2,9-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

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:

From 28 EFEB 1995 to 15 MAR 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study (OECD 474) with some restrictions (insufficient number of PCE examined, but two doses above limit dose tested)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

: insufficient number of polychromatic erythrocytes examined

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2,9-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

221-424-4

EC Name:

2,9-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

3089-17-6

Molecular formula:

C20H10Cl2N2O2

IUPAC Name:

2,9-dichloro-5,7,12,14-tetrahydro-5,12-diazapentacene-7,14-dione

Test material form:

not specified

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Age at study initiation: about 9 weeks
- Weight at study initiation: males: 31.0-38.5 g; females: 23.1-30.3 g
- Assigned to test groups randomly: yes
- Housing: seven per cage during quarantine, up to 5 per cage at randomisation
- Diet (ad libitum): Purina Certified Laboratory Chow No. 5002
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 3.3
- Humidity (%): 55 +/- 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: stock solution 250 mg/ml

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: stock solution: 8.75 g test item in corn oil, 35 ml final volume yielded an opaque, red suspension with a concentration of 250 mg/ml
Constant dosing volume: 20 ml/kg

Duration of treatment / exposure:

up to 72 h

Frequency of treatment:

single oral exposure

Post exposure period:

24, 48 and 72 h

Doses / concentrations

Remarks:

Doses / Concentrations:
1250, 2500, 5000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide, 80 mg/kg

Examinations

Tissues and cell types examined:

erythrocytes from bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
based on a range finding pre-test (no mortality or clinical symptoms of toxicity after administration of doses up to 5000 mg/kg)


DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanatized with CO2 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3-5 ml bovine serum (one tube for each animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol and stained in May-Grunwald solution followed by Giemsa. The air-dried slides were coverslipped using Depex® mounting medium.

METHOD OF ANALYSIS:
The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%.
The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes.

Evaluation criteria:

The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based an scientific judgment.

Statistics:

The analysis of the data was performed using an Analysis of Variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons was used at each harvest time to determine which dose groups, if any, were significantly different (p<0.05) from the vehicle control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): significantly less in the 2500 mg/kg group (males, 24 h harvest) and 5000 mg/kg group (females, 24 and 72 h harvest) compared to control group
- Appropriateness of dose levels and route: yes

Any other information on results incl. tables

All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately after dosing. Approximately 23, 47, and 71 hours after dosing, all animals appeared normal, but had diluted pink hair coats due to grooming and compound colored feces. All animals remained healthy until the appropriate harvest times.

The test item induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. Possibly due to toxicity induced by 2,9-Dichloroquinacridone, the PCE/NCE ratios of the 24 and 72 hour high dose females, the 24 hour males dosed with 2500 mg/kg were significantly less than the vehicle control group. The PCE/NCE ratio of the CP-treated females was also significantly less than the vehicle control group. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 2.40% ± 0.24% and 2.42% ± 0.36% for the males and females, respectively.

Treatment	Dose	Harvest time (h)	% Micronucleated PCEs, mean of 1000 per animal ± SD			Ration PCe/NCE mean ± SD
			males	females	total	males	females
Vehicle (corn oil)	20 ml/kg	24	0.02 ± 0.02	0.06 ± 0.02	0.04 ± 0.02	0.55 ± 0.07	1.03 ± 0.07
Cyclophosphamide	80 mg/kg	24	2.40 ± 0.24*	2.42 ± 0.36*	2.41 ± 0.20*	0.59 ± 0.02	0.65 ± 0.05*
Test item	1250 mg/kg	24	0.04 ± 0.02	0.06 ± 0.02	0.05 ± 0.02	0.33 ± 0.04	0.96 ± 0.07
		48	0.04 ± 0.02	0.00 ± 0.00	0.02 ± 0.01	0.62 ± 0.06	0.73 ± 0.10
		72	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.65 ± 0.08	0.64 ± 0.14
	2500 mg/kg	24	0.02 ± 0.02	0.08 ± 0.06	0.05 ± 0.03	0.28 ± 0.04*	0.89 ± 0.16
		48	0.06 ± 0.02	0.00 ± 0.00	0.03 ± 0.02	0.49 ± 0.06	0.72 ± 0.15
		72	0.08 ± 0.02	0.08 ± 0.04	0.08 ± 0.02	0.69 ± 0.02	0.69 ± 0.10
	5000 mg/kg	24	0.02 ± 0.02	0.04 ± 0.02	0.03 ± 0.02	0.49 ± 0.02	0.64 ± 0.10*
		48	0.06 ± 0.02	0.02 ± 0.02	0.04 ± 0.02	0.55 ± 0.11	0.80 ± 0.12
		72	0.02 ± 0.02	0.04 ± 0.04	0.03 ± 0.02	0.81 ± 0.05	0.58 ± 0.05*

*: significantly different from vehicle control (p < 0.05)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test item 2,9-dichloroquinacridone did not show mutagenic activity in the mouse micronucleus assay in vivo.

Executive summary:

In this mouse micronucleus assay, the test item was suspended in corn oil and applied to CD-1 mice by oral gavage at 1250, 2500, and 5000 mg/kg, based upon results of a range finding pre-test. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals dosed with the test article were euthanatized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. Vehicle and positive control groups, euthanatized approximately 24 hours after dosing, were included in the assay.

The test item did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay. The positive control group showed a significant positive response.