4,7-dichloroquinoline

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Data source

Reference

Reference Type:

publication

Title:

Evaluation of the test chemical for genotoxicity potential in vitro and in vivo

Author:

Vinod et.al

Year:

2010

Bibliographic source:

Toxicology Mechanisms and Methods, 2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

In vivo micronucleus test was performed to evaluate the genotoxic potential of the test chemical

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Isoquinol-5-ylamine

EC Number:

214-408-3

EC Name:

Isoquinol-5-ylamine

Cas Number:

1125-60-6

Molecular formula:

C9H8N2

IUPAC Name:

Isoquinol-5-ylamine

Test material form:

solid

Details on test material:

Name of the test chemical: Isoquinol-5-ylamine
Molecular Formula: C9H8N2
Molecular Weight: 144.176 g/mol
Substance Type: Organic
Physical State: Solid

Test animals

Species:

mouse

Strain:

other: C57BL/6J

Details on species / strain selection:

C57BL/6J strain

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Healthy male, C57BL/6J mice bred and raised in the Animal facility of Glenmark Research Centre (Navi Mumbai, India) were used for the study
- Age at study initiation: age 5-7 weeks
- Weight at study initiation: weighing 20 ± 2 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing:The animals were housed in polycarbonate boxes (five per cage) with paddy husk for bedding,
- Diet (e.g. ad libitum): pelleted diet (Nutrilab, Bangalore, India), ad libitum
- Water (e.g. ad libitum): fresh RO water ad libitum
- Acclimation period: The mice were acclimatized for at least 5 days prior to the treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50–65% humidity
- Air changes (per hr): no data available
- Photoperiod (hrs dark / hrs light): 12 h dark/light cycle

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: [none; no data; acetone; air; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil ; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; water]: 0.5% methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): 10 ml/kg
- Storage temperature of food:

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

daily

Post exposure period:

no data available

Doses / concentrations

Remarks:

125 and 250 mg/kgbw

No. of animals per sex per dose:

5 male mice

Control animals:

yes, concurrent vehicle

Positive control(s):

none; no data; 2-acetylaminofluorene; 2-nitrofluorene; 3-methylcholanthrene; 4-nitroquinoline-N-oxide; 7,12-dimethylbenzanthracene; 9,10-dimethylbenzanthracene; 9-aminoacridine; benzo(a)pyrene; congo red; cumene hydroperoxide; cyclohexylamine; cyclophosphamide; cyclophosphamide; ethylmethanesulphonate; ethylmethanesulphonate; ethylnitrosurea; ethylnitrosurea; furylfuramide; ICR 191; methylmethanesulfonate; mitomycin C; mitomycin C; monomeric acrylamide; N-dimethylnitrosamine; N-ethyl-N-nitro-N-nitrosoguanidine; 2-nitrofluorene; 4-nitroquinoline 1-oxide; sodium azide; triethylenemelamine: mitomycin C
- Justification for choice of positive control(s):
- Route of administration: the positive control group was injected intraperitoneally with Mitomycin-C at 5 mg/kg.
- Doses / concentrations: 5 mg/kg

Examinations

Tissues and cell types examined:

The animals were killed and their bone marrow sampled at 24 or 48 h.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The doses of micronucleus test were based on the result of preliminary dose range finding experiments in which mice were given several doses of
single administrations by oral gavage at a dose volume of 10 ml/kg. Signs of toxicity were then recorded. A dose of 250 mg/kg was selected as the highest dose for the micronucleus test, being close to the maximum tolerated dose for single administration.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):The animals were killed and their bone marrow sampled at 24 or 48 h. The cells were centrifuged at 1500 rpm
for 5 min and resuspended in fresh FBS.
DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS: Smears of cells were made on microscope slides, which were then fixed and stained with May-Grunwald and Giemsa. Finally, the slides were air-dried, rinsed with xylene, and permanently mounted with DPX. A minimum of 2000 polychromatic erythrocytes (PCE) per animal was scored in blindfold slides under 100× oil immersion for micronucleated polychromatic erythrocytes (MNPCE). The ratio of PCE-to−normochromatic erythrocytes (NCE) was determined based on a total of at least 1000 PCE+NCE.


