Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 Jul 2003 to 17 Aug 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
February 1998
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Density: 1.05 g/cm3

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD, USA
- Age at study initiation: approx. 6-8 weeks
- Weight at study initiation: 27.0 — 32.3 g (males); 22.1 — 26.3g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: up to 5 of same sex
- Diet: Harlan 2018C Certified Global Rodent Diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.7
- Humidity (%): 50 +/- 20
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: based on a solubility determination of the test article and compatibility of the vehicle with the test system
- Amount of vehicle (if gavage or dermal): dosing volume 20 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing volume 20 ml/kg bw

Duration of treatment / exposure:
1 day
Frequency of treatment:
single exposure
Post exposure period:
24 and 28 h
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 for vehicle controls, 5 in low and mid dose groups and positive control, 15 in high dose group (including 5 replacement animals per sex to ensure the availability of five animals for micronucleus analysis)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow of femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on a pre-test

DETAILS OF SLIDE PREPARATION: The bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 ml fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted

METHOD OF ANALYSIS: Bone marrow cells, polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were analyzed for the presence of micronuclei
Evaluation criteria:
the test article was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat experiment would have been recommended. The test article was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.
Statistics:
Kastenbaum-Bowman Tables

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1, 10, 100, 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Clinical signs, which were noted an the days following dose administration, included: lethargy and piloerection at 1000 mg/kg bw and above

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): No appreciable change in the ratio of polychromatic erythrocytes to total erythrocytes relative to the vehicle control was apparent in the test article-treated groups suggesting that the test article did not inhibit erythropoiesis.
- Clinical signs of toxicity in test animals: Clinical signs, which were noted an the days following dose administration, included: lethargy and piloerection at all doses tested

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test item did not induce micronuclei in erythrocytes from bone marrow in mice.
Executive summary:

The test article, 1-Chloro-2,3-dimethylbenzene, was investigated in the mouse micronucleus assay. The definitive micronucleus study consisted of seven groups, each containing 5 male and 5 female ICR mice. Animals in five of these groups were treated either with the controls (negative or positive) or with 3-Chloro-o-xylene at a dose of 500, 1000 or 2000 mg/kg bw and were euthanized 24 hours after treatment. Animals in other two groups were treated either with the negative control or test item at 2000 mg/kg and were euthanized 48 hours after treatment. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs, which were noted on the days following dose administration, included: lethargy and piloerection at all doses tested. Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after, treatment, were examined microscopically for the presence of micronuclei. No appreciable reduction in the ratio of polychromatic erythrocytes to total erythrocytes was observed in the test article-treated groups relative to the vehicle control groups suggesting that the test article did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration.The results of the assay indicate that under the conditions described in this report, a singleintraperitoneal administration of 3-Chloro-o-xylene at doses up to 2000 mg/kg did not induce asignificant increase in the incidence of micronucleated polychromatic erythrocytes in eithermale or female ICR mice. Therefore, 3-Chloro-o-xylene was concluded to be negative in the mouse micronucleus assay.