4H-Pyrazino[2,1-a]isoquinolin-4-one, 2-acetyl-1,2,3,6,7,11b-hexahydro-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-11-14 to 2006-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

470-180-7

EC Name:

-

Cas Number:

61196-40-5

Molecular formula:

C14H16N2O2

IUPAC Name:

2-acetyl-1H,2H,3H,4H,6H,7H,11bH-pyrazino[2,1-a]isoquinolin-4-one

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adult; 5-7 weeks
- Weight at study initiation: 26-32 g (DRF), 25-33 g (main study); group weights differed from the overall mean by no more than 5%
- Assigned to test groups randomly: yes, under following basis: system of random numbers
- Fasting period before study: no
- Housing: in cages that conform to the Code of Practice for the housing and care of animals used in scientific procedures
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: methyl cellulose
- Justification for choice of solvent/vehicle: not toxic to mice, good application profile
- Concentration of test material in vehicle: 12.5 - 75 mg/mL, depending on dose level
- Constant application volume of 5 mL/kg bw

Details on exposure:

In the dose-range finding study the test article was administered on a single occasion to groups of three male mice at doses of 500 and 750 mg/kg.
In the main study, the test article was administered on a single occasion to groups of six male mice at doses of 125, 250 and 500 mg/kg.

Duration of treatment / exposure:

single administration

Frequency of treatment:

once

Post exposure period:

24 and 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Male: 125 mg/kg; No. of animals: 6; Sacrifice time: 24 hours
Male: 250 mg/kg; No. of animals: 6; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 6; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 6; Sacrifice time: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamid (CPS), 40 mg/kg, dissolved in saline at 2 mg/mL.

Examinations

Tissues and cell types examined:

bone marow smeares from both femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: pre-test

DETAILS OF SLIDE PREPARATION:
Both femurs from each animal were exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 1 mL foetal calf serum into appropriately labelled centrifuge tubes (one per animal).

A further 2 mL of foetal calf serum was added and the tubes centrifuged and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube and from each tube a small volume of suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and animal number. The latter served as a code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide.

Slides were allowed to air-dry and were fixed for 5 minutes in absolute methanol, followed by rinsing several times in water. One slide from each set of two was then taken, the other was kept in reserve. After a second fixing/rinsing procedure, slides were stained for 10 minutes in filtered Giemsa stain diluted 1:6 (v/v) in distilled water. Stained slides were rinsed, and allowed to dry thoroughly before clearing in xylene for 3 minutes. When dry, the slides were mounted with coverslips.

METHOD OF ANALYSIS:
Slides from the CPA-treated mice were initially checked to ensure the system was operating satisfactorily. The slides from all control and dose groups were arranged in numerical order by sampling time and analysed by a person not connected with the dosing phase of the study.

Initially the relative proportions of polychromatic erythrocytes (PCE), seen as pale blue or blue/grey enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller yellow/orange-stained enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed.

Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored.

After completion of microscopic analysis and decoding of the data, the ratio of PCE to NCE (expressed as %PCE) for each animal and the mean for each group was calculated. The individual and group mean frequency of micronucleated PCE ± standard deviation (%MNPCE) were also determined.

%PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity.

The frequencies of micronucleated PCE in vehicle control animals were compared with the historical negative control data to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity Chi2 test.

The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine Chi2. Probability values of P<=0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
If the heterogeneity Chi2 test provided evidence of significant (p <= 0.05) variability between animals within at least one group, non-parametric analysis was more appropriate and provision made to use the Wilcoxon rank sum test.

Additional statistical analysis was not required in this study.

Evaluation criteria:

The data were evaluated as to whether exposure to the test article was associated with:
1. a statistically significant increase in the frequency of micronucleated PCE occurring at one or more dose levels.

2. an incidence and distribution of micronucleated PCE at such a point that exceeded the laboratory's historical vehicle control data.

3. a dose-response trend in the proportion of micronucleated PCE (where more than two dose levels were analysed).

The test article was to be considered positive in this assay if all of the above criteria were met.

The test article was to be considered negative in this assay if none of the above criteria were met.

