Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): 2,4,6-Trimethyl Phenol (TMP, CAS # 527-60-6)
- Lot No.: 7/31/01
- Physical state: white solid
- Storage: Room temperature, protected from light, moisture, heat and ignition sources
- Purity: 80-93%

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
- Age at study initiation: 6-8 weeks
- Weight at study initiation: male 24.7 – 32.2 g, female 24.7 – 29.2 g
- Housing: five per cage per sex in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: Harlan TEKLAD certified Rodent 7012C, ad libitum
- Water: tap water, ad libitum
- Acclimation period: no less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity (%): 50 ± 20
- Air: air-conditioned
- Photoperiod (hrs dark/hrs light) : 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by a single intraperitoneal injection at a dose volume of 20 mL/kg body weight.
Duration of treatment / exposure:
Single injection
Frequency of treatment:
Single injection
Post exposure period:
24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250, 500 mg/kg
Basis:
other: concentration injected
No. of animals per sex per dose:
- Vehicle: 10
- Low dose (125 mg/kg): 5
- Mid dose (250 mg/kg): 5
- High dose (500 mg/kg): 10
- Positive control: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 2.5 mg/mL

Examinations

Tissues and cell types examined:
- All mice were observed after dose administration for clinical signs of toxicity.
- Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based upon the results of the toxicity study, the high dose for the micronucleus test was set at 500 mg/kg, which estimated to be the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES
Bone marrow was collected 24h after dosing in all animals and also after 48 hours in the vehicle and high dose group. At the scheduled sacrifice times, five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum.

DETAILS OF SLIDE PREPARATION:
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. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained (10 x 100). 2000 PCEs per animal were scored for the presence of micronuclei. The number of micronucleated NCEs in the field of 2000 PCEs was enumerated for each animal in order to assess the quality of the differential staining procedure. The proportion of PCEs to total erythrocytes was also recorded to quantify the proliferation state of the bone marrow.
Evaluation criteria:
Validity critertia: The mean incidence of micronuclei containing PCEs must not exceed 5/1000 PCEs (0.5%) in the vehicle control. The incidence of micronuclei containing PCEs in the positive control group must be significantly increased relative to the vehicle group (p≤0.05).
Evaluation criteria: The test article was considered to induce a positive response if a dose-responsive increase in micronuclei containing PCEs was observed and one or more doses were statistically elevated relative to the vehicle control. However, values that were statistically significant but did not exceed the range of historical negative or vehicle controls were judged as not biologically significant.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Pilot study: All mice at 2000 mg/kg were found dead on the day of dosing. Clinical signs, consisting of piloerection, were observed in males at 1, 10, 100 and 1000 mg/kg. In addition, loss in body weight of approximately 10% was observed in male mice at 1000 mg/kg. Due to mortality at 2000 mg/kg, a toxicity study was performed.
- Toxicity study: Clinical signs following dose administration included: piloerection, prostration, irregular breathing in males and females at 500 and 1000 mg/kg and lethargy in males at 1000 mg/kg. Loss in body weight from approximately 2% to 14% was observed in male and female mice at 500 and 1000 mg/kg. Based upon these results, the high dose for the micronucleus test was set at 500 mg/kg, which was estimated to be the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY
- Mortality and clinical signs: No mortality was observed in all exposed animals. Clinical signs following dose administration included piloerection in male and female mice at all doses and lethargy in males and females at 250 mg/kg. In addition, prostration and irregular breathing were observed in males and females at 500 mg/kg.
- Induction of micronuclei: No appreciable reductions in the ratio of PCEs 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. The number of micronucleated PCEs per 10000 PCEs in the test article-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time.
- Ratio of PCE/NCE: No appreciable increase in the number of micronucleated NCEs in the field of 2000 PCEs per animal was found indication that an optimal differential staining was achieved.

RESULTS OF CONTROLS
Cyclophosphamide induced a significant increase in micronucleated PCEs in both male and female mice. The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or quality of the test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this test, the test substance was concluded to be negative in the mouse micronucleus assay using ICR mice.
Executive summary:

In a GLP compliant mouse micronucleus assay according to OECD 474, ICR mice were exposed to the test substance by intraperitoneal injection. Five mice per sex were treated with 125, 250 or 500 mg/kg test substance, vehicle or positive control article and sacrificed after 24 hours. iIn addition, 5 mice per sex treated with the vehicle or 500 mg/kg test substance were sacrificed after 48 hours. No mortality was observed in any male or female mice. Clinical signs following dose administration included piloerection in male and female mice at all doses and lethargy in males and females at 250 mg/kg. In addition, prostration and irregular breathing were observed in males and females at 500 mg/kg bw. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes was observed in the test article-treated groups relative to the vehicle groups suggesting that the test substance 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 28 hours after dose administration. The positive control induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice. Therefore, the test substance was concluded to be negative in the mouse micronucleus assay using ICR mice.