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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study following OECD 474 and GLP. The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. A justrification for the read-across is provided.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
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
Type:
Constituent
Details on test material:
- Storage condition of test material: stored at room temperature protected from light and moisture

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: Mice were 6-8 weeks old for each phase of the study
- Weight at study initiation: Animal body weights were within the following ranges at randomization for each of these phases:
- Pilot phase: Males: 26.9-30 g, Females: 27.2-29.0 g
- Toxicity phase: Males: 24.7 - 32.2 g, Females: 25.1-29.0 g
- Definitive Micronucleus assay: Males: 26.9-32.1 g, Females: 24.7-29.0 g

- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: 5/cage in polycarbonate cages. Heat treated hardwood chips were used for bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 degrees F +/- 3 degrees F
- Humidity (%): 50% +/- 20%
- Photoperiod (hrs dark / hrs light):12 hour light/dark cycle

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn Oil form Sigma-Aldrich
Details on exposure:
The test substance-vehicle mixture, the vehicle alone, or the positive control substance were administered as a single intraperitoneal injection at a dose volume of 20 mL/kg body weight. Mice were observed after dose administration for signs of toxicity.
Duration of treatment / exposure:
The test substance was dosed once by an intraperitoneal injection.
Frequency of treatment:
The test substance was dosed once by an intraperitoneal injection.
Post exposure period:
24 and/or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250, or 500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5/sex/dose for the low dose, mid dose, and positive control. 10/sex/dose for the high dose and vehicle control. 5 additional animals/sex were dosed with the highest dose (500 mg/kg) to ensure the avaiability of 5 animals per sex per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate dissolved in sterile water at a concentration of 2.5 mg/mL for a dose of 50 mg/kg.

Examinations

Tissues and cell types examined:
Bone marrow cells, polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were analyzed for the presence of micronuclei.
Details of tissue and slide preparation:
Marrow Collection and Slide Preparation


Scoring for Micronuclei


CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
5-10 mice /sex dosed with either corn oil, 125, 250, or 500 mg/kg of the test substance in corn oil , or 50 mg/kg cyclophosphamide (positive control) were sacrificed after 24 hours and sacrificed for the evaluated of micronuclei in the bone marrow. An additional 5 mice/sex dosed with either corn oil or 500 mg/kg of the test substance were also sacrified after 48 hours for the evaluation of micronuclei in the bone marrow .

DETAILS OF SLIDE PREPARATION:
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 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 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:
Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei can occur in both PCEs (MPCEs) and NCEs (MNCEs).
2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of MNCEs 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 per 1000 erythrocytes.
Evaluation criteria:
The incidence of MPCEs per 2000 PCEs was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables, which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as indicator of bone marrow toxicity, the proportion of PCEs to total erythrocytes was determined for each animal and treatment group.
Statistics:
All conclusions were based on sound scientific judgement; however, as a guide to interpretation of the data, the test article was considered to induce a positive response if a dose-responsive increase in MPCEs was observed and one or more doses were statistically elevated relative to the vehicle control (p<=0.05, Kastenbaum-Bowman Tables) at any sampling time. However, values that were statistically significant but did not exceed the range of historical negative or vehicle controls were judged as not biologically significant. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response or if there was evidence of a dose response with no evidence of a significant increase in any treated group relative to the control group, the Sponsor MPCEs above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at highest dose tested
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the pilot study, all male and female 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.

In the toxicity study, mortality was observed in 2/5 males and 2/5 females at 1000 mg/kg and in all males and females at doses >=1000 mg/kg of the test substance. Clinical signs following dose administration included: piloerection, prostration and irregular breathing in males and females at 500 and 1000 mg/kg and lethargy in males at 1000 mg/kg of the test substance. Loss in body weight from 2-14% 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
No mortality occurred at any dose level during the course of the study. 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.

Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs or MNCEs). 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 MPCEs per 10000 PCEs in 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 (p>0.05, Kastenbaum-Bowman Tables). In addition, no appreciable increase in the number of MNCEs in the field of 2000 PCEs per animal was found indicating that an optimal differential staining was achieved.

Any other information on results incl. tables

Cyclophosphamide induced a significant increase in MPCEs in both male and female mice (p<=0.05, Kastenbaum-Bowman Tables). The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or the 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 did not exhibit any mutagenic activity in this in-vivo micronucleus assay. A justification for the read-across has been provided.