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:
weight of evidence
Study period:
7 September, 1991-5 February, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Followed US EPA guideline 84-2 and OECD guideline 474. There was a deviation regarding the time of sacrifice of one group of animals but it was not considered to affect the scientific validity of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 84-2
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Busan 11-M1
- Substance type: No data
- Physical state: White powder
- Analytical purity: 94.3
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No.: 1-9769
- Expiration date of the lot/batch: 01/11/1993
- Stability under test conditions: No data
- Storage condition of test material: Room temperature

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males 26.6 - 37.2 g, Females 22.8 - 26.7 g
- Assigned to test groups randomly: yes
- Fasting period before study: None
- Housing: Group housed up to 5 per cagein plastic cages with hardwood chips for bedding
- Diet (e.g. ad libitum): certified laboratory rodent Chow ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3 ± 14.4°C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

IN-LIFE DATES: From: 08/10/1991 To: No data

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: better handling characteristics
- Concentration of test material in vehicle: 1.13, 2.25, 4.5 mg/ml
- Amount of vehicle (if gavage or dermal): 10 mg/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A concentration of 4.5 mg/ml was prepared for use as the dosing solution for the high dose level. Dilutions were carried out in deionised water to yield concentrations of 2.25 and 1.13 g/ml for the dosing solutions for the mid and low dose levels, respectively. All test article stocks were vortexed to prepare homogeneous suspensions of the test article.
All mice were weighed immediately prior to dose administration and the dose volume based on individual body weights

Duration of treatment / exposure:
24, 48, 72 hours
Frequency of treatment:
Once
Post exposure period:
24, 48, 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
11.3, 22.5, 45 mg/kg
Basis:
other: calculated based on body weight
No. of animals per sex per dose:
15/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): No data
- Route of administration: IP injection
- Doses / concentrations: 3 mg/mg in water

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A pilot study where the test substance was administered by IP injection at 1, 10, 100, 1000 and 5000 mg/kg at 10 ml/kg and a toxicity study where the test substance was administered by IP injection at 22, 35, 56 and 90 mg/kg at 10 ml/kg were carried out. Based on the results the LD50/7 was calculated to be approximately 55.9 mg/kg. The high dose for the micronucleus test was set at 48 mg/kg which was estimated to be approximately 80% of the LD 50/7.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): No further details

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice, the femurs were exposed, cut just above the knee and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 ml foetal 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 pipette and a small drop of bone marrow suspension was spread on to a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol and stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly-staining nuclear fragments, having sharp contour with diameters usually form 1/20 to 1/5 of the erythrocyte. The number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes counted was also recorded.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each animal and treatment group. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent control was observed at any sampling time.
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.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at the highest dose level
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: In the pilot study animal treated with 10 and 1 mg/kg survived and appeared normal throughout the observation period.
- Solubility: was tested in water and corn oil. water was found to have better handling characteristics and therefore was chosen as the vehicle
- Clinical signs of toxicity in test animals: In the toxicity study, clinical signs included ataxia, lethargy and tremors.
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No further details

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically significant increase in the test article treated animals
- Ratio of PCE/NCE (for Micronucleus assay): No change was apparent in the test article treated animals

Any other information on results incl. tables

No further data

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, Busan 11-M1 did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test.
Executive summary:

In a study conducted by Putman and Young (1992), the test substance, Busan 11-M1, was examined for its ability to increase the incidence of micronucleated polychromatic erythrocytes in bone marrowwhen administered via IP injection to male and female ICR mice. The test animals were exposed to the test substance 11.3, 22.5 and 45 mg/kg bw at 10 ml/kg bw. They were then observed for 24, 48 and 72 days following treatment to determine the effects. Under the conditions of this study, Busan 11-M1 did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test.  Based on this result, the test substance does not require classification for genotoxicity according to Regulation EC No. 1272/2008.