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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
only one treatment and one sampling after 24 h (no 2nd sampling after 48 h)
Deviations:
yes
Remarks:
one treatment only and only one sampling after 24 h (no 2nd sampling after 48 h)
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dichloro-4-nitrobenzene
EC Number:
202-764-2
EC Name:
1,2-dichloro-4-nitrobenzene
Cas Number:
99-54-7
Molecular formula:
C6H3Cl2NO2
IUPAC Name:
1,2-dichloro-4-nitrobenzene
Details on test material:
no data on purity
Specific details on test material used for the study:
Manufactured by Tokio Kasei Kogyo Co. Ltd., Japan

Test animals

Species:
mouse
Strain:
ICL-ICR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dae-Han Laboratory Animal Co., Eumsunggun Korea
- Age at study initiation: 7-8 weeks old
- Assigned to test groups randomly: yes
- Housing: six animals were housed for each group
- Diet (e.g. ad libitum): commercial pellets ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 10mL/kg
Duration of treatment / exposure:
The test substance was given once
Frequency of treatment:
Once
Post exposure period:
24 h
Doses / concentrationsopen allclose all
Dose / conc.:
692 mg/kg bw/day (nominal)
Dose / conc.:
346 mg/kg bw/day (nominal)
Dose / conc.:
173 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C, 2 mg/kg, i.p.

Examinations

Tissues and cell types examined:
bone marrow from both femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The rationale for the dose selection was the selection of half of the LD50 value as highest dose. The LD50 value from RTECS database for mice was 1384 mg/kg bw.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): From the freshly killed animal both femora 24 h after administration were removed in toto, which means that one was cutting through pelvis and tibia. The bones were then freed from muscle by the use of gauze and fingers. With the needle of appropriate size mounted, about 1mL of serum was pulled from the tube into a disposable plastic syringe. Then the needle (24 gauge) was inserted a few mm into the proximal part of marrow canal to flush the marrow cells.

DETAILS OF SLIDE PREPARATION: After centrifugation, the supernatant was removed, and cell pellet suspension of bone marrow cells was dropped onto glass slides, and then air dried. After fixation in methanol, slides were stained with 4% Giemsa in 1/15M sodium phosphate buffered saline (PBS, pH 6.8) for 30 min, washed with PBS, and then air dried for microscopic observation.

METHOD OF ANALYSIS: In scoring the preparations, micronuclei were counted in polychromatic and separately in normochromatic erythrocytes. The rate of micronucleated cells, expressed in percentage, were based on the total of polychromatic erythrocytes present in the scored optic fields. This mode of scoring, which must always be followed where the test substance markedly influences the proliferation rate in the bone marrow, prevents a distortion of the results by the influx of peripheral blood into the damaged marrow. The scoring of micronucleated normocytes not only serves to recognize the presence of artifacts (which is rare in preparations from mouse) but provides additional interesting information on the mode of action of the test substance. Generally, an incidence of more than 1 micronucleated normocyte per thousand polychromatic erythrocytes indicates an effect on cell stages past the S-phase.

Statistics:
pariwise comparison to corresponding control

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no decreased no. of immature erythrocytes observed, clinical signs of toxicity not described
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables






























































Test chemicalsDose (mg/kg)RouteSampling time (hr)MNPCE%/PCE (Mean ± SD)Ratio % of PCE/PCE+NCE (Mean ± SD)p-value
Negative control-i.p240.11±0.070.49±0.02-
-p.o240.14±0.080.50±0.1-
Positive control (Mitocycin C)2i.p243.42±0.790.50±0.010.0000
1,2-dichloro 4-nitrobenzene (99-54-7)692p.o240.17±0.060.48±0.02>0.05
346240.12±0.080.45±0.07>0.05
173240.13±0.160.43±0.03>0.05

pariwise comparison to corresponding control, significant at P < 0.05


MNPCE%/PCE: percentage of Micronucleuated polychromatic erythrocytes/1,000 polychromatic erythrocytes


PCE/PCE+NCE: polychromatic erythrocytes/1,000 erthrocytes

Applicant's summary and conclusion

Conclusions:
No significant induction ratio of percentage of micronucleated polychromatic erythrocytes/1,000 poly chromatic erythrocytes (MNPCE &/ PCE) compared to solvent control.
Executive summary:

Bone marrow micronucleus assay was performed in mice. About 7-8 weeks old male ICR mice were exposed to the test item via oral gavage. Doses of 692, 346 and 173 mg/kg bw/d were applied. The rationale for the dose selection was the selection of half of the LD50 value as highest dose. The LD50 value from RTECS database for mice was 1384 mg/kg bw. 24 h after a single substance administration, the animals were killed and the bone marrow from the femurs was analysed. No significant induction ratio of percentage of micronucleated polychromatic erythrocytes/1,000 polychromatic erythrocytes (MNPCE &/ PCE) compared to solvent control was observed.