Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Standard guideline study under GLP regulation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
12H-phthaloperin-12-one
EC Number:
230-049-5
EC Name:
12H-phthaloperin-12-one
Cas Number:
6925-69-5
Molecular formula:
C18H10N2O
IUPAC Name:
12H-isoindolo[2,1-a]perimidin-12-one

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 29 to 32,6 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: groups of five
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/- 3
- Humidity (%): > 70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil

- Concentration of test material in vehicle: 100 to 500 mg/10 mL
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single injection
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (Control) - 100 - 250 - 500 (max. tol. dose) mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophospamide
- Route of administration: intraperitoneal
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow from both femora.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: highest dose = max tolerated, lower doses at intervals of about 2x

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 h and 48 h after injection

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing of SOLVENT ORANGE 60, 24 h after dosing of the vehicle and 48 h after dosing of the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the study identification number and the animal number). The drop was spread by moving a clean slide with roundwhetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension.
The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.
Staining of the bone marrow smears. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.
Analysis of the bone marrow smears for micronuclei All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time.
Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Evaluation criteria:
Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
-

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
lethargy; rough coat; hunched posture; eyes closed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Group

Treatment

Dose (mg/kg

body weight)

 

Sampling time (h)

Number of micronucleated+ polychromatic erythrocytes per 2000 polychromatic erythrocytes (mean± S.D.)

Ratio polychromatic/ normochromatic

erythrocytes (mean± S.D.)

A

solvent control

0

 

24

1.6±1.1

1.48 ± 0.23

 

B

test item

500

24

0.8±1.1

0.95±0.18

 

C

test item

500

48

0.2±0.4

0.96±0.31

 

D

test item

250

24

0.6±0.9

0.74±0.15

 

E

test item

100

24

0.6 ± 1.3

1.25±0.15

F

CP

50

48

29.4±18.6(2

 

0.36±0.13

 

Solvent control = corn oil

CP = Cyclophosphamide

(1) Five animals per treatment group

(2) Significantly different from corresponding control group (Wilcoxon Rank SumTest,P</=0.01).

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
SOL VENT ORANGE 60 is not mutagenic in the micronucleus test under the experimental conditions described
Executive summary:

The test item was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Four groups each comprising 5 males, received an intraperitoneal injection. Two groups were dosed with 500 mg/kg body weight, one group was dosed with 250 mg/kg body weight and one group was dosed with 100 mg/kg body weight.

After dosing, the animals of the dose levels of 500 and 250 mg/kg body weight showed the following toxic signs: lethargy, rough coat, hunched posture and closed eyes. The animals of the dose levels of 100 mg/kg body weight showed lethargy one day after dosing only. A vehicle treated group served as negative control (corn oil), a group treated with an intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with the test item was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item.

The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls.

It is concluded that the test item is not mutagenic in the micronucleus test under the experimental conditions described in this report.