. It was concluded that single treatments at doses above 100 mg/kg were likely to kill some of the animals.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Trial 1 - 24 hours following last treatment
- Trial 2 - 24 and 48 hours following treatment


DETAILS OF SLIDE PREPARATION:
For both trial 1 and 2: Samples were collected 24 hours following the final treatment. Immediately following sacrifice by cervical dislocation, one femur was removed and flushed out using a 1 ml syringe fitted with a 20 gauge needle, with approximately 0.1 ml fetal calf serum containing EDTA (prepared by filling a purple-topped vacutainer with serum) into a depression dish. The cell suspension was mixed with the tip of a disposable transfer pipet, then a drop was transferred to each slide. The slides were allowed to air dry, then fixed in methanol for approximately 5 min.


METHOD OF ANALYSIS:
Slides were coded and score blind to avoid observer bias. For analysis, the slides were mounted under a #1 coverslip in Sorensen's buffer, pH 7.3. Cells were viewed under epi-fluorescence on a Nikon Optiphot microscope fitted with a 60x objective and using a Nikon type G filter block. The number of polychromatic (PE) (orange) and normochromatic (NE) (very pale green) erythrocytes were recorded for the first 1000 erythrocytes (i.e., PE+NE) had been counted. After the first 1000 erythrocytes had been counted, only PE were scored until a total of 2000 PE had been analysed. On slides were the ‰ (1 part in 1,000) MPE exceeded 20, only 1000 PEs were analysed.

Statistics:

Data were analysed using the micronucleus assay data management and analysis system software developed under contact to the USEPA.

Results and discussion

Test results

Test results 1

Sex:

female

Genotoxicity:

negative

Remarks:

Trial 1, 3 days exposure

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Sex:

male

Genotoxicity:

negative

Remarks:

Trial 1, 3 days exposure

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: a dose-range finding study was performed using 2 animals of each sex per each dose, indicated that doses above 50 mg/kg could cause animal death or distress


RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: yes, dose levels defined after a dose-range finding study
- Statistical evaluation: yes

Any other information on results incl. tables

Table 1 – summary of micronucleus induction by mmt (Trial 1, 3 day exposure)

Dose (mg/kg)	Total MPCE	Total PCE	% MPCE	SEM	Pairwise Significance	Total PCE	Total PCE+NCE	% PCE	SEM	Pairwise Significance
Females
0	38	10000	0.380	0.034		3800	5788	65.65	3.33
12.5	26	8842	0.294	0.124	0.844	3700	5311	69.67	5.26	0.755
25.0	34	10000	0.340	0.025	0.682	3920	5265	74.45	3.71	0.939
50.0	28	10000	0.280	0.078	0.891	3771	5209	72.39	2.20	0.879
CP	219	5000	4.380	0.459	0.000	2500	5539	45.13	4.23	0.000
Trend for %MPCE: p=0.849 Trend for %PCE: p=0.440
Males
0	42	10000	0.420	0.085		3400	5489	61.94	5.76
12.5	41	10000	0.410	0.086	0.544	4000	5343	74.86	2.51	0.987
25.0	29	10000	0.290	0.010	0.939	3800	5292	71.81	3.83	0.952
50.0	25	9177	0.272	0.058	0.958	3280	5465	60.02	5.18	0.376
CP	297	6000	4.950	0.396	0.000	1669	6249	26.71	4.33	0.000
Trend for %MPCE: p=0.978 Trend for %PCE: p=0.027

 

Table 2 – summary of micronucleus induction by mmt (Trial 2, single treatment)

Sex	Recovery Time	Dose (mg/kg)	‰MPCE (±SEM)	%PCE (±SEM)
Male	24	0	2.2 ± 0.3	53.71 ± 5.54
		50	3.3 ± 0.3	62.27 ± 3.70
		75	2.5 ± 0.7	53.02 ± 4.69
		100	2.7 ± 0.6	52.60 ± 5.47
Cyclophosphamide		25	11.3 ± 2.9**
Trend                                      p= 0.293         p= 0.453
	48	0	3.4 ± 0.6	70.92 ± 4.52
		75	1.8 ± 0.3	47.33 ± 4.96**
		100	3.5 ± 0.7	46.64 ± 3.94**
Trend                                  p= 0.715         p= 0.000
Female	24	0	1.6 ± 0.5	62.70 ± 3.22
		50	1.8 ± 0.6	60.64 ± 3.07
		75	2.0 ± 0.3	63.69 ± 4.35
		100	2.1 ± 0.4	42.63 ± 2.46*
Cyclophosphamide		25	12.0 ± 1.3**
Trend                                 p= 0.190        p= 0.002
	48	0	1.6 ± 0.4	57.97 ± 3.83
		75	1.6 ± 0.3	73.26 ± 4.67
		100	1.2 ± 0.3	60.02 ± 7.35
Trend                                    p= 0.190        p= 0.002
Differences between sexes p= 0.0007 Differences between times p= 0.2593

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Based on the findings, mmt administered in three consecutive days, or in one single dose did not present any adverse effect on the levels of micronucleated polychromatic erythrocytes.

Executive summary:

In a bone marrow micronucleus assay, two trials have been performed with male and female C57BL mice. In the first trial, mmt was administered to 5 males and 5 females per each dose level, 12.5, 25 and 50 mg/kg bw/day, for three consecutive days. For the second trial, the number of animals was the same, however the dose levels were 50, 75 and 100 mg/kg bw/day, and administration of the test compound was done in one single dose. For the first trial the sacrifice and sampling was performed 24-hours after the last administration, while for the second trial at 24 and 48-hours.

There was no evidence of toxicity at the concentrations tested, which were previously defined through a dose-range finding study. mmt did not cause a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any time. The positive control used was cyclophosphamide, and induced a significant increase MPCE as expected.