OTHER:

Evaluation criteria:

minimum of 2000 polychromatic erythrocytes (PCE) per animal was scored in blindfold slides under 100× oil immersion for micronucleated polychromatic erythrocytes (MNPCE).

Statistics:

Statistical significance instructural aberrations was analyzed by Fisher’s exact test. In the micronucleus test the number of MNPCE in each treated group was compared with the number in the vehicle
control group by one-way ANOVA followed by Dunnett’s test.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non genotoxic

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range:
- Solubility:
- Clinical signs of toxicity in test animals:
- Evidence of cytotoxicity in tissue analysed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): There was no increase of MNPCE in both 24 h and 48 h after both 125 and 250 mg/kg duration exposure as compared to the corresponding control.
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): The PCE/NCE ratio which was considerably reduced after the treatments with the high dose. Group mean values of micronucleated PCE were similar and not significantly different from the value for the vehicle control group, and fell within the laboratory historical negative control range
- Appropriateness of dose levels and route:
- Statistical evaluation:

Applicant's summary and conclusion

Conclusions:

There was no increase of MNPCE in both 24 h and 48 h after both 125 and 250 mg/kg duration exposure as compared to the corresponding control. Significant (p < 0.001) increases relative to the control were observed only in mice treated with the Mitomycin-C as positive control. The PCE/NCE ratio which was considerably reduced after the treatments with the high dose. Group mean values of micronucleated PCE were similar and not significantly different from the value for the vehicle control group, and fell within the laboratory historical negative control range. Hence, the test chemical can be considered to be non-genotoxic.

Executive summary:

In vivo micronucleus test was performed to evaluate the genotoxic potential of the test chemical. The study was performed according to OECD 474 Guidelines.Healthy male, C57BL/6J mice weighing 20 ± 2 g of age 5-7 weeks, bred and raised in the Animal facility of Glenmark Research Centre (Navi Mumbai, India) were used for the micronucleus test. The animals were housed in polycarbonate boxes (five per cage) with paddy husk for bedding, and maintained in a 12 h dark/light cycle at 22 ± 2°C and 50–65% humidity with access to pelleted diet (Nutrilab, Bangalore, India) and fresh RO water ad libitum. The mice were acclimatized for at least 5 days prior to the treatment. Groups of five male mice received single oral administration with the vehicle (0.5% methylcellulose) or the test chemical at 125 or 250 mg/kg, whereas the positive control group was injected intraperitoneally with Mitomycin-C at 5 mg/kg. The animals were killed and their bone marrow sampled at 24 or 48 h. The cells were centrifuged at 1500 rpm for 5 min and resuspended in fresh FBS. Smears of cells were made on microscope slides, which were then fixed and stained with May-Grunwald and Giemsa. Finally, the slides were air-dried, rinsed with xylene, and permanently mounted with DPX. A minimum of 2000 polychromatic erythrocytes (PCE) per animal was scored in blindfold slides under 100× oil immersion for micronucleated polychromatic erythrocytes (MNPCE). The ratio of PCE-to−normochromatic erythrocytes (NCE) was determined based on a total of at least 1000 PCE+NCE.There was no increase of MNPCE in both 24 h and 48 h after both 125 and 250 mg/kg duration exposure as compared to the corresponding control. Significant (p < 0.001) increases relative to the control were observed only in mice treated with the Mitomycin-C as positive control. The PCE/NCE ratio which was considerably reduced after the treatments with the high dose. Group mean values of micronucleated PCE were similar and not significantly different from the value for the vehicle control group, and fell within the laboratory historical negative control range. Hence, the test chemical can be considered to be non-genotoxic.