Data that do not fall into either of the above categories were to be judged on a case by case basis. Evidence of a dose-response is considered useful but not essential in the evaluation of a positive result. Biological relevance is taken into account, for example consistency of response within and between dose levels and any confirmatory experiments.

Statistics:

The frequencies of micronucleated PCE in vehicle control animals were compared with the historical negative control data to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity Chi2 test.

The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine Chi2. Probability values of P<=0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
If the heterogeneity Chi2 test provided evidence of significant (p <= 0.05) variability between animals within at least one group, non-parametric analysis was more appropriate and provision made to use the Wilcoxon rank sum test.

Additional statistical analysis was not required in this study.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: Clinical signs at 500 mg/kg.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Clinical signs including lethargy, palpebral closure, bradypnoea and transient unconsciousness were observed in all animals receiving 500 mg/kg indicating that the animals were systemically exposed to substantial amounts of the test material and that 500 mg/kg was a suitable estimate of the maximum tolerated dose.

Negative (vehicle) control mice exhibited group mean frequencies of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE); ratio expressed as %PCE that were within (48-hour data), or slightly below (24 hour data) historical vehicle control (normal) values. Individual frequencies of micronucleated PCE (MN PCE) in the vehicle control groups were consistent with historical vehicle distribution data.

Mice treated with the test material exhibited group mean %PCE values that were similar to the concurrent vehicle control and which were within (or close to) the historical negative control (normal) range for all dose groups. These data did not represent evidence of bone marrow toxicity (as may be evidenced by a decrease in %PCE compared to concurrent control values).

Group mean frequencies of micronucleated PCE were similar to, and not statistically different from those seen in the concurrent vehicle control group for all dose groups (24 + 48 hour data). Individual frequencies of MN PCE for all treated animals were consistent with historical vehicle control distribution data.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice treated up to 500 mg/kg (considered a suitable estimate of the maximum tolerated dose in the mouse under the experimental conditions employed). Thus, the test item was not genotoxic in vivo in the test system described.

Executive summary:

The test material was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels according to OECD TG 475. The choice of dose levels was based on an initial toxicity range-finding study in which the test material, formulated in 0.5% (wlv) methyl cellulose (0.5% MC) was administered to mice via intraperitoneal injection. The test article was administered on a single occasion to groups of three male mice at doses of 500 and 750 mg/kg. Observations were made over a 2 day period following administration and signs of toxicity recorded. The negative (vehicle) control in the study was 0.5% MC also administered via intraperitoneal injection on a single occasion. Two groups of six male mice treated with this were killed and sampled 24 or 48 hours after administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered via intraperitoneal injection as a single dose of 40 mg/kg to a group of six male mice which were killed after 24 hours. In the main study, the test item was formulated as described and administered at 125, 250 and 500 mg/kg to groups of six male mice killed 24 or 48 hours after dose administration. Clinical signs including lethargy, palpebral closure, bradypnoea and transient unconsciousness were observed in all animals at a dose level of  500 mg/kg indicating that the animals were systemically exposed to substantial amounts and that 500 mg/kg was a suitable estimate of the maximum tolerated dose.
Negative (vehicle) control mice exhibited group mean frequencies of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE); ratio expressed as %PCE that were within (48-hour data), or slightly below (24 hour data) historical vehicle control (normal) values. Individual frequencies of micronucleated PCE (MN PCE) in the vehicle control groups were consistent with historical vehicle distribution data.
Positive control animals exhibited increased numbers of micronucleated PCE such that the frequency in the positive control group was significantly greater than in the concurrent controls with all animals exhibiting marked increases in numbers of MN PCE.
The assay system was therefore considered as both sensitive and valid. Mice treated with the test material exhibited group mean %PCE values that were similar to the concurrent vehicle control and which were within (or close to) the historical negative control (normal) range for all dose groups. Group mean frequencies of micronucleated PCE were similar to, and not statistically different from those seen in the concurrent vehicle control group for all dose groups (24 + 48 hour data). Individual frequencies of MN PCE for all test item treated animals were consistent with historical vehicle control distribution data.

It is concluded that the test material did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male mice treated up to 500 mg/kg (considered a suitable estimate of the maximum tolerated dose in the mouse under the experimental conditions employed). Thus, the test item was not genotoxic in vivo in the test system described.