Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with OECD GLP (1997) regulations.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Reference substance name:
MV31 K-Salt
IUPAC Name:
MV31 K-Salt
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): MV31 K-Salt
- Substance type: Mono-constituent
- Physical state: Solid (grey ganules)
- Analytical purity: Approximately 88%
- Purity test date: 12 December 2002
- Lot/batch No.: 1268147/1-1268154/1
- Expiration date of the lot/batch: 31 December 2002
- Stability under test conditions: Acceptable for 4 hours in deionized water
- Storage condition of test material: Darnkess at approximately 20C in a fume cupboard

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstresse 27, 33178 Borchen
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: Male mean: 256.2 g, Female mean: 179.9 g
- Assigned to test groups randomly: Yes, under following basis: Randomization schemes 2002.0025 and 2002.0026
- Fasting period before study:
- Housing: Five animals per cage in transparent macrolon cages (type IV) on soft wood granulate
- Diet (e.g. ad libitum): Rat/mice diet ssniff R/M-H (V 1534), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 C
- Humidity (%): 50%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30 January 2002 To: 01 February 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: Test article solubility
- Concentration of test material in vehicle: 0 (vehicle control), 80, 250, 800 mg/kg body weight
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test article was dissolved in deionized water
Duration of treatment / exposure:
The test article was administered twice at an interval of 24 hours via oral gavage.
Frequency of treatment:
The test article was adminstered twice at an interval of 24 hours via oral gavage.
Post exposure period:
Animals from each group were killed 24 hours after the second treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle control), 80, 250, 800 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Test system and endpoint
- Route of administration: Oral gavage
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:
Femur bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary dose range finding study was conducted

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The test substance was administered twice at an interval of 24 hours via oral gavage. Animals were killed 24 hours after the second treatment and femur bone marrow was harvested.

DETAILS OF SLIDE PREPARATION: Bone marrow was flushed into a centrifuge tube with culture medium containing Hanks solution and 10% FCS at a temp of 37 C. This suspension was mixed and centrifuged for 10 minutes at 1000 rpm. All but one drop of the supernatant was drawn off. For hypotonic treatment, approximately 5 mL of 0.075 M potassium chloride solution was added and suspended. This suspension was incubated for 10 minutes in a water bath at 37 C. Approximately 1.5 mL fixative (methanol: glacial acetic ace 4:1) was then added and the suspension was bubbled mixed by air with a Pasteur pipette. After re-centrifuging for approx. 10 minutes at 1000 rpm, all but one drop of supernatant was drawn off. The sediment was carefully covered with a layer composed of approximatley 2.5 mL fixative. After at least 20 minutes the fixation was carefully removed (after re-centrifuging, when needed) and the sediment was suspended in approximately 2.5 mL fresh fixative. If needed, the mixture was then centrifuged after another 30-60 minutes, after which the liquid was removed and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a refrigerator at approximately 4 C. After re-centrifuging for 10 minutes at 1000 rpm, all but one drop of the liquid was removed and a new suspension was formed with a Pasteur pipette onto cleaned and frozen slides. The drops were then breifly passed through a Bunsen flame. Afterwards, the slides were examined under a microscope and air-dried for at least 12 hours.
Staining:
-Staining for 10 minutes in approximately 2% orcein solution
-Rinsing 3 times in distilled water
-Rinsing twice in acetone
-Brief rinse in acetone/xylene
-Approximately 2 minutes in acetone/xylene
-Approximately 5 minutes in xylene
-Minimum 10 minutes in xyxlene
-Embedding in Entellan or Eukitt

METHOD OF ANALYSIS: Analysis and metaphase counting via microscope

OTHER:
Evaluation criteria:
Criteria for a positive response: The test compound is classified as clastogenic if there is a concentration-related increase in the number of phases with aberrations (without gaps) at one or more of the concentrations tested as compared with negative controls. The test compound is classified as clastogenic if there is a concentration-related increase in the number of phases with aberrations (without gaps). The test compound is classified as non-clastogenic if the test are negative.
Statistics:
A one-sided Fisher-Exact Test was used to check the validity of the study. The study was considered valid if the proportion of the mutation frequency in the positive control was significantly higher than in the negative control (p=0.05)

Results and discussion

Test results
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: 0-1000 mg/kg
- Solubility: Soluble at all doses
- Clinical signs of toxicity in test animals: A single female rat died at the 1000 mg/kg dose, 800 mg/kg was decided upon for the highest dose in the main study

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No aberrations were observed in the study
- Appropriateness of dose levels and route: Mortality was observed at 800 mg/kg (2/5 females). No aberrations were observed at any dose. Cytotoxicity was observed in the 800 mg/kg dose groups.
- Statistical evaluation: A one-sided Fisher-Exact Test was used to check the validity of the study. The study was considered valid if the proportion of the mutation frequency in the positive control was significantly higher than in the negative control (p=0.05)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Based on the results of this test, the test article is not clastogenic
Executive summary:

The chromosome aberration potential of the test article (grey granules, purity approx. 88 Fl.%, CASRN 496805-64-2, Lot: 1268147/1-1268154/1) was evaluated in the bone marrow of Sprague Dawley rats. This study was performed in compliance with OECD GLP (1997) and the German Chemical Law (2001). The test method was based on OECD 475 (1997), US EPA OPPTS 870.5385 (1998), and EEC Directive 2000/32, L136, B.12 (2000). T-7684 was prepared in deionized water (vehicle) just prior to dosing. Rats (5/sex/group) received two doses (separated by an interval of 24 hours) of 20 mg/kg cyclophosphamide (CP, positive control) or T-7684 at 0 (vehicle), 80, 250, or 800 mg/kg via oral gavage. Another group (5/sex) was similarly dosed with 800 mg/kg test article due to due clinical signs of toxicity that were observed in the first 800 mg/kg-treated group. Bone marrow was harvested at 24 hours after the second dose. Metaphases were examined for aberrations.  In the first 800 mg/kg-treated group, 2 females were found dead. Among animals treated at 800 mg/kg, the following clinical observations were noted: decreased motor activity, stilted gait, squatting posture, coat bristling, respiratory sounds, and blood-colored encrusted snout. These observations were noted in females starting at 24 hours after the first dose and in males starting at 24 hours after the second dose. The following macroscopic findings were observed among 800 mg/kg-treated animals: remarkably light spotted liver, flatulent stomach, and remarkable dark red-brown colored content of the gastrointestinal tract. No abnormal clinical observations or necropsy findings were noted in animals treated at 80 or 250 mg/kg. At 800 mg/kg, a reduced mitotic index (4.4%) was observed when compared to the negative controls (mitotic index 10.5%), indicating the presence of cytotoxicity. No polyploid metaphases or significant increase in the aberration rate excluding and including gaps was observed in any dose group. No significant increase in the number of aberrations was observed in any dose group. Marked increases in the number of chromosome aberrations were observed in positive control group animals, indicating that the test was valid. Based on the results of this test, the test article is not clastogenic.